Explore the vast range of titles available.

Bacterial type VI secretion systems (T6SSs) inject toxic effectors into adjacent eukaryotic and prokaryotic cells. It is generally thought that this process requires physical contact between the two cells. Here, we provide evidence of contact-independent killing by a T6SS-secreted effector. We show that the pathogen Yersinia pseudotuberculosis uses a T6SS (T6SS-3) to secrete a nuclease effector that kills other bacteria in vitro and facilitates gut colonization in mice. The effector (Tce1) is a small protein that acts as a Ca 2+ - and Mg 2+ -dependent DNase, and its toxicity is inhibited by a cognate immunity protein, Tci1. As expected, T6SS-3 mediates canonical, contact-dependent killing by directly injecting Tce1 into adjacent cells. In addition, T6SS-3 also mediates killing of neighboring cells in the absence of cell-to-cell contact, by secreting Tce1 into the extracellular milieu. Efficient contact-independent entry of Tce1 into target cells requires proteins OmpF and BtuB in the outer membrane of target cells. The discovery of a contact-independent, long-range T6SS toxin delivery provides a new perspective for understanding the physiological roles of T6SS in competition. However, the mechanisms mediating contact-independent uptake of Tce1 by target cells remain unclear. Bacteria can use type VI secretion systems (T6SSs) to inject toxic effector proteins into adjacent cells, in a contact-dependent manner. Here, the authors provide evidence of contact-independent killing by a T6SS effector that is secreted into the extracellular milieu and then taken up by other bacterial cells.

Type VI secretion systems (T6SSs) are widespread multi-component machineries that translocate effectors into either eukaryotic or prokaryotic cells, for virulence or for interbacterial competition. Herein, we report that the T6SS-4 from Yersinia pseudotuberculosis displays an unexpected function in the transportation of Zn2+ to combat diverse stresses and host immunity. Environmental insults such as oxidative stress induce the expression of T6SS-4 via OxyR, the transcriptional factor that also regulates many oxidative response genes. Zinc transportation is achieved by T6SS-4-mediated translocation of a novel Zn2+-binding protein substrate YezP (YPK_3549), which has the capacity to rescue the sensitivity to oxidative stress exhibited by T6SS-4 mutants when added to extracellular milieu. Disruption of the classic zinc transporter ZnuABC together with T6SS-4 or yezP results in mutants that almost completely lost virulence against mice, further highlighting the importance of T6SS-4 in resistance to host immunity. These results assigned an unconventional role to T6SSs, which will lay the foundation for studying novel mechanisms of metal ion uptake by bacteria and the role of this process in their resistance to host immunity and survival in harmful environments.

Significance The innate immune system provides a first line of defense against invading pathogens. The inflammasome is an innate immune complex that activates inflammatory caspases upon infection, causing cell death and IL-1 cytokine release, which initiate defense against gram-negative bacterial pathogens but also mediate septic shock. Many inflammasome studies have been performed using cells from mice, but mice and humans differ in their complement of inflammatory caspases. Instead of caspase-11, humans encode the putative orthologs caspase-4 and caspase-5. Here, we show that caspase-4 plays a conserved role in inflammasome activation in response to virulent gram-negative pathogens in primary human macrophages. Our findings provide important insight into how inflammasomes are regulated in human cells. Inflammasomes are critical for host defense against bacterial pathogens. In murine macrophages infected by gram-negative bacteria, the canonical inflammasome activates caspase-1 to mediate pyroptotic cell death and release of IL-1 family cytokines. Additionally, a noncanonical inflammasome controlled by caspase-11 induces cell death and IL-1 release. However, humans do not encode caspase-11. Instead, humans encode two putative orthologs: caspase-4 and caspase-5. Whether either ortholog functions similar to caspase-11 is poorly defined. Therefore, we sought to define the inflammatory caspases in primary human macrophages that regulate inflammasome responses to gram-negative bacteria. We find that human macrophages activate inflammasomes specifically in response to diverse gram-negative bacterial pathogens that introduce bacterial products into the host cytosol using specialized secretion systems. In primary human macrophages, IL-1β secretion requires the caspase-1 inflammasome, whereas IL-1α release and cell death are caspase-1–independent. Instead, caspase-4 mediates IL-1α release and cell death. Our findings implicate human caspase-4 as a critical regulator of noncanonical inflammasome activation that initiates defense against bacterial pathogens in primary human macrophages.

Cellular ionic concentrations are a central factor orchestrating host innate immunity, but no pathogenic mechanism that perturbs host innate immunity by directly targeting metal ions has yet been described. Here, we report a unique virulence strategy of Yersinia pseudotuberculosis (Yptb) involving modulation of the availability of Mn2+, an immunostimulatory metal ion in host cells. We showed that the Yptb type VI secretion system (T6SS) delivered a micropeptide, TssS, into host cells to enhance its virulence. The mutant strain lacking TssS (ΔtssS) showed substantially reduced virulence but induced a significantly stronger host innate immune response, indicating an antagonistic role of this effector in host antimicrobial immunity. Subsequent studies revealed that TssS is a Mn2+-chelating protein and that its Mn2+-chelating ability is essential for the disruption of host innate immunity. Moreover, we showed that Mn2+ enhances the host innate immune response to Yptb infection by activating the stimulator of interferon genes (STING)-mediated immune response. Furthermore, we demonstrated that TssS counteracted the cytoplasmic Mn2+ increase to inhibit the STING-mediated innate immune response by sequestering Mn2+. Finally, TssS-mediated STING inhibition sabotaged bacterial clearance in vivo. These results reveal a previously unrecognized bacterial immune evasion strategy involving modulation of the bioavailability of intracellular metal ions and provide a perspective on the role of the T6SS in pathogenesis.

RNA degradation is an essential process that allows bacteria to regulate gene expression and has emerged as an important mechanism for controlling virulence. However, the individual contributions of RNases in this process are mostly unknown. Here, we tested the influence of 11 potential RNases in the intestinal pathogen Yersinia pseudotuberculosis on the expression of its type III secretion system (T3SS) and associated effectors (Yops) that are encoded on the Yersinia virulence plasmid. We found that exoribonuclease PNPase and endoribonuclease RNase III inhibit T3SS and yop gene transcription by repressing the synthesis of LcrF, the master activator of Yop-T3SS. Loss of both RNases led to an increase in lcrF mRNA levels. Our work indicates that PNPase exerts its influence via YopD, which accelerates lcrF mRNA degradation. Loss of RNase III, on the other hand, results in the downregulation of the CsrB and CsrC RNAs, thereby increasing the availability of active CsrA, which has been shown previously to enhance lcrF mRNA translation and stability. This CsrA-promoted increase of lcrF mRNA translation could be supported by other factors promoting the protein translation efficiency (e.g. IF-3, RimM, RsmG) that were also found to be repressed by RNase III. Transcriptomic profiling further revealed that Ysc-T3SS-mediated Yop secretion leads to global reprogramming of the Yersinia transcriptome with a massive shift of the expression from chromosomal to virulence plasmid-encoded genes. A similar reprogramming was also observed in the RNase III-deficient mutant under non-secretion conditions. Overall, our work revealed a complex control system where RNases orchestrate the expression of the T3SS/Yop machinery on multiple levels to antagonize phagocytic uptake and elimination by innate immune cells.

Pathogenic bacteria have evolved numerous virulence mechanisms that are essential for establishing infections. The enterobacterium Yersinia uses a type III secretion system (T3SS) encoded by a 70-kilobase, low-copy, IncFII-class virulence plasmid. We report a novel virulence strategy in Y. pseudotuberculosis in which this pathogen up-regulates the plasmid copy number during infection. We found that an increased dose of plasmidencoded genes is indispensable for virulence and substantially elevates the expression and function of the T3SS. Remarkably, we observed direct, tight coupling between plasmid replication and T3SS function. This regulatory pathway provides a framework for further exploration of the environmental sensing mechanisms of pathogenic bacteria.

Dissemination of bacteria following intestinal infection can lead to severe disease, including sepsis, organ damage, and death. However, the intestinal bacterial population dynamics governing the colonization of mucosal and systemic tissues and the intestinal sites that seed systemic spread are not clear. Yersinia pseudotuberculosis is a rodent and human intestinal pathogen closely related to the plague agent and provides a natural rodent-adapted model to study systemic bacterial dissemination. Our findings define the infection dynamics of enteric Yersinia and the impact of the innate immune system on Yersinia colonization of the intestine and systemic organs.

Yersinia pestis, the causative agent of plague, remains a threat to public health worldwide. From the perspective of developing safe and effective vaccines, we present a derived version of our Y. pseudotuberculosis VTnF1 live attenuated vaccine candidate that lacks the pYV virulence plasmid coding for the Type 3 Secretion system (T3SS) and carries no antibiotic resistance cassettes (VTnF1-S). This strain, named VpYV-, fails to cause disease in immunocompromised mice when given orally, and can be considered as avirulent in such conditions. It retains a tropism for Peyer's patches and mesenteric lymph nodes, whilst rarely reaching the spleen and liver. When compared to VTnF1-S, VpYV- elicited equivalent production of IgG directed to the F1 antigen, but less IgG directed to other Yersinia antigens. A single oral dose of VpYV- induced 100 % protection against bubonic and pneumonic forms of plague. Four months after vaccination, the protection induced by VpYV- had decreased more than that induced by VTnF1-S. Furthermore, VpYV- was 30 % less protective against F1-negative Y. pestis, revealing that the T3SS components encoded by pYV are mandatory to obtain a large spectrum protection. Finally, VTnF1-S and VpYV- were compared for their ability to induce immediate immune activity against co-infecting Y. pestis, which could be a potential therapeutic strategy against early-stage infections. Like the historical Y. pestis vaccine EV76, VTnF1-S was able to induce such a protection. The process involved nutritional immunity in serum, indicating a fast activation of innate immune mechanisms. By contrast, VpYV- failed to protect mice, revealing an importance of the T3SS in this mechanism. Overall, VTnF1 and its derivative strain VpYV-, offer a choice between better vaccine performance or greater vaccine safety. They represent useful tools to prevent and treat Y. pestis infection in healthy or immunocompromised individuals.

Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6-7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation.

We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.