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Learn How You Can Apply For More Scholarships
Did you ever wish you had known about something before it was too late? Yeah, us too.
Do you want to graduate college debt free? Do you want to spend your time studying and with friends instead of at a job? If you answered yes to any of those scenarios, then you don't want to miss this opportunity!
Even if you've previously won a college scholarship, the chances that it covered your entire tuition is low. Whether you've started college, are getting a Masters Degree, or even a PhD, there is still a chance to win more scholarships to help fund your education.
Don't say "I Wish I Knew" about your time in college ever again. Your future is in your hands, starting now.
Let's meet Kerry!
Kerry, is a Marketing Manager from Florida. She graduated from college with more debt than she'd like to mention. Having the opportunity to win scholarships would have enabled her to go through her college years with a lot more confidence in her future and a better start when she graduated.
What does Kerry have to say about it? "I bet everyone says the same thing, but seriously, ScholarshipOwl would have been an amazing way to get my life started. I wish I knew about it during my college years."
Let's Meet Adam!
Adam, is a Sales Manager from Washington. He left college with student loans. If he had known there was a chance that he didn't need to carry this burden, he would have definitely gone for it!
What does Adam have to say about it? "Wow, sounds like ScholarshipOwl would have changed my life. I wish I knew about it before I started college. Instead of paying back my student loans right now, I could be putting money down for my first apartment."
Let's Meet Miranda!
Miranda, is a Dentist from New Jersey. She graduated from College in 2006 with $65K+ in student debts.
What does Miranda have to say about it? "It's simple – I wish I knew about ScholarshipOwl before I started college. I wouldn't have so much debt today. But now, you have the chance to build your future."
Apply For Hundreds of Scholarships in One Application
Start applying for college scholarships today. You can be a high school student at the beginning of the application process, a current student, or applying for your Masters or PhD. You're still eligible to get not just one, but multiple scholarships in order to help cover your tuition.
You Deserve It!
We are offering everyone* a chance to get their hands on as many scholarships as possible this winter!
Don't Let Your Friends Miss Out!
Do some good for someone close to you! If you refer someone, you will be able to sign up and get automatically applied to the $1,000 'You Deserve It' Scholarship**.
FOMO when it comes to your college life is now a thing of the past. Register and see how many scholarships you can apply to with just one application.
*you must be applying to or currently in college to be eligible.
**no purchase or payment of any kind is necessary to enter or win the $1,000 'You Deserve It!' Scholarship
Disclaimer Text:
ScholarshipOwl is a personalized scholarship matching service and management tool. Applying and using the service does not guarantee the receipt of any scholarship. The people used in this video are actors and are not using their real names.


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One Awkward Identity Crisis/
You Guys! I'm seriously confused! Yesterday I took two different "Which Harry Potter Character Are You?" Quizzes and both came out cleanly as Harry. But that didn't feel right. I couldn't accept it! So today, desperate for answers, I tried again, only to find myself more lost than ever. The first test was inconclusive - telling me I'm either Luna Lovegood or Albus Dumbledore which makes NO sense becaue those two characters could NOT be more different and also (ALSO!) Dumbledore is dead (NEVER FORGET!) so, WTF, allthetests.com, you make no sense. So then I obviously had to take ANOTHER quiz and this time it told me I was Hermione, which like Yay!/Yay? because on one hand she's the best but on the other hand she's the total worst. And maybe I should have just listened to the original results, you know? I mean, Harry's the hero, that should make me happy. But somehow...I feel adrift. WHO AM I?
(And if you're thinking "Well, Liz, you're a real, adult human being who just spent the last 5 hours of her day trying to determine which fictional wizard character most represents her own personality and you should probably get a life and/or do your job, well you can just suck it and get off my blog.)
Happy Friday, Everyone! Accio Weekend!

subset: commoncrawl


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GAITHERSBURG, Md. (CBS.MW) -- An advisory panel to the U.S. Food and Drug Administration will recommend that SmithKline Beecham PLC's diabetes drug Avandia be approved for sale.
"The panel gave a strong endorsement to Avandia, saying the drug is safe and effective," said Jeremy Heymsfeld, a spokesman for SmithKline Beecham SBH, +2.78%.
"It voted unanimously that its benefits outweigh its risks," Heymsfeld said.
The FDA will decide at a later date whether it will approve the drug for sale.
The advisory panel, reviewing the safety of the drug Thursday, found no evidence that SmithKline Beecham PLC's diabetes drug Avandia causes liver toxicity, according to one analyst.
Analysts and investors have been waiting to see how the safety profiles of Avandia and another diabetes drug, Actos, developed by Eli Lilly & Co. LLY, +1.79% and Takeda Pharmaceuticals, compare with the Warner-Lambert diabetes drug Rezulin.
"The panel has found Avandia to be clearly safer where liver side effects are concerned," said Viren Mehta, an analyst at Mehta Partners.
Shares of SmithKlinerose 2 15/16 to 70 3/8.
An advisory panel is scheduled to review Actos on Friday.
Rezulin, which is used by millions to treat adult-onset diabetes, has been associated with potentially fatal liver damage.
The FDA recently recommended keeping Rezulin on the market, but the agency advocated restricting its use to combination therapy. See archived story.
As it continues its review, the FDA panel will assess Avandia's possible contribution to weight gain and to an adverse lipid, or cholesterol, profile, Mehta said.
In another development, the panel may not reach a conclusion on whether it'll back Lilly/Takeda's Actos Friday, Mehta said.
"Apparently, the advisory panel did not have a chance to get through all the data," Mehta said.
"The meeting is on, but it may not be conclusive," he said.


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In a shocking upset on April 15th, our resident Quiz Champions, the Upside Downers were knocked from first place by a new team – Schnitzel Schnatzel.
It was reported that throughout the night the scores were virtually neck and neck, with only a few points separating the teams after each round. After the last round, the decision came down to a tiebreaker – and Schnitzel Schnatzel snatched (try saying that 5 times fast!)a triumphant victory from the mighty Upside Downers!
Is this the start of a new revolution in our Quiz Nights? Will we see a new reigning champion emerge from the shadows? Stay tuned for more news, or come along on the next Quiz Night – May 20th at the Apollo Hotel Almere!


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Heather Witkop Hall | Yoga Instructor
When Heather first stepped into a yoga studio 10 years ago, she knew this was something magical. Ever since then, yoga has helped her to feel confident, empowered, and centered. The practice of yoga reminds Heather to be present in her relationships, and break through self-limiting fears in order to take control of her own life.
Heather completed her 200 hr teaching from Yen Yoga & Fitness in 2013, and has continued to be a student ever since.
She loves to incorporate pranayama and challenge poses into her classes. Heather's goal is to create a relaxed and safe environment where students can play, grow, and deepen their practice. Her hope is that students leave feeling balanced and inspired!


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Invest in a Hot Knoxville, TN Cookie Franchise Opportunity
Are you looking for a franchise opportunity in Knoxville, Tennessee? Do you want to enjoy the independence of being your own boss as well as the security of working with an established corporate support system? Are you a hard worker with drive to succeed? If you answered yes, Great American Cookies is looking for you! We are searching for someone to open our newest franchise in Knoxville, Tennessee, a city whose robust economy and useful business resources create a great location for a franchise. Knoxville has everything a Great American Cookies franchise owner is looking for; keep reading for more details about this exciting city.
When you open a Knoxville franchise, you will be able to benefit from the economy that was ranked #64 out of 200 on Forbes' list of the Best Places for Business and Careers. A huge factor in Knoxville's ranking is the low costs of living and doing business, both of which are substantially lower than the national averages. Your Knoxville franchise will benefit from these low costs, as having low overhead costs means that you have the potential for higher profits. In addition, Knoxville is growing; in 2014 alone, nearly 1,500 people migrated to the city and still unemployment managed to stay below 6%. This is because the job growth rate in Knoxville is high, keeping all the new people moving into the city employed. This growing population is a perk for your Knoxville franchise, which will directly benefit from a growing working population.
When you open a franchise in Knoxville, you won't be left on your own. The East Tennessee Economic Development Agency, or ETEDA, is an amazing resource that small businesses and franchises in eastern Tennessee can utilize. The ETEDA is a partnership between the State of Tennessee, Tennessee Valley Authority, and various local economic developers to create a network that franchise owners can use for assistance. Your Knoxville franchise will be able to get help with such things as market research, site selection, economic overview, utility reports, wage information, and more. In short, the ETEDA can assemble information that will help you decide where in Knoxville you should open your franchise, how much you can expect to pay for rent and utilities, wages your employees will expect, and more. The ETEDA is an invaluable resource for new businesses opening, and your Knoxville franchise will benefit from using this resource.
Join the Global Franchise Group Family
For inquiries, please complete the form below or
The time to invest is now! Knoxville's growth and low cost of business make it a great place for a Great American Cookies franchise.
If you're ready to invest in this dynamic and thriving city, click this LINK to learn the next step in opening your Knoxville franchise.


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trader joes
trader joes, particularly the one near my house.
i know you are not expecting to hear back from me, but you should. and while i could keep these thoughts to myself, i know i'm not alone in my sentiments and like any good ole mother, processing-out-loud is good for the mind.
so i'm writing to tell you good job. and thank you. though you are a small store and your aisles have capacity for half my family, you welcome us anyway. there are moments totaling all of 4 seconds when we form a complete line, kind of like a train, and other shoppers can get around us. but again, that's only 4 seconds of our 44 minutes in the store. on this particular day, the gentleman in scrubs waited for us to reform our "train" so he could go around. the black haired lady must have kids of her own because she gave me that 'i know the feeling, sister,' look.
but the best was the samples guy. and i use that description in the most adoring way. my kids love you. it's typically hit or miss with the appetites of my children. they don't like tofu or seaweed and turn their noses up to anything soupy. i, on the other hand, consider your food sample and communion size coffee cup a blessed treat. today was a win all the way around. pretzel bagels with cookie butter cream cheese. i know i just lost most of you there. because you are already grabbing the keys and headed to you nearby trader joes. and you should. my post will be here when you get back.
so this sample was amazing. and we clogged the entire area. because we are 4 kids and a mom and a shopping cart.
and it takes time to set out 5 samples. so we wait. and i don't drink black coffee so i spend extra time sprucing mine up a bit. and i let my 11 year old try some coffee because he thinks he's old and this communion size cup shouldn't make him addicted. and i get mom points for that. we get our samples and attempt to step aside to let others in on this treat, but then we're hogging the avocado area. we shift to block the milk and cream and butter area. it's basically ping pong.
and my 7 year old can eat and play ping pong and talk to the samples guy all at the same time. i can't do that.
"these bagels are good, but the cream cheese is really good on waffles," said the samples guy.
asher's totally listening to him now. his eyebrows raise.
"what? i love waffles!" asher says sort of loud.
"yeah, dude. blueberry waffles especially." said the samples guy, equally as loud.
"booh-yah!" asher shouts with muscle arm motions. "my favorite!"
"bomb-digitty, man!" said the samples guy while preparing more samples for other customers who were clearly in tune to the conversation.
the produce section from the butternut squash to the organic tomatoes sees the clog we have created and traffic is at a standstill. it's time to move on. with satisfied tummies and laughter, no doubt.
it's inevitable at this point. i will do my best to get what we need and clear the store so that more can come in, but this just takes time people. and it's 3 in the afternoon, so i was at least considerate of those who have had a hard day at work, and came early.
we gather around the indian frozen meals and we're not in the way here, so my daughter takes her time in thinking about her 3 food selections. these are stored like emergency foods in our home freezer for when the blain american meal doesn't look appealing to her. she's already planning them for the next 3 nights. hmmm...
i must confess that i tend to stock up on certain items. my thought is that i should get it while i'm here because it will be weeks before i am back. {this is where i am sorry. i shop at another store most of the time. it's just easier for me. but take heart. i've never written them a letter.} so i buy cans of your spaghetti sauce and bags of kettle corn and stashes of chocolate treats and jars of sauces and frozen bags here and there. i've basically just grabbed stuff that looks tasty, all with this delusional thought that i can never come back. but let's be real. it would be me and 4 kids and a shopping cart again and we all need some recovery time. so with 5 items on my shopping list and 19 items in my cart, we finally make it to the finish line. almost taking down a wine display and probably a few toes.
your folks in their hawaiian shirts always know where everything is. except okra. you don't carry okra. they never seem bothered that i brought everybody. they are always out with the people restocking and so happy. everyone i can recall seem to love their job at trader joes. that's something to be proud of. {i'll revisit this in a minute}
and then at the finish line, there's a wee wooden block. it would hold my 5 items, but not my 19. that's okay. i've got kids who love to discuss who's holding what items that don't fit on wee wooden block. i'm pretty sure i paid for everything though my children handed the clerk items from both sides of register.
my daughter, who's only been with us for 5 weeks, thinks every grocery store hands out stickers. or at least they should. she's always so happy when we are truly at the "store that hands out stickers." and while stickers are being handed out to all four kids, there's a discussion about wanting to work at trader joe's when they are of age. {see, they notice how happy every employee is!}
"sure you can work at trader joe's," i chime in. this means if i'm listening to the kids, i just missed what the clerk said to me.
"i can?" i think it was a 9 year old.
"yes!" and i think said 9 year old said it loud enough for samples guy to hear at the other end of the store.
see you real soon. my chocolate stash is already running low.
mom of 4 kids with a shopping cart


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1. City Of Nothing 4:250:00 / 4:25
2. Empty Shell 2:550:00 / 2:55
3. Complicated 4:130:00 / 4:13
4. On The Bottom 4:020:00 / 4:02
5. Stranger 3:460:00 / 3:46
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Maybe 3:210:00 / 3:21
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check our new video
Braii is an alternative music band formed in 2017 in Kyiv, Ukraine playing in post punk and indie rock genres with fresh tones of Eastern European song tradition.
The band's frontwoman Oksana Bryzgalova while being out of stage and studio talks about her work exclusively in the categories of destiny, not allowing any other future course of events except music.
Braii's worldview was partially influenced by the post-punk movement, started by Joy Division and the whole Manchester wave. Cure and other leaders of alternative rock, as well as the pioneers of the experimental scene - The Velvet Underground and the American singer and poetess Patti Smith with numerous followers of their musical and poetic radicalism, such as PJ Harvey, Mazzy Star, and others also took part in musical formation of Braii.
Band members:
Vocal, guitar - Oksana Bryzgalova, Bass - Aleksei Krivosheyev, Guitar - Andrei Shulakov , Drums - Slava Los
EP 'City Of Nothing'
A hard working young band has highly productive career results for a short period of time: in 2018 Braii released their debut EP "City of Nothing" on vinyl.
Crude, dense sound, low sensual voice, emotional and honest lyrics completely capture the attention of the listener. Stylistic references, citations and inspirations do not sound epigonistic, but on the contrary they make the record whole, well-adjusted, nostalgic and relevant at the same time.
""This is the quintessence of my life of the last seven years, absorbed by musical experiments, a symbiosis of my inner world and a reaction to what is happening outside""


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Sunny Ozell, 'I Saw the Light' [Exclusive Premiere]
Talented singer Sunny Ozell is sharing her interpretation of Hank Williams' "I Saw the Light" on her upcoming new album, but readers of The Boot get to hear it first in this exclusive premiere.
Ozell tells The Boot that it was important to her to re-create "I Saw the Light" because of the song's "inherent joy." She believes that the song speaks loudly still today because of "... the basic theme of redemption ... Who doesn't identify with that in some way?"
"If I was religious in any way, this is the sort of 'observance' I'd get into," Ozell adds.
Ozell's version of the classic tune will draw in sinners and saints alike: It's raucous and exuberant, and it'll lift the spirits of anyone who listens to it.
"We often play it live," Ozell says, "and people just adore it."
"I Saw the Light" will be included on Ozell's upcoming album, Take It With Me, which is due in the U.S. out on April 1 (following its 2015 release in the U.K). In addition to original material, the record is full of her interpretations of other lesser-known songs, and Ozell is optimistic that listeners will discover some rarities from the collective American songbook. She describes the project -- which was recorded live in four days -- as "reverent, honest and sincere."
With her music, Ozell blends influences from the blues, jazz, classic country, gospel and roots genres. By the age of four, the singer was studying the rigorous Suzuki method of classical violin, perfecting her ear training, and at 10 years old, she joined the Reno Opera Company.
"My understanding of my instrument is quite thorough at this point," Ozell explains. "I know the shape of it, the scope of it, so I can approach my work with confidence and lean into the expression that much more."
Listen to Sunny Ozell, "I Saw the Light":
More Americana, Alt-Country and Folk Acts to Watch in 2016


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Yoga Mat Poses
A lot of Yoga poses actively work as stretches, and luckily, most of these stretching poses are simple and easy to perform.
Not only great for your body, they're also a great warm up. Next time you're feeling a little stiff, follow this step-by-step guide, and you'll be feeling loose and limber in no time.


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Every six months or so we meet with one of our grantees to talk with them about the brilliant work they're doing, in a little more depth than time usually allows. It's always completely inspiring. Click on the image above to find out more about the fabulous work that Rowan - one of the projects we supported in our fourth round of funding - is doing…Read More
Our next round of funding is live!
It's Tuesday 18th September, and we're delighted to announce that we're open again for funding applications! This is our sixth round of funding since we launched in 2013, and if you're creating a project using sound or music for social good that needs funding, we'd love to hear from you.Read More
We've been featured in The Observer & NESTA's #50radicals list!!
THIS IS BRILLIANT! Our founder, Andrea - who still runs Dosoco along with Alex & Kirsten - has been listed in the The Observer as one of '50 new radicals' whose ideas are changing the world.
New Radicals is a search led by Nesta, the innovation foundation, and The Observer to find the top people, projects and organisations offering innovative ways to tackle social challenges & make the world a better place. It was launched in 2012 and runs every 2 years, and the 2018 announcement was made on Sunday 16th September in the Observer newspaper and online… you can see the Observer article here, and the full and more detailed Nesta story here.
Andrea has created three growing businesses within the arts across Cambridge. The first, running since 2010 and the reason that Dosoco came to be, is We Are Sound, which delivers extraordinary sound experiences to the general public, using the general public. The group has recently finished a run of 19 sell out sound experiences totally in the dark, garnering rave reviews and being described as "One of the stand out experiences of my life", "wonderful, moving, original, exceptional, beautiful", and "one of those gigs I will take with me to my grave". Check out the below video of those dark, haunting gigs!!!
The second is the new arts brand House of Cambridge, with its first immersive event, UNITED! - described as a "1940's immersive hangar dance on steroids with a UK film premiere in the middle, plus cocktails" - taking place at the IWM Duxford on Saturday 29th September. Andrea's vision for House of Cambridge (which she has co-founded with commercial whizz Jenny Nelder) is to encourage & draw together the city's existing, brilliant, creative brands, festivals and thinkers to create extraordinary events that would be impossible without this collaboration, and then to add some extra magic on top. The film being premiered is WWII movie Magpie, shot entirely in East Anglia and directed by Paul Cook, and you can find out more about the event here.
But the third business of course is us!!! In 5 years, we've given away just under £20K to over 30 projects across Cambridgeshire using music & sound for social good. The amounts may not seem large - between £300-£1k per project - but every single penny makes a difference and helps change people's lives for the better. And, though we're just a tiny team of volunteers, we have bags of enthusiasm and a finely-tuned process of running our grant rounds, so we are going to continue doing this for as long as we can, and give out as much money as we can.
And actually the timing of this recognition of Dosoco's work is perfect, as tomorrow we're announcing our sixth round of funding (keep an eye out here for details). How cool is that :)
Drum roll for grant round number 5
Are you involved in creative music making that makes a positive impact on people's lives, or looking for extra funding to start new projects or extend your work? If you're based in Cambridgeshire and you know of or run a local, music-based, project that needs support, we want to hear from you.Read More
£500 raised for Dosoco through the Cambridge Half Marathon
We are HUGELY grateful to the lovely Claire Debenham for running the Cambridge Half Marathon for us in March and raising a whopping £500 as a result, not to mention the fact that she was just recovering from a bout of nasty flu!Read More
Our first 'insider' case study
A Dosoco grant in 2015 enabled Cambridge Community Arts to expand its range of courses for adult learners to include guitar lessons. To date 60 people have benefited from the opportunity, with the grant supporting two of our key aims – music access and music education. In our first 'insider' case study, we take a look at the brilliant work that CCA carries out, and how our funding has helped.Read More
Our 2016 grant awards
We've just awarded six more grants, totalling over £3000, to Cambridgeshire based projects using music for social good. Click through to find out more about them and the fabulous work they're doing. And it looks like we may have just raised another £1500... more on that soon!Read More
We've been on TV
We were massively chuffed to be featured on Cambridge TV on Weds 30 March about our fourth round of grants... and delighted to hear from Beth McCabe of Cambridge Community Arts - one of the projects we funded. Click through to find out more and see the short video.Read More
Round 3, done :)
A project using sound-enhanced tennis balls to enable visually impaired people to play tennis is just one of four fantastic Cambridgeshire-based projects that we've been lucky enough to support through our third round of Dosoco funding - announced at the latest Dowsing Sound Collective gig on 25 July (pictured) - with a combined total of £2000. Click to find out more!Read More
Funding available for young musicians
We're delighted to now be open for applications for our third round of funding… and this time we're focusing on young people. Schools, families and individual young people are all encouraged to take part and applications can be as creative as possible.Read More
Our second round of grants!
We were absolutely thrilled to announce - on Sunday 14th December at The Dowsing Sound Collective's Christmas Cocktail gig in the Cambridge Corn Exchange - the recipients of our second round of grants, totalling just under £4000!Read More
Our first ever grants
We're absolutely delighted to be able to announce that we have selected and awarded our first ever round of grants! With a total of just under £3000, we are supporting five different projects, each using sound and music to 'unlock the world'. We're very proud.Read More


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About Shirts That Go
"We are here to provide to you a great line of kids t-shirts for the vehicle lover in your family. We ship within the US and Canada standard + Overseas (upon request). We have several kids train t-shirts, fire truck t-shirts, truck t-shirts, tractor trailer t-shirts, bulldozer t-shirt, motorcycle t-shirt, and a garbage truck t-shirt. For the kids and toddlers that love the trash trucks, sanitation trucks, and garbage trucks we finally have that perfect tee for you!! We are wrapping up new set of tees with a farm vehicle theme in response to the emails we receive asking about doing a farm tractor. There will be a great kids tractor t-shirt in the lineup very soon!! We recently completed the kids combine harvester t-shirt and it is ready to go!
Note that we have recently added the long sleeve t-shirts to our line up! Any of our designs can be ordered in long sleeve now! For the long sleeve we use a wonderful shirt from American Apparel which is very soft and comfortable."
Note that we have recently added the long sleeve t-shirts to our line up! Any of our designs can be ordered in long sleeve now! For the long sleeve we use a wonderful shirt from American Apparel which is very soft and comfortable."
For my review, I received the Pirate Ship T-Shirt in size 2 from Shirts That Go. I was taken aback when I took the shirt out of the package. The pirate ship has intricate details and elaborate design. The shirt itself is soft and not scratchy! It is specifically designed for the little pirate in your family! 😛
A realistic design of a classic Spanish Galleon flying the Jolly Roger! It is well printed on a 100% cotton tee! The pirate ship t-shirt is a really cool kids tshirt design inspired by the pirate ships we love and pirate lore that is a part of our culture. There is something magical about pirate stories and thoughts of those times when the world was a much larger and mysterious place. Nothing quite makes kids think of adventure like tales of pirates and the high seas.
It is made from 100% American ring spun cotton and made right here in the USA by American Apparel. Printed in USA using direct to garment (DTG) printing technology. With DTG the printed image is just as smooth as the rest of the unprinted areas of the shirt!
This would be perfect for the little boy in your life…but will also please the tomboy in your life, as well. My daughter is girly, but she also loves boy-ish things. After she grows out of this t-shirt, I can store it away for my future son!
Purchase It:
You can purchase the Pirate Ship T-Shirt for $16.99!
That's a pretty reasonable and affordable price!
Disclaimer: I received the above product(s) from Shirts That Go free of charge in exchange for an honest review. No other compensation was received. However, all opinions are strictly my own.


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Darren72 wrote:That's a lot of yeast.
RonJS wrote:Darren72 wrote:That's a lot of yeast.
That's True!
I bought that exact same brand (1 pound bag) of yeast last week and really don't bake all that much.
When that pound of yeast only cost me $2.39, (at the Restaurant Depot in Alsip http://www.restaurantdepot.com ) I don't mind....
Pie Lady wrote:I bought a strip of yeast yesterday and forgot it in the car overnight. Do you think the temperature affected it aversely?


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What is a travel vaccine and can you claim it on health insurance?
Australians love to travel. While many diseases have been controlled or eliminated in Australia, some countries we choose to visit can pose a threat to our health.
A vaccine is a dead or weakened version of a virus or bacteria that is introduced into the body. It prepares your immune system to fight and overcome the disease if you become infected with it in the future.
Most Australian travellers will need to be vaccinated, particularly the young, the elderly, pregnant women and those with chronic health conditions. The types of vaccines you need will depend on the overseas destinations you are planning to visit.
Can I claim travel vaccinations on my private health insurance?
While Medicare covers the consultation fees for your vaccination, it does not cover the vaccines themselves other than those for non-travel-related purposes (eg, influenza shots).
If you travel frequently, you may want to look into private health insurance that includes cover for travel vaccinations as part of your extras cover. Or check if you have it already in your current policy. For example, ahm Super Extras covers up to $60 for each item above the general patient PBS amount.
How much do vaccinations cost?
Vaccination costs vary depending on the type of vaccine and the administrator. Single vaccines can range from $45 to $85 each, with new vaccines costing more. You may need follow-up doses, which is why it is important to get your vaccinations at least six to eight weeks before you plan to travel.
What vaccinations might you need?
The types of vaccines you need depends on a number of factors:
- Where you are going
- How long you will be travelling
- The season you will be travelling
- Where you will be staying
- What you will be eating
- Your age and general health
The diseases you are most likely to encounter when travelling in developing countries or tropical regions include:
- Cholera. Cholera is a bacterial infection that causes severe diarrhoea, vomiting and dehydration. It can also be fatal. Cholera vaccine is a liquid taken orally in two doses one and six weeks apart.
- Hepatitis A. Hepatitis A is a virus that causes liver disease. Vaccination involves two injections at least two weeks prior to travelling.
- Hepatitis B. Hepatitis B is a virus that causes liver disease and cancer. Vaccination consists of three injections over the course of a month.
- Japanese encephalitis. Japanese encephalitis is a virus transmitted by mosquitoes that causes swelling of the brain, convulsions and coma. The vaccine is a course of three injections over four weeks.
- Meningococcal meningitis. Meningococcal meningitis is a bacterial infection that causes thinning of the lining around the brain and spinal cord. Vaccination involves a single injection before travelling.
- Rabies. Rabies is a virus that is transferred by animal bites and scratches, which causes inflammation of the brain and spinal cord. Vaccination is three injections over the course of a month.
- Typhoid. Typhoid is a bacterial infection that causes extreme fever, headache and diarrhoea. The vaccine is a single injection or three capsules taken two days apart.
- Yellow fever. Yellow fever is a mosquito-borne virus that causes bleeding, jaundice, and kidney and liver failure. The vaccine is a single injection given at least 10 days prior to travel.
- Malaria. Malaria is an infection transmitted by mosquitoes that causes fever, headache, vomiting and disruption of the blood supply to vital organs. There is no vaccine, so you should take preventative measure to avoid mosquito bites.
Countries you may need vaccinations for
Because local destinations in South East Asia and the South Pacific cost less for Australians to visit, countries where we require vaccinations can include:
- Indonesia. Vaccinations are essential for visitors to Indonesia and Bali. Recommended vaccines include Hepatitis A and B, influenza and typhoid. Rabies, Japanese encephalitis and cholera vaccination may also be required for higher risk travellers.
- Thailand. While most of Thailand is low risk, vaccinations for Hepatitis A and B, typhoid and influenza are recommended.
- India. A high-risk location with vaccinations recommended for many of the main diseases, including rabies and Japanese encephalitis.
- Vietnam. Vaccination for common diseases such as Hepatitis A and B, typhoid and influenza are recommended and also Japanese encephalitis with certain travel itineraries.
- Cambodia. Cambodia is a medium-risk location with vaccinations recommended for common diseases such as Hepatitis A and B, typhoid and influenza plus Japanese encephalitis for higher risk travellers.
- China. Vaccination is recommended for Hepatitis A, influenza and possibly typhoid, plus rabies, Japanese and tick-borne encephalitis, and cholera, depending on the time of year and the parts of China you're visiting.
- Fiji. Some vaccinations are recommended for those heading to Fiji including Hepatitis A and B and influenza, plus typhoid for visitors to some of the more remote islands.
Are there any side effects?
Even though the Therapeutic Goods Administration (TGA) approves all vaccines used in Australia, there is always a small possibility of side effects occurring in some travellers for whom vaccines may not be appropriate.
These can include people with weakened immune systems due to infection, those taking medicines to suppress their immune system and people who are allergic to particular types of vaccines or any of their ingredients.
Generally, any side effects experienced from vaccination are very minor and short-lived. Symptoms may include mild fever, tenderness at the site of an injection, headache, nausea, tiredness and muscle or joint pain.
Compare extras policies from Australian funds
Picture: Shutterstock


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Foreign pension checking service
Foreign pension checking service
We provide a pension confirmation service for British Nationals who are drawing a pension from a foreign country
To get your form stamped and signed by us, come to the Register Office between 9am and 11am Tuesdays or Thursdays.
The address is: Old Council House, Corn Street, Bristol, BS1 1JG. The entrance is on Broad Street. You can find an access guide on the DisabledGo website.
You'll need
We need two things. Bring with you:
- proof of address, like a utility bill from the last 3 months
- a valid passport or a photo driving licence
This service costs £10. You must pay when you're at the Register Office.
Terms and conditions
See our Register Office terms and conditions (pdf, 295KB) (opens new window) .


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The Grand Mufti of Saudi Arabia has ordered "sinful" statues depicting horses to be demolished from the centre of a roundabout in the kingdom's far southwest.
(Above statue is not the actual one that was demolished in the photo below. Couldn't find the real one)
Arabian Business Sheikh Abdul Aziz Al-Sheikh, the kingdom's top cleric, ordered the governor of Jazan, a coastal city, to smash the statues because they are forbidden in Islam.


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Updated: February 13, 2007 11:36:12 AM
Type: Adware
Publisher: Mindset Interactive, netpalnow.com
Risk Impact: Medium
File Names: Netpal.dll,or variants of this file name,such as N3tpa1.dll.
Systems Affected: Windows
Adware.Netpal is a Browser Helper Object that tracks Web-browsing habits, and may display targeted pop-up advertisements.
- Existence of any files detected as Adware.Netpal.
- Existence of registry keys that adware created (see the "Technical Details" section for details).
- Internet Explorer may be unusually slow or unstable.
Must be manually installed, or as a component of another program. It may be bundled with other adware applications, such as Adware.FOne.
Antivirus Protection Dates
- Initial Rapid Release version October 02, 2014 revision 022
- Latest Rapid Release version May 07, 2019 revision 006
- Initial Daily Certified version December 18, 2003
- Latest Daily Certified version May 07, 2019 revision 008
- Initial Weekly Certified release date December 23, 2003
Click here for a more detailed description of Rapid Release and Daily Certified virus definitions.
Technical Description
When the Adware.Netpal .dll file is loaded, it performs the following actions:
- Creates the following registry keys:
- May create the registry key:
to store configuration data.
- Monitors Web-browsing habits, including visited URL's.
- Periodically contacts a server to send the information it collects, download configuration data, or download advertisements.
- May query the registry for personal information, including your email addresses as configured in Microsoft Outlook.
- May change the default Internet Explorer home page and search page.
The following instructions pertain to all Symantec antivirus products that support Security Risk detection.
- Update the definitions.
- Close all open browser windows.
- Unregister the N3tpa1.dll file.
- Run a full system scan and delete all the files detected as Adware.Netpal.
- Delete the keys that were added to the registry.
1. Updating the definitions
To obtain the most recent definitions, start your Symantec program and run LiveUpdate.
2. Closing all the open browser windows
Since Adware.Netpal functions as a Microsoft Internet Explorer plug-in, it is necessary to close all the open browser windows to remove it. If you are reading this writeup in Internet Explorer, print it using our printer friendly option at the top of the page, or write down the following instructions, and then close all the open browser windows.
3. Unregistering the n3tpa1.dll file
- Start Norton AntiVirus.
- Run a full system scan.
- If a .dll file is detected as Adware.Netpal, write down the file name, but do not attempt to delete it at this time.
- Click Start, and then click Run. (The Run dialog box appears.)
- Type, or copy and paste, the following text:
Note: If the file name of the .dll file that was detected as Adware.Netpal is different than n3tpa1.dll, make the appropriate substitution when typing the command that follows.
regsvr32 /u "n3tpa1.dll"
then click OK.
- If a dialog box confirming this action appears, click OK.
4. Scanning for and deleting the files
- Start Norton AntiVirus and make sure that it is configured to scan all the files. For more information, read the document, "How to configure Norton AntiVirus to scan all files."
- Run a full system scan.
- If any files are detected as Adware.Netpal, click Delete.
Note: If your Symantec antivirus product reports that it cannot delete a detected file, write down the path and file name. Then use Windows Explorer to locate and delete the file.
WARNING: Symantec strongly recommends that you back up the registry before making any changes to it. Incorrect changes to the registry can result in permanent data loss or corrupted files. Modify the specified keys only. Read the document, "How to make a backup of the Windows registry ," for instructions.
Note: This is done to make sure that all the keys are removed. They may not be there if regsvr32 removed them.
- Click Start, and then click Run. (The Run dialog box appears.)
- Type regedit
Then click OK. (The Registry Editor opens.)
- Navigate to and delete the following keys:
- Exit the registry editor.


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Start watching with your public library card or university login
Gifts From The Mountain - Tools for Dealing with Life's Complexities
We live in a non-stop world where the demands of a hectic life force us to move faster and faster. Join author Eileen McDargh in Gifts from the Mountain, where she discusses six fundamental truths to help us deal with life's complexities.
Concepts from the film include:
- Look For Viewing Points
- Every Ounce Counts
- Don't Cross The Creek Until You Come To It
- Watch For Wild Onions
- The Easy Trail Can Be The Most Dangerous
- Celebrate How Far You've Come
We all climb some kind of mountain every day. By slowing down and considering our surroundings, we can better appreciate the journey, and lead more productive, positive and purposeful lives.


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Complete a primary source analysis of this image using the Library's primary source analysis tool. Then click the image above to see if the information found in the curator's note there answers any of your questions. paper (.pdf) digital padlet Find out more about the woman in this image by investigating this related primary source set. Were you able to find answers to any of your analysis questions? Which ones? Describe what else you learned. Can you find a discrepancy between … [Read more...]
Featured Source: Woman fingerprinted
February 24, 2015 by Leave a Comment


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Meeting with author is an affair to remember at the WI
Members of Hepscott WI enjoyed an entertaining evening with contemporary romantic novelist Daisy James.
Daisy began by leading us through the stages of writing a novel to its final publication, from developing the characters and swift flowing plot lines to a happy ending.
She admitted that in visualising her heroes it always seems to be George Clooney who springs to mind.
In her latest novel, to be published in May, members may recognise WI Adviser Jenny McLean, who won a competition to appear as a character.
Daisy read an extract from her recent book If The Dress Fits, and answered the many questions asked by her audience.
She ended her talk by leading us through a fun literary bingo game based on her own version of Cinderella and featuring a lot of numbers.
On Tuesday, Dr Ray Lowry, retired doctor, dentist and comedy script writer, provided rib-tickling laughter connected with Funny Bones And Wisdom Teeth.


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We are having one of those days that I love. It rained over night, which meant no need to water. Throughout the day, clouds gather, a gentle rain falls, the sun comes out...rinse, repeat - over and over again. While the humidity is high, the temperatures are moderate, so lots can get done.
Today was pruning and tying day, and also offered a chance to take stock on progress. The news is largely very good, but as with all gardens and all seasons, there is a spark of sadness here and there. I wonder how many of you find gardening to be as emotional as I do?
As of early afternoon, all plants are appropriately tied to their supporting stakes. All damaged foliage has been removed. All plants have been assessed and graded, and the information now resides in my seasonal Excel spreadsheet that represents my garden log. The first ripe tomato awaits consumption on our kitchen counter; it is an early set fruit on one of our dwarf cherry tomato candidates in the Teensy line.
We are off later this week to Burlington, Vermont, for my speaking appearances at the Mother Earth News Fair, taking on a few days vacation on either end (it is so, so hard to leave the garden at this time of year, but it will be in the capable hands of neighbors for watering).


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Today, after a glorious career that includes two beautiful children, Mark's ability to procreate was officially retired. That's right, folks, my husband loves me so much he went under the knife just so I'd stop having nightmares about unplanned pregnancy. The other night I didn't get any sleep because I kept dreaming that I woke up 12 weeks pregnant. With twins. Boys. GAH! The dreams got so "real" that I finally vaulted out of bed at six am to take a pregnancy test. The single pink stripe sent me back to bed where I actually slept peacefully for a while.
Not that I don't think babies (even of the boy variety) are lovely, I just don't want any more children. And since we are in total agreement on this point, we decided to make it official. He claims that it really wasn't that bad, with just some discomfort. There was a moment when the doctor picked up the knife to begin and he thought, "do I really want to do this?" and then he thought of me in labor and the stupid cyst ER incident and thought, "After all she's been through, I can handle this." And so it is done. He's hanging out on the couch with a big bowl of ice cream as I type.
Last night while helping Lily brush her teeth, I was explaining to her that Daddy would be having a little surgery. This meant that when she came home from Grandma's house, she'd need to be very gentle with him as he'd have an owie. "It'll take a couple of days to heal and you'll need to be very nice to Daddy so it doesn't hurt too much." I explained.
"Daddy will have an owie?"
"A big owie?"
"It'll be a small cut, but it might make him hurt for a couple of days."
She thought about this for a moment and then nodded her head in a final kind of way, "I'm gonna kiss it."
So glad I hadn't just taken a sip of water; it would have been a perfect moment for a spit-take.
When I told my mom this story she made an excellent suggestion. "I think Mark's gong to need a band-aid on his knee."
Yes indeed, Grandma. Yes indeed.


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Emily has been an active exhibitor in the Southern California dressage community since a young age and is currently training out of Circle P Ranch in Escondido, CA. She is a recipient of numerous CDS and USDF awards as well as a member of the 2007 gold medal team at the North American Young Rider Championships. She has experience at all levels, from the developing horse through Grand Prix. She also has a B.S. in Animal Science from Cal Poly Pomona giving her an extensive knowledge of proper equine care. Emily welcomes all levels and breeds and looks forward to helping you and your horse achieve your goals. Full, half, and quarter training options are available and if you do not own a horse yourself, she even has a lesson horse available!
For more information please visit her website at www.emilytearsdressage.com or you can contact her via phone (949) 233-3171 or email her at email@example.com.


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Enterotoxigenic Escherichia coli (ETEC) remains a formidable cause of diarrheal illness worldwide. At present, there is no vaccine that provides broad-based protection against ETEC. A ′phoA-based self-cloning mutagenesis system, TnphoA.ts, employed to identify novel ETEC surface antigens, led to identification of an ETEC two-partner secretion locus (etpBAC) on the pCS1 virulence plasmid of prototype strain H10407. Cloning and expression of etpBAC in recombinant E. coli LMG194(pJY019) resulted in secretion of a high-molecular-weight (HMW) glycosylated exoprotein. This glycoprotein, EtpA, exhibits linear peptide sequence and predicted structural homologies with known HMW adhesins produced by other two-partner secretion loci. Antibodies directed against recombinant EtpA (anti-rEtpA.6H) recognized an HMW protein in culture supernatants of ETEC strains H10407 and LMG194(pJY019) but not in culture supernatant of strain H10407-P, which lacks the 92-kb pCS1 plasmid, or an isogenic etpA mutant. etpA mutants were deficient in adherence to intestinal epithelial cells in vitro, and anti-rEtpA.6H antibodies inhibited association of H10407 with target epithelial cells. Cloning and expression of etpB in recombinant E. coli were sufficient to confer adherence. Screening of multiple ETEC isolates for the etpBAC locus by colony hybridization and by EtpA immunoblotting suggested that EtpA is one of the most common antigens secreted by these pathogens. Together, these results indicate that the newly identified ETEC two-partner secretion locus directs the secretion of a high-molecular-weight glycosylated protein, EtpA, that in concert with the putative EtpB transporter participates in adherence of H10407 to epithelial cells, thereby expanding the repertoire of potential ETEC virulence proteins and vaccine candidates.
Enterotoxigenic Escherichia coli (ETEC) remains one of the principal causes of infectious diarrhea in travelers and in children in developing countries (72). ETEC is also an emerging cause of diarrheal illness in industrialized countries, including the United States (3, 6, 75). The organisms belonging to this taxon have genetically diverse E. coli pathotypes characterized by the production of heat-labile enterotoxin and/or heat-stable enterotoxin. In the classic paradigm of ETEC infection, these organisms adhere to the small intestinal mucosa via fimbrial colonization factor (CF) molecules, where they elaborate heat-labile enterotoxin and/or heat-stable enterotoxin. While early studies indicated that immunization with CFs provides protection against ETEC infection, development of a broadly protective vaccine has been hampered by the antigenic heterogeneity of these molecules. However, the recent identification of additional ETEC surface molecules (32, 56, 68) suggests that the present pathogenesis paradigm is incomplete and consequently that there may be additional antigens that can be exploited in vaccine development.
Surface expression of virulence molecules by ETEC and other gram-negative pathogens requires export through the inner and outer membranes. In general, gram-negative export of virulence proteins occurs via a number of different types of secretion systems (types I to V). These systems range from very complex multimeric structures that require coordinated assembly of multiple proteins to simple systems consisting of a single protein, such as the autotransporters (type V) (42). Autotransporters consist of three essential domains: an amino-terminal signal peptide, a passenger domain, and a carboxy-terminal transporter. In classical autotransporter molecules (type Va) the carboxy-terminal transporter is thought to form a hydrophilic channel composed of β strands in the outer membrane. This pore in the outer membrane is required to export the amino-terminal passenger domain to the bacterial surface. To date, two autotransporters, TibA and EatA, have been described in ETEC (27, 68).
A second set of type V secretion molecules (type Vb) are also involved in the secretion of large virulence proteins from gram-negative pathogens. In these systems the secreted exoproteins (generically referred to as TpsA proteins) and their requisite transporters (TpsB) are encoded on separate genes. Therefore, they have been referred to as "unlinked autotransporters" (41) or, perhaps more descriptively, two-partner secretion (TPS) pathways (42, 46). A TPS pathway has been described in a variety of pathogens, including Bordetella pertussis (24, 57, 89), Serratia marcescens (69, 79), Proteus mirabilis (87), and Haemophilus influenzae (4, 5). Like the autotransporter passenger domains, the exoproteins secreted via the TPS systems play different roles, and a number of the proteins characterized to date function as adhesins (21, 35, 57, 73, 83, 88) or cytolysins (12, 69, 87).
Some TPS proteins, such as filamentous hemagglutinin (FHA) of B. pertussis, and the high-molecular-weight (HMW) adhesins of H. influenzae, have been the subjects of considerable structural and functional characterization. However, there appear to be many potential TPS systems in other pathogens which have yet to be characterized (45, 46). Indeed, genes for many potential two-partner secretion pathways have been identified in the sequences of the genomes of a variety of pathogens (13, 46). Here we describe identification of a two-partner secretion system in ETEC using a ′phoA-based plasposon (23), TnphoA.ts (Fig. 1), which was developed to facilitate identification of secreted proteins.
Media and strains.A complete list of the plasmids and bacterial strains employed in this study is shown in Table 1. 5-Bromo-4-chloro-3-indolyl-phosphate (p-toluidine salt) (XP) was prepared as a 20-mg/ml stock solution in N,N-dimethylformamide. Plates for detection of gene fusions with phoA contained XP at a final concentration of 40 μg/ml and 100 μg/ml ampicillin in a base medium composed of tryptone (1%), yeast extract (0.5%), and agar (1.5%) (58).
Construction of a TnphoA-based plasposon.To construct a plasmid that would expedite isolation of TnphoA fusions to potential virulence genes in ETEC, we started with two existing plasmids, pmini-OphoA, which contains a Tn5-based transposon with the cognate transposase outside the inverted repeats (10), and pST76-A, a temperature-sensitive suicide plasmid (70). First, primers jf020904.1 (5′-GATCAGCCAGTGCCAAGC-3′) and jf020904.2 (5′-AAACCGGTTACCGATACAAGAGCCATAAGA-3′) were used to produce a 3,077-bp PCR product from pST76-A. The resulting amplicon was cloned into the EcoRV site of pSTBlue-1. The resulting plasmid, pJFF025, was then digested with BstEII and SacI, yielding a 2,983-bp fragment which was cloned into the corresponding sites of pmini-OphoA to construct the final plasposon. In this plasposon, pTnphoA.ts, the gentamicin resistance cassette and the pMB1/ColE1 origin of replication located between the inverted repeat elements of pmini-OphoA were replaced by the beta-lactamase gene and the conditional temperature-sensitive origin of replication from pST76-A.
Generation of TnphoA.ts mutants in ETEC.ETEC strain H10407 was transformed with pTnphoA.ts by electroporation. Ampicillin-resistant colonies obtained after overnight growth on Luria agar plates containing 100 μg/ml of ampicillin at 30°C were pooled and used to inoculate Luria broth (LB) containing ampicillin (100 μg/ml), which was incubated overnight at 30°C. After 1:2 dilution in 50% glycerol, this stock (jf1181) was frozen at −80°C, and it was subsequently used as the starting material for generation of mutants.
Briefly, 2-ml aliquots of LB containing ampicillin (100 μg/ml) were inoculated with jf1181 and incubated overnight at 30°C and 250 rpm. The following morning cultures were diluted 1:100 in fresh medium and grown for 90 min at 30°C, after which the cultures were again diluted 1:100 and 100-μl aliquots were plated onto multiple XP plates. Plates inoculated in this way were then incubated at 37°C overnight. Blue colonies selected from these plates were streak purified on fresh XP plates, and isolated colonies were subsequently used to inoculate 96-well plates containing LB with ampicillin. Total genomic DNA isolated from these colonies was digested with a restriction endonuclease (e.g., MluI), ligated, and used to transform E. coli DH10BT1 to ampicillin resistance after growth at 30°C. Plasmid DNA isolated from the resulting Ampr transformants was digested with the corresponding enzyme and subjected to agarose gel electrophoresis. Plasmids exhibiting a single band at ≥5 kb were then used as templates in DNA sequencing reactions. The DNA sequence upstream from the ′phoA region of the transposon was then screened for unique sequences not present in the E. coli K-12 strain MG1655 genome (9) by BLASTN searches.
DNA sequencing of the etpBAC locus.The focus of these studies, TnphoA.ts mutant 220, was 1 of approximately 250 colonies generated in an initial screening analysis. Plasmid DNA from this mutant was generated as described above following initial digestion of the genomic DNA with MluI. Digestion of the resulting plasmid, pJML001, yielded a single HMW band at approximately 12 kb. To obtain DNA sequences immediately upstream and downstream of the transposon insertion cloned in pJML001, we employed primers TnphoA.179 (5′-CCATCCCATCGCCAATCA-3′) and TnphoA.ts1 (5′-CGAAATTAATACGACTCA-3′), respectively. Additional DNA sequence from this locus was obtained by a combination of primer walking and generation of additional templates using in vitro Tn7 transposition of the GPS1.1 and GPS2.1 transprimers (New England Biolabs) into pJML001. Templates generated in this way were then sequenced using primers N (5′-ACTTTATTGTCATAGTTTAGATCTATTTTG-3′) and S (5′-ATAATCCTTAAAAACTCCATTTCCACCCCT-3′). To sequence through the repeat regions of etpA, this gene was first amplified using primers jf011005.1 (5′-CGGAATTCAATGAACCGTATATATAAA-3′) and jf011005.2 (5′-GAAGATCTTTGCCAGTACACCTCACT-3′) and cloned into the EcoRV site of pT7Blue-3 to create pJY017. This plasmid contained unique vector BamHI and SphI sites that were located 18 and 39 bp downstream from the 3′ end of etpA, respectively. Following restriction endonuclease digestion with these enzymes, approximately 2.5 μg of the linearized DNA was digested with exonuclease III at 37°C, which removed aliquots at 30-s intervals. Each aliquot was then treated with S1 nuclease, repaired with the Klenow fragment, and ligated with T4 DNA ligase. The resulting plasmids having progressive deletions from the 3′ end of etpA were then sequenced using vector primer R-20 (5′-CAGCTATGACCATGATTACG-3′). All sequencing was performed with an Applied Biosystems 373 automated sequencer and were edited and assembled with Sequencher 3.0 (GeneCodes, Ann Arbor, MI).
DNA and protein sequence comparisons: Clustal alignment.Protein sequences were aligned by using ClustalW (http://www.ch.embnet.org/software/ClustalW.html), and resulting alignments were shaded for similarity using Boxshade 3.2.1 (http://www.ch.embnet.org/software/BOX_form.html).
Molecular cloning.To generate a recombinant expression plasmid containing orf1 (etpB), primers jf102604.1 (5′-CCCAAGCTTGTGGTGAAATTCATGTCA-3′) and jf102604.3 (5′-GCGAATTCGAACGTTTTCAGGGCTGA-3′) were used to amplify an 1,819-bp fragment which was cloned into the EcoRV site of pT7Blue-3 to create pJMF1001. The corresponding HindIII/EcoRI insert fragment from pJMF1001 was then directionally cloned into the corresponding sites in pFLAG-CTC (Sigma). The resulting plasmid, pJMF1002, contained etpB minus its native stop codon to form an in-frame gene fusion with the region of pFLAG-CTC encoding the FLAG epitope tag. pJMF1002 was sequenced with primers N-26 (5′-CATCATAACGGTTCTGGCAAATATTC-3′) and C-24 (5′-CTGTATCAGGCTGAAAATCTTCTCTC-3′) to verify the construct.
To construct an etpA-polyhistidine tag gene fusion, the etpA gene minus its native stop codon was amplified using primers jf092804.1 (5′-CGGGATCCATGAACCGTATATATAAA-3′) and jf092804.2 (5′-CCGCTCGAGTTGCCAGTACACCTCACT-3′). Following digestion with BamHI and XhoI, the resulting 3,269-bp amplicon was cloned into the corresponding sites on pET30a(+) in frame with the polyhistidine tag coding region to create pJY005. DNA sequencing of pJY005 was performed using the T7 and SP6 vector primers to ensure that the insert encoding the etpA fragment was in frame with the polyhistidine tag.
To construct an EtpA expression plasmid, a 5,308-bp EcoRI-BglII etpA fragment from pJY017 was directionally cloned into the corresponding sites of pFLAG-CTC, placing etpA in frame with a FLAG epitope coding sequence to create pJY035.
Finally, primers jf031505.1 (5′-AATAATCTCGAGAATGGTGGTGAAATTCATG-3′; XhoI site underlined) and jf031505.2 (5′-AATAATAAGCTTTCATTCGGCACCCTCCTC-3′; HindIII site underlined) were used to amplify the entire etpBAC locus for cloning into the corresponding restriction sites on pBAD/Myc-His A to create pJY019.
Construction of deletion mutants.To construct an isogenic etpA mutant, an amplicon with an in-frame etpA deletion was first constructed by PCR. Primers jf092504.1 (5′-GCATGCCGCGTACCGGCAGACAGT-3′) and jf092704.4 (5′-ATGAACCGTATATATAAAATTACTCTTACCGGGAAC-3′) were used to generate a 1,167-bp amplicon which included the first 6 codons and last 12 codons of the predicted etpA open reading frame (ORF) and 1,065 bp of DNA sequence downstream from the stop codon of this gene. Next, the reverse complement of jf092704.4, primer jf092704.5 (5′-GTTCCCGGTAAGAGTAATTTTATATATACGGTTCAT-3′), and primer jf100104.5 (5′-GCCTCTCCCTGCTGCT GA-3′) were used to generate a 1,308-bp amplicon with the reverse complement of the etpA deletion and 1,272 bp of upstream flanking sequence. The products of these two reactions were combined as the template DNA in a third PCR using the jf100104.5 and jf092504.1 flanking primers, which resulted in a 2,400-bp amplicon that was cloned into pT7Blue-3, yielding pJY001. pJY001 was then sequenced with primers U-19 (5′-GTTTTCCCAGTCACGACG T-3′) and R-20 (5′-CAGCTATGACCATGATTACG-3′) to verify the deletion constructed. Next, a 2,113-bp SphI insert fragment from pJY001was cloned into the corresponding site of pCVD442 to obtain the suicide plasmid pJY028. SM10λpir(pJY028) was then used to introduce the suicide plasmid into H10407-S to construct the in-frame deletion by double homologous recombination, as previously described (25). Resulting Amps Smr colonies were then examined by PCR and Southern blotting to confirm construction of the in-frame deletion.
To construct an etpC deletion mutant, a λ-Red recombinase-based method was employed, as previously described by Datsenko and Wanner (22). Briefly, the λ-Red recombinase expression plasmid pKD46 was introduced into wild-type strain H10407 by electroporation, followed by selection on ampicillin (100 μg/ml) at 30°C. Primers jf093005.1 (5′-GCTGCAGGGCGCACGCTACCGGTTAATTTTTCGTATTCAGGAGTGTAGTGTAGGCTGGAGCTGCTTC-3′) and jf093005.2 (5′-TTCGGCACCCTCCTCCTGACGATGGCTACGCGCCTCCCACAACTGCCAATT CCGGGGATCCGTCGACC-3′) were used to amplify a 1,398-bp PCR product containing etpC flanking sequences (underlined) and a cassette containing kanamycin resistance and FLP recombinase target sites from pKD13. The resulting amplicon was electroporated into H10407(pKD46), and this was followed by selection on kanamycin (25 μg/ml) at 37°C. Kmr Amps colonies were then tested by performing a three-primer PCR with etpC flanking primers jf091504.1 (5′-TGATGAAGAGGGCAACTG-3′) and jf031505.2 (5′-TCATTCGGCACCCTCCTC-3′) and primer K2 (5′-CGGTGCCCTGAATGAACTGC-3′) (22).
Southern hybridization.An etpA gene probe was amplified from H10407 using primers jf011005.1 (5′-ATGAACCGTATATATAAA-3′) and jf011005.2 (5′-TTGCCAGTACACCTCACT-3′), and the resulting amplicon was then labeled by random primer labeling with digoxigenin (DIG)-UTP (44). Hybridization with plasmid DNA digested with SphI or genomic DNA digested with MluI was carried out under stringent conditions without formamide.
Colony hybridization.In colony hybridization studies to screen for the presence of etpBAC, DNA probes were amplified using the following primer sets: for etpA (3,257 bp), jf011005.1 (5′-ATGAACCGTATATATAAA-3′) and jf011005.2 (5′-TTGCCAGTACACCTCACT-3′); for etpB (1,812 bp), jf102604.1 (5′-CCCAAGGTGGTGAAATTCATGTCA-3′) and jf102604.2 (5′-GGAATTCTCAGAACGTTTTCAGGGCTGA-3′); and for etpC (1,495 bp), jf092104.1 (5′-CCTGAACGCGGATAACCT-3′) and jf031505.2 (5′-AATAATAAGCTTTCATTCGGCACCCTCCTC-3′). Products were labeled by random primer labeling with digoxigenin and used in hybridization studies carried out as previously described (68, 77) under stringent conditions without formamide.
Adhesion assays.The Caco-2 (33) and HCT-8 (86) intestinal cell lines used in the adhesion assays were obtained from the American Type Culture Collection. Each cell line was propagated by using American Type Culture Collection protocols. On the day prior to assays, cells were split at a ratio of approximately 1:2, and the resulting cell suspensions were used to inoculate individual wells of 96-well tissue culture plates. Bacteria were grown overnight in 2 ml of LB with appropriate antibiotics and then diluted 1:100 on the morning of the experiment and grown for 90 min to the mid-logarithmic growth phase. Organisms were then added to wells of the tissue culture plates in triplicate at a multiplicity of infection (MOI) of approximately 10:1 (26). After 1 h the monolayers were washed with tissue culture medium and then exposed to 0.1% Triton X-100 in phosphate-buffered saline for 5 min. Triton X-100 lysates containing total cell-associated bacteria were then diluted 1:10 in phosphate-buffered saline and plated onto Luria agar. To determine the number of intracellular bacteria, monolayers were washed three times with RPMI and then incubated with medium containing gentamicin (100 μg/ml) for 2 h. After washing to remove gentamicin, bacteria released by Triton X-100 lysis were plated onto Luria agar. The number of bacteria that remained cell associated relative to the inoculum was then determined by determining the number of CFU. The data were expressed as the percentage of organisms recovered per CFU/inoculum. Alternatively, monolayers were fixed with methanol and stained with Giemsa stain, and the number of adherent bacteria/cell was determined by microscopic examination. In antibody inhibition experiments, antibodies were added to wells at the appropriate dilution immediately prior to introduction of the bacteria.
Antibody production.Following expression of E. coli BL21(DE3)(pJY005) and purification of recombinant polyhistidine-tagged EtpA (rEtpA.6H) by Ni affinity chromatography, the resulting 110-kDa protein was used to immunize two New Zealand White rabbits, as previously described (31). Polyclonal rabbit antisera were absorbed on an E. coli lysate column (Pierce Biotechnology) and subsequently incubated with lyophilized E. coli AAEC191-A to remove cross-reacting antibodies. Additional preparative purification of antibodies from both preimmune and immune sera was performed using protein G column chromatography (HiTrap Protein G HP; Amersham Biosciences).
For affinity purification of anti-EtpA antibodies we employed rEtpA.6H protein immobilized on nitrocellulose membranes. Following separation by sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE), recombinant protein was transferred to nitrocellulose, and the membrane was blocked with 1% bovine serum albumin in Tris-buffered saline for 1 h at room temperature. Nitrocellulose strips with the immobilized antigen were then incubated with a 1:10 dilution of anti-EtpA rabbit polyclonal antibody overnight at 4°C. The antibody was eluted with 1 ml of 0.1 M glycine (pH 2.7) for 15 min at room temperature. The eluate was sterile filtered and concentrated 50-fold using a 10,000-molecular-weight cutoff centrifugal concentration filter.
Immunoblotting and detection of glycoproteins.To identify FLAG epitope-tagged EtpA molecules in culture supernatants, proteins were first concentrated by precipitation with trichloroacetic acid (TCA). Briefly, 750 μl of cold TCA (20%) was added to an equal volume of supernatant from an overnight culture, incubated on ice for 30 min, and centrifuged at 4°C and 15,000 × g for 15 min, and the supernatant was discarded. The resulting pellet was washed twice with 500 μl of ice-cold acetone, air dried, and resuspended in 10 μl of 1 M Tris (pH 7.4). Concentrated proteins were separated by SDS-PAGE (54) and transferred to nitrocellulose. Mouse monoclonal antibody M2 (Sigma) directed against the FLAG octapeptide (N-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-C) was used at a dilution of 1:1,000, followed by horseradish peroxidase-labeled goat anti-mouse antibodies (Pierce) at a dilution of 1:80,000. To detect native EtpA secreted into culture supernatants, immunoblotting was performed using preabsorbed rabbit polyclonal anti-rEtpA.6H sera (1:2,000) and goat anti-rabbit immunoglobulin G(Fc)-horseradish peroxidase (1:60,000; Pierce). All blocking and incubation steps were performed at room temperature in Tris-buffered saline (pH 7.4) containing 0.05% Tween 20 and 5% milk. Detection was carried out with a luminal-based chemiluminescent substrate (SuperSignal; Pierce).
After separation by SDS-PAGE and transfer to nitrocellulose, glycoproteins in culture supernatants were detected using DIG-glycan reagents (39, 50, 66) (Roche). Briefly, carbohydrate residues were first oxidized with 10 mM sodium metaperiodate in sodium acetate buffer (pH 5.5) and then labeled with DIG- 0-3-succinyl-ε-aminocaproic acid hydrazide. DIG-labeled proteins were then detected with alkaline phosphatase conjugated to anti-digoxigenin antibodies using a 4-nitroblue tetrazolium chloride-5-bromo-4-chloro-3-indolyl-phosphate solution as the substrate.
Fractionation of bacterial cells.To obtain whole-cell lysates of bacteria, overnight cultures were centrifuged to pellet bacteria, and the pellets were solubilized in nonionic detergent in 20 mM Tris-HCl (pH 7.5) (B-PER; Pierce). The bacterial membranes were fractionated by solubilization of inner membranes in Triton X-100, followed by isolation of outer membrane proteins by ultracentrifugation as previously described (81).
Nucleotide sequence accession number.Nucleotide data for the etpBAC locus have been deposited in the GenBank database under accession number AY920525.
Identification of a two-partner secretion locus by TnphoA.ts mutagenesis.Initial DNA sequencing of the region interrupted in TnphoA.ts mutant 220 using primers TnphoA.179 and TnphoA.ts1 revealed no homology to E. coli K-12. Further sequencing of this locus revealed three candidate ORFs in the same transcriptional orientation, as shown in Fig. 2. BLASTP analysis (2) of the predicted proteins suggested that this locus may encode a two-partner secretion pathway (46) (Table 2).
The middle ORF, etpA, interrupted by the transposon insertion in mutant 220, encodes a predicted protein having a molecular mass of approximately 177 kDa which exhibits homology with a family of secreted high-molecular-weight proteins exemplified by the HMW adhesins previously identified in nontypeable H. influenzae strains (83) (Table 2). EtpA exhibits significant homology with HmwA-like putative adhesin proteins from Yersinia pestis (67) and Yersinia pseudotuberculosis (16) and the RscA protein of Yersinia enterocolitica (64). EtpA has a putative secretion domain in its amino terminus (Table 2). This domain is found at the amino-terminal end of a variety of known adhesins, including HMW 1A (4, 5), and filamentous hemagglutinin of B. pertussis (74). In this conserved amino-terminal domain is an Asn-Pro-Asn-Gly-Val sequence (Fig. 3) similar to the Asn-Pro-Asn-Gly-Ile motif found in many exoproteins secreted via a two-partner secretion pathway (46). Mutagenesis of this similar motif in other TpsA proteins interferes with their secretion (82) and/or release (84) from the bacterial cell.
The carboxy-terminal region of EtpA (from S648 to G1706) consists of a series of repeated domains (Fig. 3). Four repeats that are approximately 226 amino acids long are preceded by a single 173-residue partial repeat beginning at S684.
Additional in silico analysis of EtpA suggested that there is structural similarity to a number of bacterial virulence proteins and predicted that EtpA assumes a β-helical conformation. Examination of the predicted EtpA peptide with BetaWrap (http://betawrap.lcs.mit.edu/), a program which scores primary amino acid sequences for compatibility with right-handed beta-helix supersecondary structure, yielded highly significant results (P = 7.8 × 10−05). Interestingly, this predicted structure appears to be common among bacterial virulence proteins, including the passenger domain of the B. pertussis autotransporter pertactin (28), but it is found only infrequently in most other protein sequences (11). Similarly, analysis of the EtpA peptide with the 3D-PSSM algorithm (http://www.sbg.bio.ic.ac.uk/servers/3dpssm/) (47) yielded similar structural predictions, again suggesting that there is a β-helical structure similar to that of pertactin.
The etpB gene, immediately upstream from etpA, encodes a predicted 61.7-kDa protein that exhibits considerable homology with Enf (Table 2), a putative afimbrial adhesin of enteroaggregative E. coli serotype O111:H12 (63). In addition, the predicted EtpB protein exhibits similarity to RscB from Y. enterocolitica and to the prototypical TpsB transporter proteins Hmw1B from H. influenzae and FhaC from B. pertussis, as demonstrated in the ClustalW alignment shown in Fig. S1 in the supplemental material. A SignalP (7) search of the amino terminus of EtpB predicted a signal peptide cleavage site between amino acid residues 24 and 25, suggesting that the molecular mass of the mature protein in the outer membrane is approximately 59.2 kDa. This is similar to the size of the mature 58-kDa Enf protein. Further analysis of the EtpB peptide using the 3D-PSSM algorithm supported the possibility that this structure forms a β-barrel in the outer membrane.
The final 1,913-bp ORF in this locus, etpC, encodes a putative 72-kDa protein with homology to RscC of Y. enterocolitica (64) and Hmw1C (4, 5). The predicted EtpC protein contains a domain that is highly conserved in a family of proteins involved in posttranslational modification of other proteins, which work as O-linked N-acetylglucosamine transferases to catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to target Ser/Thr residues (38).
Flanking the locus described above are potential mobility elements, including an upstream ORF with homology to ORF 121 from IS91 (62). Downstream from this locus is a copy of IS1414 (61), which contains a copy of astA, the gene for EAST1 (78) in the putative transposase. Because of the close proximity of these potential mobility elements to the etpBAC locus and because other organisms may have more than one closely related TPS locus, such as HMW1 and HMW2 of H. influenzae (5), we sought to determine whether a single copy of the etpBAC locus was present. As shown in Fig. 2, Southern blotting of both plasmid and total genomic DNA from H10407 yielded single bands when the DNA was hybridized with an internal etpA gene probe. These bands were absent after hybridization with DNA obtained from H10407-P, which lacks the large 92-kb pCS1 virulence plasmid, suggesting that a single copy of etpBAC is in this extrachromosomal element.
Taken together, these data suggested that the genes in this locus are dedicated to the formation of a TPS pathway. Therefore, additional studies were undertaken to explore the validity of this suggestion.
Secretion of EtpA.Because the predicted peptide encoded by etpA exhibits significant homology with a number of secreted proteins, we performed experiments to examine the secretion of EtpA. As shown in Fig. 4, antisera raised against a recombinant polyhistidine-tagged EtpA fragment (rEtpA.6H) reacted with an HMW specimen in immunoblots of TCA-precipitated culture supernatants from the H10407 wild-type strain but not in immunoblots of TCA-precipitated culture supernatants from jf1289, an isogenic etpA deletion mutant, suggesting that EtpA is in fact secreted. Restoration of the etpA gene in trans as a FLAG epitope fusion on expression plasmid pJY035 restored secretion of a protein that could be detected either with anti-EtpA antisera (Fig. 5A) or with monoclonal antibodies directed against the FLAG epitope (not shown). Supernatants from H10407-P did not contain a similar reactive protein. Furthermore, cloning and expression of the etpBAC locus in a recombinant E. coli background, LMG194(pJY019), were sufficient to produce the HWM protein in culture supernatant that was recognized by anti-EtpA antisera on Western blots (Fig. 4A).
Although EtpA could be identified easily in concentrated supernatant from H10407, only a small amount of this protein was detected on immunoblots of whole-cell lysates, and we were not able to detect EtpA in outer membrane preparations of this strain (not shown). These findings suggest that the secretion of EtpA is quite efficient and that this exoprotein may be only transiently associated with the bacterial cell surface.
etpBAC locus directs secretion of a high-molecular-weight glycosylated exoprotein.Based on their sequence homology to proteins involved in secretion of high-molecular-weight glycoproteins by H. influenzae, we predicted that the proteins encoded by the etpBAC locus would also result in the export of a large glycosylated molecule (35). Indeed, as shown in Fig. 4B, we identified HMW glycoprotein in culture supernatants from LMG194(pJY019) and the H10407 parent strain. This glycoprotein was absent in TCA-precipitated supernatants obtained from H10407-P and the isogenic etpA deletion mutant, jf1289.
Role of the etpBAC locus in adherence of ETEC to cultured intestinal epithelial cells.Because EtpA exhibits homology with a number of previously identified TpsA exoproteins that have been shown to function as adhesins, we tested the ability of etpA mutants to adhere to cultured epithelial cells. As shown in Fig. 5A, jf1289 did not adhere as efficiently to cultured HCT-8 cells as the H10407 wild-type strain adhered (P = 0.02), while complementation of the etpA deletion in trans with pJY035 encoding a FLAG epitope-tagged version of EtpA restored this phenotype to wild-type levels (P = 0.015 for a comparison of the etpA deletion strain and the complemented mutant). To ensure that bacteria were actually cell associated and not adherent to the wells, the experiments included controls in which the same number of organisms was used to inoculate wells containing no target epithelial cells. We routinely failed to recover any bacteria from these wells (data not shown). In addition, Giemsa staining of monolayers (Fig. 5B) following infection with either the wild type or the etpA mutant at an MOI of 10 resulted in identification of approximately 0.8 ± 0.02 wild-type bacterium per epithelial cell, compared to 0.12 ± 0.04 organism/cell for the mutant (P = 0.002). An increase in the MOI to 100:1 resulted in 3.3 ± 1.1 wild-type organisms/cell, compared to 0.29 ± 0.06 mutant bacterium/cell (P = 0.046).
To further determine whether EtpA might play a role in mediating adherence to epithelial cells, we tested the ability of antibodies directed against recombinant EtpA (rEtpA) to inhibit adherence by the ETEC strain H10407 prototype. As shown in Fig. 5C, anti-rEtpA.6H antisera dramatically inhibited association of the wild-type strain with target HCT-8 cells compared to the results obtained with identical concentrations of preimmune sera from the same rabbit. Similar results were obtained using Caco-2 cells or using affinity-purified anti-EtpA antibodies (data not shown). These data suggest that EtpA, like its molecular TpsA homologues, promotes pathogen-host cell interaction. Expression of the entire etpBAC locus (on pJY019) in recombinant E. coli strain LMG194 was sufficient to promote adherence to HCT-8 cells. After induction of LMG194(pJY019) with 0.0002 mM arabinose, 0.44% ± 0.04% of the organisms added to HCT-8 monolayers were recovered in adherence assays, which was significantly more than the value for the recombinant vector control strain, LMG194(pBAD/Myc-His A), for which no bacteria were recovered (P = 0.001), but was less than the value for wild-type ETEC strain H10407 (0.89% ± 0.08) (P = 0.01).
Each of the bacterial glycoprotein adhesins described to date appears to require glycosylation to effectively promote adherence in vitro (8, 35, 56). To investigate the potential role of posttranslational modification of EtpA by glycosylation in the adherence phenotype, we constructed a deletion in etpC. As predicted by the homology of EtpC to HmwC, a protein involved in glycosylation of the HmwA adhesin of H. influenzae, etpC deletion mutants produced nonglycosylated EtpA (Fig. 6A and B). Interestingly, we found that the adherence phenotype of etpC glycosylation mutants was strikingly dependent on the target intestinal cell line employed. Consistent with the observation that glycosylation of HmwA is required for adherence in vitro, we found that etpC deletion mutants were less likely to associate with Caco-2 cells than the wild type was (Fig. 6C). However, the same mutants were actually hyperadherent when HCT-8 cells were used in the assays. This hyperadherent phenotype was due entirely to an increase in adherence as the numbers of organisms that survived gentamicin treatment were quite small and were not significantly different for the wild type and the mutant (6.31E-02% ± 1.80E-02% and 4.85E-02% ± 8.10E-03%, respectively). These data suggest that EtpA may mediate adherence to different receptors in a process that is differentially modulated by glycosylation.
As noted above, the predicted protein encoded by etpB exhibits considerable homology with Enf, a recently identified outer membrane protein adhesin of enteroaggregative E. coli (63). Indeed, when etpB was cloned and expressed in a recombinant E. coli background, rEtpB was largely localized to the outer membrane (Fig. 7A) and conferred the ability to adhere to cultured epithelial cells of gastrointestinal origin. A laboratory strain of E. coli (NovaBlue) bearing pJMF1002 exhibited marked increases in adherence to Caco-2 cells compared to the control strain bearing the expression plasmid vector pFLAG-CTC (Fig. 7B), suggesting that EtpB, independent of its possible function as a TpsB transporter for EtpA, might promote adherence similar to that promoted by the highly homologous Enf afimbrial adhesin. Interestingly, a BLAST-X search of the 192 bp of DNA sequence downstream from the enfA gene available in GenBank accession no. AF196977 indicated the beginning of an ORF that, when translated, exhibited homology to TpsA exoproteins, including the HMW adhesins of H. influenzae (data not shown), raising the possibility that Enf might also be part of a TPS pathway.
Multiple ETEC strains secrete the EtpA glycoprotein.The identification of genes encoding potential mobility elements adjacent to etpBAC led us to examine the prevalence of this locus in other E. coli strains. In screening for the etpBAC locus by DNA hybridization, we examined a variety of diarrheagenic clinical E. coli isolates from different pathotypes, including enterotoxigenic E. coli, enteropathogenic E. coli, enteroaggregative E. coli, enteroinvasive E. coli, and enterohemorrhagic E. coli. We detected hybridization signals from many of the ETEC strains in our initial collection with all three probes (etpB, etpA, and etpC) (Table 3), while no signal was detected in colony hybridizations of enteroaggregative E. coli, enteroinvasive E. coli, enteropathogenic E. coli, or enterohemorrhagic E. coli strains (Table 4). These findings suggested that the locus is unique to ETEC.
Immunoblotting studies to detect EtpA in concentrated supernatants from these strains corroborated the results of our hybridization analysis, since only strains that hybridized with all three probes secreted EtpA (Fig. 8). Although some ETEC strains, such as B7A and DS300-1, yielded a positive signal with the etpA gene probe, there was no detectable hybridization with either the etpB or etpC probes, and these strains did not secrete EtpA. Production of HMW glycoprotein by ETEC paralleled the results of immunoblotting experiments since all of the strains exhibiting immunoreactivity with anti-EtpA antibodies also produced a glycoconjugate having the same molecular weight (Fig. 9). Interestingly, several EtpA-negative strains, such as ETEC-10, ETEC-13, ETEC-15, and ETEC-26, yielded a weakly reactive species as determined by DIG-glycan analysis, raising the possibility that these strains may produce other exported HMW glycoproteins.
Altogether, 59% (10/17) of CFAI-expressing strains, 56% (10/18) of CFAII-expressing strains, and 75% (3/4) of strains expressing CS14 or CS17 expressed EtpA or reactive proteins with a molecular weight identical to that of EtpA. However, strains expressing CS4, CS5, or CS6 (CFAIV strains) were negative as determined by Western blotting. In supernatant from a single ETEC strain, DS97-2 (CFAI, heat-stable enterotoxin, serotype O153:nm), anti-EtpA antibodies identified a protein whose molecular weight was significantly different than that of EtpA.
Although a large number genes encoding potential TPS systems have been identified through DNA sequencing of microbial genomes, only a limited number of TPS molecules have been fully characterized so far. Of the few TPS systems that have been characterized, most have been shown to play a role in adhesion (5, 74, 76) or cytolysis (43, 69, 87), while the hxuCBA locus of H. influenzae appears to be involved in iron acquisition (18-20).
Here, we describe identification and initial molecular characterization of a TPS system in a prototypical ETEC strain. Similar to the other TPS systems described to date, the etpBAC locus supports the secretion of a high-molecular-weight exoprotein. The homology of the proteins encoded at this locus to known TpsA and TpsB proteins predicts that EtpB is an outer membrane transporter (TpsB) required for the secretion of EtpA exoprotein (TpsA). This prediction is supported by our findings that both native EtpA and rEtpA proteins were found predominately in culture supernatants and that cloning and expression of the etpBAC locus were sufficient to export EtpA from a recombinant E. coli strain.
Furthermore, based on the homology of EtpC with Hwm1C, a protein previously shown to be involved in the glycosylation of the H. influenzae adhesin HMW1 (35), we predicted that EtpA is also glycosylated. Indeed, in culture supernatants of H10407 and LMG194(pJY019) we were able to identify native EtpA and rEtpA, respectively, using either antibodies which recognize EtpA or DIG-glycan reagents. These findings appear to contradict previous studies which described the first report of glycoprotein production in E. coli, as they suggested that the TibA outer membrane protein is the only glycosylated protein produced by H10407 (56). However, in the previous studies the workers focused on analysis of whole-cell lysates of H10407 rather than on secreted proteins, and the results may simply indicate that very little of the EtpA glycoprotein remains cell associated when cells are grown under some laboratory conditions. Indeed, we were not able to identify EtpA in outer membrane preparations from the wild-type strain, suggesting that it does not remain tightly associated with the outer membrane.
Interestingly, while production of glycoproteins by eukaryotic cells is essential for a number of important biological processes, glycosylation is now increasingly recognized in prokaryotes, particularly bacterial mucosal pathogens (80, 85). Several of the bacterial glycoproteins described to date, including TibA (55, 56), the related E. coli autotransporter AIDA-I (8, 53), HMW1 (35), and a 40-kDa Chlamydia trachomatis major outer membrane protein (52), promote adherence to epithelial cells, and in each instance modification of the protein by glycosylation is required to demonstrate the adherence phenotype. Our studies confirmed that EtpC is required for glycosylation of EtpA, similar to the previously described system in H. influenzae (35). Interestingly, the effect of glycosylation on EtpA-mediated adherence appears to be quite complex. Similar to previous reports of the importance of glycosylation in the adherence phenotype, etpC was required for adherence when Caco-2 intestinal monolayers were used as the target. In contrast, the etpC mutant was shown be significantly hyperadherent compared to the wild-type strain when HCT-8 intestinal cells were employed, suggesting that glycosylation may modulate binding to different molecules on the surfaces of different cell lines. Therefore, while the etpBAC locus may have many similarities to previously described TPS operons, the studies described here revealed important differences in the biology of the different exoprotein adhesin molecules.
The predicted EtpA peptide sequence contains at its amino-terminal end a putative secretion module associated with other TpsA exoproteins, including FHA, a 230-kDa adhesin of B. pertussis. The recent determination of the crystal structure of the filamentous hemagglutinin secretion domain (17) provides insight into the possible mechanism of export via TPS, suggesting that this region is probably involved in formation of the ultimate β-helical structure of the TpsA proteins. The highly conserved Asn-Pro-Asn-Gly motif found in this region of EtpA, which is essential for secretion of other TpsA proteins (36, 82), may be involved in stabilization of the β-helix through formation of type I β-turns (17).
The carboxy-terminal portion of EtpA is comprised of multiple repeat regions. While a combination of intragenic duplication and recombination is thought to underlie the formation of repeats (2), the molecular significance of the repeat regions in EtpA is not clear yet. Repeat regions of proteins are often involved in protein-protein interactions. Indeed, the passenger domain of the ShdA autotransporter of Salmonella enterica serovar Typhimurium has repeat regions in the carboxy-terminal region of the molecule which are involved in its interaction with fibronectin (49).
The homology of EtpB to the afimbrial adhesin Enf from enteroaggregative E. coli serotype O111:H12 led us to investigate whether EtpB had similar properties. Like Enf, the cloned recombinant EtpB outer membrane protein was able to promote adherence to epithelial cells. Together, these findings indicate that EtpB (and perhaps Enf) may have two functions, transporting cognate TpsA exoproteins and functioning independently to promote adherence.
Adherence to epithelial cells plays an important role in the pathogenesis of many mucosal pathogens, and many organisms possess multiple adhesins (65). Here we found that a newly identified TPS system of a prototypical ETEC strain contributes to the interaction of ETEC with target epithelial cells. Our findings that an isogenic etpA mutant strain was demonstrably less adherent than the parent organism and that antibodies raised against recombinant EtpA prevented adherence to cultured epithelial cells strongly suggest that this exoprotein contributes to the adherence phenotype observed in vitro. However, the precise role of the etpBAC locus and the role of the EtpA exoprotein remain undefined. Indeed, the present studies were complicated by the fact that the expression of known fimbrial adhesins, such as CFAI, may not be optimal under the conditions which we employed.
The mechanism by which EtpA might facilitate adherence is not obvious from the present data. Indeed, the role played by FHA, an homologous TpsA adhesin from B. pertussis, is actually quite complex. FHA, which can be found on the bacterial surface and in the external milieu (21), appears to play a multifunctional role in adherence of B. pertussis to host cells, binding to multiple ligands via different functional domains, including binding to lactosylceramide (71) via the carbohydrate binding domain, to sulfated glycoconjugates via the heparin binding domain (37), or to the integrin CR3 via its RGD sequence (73).
Like the recently described eatA autotransporter gene, the etpBAC locus also resides on the pCS1 virulence plasmid of ETEC strain H10407. Several lines of evidence support this conclusion. First, DNA sequencing of the etpBAC locus showed that immediately downstream of etpC is an IS1414 copy bearing an EAST1 gene which was previously found on pCS1 in H10407 (90). In addition, H10407-P, which lacks pCS1, was negative for etpA as determined by PCR, Southern blotting, and colony hybridization studies. Finally, both blotting and detection of concentrated supernatant proteins from H10407-P with either anti-rEtpA.6H antibodies or DIG-glycan reagents failed to detect the high-molecular-weight EtpA protein. Conjugal transfer of pCS1 (91), which contains the etpBAC locus as well as CFAI, heat-stable enterotoxin, and EatA genes (68), may account for the relatively high prevalence of the etpBAC locus in CFAI-expressing strains. The finding that there are potential mobility elements adjacent to this locus may account for its identification in ETEC strains having multiple CF types.
The studies described here, along with other recently described studies of new potential virulence loci of ETEC, highlight the previously unappreciated complexity of the ETEC genome and suggest that there is much to be learned about the pathogenesis of this organism. While the precise role of the etpBAC locus in the pathogenesis of ETEC remains to be completely defined, this locus is an enticing target for further investigation given the apparent structural and functional homology of its encoded proteins to established virulence molecules that have recently proven to be valuable in development of vaccines for other pathogens.
This work was supported by funds from the Department of Veteran's Affairs (to J.M.F.), by Public Health Service grant RR16190-04 (to J.M.F), and in part by NIH Medical Student Research Program grant T35 DK07405.
We thank Erik Dhondt and Didier Favre of the Division of Live Bacterial Vaccines at Berna Biotech for supplying their collection of ETEC strains, Donald E. Woods for supplying the pmini-OphoA plasmid, and Jing Yu and Bradley Postlethwaite for their contributions. We also thank Harry Courtney and David Hasty for their thoughtful reviews of the manuscript.
- Received 7 November 2005.
- Returned for modification 21 December 2005.
- Accepted 10 January 2006.
Editor: A. D. O'Brien
↵† Supplemental material for this article may be found at http://iai.asm.org/.
- American Society for Microbiology


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