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Sebaceous cysts are cysts which form inside the skin and are filled with semi solid substance called the sebum. Sebum is normally produced by sebaceous glands. These lumps develop just beneath the surface of the skin. They are basically painless and can be easily moved. In some cases a sebaceous cyst may get infected and inflamed which causes redness of the skin above the cyst and pain. Sebaceous cysts may affect any part of the skin containing sebaceous glands. However, they predominantly affect the scalp, face, neck, chest, back and shoulders. In rare cases these cysts may even form in genital area. Differential diagnosis of sebaceous cysts includes epidermoid and pilar cysts.
Presentation and Causes of Sebaceous Cysts
Sebaceous cysts are located beneath the skin. They represent sacs filled with the sebum. This substance has a foul odor. Characteristic bumps are round in shape and may drastically vary in size. They are most commonly found on the scalp. Apart from sebum sebaceous cyst may also contain fibrous tissue and small amounts of fluid. If a sebaceous cyst gets inflamed it fills with puss and bloody material.
Sebaceous cysts generally develop as a consequence of skin trauma, swollen hair follicles and blockage of sebaceous glands. In some people the occurrence of sebaceous cyst is associated with high levels of testosterone. There have also been cases of sebaceous cysts caused by the attack of human botfly. And finally, some people have genetic predisposition to sebaceous cysts.
Treatment for Sebaceous Cysts
Sebaceous cysts are not serious and cause no harm. They are usually painless unless they get inflamed. The major problem related to them is their unsightly appearance. This especially bothers people in whom sebaceous cysts are located on visible areas of the body. Small sebaceous cysts usually require no treatment unless they are considered an aesthetic problem.
Inflammation of sebaceous cysts requires treatment. Patient suffering from large sebaceous cysts, inflamed ones and sebaceous cyst which are aesthetic problem are treated surgically. The sebaceous cyst is surgically removed and the surgeon pays close attention to remove the entire cyst this way preventing its reoccurrence. Reoccurrence of sebaceous cysts is common for people who are prone to their formation. Depending of the size of the cyst and its location the surgeon may perform a traditional wide excision, punch biopsy or minimal excision. If the cyst is inflamed patients are given antibiotics prior and after the surgery.
Inflammation of small sebaceous cysts can be also reduced by injection of steroids right into the cyst. In some cases sebaceous cysts can be cancerous.


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Traditionally, tourists start discovering Valletta landmarks from the famous Triton fountain, which is a true symbol of the sea, closely associated with ancient Greek traditions. Located right in the center of a large square near the City Gate, the impressive fountain catches the eye with its spectacular scale, pronounced dynamics, expressive and sophisticated composition. The water element is harmoniously combined with a green area of a boulevard called Maglio Gardens adjoining the square with the fountain. It promises both pleasant rest in the coolness of its clear water jets and a nice photo shoot as a memento of Valletta.
The Triton fountain was constructed in 1959, designed by Vincent Apap, a distinguished Maltese sculptor, who won the national competition for designing a fountain to decorate the main city bus station. He was inspired by famous fountains located in Rome. Travertine slabs were used for the fountain base, while the figures of tritons were casted in bronze in an Italian foundry. The construction took more than 4 years and cost about £70-100,000.
Initially, the fountain composition included only three tritons and a plate they hold without any central pillar, and the water passed through the tritons. Unfortunately, the fountain was used as a stage for various performances and in the late 70s its plate and two of three tritons were damaged. In the late 80s the fountain was repaired by installing a stylized bronze pillar on the plate center. It was designed by the same sculptor and was decorated by sea gulls in the same marine style. The pillar serves as a support to the plate and also hides a new water supply system, due to which the original fountain design was somewhat changed.
In 2011, the fountain nearly changed its location, since it was planned to move it within a large-scale project of reconstruction of the City Gate and the surrounding area. After combative debate between the government of Malta and the concerned community, it was decided to leave the fountain on its traditional place. In 2015, the government announced the decision to spend €500,000 for the fountain restoration, but by the middle of 2016 the work has not yet begun. In the meantime, the Triton fountain continues to delight the eye of both locals and tourists with its beauty, which is especially striking in the light of the evening illumination.
2013.09.28 review from Foursquare
The Triton Fountain is seen in a brief overhead shot in World War Z (2013) as Jerusalem (which Malta serves as for the film) is overrun by zombies and Gerry's pilot abandons the city in his plane.
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Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages by Legionella pneumophila. In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replication between 4 and 8 h postentry and to grow to large numbers in the Legionella-containing vacuole (LCV), as evident at 12 h. At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum (ER)-derived vesicles to LCVs. The mutant did not lose expression or translocation of six type IV secretion effectors (e.g., SidM) that are well known for mediating Rab1B association with the LCV, indicating that T2S promotes the interaction between the LCV and Rab1B via a novel mechanism. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA (shRNA) treatment, indicating that T2S also potentiates events beyond Rab1B association. In support of this, a sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant (and a sidM mutant) and showed a growth difference of as much as a 400-fold compared to the wild type. Together, these data reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes.
Legionella pneumophila is widespread in natural and man-made water systems and is best known as the etiologic agent of Legionnaires' disease (1–3). Recently, the incidence of pneumonias caused by Gram-negative L. pneumophila has roughly tripled in the United States and elsewhere (4–6). In water, L. pneumophila grows primarily as an intracellular parasite of amoebae (7–11). Human infection occurs mainly by the inhalation of contaminated water droplets originating from a wide range of aerosol-generating devices (12, 13). Yet the first case of person-to-person spread was reported in 2016 (14). In the lungs, L. pneumophila invades and grows in the resident macrophage population and ultimately triggers severe inflammation (3). In both amoebae and macrophages, the bacterium evades the host's degradative lysosomal pathway and replicates to large numbers within a membrane-bound vacuole, the Legionella-containing vacuole (LCV) (15, 16). The Dot/Icm type IV secretion (T4S) system of L. pneumophila plays a major role in pathogenesis (17–19). Indeed, the Dot/Icm apparatus translocates myriad proteins across the LCV membrane and into the host cell cytoplasm, and then the delivered effectors proceed to modulate a wide variety of host processes. One of the earliest and most notable events that has been ascribed to the Dot/Icm system is the recruitment of host GTPases and endoplasmic reticulum (ER)-derived vesicles to the LCV for the purpose of potentiating bacterial replication (20).
L. pneumophila possesses another protein secretion system that also has a major role in the natural history of disease, i.e., the Lsp type II secretion (T2S) system (21–23). In T2S, substrates are first transported across the bacterial inner membrane and then, upon the action of the T2S pilus-like apparatus, exit the cell through a dedicated pore in the outer membrane (24). Based on proteomic and enzymatic assays, we determined that the T2S system of L. pneumophila secretes more than 25 proteins, including 18 known enzymes and various novel proteins (25, 26). Within water systems, T2S enhances the survival of L. pneumophila at low temperatures and is essential for infection of amoebae belonging to the genera Acanthamoeba, Naegleria, Vermamoeba (formerly Hartmannella), and Willaertia (26, 27). Among the secreted proteins that promote intracellular infection of amoebae are the novel proteins NttA and NttC as well as the PlaC acyltransferase, ProA metalloprotease, and SrnA RNase (26, 28–30). L. pneumophila T2S also promotes biofilm formation and sliding motility (31, 32). In the lungs, T2S promotes infection of macrophages, suppression of innate immunity, and destruction of tissue (23, 33, 34). Lsp mutants of L. pneumophila that lack T2S, but not their complemented derivatives, are impaired ∼10-fold for infection of U937 cells, a human macrophage-like cell line, and MH-S cells, a murine macrophage line (33–35). However, the basis for the importance of T2S in macrophage infection has remained undefined. We now report that T2S is required for the optimal association of the host GTPase Rab1B with the LCV, coincident with the initiation of intravacuolar replication between 4 and 8 h after bacterial entry into the macrophage. We further show that T2S continues to promote replication at 12 h and beyond, in a manner that is independent of Rab1B recruitment.
Bacterial strains and media.L. pneumophila strain 130b (American Type Culture Collection [ATCC] strain BAA-74) served as our wild-type (WT) strain (36). The lspF mutant NU275, the lspE mutant NU361, and the complemented lspF mutant NU275(pMF) were also described previously (33, 37), as were the chiA mutant NU318, the lapA mutant NU320, the lipA mutant NU262, the map mutant NU254, the rcp mutant NU260, the plaC mutant NU367, the plcA mutant NU268, the proA mutant AA200, and the srnA mutant NU328 (25, 28–30, 38–41). To help monitor the behavior of L. pneumophila in macrophages, we introduced a green fluorescent protein (GFP)-expressing plasmid, pMGFP, into strains 130b and NU275. To that end, a DNA fragment encoding GFP was PCR amplified from pMMB-GRN (42) by use of the primers RW1 and RW2 (see Table S1 in the supplemental material). The EcoRI/HindIII-digested PCR product was then cloned into EcoRI/HindIII-digested pMMB2002 (33), yielding pMGFP. Finally, the plasmid was electroporated into the L. pneumophila strains (43). Legionellae were routinely grown at 37°C on buffered charcoal-yeast extract (BCYE) agar, which, when appropriate, was supplemented with chloramphenicol at 3 μg/ml, kanamycin (Kn) at 25 μg/ml, or gentamicin at 2.5 μg/ml (33). GFP-expressing bacteria were grown on BCYE agar supplemented with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) to induce expression of GFP. To monitor the extracellular growth of L. pneumophila, bacteria were incubated in buffered yeast extract (BYE) broth with shaking at 37°C, and the optical densities of cultures were determined at 660 nm (26). Escherichia coli strain DH5α (Life Technologies, Carlsbad, CA) was the host for recombinant plasmids and was grown in Luria-Bertani medium with Kn (50 μg/ml), chloramphenicol (30 μg/ml), or ampicillin (100 μg/ml). Unless otherwise noted, chemicals were obtained from Sigma-Aldrich (St. Louis, MO).
Mutant construction.Mutants of L. pneumophila strain 130b containing nonpolar deletions in sidM or dotA were constructed using a form of allelic exchange. To begin, mutagenized alleles were generated using overlap extension PCR (OE-PCR) as previously done (44, 45). The 5′ and 3′ regions flanking the open reading frames (ORFs) of sidM and dotA were PCR amplified from 130b DNA by use of primers RW3 and RW4 for 5′ sidM, RW5 and RW6 for 3′ sidM, RW9 and RW10 for 5′ dotA, and RW11 and RW12 for 3′ dotA (see Table S1 in the supplemental material), as well as HiFi Taq polymerase (Life Technologies), and the Kn resistance cassette flanked by Flp recombination target sites was similarly PCR amplified from pKD4 (44) by use of primers RW7 and RW8 for sidM and RW13 and RW14 for dotA (see Table S1). We then performed two-step OE-PCR to combine the 5′ and 3′ regions of sidM or dotA with the respective Kn resistance cassette. PCR products corresponding to the correct target sizes were gel purified and ligated into pGEM-T Easy (Promega, Fitchburg, WI) to yield either pGsidM::Kn or pGdotA::Kn. After transforming (46) strain 130b with the newly made plasmids, bacteria containing an inactivated sidM or dotA gene were obtained by plating on BCYE agar containing Kn and verified by PCR using primers RW19 and RW20 or RW21 and RW22 (see Table S1). Next, following electroporation (43) of pBSFLP (44) into the sidM mutant and subsequent plating on BCYE agar containing 1 mM IPTG and gentamicin, the colonies obtained were patched onto ordinary BCYE agar in order to promote the loss of pBSFLP. Clones that were sensitive to gentamicin and Kn were isolated, and the loss of the plasmid and the chromosomal antibiotic cassette (leaving only an unmarked deletion) was confirmed by PCR. The new sidM deletion mutant was designated NU427, and the dotA deletion mutant was designated NU428. To construct a double mutant lacking both sidM and T2S, we introduced pGlspF::Kn (33) into NU427 by transformation (46) and then selected for the acquisition of Kn resistance. The genotype of the sidM lspF double mutant, designated strain NU429, was verified by PCR using primers RW47 and RW48 (see Table S1). A complemented derivative of the sidM lspF mutant was obtained by introducing the lspF-encoding plasmid pMF (33) into NU429 by electroporation.
Macrophage cell lines and intracellular infection assays.In order to assess L. pneumophila growth within mammalian cells, we utilized bone marrow-derived (BMD) macrophages that were obtained from A/J mice (Jackson Laboratory, Bar Harbor, ME) and human U937 cells (ATCC CRL-1593.2) terminally differentiated into macrophages by treatment with phorbol myristate acetate (47). To aid in determining the effect of Rab1B on L. pneumophila growth, we generated a stable U937 Rab1B-knockdown cell line. Silencing was achieved by transduction with a vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentivirus that carries a GFP reporter gene and encodes a short hairpin RNA (shRNA) molecule targeting Rab1B, i.e., clone V2LHS_137920 from OpenBioSystems (GE Healthcare, Chalfont, United Kingdom). As a control, we generated a lentivirus encoding a nontargeting shRNA molecule ("scramble"), i.e., clone RHS4346 (GE Healthcare). Lentiviral vectors were prepared from HEK293T cells (ATCC CRL-3216) as previously described (48), and then a 2.5-ml suspension of undifferentiated, replicating U937 cells (approximately 2 × 106) in RPMI medium (Corning, Corning, NY) with 10% fetal bovine serum (FBS) (Atlanta Biologicals, Flowery Branch, GA) was mixed with 750 μl unconcentrated virus in the presence of 4 μg/ml Polybrene (Santa Cruz Biotechnology, Dallas, TX) in a 6-well dish (Falcon; Becton Dickinson, Franklin Lakes, NJ). The resulting cell suspension was transduced upon centrifugation at 1,000 × g for 90 min at 32°C followed by incubation at 37°C. After 16 h, the medium was replaced with fresh RPMI medium containing FBS (RPMI-FBS) to remove the Polybrene. After 3 days of further incubation at 37°C, transduced knockdown cells were selected by incubation in the presence of 1 μg/ml puromycin (Corning) for 2 or 3 passages, at which time all cells in the suspension were GFP positive. Silencing of Rab1B was confirmed by immunoblot analysis. To that end, cell pellets were lysed in Laemmli sample buffer and analyzed via SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Thermo Scientific, Waltham, MA), blocked in Tris-buffered saline with 0.1% Tween 20 (TBS-T) and 5% milk (Bio-Rad Laboratories, Hercules, CA), and probed with a rabbit polyclonal anti-Rab1B antibody (sc-599; Santa Cruz Biotechnology) at 1:250 for 12 h at 4°C. Proteins were finally detected using an anti-rabbit IgG–horseradish peroxidase conjugate (Cell Signaling Technology, Danvers, MA) and an enhanced chemiluminescence (ECL) detection reagent (GE Healthcare). Densitometry analysis of the blots was performed using Image Lab software (Bio-Rad).
In the standard infection assay done at 37°C (34), 2.5 × 105 BMD macrophages or 1 × 106 differentiated U937 cells were seeded in triplicate into 24-well plates (Falcon) in RPMI-FBS and inoculated with L. pneumophila that had been grown to post-exponential phase on BCYE agar for 3 days. As described before, bacteria were added at a multiplicity of infection (MOI) (i.e., ratio of added bacteria to host cells) equal to 1 for BMD macrophages or 0.5 for U937 cells. Following a 2-h incubation to allow for bacterial uptake, the monolayers were washed extensively to remove remaining extracellular legionellae, and then the infection was continued in the presence of fresh RPMI-FBS for up to 3 days. In infection assays using GFP-expressing bacteria, RPMI-FBS was supplemented with 1 mM IPTG. For CFU determination, the macrophages were lysed with 0.1% saponin, and then dilutions of the lysates were plated onto BCYE medium. As a variation on this protocol, monolayers were inoculated at an MOI of 1 or 50, and then the bacteria were centrifuged onto the macrophages (250 × g for 5 min) in order to hasten and synchronize the infection event, as done previously (49–51). After 5 min of incubation at 37°C to permit bacterial uptake (49), monolayers were washed with RPMI medium and then exposed to 200 μg/ml gentamicin (Corning) in 1 ml RPMI-FBS for 1 h to kill any remaining extracellular legionellae. After another series of three washes to remove the antibiotic, the infected monolayers (n = 4) were incubated for another 2 to 72 h and then lysed, and the numbers of CFU were determined by plating. By measuring the levels of lactate dehydrogenase released into the tissue culture supernatants as previously described, using a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega) (52), we confirmed that the infections done using an MOI of 50 did not result in contact-dependent cytotoxicity for the host cells (data not shown).
Assessment of bacterial entry into macrophages.To assess the extent of L. pneumophila entry into macrophages, a gentamicin protection assay was done (36, 53). Briefly, the bacterial strains were added to the BMD macrophages at an MOI equal to 1 or 50, and then the infection was synchronized as described above. Following gentamicin treatment to kill residual extracellular legionellae and a final wash step as detailed above, the infected monolayers (n = 4) were lysed and the numbers of internalized CFU were determined by plating. Entry was measured as the percentage of intracellular CFU recovered from the monolayers compared to the number of CFU in the inoculum. As a second means of monitoring bacterial entry, we utilized a trypan blue quenching-based entry assay which has been described previously for L. pneumophila (54–56). To that end, BMD macrophages (5 × 104) or differentiated U937 cells (1 × 105) were seeded into black, clear-bottomed, 96-well microtiter plates (Falcon), and bacteria suspended in RPMI medium lacking phenol red (in order to reduce background fluorescence) were added at an MOI of 50 to six replicate wells. After synchronizing the infections as described above, a fluorescence reading (excitation, 485 nm; emission, 530 nm) was taken using a Synergy H1 hybrid microplate reader (BioTek, Winooski, VT) in order to determine the initial amount of fluorescent legionellae in the infected wells. Following the removal of extracellular bacteria by washing, 50 μl of trypan blue solution (54) was added for 1 min to quench any residual extracellular fluorescence. Finally, another fluorescence reading was taken to determine the amount of trypan blue-resistant fluorescence (i.e., intracellular legionellae) remaining in the wells. Entry was defined as the percentage of resistant fluorescence over total fluorescence.
Phagosome-lysosome fusion assays.As a first method for assessing the fusion of LCVs with macrophage lysosomes, we examined infected monolayers by using the marker LysoTracker Red, as previously done for the study of L. pneumophila (57–59). BMD macrophages (1 × 105) or differentiated U937 cells (2 × 105) were seeded onto sterile circular glass coverslips (Fisher Scientific, Waltham, MA) which had been placed within a 24-well dish and then infected at an MOI of 50 with synchronization. As a positive control, macrophages were also exposed to WT bacteria that had been heat killed by incubation at 65°C for 10 min (60). Following removal of extracellular bacteria by washing, LysoTracker Red (Life Technologies) was added to 75 nM in RPMI-FBS, and after 2 h, the colocalization of bacteria and LysoTracker Red was assessed using immunofluorescence microscopy. To that end, infected macrophages were fixed in 2% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), permeabilized in ice-cold methanol, and then blocked in PBS containing 2% goat serum (Life Technologies) as previously described (61). To then detect intracellular bacteria, the coverslips were incubated with the mouse monoclonal anti-Legionella antibody MAB2 (62, 63) at 1:10,000 for 1 h at 37°C, washed, and then treated with goat anti-mouse–Oregon Green (Life Technologies) at 1:10,000 for 1 h at 37°C. After final washes, coverslips were mounted on glass slides (Fisher Scientific) by use of ProLong Diamond antifade mounting reagent with DAPI (4′,6-diamidino-2-phenylindole) (Life Technologies). Finally, the infected cells were inspected with an Evos FL cell imaging system, using the manufacturer's DAPI, Texas Red, and GFP filter cubes (Advanced Microscopy Group, Life Technologies). As a second method for monitoring phagosome-lysosome fusion, we utilized the Texas Red ovalbumin (TROv) (Life Technologies) detection assay, which has been applied previously to the study of L. pneumophila (64, 65). Following prelabeling with 50 nM TROv for 30 min and a 30-min chase with fresh RPMI-FBS, BMD macrophages were infected at an MOI of 50 with synchronization, and at 2, 4, and 6 h, the infected cells were examined for colocalization of TROv and bacteria by immunofluorescence microscopy as detailed above. For the LysoTracker Red- and TROv-based assays, at least 100 infected cells were scored per strain per experiment.
Microscopic assessment of intravacuolar replication.GFP-expressing bacteria were added to 7.5 × 104 BMD macrophages on coverslips at an MOI of 5, and then the infection was synchronized as noted above. Following the usual wash step, the infected monolayers were replenished with RPMI-FBS supplemented with 1 mM IPTG in order to induce GFP expression and then incubated for another 4, 8, or 12 h. Finally, the infected macrophages were processed for immunofluorescence microscopy, and the number of GFP-expressing legionellae per LCV was determined by visual inspection (66–68). However, to precisely distinguish intracellular legionellae from any residual extracellular bacteria, the monolayers were treated with anti-L. pneumophila antibody and then with goat anti-mouse–Alexa Fluor 594 (Life Technologies) at 1:250 for 1 h in the absence of permeabilization. Thus, only LCVs containing bacteria expressing GFP but not labeled with Alexa Fluor 594 were scored. For this assay, we counted the number of bacteria within at least 300 vacuoles per strain per experiment, using an Evos FL cell imaging system with DAPI and GFP filter cubes.
Measurement of the association of Rab1B with the LCV.BMD macrophages (1 × 105) or differentiated U937 cells (2 × 105) were infected with L. pneumophila strains at an MOI of 50, as detailed above, and then incubated for another 1, 2, 4, 6, and 8 h. The samples were then fixed and permeabilized as described above. In addition to being treated with the anti-L. pneumophila monoclonal antibody to detect intracellular legionellae in LCVs, the coverslips were incubated with rabbit polyclonal anti-Rab1B at 1:100 for 1 h at 37°C followed by goat anti-rabbit–Alexa Fluor 594 (Life Technologies) at 1:250 for 1 h at 37°C. The colocalization of LCV and Rab1B was then assessed using immunofluorescence microscopy, with at least 100 infected cells scored per strain per experiment, using an Evos FL cell imaging system with DAPI, Texas Red, and GFP light cubes.
qRT-PCR analysis.Bacterial RNA was isolated from infected BMD macrophages by use of TRIzol reagent (Life Technologies) according to the manufacturer's instructions. Briefly, after removal of the culture medium, the monolayers were lysed with TRIzol, and RNA was extracted via phase separation (69). Samples were then treated with RQ1 DNase (Promega) for 45 min at 37°C. The purity and concentration of the RNA were confirmed by spectrophotometry (NanoDrop; Thermo Scientific). cDNA was synthesized from the DNase-treated RNA in a 20-μl reaction mixture as previously described (47). The PCR primers used to detect transcripts from the L. pneumophila chiA (RW37 and RW38), gyrB (RW39 and RW40), lspF (RW41 and RW42), plaC (RW43 and RW44), and srnA (RW45 and RW46) genes are listed in Table S1 in the supplemental material. Quantitative reverse transcription-PCR (qRT-PCR) was done as described before (47), using a SYBR green probe and a CFX96 Touch real-time PCR detection system (Bio-Rad), with the only modification being the addition of 5 more amplification cycles. The primers used for qRT-PCR analysis were designed using the Primer-BLAST tool on the NCBI website (70) for the sidM (RW23 and RW24), ankX (RW25 and RW26), lidA (RW27 and RW28), lepB (RW29 and RW30), lem-3 (RW31 and RW32), sidD (RW33 and RW34), and dotA (RW35 and RW36) genes, and they are listed in Table S1. The relative RNA abundance was determined using the −ΔCT method (CT for reference − CT for gene of interest) (71), with gyrB used as the reference gene (47, 72, 73).
T4S translocation assay.In order to study the translocation efficiency of the T4S system in WT- versus mutant-infected macrophages, effector protein genes were cloned just downstream of the β-lactamase (Bla) gene in pXDC61, a derivative of pMMB2002 (74), creating fusion proteins with the Bla reporter at the N terminus. Plasmids pXlidA, pXlepB, pXralF, pXsidM, and pXankX, encoding Bla fusions with LidA, LepB, RalF, SidM, and AnkX, respectively, were a kind gift from Howard Shuman, University of Chicago (54). To also study the translocation of the T4S effectors Lem3 and SidD, we constructed plasmids pXlem3 and pXsidD by using previously described methods (74). Thus, the lem-3 and sidD ORFs were amplified by use of primer pairs RW15-RW16 and RW17-RW18, respectively (see Table S1 in the supplemental material). The lem-3 PCR product was digested with KpnI and XbaI and then cloned into KpnI/XbaI-digested pXDC61, whereas the sidD product was digested with SmaI and XbaI and then cloned into similarly treated pXDC61. The in-frame insertions of Bla were confirmed by PCR and DNA sequence analysis. Finally, pXlidA, pXlepB, pXralF, pXsidM, pXankX, pXlem3, and pXsidD were introduced into WT strain 130b, the lspF mutant NU275, and the dotA mutant NU428 by electroporation. Assessments of protein translocation out of the LCV and into the host cell cytosol were performed as previously described (54, 74–76). Briefly, BMD macrophages were seeded into black, clear-bottomed microtiter plates at 5 × 104 cells per well for 72 h prior to infection. L. pneumophila strains carrying the various Bla fusions were grown for 24 h on BCYE agar containing chloramphenicol and 0.5 mM IPTG to induce production of the hybrid proteins. Bacteria were then added to the macrophages at an MOI of 50, and the infection was synchronized by centrifugation (600 × g, 10 min). Following a 1-h incubation, the infected cells were exposed to the Bla substrate CCF2-AM (Life Technologies) in the presence of 15 mM probenecid (Santa Cruz Biotechnology), which promotes retention of CCF2-AM. Finally, after a further 1 h of incubation, the levels of fluorescence generated in the macrophage cytoplasm due to the cleavage of CCF2-AM by the translocated Bla fusions were quantified on a BioTek fluorescence microplate reader, with excitation at 405 nm and emission at 460 nm and 530 nm (74). Translocation was measured as the 460-nm/530-nm emission ratio adjusted for background fluorescence. To confirm the expression of the SidD fusion protein, which did not show evidence of translocation (see Fig. 6 and below), bacteria containing pXsidD were examined by immunoblot analysis using anti-Bla antibodies (QED Bioscience, San Diego, CA) at 1:200 in TBS-T containing 5% nonfat dry milk.
Assessment of bacterial lysis within infected macrophages.The level of lysis of intracellular Legionella was determined essentially as previously described (67). BMD macrophages were infected with GFP-expressing L. pneumophila strains at an MOI of 50 as described above. At 4 h, the RPMI medium was removed from the wells and replaced with 1 ml of fresh medium containing 0.2% saponin in order to lyse the infected macrophages but not the intact bacteria contained within them. After dislodging the macrophages with a cell scraper (Falcon), the entire content of each well was centrifuged in a microcentrifuge tube at 8,000 × g for 10 min at 4°C. The resultant pellet, containing intact bacteria and large cellular debris, was resuspended and lysed in Laemmli buffer. The soluble proteins in the supernatant were isolated and concentrated via methanol-chloroform extraction (77) and then reconstituted in Laemmli buffer. Cellular and soluble fractions were subjected to SDS-PAGE on a 4 to 20% gradient gel (Bio-Rad), and the separated proteins were transferred to a PVDF membrane. After incubating for 12 h at 4°C in TBS-T containing 5% nonfat dry milk, the membranes were treated with rabbit anti-GFP antibody (Cell Signaling Technology) at a dilution of 1:1,000 in TBS-T containing 5% nonfat dry milk for 12 h at 4°C. As a loading control, membranes were also treated with mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) (Santa Cruz Biotechnology) at 1:400, which detects a host cytoplasmic protein that is abundant in the soluble fraction. Following washes, the blots were incubated with anti-rabbit or anti-mouse IgG–horseradish peroxidase conjugate (Cell Signaling Technology) at a dilution of 1:1,000 in TBS-T containing 5% dry milk for 1 h at room temperature and then developed using ECL detection reagent, and the immunoblot was analyzed using Image Lab software as noted above.
T2S promotes L. pneumophila infection of A/J mouse macrophages.We previously demonstrated that the L. pneumophila T2S system is required for optimal infection of human macrophages, as an lspF mutant of strain 130b that lacks T2S, but not a complemented lspF mutant, exhibited an approximately 10-fold reduction in CFU in infected U937 cell monolayers (33). We also determined that T2S promotes L. pneumophila replication and persistence in the lungs of A/J mice (25, 33, 34). Thus, to begin this study, we compared WT strain 130b, its lspF mutant, and the complemented mutant for the ability to replicate within monolayers of BMD macrophages from A/J mice over a 3-day incubation period. In this assay, initially infected macrophages are lysed by 24 h, and then one or two rounds of reinfection follow over the next 24 to 48 h. After inoculation at an MOI equal to 1, significantly fewer mutant CFU were recovered from the monolayers at 24, 48, and 72 h postinoculation (Fig. 1A). When the MOI was increased to 50, fewer mutant bacteria were again observed (Fig. 1B). Importantly, the complemented lspF mutant, carrying an intact copy of lspF on a plasmid, behaved like the WT strain did (Fig. 1A and B), confirming that the mutant defect was due to the loss of T2S. Together, these data confirmed that T2S promotes L. pneumophila infection of A/J macrophages and does so over a range of infectious doses and during multiple rounds of infection. Since the lspF mutant defect was seen in the first 24 h, as is the case for infected U937 cells (33), we posited that T2S promotes the initial round of infection, helping to mediate bacterial entry, resistance to immediate intracellular killing, and/or replication itself.
T2S is dispensable for entry of L. pneumophila into macrophages.As the first step toward explaining the role of T2S in intracellular infection, we assessed the contribution of T2S to L. pneumophila entry into host cells. BMD macrophages were briefly exposed to WT 130b and the lspF mutant at an MOI of 1 or 50, and after the removal of residual extracellular bacteria by treatment with gentamicin, intracellular bacteria were counted by plating. At either MOI, the entry levels of WT and mutant bacteria were comparable (Fig. 2A). The observation that 1 to 3% of the inoculum entered into the macrophages is similar to past observations made with L. pneumophila and this type of gentamicin protection assay (53). To confirm our results, we utilized a trypan blue quenching method that was recently adapted to assess L. pneumophila entry into host cells (54–56). In this case, we infected the BMD macrophages with GFP-expressing bacteria at an MOI equal to 50, and after treatment with trypan blue to quench residual extracellular bacteria, the level of trypan blue-resistant fluorescence remaining associated with the monolayer (i.e., intracellular bacteria) was assessed using a fluorescence microplate reader. By this method, the entry levels of WT and mutant bacteria were again comparable (Fig. 2B). Moreover, the overall levels of entry detected were similar to those obtained using the gentamicin protection assay. When the assay was done using U937 cells, there appeared to be an overall increase in bacterial entry, which is compatible with the results of a previous study that compared U937 cells and BMD macrophages (67). However, there was still no difference between the WT and the lspF mutant (Fig. 2B). In sum, these data showed that the lspF mutant is not impaired for entry into A/J macrophages or human U937 cells, indicating that T2S is dispensable for L. pneumophila entry into macrophages.
T2S is dispensable for L. pneumophila evasion of the host degradative pathway.Shortly after its entry into macrophages, L. pneumophila evades trafficking to the endolysosomal pathway and thereby avoids acidification and degradation (78, 79). Thus, as the next step toward defining the role of T2S in intracellular infection, we compared the WT and lspF mutant strains for the ability to escape this degradative pathway in BMD macrophages, as measured by bacterial colocalization with LysoTracker Red at 2 h postinoculation. As expected (79, 80), WT 130b evaded the lysosomal compartment (3%), whereas the heat-killed WT strain did not (83%) (Fig. 3A). The lspF mutant displayed a very low level of colocalization that was identical to that of the WT (Fig. 3A). To confirm these results, we monitored colocalization using a different marker, namely, TROv (81). Again, the WT and mutant strains exhibited equivalent abilities to evade lysosomes (4%), whereas heat-killed bacteria readily trafficked to the degradative compartment (89%) (Fig. 3B). The capacity of the lspF mutant to evade degradation was evident not only at 2 h but also at 4 and 6 h postinoculation. Together, these data indicated that the T2S mutant was not preferentially delivered to degradative lysosomes during infection of murine macrophages. The same outcomes were obtained when we assessed the colocalization of bacteria and LysoTracker Red in human U937 cells, i.e., 4% for the WT and mutant strains and 75% for the heat-killed WT (Fig. 3C). Thus, we concluded that T2S is dispensable for the ability of L. pneumophila to evade the macrophage's degradative pathway, and we suspected that the lspF mutant is defective in the later, replicative phase of intracellular infection.
T2S, although dispensable for early intracellular survival, is required for intravacuolar replication.To begin to determine when T2S becomes important during infection, we exposed BMD macrophages to WT 130b and the lspF mutant and then enumerated intracellular CFU at 0, 2, 4, and 12 h postinoculation. Between 0 and 4 h, WT numbers declined approximately 3-fold (Fig. 4A), indicating killing by the macrophages, which was likely due at least in part to the low level of lysosome association that we observed (Fig. 2B). Compatible with the results obtained using LysoTracker Red and TROv, there were no significant differences between the WT and mutant strains in terms of numbers of CFU recovered at 2 and 4 h (Fig. 4A), supporting the view that T2S is dispensable for early intracellular survival. Between 4 and 12 h, the WT clearly began to replicate, showing a 17-fold increase in the number of CFU recovered (Fig. 4A). The fact that L. pneumophila begins replication at approximately 4 h postentry has been observed previously in studies using various other WT strains and macrophage hosts (67). At 12 h, the lspF mutant had 3-fold fewer CFU than the WT, revealing the emergence of a significant mutant phenotype between 4 and 12 h (Fig. 4A). To more precisely determine when the mutant begins to display its growth defect, we repeated the infection assay but enumerated intracellular bacteria at both 8 and 12 h postinoculation. At the 8-h time point, the mutant displayed 2-fold fewer CFU than the WT (Fig. 4B), implying that T2S begins to promote intracellular replication sometime between 4 and 8 h after entry into the macrophage. Once again, the mutant showed approximately 3-fold fewer CFU at 12 h (Fig. 4B). The complemented lspF mutant behaved as the WT did at both 8 and 12 h (Fig. 4B), confirming that the mutant's growth defect at these early time points was due to the loss of T2S. To support the results obtained from the CFU recovery assay, we infected BMD macrophages with GFP-expressing WT and lspF mutant bacteria and used immunofluorescence microscopy to count the numbers of legionellae within individual LCVs. As a prelude to this analysis, we confirmed that the expression of GFP did not alter the ability of either the WT or the lspF mutant to grow in the macrophage host (see Fig. S1 in the supplemental material). Based on the microscopic counting method, there was little evidence of intracellular replication at 4 h postinoculation, since ≥90% of WT LCVs had only a single bacterium, and compatible with the results of the CFU recovery assay, the lspF mutant behaved comparably to the WT at this early time point (Fig. 4C). By 8 h, the WT had obviously begun to replicate, as demonstrated by the marked reduction in the number of LCVs harboring 1 bacterium and by >70% of the LCVs having at least 3 bacteria (Fig. 4C). As in the CFU recovery assay, the mutant phenotype emerged by the 8-h time point, i.e., more mutant LCVs contained only 1 bacterium, and there were also fewer mutant LCVs that had ≥10 bacteria, although the latter difference was not quite statistically significant (Fig. 4C). When we repeated the immunofluorescence detection experiment by assaying at both 8 and 12 h postentry, the mutant growth defect was even more apparent at 12 h (Fig. 4D). Whereas the most common WT LCVs (at 56%) were those that contained ≥10 bacteria, approximately 40% of mutant LCVs still contained 1 bacterium, and only about 29% of the mutant LCVs had ≥10 bacteria. For both the WT and the mutant, the proportion of LCVs containing 1 bacterium did not decrease further between 8 and 12 h (Fig. 4D), suggesting that the replicative capacity of the LCV is defined sometime between 4 and 8 h. Together, these data indicated that the T2S mutant was impaired not only in the ability to initiate replication between 4 and 8 h but also in the capacity to grow to large numbers within LCVs at 12 h. Thus, we concluded that L. pneumophila T2S first promotes intravacuolar growth sometime between 4 and 12 h after bacterial entry, which explains, at least in part, its contribution to growth during more extended incubation periods, such as in Fig. 1.
T2S promotes the association of host Rab1B with the LCV.A hallmark of the LCV is its association with various smooth vesicles derived from the endoplasmic reticulum (ER)-to-Golgi trafficking pathway (82–85). These host vesicles are thought to deliver membrane material, foodstuffs, and other "cargo" that facilitate intravacuolar growth of L. pneumophila (86). Among the host GTPases that modulate ER-to-Golgi vesicle traffic, Rab1B is well known for being recruited rapidly and substantially to LCVs in A/J BMD macrophages and U937 cells as well as murine RAW264.7 macrophages, human A549 lung epithelial cells, and human HEK293 embryonic kidney cells (61, 66, 67, 87–98). Expression of a dominant negative form of Rab1 restricts L. pneumophila growth in Chinese hamster ovary (CHO) cells and COS1 cells, suggesting that Rab1B recruitment enhances bacterial growth (61, 66). Thus, we explored the possibility that T2S is needed for the association of Rab1B with the LCV. To begin to test this, we infected BMD macrophages with WT 130b and its lspF mutant and then monitored the association of endogenous Rab1B with the LCVs by using immunofluorescence microscopy. At 1 and 2 h postinoculation, WT and mutant LCVs exhibited identical levels of Rab1B association, which reached a maximum of 60% (Fig. 5A, left panel), indicating that T2S is not required for the initial recruitment of Rab1 to the LCV. Yet when we examined the infected cells at 4 h, there was a significant (37%) reduction in Rab1B association with the mutant LCVs but not the WT LCVs (Fig. 5A, left panel; see Fig. S2 in the supplemental material for representative images showing the presence versus absence of colocalization), suggesting that the lspF mutant had an impaired ability to continue Rab1B recruitment between 2 and 4 h or to retain the Rab1B that had already been recruited. To determine if the difference between the WT and mutant strains changed over time, we analyzed the Rab1B association at 4, 6, and 8 h postentry. Compatible with previous observations (89, 92), Rab1B association with LCVs containing the WT declined between 4 h and 8 h (Fig. 5A, right panel). At the 6- and 8-h time points, the lspF mutant LCVs displayed levels of Rab1B association that were now comparable to those with the WT LCVs. Together, these data indicated that the lspF mutant had a transient defect in Rab1B association that was seen 4 h after bacterial entry into BMD macrophages. A second, independently derived T2S mutant (i.e., the lspE mutant NU361) exhibited the same inability to associate with Rab1B (Fig. 5B), indicating that the defect we observed was due to the loss of T2S, not to a spontaneous second-site mutation(s). Further support for a role for T2S in Rab1B association was obtained when reintroduction of an intact copy of lspF into the lspF mutant restored Rab1B association to WT levels (Fig. 5B).
The observation that the lspF mutant showed neither increased trafficking to lysosomes nor impaired survival at 4 h postinoculation (Fig. 3B and 4A and C) implied that its reduction of Rab1B association was not an artifact of there being more dead or damaged bacteria within the LCVs. Nonetheless, to directly determine whether the lspF mutant undergoes increased lysis, we infected BMD macrophages with WT and mutant strains expressing cell-associated GFP and then, at 4 h postinoculation, assayed levels of GFP released from the bacteria into the soluble fraction of the host cells. Similarly low levels of released GFP were seen for the lysates obtained from WT- versus mutant-infected cells (see Fig. S3 in the supplemental material), affirming that the T2S mutant does not undergo more lysis than the WT. The T2S mutant defect in Rab1 trafficking was also not simply a nonspecific effect of impaired bacterial growth, since it occurred at 4 h, prior to the onset of the mutant replication defect (Fig. 3). In support of this conclusion, a previously described rcp mutant of strain 130b (40), which has normal T2S but has a replication defect that is comparable to that of the lspF mutant, displayed a level of Rab1B association that was identical to that of the WT (see Fig. S4). Moreover, there are various types of L. pneumophila mutants that are also defective for intracellular growth but do not show any alteration in Rab1 association (67, 99). Thus, we concluded that the T2S mutant defect in Rab1B association is physiologically relevant and worthy of further investigation.
As noted above, at least 15 studies have documented the association between Rab1B and LCVs; however, only 3 of these studies used human macrophages, and in no case was there a direct comparison between murine and human host cells (66, 93, 97). Therefore, we infected U937 cells with WT 130b and its lspF mutant and monitored the association of Rab1B with the LCVs, using the same immunofluorescence assay as that used to assess BMD macrophages. Approximately 60% of WT LCVs had a Rab1B association at 4 h (Fig. 5C; see Fig. S2 in the supplemental material), documenting that Rab1B associations are comparable between murine and human macrophages. The lspF mutant, once again, displayed a reduction in the number of Rab1B+ LCVs (Fig. 5C; see Fig. S2), demonstrating that the role of T2S in promoting Rab1B association exists in both murine and human macrophages.
The role of T2S in Rab1B association is not due to an effect on the expression or translocation of key T4S effectors.The L. pneumophila Dot/Icm T4S system is very well known for its role in modulating the association of Rab1B with the LCV (20, 100). The T4S apparatus translocates myriad effectors across the LCV and into the host cell cytosol, and a subset of those effectors (i.e., AnkX [LegA8], Lem3, LepB, LidA, SidD, and SidM [DrrA]) mediate the tethering of Rab1B to the LCV membrane or the later release of the Rab1 protein (87–89, 91, 92, 101–108). Therefore, we considered the possibility that the lspF mutant's impaired association with Rab1B might be due to an artifact of an absent T2S system relevant to the expression or translocation function of the T4S system. To assess effects on gene expression, we infected BMD macrophages with WT and lspF mutant bacteria for 4 h and then, using quantitative RT-PCR analysis, analyzed the levels of ankX, lem-3, lepB, lidA, sidD, and sidM transcripts as well as mRNA levels for dotA, which encodes a key component of the T4S apparatus (79). In all cases, the levels of transcripts were comparable between WT and mutant samples (Fig. 6A), indicating that the lspF mutation and the resultant lack of T2S had neither indiscriminately diminished the expression of T4S nor specifically dampened key effector genes. Incidentally, we did observe that the T4S genes were expressed to differing degrees at the 4-h time point, with lepB and lidA being expressed most highly and ankX being the least expressed gene (Fig. 6A). Next, to investigate a potential effect of the lspF mutation on the translocation function of the Dot/Icm system, we used a β-lactamase (Bla) reporter assay that has been used extensively to monitor L. pneumophila T4S within infected macrophages (74, 75, 109, 110). To that end, plasmids expressing a fusion between AnkX, Lem3, LepB, LidA, SidD, or SidM and the Bla reporter were introduced into the WT and its lspF mutant, and after infection of BMD macrophages, we monitored the levels of Bla delivered into the host cell cytosol. As a negative control, we also introduced the plasmids into a dotA mutant of strain 130b. As expected (74), WT 130b, but not the dotA mutant, exhibited translocation of AnkX, Lem3, LepB, LidA, and SidM, albeit to differing degrees (Fig. 6B). We did not detect a significant level of SidD translocation, although immunoblot analysis confirmed that the fusion protein was made by the strains (data not shown). Most importantly, the lspF mutant also exhibited effector translocation (Fig. 6B). The translocation of RalF, a T4S effector that acts on a different host GTPase, was also apparent in both the WT and the mutant (Fig. 6B). Together, these data showed that the loss of T2S in the lspF mutant had not broadly eliminated translocation of T4S effectors nor abolished the translocation of effectors that specifically modulate Rab1B. Thus, the effect of T2S on Rab1B association that we observed likely occurred independently of the expression and translocation of the Dot/Icm effectors that are known to influence Rab1B association with the LCV. Hence, we hypothesize that the L. pneumophila T2S system elaborates a secreted protein(s) that promotes the interaction between the LCV and Rab1B in a novel fashion. Compatible with this scenario, transcripts encoding components of the T2S apparatus as well as various T2S-dependent proteins were easily detected in infected host cells at the 4-h time point, when the effect of T2S on Rab1B is evident (see Fig. S5 in the supplemental material). As an initial attempt to determine which T2S substrate(s) promotes Rab1B association, we examined eight previously characterized mutants that lack individual T2S substrates. However, mutants of strain 130b defective for either chiA, lapA, lipA, map, plaC, plcA, proA, or srnA displayed the same level of Rab1B association as that of the wild type (see Fig. S6), indicating that the ChiA chitinase, LapA aminopeptidase, LipA lipase, Map phosphatase, PlaC acyltransferase, PlcA phospholipase C, ProA metalloprotease, and SrnA RNase are not required for optimal Rab1B association.
Depletion of Rab1B within human macrophages further restricts the growth of an L. pneumophila T2S mutant.Before considering further the identification of a T2S-dependent protein(s) that modulates Rab1B, we sought to discern the degree to which the impaired ability to harness the Rab1 protein explains the growth defect of the lspF mutant. The fact that impaired Rab1B association preceded the appearance of fewer mutant bacteria by only a few hours was compatible with the two phenotypes being connected. In order to directly test this hypothesis, we generated a stable U937 cell line expressing shRNA targeted against human Rab1B. We focused on the U937 cell model because it would not only help us to understand the T2S mutant but also address, for the first time, the need for Rab1B recruitment for the growth of WT L. pneumophila in human macrophages. The only past study to assess the impact of depleted Rab1 on L. pneumophila utilized knockdown of Drosophila Kc167 cells, and in that case, loss of the protein did not limit growth (111). A similar conclusion was reached when sidM mutants grew to WT levels within A/J BMD macrophages and amoebae despite their inability to recruit Rab1B (87, 88). Upon differentiation of the treated U937 cells into macrophage-like cells and removal of antibiotic selection, we attained excellent (>80%) knockdown of Rab1B at the protein level (Fig. 7A). We then compared Rab1B-depleted U937 cells and a U937 cell line containing a nontargeting shRNA ("scramble") for the ability to sustain the growth of WT 130b and the lspF mutant. The WT grew comparably in the two cell lines and, if anything, grew slightly better in the Rab1B-depleted cells (Fig. 7B; see Fig. S7 in the supplemental material), demonstrating that full expression of Rab1B is not required for the growth of L. pneumophila in human macrophages. Supporting this conclusion, we observed that our sidM mutant, which lacked the ability to recruit Rab1B (Fig. 5C), grew to WT levels in the depleted U937 cells (Fig. 7C; see Fig. S7). We surmise that in the case of the WT, other host GTPases and their associated vesicles compensate for the lack of Rab1B recruitment, as previously postulated (112). Since the growth of the sidM mutant was also unaffected by depletion of Rab1B in U937 cells (Fig. 7B), we inferred that the sidM mutant can still recruit other vesicles from the human host cell in order to support its growth (112). Most importantly, in contrast to the WT and the sidM mutant, the lspF mutant showed a 21% reduction in growth in Rab1B-depleted U937 cells compared to scramble U937 cells (Fig. 7B; see Fig. S7). The complemented lspF mutant behaved as the WT did in the Rab1B-depleted cells (Fig. 7B), confirming that the mutant's phenotype was due to the loss of T2S. In sum, our knockdown experiments indicate that the association of Rab1B with the LCV in macrophages is not absolutely necessary for the growth of WT L. pneumophila but can be important for a strain that lacks T2S. The same data also indicate that the lspF mutant lacks a function(s) beyond Rab1 recruitment and that this other defect(s) affects growth.
T2S promotes L. pneumophila growth through both Rab1B-dependent and Rab1B-independent processes.In order to validate the data obtained from examining Rab1B-depleted U937 cells, we constructed a sidM lspF double mutant by introducing an lspF mutation into the chromosome of the sidM mutant and then proceeded to test the new mutant for its relative infectivity for macrophages. Because mutation of sidM abolished Rab1B recruitment in both U937 cells and BMD macrophages (Fig. 5D; see Fig. S8 in the supplemental material), we reasoned that the double mutant would recapitulate the larger mutant defect observed in the Rab1B-knockdown cells. As a prelude to the infection assays, we first determined that the double mutant grows normally under standard extracellular growth conditions (see Fig. S9). As predicted, the sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant in both the human and murine macrophages, with the difference between the two mutants being as much as 40-fold at 72 h in the BMD macrophages (Fig. 8). Thus, the double mutant was impaired relative to WT strain 130b (and the sidM mutant) at all time points examined, with its defect relative to the WT reaching as much as 400-fold. Providing an intact copy of lspF on a plasmid restored the growth of the double mutant to WT levels (Fig. 8). These results confirmed that T2S promotes intracellular growth and that it does so partly, if not mainly, through a Rab1B-independent process.
The results presented here significantly advance our understanding of the role of T2S in intracellular infection of macrophages by L. pneumophila. Although the T2S system was dispensable for early infection events, including bacterial entry into the host cell, evasion of phagosome-lysosome fusion, and resistance to initial intracellular killing, it was necessary for optimal initiation of intravacuolar replication between 4 and 8 h postentry as well as for the capacity to grow to large numbers within LCVs between 8 and 12 h and beyond. Based upon our further analysis of multiple lsp mutants and a complemented derivative in human and murine macrophages, we determined that the role of T2S in early replication involves at least the promotion of Rab1B association with the LCV. Because the loss of T2S in the lspF mutant did not simply eliminate the transcription or translocation of T4S effectors, including ones known to modulate Rab1B recruitment, we posit that T2S elaborates a secreted protein(s) that promotes the interaction between the LCV and host Rab1B. In support of our data, a past study detected lspE and lspL mutants, which, like our lspF mutant, lack the type II secretion apparatus, in a screen of transposon-mutagenized L. pneumophila mutants for strains with impaired Rab1 association (88). Because it was focused on T4S, that study did not attempt complementation of the lsp mutants or investigate, to any degree, the lsp mutants' defects in intracellular infection. Thus, our study represents the first documentation of a role for T2S in the association of the LCV with Rab1B. Although T2S was recently shown to be important for the intracellular survival or growth of Burkholderia cenocepacia, Chlamydia trachomatis, and Yersinia enterocolitica (113–116), the current study is also the first demonstration of a link between T2S and Rab1 proteins associated with vesicular trafficking.
The recruitment of the Rab1B GTPase to the LCV and the role that T4S effectors play in that process have been the focus of an extraordinary amount of research, revealing a remarkable array of enzymatic reactions. Rab1B and the other Rab proteins are master regulators of intracellular trafficking, controlling the specificity, direction, timing, and coordination of vesicle transport (117). Rab1B is first activated and recruited to the LCV via T4S effectors, and then activated Rab1B facilitates the tethering of ER-derived vesicles at the LCV (61). In the first step of Rab1B activation, the T4S effector SidM (DrrA) promotes recruitment by acting as a guanine dissociation inhibitor (GDI) displacement factor, guanine nucleotide exchange factor (GEF), and AMPylation factor (87, 88, 101). The effector LidA, with its high affinity for active Rab1B, further enhances the recruitment of Rab1 to the LCV, while AnkX, by virtue of its phosphocholination activity, blocks possible inactivation of Rab1B by other factors (87, 102). Ultimately, SidD antagonizes SidM function with a deAMPylation activity, and Lem3 reverses the effect of AnkX, thus allowing Rab1B to be inactivated by LepB, a GTPase activation protein (GAP) (89, 91, 92, 103). With the hydrolysis of GTP to GDP, the Rab1 protein is released from the LCV and extracted back into the cytoplasmic inactive pool. Looking beyond the well-characterized SidM, LidA, AnkX, SidD, Lem3, and LepB proteins, recent reports implicate additional T4S effectors as interactors/modifiers of Rab1B (68, 118, 119). The fact that so many T4S effectors are dedicated to Rab1B recruitment indicates that the association of this Rab protein and ER-derived vesicles with the LCV is important for L. pneumophila growth. Without diminishing the role of T4S in Rab1B recruitment, our study signals that the recruitment and retention of this host GTPase at the LCV are not strictly the purview of T4S.
Because the lspF mutant's defect in Rab1B association was manifest at 4 h postentry, but not at 1 and 2 h postentry, we suspect that T2S does not act at an early stage of Rab1B recruitment but is needed for ongoing recruitment or retention. Indeed, T2S may promote L. pneumophila's association with Rab1B by enhancing later stages of Rab1B recruitment and tethering to the LCV or by inhibiting the release of recruited Rab1B. In considering further how T2S facilitates the association of Rab1B with the LCV, several intriguing scenarios can be envisioned. In one general scenario, a protein(s) secreted via T2S is translocated out of the LCV and gains access to the macrophage cytoplasm, where it acts directly on Rab1B so as to promote or prolong its association with the LCV. Alternatively, a translocated type II secreted protein indirectly influences Rab1B, either by modulating another factor that acts on Rab1B, such as a host protein or a T4S effector, or by influencing the nature of the cytoplasmic face of the LCV which serves to tether Rab1B. Compatible with this scenario is the fact that the L. pneumophila T2S system secretes a wide range of enzyme activities as well as many novel proteins of undefined function (23, 26, 30). In a second type of situation, the protein(s) secreted by the T2S system remains localized within the lumen of the LCV but proceeds to modify ("from within") the nature of the LCV and its membrane such that it is more receptive to association with Rab1B. Clearly, future investigations need to determine the subcellular location of type II secreted proteins in infected macrophages as well as to characterize the secreted protein(s) that influences Rab1B.
The simple fact that the lspF mutant has a growth defect in both human and murine macrophages yet a sidM mutant that completely lacks Rab1B recruitment grows similarly to the WT indicates that T2S must promote growth beyond facilitating Rab1B association with the LCV. By demonstrating that the magnitude of the lspF mutant defect is increased upon depletion of Rab1B in human macrophages, we confirmed that T2S also promotes intracellular growth by means that are independent of Rab1B. Once again, there are many potential explanations for this additional role of T2S. The magnitude of the defect linked to the loss of T2S was greatly exacerbated by the additional loss of SidM (i.e., the sidM lspF mutant had a larger defect than the lspF mutant), suggesting that T2S facilitates a process whose function is complementary to that of Rab1B-mediated vesicle recruitment. Indeed, simultaneously targeting components of multiple early secretory pathways via small interfering RNA (siRNA)-mediated depletion can result in dramatic inhibition of L. pneumophila growth (111). In a similar vein, a bacterial mutant that lacks the ability to harness a single vesicular pathway and thus still grows to WT levels in normal host cells (e.g., a lidA mutant lacking Rab1 recruitment) exhibits a growth defect in host cells that are defective (via siRNA depletion) for another aspect of vesicle trafficking (120). Thus, we posit that T2S promotes the recruitment of various types of host vesicles to the LCV. On the other hand, it is also possible that T2S-dependent proteins which come to reside in the host cell cytoplasm promote intracellular growth by means that are unrelated to vesicular trafficking, such as promoting the association of the LCV with mitochondria or the regulation of protein turnover and the ubiquitin pathway, among other things (15, 16). Finally, it is also possible that the Rab1B-independent effect of T2S is due to events that occur strictly within the LCV, such as the acquisition of nutrients and the stabilization of the LCV membrane. None of the L. pneumophila mutants lacking individual type II secreted proteins that have been tested in macrophages have exhibited a growth defect (23), suggesting that an as yet undefined secreted protein(s) is responsible for the role of T2S in intramacrophage growth. Compatible with this hypothesis, our proteomic and in silico analyses indicate that the L. pneumophila T2S system may secrete as many as 60 proteins (25).
In summary, we have shown that the T2S system of L. pneumophila promotes intravacuolar growth and enhances the association of Rab1B with the LCV, a process that we have shown can promote growth in human and murine macrophages. Our Rab1B depletion experiments and analysis of various bacterial mutants further indicate that T2S also promotes intramacrophage replication through Rab1B-independent mechanisms that likely involve a novel secreted protein(s). Finally, we strongly suspect that the L. pneumophila T2S system evolved in part to facilitate similar events in infected amoebae, as the intracellular life cycles of L. pneumophila within macrophages and environmental protozoa are remarkably similar (121). Indeed, in both hosts, the LCV associates with secretory vesicles, rough ER, mitochondria, and the ubiquitination machinery, among other things, and proteomic analyses of LCVs purified from amoebae revealed the presence of small GTPases, including Rab1 homologs (122, 123).
We thank past and present members of the Cianciotto lab for their helpful advice. We acknowledge Ombeline Rossier for construction of pMGFP and Meghan Pearce for introducing the plasmid into L. pneumophila strains. We thank Howard Shuman for providing pXlidA, pXlepB, pXralF, pXsidM, and pXankX, Michele Swanson for pMMB-GRN, and Richard Kohler for anti-L. pneumophila MAB2. We also thank Laimonis Laimins for providing lentiviral packaging plasmids and the nontargeting shRNA control plasmid and Eva Gottwein for providing HEK293T cells.
R.C.W. was supported in part by NIH training grant T32 AI0007476. This study was funded by NIH grant R01 AI043987 awarded to N.P.C.
- Received 29 August 2016.
- Accepted 31 August 2016.
- Accepted manuscript posted online 6 September 2016.
Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00750-16.
- Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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Welcome to My Tour de Cure Web Page!
This is my seventh year riding in the Tour de Cure.
I am riding because the American Diabetes Association's Tour de Cure is so much more than a cycling event to me. It is my own personal challenge, biking has become a passion and I would like to help make a difference.
I am committed to ride 100 miles this year in this event. I ride year round in the cold, the heat, and someday's in the rain. There are 26 million people in the United States and though out the world that have diabetes, I personally know some of them, they are my family my friends, AND MYSELF.
By making a donation small or large, you will be helping provide community-based education programs, protect the rights of people with diabetes and fund critical research for a cure. I know firsthand how the education and information has helped. I have a great support team from my family, friends, team mates, my doctors and you. Without all of these people and you in my life I would be lost.
This year I ride with an Insulin Pump and Censor. I'm feeling the best that I have felt in years. I did not realize how shitty I was feeling. This technology is because of your donations. You have helped me personally and made my life allot better. I have battled this monster since I was 17 years old when it was first detected, I may have had it longer.
WE will pedal for a cure together.
I truly appreciate your support. Together we can Stop Diabetes!
Thank you for your help though a donation, for being there in my life. All of you are my support team and I love you. With a big hug and my heart, thank you.
If you think this page contains objectionable content, please inform the system administrator.


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Drawn with MICRON ~3 hours of work
This last September, the Joshua Center for Neurological Disorders held the 29th annual PorkOut auction dinner. I entered another drawing of a pig into the auction, along with another "drawing of the highest bidder's choice" and raised $100 more than last year. The pig raised $500, the other item raised $600 (split among two bidders). […]
I am in AP studio art this year. It requires me to create 24 drawings (12 anything, 12 are a concentration) and this is one. It's based off a photograph I took in Steamboat, Colorado over the summer, along the trails at Fish Creek Falls. The paper is 4×6″ done in graphite.
January 3, 2014
I've worked a month and a half on this drawing. One of my best yet. I took this photograph on a hike to the top of Han's Peak in Steamboat, Colorado. He's wearing my pair of mirrored Premium Knockaround sunglasses, so I thought it would be a cool picture. I set a high exposure and […]
April 30, 2013
I did this in graphite in my art class between yesterday and today. I plan on working more on it.
April 25, 2013
This is a pied crow.
April 24, 2013
I drew all of these in Drawing class at school. The first two are done in India ink, and the last one in a sort of oil pastel.


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Today, every computer, tablet and phone comes with audio input and output. Apps, games, and user interfaces would be unthinkable without sound. Yet, C++ currently provides no audio facilities out of the box. The literature on audio in C++ is sparse. For even the simplest possible audio functionality, programmers need to deal with a confusing landscape of complex platform-specific APIs and proprietary 3rd party libraries, even though the low-level mechanics of audio I/O is very similar on all these platforms.
In this talk, we will first show how audio signals can be digitally represented in C++, covering basic concepts such as channels, audio buffers, PCM, and sample rate. We explain the principles of communicating with your sound card and playing, recording, reading and writing audio data. We will then present a proposal to add a minimal, modern, low-level audio API to the C++ standard library, implementing these principles. Finally, we will show some simple example apps, using our implementation of this API to make some actual sound!


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Developer could build two hotels downtown
Developers of a $22 million hotel project for the southern end of downtown said Tuesday they could build a second hotel if market conditions warrant an addition.
The Midland City Council, by a 4-3 vote, approved tax abatements – which are not to exceed $794,908 --with RIMT Property, LP. The vote also allows for vacating an 80-foot-by-300-foot portion of Indiana Avenue right-of-way.
The developer is seeking to bring a Residence Inn to the southern piece of property between Big Spring and Marienfeld streets.
Developers told the council that the Residence Inn could be the first of two projects if the Midland market shows a need for a second hotel. If there is a need, developers told city leaders of the potential for a "full-service hotel" with possible upscale restaurant on the north side of Indiana. For now, RIMT Property officials said there is a need to allow the market to sort itself out.
As it stands now, vacating Indiana Avenue will allow for a private drive into the hotel property. At some point, developers expect to have a roundabout that will help with traffic control should a second hotel be built.
Handing over that portion of Indiana and the abatement costs were discussion items. Developers said it seemed like an appropriate request considering water and sewer lines will have to be moved and that could cost as much as $1.5 million.
City officials said a 110-room Residence Inn could raise around $2.6 million in hotel-motel tax revenue over a 10-year period.
Those voting against the abatement and vacation of city property were Spencer Robnett, Jack Ladd Jr. and John Norman.
Council notes
The Midland City Council held off on votes for members to the Midland Development Corp. and Parks Department boards for the second-straight meeting. City leaders decided they want to increase the MDC board from five to seven members. The wait also will allow time for council members to speak with city staff about city boards. It also provides more time to study the number of resumes the city received in the past month.
Clarification on airport drilling story
The Midland City Council approved the city manager to negotiate a land swap with Midland College that will allow a local oilman to drill underneath Midland Airpark. In a preview story, it was written that some mineral revenue could go into the general fund. It should be noted that mineral revenue from drilling directly under airpark goes into an Airport fund, and money can't be moved from that fund to the General Fund. Drilling under other city property would create mineral revenue for the General Fund.


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Web Hosting transfer do i need transfer my domain?
Article ID: 3777 Published: 7 Sep 2015 Updated: 21 Mar 2018 Authored by: Aust Domains Support
We do not require the domain name to be transferred to us, this is your decision. It can stay with your current domain provider.
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To protect the safety of our students and limit the spread of the Corona Virus, the CPI Campus is closed to all students and visitors through April 30th. We will continue to evaluate conditions and may extend the duration of the closure. If you have questions, please direct them to firstname.lastname@example.com.
Thank you for your understanding during this difficult time and please stay safe and healthy.


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GREAT LOCATION! This well maintained 4 level split home offers 4 bedrooms and is ready for immediate possession! Newer shingles, furnace and hot water tank. Some new windows and newer patio door. Master bedroom with 3 piece ensuite. Large living room, dining are and eat-in kitchen. Lower level family room. Rec room in basement, bar, utility room and lots of storage space! Private south facing backyard with mature trees. Oversized double attached garage. See it today! Visit REALTOR® website for additional information.
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Data was last updated April 7th, 2020 at 10:16pm MDT.


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CM inaugurates Modern Police Control room
With the multi channel telephonic system in it, the operator can take more calls at a time and be in touch with the caller and the PCR van present at the nearest location of the caller. This would lessen the response time and help the police to alleviate crime rates and maintain law and order in the Capital.
"I think this will do a great deal of service for simplifying and make it quicker the security and the safety of the citizens of the city", said Chief Minister Naveen Patnaik.
Set up with the help of Interact Public Security Systems (IPSS), the control room is equipped with all the modern facilities. A total 30 trained constables and five officers are entrusted to operate the system.
Meanwhile, the commissioner of police informed that this kind of system would soon be initiated at Cuttack and other cities of the state.
"It is a platform where we can implement an integrated command and control system in future when we will install CCTVs and focus the whole city by the cameras", said police commissioner Bijay Kumar Sharma.


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When it comes to late night talk show hosts there is no shortage of dreamy men to crush on. There's the towering splendour of Conan O' Brien and his shock of red hair, the fiery passion of Jon Stewart, even Jimmy Fallon was a cutie in his SNL days (now he seems more like a hyper-caffeinated fan boy).
But there's only one man in late night for me and that man is Craig Ferguson. As you may have guessed, I have an inappropriate crush on him. He's the reason I can't get a good night's sleep. Every evening I tell myself to go to bed at a decent hour but instead I let the hours slip by, lured by the promise of another hilarious episode of the Late Late Show.
At exactly 12:37 a.m. Craig and his suaveness appear on my screen. For ten years Craig's "I don't give a fuck attitude" has cultivated a relaxed and inviting atmosphere. Craig is so smooth and charismatic that his guests truly open up to him. His is the only show where it seems like he's just having a casual conversation with a guest instead of setting them up for a well-rehearsed anecdote. Sometimes it's hard not to get lost in his dreamy blue eyes and thick Scottish accent and imagine that he's interviewing me instead.
But that's not the only thing that draws me to Craig. As if he was playing right into my liberal arts educated mind, Craig insists that his offbeat humour and wacky antics are his way of "deconstructing the genre". One of those antics included pretending that puppets are hosting the show:
Another was eschewing the usual bandleader sidekick and instead acquiring a robot skeleton named Geoff Peterson.
As well as a fake horse named Secretariat.
And if that wasn't enough, Craig likes to put his cultural literacy on display with frequent humorous references to everything from classic literature to postmodern art. His recent rant about Rothko has become yet another in a long line of inside jokes on his program. The whole show becomes a strange irreverent dream that is a breath of fresh air in a timeslot that's usually overflowing with vapid celebrity interviews.
But what attracts me the most to Craig is his ability to bare his soul and show compassion in a genre that usually treats celebrities as punch lines. When Britney Spears was going through her well-publicized meltdown of 2007, he opened the show with a serious monologue that refused to take any low hits at her. He began explaining his own story about his addiction to drugs and alcohol and his path to sobriety. His ability to be so candid while also encouraging his audience to show some benevolence is what ultimately won me over.
It's clear to me that Craig Ferguson is the whole package. He's hilarious, sincere and has the perfect mix of high and low culture preferences. Add in his tall stature, salt and pepper hair and dashing good looks and he's the kind of man I just can't resist. As Craig ends his tenure on the Late Late Show, I'll be sad to say goodbye. His will no longer be the last face I see before going to sleep at night. But he'll always have a place in my heart.
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As Facebook gets more and more into our lives, we present the most common mistakes on Facebook that users do.
1. Sharing personal information with strangers
Would you share your private life with a complete stranger? Would you share your personal information (age, address, phone number, etc.) and photos with him? Why should you do that on Facebook? Having lots of strangers in your friend list implies you are going to get a load of unwanted things such as invitations to stupid events or loads of spam and annoying posts and mails. Even worse suppose a thief knows your address and sees your status saying that you are on vacation.
2. Posting Weird Pictures
Uploading embarrassing pictures is one of the most popular self inflicted dangers of using Facebook. This sort of mistake can lead to all kinds of repercussions for example, losing your job. Therefore, avoid posting private and embarrassing images of yourself.
3. Adding co-workers to your friend list
Making your co-workers friends on Facebook isn't always a good idea. It is best to keep your private and work life separate. There have been many incidents of people who faced serious problems at their work, just because their boss didn't approve their comments or photos uploaded. The best way is to create a separate group of your co-workers friends, in case you can't or don't want to unfriend them. It is more important to keep your job rather than post something on Facebook.
4. Adding Random Friends
It is still quite surprising that some people out there accept every friend request they receive. By confirming Facebook 'friendship' without actually knowing the person involved, you might just end up making ban and wrongly informed choices.
5. Using obvious passwords
People use passwords that can easily remember. The most common are birth dates, names, phone numbers or a combination of them. Remember that you already have most of them in your profile. It's better to choose passwords which combine letters, numbers and symbols.
6. Not protecting your children
Many users often upload photos of their children mentioning their names, age etc. Parents need to know that on a social network like Facebook, safety is a shared responsibility between users and the site. Anyone can share your childrens' photos or download and use them for any purpose. There are many sick minds out there. Be patient! Your children will grow up and will create their own Facebook profiles (hopefully avoiding these mistakes).
7. Not reading the terms of use when using Facebook apps
When using a Facebook app, be careful when you allow access to your Profile data. In most cases your data will be used for marketing reasons. There is no worry about this, but there is always a chance to get spammed.
8. Downloading suspicious files
According to statistics, approximately 83% of people who use social networking sites download files from other people's profiles. Since these files are usually coming from unknown individuals, some of them contain viruses that either corrupt your computer, or attempt to collect personal information that can be used against you. The first step in prevention is to only download files from people you trust. It's also important to keep your antivirus software up to date and turned on when you are online.
9. Revealing Whereabouts
Another popular Facebook mistake is declaring your whereabouts or what you are doing particularly if you have something to hide. Sometimes, using social networking sites such as Facebook might seem so simple and harmless such that some users actually forget that their activities may be viewable by others such as an employer, your girlfriend or burglars interesting at your house.
10. Using Facebook all day long
Facebook and all social networks are very addictive especially for children. It is recommended by experts to take regular breaks or use it only in specific hours during the day. Many people prefer to communicate through Facebook rather than go out for a coffee with their friends. In extreme cases this may lead to isolation and antisocial behavior.
Nonetheless Facebook remains one of the most entertaining activities on the internet and we like sharing on it.


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299 1 Bedroom Apartments for rent in Pearland, TX
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April 2020 Pearland Rent Report
Welcome to the April 2020 Pearland Rent Report. Pearland rents increased over the past month. In this report, we'll evaluate trends in the Pearland rental market, including comparisons to cities throughout the metro, state, and nation.
April 2020 Pearland Rent Report
Welcome to the April 2020 Pearland Rent Report. Pearland rents increased over the past month. In this report, we'll evaluate trends in the Pearland rental market, including comparisons to cities throughout the metro, state, and nation.
Pearland rents increased moderately over the past month
Pearland rents have increased 0.3% over the past month, and have increased moderately by 2.0% in comparison to the same time last year. Currently, median rents in Pearland stand at $1,115 for a one-bedroom apartment and $1,364 for a two-bedroom. This is the second straight month that the city has seen rent increases after a decline in January. Pearland's year-over-year rent growth is on par with the state average of 2.0%, but exceeds the national average of 1.9%.
Rents rising across the Houston Metro
Throughout the past year, rent increases have been occurring not just in the city of Pearland, but across the entire metro. Of the largest 10 cities that we have data for in the Houston metro, 8 of them have seen prices rise. Here's a look at how rents compare across some of the largest cities in the metro.
- Sugar Land has seen rents fall by 0.5% over the past month, the biggest drop in the metro. It's also the most expensive city in the Houston metro with a two-bedroom median of $1,574.
- Conroe has seen the fastest rent growth in the metro, with a year-over-year increase of 3.2%. The median two-bedroom there costs $1,107, while one-bedrooms go for $905.
- Pasadena has the least expensive rents in the Houston metro, with a two-bedroom median of $1,019; rents were up 0.3% over the past month and 2.3% over the past year.
Pearland rents more affordable than many large cities nationwide
As rents have increased moderately in Pearland, a few large cities nationwide have seen rents grow more modestly, or in some cases, even decline. Pearland is still more affordable than most large cities across the country.
- Rents increased moderately in other cities across the state, with Texas as a whole logging rent growth of 2.0% over the past year. For example, rents have grown by 3.5% in Austin, 2.3% in Dallas, and 1.3% in San Antonio.
- Pearland's median two-bedroom rent of $1,364 is above the national average of $1,197. Nationwide, rents have grown by 1.9% over the past year compared to the 2.0% increase in Pearland.
- While Pearland's rents rose moderately over the past year, the city of San Francisco saw a decrease of 0.2%.
- Renters will find more reasonable prices in Pearland than most large cities. For example, San Francisco has a median 2BR rent of $3,102, which is more than twice the price in Pearland.
For more information check out our national report. You can also access our full data for cities and counties across the U.S. at this link.
Methodology - Recent Updates:
Data from private listing sites, including our own, tends to skew toward luxury apartments, which introduces sample bias when estimates are calculated directly from these listings. To address these limitations, we've recently made major updates to our methodology, which we believe have greatly improved the accuracy and reliability of our estimates.
Read more about our new methodology below, or see a more detailed post here.
Apartment List is committed to making our rent estimates the best and most accurate available. To do this, we start with reliable median rent statistics from the Census Bureau, then extrapolate them forward to the current month using a growth rate calculated from our listing data. In doing so, we use a same-unit analysis similar to Case-Shiller's approach, comparing only units that are available across both time periods to provide an accurate picture of rent growth in cities across the country.
Our approach corrects for the sample bias inherent in other private sources, producing results that are much closer to statistics published by the Census Bureau and HUD. Our methodology also allows us to construct a picture of rent growth over an extended period of time, with estimates that are updated each month.
Read more about our methodology here.
About Rent Reports:
Apartment List publishes monthly reports on rental trends for hundreds of cities across the U.S. We intend these reports to be a source of reliable information that help renters and policymakers make sound decisions, and we invest significant time and effort in gathering and analyzing rent data. Our work is covered regularly by journalists across the country.
We are continuously working to improve our methodology and data, with the goal of providing renters with the information that they need to make the best decisions.


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