Reciprocal Regulation Of Yersinia Pestis Biofilm Formation And Virulence By RovM And RovA

Yersinia pestis, the causative agent of plague, is a kind of Gram-negative bacillar bacterium. In addition, Y. pestis presents different morphous in different culture conditions or different host environments. The chromosome genome size of Y. pestis is about 4.65 Mb, which contains about 4000 genes. Most of the Y. pestis strains contain three plasmids: p PCP1, p CD1, and p MT1. Abundant virulence genes are located in the three plasmids and play important roles in the survival, transmission and pathogenicity of Y. pestis. In the early stage of infection, Y. pestis can quickly spread to the demic lymph and cause the bubonic plague. Once Y. pestis spreads into the blood, it will propagate abundantly and cause the septicemic plague. Then the pathogen furtherly diffuse to the spleen, liver, lung and other organs and organizations; finally, the pneumonic plague will appear in a part of patients, at that time, the pathogen can directly and easily spread to other people through the air with high mortality rate. Y. pestis has caused at least three plague pandemics in human history, which has brought a great disaster to human beings. Simaltaneously, it has influenced the politics, economic and culture all over the world. Moreover, the plague has been listed as a infectious disease of resurgence by the World Health Organization. Therefore, the research about the prevention and treatment of plague is still very important.Y. pestis, differs from its progenitor Yersinia pseudotuberculosis, because the former can produce biofilms in the flea proventriculus for its biofilm-dependent transmission. Y. pestis biofilms can attach to and block flea’s proventriculus. The inability to take in a blood meal when the proventriculus is blocked makes fleas feel hungry and bite repeatedly and thereby promoting Y.pestis to be spread into new individuals of mammalian reservoirs. Therefore, the formation of Y. pestis biofilm is of significance to the transmission of Y. pestis.Y. pestis has complex and precise regulatory networks that control biofilm formation and virulence, and thus the regulators and target genes involved in these processes should be treated importantly. The Y. pestis hms HFRS operon is responsible for the synthesis and transport of exopolysaccharide, the primary dry component of the biofilm matrix. 3,5’-cyclic diguanylic acid(c-di-GMP) is a signaling molecule that promotes biofilm exopolysaccharide production in bacteria. In Y. pestis, Hms T and Hms D(hms D is located in the three-gene operon hms CDE) are the only two diguanylate cyclases that catalyze c-di-GMP synthesis, while Hms P is the sole phosphodiesterase catalyzing c-di-GMP degradation. Lipopolysaccharide(LPS) is an integral component of the outer membrane of Gram-negative bacteria. Generally, it is composed of three domains: lipid-A, core oligosaccharide, and O-specific antigen or O side chain. However, the LPS from Y. pestis contains only lipid-A bound to the core oligosaccharide by 3-deoxy-D-manno-octulosonic acid(Kdo). In Y. pestis, waa A, waa E, and coa D constitute a three-gene operon. The waa A gene encodes a Kdo transferase involved in the attachment of lipid-A to the core oligosaccharide, and waa A mutants show a severely biofilm-defective phenotype of Y. pestis. Y. pestis psa loci are composed of two adjacent operons: psa ABC and psa EF. psa A is the structural gene of the p H6 antigen, a Psa A polymer fimbrial structure; Psa B and Psa C constitute a chaperone/usher machinery that mediates the secretion and assembly of p H6 antigen on the cell surface. psa ABC expression is greatly stimulated following a rise in temperature from 26°C to 37°C and in acidic environments, while psa EF encodes the transcriptional activators of psa ABC. p H6 antigen is an adhesin that mediates the entry of bacteria into human pulmonary epithelial cells. It is also critical for contact of Y. pestis with eukaryotic cells that promotes the delivery of Yops(effectors of plasmid p CD1-encoding type III secretion system) to target host cells. A further p H6 function is as an anti-phagocytic factor, independent of Yops and the F1 capsule, which blocks bacterial uptake.Rov A is required for full virulence in all three pathogenic Yersinia species. Rov A also plays a critical role in modulating construction and functioning of the Y. pestis cell membrane. Most importantly, Y. pestis Pho P and Rov A bind to the promoter-proximal regions of psa ABC and psa EF to repress and stimulate their transcription, respectively. Pho P directly represses rov A transcription and, thereby directly and indirectly negatively regulating psa genes though acting on both psa genes and rov A. Rov M is a Lys R-type transcription factor that directly inhibits rov A transcription in Y. pseudotuberculosis, which is genetically closely related to Y. pestis. A set of Lys R-family regulators, such as Pec T in Erwinia chrysanthemi, Hex A in E. carotovora, Lrh A in Escherichia coli, and Yfb A in Y. pestis, were found to modulate bacterial virulence and biofilm formation. However it remains unknown whether Rov M controls virulence and biofilm gene expression in Y. pestis. Similarly, an array of Mar R-family regulators, such as Tca R in Staphylococcus epidermidis, Asr R in Enterococcus faecium, Rca R in Streptococcus mutans, and Sar Z/Sar A in Staphylococcus aureus, were reported to affect biofilm formation. Rov A is another Mar R-family regulator, but it is not clear whether Y. pestis Rov A is involved in the modulation of biofilm production.Data presented here show that Y. pestis Rov M inhibits virulence in mice, most likely through the direct repression of the transcription of rov A, which encodes a transcriptional activator of multiple virulence genes including the psa loci. Additionally, Rov M promotes biofilm/c-di-GMP production by directly stimulating the transcription of hms T and hms CDE, and up-regulates hms HFRS transcription and simultaneously down-regulates hms P transcription, both in an indirect manner. Rov M appears to be a master activator of Y. pestis biofilm production because it is able to modulate all four major biofilm gene loci, hms T, hms CDE, hms HFRS, and hms P. By contrast, Rov A inhibits Y. pestis biofilm formation through directly repressing hms T transcription and indirectly down-regulating waa AE-coa D, YPO1635-pho PQ-YPO1632, and pho PQ-YPO1632. waa AE-coa D transcription is known to be directly activated by Pho P. Y. pestis Rov A cannot bind to the promoter region of either YPO1635-pho PQ-YPO1632 or pho PQ-YPO1632. Rov A probably represses the transcription of YPO1635-pho PQ-YPO1632 and pho PQ-YPO1632 via acting on other protein or non-coding RNA regulators. The up-regulation of rov M accompanied with the down-regulation of rov A has previously been reported in the flea gut. As shown in this work, rov M expression is significantly up-regulated at 26°C(flea gut temperature) relative to 37°C(warm-blooded host temperature). Rov M appears to sense temperature signals to up-regulate its own gene expression and in turns inhibits rov A expression in the flea gut. The elevated Rov M production in the flea gut will promote the synthesis of attached biofilms and inhibit virulence gene expression, facilitating the establishment of a transmissible infection in fleas. Then, once the bacterium shifts to a lifestyle in the warm-blooded hosts, Rov M production is decreased and Rov A synthesis initiated, which in turn drives virulence factor synthesis and inhibits biofilm gene expression. Notably, cellular Rov M levels were not shown to change following a temperature shift from 37°C and 26°C in Y. pseudotuberculosis, and the temperature-dependent rov M expression might be an example of favorable evolution to promote Y. pestis transmission via fleas.