Abstract
BackgroundIn the enteropathogenic Yersinia species, RovA regulates the expression of invasin, which is important for enteropathogenic pathogenesis but is inactivated in Yersinia pestis. Investigation of the RovA regulon in Y. pestis at 26°C has revealed that RovA is a global regulator that contributes to virulence in part by the direct regulation of psaEFABC. However, the regulatory roles of RovA in Y. pestis at 37°C, which allows most virulence factors in mammalian hosts to be expressed, are still poorly understood.Methodology/Principal FindingsThe transcriptional profile of an in-frame rovA mutant of Y. pestis biovar Microtus strain 201 was analyzed under type III secretion system (T3SS) induction conditions using microarray techniques, and it was revealed that many cell-envelope and transport/binding proteins were differentially expressed in the ΔrovA mutant. Most noticeably, many of the T3SS genes, including operons encoding the translocon, needle and Yop (Yersinia outer protein) effectors, were significantly up-regulated. Analysis of Yop proteins confirmed that YopE and YopJ were also expressed in greater amounts in the mutant. However, electrophoresis mobility shift assay results demonstrated that the His-RovA protein could not bind to the promoter sequences of the T3SS genes, suggesting that an indirect regulatory mechanism is involved. Transmission electron microscopy analysis indicated that there are small loose electron dense particle-like structures that surround the outer membrane of the mutant cells. The bacterial membrane permeability to CFSE (carboxyfluorescein diacetate succinimidyl ester) was significantly decreased in the ΔrovA mutant compared to the wild-type strain. Taken together, these results revealed the improper construction and dysfunction of the membrane in the ΔrovA mutant.Conclusions/SignificanceWe demonstrated that the RovA regulator plays critical roles in the construction and functioning of the bacterial membrane, which sheds considerable light on the regulatory functions of RovA in antibiotic resistance and environmental adaptation. The expression of T3SS was upregulated in the ΔrovA mutant through an indirect regulatory mechanism, which is possibly related to the altered membrane construction in the mutant.
Highlights
There are three Yersinia species that are pathogenic to humans
To address the role of RovA in pathogenesis of Y. pestis, Cathelyn et al defined the RovA regulon in Y. pestis strain CO92, and they demonstrated that RovA is a global regulator that is indispensable for dissemination and colonization of the spleen and lungs in mice infected by the s.c. route and that it can directly bind to the promoters of psaA and psaE to contribute to the virulence of Y. pestis [6]
Bubbles could be observed at the surface of some rovA mutant cells (Figure 7 G and F), and some cells were totally lysed and surrounded by remaining electron-dense materials (Figure 7 J). These results indicated that cell membrane construction and cell integrity in Y. pestis showed significant dependence on RovA whether the bacteria were grown under T3SS-inducing or non-inducing conditions, which suggests that RovA could play a critical regulatory role in the construction of the cell membrane and is necessary for the maintenance of the stability of the cell membrane
Summary
There are three Yersinia species that are pathogenic to humans. RovA is a member of the MarR/SlyA family of global regulators, and proteins of this family are structurally conserved and are considered to be ubiquitous among bacteria [4]. Members of the MarR/SlyA family of proteins regulate a wide variety of functions including antibiotic resistance, environmental adaptation, production of antimicrobial agents and virulence factors. In human pathogenic Yersinia species, RovA has been shown to coordinate multiple metabolic, stress and virulence genes in response to environmental signals in the infected host [5]. Yersinia RovA was first identified as a regulator of invasion factor expression in a transposon mutagenesis screen of Y. enterocolitica using an inv::phoA reporter to monitor inv expression [5]. Inv, an important adhesion and invasion factor for the virulence of enteropathogenic Yersinia species, is inactive in Y. pestis. The regulatory roles of RovA in Y. pestis at 37uC, which allows most virulence factors in mammalian hosts to be expressed, are still poorly understood
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