Acquisition of omptin reveals cryptic virulence function of autotransporter YapE in Yersinia pestis.

Autotransporters, the largest family of secreted proteins in Gram-negative bacteria, perform a variety of functions, including adherence, cytotoxicity and immune evasion. In Yersinia pestis the autotransporter YapE has adhesive properties and contributes to disease in the mouse model of bubonic plague. Here, we demonstrate that omptin cleavage of Y. pestis YapE is required to mediate bacterial aggregation and adherence to eukaryotic cells. We demonstrate that omptin cleavage is specific for the Y. pestis and Y. pseudotuberculosis YapE orthologues but is not conserved in the Yersinia enterocolitica protein. We also show that cleavage of YapE occurs in Y. pestis but not in the enteric Yersinia species, and requires the omptin Pla (plasminogen activator protease), which is encoded on the Y. pestis-specific plasmid pPCP1. Together, these data show that post-translation modification of YapE appears to be specific to Y. pestis, was acquired along with the acquisition of pPCP1 during the divergence of Y. pestis from Y. pseudotuberculosis, and are the first evidence of a novel mechanism to regulate bacterial adherence.

The dependence of the Yersinia pestis capsule on pathogenesis is influenced by the mouse background.

Yersinia pestis is a highly pathogenic Gram-negative organism and the causative agent of bubonic and pneumonic plague. Y. pestis is capable of causing major epidemics; thus, there is a need for vaccine targets and a greater understanding of the role of these targets in pathogenesis. Two prime Y. pestis vaccine candidates are the usher-chaperone fimbriae Psa and Caf. Herein we report that Y. pestis requires, in a nonredundant manner, both PsaA and Caf1 to achieve its full pathogenic ability in both pneumonic and bubonic plague in C57BL/6J mice. Deletion of psaA leads to a decrease in the organ bacterial burden and to a significant increase in the 50% lethal dose (LD50) after subcutaneous infection. Deletion of caf1 also leads to a significant decrease in the organ bacterial burden but more importantly leads to a significantly greater increase in the LD50 than was observed for the ΔpsaA mutant strain after subcutaneous infection of C57BL/6J mice. Furthermore, the degree of attenuation of the Δcaf1 mutant strain is mouse background dependent, as the Δcaf1 mutant strain was attenuated to a lesser degree in BALB/cJ mice by the subcutaneous route than in C57BL/6J mice. This observation that the degree of requirement for Caf1 is dependent on the mouse background indicates that the virulence of Y. pestis is dependent on the genetic makeup of its host and provides further support for the hypothesis that PsaA and Caf1 have different targets.

Mucosal lipocalin 2 has pro-inflammatory and iron-sequestering effects in response to bacterial enterobactin.

Nasal colonization by both gram-positive and gram-negative pathogens induces expression of the innate immune protein lipocalin 2 (Lcn2). Lcn2 binds and sequesters the iron-scavenging siderophore enterobactin (Ent), preventing bacterial iron acquisition. In addition, Lcn2 bound to Ent induces release of IL-8 from cultured respiratory cells. As a countermeasure, pathogens of the Enterobacteriaceae family such as Klebsiella pneumoniae produce additional siderophores such as yersiniabactin (Ybt) and contain the iroA locus encoding an Ent glycosylase that prevents Lcn2 binding. Whereas the ability of Lcn2 to sequester iron is well described, the ability of Lcn2 to induce inflammation during infection is unknown. To study each potential effect of Lcn2 on colonization, we exploited K. pneumoniae mutants that are predicted to be susceptible to Lcn2-mediated iron sequestration (iroA ybtS mutant) or inflammation (iroA mutant), or to not interact with Lcn2 (entB mutant). During murine nasal colonization, the iroA ybtS double mutant was inhibited in an Lcn2-dependent manner, indicating that the iroA locus protects against Lcn2-mediated growth inhibition. Since the iroA single mutant was not inhibited, production of Ybt circumvents the iron sequestration effect of Lcn2 binding to Ent. However, colonization with the iroA mutant induced an increased influx of neutrophils compared to the entB mutant. This enhanced neutrophil response to Ent-producing K. pneumoniae was Lcn2-dependent. These findings suggest that Lcn2 has both pro-inflammatory and iron-sequestering effects along the respiratory mucosa in response to bacterial Ent. Therefore, Lcn2 may represent a novel mechanism of sensing microbial metabolism to modulate the host response appropriately.

Herpesvirus latency confers symbiotic protection from bacterial infection.

All humans become infected with multiple herpesviruses during childhood. After clearance of acute infection, herpesviruses enter a dormant state known as latency. Latency persists for the life of the host and is presumed to be parasitic, as it leaves the individual at risk for subsequent viral reactivation and disease. Here we show that herpesvirus latency also confers a surprising benefit to the host. Mice latently infected with either murine gammaherpesvirus 68 or murine cytomegalovirus, which are genetically highly similar to the human pathogens Epstein-Barr virus and human cytomegalovirus, respectively, are resistant to infection with the bacterial pathogens Listeria monocytogenes and Yersinia pestis. Latency-induced protection is not antigen specific but involves prolonged production of the antiviral cytokine interferon-gamma and systemic activation of macrophages. Latency thereby upregulates the basal activation state of innate immunity against subsequent infections. We speculate that herpesvirus latency may also sculpt the immune response to self and environmental antigens through establishment of a polarized cytokine environment. Thus, whereas the immune evasion capabilities and lifelong persistence of herpesviruses are commonly viewed as solely pathogenic, our data suggest that latency is a symbiotic relationship with immune benefits for the host.

RovA, a global regulator of Yersinia pestis, specifically required for bubonic plague.

The pathogenic species of Yersinia contain the transcriptional regulator RovA. In Yersinia pseudotuberculosis and Yersinia enterocolitica, RovA regulates expression of the invasion factor invasin (inv), which mediates translocation across the intestinal epithelium. A Y. enterocolitica rovA mutant has a significant decrease in virulence by LD(50) analysis and an altered rate of dissemination compared with either wild type or an inv mutant, suggesting that RovA regulates multiple virulence factors. Here, we show the involvement of RovA in the virulence of Yersinia pestis, which naturally lacks a functional inv gene. A Y. pestis DeltarovA mutant is attenuated approximately 80-fold by LD(50) and is defective in dissemination/colonization of spleens and lungs after s.c. inoculation. However, the DeltarovA mutant is only slightly attenuated when given via an intranasal or i.p. route, indicating a more important role for RovA in bubonic plague than pneumonic plague or systemic infection. Microarray analysis was used to define the RovA regulon. The psa locus was among the most highly down-regulated loci in the DeltarovA mutant. A DeltapsaA mutant had a significant dissemination defect after s.c. infection but only slight attenuation by the pneumonic-disease model, closely mimicking the virulence defect seen with the DeltarovA mutant. DNA-binding studies revealed that RovA specifically interacts with the psaE and psaA promoter regions, indicating a direct role for RovA in regulating this locus. Thus, RovA appears to be a global transcription factor in Y. pestis and, through its regulatory influence on genes such as psaEFABC, contributes to the virulence of Y. pestis.

Protective role of interleukin-6 during Yersinia enterocolitica infection is mediated through the modulation of inflammatory cytokines.

Yersinia enterocolitica is a gram-negative enteric pathogen responsible for a number of gastrointestinal disorders. A striking feature of the pathology of a Y. enterocolitica infection is inflammation. Recently, we demonstrated a role for interleukin-1alpha (IL-1alpha) in the establishment of intestinal inflammation in response to a Y. enterocolitica infection. A cytokine directly affected by IL-1 levels is IL-6. A previous report suggested that IL-6 plays an anti-inflammatory role during Y. enterocolitica infection, and in other systems IL-6 has been shown to be proinflammatory. Therefore, a closer examination of the roles of IL-6 and inflammatory cytokines in the control of Y. enterocolitica infection in IL-6(-/-) mice was undertaken. Y. enterocolitica organisms were more virulent in the IL-6(-/-) mice (60-fold decreased 50% lethal dose) and colonized systemic tissues more rapidly and to a higher level than in the wild-type mice. One role of IL-6 during a Y. enterocolitica infection may be the downmodulation of the inflammatory response. The IL-6(-/-) mice have a more robust T(H)1 T-cell response, as well as hyperinflammatory pathologies. These phenotypes appear to be due to the misregulation of tumor necrosis factor alpha, monocyte chemotactic protein 1, IL-10, transforming growth factor beta1, and gamma interferon in the IL-6(-/-) mouse. These data provide further insight into the intricate cytokine signaling pathways involved in the regulation of inflammatory responses and the control of bacterial infections.