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

Adhesin mutants have variable, strain dependent effects on translocation into professional phagocytes.

<p>Splenocytes were infected with the indicated ETEM-expressing strains at an MOI of 1∶1 for (<b>A</b>) 1 h with IP2666, (<b>B</b>) 45 min with IP32953 strains or (<b>C</b>) 45 min with YPIII strains. Professional phagocytes were distinguished by cell-type surface marker staining using flow cytometry. The left Y-axis represents the percentage of each cell type in the spleen (white bars) while the right Y-axis represents the percentage of each cell type present in the Blue⁺ population (grey bars). Experiment was repeated 3–8 times (ND, not determined; *P<0.05, **P<0.01 and ***P<0.001 compared to WT).</p

Complement depletion restores virulence and translocation of Yops by the <i>ΔailΔinvΔyadA</i> mutant.

<p>CVF-treated mice were infected IV with 800 CFU of IP2666 WT-ETEM (1X-WT), 800 CFU of <i>ΔailΔinvΔyadA-ETEM</i> (1X-<i>ΔailΔinvΔyadA</i>), or 30,000 CFU of <i>ΔailΔinvΔyadA-ETEM</i> (37.5X-<i>ΔailΔinvΔyadA</i>). (<b>A</b>) Animals were monitored for morbidity and mortality for a period of 15 days post infection and survival was plotted. (<b>B–D</b>) Four days post-infection, spleens were isolated to determine CFUs (<b>B</b>) and the percentage of Blue⁺ cells present in the organ compared to the log₁₀CFU (<b>C</b>). (<b>B–C</b>) Each symbol represents data from one mouse; the bar in (<b>B</b>) represents the geometric mean. (<b>C</b>) The black line represents values from mice infected with 800 CFU WT-ETEM as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003415#ppat-1003415-g004" target="_blank">Fig. 4B</a>, open-grey squares with grey line represents CVF-treated mice infected with 800 CFU WT-ETEM and open-grey triangles with dashed grey dotted line represent values from 800 CFU <i>ΔailΔinvΔyadA</i>-ETEM. Linear regression analysis determined that the percentage of Blue⁺ cells is the same in both WT infections regardless of CVF. In contrast, the CVF+1X-<i>ΔailΔinvΔyadA</i> injected Yops into significantly more cells than 1X-WT and CVF+1X-WT (with P<0.0001 for both). (<b>D</b>) The distribution of cell types found in the organ (white bars, left y-axis) versus the distribution of cell types found in the Blue⁺ population (gray bars, right y-axis) for each infection condition was compared. (<b>B and D</b>) (**P<0.01 and ***P<0.001). Data from mice infected with 800 CFU of WT-ETEM and 30,000 CFU of <i>ΔailΔinvΔyadA</i> in panels <b>B–D</b> are from the same mice analyzed in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003415#ppat-1003415-g004" target="_blank">Fig. 4B–C</a>.</p

Serum directs Yop translocation into professional phagocytes.

<p>(<b>A–C</b>) Splenocytes in 5% HIS, Fn (40 µg/ml), BSA (5 mg/ml) or SFM were infected for 1 h with IP2666 WT-ETEM at an MOI of 0.2∶1, 0.5∶1 or 1∶1 and the percentage of Blue⁺ cells was determined by FACS. (<b>B–C</b>) Splenocytes infected for 1 h with IP2666 WT-ETEM at an MOI of 0.2∶1 or 1∶1 in SFM or an MOI of 1∶1 in HIS were analyzed for (<b>B</b>) the percentage of Blue⁺ cells in each cell type population or (<b>C</b>) the percentage of each cell type in the Blue⁺ population (gray bars) compared to the percentage of each cell type in the spleen (white bars). (<b>D</b>) Splenocytes were infected for 1 h at an MOI of 1∶1 with IP2666 WT-ETEM, <i>ΔailΔinvΔyadA-ETEM</i> or <i>ΔyopB-ETEM</i> and the percentage of Blue⁺ cells was determined. (<b>E–F</b>) Splenocytes were infected with the indicated IP2666 GFP⁺ strains at an MOI of 0.5∶1 and (<b>E</b>) the percentage of cells bound to GFP⁺ bacteria determined by fluorescence intensity in the FITC channel of the total splenocyte population, or (<b>F</b>) the percentage of specific cell types bound by GFP⁺<i>Yptb</i>. Experiment was repeated 5–8 times (*P<0.05, **P<0.01 and ***P<0.001).</p

<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

<i>ΔailΔinvΔyadA</i> is reduced in virulence and for Yop translocation during animal infections.

<p>Mice were infected IV with 800 CFU of IP2666 WT-ETEM (1X-WT) or <i>ΔailΔinvΔyadA-ETEM</i> (1X-<i>ΔailΔinvΔyadA</i>), or 30,000 CFU of <i>ΔailΔinvΔyadA-ETEM</i> (37.5X<i>ΔailΔinvΔyadA</i>) or <i>ΔyopB-ETEM</i> (37.5X-<i>ΔyopB</i>). (<b>A</b>) Animals were monitored for morbidity and mortality for a period of 15 days post infection and survival was plotted. (<b>B–C</b>) Four days post-infection, spleens were isolated and single cell suspensions were generated to enumerate CFU and percentage of Blue⁺ cells from mice infected with 800 CFU of WT-ETEM and 30,000 CFU of <i>ΔailΔinvΔyadA-ETEM</i>. (<b>B</b>) Black-filled squares with a solid black line represent values from mice infected with WT-ETEM and grey-filled diamonds with a dashed grey line represent values from mice infected with <i>ΔailΔinvΔyadA-ETEM</i>. Linear regression analysis determined that the percentage of Blue⁺ cells is significantly higher in animals infected with <i>WT-ETEM</i> than with <i>ΔailΔinvΔyadA-ETEM</i> (P = 0.0001). (<b>C</b>) The percentage of each cell type in the organ (white bars) compared with the percentage of each cell type present in the Blue⁺ population (grey bars) from spleens infected with WT-ETEM or <i>ΔailΔinvΔyadA-ETEM</i>. Only mice with greater than 4.8×10⁴ CFU were analyzed (***P<0.001).</p

<i>ΔailΔinvΔyadA</i> strains are defective for Yop translocation into isolated splenocytes.

<p>Splenocytes were infected with the indicated ETEM-expressing strains (<b>A–C</b>) at an MOI of 1∶1 for (<b>A</b>) 1 h with IP2666 strains, (<b>B</b>) 45 min with IP32953 strains or (<b>C</b>) 45 min with YPIII strains, or (<b>D–E</b>) with the indicated IP2666 strains for (<b>D</b>) 4 h at an MOI of 1∶1 or (<b>E</b>) 1 h at an MOI of 20∶1. (<b>A–E</b>) CCF4 conversion from green to blue was measured by flow cytometry and the relative percentage of Blue⁺ cells was determined by setting WT to 1 and normalizing the percentage of Blue⁺ cells of the adhesin mutants to WT. Experiments were repeated 3–8 times (<b>A–E</b>: *P<0.05, **P<0.01 and ***P<0.001 compared to WT; and <b>A–C</b>: ⁺P<0.05, ⁺⁺P<0.01 and ⁺⁺⁺P<0.001 compared to <i>ΔyopB</i>).</p

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

A Single Bacterial Immune Evasion Strategy Dismantles Both MyD88 and TRIF Signaling Pathways Downstream of TLR4

During bacterial infections, Toll-like receptor 4 (TLR4) signals through the MyD88-dependent pathway to promote rapid pro-inflammatory responses, but also signals via the TRIF-dependent pathway, which promotes Type-I interferon responses and acts with MyD88 signaling to potentiate inflammatory cytokine production. Bacteria can inhibit the MyD88 pathway, but if the TRIF pathway is also targeted is unclear. We demonstrate that, in addition to MyD88, Yersinia pseudotuberculosis inhibits TRIF signaling through the Type III secretion system effector YopJ. Suppression of TRIF signaling occurs during dendritic cell (DC) and macrophage infection, and prevents expression of Type-I IFN and pro-inflammatory cytokines. YopJ-mediated inhibition of TRIF prevents DCs from inducing Natural Killer cell production of antibacterial interferon-γ. During infection of DCs, YopJ potently inhibits MAPK pathways but does not prevent activation of IKK- or TBK1-dependent pathways. This singular YopJ activity efficiently inhibits TLR4 transcription-inducing activities, thus illustrating a simple means by which pathogens impede innate immunity

Kinase Activities of RIPK1 and RIPK3 Can Direct IFN-beta Synthesis Induced by Lipopolysaccharide

The innate immune response is a central element of the initial defense against bacterial and viral pathogens. Macrophages are key innate immune cells that upon encountering pathogen-associated molecular patterns respond by producing cytokines, including IFN-beta. In this study, we identify a novel role for RIPK1 and RIPK3, a pair of homologous serine/threonine kinases previously implicated in the regulation of necroptosis and pathologic tissue injury, in directing IFN-beta production in macrophages. Using genetic and pharmacologic tools, we show that catalytic activity of RIPK1 directs IFN-beta synthesis induced by LPS in mice. Additionally, we report that RIPK1 kinase-dependent IFN-beta production may be elicited in an analogous fashion using LPS in bone marrow-derived macrophages upon inhibition of caspases. Notably, this regulation requires kinase activities of both RIPK1 and RIPK3, but not the necroptosis effector protein, MLKL. Mechanistically, we provide evidence that necrosome-like RIPK1 and RIPK3 aggregates facilitate canonical TRIF-dependent IFN-beta production downstream of the LPS receptor TLR4. Intriguingly, we also show that RIPK1 and RIPK3 kinase-dependent synthesis of IFN-beta is markedly induced by avirulent strains of Gram-negative bacteria, Yersinia and Klebsiella, and less so by their wild-type counterparts. Overall, these observations identify unexpected roles for RIPK1 and RIPK3 kinases in the production of IFN-beta during the host inflammatory responses to bacterial infection and suggest that the axis in which these kinases operate may represent a target for bacterial virulence factors