Cell Membrane Is Impaired, Accompanied by Enhanced Type III Secretion System Expression in Yersinia pestis Deficient in RovA Regulator

BACKGROUND: In 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 FINDINGS: The 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/SIGNIFICANCE: We 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

The Yersinia pestis type III secretion system: expression, assembly and role in the evasion of host defenses

Yersinia pestis, the etiologic agent of plague, utilizes a type III secretion system (T3SS) to subvert the defenses of its mammalian hosts. T3SSs are complex nanomachines that allow bacterial pathogens to directly inject effector proteins into eukaryotic cells. The Y. pestis T3SS is not expressed during transit through the flea vector, but T3SS gene expression is rapidly thermoinduced upon entry into a mammalian host. Assembly of the T3S apparatus is a highly coordinated process that requires the homo- and hetero-oligomerization over 20 Yersinia secretion (Ysc) proteins, several assembly intermediates and the T3S process to complete the assembly of the rod and external needle structures. The activation of effector secretion is controlled by the YopN/TyeA/SycN/YscB complex, YscF and LcrG in response to extracellular calcium and/or contact with a eukaryotic cell. Cell contact triggers the T3S process including the secretion and assembly of a pore-forming translocon complex that facilitates the translocation of effector proteins, termed Yersinia outer proteins (Yops), across the eukaryotic membrane. Within the host cell, the Yop effector proteins function to inhibit bacterial phagocytosis and to suppress the production of pro-inflammatory cytokines

The Yersinia Ysc-Yop 'type III' weaponry

'Type III secretion'--the mechanism by which some pathogenic bacteria inject proteins straight into the cytosol of eukaryotic cells to 'anaesthetize' or 'enslave' them--was discovered in 1994. Important progress has been made in this area during the past few years: the bacterial organelles responsible for this secretion--called 'injectisomes'--have been visualized, the structures of some of the bacterial protein 'effectors' have been determined, and considerable progress has been made in understanding the intracellular action of the effectors. Type III secretion is key to the pathogenesis of bacteria from the Yersinia genus

The type III secretion injectisome

The type III secretion injectisome is a complex nanomachine that allows bacteria to deliver protein effectors across eukaryotic cellular membranes. In recent years, significant progress has been made in our understanding of its structure, assembly and mode of operation. The principal structural components of the injectisome, from the base located in the bacterial cytosol to the tip of the needle protruding from the cell surface, have been investigated in detail. The structures of several constituent proteins were solved at the atomic level and important insights into the assembly process have been gained. However, despite the ongoing concerted efforts of molecular and structural biologists, the role of many of the constituent components of this nanomachine remain unknown