Matthew B. Lawrenz

Y ersinia pestis is a vector-borne pathogen that evolved from the enteric pathogen Yersinia pseudotuberculosis . Despite different routes of infection, both pathogens need to overcome host-mediated zinc limitation. Y. pestis relies on the transporter ZnuABC and the metallophore yersiniabactin, but surprisingly, disruption of these systems in Y. pseudotuberculosis did not limit growth during zinc limitation, suggesting the presence of additional mechanisms. Here, we identify the opine-type metallophore yersinopine, encoded by the cnt operon, as a primary zinc uptake system in Y. pseudotuberculosis . While a znuA ybtX mutant was not attenuated for growth in metal-limited medium, cnt znuA and cnt ybtX mutants both had significant growth defects. Moreover, a znuA ybtX cnt mutant was completely attenuated for growth, which was rescued by zinc but not iron supplementation. While the cnt operon is present in Y. pestis , growth of analogous cnt mutants was not altered. Comparison of the cnt operons indicated the presence of two frameshift mutations in the predicted yersinopine importer cntQ in Y. pestis , suggesting that it is a pseudogene. Indeed, introduction of the same mutations into cntQ in Y. pseudotuberculosis znuA ybtX phenocopied a znuA ybtX cnt mutant. Finally, yersinopine also contributed to Y. pseudotuberculosis fitness in the presence of calprotectin and during infection in the murine model of yersiniosis. These data establish yersinopine as a primary zinc acquisition system in Y. pseudotuberculosis that is not required for Y. pestis to overcome zinc-mediated nutritional immunity and show divergent evolution in zinc acquisition strategies in these closely related species. IMPORTANCE: Zinc sequestration by the host is a major component of nutritional immunity that restricts bacterial growth during infection. Here, we identify the opine metallophore yersinopine as a primary zinc acquisition system in Yersinia pseudotuberculosis . Yersinopine promotes fitness during calprotectin-mediated zinc starvation and contributes to virulence during infection. In contrast, yersinopine does not support zinc uptake in Yersinia pestis due to inactivating mutations in the protein required for uptake of zinc-bound yersinopine. These findings establish yersinopine-dependent zinc acquisition as a critical virulence factor in Y. pseudotuberculosis and reveal a previously unrecognized evolutionary divergence in metal acquisition strategies among closely related Yersinia species.

The aryl hydrocarbon receptor (AHR) plays a central role in orchestrating gut barrier and mucosal immune functions in the pathogenesis of inflammatory bowel disease (IBD). Nevertheless, activation of the AHR by diverse ligands yields varied outcomes, and the downstream pathways responsible for these effects remain unknown. Here, we report that selective activation of AHR in mouse intestinal epithelial cells (IEC) by the microbial metabolite, urolithin A (UroA), triggers the Nod-like receptor pyrin domain-containing protein 6 (NLRP6) inflammasome, resulting in the release of interleukin (IL)−18 but not IL-1β. Further, we show that UroA-induced IL-18 in IECs is critical for IL-22, mucin 2 and REG3γ production, as well as protection against colitis. Moreover, UroA significantly upregulates IL-18 and IL-22 levels in IECs and type-3 innate lymphoid cells, respectively, in intestinal biopsies from patients with IBD patients. These results demonstrate that activation of AHR by UroA modulates intestinal barrier function through an NLRP6-IL-18-IL-22 pathway in both healthy and IBD conditions.

Yersinia pestis is the etiologic agent of the plague. A hallmark of plague is subversion of the host immune response by disrupting host signalling pathways required for inflammation. This non‐inflammatory environment permits bacterial colonization and has been shown to be essential for disease manifestation. Previous work has shown that Y. pestis inhibits phagocytosis and degranulation by neutrophils. Manipulation of these key vesicular trafficking pathways suggests that Y. pestis influences extracellular vesicle (EV) secretion, cargo selection, trafficking and/or maturation. Our goals were to define the EV population produced by neutrophils in response to Y. pestis and determine how these vesicles might influence inflammation. Towards these goals, EVs were isolated from human neutrophils infected with Y. pestis or a mutant lacking bacterial effector proteins known to manipulate host cell signalling. Mass spectrometry data revealed that cargoes packaged in EVs isolated from mutant infected cells were enriched with antimicrobial and cytotoxic proteins, contents which differed from uninfected and Y. pestis infected cells. Further, EVs produced in response to Y. pestis lacked inflammatory properties observed in those isolated from neutrophils responding to the mutant. Together, these data demonstrate that Y. pestis actively inhibits the production of antimicrobial EVs produced by neutrophils, likely contributing to immune evasion.

Yersinia pestis is the etiological agent of human plague. However, certain evolutionarily divergent subspecies have different host specificities and virulence capacity compared to the more commonly studied strains with pandemic potential. This resource examines 10 diverse isolates representing some of the most understudied subspecies commonly referred to as Yersinia pestis Pestoides.

Yersinia pestis is the etiologic agent of the plague. A hallmark of plague is subversion of the host immune response by disrupting host signaling pathways required for inflammation. This non-inflammatory environment permits bacterial colonization and has been shown to be essential for disease manifestation. Previous work has shown that Y. pestis inhibits phagocytosis and degranulation by neutrophils. Manipulation of these key vesicular trafficking pathways suggests that Y. pestis influences EV secretion, cargo selection, trafficking, and/or maturation. Our goal was to define the EV population produced by neutrophils in response to Y. pestis and determine how these vesicles might influence inflammation. Toward these goals, EVs were isolated from human neutrophils infected with Y. pestis or a mutant lacking bacterial effector proteins known to manipulate host cell signaling. Mass spectrometry data revealed that cargoes packaged in EVs isolated from mutant infected cells were enriched with antimicrobials and cytotoxic proteins, contents which differed from uninfected and Y. pestis infected cells. Further, EVs produced in response to Y. pestis lacked inflammatory properties observed in those isolated from neutrophils responding to the mutant. Together, these data demonstrate that Y. pestis actively inhibits the production of antimicrobial EVs produced by neutrophils, likely contributing to immune evasion.

Leukotriene B4 (LTB₄) is an inflammatory lipid produced in response to pathogens that is critical for initiating the inflammatory cascade needed to control infection. However, during plague, Yersinia pestis inhibits the timely synthesis of LTB₄ and subsequent inflammation. Using bacterial mutants, we previously determined that Y. pestis inhibits LTB₄ synthesis via the action of the Yop effector proteins that are directly secreted into host cells through a type 3 secretion system (T3SS). Here, we show that the T3SS is the primary pathogen associated molecular pattern (PAMP) required for production of LTB₄ in response to both Yersinia and Salmonella. However, we also unexpectantly discovered that T3SS-mediated LTB₄ synthesis by neutrophils and macrophages require the activation of two distinctly different host signaling pathways. We identified that phagocytosis and the NLRP3/CASP1 inflammasome significantly impact LTB₄ synthesis by macrophages but not neutrophils. Instead, the SKAP2/PLC signaling pathway is required for T3SS-mediated LTB₄ production by neutrophils. Finally, while recognition of the T3SS is required for LTB₄ production, we also discovered that a second unrelated PAMP-mediated signal activates the MAP kinase pathway needed for synthesis. Together, these data demonstrate significant differences in the host factors and signaling pathways required by macrophages and neutrophils to quickly produce LTB₄ in response to bacteria. Moreover, while macrophages and neutrophils might rely on different signaling pathways for T3SS-dependent LTB₄ synthesis, Y. pestis has evolved virulence mechanisms to counteract this response by either leukocyte to inhibit LTB₄ synthesis and colonize the host.

Leukotriene B4 (LTB ₄ ) is critical for initiating the inflammatory cascade in response to infection. However, Yersinia pestis colonizes the host by inhibiting the timely synthesis of LTB ₄ and inflammation. Here, we show that the bacterial type 3 secretion system (T3SS) is the primary pathogen associated molecular pattern (PAMP) responsible for LTB ₄ production by leukocytes in response to Yersinia and Salmonella , but synthesis is inhibited by the Yop effectors during Yersinia interactions. Moreover, we unexpectedly discovered that T3SS-mediated LTB ₄ synthesis by neutrophils and macrophages require two distinct host signaling pathways. We show that the SKAP2/PLC signaling pathway is essential for LTB ₄ production by neutrophils but not macrophages. Instead, phagocytosis and the NLRP3/CASP1 inflammasome are needed for LTB ₄ synthesis by macrophages. Finally, while recognition of the T3SS is required for LTB ₄ production, we also discovered a second unrelated PAMP-mediated signal independently activates the MAP kinase pathway needed for LTB ₄ synthesis. Together, these data demonstrate significant differences in the signaling pathways required by macrophages and neutrophils to quickly respond to bacterial infections. Significance: The production of inflammatory lipid mediators by the host is essential for timely inflammation in response to invasion by bacterial pathogens. Therefore, defining how immune cells recognize pathogens and rapidly produce these lipids is essential for us to understand how our immune system effectively controls infection. In this study, we discovered that the host signaling pathways required for leukotriene B4 (LTB ₄ ) synthesis differ between neutrophils and macrophages, highlighting important differences in how immune cells respond to infection. Together, these data represent a significant improvement in our understanding of how neutrophils and macrophages rapidly react to bacteria and provide new insights into how Yersinia pestis manipulates leukocytes to evade immune recognition to cause disease.

Introduction: Yersinia pestis is the gram-negative, facultative intracellular bacterium that causes the disease known as plague. Due to the risk for aerosol transmission, a low infectious dose, and the acute and lethal nature of pneumonic plague, research activities with Y. pestis require Biosafety Level 3 (BSL-3) facilities to provide the appropriate safeguards to minimize accidental exposures and environmental release. However, many experimental assays cannot be performed in BSL-3 due to equipment availability, and thus require removal of samples from the BSL-3 laboratory to be completed. Objectives: To remove samples from BSL-3 containment and safely handle them at lower containment requires effective inactivation of any viable organisms from the samples prior to removal. While commonly used inactivation methods have been published for various select agents, there is an absence in the literature of a single source providing detailed examples for inactivation methods for Y. pestis . Our objective here is to provide examples of dose-dependent kill curves for commonly used inactivation approaches against Y. pestis. Methods: Time- and dose-dependent kill curves using heat, methanol, and formaldehyde inactivation methods, and common nucleic acid extraction procedures. Results/Conclusions: We show data demonstrating the complete inactivation of Y. pestis using these methods. While not all-inclusive, this study provides data and examples that can be used by other researchers to develop their own in-house validated inactivation protocols for Y. pestis .