Yersinia pestis insecticidal-like toxin complex (Tc) family proteins: characterization of expression, subcellular localization, and potential role in infection of the flea vector

BACKGROUND: Toxin complex (Tc) family proteins were first identified as insecticidal toxins in Photorhabdus luminescens and have since been found in a wide range of bacteria. The genome of Yersinia pestis, the causative agent of bubonic plague, contains a locus that encodes the Tc protein homologues YitA, YitB, YitC, and YipA and YipB. Previous microarray data indicate that the Tc genes are highly upregulated by Y. pestis while in the flea vector; however, their role in the infection of fleas and pathogenesis in the mammalian host is unclear. RESULTS: We show that the Tc proteins YitA and YipA are highly produced by Y. pestis while in the flea but not during growth in brain heart infusion (BHI) broth at the same temperature. Over-production of the LysR-type regulator YitR from an exogenous plasmid increased YitA and YipA synthesis in broth culture. The increase in production of YitA and YipA correlated with the yitR copy number and was temperature-dependent. Although highly synthesized in fleas, deletion of the Tc proteins did not alter survival of Y. pestis in the flea or prevent blockage of the proventriculus. Furthermore, YipA was found to undergo post-translational processing and YipA and YitA are localized to the outer membrane of Y. pestis. YitA was also detected by immunofluorescence microscopy on the surface of Y. pestis. Both YitA and YipA are produced maximally at low temperature but persist for several hours after transfer to 37°C. CONCLUSIONS: Y. pestis Tc proteins are highly expressed in the flea but are not essential for Y. pestis to stably infect or produce a transmissible infection in the flea. However, YitA and YipA localize to the outer membrane and YitA is exposed on the surface, indicating that at least YitA is present on the surface when Y. pestis is transmitted into the mammalian host from the flea

Neutrophils Are Resistant to Yersinia YopJ/P-Induced Apoptosis and Are Protected from ROS-Mediated Cell Death by the Type III Secretion System

The human innate immune system relies on the coordinated activity of macrophages and polymorphonuclear leukocytes (neutrophils or PMNs) for defense against bacterial pathogens. Yersinia spp. subvert the innate immune response to cause disease in humans. In particular, the Yersinia outer protein YopJ (Y. pestis and Y. pseudotuberculosis) and YopP (Y. enterocolitica) rapidly induce apoptosis in murine macrophages and dendritic cells. However, the effects of Yersinia Yop J/P on neutrophil fate are not clearly defined.In this study, we utilized wild-type and mutant strains of Yersinia to test the contribution of YopJ and YopP on induction of apoptosis in human monocyte-derived macrophages (HMDM) and neutrophils. Whereas YopJ and YopP similarly induced apoptosis in HMDMs, interaction of human neutrophils with virulence plasmid-containing Yersinia did not result in PMN caspase activation, release of LDH, or loss of membrane integrity greater than PMN controls. In contrast, interaction of human PMNs with the virulence plasmid-deficient Y. pestis strain KIM6 resulted in increased surface exposure of phosphatidylserine (PS) and cell death. PMN reactive oxygen species (ROS) production was inhibited in a virulence plasmid-dependent but YopJ/YopP-independent manner. Following phagocytic interaction with Y. pestis strain KIM6, inhibition of PMN ROS production with diphenyleneiodonium chloride resulted in a reduction of PMN cell death similar to that induced by the virulence plasmid-containing strain Y. pestis KIM5.Our findings showed that Yersinia YopJ and/or YopP did not induce pronounced apoptosis in human neutrophils. Furthermore, robust PMN ROS production in response to virulence plasmid-deficient Yersinia was associated with increased PMN cell death, suggesting that Yersinia inhibition of PMN ROS production plays a role in evasion of the human innate immune response in part by limiting PMN apoptosis

Microduplications of 16p11.2 are associated with schizophrenia

Recurrent microdeletions and microduplications of a 600-kb genomic region of chromosome 16p11.2 have been implicated in childhood-onset developmental disorders1,2,3. We report the association of 16p11.2 microduplications with schizophrenia in two large cohorts. The microduplication was detected in 12/1,906 (0.63%) cases and 1/3,971 (0.03%) controls (P = 1.2 × 10−5, OR = 25.8) from the initial cohort, and in 9/2,645 (0.34%) cases and 1/2,420 (0.04%) controls (P = 0.022, OR = 8.3) of the replication cohort. The 16p11.2 microduplication was associated with a 14.5-fold increased risk of schizophrenia (95% CI (3.3, 62)) in the combined sample. A meta-analysis of datasets for multiple psychiatric disorders showed a significant association of the microduplication with schizophrenia (P = 4.8 × 10−7), bipolar disorder (P = 0.017) and autism (P = 1.9 × 10−7). In contrast, the reciprocal microdeletion was associated only with autism and developmental disorders (P = 2.3 × 10−13). Head circumference was larger in patients with the microdeletion than in patients with the microduplication (P = 0.0007)

Yersinia pestis Type III Secretion System-Dependent Inhibition of Human Polymorphonuclear Leukocyte Function▿

Human polymorphonuclear leukocytes (PMNs, or neutrophils) are the primary innate host defense against invading bacterial pathogens. Neutrophils are rapidly recruited to sites of infection and ingest microorganisms through a process known as phagocytosis. Following phagocytosis by human PMNs, microorganisms are killed by reactive oxygen species (ROS) and microbicidal products contained within granules. Yersinia pestis, the causative agent of plague, is capable of rapid replication and dissemination from sites of infection in the host. Although Y. pestis survives in macrophages, the bacterial fate following interaction with human PMNs is less clear. The ability of Y. pestis to inhibit phagocytosis by human PMNs was assessed by differential fluorescence microscopy and was shown to be dependent on expression of the type III secretion system (TTSS). Previous studies have demonstrated that TTSS expression in enteropathogenic Yersinia spp. also inhibits the respiratory burst in PMNs and macrophages, and we show here that human PMN ROS production is similarly repressed by Y. pestis. However, exclusion of uningested TTSS-expressing Y. pestis with gentamicin revealed that intracellular bacteria are eliminated by human PMNs, similar to bacteria lacking the TTSS. In summary, our results suggest that the Y. pestis TTSS contributes to extracellular survival following interactions with human PMNs and that the intracellular fate is independent of TTSS inhibition of neutrophil ROS production

Yersinia pestis Two-Component Gene Regulatory Systems Promote Survival in Human Neutrophils▿

Human polymorphonuclear leukocytes (PMNs, or neutrophils) are the most abundant innate immune cell and kill most invading bacteria through combined activities of reactive oxygen species (ROS) and antimicrobial granule constituents. Pathogens such as Yersinia pestis resist destruction by the innate immune system and are able to survive in macrophages and neutrophils. The specific molecular mechanisms used by Y. pestis to survive following phagocytosis by human PMNs are incompletely defined. To gain insight into factors that govern Y. pestis intracellular survival in neutrophils, we inactivated 25 two-component gene regulatory systems (TCSs) with known or inferred function and assessed susceptibility of these mutant strains to human PMN granule extracts. Y. pestis strains deficient for PhoPQ, KdpED, CheY, CvgSY, and CpxRA TCSs were selected for further analysis, and all five strains were altered for survival following interaction with PMNs. Of these five strains, only Y. pestis ΔphoPQ demonstrated global sensitivity to a panel of seven individual neutrophil antimicrobial peptides and serine proteases. Notably, Y. pestis ΔphoPQ was deficient for intracellular survival in PMNs. Iterative analysis with Y. pestis strains lacking the PhoP-regulated genes ugd and pmrK indicated that the mechanism most likely responsible for increased resistance to killing is 4-amino-4-deoxy-l-arabinose modification of lipid A. Together, the data provide new information about Y. pestis evasion of the innate immune system

Induction of caspase activity is YopJ/YopP dependent but delayed in human MDMs compared to J774A.1 cells.

<p>HMDMs and J774A.1 cells were incubated alone or with <i>Y. pestis</i> KIM5, KIM5 YopJ-YopP, KIM5Δ<i>yopJ</i>, KIM6 and <i>Y. enterocolitica</i> 8081v grown at 37°C. Caspase-3, -8, -9, and -2 activity was measured after 6 h of incubation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009279#s2" target="_blank"><i>Materials and Methods</i></a> and is expressed in relative fluorescence units (RFLU). Results are expressed as the mean ± SEM of three experiments. *, represents difference from HMDM and J774A.1 controls (<i>P</i><0.05).</p

Role of YopJ and YopP in human PMN cell death.

<p>PMNs were incubated alone or with <i>Y. pestis</i> KIM5, KIM5 YopJ-YopP, KIM5Δ<i>yopJ</i>, KIM6 and <i>Y. enterocolitica</i> 8081v grown at 37°C. The percentage of PMN cell death following incubation with each strain for the indicated time was determined by comparison to detergent lysed controls. Indicators of cell death measured were release of (<b>A</b>) LDH into the supernatant and (<b>B</b>) EthD-1 uptake into PMN cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009279#s2" target="_blank"><i>Materials and Methods</i></a>. Results are expressed as the mean ± SEM of three experiments. *, represents difference from PMN controls (<i>P</i><0.05).</p

Analysis of PMN phosphatidylserine (PS) externalization.

<p><i>Y. pestis</i> KIM5, KIM5 YopJ-YopP, KIM5Δ<i>yopJ</i>, KIM6 and <i>Y. enterocolitica</i> were grown at 37°C, combined with PMNs, and incubated for the times indicated. PS externalization was determined by annexin V-FITC and flow cytometry following interaction with <i>Yersinia</i> Results are expressed as the mean ± SEM of at least three experiments. *, represents difference from PMN controls (<i>P</i><0.05).</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p