The Yersinia YopE and YopH type III effector proteins enhance bacterial proliferation following contact with eukaryotic cells

BACKGROUND: Several bacterial pathogens express antihost factors that likely decrease both their maximal growth rate (due to metabolic costs) as well as their mortality rate (by neutralizing host defenses). The pathogenic yersiniae make a huge metabolic investment expressing virulence proteins (referred to as Yops) that are directly injected into eukaryotic cells and that modulate host defense responses such as phagocytosis and stress-activated signaling pathways. Although host-cell contact enhanced Yop expression as well as the cellular activities of several Yops have recently been described, a clear link between these phenomena and bacterial survival and/or proliferation remains to be established RESULTS: We show that the proliferation of Y. pseudotuberculosis is compromised when the bacterium is growing in association with eukaryotic cells compared to free-living bacteria. One factor likely limiting Yersinia proliferation is the metabolically taxing expression of yopE which we show using flow cytometry increases in individual bacteria following their contact with cultured macrophage-like cells. An additional factor limiting Y. pseudotuberculosis proliferation are host cell defense systems which can be significantly ameliorated by disrupting the host cell cytoskeletal system by either exogenously added toxins or by the bacterial-mediated injection of YopE or YopH. CONCLUSIONS: Our results demonstrate that despite their metabolic costs the Yop virulence proteins play an important role in enabling Y. pseudotuberculosis to survive and proliferate when confronted with the antimicrobial activities of the eukaryotic cell

Chromosomally-Encoded Yersinia pestis Type III Secretion Effector Proteins Promote Infection in Cells and in Mice

Yersinia pestis, the causative agent of plague, possesses a number of virulence mechanisms that allows it to survive and proliferate during its interaction with the host. To discover additional infection-specific Y. pestis factors, a transposon site hybridization (TraSH)-based genome-wide screen was employed to identify genomic regions required for its survival during cellular infection. In addition to several well-characterized infection-specific genes, this screen identified three chromosomal genes (y3397, y3399, and y3400), located in an apparent operon, that promoted successful infection. Each of these genes is predicted to encode a leucine-rich repeat family protein with or without an associated ubiquitin E3 ligase domain. These genes were designated Yersinia leucine-rich repeat gene A (ylrA), B (ylrB), and C (ylrC). Engineered strains with deletions of y3397 (ylrC), y3399 (ylrB), or y3400 (ylrA), exhibited infection defects both in cultured cells and in the mouse. C-terminal FLAG-tagged YlrA, YlrB, and YlrC were secreted by Y. pestis in the absence but not the presence of extracellular calcium and deletions of the DNA sequences encoding the predicted N-terminal type III secretion signals of YlrA, YlrB, and YlrC prevented their secretion, indicating that these proteins are substrates of the type III secretion system (T3SS). Further strengthening the connection with the T3SS, YlrB was readily translocated into HeLa cells and expression of the YlrA and YlrC proteins in yeast inhibited yeast growth, indicating that these proteins may function as anti-host T3S effector proteins

Murine Neonates Are Highly Resistant to Yersinia enterocolitica following Orogastric Exposure

Neonates are considered highly susceptible to gastrointestinal infections. This susceptibility has been attributed partially to immaturity in immune cell function. To study this phenomenon, we have developed a model system with murine neonates, using the natural orogastric route of transmission for the enteropathogen Yersinia enterocolitica. The susceptibilities of 7-day-old and adult mice to orogastric Y. enterocolitica infection were assessed in 50% lethal dose experiments. Remarkably, neonatal mice of either the BALB/c or C57BL/6 mouse strain showed markedly enhanced survival after infection compared to adult mice. The resistance of neonates was not due to failure of the bacteria to colonize neonatal tissues; Y. enterocolitica was readily detectable in the intestine and mesenteric lymph nodes (MLN) for at least 1 week after infection. In adult mice, Y. enterocolitica rapidly disseminated to the spleen and liver. In striking contrast, bacterial invasion of the spleen and liver in neonates was limited. Using flow cytometry and histology, we found substantial increases in the percentages of neutrophils and macrophages in the neonatal MLN, while influx of these cells into the adult MLN was limited. Similar results were obtained using two different high-virulence Y. enterocolitica strains. Importantly, depletion of neutrophils with a specific antibody led to increased translocation of the bacteria to the spleens and livers of neonates. Together, these experiments support the hypothesis that the neonatal intestinal immune system can rapidly mobilize innate phagocytes and thereby confine the bacterial infection to the gut, resulting in a high level of resistance

Polynucleotide Phosphorylase and the T3SS

Low temperatures as well as encounters with host phagocytes are two stresses that have been relatively well studied in many species of bacteria. The exoribonuclease polynucleotide phosphorylase (PNPase) has previously been shown to be required by several species of bacteria, including Yersinia, for low-temperature growth. We have shown that PNPase also enhances the ability of Yersinia to withstand the killing activities of murine macrophages. We have gone on to show that PNPase is required for the optimal functioning of Yersinia’s type three secretion system (T3SS), an organelle that injects effector proteins directly into host cells. Surprisingly, the PNPase-mediated effect on T3SS activity is independent of PNPase’s ribonuclease activity and instead requires only its S1 RNA-binding domain. In stark contrast, the catalytic activity of PNPase is strictly required for enhanced growth at low temperature. Preliminary experiments suggest that the RNA-binding interface of the S1 domain is critical for its T3SS-enhancing activity. Our findings indicate that PNPase plays versatile roles in promoting Yersinia’s survival in response to stressful conditions

RNase E Regulates the Yersinia Type 3 Secretion System▿

Yersinia spp. use a type 3 secretion system (T3SS) to directly inject six proteins into macrophages, and any impairment of this process results in a profound reduction in virulence. We previously showed that the exoribonuclease polynucleotide phosphorylase (PNPase) was required for optimal T3SS functioning in Yersinia pseudotuberculosis and Yersinia pestis. Here we report that Y. pseudotuberculosis cells with reduced RNase E activity are likewise impaired in T3SS functioning and that phenotypically they resemble Δpnp cells. RNase E does not affect expression levels of the T3SS substrates but instead, like PNPase, regulates a terminal event in the secretion pathway. This similarity, together with the fact that RNase E and PNPase can be readily copurified from Y. pseudotuberculosis cell extracts, suggests that these two RNases regulate T3SS activity through a common mechanism. This is the first report that RNase E activity impacts the T3SS as well as playing a more general role in infectivity

Induction of the Yersinia Type 3 Secretion System as an All-or-None Phenomenon

Pathogenic Yersinia spp. possess a protein secretion system, designated as type 3, that plays a clear role in promoting their survival vis-à-vis the macrophage. Inductive expression of the Yersinia type 3 secretion system (T3SS), triggered either by host cell contact, or, in the absence of host cells, by a reduction in extracellular calcium ion levels, is accompanied by a withdrawal from the bacterial division cycle. Here, we analyzed Ca 2+-dependent induction of the T3SS at the single-cell level to understand how Yersinia coordinates pro-survival and growth-related activities. We utilized a novel high-throughput quantitative microscopy approach as well as flow cytometry to determine how Ca 2+ levels, T3SS expression, and cellular division are interrelated. Our analysis showed that there is a high degree of homogeneity in terms of T3SS expression levels among a population of Y. pseudotuberculosis cells following the removal of Ca 2+, and that T3SS expression appears to be independent of the cellular division cycle. Unexpectedly, our analysis showed that Ca 2+ levels are inversely related to the initiation of inductive T3SS expression, and not to the intensity of activation once initiated, thus providing a basis for the seemingly graded response of T3SS activation observed in bulk-level analyses. The properties of the system described here display both similarities to and differences from that of the lac operon first described 50 years ago by Novick and Weiner

Use of Polymerase Chain Reaction To Detect the Take-All Fungus, Gaeumannomyces graminis, in Infected Wheat Plants

Gaeumannomyces graminis, the causative agent of take-all disease of wheat, barley, and oats, was detected in infected wheat seedlings by using the polymerase chain reaction to amplify Gaeumannomyces-specific DNA fragments. Nested primers and two rounds of amplification were used to amplify two fragments, approximately 287 and 188 bp in size, from G. graminis-infected wheat seedlings. The use of nested primers greatly decreased the number of nonspecific amplification products. Polymerase chain reaction products were not obtained with DNA from seedlings infected with several other phytopathogenic fungi or with DNA from uninfected seedlings. Amplified products were visualized on agarose gels, and their identities were confirmed by DNA hybridization. This method did not require culturing the fungus and has potential for detecting G. graminis in infested wheat, barley, or oat fields

Yersinia enterocolitica

Mucosal immunity to gastrointestinal pathogens in early life has been studied only slightly. Recently, we developed an infection model in murine neonates using the gastroenteric pathogen Yersinia enterocolitica. Here, we report that oral infection of neonatal mice with low doses of virulent Y. enterocolitica leads to vigorous intestinal and systemic adaptive immunity. Y. enterocolitica infection promoted the development of anti-LcrV memory serum IgG1 and IgG2a responses of comparable affinity and magnitude to adult responses. Strikingly, neonatal mesenteric lymph node CD4(+) T cells produced Yersinia-specific gamma interferon (IFN-γ) and interleukin-17A (IL-17A), exceeding adult levels. The robust T- and B-cell responses elicited in neonates exposed to Y. enterocolitica were associated with long-term protection against mucosal challenge with this pathogen. Using genetically deficient mice, we found that IFN-γ and CD4(+) cells, but not B cells, are critical for protection of neonates during primary Y. enterocolitica infection. In contrast, adults infected with low bacterial doses did not require either cell population for protection. CD4-deficient neonatal mice adoptively transferred with CD4(+) cells from wild-type, IFN-γ-deficient, or IL-17AF-deficient mice were equally protected from infection. These data demonstrate that inflammatory CD4(+) T cells are required for protection of neonatal mice and that this protection may not require CD4-derived IFN-γ, IL-17A, or IL-17F. Overall, these studies support the idea that Y. enterocolitica promotes the development of highly inflammatory mucosal responses in neonates and that intestinal T-cell function may be a key immune component in protection from gastrointestinal pathogens in early life