Invasin and beyond: regulation of Yersinia virulence by RovA

doi:10.1016/j.tim.2004.04.006

Trends in Microbiology

Volume 12, Issue 6, June 2004, Pages 296-300

https://doi.org/10.1016/j.tim.2004.04.006 Get rights and content

Abstract

RovA, a member of the MarR/SlyA family of winged-helix transcription factors, regulates expression of invasin, the major adhesion and invasion factor in Yersinia enterocolitica and Yersinia pseudotuberculosis. Disruption of rovA increases the LD₅₀ of the organism when inoculated using the oral route. However, when administered by intraperitoneal injection only a slight difference in LD₅₀ between mutant and wild-type organisms is apparent. The study of RovA and the genes it regulates provides a unique opportunity to gain insight into the initial stages of a Yersinia infection.

Section snippets

Identification of RovA: a regulator of inv expression

A regulator of inv expression was identified by a transposon mutagenesis screen of Y. enterocolitica that used an inv∷phoA reporter to monitor inv expression [27]. Mutants that exhibited decreased alkaline phosphatase activity mapped to a single gene on the Y. enterocolitica chromosome, which was named rovA for regulator of virulence A. Subsequent work using a genetic complementation strategy in E. coli also identified RovA as a regulator of inv in Y. pseudotuberculosis [28]. The rovA gene

RovA is required for inv expression

Y. enterocolitica rovA mutants demonstrate decreased levels of invasin during growth in vitro and in the Peyer's patches of infected mice. Correlating with this decreased level of invasin, the rovA mutant also exhibits a decreased ability to invade Chinese hamster ovary (CHO) cells [27]. Work in Y. pseudotuberculosis has confirmed the regulation of inv by RovA and has also demonstrated that rovA directly interacts with DNA upstream of the inv gene [28]; RovA was reported to bind at a region

Regulation of rovA expression

To date, the signals that mediate rovA regulation are unknown. Expression of rovA in Y. pseudotuberculosis follows a similar temperature-dependent regulatory pattern as inv during in vitro growth, indicating that a change in temperature might result in alteration of rovA expression [28]. Furthermore, post-transcriptional regulation of RovA occurs in a temperature-dependent manner, adding support for temperature playing a role in rovA regulation [28]. It is also possible that the involvement of

RovA is important for virulence

As described previously, Yersinia that are unable to express invasin are only mildly attenuated in the mouse model [24]. The inv mutant is delayed in initiating infection in the Peyer's patches; however, inactivation of inv does not affect the outcome of infection or alter the dose at which 50 percent of the mice succumb to infection (LD₅₀) compared with wild-type bacteria. Interestingly, although the rovA mutant maintains low-level expression of invasin, a much more attenuated phenotype was

A model for RovA regulation

Combining the current data about rovA with information from studies of other MarR/SlyA members, we can speculate about the role of RovA during infection and postulate a model to describe rovA regulation in vivo (Figure 1). Preliminary data suggest that levels of RovA in Yersinia are maintained at a low level under non-inducing conditions [28]. Upon entry into the host, however, we hypothesize that the levels of RovA increase. In vivo data demonstrate that Y. enterocolitica requires rovA for

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This is consistent with previous findings that (p)ppGpp synthases influences bacterial phenotypes by controlling the expression of many other regulators such as the cAMP receptor protein CRP, the flagellar master regulator FlhDC, and the integration host factor IHF (Aviv et al., 1994; Johansson et al., 2000; Lemke et al., 2009). The MarR-type global regulator RovA of Yersinia has been shown to control the expression of multiple metabolic, stress and virulence genes that are required for environmental adaptation and pathogenesis (Nagel et al., 2001; Ellison et al., 2004; Cathelyn et al., 2006). Expression of Yersinia RovA is directly repressed by RovM, which itself is controlled by the Csr system in response to nutrition status (Heroven and Dersch, 2006).
The type VI secretion system (T6SS) is a versatile molecular machinery widely distributed in Gram-negative bacteria. The activity of the T6SS is tightly regulated by various mechanisms, including quorum sensing (QS), iron concentration, and transcriptional regulators. Here we demonstrated that the stringent response regulator, RelA, contributes to bacterial resistance to multiple environmental stresses in Yersinia pseudotuberculosis. We also revealed that the stress resistance function of stringent response (SR) was partially mediated by the general stress response T6SS4 system. RelA positively regulates the expression of T6SS4 to combat various stresses in response to nutrition starvation collectively mediated by the RovM and RovA regulators. These findings revealed not only the important role of T6SS4 in SR induced stress resistance, but also a new pathway to regulate T6SS4 expression in response to starvation stress.
Role of β1 integrins and bacterial adhesins for Yop injection into leukocytes in Yersinia enterocolitica systemic mouse infection
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The last two C-terminal domains are involved in host cell binding (Hamburger et al., 1999). In contrast to YadA, Inv expression is regulated by RovA and is induced at a low temperature (26 °C) (Ellison et al., 2004). In cell culture experiments, Inv has been demonstrated to promote host cell adhesion and invasion (Wong and Isberg, 2005).
Injection of Yersinia outer proteins (Yops) into host cells by a type III secretion system is an important immune evasion mechanism of Yersinia enterocolitica (Ye). In this process Ye invasin (Inv) binds directly while Yersinia adhesin A (YadA) binds indirectly via extracellular matrix (ECM) proteins to β1 integrins on host cells. Although leukocytes turned out to be an important target of Yop injection by Ye, it was unclear which Ye adhesins and which leukocyte receptors are required for Yop injection. To explain this, we investigated the role of YadA, Inv and β1 integrins for Yop injection into leukocytes and their impact on the course of systemic Ye infection in mice. Ex vivo infection experiments revealed that adhesion of Ye via Inv or YadA is sufficient to promote Yop injection into leukocytes as revealed by a β-lactamase reporter assay. Serum factors inhibit YadA- but not Inv-mediated Yop injection into B and T cells, shifting YadA-mediated Yop injection in the direction of neutrophils and other myeloid cells. Systemic Ye mouse infection experiments demonstrated that YadA is essential for Ye virulence and Yop injection into leukocytes, while Inv is dispensable for virulence and plays only a transient and minor role for Yop injection in the early phase of infection. Ye infection of mice with β1 integrin-depleted leukocytes demonstrated that β1 integrins are dispensable for YadA-mediated Yop injection into leukocytes, but contribute to Inv-mediated Yop injection. Despite reduced Yop injection into leukocytes, β1 integrin-deficient mice exhibited an increased susceptibility for Ye infection, suggesting an important role of β1 integrins in immune defense against Ye. This study demonstrates that Yop injection into leukocytes by Ye is largely mediated by YadA exploiting, as yet unknown, leukocyte receptors.
Unique virulence properties of Yersinia enterocolitica O:3-An emerging zoonotic pathogen using pigs as preferred reservoir host
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This is in strong contrast to other Y. enterocolitica serotypes, in particular to serotype O:8, in which invasin synthesis is strongly repressed at 37 °C due to (i) rapid degradation of the invA transcriptional activator RovA and (ii) silencing of invA transcription by the nucleoid-associated histone-like protein H-NS (Lawrenz and Miller, 2007). We unraveled the underlying mechanism and found that all O:3 strains acquired an insertion of the mobile element IS1667 at position −143 of the invA promoter between the two adjacent RovA binding sites which are important for the transcriptional activation of invA (Ellison et al., 2004; Heroven et al., 2004; Fig. 2). This IS1667 element harbors an additional s70-dependent promoter (PIS1667) that is mainly responsible for constitutive invA expression.
Enteropathogenic Yersinia enterocolitica bioserotype 4/O:3 are the most frequent cause of human yersiniosis worldwide with symptoms ranging from mild diarrhea to severe complications of mesenteric lymphadenitis, liver abscesses and postinfectious extraintestinal sequelae. The main reservoir host of 4/O:3 strains are pigs, which represent a substantial disease-causing potential for humans, as they are usually asymptomatic carriers. Y. enterocolitica O:3 initiates infections by tight attachment to the intestinal mucosa. Colonization of the digestive tract is frequently followed by invasion of the intestinal layer primarily at the follicle-associated epithelium, allowing the bacteria to propagate in the lamina propria and disseminate into deeper tissues. Molecular characterization of Y. enterocolitica O:3 isolates led to the identification of (i) alternative virulence and fitness factors and (ii) small genetic variations which cause profound changes in their virulence gene expression pattern (e.g. constitutive expression of the primary invasion factor InvA). These changes provoke a major difference in the virulence properties, i.e. reduced colonization of intestinal tissues in mice, but improved long-term colonization in the pig intestine. Y. enterocolitica O:3 strains cause also a considerably lower level of proinflammatory cytokine IL-8 and higher levels of the anti-inflammatory cytokine IL-10 in porcine primary macrophages, as compared to murine macrophages, which could contribute to limiting inflammation, immunopathology and severity of the infection in pigs.
Gene expression profiling of Yersinia pestis with deletion of lcrG, a known negative regulator for Yop secretion of type III secretion system
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Yersinia pestis injects a set of virulent proteins into the cytosol of eukaryotic cells by a type III secretion system (T3SS). LcrG is a known negative regulator for secretion of Yersinia outer-membrane proteins (Yops) by blocking the secretion apparatus (Ysc) from the inner membrane. To further understand the effect of lcrG deletion on Y. pestis T3SS regulation, transcriptional profiles from the ΔlcrG mutant and wild-type Y. pestis strains were compared. The results showed that although the ΔlcrG mutant was markedly attenuated (600-fold increase of LD₅₀ in s.c. challenged BALB/c mice), transcriptions of almost all the type III genes were upregulated significantly in the ΔlcrG mutant. The immunoblotting analysis of YopM and LcrV demonstrated that their expressions were also increased in the ΔlcrG mutant in comparison to the wild-type strain. We speculate that, in addition to the negative regulation of the Yop secretion, LcrG could possibly play a negative regulatory role in the transcription of T3SS genes through indirect mechanisms. Furthermore, this report also revealed significant transcriptional changes in the genes encoding cell-envelope-related proteins and a virulence-related transcription factor RovA in the ΔlcrG mutant.
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Trends in Microbiology

Invasin and beyond: regulation of Yersinia virulence by RovA

Abstract

Section snippets

Identification of RovA: a regulator of inv expression

RovA is required for inv expression

Regulation of rovA expression

RovA is important for virulence

A model for RovA regulation

Clin. Lab. Med.

J. Pediatr.

FEMS Microbiol. Lett.

Int. J. Med. Microbiol.

FEMS Microbiol. Lett.

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Yersinia pestis and the plague

Am. J. Clin. Pathol.

Yersinia enterocolitica: the charisma continues

Clin. Microbiol. Rev.

Yersinia enterocolitica septicemia

Am. J. Clin. Pathol.

Yersinia enterocolitica infection in resistant and susceptible strains of mice

Infect. Immun.

The Yersinia Ysc-Yop virulence apparatus

Int. J. Med. Microbiol.

Invasin production by Yersinia pestis is abolished by insertion of an IS200-like element within the inv gene

Infect. Immun.

Evidence for two genetic loci from Yersinia enterocolitica that can promote invasion of epithelial cells

Infect. Immun.

Role of the Yersinia outer membrane protein YadA in adhesion to rabbit intestinal tissue and rabbit intestinal brush border membrane vesicles

APMIS

Increased virulence of Yersinia pseudotuberculosis by two independent mutations

Nature

YadA mediates specific binding of enteropathogenic Yersinia enterocolitica to human intestinal submucosa

Infect. Immun.

Pathogenesis of defined invasion mutants of Yersinia enterocolitica in a BALB/c mouse model of infection

Infect. Immun.

The Yersinia pseudotuberculosis adhesin YadA mediates intimate bacterial attachment to and entry into Hep-2 cells

Infect. Immun.

Thermoregulation-dependent expression of Yersinia enterocolitica protein 1 imparts serum resistance to Escherichia coli K-12

J. Bacteriol.