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Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host–parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms.
By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice.
Findings and Conclusions
We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.
Author Summary
The parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical disease. A major finding of the T. cruzi genome project was the discovery of a multigene family that encodes mucin-associated surface proteins (MASP), a highly polymorphic family expressed at the surface of infective forms of the parasite. We speculate that MASP may contribute to the ability of T. cruzi to infect several host cells and/or participate in host immune evasion mechanisms. To begin investigating this hypothesis, we analyzed the MASP expression profile in trypomastigotes derived from different host cells and in bloodstream parasites after sequential passages in mice. We also investigated the MASP antigenic profile in acutely infected mice. We observed more pronounced MASP expression changes by comparing bloodstream and tissueculture trypomastigotes and between bloodstream forms from sequential passages in infected mice. We also found that MASP peptides could trigger different IgG and IgM antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during the course of an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.
Citation: dos Santos SL, Freitas LM, Lobo FP, Rodrigues-Luiz GF, Mendes TAdO, Oliveira ACS, et al. (2012) The MASP Family of Trypanosoma cruzi: Changes in Gene Expression and Antigenic Profile during the Acute Phase of Experimental Infection. PLoS Negl Trop Dis 6(8): e1779. https://doi.org/10.1371/journal.pntd.0001779
Editor: Eric Dumonteil, Universidad Autónoma de Yucatán, Mexico
Received: February 8, 2012; Accepted: July 2, 2012; Published: August 14, 2012
Copyright: © dos Santos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Instituto Nacional de Ciência e Tecnologia de Vacinas (INCTV). DCB, RTF, SMRT, RTG and EC are CNPq research fellows. SLS, LMF and FPL received scholarships from CAPES, and TAOM and GLR received scholarships from FAPEMIG and CAPES, respectively. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Trypanosoma cruzi is the etiological agent of Chagas disease, a major public health problem in Central and South America. Currently there are approximately 10 million people infected and 40 million people at risk of acquiring the disease [1], [2]. Trypomastigotes are the bloodstream circulating form that infect a wide variety of nucleated host cells and subsequently differentiate into the intracellular replicative amastigote forms. After several rounds of binary division, amastigotes differentiate into trypomastigotes, which are released into the extracellular medium and bloodstream. The repetitive cycle of cell infection triggers the acute phase of Chagas disease, characterized by high blood parasitaemia, broad tissue parasitism, and a strong host immune response. The chronic phase is achieved after the host immune system controls the parasitaemia but fails to completely eliminate the parasite [3].
The annotation of the T. cruzi genome revealed a new multigene family composed of approximately 1,300 genes, which became known as mucin-associated surface protein (MASP) as they were not randomly distributed throughout the genome but instead clustered with genes encoding mucins and other surface protein families [4]. A previous study on the molecular characterization of a few members found that MASP proteins are expressed on the surface of the circulating infective forms of the parasite and can be shed into the extracellular medium [5]. MASP expression in the trypomastigote stage was also demonstrated by recent proteomic studies [6], [7]. Moreover, the MASP family is characterized by a strikingly variable and repetitive central region composed of peptides shared among its members, thus contributing to the extended repertoire of parasite polypeptides that could be exposed to the host cells and immune system [5]. The MASP repertoire of peptides could also contribute to cell-type-specific interactions, because the polymorphism of T. cruzi surface proteins has been suggested to be an important factor in the parasite's ability to infect multiple cell types [8], [9]. Taken together, this evidence has prompted us to examine a possible role of the MASP family in host–parasite interactions, such as the host-cell-dependent expression profile or/and immune evasion mechanisms. We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed when comparing bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Furthermore, in this work we describe the antibody recognition of several MASP peptides. Taken together, our findings prompt us to speculate that variations in the large repertoire of antigenic peptides derived from the MASP family may favor the parasite's escaping the host immune response during the acute phase of infection.
Trypomastigote culture, collection, and RNA extraction
The following steps for CL Brener trypomastigote culture and collection are detailed in Figure 1.
A: Mouse groups were sequentially infected with CL Brener clone. Blood collections were performed after 14–15 days. Bloodstream parasite forms were collected after two and ten passages. Part of the trypomastigotes isolated from mouse blood was used in LLC-MK2 and L6 cell infection (bloodstream parasite forms after two passages in mice). Trypomastigotes were collected after four sequential passages in culture. B: The same group of culture trypomastigotes was used in the infection of LLC-MK2 (epithelial) and L6 (myoblast) cell types. Trypomastigotes were collected after 14 sequential passages in culture.
In vitro culture of trypomastigotes.
CL Brener clone trypomastigotes obtained from one culture flask were used to infect two cell lineages, the rhesus-monkey epithelial cells LLC-MK2 and the rat myoblasts L6, both grown at 37°C, 5% CO2 in RPMI medium supplemented with 1% fetal bovine serum.
In vivo production of trypomastigotes.
To obtain bloodstream trypomastigotes, 10 Swiss mice were infected intraperitoneally with 50,000 CL Brener trypomastigotes, and then bled after 14–15 days when they reached the peak of parasitaemia. Bloodstream parasite forms were collected after two and 10 passages in mice, whereas infected mouse serum was obtained after passages 2, 10, and 12.
In vitro culture of trypomastigotes after the in vivo step.
Part of the bloodstream trypomastigotes produced by in vivo infection of Swiss mice after two passages as described above, were used to infect LLC-MK2 and L6 cells. After four passages in culture, the trypomastigotes were collected.
RNA extraction.
Tissue culture and bloodstream trypomastigote samples were purified by centrifugation at 400× g and washed three times with PBS before total RNA extraction using the RNeasy kit (Qiagen), following manufacturer's recommendations. Only parasite samples containing >90% trypomastigotes were used in this study. Cell cultures were PCR-tested weekly for Mycoplasma sp. using genus-specific primers [10]. Only PCR-negative cell cultures were used in the experiments. Negative sera from Swiss mice were also obtained by bleeding of uninfected animals maintained in the same experimental conditions.
Quality control of total RNA and cDNA synthesis
cDNA samples were generated by reverse transcriptase Super Script II (Invitrogen) reactions using random primers and 3–4 µg total RNA. cDNA quality was tested by PCR using specific primers for aromatic L-alpha-hydroxy acid dehydrogenase (AHADH2) (AHADH2_F 5′ CCAAATGTTTCGCCACTCG 3′ and AHADH2_R 5′ CACGCTGCGGAGGGATCTC 3′) and for the DNA repair gene RAD51 (RAD51_F 5′ GGCTGTCAAGGGTATCA 3′ and RAD51_R 5′ AACCACTGCGGATGTAA3′), a gene with low expression in trypomastigotes [11]. Approximately 200 ng of each cDNA or 250 ng of total RNA (negative control) were used in PCR reactions, with 200 µM dNTPs, 2 µM primers, and 1.25 U Taq DNA polymerase in 50 mM KCl, 10 mM Tris-HCl pH 8.4, 1% X-100 Triton, and 1.5 mM MgCl2. To ensure that the samples were not contaminated with genomic DNA, total RNA samples were tested by PCR for the T. cruzi gene AHADH2 using the primers described above. DNA contaminated samples were treated with DNase I (Fermentas), following manufacturer's recommendations, and submitted to RNA clean-up using the RNeasy kit (Qiagen). Treated samples were also PCR-tested. PCR amplification was observed in agarose ethidium bromide or polyacrilamid silver stained gels.
Construction and sequencing of expression libraries
To construct expression libraries, several primer combinations were tested in silico by electronic PCR (e-PCR) (http://www.ncbi.nlm.nih.gov/projects/e-pcr/) and experimentally to amplify the majority of the MASP genes. A semi-nested PCR was set up using the following primer combinations: SL 5′ AACGCTATTATTGATACAGTTTCTGTACTATATTG 3′ for the 35 pb spliced leader region and 3′UTR1 (reverse) 5′ GTGTGCTTCGTGGGGTGAGGTG 3′ for the 3′UTR in the first reaction and SL and 3′UTR2 5′ CTCACTCTCACGCGGCCACCACCACCG 3′ also for 3′UTR (more internal localization) in the second reaction. The semi-nested RT-PCR reactions with these primers were performed with 2 µL of amplified product of the first reaction or 400 ng of the cDNA with 200 µM dNTPs, 2 µM of primers, and 3.75 U High Fidelity Taq DNA polymerase (Invitrogen). The amplicons between 500 and 2,000 bp of the second reaction were cloned in pGEM-T (Promega), and at least 96 ampicillin-resistant clones were selected and cultured. After plasmid extraction, a PCR of each clone was performed using primers for the insert flanking regions M13F (forward) 5′ CGCCAGGGTTTTCCCAGTCACGAC 3′ and M13R (reverse) 5′ TCACACAGGAAACAGCTATGAC 3′. Amplified products were precipitated with 20% polyethylene glycol 8000 and 2.5 M NaCl and submitted to sequencing at one end in the ABI Prism 3730xl DNA Analyser (Applied Biosystems) by Macrogen Inc (Korea). The libraries constructed for each trypomastigote sample are presented in Table 1. The EST sequences have been deposited in the GenBank database under Accession Number JK743993 - JK744782.
Sequence and hierarchical cluster analyses
Sequences from the expression libraries were processed by an in-house pipeline. The EST sequences were processed by the Phred algorithm and then filtered after a cross-match with the vector sequence (pGEM-T- Promega). The MASP genes were identified using BLASTN algorithm against two T. cruzi sequence databases: one with all T. cruzi predicted features of the genome (coding sequences, retroelements and structural RNAs), and the other with T. cruzi contigs. The e-value (expected value) cutoff of the BLASTN searches used was 10−10, and the minimum identity between the ESTs and the database entries was 70%. The uncertainty of the hierarchical cluster analysis of all expression libraries was calculated using the Pvclust package [12] using the R software platform [13]. Pvclust calculates probability values (p-values) for each cluster using bootstrap re-sampling techniques. AU (approximately unbiased) p-value was used, which is calculated by multi-scale bootstrap re-sampling and has superiority in bias over BP (bootstrap probability) according to the authors [12].
Representation of MASP genes and transcripts in multidimentional scaling plots
MASP sequence variability and expression profile were visualized in a multidimentional scaling plot (MDS). To this end, we calculated the pairwise distance of all 1,377 annotated MASP genes and generated the distance matrixes using the package PHYLIP [14]–[15]. To provide a visual representation of the distance matrix, we used an MDS plot with two dimensions (2D). The k-means method [16] was used to define six clusters or subgroups. Each dot in the MDS represents a MASP member and its graphic localization, the sequence similarity among the genes. The expressed MASP genes were plotted in the MDS graphics, where the size of the dots represents the frequency of the MASP genes in the library and the colors represent their group classification. The databases generated by the e-PCR predictions were also plotted in the MDS graphics. The MDS, hierarchical clustering, statistical analyses, and graphing were performed using the R software platform [13].
The genes targeted for Real Time PCR (qRT-PCR) were selected after comparative analysis of the expression libraries. Primers were designed using Allele ID 7 (Premier Biosoft, demo version), and NCBI T. cruzi database BLAST searches were performed to exclude primers with cross homology with other MASP members and other T. cruzi genes. The design also included the template's secondary structure test at 60°C. The MASP complete genes analyzed by qRT-PCR were: MASP2 (Tc00.1047053510359.460), MASP4 (Tc00.1047053508541.110), MASP14 (Tc00.1047053504039.230), MASP16 (Tc00.1047053510693.190), MASP23 (Tc00.1047053511089.19), and MASP27 (Tc00.1047053506615.100). The primer sequences used in the analysis for each MASP gene are listed in Table S1. Reactions in triplicate were prepared with 1 µM forward and reverse primers, SYBR Green Supermix (Applied Biosystems), and each of the diluted template cDNAs (1∶4 in DNAse free water) and were performed using cycling conditions as recommended by the manufacturer (Applied Biosystem). Standard curves were used for the calculation of relative quantity (Rq) values of each sample for each target. qRT-PCRs for MSH2 and RAD51 genes (Table S1) were performed, and the average value between them was used to normalize the MASP gene results. The results were analyzed by a one-way ANOVA test, and graphics were constructed in GraphPad Prism 5.0 (GraphPad Inc.).
MASP epitope prediction, synthesis of SPOT peptide arrays, and immunoblotting
MASP predicted protein sequences derived from expressed genes were submitted to B-cell linear epitope prediction using the BepiPred algorithm [17], and the output was parsed by an in-house PERL script to select 15-mer amino acid peptides whose prediction score according to their quality as an epitope was >1.3. One or two peptides from specific MASP genes identified in the expression libraries were selected to be synthesized in pre-activated cellulose membranes according to the SPOT synthesis technique [18]. The SPOT synthesis was employed using a method for the preparation of approximately 5 nmol of immobilized peptides. The assembly of the peptides was performed utilizing the previously described Fmoc-chemistry [18]. Briefly, 0.5 mM of each activated Fmoc (9-fluorenylmethoxycarbonyl) amino acid was automatically spotted on pre-activated membranes using the MultiPep SPOT synthesizer (Intavis AG). Each cycle of amino acid coupling was followed by a 10% acetic anhydride blocking and deprotection of Fmoc amino acids by adding 25% 4-methyl piperidine. The coupling and deprotection of Fmoc amino acids were confirmed after each cycle by staining the membrane with 2% bromophenol blue. After the synthesis, the side chain deprotection was performed by adding a 25∶25∶1.5∶1 solution of trifluoroacetic acid, dichloromethane, triisopropylsilane and water. The side-chain deprotection was also confirmed by staining with 2% bromophenol blue. The synthesized peptides are listed in Table S2. Membranes were blocked with 5% BSA and 4% sucrose in PBS overnight and incubated for 1.5 hours with pools of diluted mice sera (1∶500 for IgG or 1∶5,000 for IgM) in blocking solution. After washing three times for 10 minutes in PBS-T (PBS; 0.1% Tween 20), membranes were incubated for 1.5 hours with secondary HRP-conjugated anti-mouse IgM or IgG antibody (Sigma-Aldrich), diluted 1∶2,000 in blocking solution. After a second washing, membranes were revealed by ECL Plus Western blotting (GE Healthcare), in the Gel Logic 1500 Imaging system (KODAK). Synthetic peptides corresponding to epitopes of a trans-sialidase [19] and L7A ribosomal protein [20] were included in the experiments as positive controls. As negative controls, membranes were submitted to the same experimental conditions using sera of uninfected Swiss mice. Densitometry measurements and analysis of each peptide were performed using Image Master Platinum (GE), and the relative intensity ratio (RI) cutoff for positivity was determined as 2.0. Reactive spots in the positive blottings (using the infected mouse serum pool) were only selected for analysis when not reactive in the negative blotting. Graphics were constructed in GraphPad Prism 5.0 (GraphPad Inc.).
Seven peptides with the highest RI values and a peptide derived from trans-sialidase SAPA (shed acute phase antigen) [21] (Table S3) were submitted to soluble synthesis (Peptide 2.0) and ELISA experiments. Flexible ELISA polyvinylchloride plates (BD Falcon) were sensitized with 2 µg of soluble peptides or trypomastigote extract in water at 37°C overnight. After blocking with 2.5% BSA in PBS for 2 hours at 37°C, the plates were incubated with sera from uninfected and infected mice (dilution 1∶100) for 1.5 hours at 37°C. After washing in 0.05% Tween 20-PBS, the plates were incubated with secondary antibodies anti-mouse IgM or IgG (dilution 1∶2000; Sigma). After several washes in PBS-0.05% Tween 20, the plates were revealed with OPD (o-phenylenediamine; Sigma), in citric acid buffer (50 mM Na2HPO4, 27 mM, citric acid, pH 5.0) and hydrogen peroxide and read at 492 nm. The reactivity of the trypomastigote extract was used in the normalization of ELISA results using the peptides. The results were analyzed by one-way ANOVA test and graphics were constructed with GraphPad Prism 5.0 (GraphPad Inc.). Trypomastigote total extracts were obtained by ultrasound lysis of purified and PBS-washed parasite. Protein quantification was determined by the BCA™ Protein Assay Kit (Pierce).
Avidity ELISA
Low-affinity antibodies were eluted by adding an incubation step with 6 M urea for 5 minutes at room temperature after the mouse serum incubation. Avidity index was expressed as (mean OD of urea-treated sera/mean OD urea-untreated sera)×100%. Affinity indexes <40% or >40% were considered low and intermediate affinity levels, respectively [22]. The three mouse serum pools (after 2, 10, and 12 passages) were tested in triplicate.
Invasion assay
Trypomastigotes derived from L6 cells after 17 passages were purified by centrifugation at 400× g followed by incubation at 37°C for 4 h to allow motile trypomastigotes to swim up. The trypomastigotes were then used to infect LLC-MK2 and L6 cells as follows. LLC-MK2 or L6 cells ressuspended in RPMI supplemented with 10% FBS were plated (4×104 cells/well) in 24-well plates containing coverslips and incubated at 37°C and 5% CO2 for 36 hours prior to infection. Infection was performed by exposing cells to purified trypomastigotes for 30 min at 37°C at a multiplicity of infection (MOI) of 50. Cells were then washed five times with PBS to remove extracellular parasites and fixed with 4% (wt/vol) paraformaldehyde/PBS overnight at 4°C. After fixation, coverslips with attached cells were washed three times in PBS, incubated for 20 min with PBS containing 2% BSA, and processed for an inside/outside immunofluorescence invasion assay using an anti-T. cruzi rabbit polyclonal antibody and a secondary anti-rabbit IgG antibody conjugated with Alexa Fluor 546 (Life technologies) according to a previous protocol [23]. In this step, all extracellular, non-washed parasites were stained. Coverslips were then washed twice with PBS, incubated with 10 g/ml DAPI (4′,6′-diamidino-2-phenylindole; Sigma) in PBS for 2 min, washed three times in PBS and mounted on slides using a fluorescence mounting solution containing 1 mg/ml of PPD (p-phenylenediamine) in Glycerol/Tris-HCl. At least 250 cells (10 fields) were analyzed per coverslip in triplicate, and invasion rates were calculated as the number of intracellular parasites/100 host cells. Graphs were plotted using GraphPad Prism 5.0 (GraphPad Inc.) and statistically significant differences were determined using Student's t test.
Ethics statement
All animal procedures were approved by the animal-care ethics committee of the Federal University of Minas Gerais (Protocol # 232/2009) and were performed under the guidelines from COBEA (Brazilian College of Animal Experimentation) and strictly followed the Brazilian law for "Procedures for the Scientific Use of Animals" (11.794/2008).
MASP subgroups and library construction
Prior to investigating the MASP expression profile, we performed sequence clustering analysis of the family to identify subgroups, whose expression profiles were then analyzed. To this end, we performed pairwise alignments of the coding sequences of all MASP genes, resulting in a distance matrix that was used to generate a multidimensional scaling (MDS) plot (Figure S1). The k-means method was used to define six clusters or subgroups (Figure S1A) (Table S4). Due to the extensive sequence variability of the MASP family, we performed a series of electronic PCR analyses to select the primers suitable to amplify most MASP transcripts. These analyses suggest that most of the MASP complete genes (77.8% or the equivalent to 630 genes) and eventually those derived from pseudogenes (55.9%, 317 members) could be amplified in a semi-nested RT-PCR using primers for the spliced leader sequence (SL) and MASP 3′UTR (3′UTR1 and 3′UTR2) (Figure S1B). The SL primer was used in both reactions of the semi-nested RT-PCR to guarantee that the amplified transcripts were mature, whereas the 3′UTR1 and 3′UTR2 primers were derived from the MASP 3′UTR, which is the most conserved region of MASP transcripts [5]. In addition, because of the mosaic structure of MASP genes having shared fragments among the members [4], this combination of primers has the advantage of generating amplification products containing the entire MASP coding region and therefore would allow an unequivocal identification of the expressed genes. Using these primers, five of the six different MASP subgroups can be amplified (Figure S1).
MASP expression profile in culture and bloodstream trypomastigotes
We hypothesized that the large repertoire of MASP peptides may contribute to ability of T. cruzi to infect and/or survive within several host-cell types and/or participate in host immune evasion mechanisms [5]. To begin investigating this hypothesis, we constructed seven expression libraries from tissue culture trypomastigotes derived from two cell types (epithelial cells and myoblasts), and from bloodstream trypomastigotes recovered after sequential passages in mice (Table 1). For all libraries, the amplification profile by semi-nested RT-PCR presented a smear in both reactions (Figure S2), indicating the co-expression of several MASP transcripts with different lengths. Fragments ranging from 500 to 2,000 bp were cloned into pGEM-T vector (Promega) and a total of 960 clones were sequenced. Based on the percentages of valid sequences, ranging from 76.4% to 90.6%, the libraries were considered to be of good quality (Table 2). The T. cruzi genes corresponding to the best hit of each valid EST sequence are shown in the Table S5. We retrieved a total of 94 MASP complete genes by analyzing the content of the libraries altogether, even though the number of sampled transcripts per library was restricted to 20 to 32 genes. Although the proportion of sequences with non-MASP best hit ranged from 50% to 83.9%, the majority of these genes are short hypothetical proteins that appear to be unreal because they were predicted within the MASP 3′UTR, which contains the annealing sites of the reverse primers used to construct the libraries. Other non-MASP hits include chimeric sequences containing the MASP C-terminal coding sequence and the downstream MASP 3′UTR and TcMUC mucin genes and retroelements followed by fragments of the MASP 3′UTR. We also detected the transcription of pseudogenes, ranging from 4.8% to 37.8% of the MASP hits in all the libraries. Because the library of bloodstream trypomastigotes after 10 passages (Blood.Tryp.10p) presented a higher proportion of pseudogenes (37.8% versus an average of 12.3% for all other libraries), a larger number of sequences from this library was analyzed (Table 2). We analyzed 96 more sequences of the Blood.Tryp.10p expression library than of the other libraries, and the number of MASP genes sampled in this library was similar to those of the other libraries. We mapped the MASP cDNAs in the MDS distribution to represent a visual analysis of the expressed members. A transparency was applied to the dots so that differences in their size would represent differences in the level of MASP expression (Figure 2).
Figure 2A: MDS distribution of sequences from the MASP expression libraries of bloodstream forms from sequential passages and of culture trypomastigotes after the in vivo step. Figure 2B: MDS distribution of sequences from the MASP expression libraries of culture trypomastigotes derived from distinct cell types. Black bordered genes: MASP complete genes analyzed by qRT-PCR. Tryp.0.llcmk2: culture trypomastigotes prior to infection of the two cell types; Tryp.14p.llcmk2: culture trypomastigotes derived from LLC-MK2 cells after 14 passages; Tryp.14p.l6: culture trypomastigotes derived from L6 cells after 14 passages; BloodTryp.2p: bloodstream forms after 2 passages; BloodTryp.10p: bloodstream forms after 10 passages; Tryp.4p.llcmk2: culture trypomastigotes after in vivo step derived from LLC-MK2 cells after 4 passages; Tryp.4p.l6: culture trypomastigotes after in vivo step derived from L6 cells after 4 passages.
In a previous study, we had identified several MASP transcripts in a cDNA library constructed from tissue culture trypomastigotes derived from Vero cells [5]. Here, we investigated whether MASP is also expressed in bloodstream trypomastigotes during the acute phase of experimental infection. In addition, we compared two expression libraries constructed from bloodstream trypomastigote forms after sequential passages in mice. As shown in Figure 2A, bloodstream trypomastigotes derived from a given passage co-express MASP genes belonging to all five different groups, indicating a broad expression of different MASP genes. MASP expressed genes for which we could design specific primers were analyzed by qRT-PCR. Significant differential expression was observed by qRT-PCR for MASP2, MASP16, and MASP27 between bloodstream trypomastigotes from sequential passages (Figure 3A). MASP2 and MASP27 were significantly more expressed in bloodstream trypomastigotes after 10 passages in mice compared to trypomastigotes after two passages. In contrast, MASP16 was significantly more expressed in bloodstream forms after two passages in mice. In addition to the temporal changes in the expression of a given gene after sequential passages in mice, we also found that the level of expression of distinct MASP transcripts varies significantly in the trypomastigote population. For instance, MASP27 is 100 times more expressed than MASP 23 in all libraries (Figure 3). These results indicate that the expression profile of distinct MASP genes is heterogeneous and may vary after sequential passages in mice.
Relative quantity (Rq) calculations were based on specific standard curves for each MASP gene. Rq values of each cDNA sample (MASP Rq) were normalized with the average of two constitutive genes MSH2 (MSH2 Rq) and RAD51 (RAD51 Rq). Figure 3A: Expression analysis by qRT-PCR of six MASP genes in bloodstream forms from sequential passages and of culture trypomastigotes after the in vivo step. Figure 3B: Expression analysis by qRT-PCR of six MASP genes in culture trypomastigotes derived from distinct cell types. Tryp.0.llcmk2: culture trypomastigotes prior to infection of the two cell types; Tryp.14p.llcmk2: culture trypomastigotes derived from LLC-MK2 cells after 14 passages; Tryp.14p.l6: culture trypomastigotes derived from L6 cells after 14 passages; BloodTryp.2p: bloodstream forms after 2 passages; BloodTryp.10p: bloodstream forms after 10 passages; Tryp.4p.llcmk2: culture trypomastigotes after in vivo step derived from LLC-MK2 cells after 4 passages; Tryp.4p.l6: culture trypomastigotes after in vivo step derived from L6 cells after 4 passages.
To investigate whether tissue culture trypomastigotes had a distinct MASP expression profile compared with the bloodstream forms, part of the trypomastigote population collected after two passages in mice was used to infect myoblast (L6) and epithelial cells (LLC-MK2), and after 4 passages in culture, the RNA was extracted for library construction. Both libraries, Tryp.4p.llcmk2 and Tryp.4p.l6, showed a very similar pattern of expression (Figure 2A). In fact, no significant difference in gene expression between these two libraries was observed for those genes analyzed by qRT-PCR (Figure 3A). In contrast, we detected differences in the expression profile of bloodstream forms after two passages (BloodTryp.2p) compared with both Tryp.4p.llcmk2 and Tryp.4p.l6 libraries (Figure 2A). This was confirmed by qRT-PCR: MASP2 and MASP27 were more expressed in Tryp.4p.l6 compared to the BloodTryp2 library, whereas MASP16 was more expressed in bloodstream forms after two passages compared with L6- and LLC-MK2-derived trypomastigotes after four passages (Figure 3A).
Because we did not detect significant changes in MASP expression when comparing trypomastigotes after four passages in the two types of host cells (myoblast and LLC-MK2 cells), we decided to analyze the MASP expression profile after a larger number of passages in these two types of tissue culture cells (14 passages). Similar to what was observed in the other libraries, in both Tryp.14p.llcmk2 and Tryp.14p.l6 libraries, we also detected co-expression of MASP genes in five different groups. Furthermore, MASP27 is one of the most represented genes in the sequenced clones, while MASP23 was not sampled in these libraries (Figure 2B). This data were validated by qRT-PCR since we detected remarkably high levels of expression of MASP27 compared to the other analyzed genes, while MASP23 was approximately 100 times less expressed in all libraries (Figure 3B). More importantly, more notable differences in MASP expression in trypomastigotes derived from both host cells were observed after 14 passages compared to the expression profile after 4 passages. We observed by qRT-PCR that MASP2, MASP14, and MASP16, belonging to the Red subgroup, were more expressed in L6-derived trypomastigotes compared to LLC-MK2-derived trypomastigotes after 14 passages (Figure 3B). Whether these specfiic MASP members are implicated in trypomastigote invasion, replication, and/or survival within L6 cells remain to be investigated. Nevertheless, by performing invasion assay, we confirmed an association between the MASP profile and the infectivity of L6-derived trypomastigotes (Figure S3). Specifically, we evaluated the rate of invasion of L6 and LLC-MK2 cells by the same population of trypomastigotes that were maintained for 17 consecutive passages in L6 cells. We found a higher rate of invasion of L6 cells compared with LLC-MK2, reinforcing our findings and suggesting that successive passages of trypomastigotes in a given host cell may configure a specific expression profile that optimizes the rate of invasion.
The differences observed by qRT-PCR are in agreement with the hierarchical clustering analysis of all expression libraries using the pvclust package (Figure 4). The dendrogram derived from the pvclust analysis shows that, after four passages, tissue culture trypomastigotes derived from either L6 or LLC-MK2 cells are very similar. In contrast, tissue culture trypomastigotes libraries are distantly clustered from bloodstream trypomastigote libraries. Taken together, these results indicate that tissue culture and in vivo infection may selectively configure a distinct MASP expression profile in trypomastigotes.
The uncertainty of the hierarchical cluster analysis of all expression libraries was calculated using Pvclust package [10]. AU values (in red): approximately unbiased p-value.
MASP antigenic profile in the acute phase of the experimental infection
To investigate the MASP antigenic profile in the acute phase of the experimental infection, we performed B-cell linear epitope prediction on the MASP proteins derived from expressed members using the Bepipred algorithm [17]. Only predicted epitopes exclusive for MASP proteins were selected. A total of 110 peptides for 64 MASP expressed genes, from a total of 94 genes identified in the expression libraries, are member specific and were analyzed by immunoblotting. We found that 74 to 88% of the analyzed MASP members were recognized by sera of acutely infected mice, having at least one reactive peptide (RI>2.0) against one serum pool (Figure 5). Additionally, 21 to 33% of the MASP members were recognized by all three serum samples. The remaining peptides were not reactive or were excluded from the analysis after the normalization step with sera of uninfected mice.
Blotting representative images using sera pools (n = 10) after 2, 10, and 12 passages in mice of CL-Bnener T. cruzi; predicted peptides were covalently synthesized in pre-activated cellulose membranes according to the SPOT synthesis technique (Frank et al., 1992); circled spots: peptides with the highest Rd values.
Following the peptide screenings on SPOT arrays, seven peptides with high RI values were submitted for soluble synthesis to be used in ELISA experiments (Table S3). As expected, IgM and IgG antibodies from infected mice were reactive against all MASP peptides and also against SAPA, a known T. cruzi epitope derived from a repetitive region of the trans-sialidase enzyme [21] (Figure 6). MASP genes with low expression levels had reactive peptides, such as peptide D10, derived from MASP23. However, it is important to emphasize that mRNA and protein levels in T. cruzi are not always linearly associated, and therefore we cannot assume that the level of expression of the MASP23 protein is also low. A variable level of reactivity against MASP peptides was also observed between the sequential passages in mice. Furthermore, variable levels of recognition by the two immunoglobulin types (IgG and IgM) of each peptide were observed. High levels of IgM were observed against peptide B5, derived from MASP27, after ten passages in mice. It is worth noting that, as mentioned before, MASP27 had the highest expression levels in all cDNA libraries. High levels of IgM were also observed against the peptides H5 and J10 after two and 12 passages in mice. Both peptides are derived from MASP genes with low expression levels. The other peptides tested by ELISA that were derived from genes expressed at low levels, displayed a distinct level of antibody recognition. SAPA reactivity by IgM had high values, and the results also indicated that there was a differential recognition by IgM antibodies between the sequential passages in mice. The affinity levels of the IgG and IgM antibodies against MASP peptides were also measured (Figure S4). The IgG antibodies against all tested MASP peptides presented intermediate affinity, ranging from 52 and 58.3%, which were higher levels than that of the affinity of antibodies against SAPA (40%). In contrast, IgM antibodies against MASP peptides presented variable levels of affinity. IgM antibodies against C5 peptides presented intermediate affinity (54.9%), whereas antibodies against MASP peptides C3, B5, D10, H1, and SAPA presented low affinity, ranging from 17.8 to 33.2%. The distinct antibody affinity levels are most likely related to differences in peptide composition rather than to the total antibody levels against the peptides. For instance, antibodies against the peptides C5 had the highest affinity index, despite the fact that the total antibody against this peptide had the lowest level (Figure S4).
Seven MASP peptides and SAPA were submitted to soluble synthesis (Peptide 2.0) and ELISA experiments. Flexible ELISA plates (Falcon) were sensitized with 2 µg of soluble peptides, incubated with uninfected and infected mouse serum pools from three passages, IgG or IgM secondary antibodies, and revealed with OPD solution. Absorbance values were normalized with values resulting from ELISA of each passage against total trypomastigote extract.
Overall, these results showed that different members of the MASP family are expressed during acute T. cruzi infection and constitute parasite antigens recognized by IgG and IgM antibodies. The results also indicated that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice.
A key T. cruzi strategy to survive in a mammalian host is its ability to actively invade a wide variety of non-phagocytic host cells. The acute phase of Chagas disease is characterized by intense parasitaemia and tissue parasitism involving the infection of heart, skeletal and smooth muscle cells, as well as liver, fat, and brain cells [24]–[27]. Furthermore, during the chronic phase, there are several reports on differences of T. cruzi tropism to host tissue, which is associated with the pathogenesis of Chagas disease [28]–[31]. Therefore, an important aspect to understanding T. cruzi infection is the identification of molecular components of both parasite and host cells that play a role in the infection of a broad range of cell types. In this regard, several studies have investigated changes in gene expression during T. cruzi infection. However, these studies have focused mainly on the modulation of gene expression of the host cells [32]–[40]. Although several trypomastigote surface proteins have been implicated in host-cell recognition/invasion [41]–[46], so far there is no clear association between a T. cruzi expression profile and its ability to invade/proliferate in a given host cell.
In our previous work on the molecular characterization of the MASP family [5], we speculated that these proteins may be involved in host–parasite interactions because of their surface localization on the infective circulating trypomastigote forms. Moreover, the MASP family is highly polymorphic and can be secreted by the parasite, thus contributing to a large T. cruzi polypeptide repertoire that could be exposed to the host cells and the host's immune system [5]. In fact, it has been shown recently that a MASP protein is able to induce endocytosis in Vero cells [47], a process whereby the trypomastigote forms of the parasite actively invade host cells [48]. As an attempt to investigate whether MASP members could be implicated in interactions with specific cell types, we investigate the MASP expression profile in trypomastigotes derived from epithelial (LLC-MK2) and myoblast (L6) cell lines. We selected these cell types because LLC-MK2 is widely used for maintaining T. cruzi in in vitro culture, whereas myoblasts give raise to muscle cells, which are target by T. cruzi during acute and chronic phase of Chagas disease [26], [49]. We did not detected significant changes in the MASP expression profile between these two host cells after 4 passages in tissue culture. However, differential expression of MASP genes were detected by sequencing and by qRT-PCR analyses between trypomastigote forms derived from these two host cells after a larger number of tissue culture passages: MASP2, MASP14 and MASP16 were significantly more expressed in myoblasts compared to epithelial cells after 14 passages (Figure 3B). This is an indirect evidence that different MASP genes may be implicated in the interaction with distinct cell types. As indicated before, it was recently demonstrated that one MASP member (named MASP52, Tc00.1047053504239.220) is secreted by trypomastigote forms upon contact with non-phagocytic Vero cells and is able to induce endocytosis [47]. This MASP gene was not sampled in our sequenced clones. Nevertheless, an association between the selection of a MASP profile and the infectivity of L6-derived parasites is suggested by the invasion assay experiment (Figure S3). Whether peptides derived from MASP2, MASP14 and MASP16 are involved in myoblast recognition/invasion and/or parasite proliferation/survival within myoblast cells remains to be investigated.
We have also investigated whether the transition from in vivo to in vitro infection would affect the MASP expression profile. To this end, the same trypomastigote population that was recovered from passage 2 in mice was used to infect myoblast and epithelial cells, and after four passages in each cell type, the MASP expression profile was analyzed. We found noticeable differences in the MASP family expression profile when tissue-culture and bloodstream forms were compared (Figures 2A, 3A and 4). Significant differential expression of the genes MASP2, MASP16 and MASP27 was observed by qRT-PCR in tissue-culture derived trypomastigotes after four passages (Tryp.4p.llcmk2 and Tryp.4p.l6) compared to bloodstream trypomastigotes after two passages (BloodTryp.2p) (Figure 3A). These observations suggest that selective pressure driven by tissue culture or in vivo infection may induce distinct MASP expression profiles. Differences in infections using bloodstream- and tissue culture-derived trypomastigotes have already been reported. Specifically, it has been shown that the rate of in vitro infection of distinct host cell types does not correlate with the level of parasitaemia of experimentally infected mice [50]. We also found a heterogeneous MASP expression profile when bloodstream trypomastigotes recovered from mice after 2 and 10 passages were compared: MASP2 and MASP16 were significantly more expressed in bloodstream forms after 10 passages compared with bloodstream forms from passage 2, and the inverse was observed for MASP16 (Figure 3A). In addition, a large number of MASP pseudogenes were expressed in bloodstream forms after 10 passages, which was not observed for any other library (Table 2). Sequencing a larger number of clones of this library did not significantly change this profile. Pseudogenes may contribute to the diversity of the sequence repertoire through recombination. Indeed, the involvement of pseudogenes in the generation of variant surface glycoprotein (VSG) diversity has already been described in T. brucei [51], and it was hypothesized that this mechanism may explain the large number of VSG pseudogenes in the T. brucei genome [52]. Whether there is a selective pressure imposed by the vertebrate host immune system to increase the sequence diversity of the MASP family in the circulating trypomastigote forms at the expenses of generating pseudogenes remains to be investigated.
It is well established that several T. cruzi surface proteins are co-expressed at a given time in the parasite population, leading to the phenomenon of antigenic variability [53]. Here, we found that, in fact, several MASP genes are co-expressed, although the level of expression of each transcript is very variable in a given library (Figure 3), indicating a heterogeneous expression of the family. This expression profile was also observed in the proteomic study of the trypomastigote form of the Y strain [7]. In this study, 37 unique MASP peptides found in 167 MASP proteins were identified at different expression levels in a parasite population derived from LLC-MK2 host cells. Although the conditions and strains used in both studies were different, it is interesting that two MASP members sampled in our expression libraries from trypomastigotes derived from LLC-MK2 host cell were also represented in this proteomic study (Tc00.1047053508253.10 and Tc00.1047053510163.30). We have previously analyzed the MASP expression in individual trypomastigotes by performing immunofluorescence using non-permeabilized cells and an affinity-purified antibody specific to a MASP subgroup. Only a few trypomastigotes were labeled with the antibody, suggesting that, at least for some MASP proteins, their expression on the surface of trypomastigotes is not uniform in the parasite population [5]. The present study added another layer of complexity to the expression of the MASP family since we detected temporal changes in gene expression of the same gene after sequential passages in mice and also in trypomastigotes derived from epithelial and myoblast cells after a large number of in vitro passages (Figures 2 and 3).
How the parasite modulates the expression of MASP genes during the infection is an open question. We did not detect a correlation between chromosomal location of the MASP genes sampled in our cDNA libraries and their expression levels (data not shown), suggesting that there is no apparent bias regarding the chromosomal location of expressed MASP genes. It is well established that the control of gene expression in Trypanosomatids operates almost exclusively at a post-transcription level, primarily mediated by regulatory elements with the 3′UTR of the transcripts that modulate the mRNA stability by means of interactions with regulatory proteins [reviewed in 54]. We have previously shown that the 3′UTR of MASP transcripts is highly conserved among the family members [5] and therefore may not be involved in the differential expression of the distinct MASP genes. Nevertheless, subtle nucleotide differences in these regions and/or alternative polyadenylation sites among the different transcripts may favor or abolish specific interactions with regulatory proteins. How the parasite changes the expression of the same MASP gene under different in vitro and in vivo conditions is also intriguing. In this case, it is possible that the host cell and/or the host immune system may configure a specific MASP expression profile.
Another possible MASP function that may explain the high level of polymorphism of the family would be its involvement in immune evasion mechanisms. In addition to its extreme polymorphism, localization at the trypomastigote surface, and shedding properties, another MASP feature that reinforces this hypothesis is the large repertoire of distinct repetitive motifs of the MASP proteins [5]. It has been shown that several parasitic repetitive proteins are targets for strong B-cell responses [53], [55]–[56]. In fact, in silico predictions performed by our group on the entire MASP proteome suggest the occurrence of a large repertoire of B-cell epitopes in the family (data not shown). In the present study, we validated these predictions by showing that several peptides derived from MASP-expressed members reacted with sera from acutely infected mice (Figures 5 and 6). The antibody recognition of several MASP peptides supports the interaction of the MASP family with the host immune system during acute T. cruzi infection.
We have also investigated whether the MASP antigenic profile changes during acute infection. Indeed, variable antigenic profiles between the trypomastigotes isolated from sequential passages in experimentally infected mice were observed by immunoblotting and ELISA (Figures 5 and 6). The MASP family, along with other T. cruzi surface proteins, may contribute to the polyclonal lymphocyte activation that leads to hypergammaglobulinemia and the delayed specific humoral immune response, that are characteristic of the acute phase of Chagas disease. These phenomena are suggested to be an immune evasion mechanism [57]–[59]. Polyclonal lymphocyte B activation could scatter the immune response, preventing the development of a specific and neutralizing response against the parasite and its complete elimination. T-cell independent responses may contribute to hypergammaglobulinemia [57]. Indeed, the SAPA repetitive C-terminal region of the trans-sialidase protein was reported to be a T-cell independent B mitogen and inducer of non-specific Ig secretion [56]. It is possible that MASP peptides could mediate both specific T-dependent or unspecific T-independent immune responses, a hypothesis that is partially supported by the differential recognition of MASPs by the two immunoglobulin types (IgM and IgG) and the difference in the antibody affinity levels against each of the synthetic peptides. We speculate that variations in the large repertoire of antigenic peptides derived from the MASP family may contribute to the mechanism of immune evasion during the acute phase of the infection.
This is the first report on the antigenic properties of the MASP family, supported by the description of the antibody recognition of expressed MASP peptides in the acute phase of the experimental infection. MASP expression in bloodstream trypomastigotes is also first described in this study, as well as the differential expression of its members in trypomastigotes derived from distinct host cells and during acute experimental infection. The MASP expression profile is likely to be even more complex than reported here due to the limitations of our approach. The construction of the expression libraries in our investigation was limited by the similarity of the MASP 3′UTRs, and by the limited number of clones sequenced. Nevertheless, this study revealed a much more complex pattern of MASP expression than was previously described [5]. The use of an RNA-seq approach to study the transcription of bloodstream, tissue-culture derived parasites and infected host cells will reveal a comprehensive picture of the expression of genes involved in T. cruzi–host cell interactions.
Supporting Information
Figure S1.
Multidimensional scaling (MDS) plot of MASP genes. Pairwise alignments of the 1,377 MASP genes were performed and the distance matrix was used to generate a multidimensional scaling (MDS) plot. K-means method was used to define the clusters or groups. A: MDS distribution of all 1,377 MASP genes; B: MDS distribution of the 947 MASP genes amplified by e-PCR allowing 2 gaps and 2 mismatches in the primer annealing sequences. Pseudogenes are shown in purple color.
Figure S2.
Construction of the MASP expression libraries: RNA and cDNA quality controls and semi-nested RT-PCR for MASP representative agarose (A, B, D, and E) or polyacrilamide (C), electrophorese gels. A. 1 to 7: PCR for AHADH2 gene using total RNA as template; 1a, 2a, and 3a: same RNA samples before DNAse I digestion, DNA contaminated. B. 1 to 7: PCR for AHADH2 gene using the cDNAs as templates corresponding to each RNA sample; CNRT: PCR using a negative cDNA (with no RNA) as template; C. 1 to 7: PCR for RAD51 gene using the cDNAs as templates corresponding to each RNA sample. D. 1 to 7: First PCR reaction for MASP family using each cDNA as template, with primers SL and 3′UTR1. E. 1 to 7: Second PCR reaction for MASP family using each PCR sample as template, with primers SL and 3′UTR2; gDNA: PCR using 10 ng T. cruzi genomic DNA as template; CN: negative control of PCR reaction, with no DNA template.
Figure S3.
Invasion assay. LLC-MK2 or L6 cells were infected with trypomastigotes derived from L6 cells after 17 passages, fixed, and processed for immunofluorescent detection of intracellular parasites. The data correspond to the mean of triplicates ± SD and were analyzed using the Student's t test. The results are representative of one of two experiments that yielded similar results.
Figure S4.
Affinity ELISA of MASP peptides. After the incubation with sera pool of mice infected with T. cruzi after two passages, a single wash step was added to the peptide ELISA protocol with 6 M urea. The results of absorbance were compared to the respective non-washed samples (A and C) in the same experiment. Affinity levels <40% and >40% were considered intermediate and low affinity indexes, respectively (dotted lines). A and B: total reactivity (A) and affinity levels (B) of IgG antibodies against the MASP peptides; C and D: total reactivity (C) and affinity levels (D) of IgM antibodies against the MASP peptides.
Table S1.
List of primers used in the Real Time RT-PCR analysis.
Table S2.
List of MASP peptides analyzed by immunoblotting.
Table S3.
List of soluble peptides used in ELISA experiments.
Table S4.
List of members of each MASP group.
Table S5.
EST annotation.
We thank Michele Silva de Matos, Jefferson Bernardes and Afonso da Costa Viana for technical assistance.
Author Contributions
Conceived and designed the experiments: SLS LMF LOA DCB. Performed the experiments: SLS LMF FPL GFRL TAOM ACSO LOA. Analyzed the data: SLS LMF FPL DCB. Contributed reagents/materials/analysis tools: LOA EC RTG SMRT RTF DCB. Wrote the paper: SLS LMF LOA SMRT DCB.
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text:
After the flight – January 25th in Kuwait (John Baker)
After the flight – The "night" of January 25th in Kuwait…
After we deplaned and went through the short painless visa process we headed to baggage claim and got our stuff. Three out of the four members of the dance team have bags that are bigger than they are. Girls. After we went through customs, which consisted of having our bags put through some sort of security scanner, we were out the door and into a van driving to our next destination…the hotel.
While riding down the freeway, the first thing I notice is how Western this place has become. H2s, Corvettes, and other popular American cars race down the road at break neck speeds. A member of our security detail informs us that the police do not enforce speeding on any of the roads so the goal is to drive as fast as you can without getting into a fatal accident. (I would learn later that this ideal is known in the world of Islam as "Insha'Allah" which translates to God willing. The people truly believe that when it is their time to die, they will die, so they drive with a strange reckless abandon, putting all their faith that Allah will deliver them to safety. They also have a "thirty minute rule" when it comes to dealing with the victims of an accident. The police and medical response units will only intercede thirty minutes after a crash. If you die before that, "Insha'Allah", it is what God wills. If you last thirty minutes, you will receive medical attention.) We pass a giant mall that looks like a basketball arena and myriad giant palace-like houses that seem to line the freeway. We eventually pass a real palace on the way to the hotel and the best way to describe it (since it is against the law to photograph the building) is with one word: majestic.
Our hotel is super nice, my room, or rather rooms, has a bedroom, two bathrooms, a dining room and a family style room with a big flat screen TV. Much of the programming is in English, so I feel right at home. For all I know this could be transplanted right into the US and no one could tell the difference.
After dropping the bags off at the room and changing into some athletic gear, I hit the gym with Coghlan in hopes of working up a sweat so that I can fall asleep. Being 11 hours in the future is just plain weird. The gym is extremely nice, it is on par with the large health clubs in the states, basically an upscale 24 Hour Fitness. I workout for around half an hour and successfully shake off the stiffness I have developed over twenty plus hours of traveling.
Post workout the four guys on the trip got together for some dinner. We figured that Chinese food should be good in Asia so that is what we had. Basically middle-eastern PF Chang's, I will say now that we won't be eating there again.
John Baker
@manbearwolf on Twitter
1 Comment
Thanks for the update Bake, we fans are proud of you guys and what you're doing. Can't wait until the season starts.

subset: commoncrawl


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Vancouver Foodie Tours
- 1592 Johnston St
- Vancouver, BC V6H 3S2
- Phone: 604.295.8844 , Toll Free: 1.877.804.9220
With over 1200 5-star reviews, come and experience TripAdvisor's #1 tour in Vancouver! Vancouver Foodie Tours offers food tasting and educational walking tours, introducing locals and visitors to one-of-a-kind dishes that define Vancouver's culinary scene. Guests indulge in a feast for their eyes, belly and mind, tasting signature dishes at award-winning Vancouver gourmet restaurants, food shops and food trucks. Participants learn about the origin of ingredients, how dishes are prepared, as well as the history, culture and architecture of Vancouver. This is the best way to see and taste Vancouver!
We offer four delectable Foodie Tours : Granville Island Market Tour, World's Best Food Truck Tour, Guilty Pleasures Gourmet Tour and Gastronomic Gastown Tour.
Granville Island Market Tour
Noisy and chaotic, yet irresistibly atmospheric, Granville Island is Canada's second most visited destination, behind Niagara Falls, and Vancouver's destination for lovers of food, art and culture. Offering fresh, local, seasonal produce, alongside a slice of local life, this is where guests rub elbows with local chefs shopping for the day's menu. During the tour, guests will savour a cornucopia of fine foods from independent farmers and food artisans throughout Vancouver and beyond, from smoked salmon and cheeses to locally made charcuterie, melt-in-your-mouth donuts and chocolates. Visiting the Granville Island Public Market will be one of the most authentic way to experience Vancouver — after all, what better way to feel the pulse of a city than to see its merchants in action! Click to read more
Gastronomic Gastown Tour
This culinary exploration meanders through the cobblestone streets and alleyways of Vancouver's oldest neighbourhood, laden with quaint restaurants and legendary drinking establishments, to partake in local specialties. 'Vancouver's Best Pizzeria,' resurrected inside a centuries-old tack shop, serves up VPN-Certified Neapolitan-style pizza that runs four generations deep. We explore the original site of Vancouver to pay tribute to the father of Gastown with comfort food and cheerful pairings! The picturesque route is tough competition for a very sweet ending – out-of-this-world dessert ideal for picture-perfect reminders of your trip through Gastown. Click here to read more
Guilty Pleasures Gourmet Tour
The Guilty Pleasures Gourmet Tour showcases over four of downtown Vancouver's award-winning restaurants and food shops on a three-hour food tasting and educational walking tour. Guests sample gourmet food paired with optional wines and beer, meet award-winning food artisans and learn about Vancouver's food trends, culture and architecture. This is the ticket to Vancouver's most exciting food destinations. Click to read more
World's Best Food Truck Tour
Some of the world's most exciting food comes from passionate people who sell their fare from food trucks and carts—allowing them to connect with their customers on a direct level. This tour celebrates Vancouver's ever-growing food truck landscape —Travel + Escape named it the third best street food city in North America—by taking foodies on a global adventure to some of the hottest curbside kitchens in the city. Guests taste signature dishes (from decadent delights to healthy options and globally-inspired fare), meet talented 'cartrepreneurs', watch food being prepared before their eyes and learn where the ingredients originated. Click to read more


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The SHAKESPEARE System – With creative marketing, self-published independent author Matthew Mather quickly reached #1 on the Amazon Science Fiction best-seller list with his first novel, The Atopia Chronicles, surpassing 40,000 copies shipped in its first ten weeks. He is now sharing his SHAKESPEARE system of 11 tips to help new authors achieve their own self-publishing success.
Matthew Mather is a great example of a self-publishing success story in this new era of publishing, where electronic books outsell physical books by a margin of more than two to one. The rapidly growing popularity of ereaders, tablets, smartphones and sites that sell digital books gives independent authors a chance to hit the mainstream without a traditional publishing deal.
Mather's The Complete Atopia Chronicles was his first novel, and within days of its release at the start of August 2012 it shot to #1 on Amazon science fiction with over 40,000 downloads in less than ten weeks, and still remains in the top ten. While luck always plays a role, in this case it wasn't purely luck, but a focused and directed marketing campaign that helped drive it to the top --- the SHAKESPEARE system:
1.Serialize - As attention spans shorten in the online (and real) world, readers don't trust a new author enough to read 400 pages to get the point. For a new author, a winning approach is to serialize, to create your work as a set of progressively longer stories that connect together through cliffhangers to get a reader hooked. And speaking of that…
2.Hook - The first short story needs to be punchy and tell a complete story in itself while leaving the reader wanting to know more. You must also hook the reader on the first page somehow, create a mystery, a reason and need to keep reading.
3.Amazon - To start, focus only on Amazon. I'm not here to promote Amazon, but the first rule of entrepreneurism is to focus, focus, focus. The large majority of revenue in digital books comes from Amazon, with a small minority coming from all of the other players combined. So when you start, focus solely on Amazon; getting reviews, moving up in the ranking. By focusing exclusively on Amazon, you force people to buy from one place and thus drive up your rankings in this one spot. Once you have achieved some success there, expand to other platforms (FYI the easiest way to get on other platforms is just to use Smashwords).
4.Key networks - Make sure to use your personal social networks to maximum effect. Post on Facebook and ask people to re-post your postings for free book offers. Make sure to email everyone at work on the "internal" email (ask your boss first, of course!) Use your LinkedIn network to mention that you have a book out. What other networks are you a part of?
Try emailing top-selling authors in your category when you release the first installments of your work. Ask them to read the first one (by starting with serialized shorts, it makes it easier for other authors to try reading your work), or just ask them to post on their blog or Facebook. When I released Atopia, I had about five or six top-selling authors who posted to their readers for me!
5.Empathize - It is critical to create a character that you introduce readers to right away that they can empathize with. People read still primarily because they want to feel an emotional involvement with a character they meet in your writing. Keep this front and center of your mind when writing.
6. Select Program on Amazon - Use the Amazon Select Program: You can offer your book for $0 (free) for 5 days each 3 month period. Used effectively, this is an extremely potent tool for reaching an audience.
There are at least 40 websites I use to promote a "free weekend" for my books (email me for a list) - these sites are mostly specific to books that go free on Amazon Select and are mostly free to use for promotion.
If you can plan it ahead of time, write out all of the parts of your serialized work ahead of time, and then each two weeks release one of them, promoting it on Amazon select for free and on the promotional websites. I can usually get 4000+ downloads of a free book when I do this.
7. Perceived Value
Create perceived value by offering a deal. For instance, try and divide your 'whole' work into 6 parts, and sell each for $0.99, and then offer the whole 'collection' at half price, e.g. $2.99 for all six. This creates perceived value on the part of the buyer when you start to sell the whole collection
8. Editing
If your work is not edited well, you will get killed in the reviews and by word of mouth. Go on Craigslist and find some recently-graduated (and unemployed) English lit majors to edit your book on the cheap. A "real" editor can be extremely expensive; while the quality may not be perfect, hiring unemployed English-lit majors can save you hundreds, even thousands of dollars. There is no excuse to not get an external editor of some kind, and not getting one will kill your chances of success.
9. All free posting websites - Craigslist and other free online classified ads are secret weapons for new authors. It is incredibly difficult to get outside feedback when you are a new writer. My solution? Post an ad saying you'll pay someone $10 or $20 to read your book and give you honest feedback. Note that this is not for line editing, but for high level feedback to make your story more engaging in an iterative process.
Bonus: Get 20 people to read your book like this; these people will probably become your biggest promoters and will be happy to write reviews and Facebook and tweet about your book when released.
Free PR – When you release your book, create several press releases about different aspects of the book, what it is about, why people would like it.
When you release each of the story segments, put these press releases up on the free press release websites. There are about a dozen high quality free release sites out there. Highlight that the short story is free that week.
10. Reviews
It is crucial to get reviews as this has a direct impact on the Amazon ranking and recommendation system. YOU CANNOT do fake reviews. Apart from the ethical issues, Amazon has an impressive array of technical tools to make this very difficult. Instead, be honest and creative; ask friends, family, co-workers; and see my point regarding Craigslist and getting people ready to punt for your project.
11. Engage (Bonus Tip)
Find any and all ways to engage with your audience once you start to get readers. Do a video blog on YouTube about the process, do a regular blog showing progress on your next books and stories, get people to like your Facebook page. Just get engaged with them somehow!
Self-published writers like Amanda Hocking, B.V. Larson and Hugh Howey have all succeeded beyond the highest projections of publishing houses, with the 50 Shades of Grey series, the Star Force series and Wool. Often overlooked, independent writing has a long and illustrious history, including legendary writers like Edgar Allen Poe, Henry David Thoreau, Mark Twain, Beatrix Potter and Rudyard Kipling. Mather believes the digital age will provide opportunities for an unprecedented number of independent authors to succeed as self published authors.
Matthew Mather
The Atopia Chronicles The Atopia Chronicles are an exploration of the meaning of love, life and the pursuit of happiness in a world teetering on the brink of eco-Armageddon. What could be worse than letting billions die? In the future, be careful what you wish for. Here's a link to the novel. (Kindle Edition)
Matthew Mather started his career at the McGill Center for Intelligent Machines, founded one of the world's first tactile feedback companies, and won the $2M award for "Best New Videogame in Canada" in 2007. He's worked professionally in computational nanotechnology, weather prediction systems, genomics and cybersecurity. Matthew lives in Montreal, Canada.
Don't forget ... we encourage you to share your discoveries with other readers. Just send and email, contribute your own article, join the Design Cafe forums, or follow DTG on Facebook!


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A Pair Of Complete 05-07 Chevy Corvette LED Style Tail Lights From K2Motor. Add The Finishing Touch To Your Vehicle With Our Extensive-Line Of High Quality LED Tail Lights. A Revolutionary Altezza LED Tail Light Not Only Improves The Appearance Of Your Vehicle, But Also Customizes Your Vehicle With Unique Styling. Our Exclusive Line Of Altezza Style Tail Lights Feature Hassle Free Plug & Play Installation. Buy Yours Today And Get Free U.S. Domestic Shipping!
- Fits: 2005-2007 Chevy Corvette
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RETURNS & EXCHANGES - Our Policies and Promises to Our Customers.
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OpenTracker1+ SMT
OpenTracker1+ SMT (OT1+ SMT) - Assembled and tested OpenTracker+ surface-mount (SMT) board. This version of the OT1+ is perfect for installation inside a radio or any other application where size and weight are a concern. Without pins installed, the board measures 1.22 x 0.72 x 0.2" (31 x 18 x 5 mm).
All off-board connections are through a double row of holes, in a standard 24-pin DIP footprint. Two pin header strips are provided (not installed) to allow the board to be mated with another PCB or installed in a solderless breadboard.
This board does not include a built-in temperature sensor or LEDs.
Pin - Name - Function
- 5VIN Regulated 5-volt input
- RXD RS-232 data in (from GPS or computer)
- 1WIRE Dallas 1-wire bus for external sensors
- AOUT Audio out to radio
- ADC5 Analog input
- T1CH1 Timer channel
- ADC6 Analog input
- RST Reset (active low)
- ADC7 Analog input
- IRQ Transmit-now, profile select, or counter
- ADC8 Analog input
- ADC9 Analog input
- AIN Audio input from radio
- ADC4 Analog input
- ADC3 Analog input
- TXD RS-232 data out
- RED Red LED output
- GREEN Green LED output
- ADC2 Analog input
- ADC1 Analog input
- PTT Push-to-talk signal to radio
- REGOUT Regulator output - 5 volts
- GND Ground
- REGIN Regulator input - 6.5 to 28 volts
There is no on-board connection between the regulator output and the 5 volt supply. This allows the regulator section to be bypassed if an external 5 volt source is available. To use the regulator, either connect the REGOUT and 5VIN pins externally or place a small blob of solder on the two jumper pads on the bottom of the PCB.
When the telemetry option is enabled, the device will transmit an APRS telemetry packet containing 8-bit analog values for inputs ADC1-5 and digital values for inputs T1CH1 and ADC6-9


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Home > About Us > Building Lasting Unforgettable Experiences > BLUE Stars - July 2014
BLUE Stars - July 2014
Building Lasting Unforgettable Experiences... At WILLCARE, that's what we do best! We strive to provide exceptional customer service for all our customers, including patients, their families, physicians, vendors, referral sources, and co-workers. We view every interaction as a chance to leave our customers with positive experiences that they will never forget.
Our BLUE program (short for Building Lasting Unforgettable Experiences of course!) recognizes those WILLCARE team members who've gone above and beyond to provide fantastic customer service. Once a month, the WILLCARE employees that best embody the spirit of BLUE are selected to be our BLUE Stars. Learn more about our July 2014 winner below.


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If you are or know of a kitty who needs comforting, healing, prayers and good strong positive energy please leave a message. And if you would like to send healing purrs to the cats in our circle, we thank mew :-)
"Until one has loved an animal, a part of one's soul remains unawakened." - Anatole France


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Chicago Illinois Electricians that start with E1 - 2 of 2
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Electrical Services Inc Chicago, IL 60631(773) 774-5190
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Recent Reviews
Bonavia's Advanced Electric
Code update"I had them out to my home, due to local inspector not approving my work from another contractor. Bonavia fixed everything and coordinated with the inspector. They got the job done fast and passed with flying colors! Very reasonable as well. That's always a ggod thing."
Bonavia's Advanced Electric
Happy Homeowner"I recently had a problem with my lights flickering really bad so I called my friend for a referral. He recommended Joe Bonavia (owner) I called he came out and fixed it right away. I couldn't believe how reasonable he was, especially because I was in a bind. Fast, friendly service! Definitely would recommend."
Bookless John Wm
Serious Electrician"John knows what he's doing and has an understanding of the problems associated with historic building and old wiring. And he is such a nice guy."
Berry Electric Contracting Company
One Employee does not a Co. make!"Mr. P. Tippett...in response to the above.While I can understand Your feelings toward the said employee.How does that tell you whether Berry Electric is,..or is not a GOOD Co.Has Berry Electric finished any contracting jobs for you or your firm?\n\nIf so..was it Commercial or Residential?Did they pass all local inspections legally and on schedule?Was the job managed by Master or Journeyman Electricians?Was the job maned up daily?How close was the final price compared to the bid you received?\n\nAnd then again...was the said employee a new hire? Was he fired the next day after the incident?Did management respond to customer or personnel complaints swiftly and appropriately? \n\n"
Berry Electric Contracting Company
Sucks - Look for another company!"Company workers can't even drive a vehicle compentently nor can they move a shopping cart without ramming it into a vehicle 8 times and have foul tempers to boot! Store director at Jewel/Osco even had to apologize for the behavior of Berry Electrics companies employee. Several people saw what the driver of the Berry Electric truck did for no reason. I was walking out of the store at the time to witness this idiots temper tantrum."