pTTSS Mutants Are Adjacent to B and T Lymphocytes Whereas WT Is Adjacent to B and T Lymphocytes and CD11b⁺ Cells

<p>BALB/c mice were intragastrically inoculated with 2 × 10⁹ WT IP2666 or IP2666 pIB1⁻. At 4 d post-inoculation, MLN were harvested, sectioned, and examined by fluorescence microscopy. Staining with antibodies to <i>Yptb</i> (red) (A), (D), (G), and (J), to CD4-CD8-B220 (green) (B) and (E), or to CD11b (green) (H) and (K) was performed, and images merged (C), (F), (I), and (L). Pictures show the cortex–paracortex and are representative of multiple fields and samples for WT-infected mice and pIB1 mice stained with CD4-CD8-B220. Fewer fields had pIB1 bacteria and CD11b+ cells. Scale bars correspond to 22 μm for 900× magnification.</p

<i>Yptb</i> pTTSS Mutants Remain Extracellular in the MLN and Are Not Efficiently Internalized by B or T Lymphocytes in Culture

<div><p>(A) Single-cell suspensions of <i>Yptb</i>-infected MLN were assayed for the presence of intracellular bacteria using a gentamicin protection assay. BALB/c mice were intragastrically inoculated with WT YPIII (<i>n</i> = 6 mice), YPIII <i>yscBL</i> (<i>n</i> = 5 mice), WT IP2666 (<i>n</i> = 5 mice), IP2666 <i>pIB1⁻</i> (<i>n</i> = 7 mice), or S. typhimurium (<i>n</i> = 4 mice). Four days post-inoculation with IP2666 strains or 5 d post-inoculation with YPIII strains, the percentage of intracellular bacteria was assessed by generating a single-cell suspension of the MLN and treating half the suspension with gentamicin and half without gentamicin (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020086#s4" target="_blank">Materials and Methods</a>).</p><p>(B) Murine macrophage RAW264.7 cells, human T cells SUP-T1, and B and T lymphocytes isolated from MLN (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020086#s4" target="_blank">Materials and Methods</a>) were infected with WT YPIII, YPIII <i>yscBL,</i> WT IP2666, or IP2666 pIB1⁻ strains at MOI of 10:1 for 30 min, and then treated with gentamicin for 90 min. The data are presented as 100 times the number of gentamicin-resistant bacteria divided by the number of input bacteria. Data from one representative experiment done in triplicate is shown. All experiments were performed at least three times.</p></div

MLN Histopathology during WT or pIB1⁻ Infection

<div><p>BALB/c mice were intragastrically inoculated with 2 × 10⁹ WT or IP2666 pIB1^−<i>,</i> and MLN were processed for H&E staining 4 d later. MLN sections from uninfected (Uninf.) (A–D), WT infected (E–H), and pIB1⁻ infected (I–L) are shown at 15×, 40×, 90×, and 450× magnification. White boxes indicate magnified areas in the next slide. White arrow points to the bacterial foci, black arrow to neutrophils. Scale bars correspond to 133 μm for 15× magnification, 50 μm for 40× magnification, 22 μm for 90× magnification, and 4.4 μm for 450× magnification. Pictures shown are representative of multiple fields and samples from 16 MLN infected with WT or 14 MLN infected with pIB1⁻.</p><p>C, cortex; gc, germinal center; M, medulla; P, paracortex.</p></div

pTTSS Is Not Required for MLN Colonization

<div><p>BALB/c or C57BL6/J mice were intragastrically inoculated with 2 × 10⁸ WT YPIII, YPIII <i>yscBL,</i> YPIII <i>yscNU,</i> YPIII <i>yscBU,</i> or YPIII <i>pIB1⁻,</i> and colonization levels of the ileum (A), (F), and (K), cecum (B), (G), and (L), PP (C), (H), and (M), MLN (D), (I), and (N), and spleen (E), (J), and (O) were determined at 6 h (A–E), 2 d (F–J), and 5 d (K–O) post-inoculation. BALB/c mice were intragastrically inoculated with 2 × 10⁸ mouse commensal <i>E. coli,</i> and colonization levels of tissues were studied at 6 h (A–E) and 2 d post-inoculation (F–J). BALB/c mice were intragastrically inoculated with 2 × 10⁹ of WT IP2666, IP2666 <i>yscNU,</i> or IP2666 <i>pIB1^−,</i> and colonization levels were determined at 4 d post-inoculation ( [K–O], middle section).</p><p>Data are from 4–12 mice from at least two different experiments. All data was combined for each strain, tissue, and time point. Each square represents the log cfu/g tissue from one mouse; open squares indicate that less than 10 cfu were recovered per tissue, and bars represent the geometric mean. Asterisks (*) and black circles (•) indicate statistically significant differences between the WT and the mutants, calculated by the <i>t</i> test (*<i>p</i> < 0.01) or by Mann-Whitney (•<i>U</i> < 0.05), respectively.</p></div

Invasin and cTTSS Are Not Essential for <i>Yptb</i> Survival in the MLN

<div><p>(A) BALB/c mice were intragastrically inoculated with 2 × 10⁸ YPIII pIB1 or YPIII <i>pIB1⁻inv^−,</i> and colonization levels of the ileum, PP, MLN, and spleen were determined 5 d post-inoculation.</p><p>(B) BALB/c mice were i.p. inoculated with 2 × 10⁶ YPIII pIB1 or YPIII pIB1⁻<i>inv⁻,</i> and their colonization levels in the MLN and spleen were determined 3 d post-inoculation.</p><p>(C) BALB/c mice were inoculated intragastrically with 2 × 10⁸ YPIII pIB1⁻ or YPIII pIB1⁻cTTSS, and colonization levels of the ileum, PP, MLN, and spleen were determined. Data are from 6–11 mice from at least two different experiments. Each square represents log ₁₀ cfu/g tissue recovered from one mouse. Bars represent the geometric mean. Open squares indicate than less than 10 cfu were recovered per tissue. Asterisks (*) and black circles (•) indicate statistically significant differences between the pIB1⁻ and either pIB1⁻<i>inv</i>⁻ or pIB1⁻cTTSS strains, calculated by the <i>t</i> test (*<i>p</i> < 0.01) or by Mann-Whitney (•<i>U</i> < 0.05), respectively.</p></div

<i>Yptb</i> Localizes in the Cortex and Paracortex of the MLN

<div><p>BALB/c mice were intragastrically inoculated with 2 × 10⁹ WT or IP2666 pIB1⁻. At 4 d post-inoculation, MLN were harvested and stained for <i>Yptb</i> followed by hematoxylin staining. Picture sections of uninfected (A–C), WT infected (D–I), and pIB1⁻ infected (J–L) MLN were taken at 150×, 900×, and 4,500× magnification. White boxes indicate magnified areas in the next slide. Arrows point to <i>Yptb</i> microcolonies. Scale bars correspond to 133 μm for 15× magnification, 22 μm for 90× magnification, and 4.4 μm for 450× magnification. Pictures shown are representative of multiple fields and samples from MLN infected with WT or pIB1⁻.</p><p>C, cortex; gc, germinal center; M, medulla; P, paracortex.</p></div

B and T Lymphocytes Are Important for pIB1⁻ Colonization in the MLN

<div><p>BALB/c or <i>Rag1^−/−</i> mice were intragastrically inoculated with 2 × 10⁹ WT IP2666 or IP2666 pIB1⁻.</p><p>(A) Size and weight of MLN from BALB/c or <i>Rag1^−/−</i> mice that were either uninfected or infected with WT IP2666 or IP2666 pIB1⁻ were measured.</p><p>(B) and (C) Colonization of the MLN or luminal content of the ileum 4 d post-intragastric inoculation of BALB/c or isogenic <i>Rag1^−/−</i> mice was determined. Each square indicates the cfu from one mouse calculated as log cfu/MLN (B) or the log cfu/g of luminal content of the ileum (C); bars represent the geometric mean. Each experiment was performed with two to three mice and repeated three times. Asterisks (*) and black circles (•) indicate statistically significant differences between the number of pIB1⁻ recovered from BALB/c mice versus <i>Rag1^−/−,</i> calculated by the <i>t</i> test (*<i>p</i> < 0.05) or by Mann-Whitney (•<i>U</i> < 0.05), respectively.</p></div

<i>ΔyadA</i> mutants associate more frequently with B and T cells than WT.

<p>(<b>A–C</b>) Splenocytes were infected at an MOI of 0.5∶1 with the indicated IP2666 GFP-expressing strains and cell types were distinguished by cell-type surface marker staining using flow cytometry. (<b>A</b>) The percentage of cells bound to GFP⁺ bacteria was determined by fluorescence intensity in the FITC channel of the total live splenocyte population. (<b>B and C</b>) The percentage of B and T cells (<b>B</b>) and phagocytes (<b>C</b>) bound by GFP⁺ bacteria. Experiment was repeated 5–8 times (*P<0.05, **P<0.01 and ***P<0.001 compared to WT).</p

Adhesins and Host Serum Factors Drive Yop Translocation by <i>Yersinia</i> into Professional Phagocytes during Animal Infection

<div><p><i>Yersinia</i> delivers Yops into numerous types of cultured cells, but predominantly into professional phagocytes and B cells during animal infection. The basis for this cellular tropism during animal infection is not understood. This work demonstrates that efficient and specific Yop translocation into phagocytes by <i>Yersinia pseudotuberculosis</i> (<i>Yptb</i>) is a multi-factorial process requiring several adhesins and host complement. When WT <i>Yptb</i> or a multiple adhesin mutant strain, <i>ΔailΔinvΔyadA</i>, colonized tissues to comparable levels, <i>ΔailΔinvΔyadA</i> translocated Yops into significantly fewer cells, demonstrating that these adhesins are critical for translocation into high numbers of cells. However, phagocytes were still selectively targeted for translocation, indicating that other bacterial and/or host factors contribute to this function. Complement depletion showed that complement-restricted infection by <i>ΔailΔinvΔyadA</i> but not WT, indicating that adhesins disarm complement in mice either by prevention of opsonophagocytosis or by suppressing production of pro-inflammatory cytokines. Furthermore, in the absence of the three adhesins and complement, the spectrum of cells targeted for translocation was significantly altered, indicating that <i>Yersinia</i> adhesins and complement direct Yop translocation into neutrophils during animal infection. In summary, these findings demonstrate that in infected tissues, <i>Yersinia</i> uses adhesins both to disarm complement-dependent killing and to efficiently translocate Yops into phagocytes.</p></div

A model of factors that drive Yop translocation during mouse infection.

<p>(<b>A</b>) During infection, <i>Yptb</i> expresses adhesins Ail, Invasin and YadA, which mediate binding to host-cell receptors directly or indirectly via ECM components or complement, leading to TTSS engagement and Yop delivery into host cells. (<b>B</b>) In a <i>ΔailΔinvΔyadA</i> mutant, another unknown <i>Yptb</i> adhesin becomes accessible and mediates Yop delivery. Factors such as complement dampen proper engagement of the unknown adhesin with host cells leading to fewer Yop translocated cells. (<b>C</b>) In the absence of complement, an unknown adhesin becomes accessible in the <i>ΔailΔinvΔyadA</i> mutant and mediates TTSS engagement and Yop delivery into host cells.</p