Interest in arthropod-borne pathogens focuses primarily on how they cause disease in humans. How they produce a transmissible infection in their arthropod host is just as critical to their life cycle, however. Yersinia pestis adopts a unique life stage in the digestive tract of its flea vector, characterized by rapid formation of a bacterial biofilm that is enveloped in a complex extracellular polymeric substance. Localization and adherence of the biofilm to the flea foregut is essential for transmission. Here, we review the molecular and genetic mechanisms of these processes and present a comparative evaluation and updated model of two related transmission mechanisms.

“Fleaing” the Plague: Adaptations of Yersinia pestis to Its Insect Vector That Lead to Transmission*

Hallmarks of Yersinia pathogenesis include the ability to form biofilms on surfaces, the ability to establish close contact with eukaryotic target cells and the ability to hijack eukaryotic cell signaling and take over control of strategic cellular processes. Many of these virulence traits are already well-described. However, of equal importance is knowledge of both confined and global regulatory networks that collaborate together to dictate spatial and temporal control of virulence gene expression. This review has the purpose to incorporate historical observations with new discoveries to provide molecular insight into how some of these regulatory mechanisms respond rapidly to environmental flux to govern tight control of virulence gene expression by pathogenic Yersinia.

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Environmental Regulation of Yersinia Pathophysiology.

The waterborne pathogen Legionella pneumophila grows as a biofilm, freely or inside amoebae. Cyclic-di-GMP (c-di-GMP), a bacterial second messenger frequently implicated in biofilm formation, is synthesized and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively. To characterize the c-di-GMP-metabolizing enzymes involved in L. pneumophila biofilm regulation, the consequences on biofilm formation and the c-di-GMP concentration of each corresponding gene inactivation were assessed in the Lens strain. The results showed that one DGC and two PDEs enhance different aspects of biofilm formation, while two proteins with dual activity (DGC/PDE) inhibit biofilm growth. Surprisingly, only two mutants exhibited a change in global c-di-GMP concentration. This study highlights that specific c-di-GMP pathways control L. pneumophila biofilm formation, most likely via temporary and/or local modulation of c-di-GMP concentration. Furthermore, Lpl1054 DGC is required to enable the formation a dense biofilm in response to nitric oxide, a signal for biofilm dispersion in many other species.

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New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling.

Vibrio parahaemolyticus, the leading cause of seafood-associated gastroenteritis worldwide, requires the two type-III secretion systems (T3SS1 and T3SS2) and a thermostable direct hemolysin (encoded by tdh1 and tdh2) for full virulence. The tdh genes and the T3SS2 gene cluster constitute an 80 kb pathogenicity island known as Vp-PAI located on the chromosome II. Expression of T3SS1 and Vp-PAI is regulated in a quorum sensing (QS)-dependent manner but its detailed mechanisms remain unknown. Herein, we show that three factors (QS regulators AphA and OpaR and an AraC-type transcriptional regulator QsvR) form a complex regulatory network to control the expression of T3SS1 and Vp-PAI genes. At low cell density (LCD), whereas Vp-PAI expression is repressed, T3SS1 genes are induced by AphA, which directly binds (an operator region of) the exsBAD-vscBCD operon. At high cell density (HCD), the bacterium turns off T3SS1 expression by replacing AphA with OpaR, triggering the induction of Vp-PAI. Furthermore, QsvR binds to the regulatory regions of all the tested T3SS1 and Vp-PAI genes to activate their transcription at HCD. Taken together, our data highlight how multiple QS regulators contribute to the pathogenicity of V. parahaemolyticus by precisely controlling the expression of major virulence determinants during different stages of growth.

QsvR integrates into quorum sensing circuit to control Vibrio parahaemolyticus virulence.

Pseudomonas putida (P. putida) MnB1 is a widely used model strain in environment science and technology for determining microbial manganese oxidation. Numerous studies have demonstrated that the growth and metabolism of P. putida MnB1 are influenced by various environmental factors. In this study, we investigated the effects of hydrogen peroxide (H2O2) and manganese (Mn2+) on proliferation, Mn2+ acquisition, anti-oxidative system, and biofilm formation of P. putida MnB1. The related orthologs of 4 genes, mco, mntABC, sod, and bifA, were amplified from P. putida GB1 and their involvement were assayed, respectively. We found that P. putida MnB1 degraded H2O2, and quickly recovered for proliferation, but its intracellular oxidative stress state was maintained, with rapid biofilm formation after H2O2 depletion. The data from mco, mntABC, sod and bifA expression levels by qRT-PCR, elucidated a sensitivity toward bifA-mediated biofilm formation, in contrary to intracellular anti-oxidative system under H2O2 exposure. Meanwhile, Mn2+ ion supply inhibited biofilm formation of P. putida MnB1. The expression pattern of these genes showed that Mn2+ ion supply likely functioned to modulate biofilm formation rather than only acting as nutrient substrate for P. putida MnB1. Furthermore, blockade of BifA activity by GTP increased the formation and development of biofilms during H2O2 exposure, while converse response to Mn2+ ion supply was evident. These distinct cellular responses to H2O2 and Mn2+ provide insights on the common mechanism by which environmental microorganisms may be protected from exogenous factors. We postulate that BifA-mediated biofilm formation but not intracellular anti-oxidative system may be a primary protective strategy adopted by P. putida MnB1. These findings will highlight the understanding of microbial adaptation mechanisms to distinct environmental stresses.

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bifA Regulates Biofilm Development of Pseudomonas putida MnB1 as a Primary Response to H2O2 and Mn2.

The hmsHFRS operon is responsible for biosynthesis and translocation of biofilm matrix exopolysaccharide. Yersinia pestis expresses the two sole diguanylate cyclases HmsT and HmsD and the sole phosphodiesterase HmsP, which are specific for biosynthesis and degradation, respectively, of 3',5'-cyclic diguanosine monophosphate (c-di-GMP), a second messenger promoting exopolysaccharide production. In this work, the phenotypic assays indicates that Y. pestis sRNA HmsB enhances the production of c-di-GMP, exopolysaccharide, and biofilm. Further gene regulation experiments disclose that HmsB stimulates the expression of hmsB, hmsCDE, hmsT, and hmsHFRS but represses that of hmsP. HmsB most likely acts as a major activator of biofilm formation in Y. pestis. This is the first report of regulation of Yersinia biofilm formation by a sRNA. Data presented here will promote us to gain a deeper understanding of the complex regulatory circuits controlling Yersinia biofilm formation.

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Shi Qu

Papers

HmsB enhances biofilm formation in Yersinia pestis