IgA and IgG serum antibody responses to CS5 and CS6 in individual patients.

<p>The responses to CS5 (broken lines) and CS6 (solid lines) were measured by ELISA in sera from patients infected with strains E1777, E1785 and E1779, on different days after hospitalization. Serum from the patient infected with strain E2265 was not available.</p

Phenotypic CS5 levels in LB alone and LB supplemented with bile or individual bile salts.

<p>CS5 expression was quantified by inhibition ELISA after overnight culture to stationary phase of strains E1777, E1779, E1785, E2265, and E3003. CS5 surface expression tifwas induced by NaGCH, but not by the corresponding unconjugated bile salt NaCH or its tauro-conjugated counterpart TCA (representative data).</p

Dose-dependent induction of phenotypic CS5 expression by NaGCH.

<p>Expression of CS5 was determined by inhibition ELISA in strain E1777 after overnight culture to stationary phase in LB medium supplemented with NaGCH or crude bile. Bars indicate means and standard errors of the means of two measurements in one experiment.</p

Levels of <i>csfD</i> and <i>cssB</i> transcription compared to in LB alone after one hour of culture.

<p>Level of <i>csfD</i> (A) and <i>cssB</i> (B) transcription in LB supplemented with crude bile or individual bile salts standardized to the level of transcription in LB medium alone, after one hour of culture. Transcription was measured by reverse transcriptase real time PCR. Bars show means and standard errors of the means of three separate experiments (strains E1785, E2265, and E3003, respectively). *, P = <0.05</p

Strains used in the study and results from culturing^a of clinical stool specimens.

a<p>Culture was performed on MacConkey agar plates to detect <i>E. coli</i> and on Taurocholate-tellurite-gelatin agar (TTGA) for detection of vibrios; all cultures were performed overnight at 37°C.</p>b<p>Department of Microbiology and Immunology, University of Gothenburg, enterotoxigenic <i>Escherichia coli</i> (ETEC) strain collection number.</p

Primers used for PCR and real-time Reverse Transcriptase PCR (rt RT-PCR).

a<p>CS5 minor subunit<b>;</b> GenBank accession number <u>AJ224079</u>.</p>b<p>CS6 structural subunit CssB; GenBank accession number <u>UO4846</u>.</p>c<p>Primers described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035827#pone.0035827-Nicklasson1" target="_blank">[44]</a>.</p>d<p><i>E. coli</i> D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH); GenBank accession number <u>U014639.1.</u></p

Transcriptional <i>csfD</i>:<i>ccsB</i> ratio <i>in vivo</i> and <i>in vitro</i>.

<p>Transcription of <i>csfD</i> and <i>cssB</i> was quantified by real time reverse transcriptase PCR (rt RT-PCR) in <i>in vivo</i> bacterial samples (strains E1777, E1785, E2265; closed bar) and after one (strains E1785, E2265, E3003; open bars) and two (E1777, E1779; hatched bars) hours of <i>in vitro</i> culture in LB medium alone and in LB supplemented with 0.15% crude bile or 0.2% individual bile salts. Bars show the means and standard errors of the means of two (after two hours of culture) or three (<i>in vivo</i> and after one hour of culture) separate experiments.</p

Conservation of novel pathotype-specific antigens EtpA and EatA among phylogenically distinct strains expressing different colonization factors.

<p>a. Heatmap of EtpA and EatA showing the proportion of strains positive for expression of these antigens among different CF groups. CF antigen designation is shown at left of the heatmap. nd = no CF antigen detected. Below is the heatmap key depicting colors associated with each degree of antigen positivity. Density line in yellow depicts the relative number of map features assigned at each proportion. Primary data used to construct the heatmap can be found in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0003446#pntd.0003446.s007" target="_blank">S2 Dataset</a>. b. Imunoblot detection of EtpA and EatA expression among strains from different phylogenies. The upper immunoblot demonstrates EtpA production in the prototype H10407 strain, ThroopD isolated in Dallas, TX in 1975, the Juruá_18/11 (Amazon, 1998), and phylogentically dispersed strains from icddr,b. The <i>etpA</i> mutant is included as a negative control. The lower blot demonstrates EatA production by H10407, phylogenically distributed strains from icddr,b and Envira_10/1, an additional isolate from cholera-like outbreaks in the Amazon. The <i>eatA</i> mutant is included as a negative control. c. Phlyogram showing the phylogenetic distribution of selected ETEC strains (designations in blue) and reference E. coli strains (designations in black). Red circles and gold stars represent eatA+, and etpA+ strains, respectively.</p

Distribution of EtpA and EatA among strains expressing different colonization factors.

<p>Distribution of EtpA and EatA among strains expressing different colonization factors.</p

Relationship of strain subsets used in antigen expression studies, and strains with available whole genome sequences.

<p>All of the strains in the collection (n = 181) were examined for production of three secreted ETEC virulence proteins EtpA, EatA, and YghJ by immunoblotting of culture supernatants with the respective antibodies. A subset of these strains (n = 91) were recently sequenced at the Genome Sequencing Center for Infectious Diseases (GSCID).</p