The Regulatory Mechanism Of Plasmid PPCP1-derived SRNA HmsA On Biofilm Formation And Virulence In Yersinia Pestis

Broadly speaking, small regulatory RNAs(s RNAs) are non-coding RNAs except for r RNA and t RNA acting as crucial regulators of gene expression in bacteria. The s RNAs are generally encoded in the intergenic, antisense and untranslated region with the size of 50 to 500 nts. Compared to the regulatory protein, s RNAs are economically synthesized and rapidly act on their targets. It is shown that they play crucial roles in the post-transcriptional regulation to enable the bacterial cells to duely adjust their physiology to environmental changes. Typically, s RNA functions by binding to their target m RNAs or proteins and thus affects m RNA translation or protein activity. Based on the positional derivation and action modes,s RNA can be divided into two categories: cis-encoded s RNA and trans-encoded s RNA. The trans-encoded s RNAs generally function with the help of Hfq chaperon.Yersinia pestis is the cautive pathogen of plague. It usually causes the plague epidemic among rodents, the reservoir host of Y. pestis, and spreads with the transmission by fleas. Human is occasionally infected by flea biting or by directly contacting with infected animals. Y. pestis mainly causes bubonic plague, pneumonic plague and septicemia plague. The biofilm formation in fleas is crucial for the survival and transmission of Y. pestis. The protein-implicated regulatory networks of biofilm formation in Y. pestis have been extensively studied, but there are few reports about regulation of biofilm formation in Y. pestis. How s RNAs influence the virulence in Y. pestis is also poorly understood. Our previous study showed that a chromosome-encoded s RNA Hms B positively regulated the biofilm formation of Y. pestis. Here we focused on an s RNA Hms A, encoded on p PCP1 plasmid of Y. pestis. The p PCP1 plasmid is laterally acquired during the evolution of Y. pestis, which plays a key role in virulence of Y. pestis. Therefore, it is meaningful to investigate the role of Hms A in biofilm formation and virulence in Y. pestis.As predicted by RNA sequencing, Hms A is located at 9,435-9,500 nt on plasmid p PCP1. The primer extension and Northern blotting assays were used to verify the presence, size and the transcription initiation site of Hms A. By using Northern Blotting, we monitored the expression of Hms A in Y. pestis wild-type strain 201(WT) and Δhfq mutant. The results showed that Hms A expression is Hfq-dependent, suggesting that Hms A might function as a trans-acting s RNA in Y. pestis.To construct the hms A deletion mutant(Δhms A), full length of the hms A gene was deleted from Y. pestis genome by using λ-Red recombinant system. No significant difference was found in the growth curve between WT and Hms A mutants(Δhms A) strain. An Hms A overexpressed strain(Δhms A: Hms A) was constructed by transforming the Hms A-overexpressed plasmid into Δhms A. By using the canonical experiments such as colony morphology, crystal violet staining, Caenorhabditis elegans killing assays, we proved that Hms A promotes the biofilm formation in Y. pestis. Besides that, the intracellular concentration of the c-di-GMP second messenger controlling EPS, a major component of biofilm, were shown decreased upon deletion of the hms A gene. Bal B/c mice were inoculated with WT and Δhms A strain via the intravenous, subcutaneous or intraperitoneal route, the survival dynamics of mice were observed within two weeks. The results showed that the virulence of Δhms A strain was higher than that of WT strain, which confirmed that Hms A has an impact on the virulence of Y. pestis.To determine whether Hms A regulate the biofilm- or virulence-associated genes, we used primer extension, Northern blotting, q RT-PCR, and β-galactosidase(Lac Z) reporter gene fusion assays to monitor the abundance changes and promoter activities of hms HFRS, hms CDE, hms T, hms P, hms B and fur. The results showed that Hms A positively regulated hms HFRS operon and s RNA Hms B at the transcriptional level but might have a positive role in the posttranscriptional regulation of hms T gene and hms CDE operon. No abundance changes in hms P and fur gene were found. By using the same methods, we found that Hms A negatively regulated rov A gene, psa ABC and psa EF operons and positively regulated rov M gene, which is consistent with the phenotype of enhanced virulence.of Y. pestis Δhms A mutant. Therefore, we hypothesized that Hms A might act on several key regulators to influence the biofilm formation and virulence of Y. pestis.It is well known that trans-acting s RNAs generally regulate their target genes at the posttranscriptional level. To determine the direct target genes of Hms A, the GFP reporter gene-based translational fusion system was introduced.. The system has been successfully applied to identification of direct targets of E. coli s RNAs. In this study we utilized a paradigmatic s RNA-target m RNA pairs(Mic F-omp F) as a model to explore the possibility of its application in Y. pestis. The translational activity of omp F was found slightly upregulated in Y. pestis WT strain upon Mic F overexpression, which contradicted the previously established model. Interestingly, the translational repression of omp F target fusions was restored in the intrinsic plasmids-cured Y. pestis strain, suggesting intrinsic plasmids influence the s RNA-mediated regulation of m RNA in Y. pestis. Further examination showed that plasmid p PCP1 is likely the main contributor to this phenomenon. These results suggested that the system could be used to validate the s RNA-mediated post-transcriptional regulation of target m RNAs in Y. pestis but the possible roles of intrinsic plasmids should be considered. By using this system, we confirmed that hms T and hms CDE might be potentially direct targets of Hms A.It is the first attempt to investigate the roles of the s RNA encoded on laterally acquired plasmid in Y. pestis biofilm and pathogenesis. Our study indicated that p PCP1-deriving Hms A encoded by plasmid p PCP1 contributes to biofilm formation and virulence of Y. pestis, suggesting that s RNA might involve in acquisition of the causative ability of fatal disease in mammalians and biofilm formation in fleas during evolution from Y. pseudotuberculosis to Y. pestis. Several biofilm- and virulence-associated genes were shown regulated by Hms A at the transcriptional or posttranscriptional level. We successfully established the GFP-based translation system in Y. pestis and attempted to validate its direct targets(hms T and hms CDE). The precise regulatory mechanism of Hms A-mediated regulation on Y. pestis biofilm and virulence remains to be further investigated.