Sequence similarity of TAD locus in strains of <i>P. acnes</i>, “<i>P. humerusii</i>” and <i>C. leptum</i>.

<p>A sequence alignment of the partial TAD-like loci of <i>P. acnes</i> 15.1.R1, “<i>P. humerusii</i>” P08 [GenBank: AFAM00000000] and <i>C. leptum</i> DSM753 [GenBank:ABCB00000000] is shown. High sequence similarity (shown in red, >95% nucleotide identity) exists between these loci in different species. The 5′ end of the “<i>P. humerusii</i>” contig, which has a lower G+C content, matches to a genomic region in the KPA chromosome (PPA1278–PPA1304). The location of CRISPR spacers is indicated; the protospacer “A” refers to a spacer found in the CRISPR region of <i>P. avidum</i>. The G+C content is depicted above the open reading frames (sliding window 500 bp); the percentages refer to the average G+C content of the contigs. The figure was made using the Artemis Comparison Tool (ACT, Sanger).</p

The CRISPR/<i>cas</i> gene locus in <i>P. acnes</i> J139.

<p>Shown is the gene order in strain J139 (locus tags: HMPREF9206_0746 (<i>cas3</i>) to HMPREF9206_0752 (<i>cas1</i>); <i>cas2</i> has not been correctly annotated in the draft genome [GenBank: ADFS00000000]). The gene cluster is identical in all analyzed <i>P. acnes</i> genomes. The CRISPR loci are composed of 1–9 repeats and 1–8 spacers.</p

Tight adherence (TAD) gene cluster in <i>P. acnes</i> strain 15.1.R1.

<p>The gene cluster [GenBank: JQ612072] encoding proteins similar to TadZABC is shown. This cluster of 20.2 kb is part of the 54 kb mobile genetic element in strain <i>P. acnes</i> 15.1.R1 and other type I <i>P. acnes</i> strains. The annotation is based on sequence similarity, employing the tools BLASTP and InterPro. Genes without annotation are considered hypothetical. CRISPR spacers that are identical or similar to sequences of this mobile genetic element are marked; the numbers refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034171#pone-0034171-t001" target="_blank">Table 1</a>.</p

CRISPR spacers identified in type II <i>P. acnes</i> strains.

*<p>numbers in brackets indicate additional spacers present in the respective strain (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034171#pone.0034171.s005" target="_blank">Table S2</a> for a complete overview).</p

Phylogenetic tree of <i>P. acnes</i> strains used in this study and distribution of mobile genetic elements.

<p>The tree was constructed based on 4,287-bp concatamer of partial sequences of nine housekeeping genes, and sequences types (STs) were assigned employing the Aarhus MLST scheme (<a href="http://pacnes.mlst.net/" target="_blank">http://pacnes.mlst.net/</a>). The tree was constructed using the Minimum Evolution algorithm in MEGA version 5.05. The bootstrap values were based on 500 replicates and only values exceeding 50 are shown. Clonal complexes (CCs) were determined according to an eBURST analysis described in Kilian et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034171#pone.0034171-Kilian1" target="_blank">[10]</a>. The distribution of three genomic regions within this strain collection was checked by PCR, namely the CRISPR locus (red circle), the TAD locus (green square), and the bacteriocin (bcn) island (blue triangle).</p

Use of a novel antigen expressing system to study the <i>Salmonella enterica</i> serovar Typhi protein recognition by T cells

<div><p><i>Salmonella enterica</i> serovar Typhi (<i>S</i>. Typhi), the causative agent of the typhoid fever, is a pathogen of great public health importance. Typhoid vaccines have the potential to be cost-effective measures towards combating this disease, yet the antigens triggering host protective immune responses are largely unknown. Given the key role of cellular-mediated immunity in <i>S</i>. Typhi protection, it is crucial to identify <i>S</i>. Typhi proteins involved in T-cell responses. Here, cells from individuals immunized with Ty21a typhoid vaccine were collected before and after immunization and used as effectors. We also used an innovative antigen expressing system based on the infection of B-cells with recombinant <i>Escherichia coli</i> (<i>E</i>. <i>coli</i>) expressing one of four <i>S</i>. Typhi gene products (i.e., SifA, OmpC, FliC, GroEL) as targets. Using flow cytometry, we found that the pattern of response to specific <i>S</i>. Typhi proteins was variable. Some individuals responded to all four proteins while others responded to only one or two proteins. We next evaluated whether T-cells responding to recombinant <i>E</i>. <i>coli</i> also possess the ability to respond to purified proteins. We observed that CD4⁺ cell responses, but not CD8⁺ cell responses, to recombinant <i>E</i>. <i>coli</i> were significantly associated with the responses to purified proteins. Thus, our results demonstrate the feasibility of using an <i>E</i>. <i>coli</i> expressing system to uncover the antigen specificity of T-cells and highlight its applicability to vaccine studies. These results also emphasize the importance of selecting the stimuli appropriately when evaluating CD4⁺ and CD8⁺ cell responses.</p></div

Ability of the <i>Hly</i> gene to improve antigen processing.

<p>B-LCL cells were infected with either <i>E</i>. <i>coli</i> strain BL21 (BL21) or its <i>Hly</i>-recombinant <i>E</i>. <i>coli</i> (Hly) counterpart at 1:30 or 1:100 multiplicity of infection (MOI). After 2 hours, the cells were washed and exposed to gentamicin for an additional 2 hours to kill and detach extracellular bacteria. After further washings, the ability of the <i>Hly</i> gene to improve antigen processing was assessed by detecting <i>E</i>. <i>coli</i> antigens at the B-cell surface over time by flow cytometry (up to 120 minutes). Cells exposed to media only were used as control (uninfected).</p

CD4+ T cell responses to <i>S</i>. Typhi proteins presented by targets infected with recombinant <i>E</i>. <i>coli</i>.

<p><i>Ex vivo</i> PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets infected at 1:30 MOI with one of the four recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi/Hly (<i>Hly</i>/<i>SifA</i> (SifA), <i>Hly</i>/<i>FliC</i> (FliC), <i>Hly</i>/<i>GroEL</i> (GroEL) and <i>Hly</i>/<i>OmpC</i> (OmpC)) or only <i>Hly</i> (control) proteins. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD4+ T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.</p

Correlation between T cell subset responses to B-LCL targets exposed to recombinant <i>S</i>. Typhi proteins or infected with recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi proteins.

<p><i>Ex vivo</i> PBMC were analyzed as described in Figs <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005912#pntd.0005912.g001" target="_blank">1</a> & <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0005912#pntd.0005912.g002" target="_blank">2</a>. Shown are the correlation between T cell subset responses to either of the four proteins (SifA, FliC, GroEL and OmpC) after stimulation by B-LCL targets exposed to recombinant <i>S</i>. Typhi proteins or infected with recombinant <i>E</i>. <i>coli</i> expressing <i>S</i>. Typhi proteins. Samples are representative of two individuals collected 42 days after vaccination. Coefficients of determination “R2” and “p” values are shown. p values of <0.05 were considered statistically significant. Dashed lines represent 95% confidence intervals.</p

CD8+ T cell responses to <i>S</i>. Typhi proteins presented by targets exposed to one of the four recombinant <i>S</i>. Typhi proteins.

<p><i>Ex vivo</i> PBMC from a volunteer collected 42 days after immunization were co-cultured for 16–18 hrs. with autologous B-LCL targets exposed to 0.5ug/ml with one of the four recombinant <i>S</i>. Typhi proteins: SifA, OmpC, FliC, and GroEL. Untreated B-LCL targets (media) were used as controls. After incubation, cells were stained and the ability of the PBMC to express one or more cytokines (IL-17A, IFN-γ and TNF-α) and/or CD107a/b molecules was evaluated by flow cytometry. Shown are the CD8⁺ T cell responses from a representative volunteer. Numbers represent the percentage of positive cells.</p