Hfq Regulates Biofilm Gut Blockage That Facilitates Flea-Borne Transmission of Yersinia pestis

Abstract

The plague bacillus Yersinia pestis can achieve transmission by biofilm blockage of the foregut proventriculus of its flea vector. Hfq is revealed to be essential for biofilm blockage formation and acquisition and fitness of Y. pestis during flea gut infection, consistent with posttranscriptional regulatory mechanisms in plague transmission.Y ersinia pestis, the etiological agent of bubonic plague, is transmitted to humans by fleabite. Colonization and biofilm formation by Y. pestis in the flea gut are essential steps for proventricular foregut blockage and facilitate subsequent transmission of Y. pestis during "frustrated feeding" by an infected flea (16,20). The known transmission factors hmsHFRS, gmhA, and ymt are required for flea-borne transmission but are dispensable for infection within the mammalian host (8,(16)(17)(18)(19). Whole-genome comparative transcriptional profiling revealed, however, that these three genes are not differentially transcribed during Y. pestis biofilm formation and gut blockage of the flea relative to temperature-matched in vitro conditions (40). Clearly, the transcriptome provides only a first order of gene regulation, while a second order of posttranscriptional regulation may shape the unique flea-associated biofilm that allows transmission. This is in keeping with the emerging paradigm for extensive posttranscriptionally regulated virulence in numerous bacterial pathogens (6,25,29,(32)(33)(34) The hfq gene is abundantly transcribed during Y. pestis biofilm proventricular blockage (40), predicting a second order of posttranscriptional regulation directed by Hfq during Y. pestis flea infection. To investigate the role of Hfq in mediating flea gut blockage, an hfq deletion mutant in the Y. pestis KIM6ϩ strain was constructed using the lambda red recombinase system (9). This strain contains the hmsHFRS gene locus required for the synthesis of extracellular matrix polysaccharide (EPS) essential for biofilm formation and flea foregut blockage (18,31,39). We hypothesized that the ⌬hfq mutant growing in vitro at the 21°C insect host temperature or within the flea gut would be impaired in its growth and biofilm gut blockage capability essential for plague survival and transmission.Growth of a Y. pestis KIM6؉ hfq deletion mutant. Functional mutations of hfq frequently result in compromised bacterial growth fitness (6, 13), including in Y. pestis growing at the mammalian host temperature of 37°C versus laboratory growth at 28°C (3, 13). Here, at 21°C, the insect host temperature, the ⌬hfq mutant, relative to its isogenic wild type, exhibited a significantly impaired specific growth rate and a decrease in stationary-phase cell density (Fig. 1A) similar to those seen with growth at 37°C (3, 13) and at 28°C (Fig. 1B) in brain heart infusion broth (BHI). The ⌬hfq mutant, complemented with the native gene and promoter region of hfq on a high-copy-number plasmid, pCR4-TOPO (Invitrogen), sufficiently rescued this growth fitness defect in BHI broth. However, the ⌬hfq mutant showed no significant...

YfbA, a Yersinia pestis Regulator Required for Colonization and Biofilm Formation in the Gut of Cat Fleas

“…It seems plausible that yfbA may control the expression of a still unidentified biofilm or colonization factor. Similar phenotypes have been reported for two regulatory factors, PhoP (a two-component response regulator) and Hfq (a bacterial RNA binding protein) when studying biofilm formation in the rat flea (55,56).…”

“…Hfq is a cytoplasmic protein that binds many different small RNAs and controls their stability and regulatory attributes (57,58). Y. pestis hfq is expressed both in the mammalian host and in flea intestines (47,48); however, a specific RNA(s) that supports bacterial adaptation to the flea gut is not yet known (56). Y. pestis ymt encodes cytoplasmic phospholipase D, which is not required for laboratory growth or the pathogenesis of plague in mice (16).…”

Identification and characterization of small RNAs inYersinia pestis

“…Hfq is also required for the virulence of many pathogenic bacteria, 8 including Y. pestis 9 and Y. pseudotuberculosis , 10 suggesting that sRNAs are key regulators of virulence genes in these species. Moreover, Hfq is required for efficient biofilm formation and gut blockage in the flea, important processes for transmission to a mammalian host, 11 and for the growth of some Y. pestis strains, but not Y. pseudotuberculosis , at 37°C 12 . Strikingly, Hfq amino acid sequence is 100% identical across eight sequenced strains of Y. pestis and four of Y. pseudotuberculosis , suggesting that regulation by sRNAs rather than Hfq itself contributes to the difference in viability of Y. pestis and Y. pseudotuberculosis hfq mutants at 37°C.…”

Genome-Wide Analysis of Small RNAs Expressed by Yersinia pestis Identifies a Regulator of the Yop-Ysc Type III Secretion System

“…Early work in this field established the significant impact of sRNAs on the regulation of outer membrane protein synthesis in E. coli, and more recently, the posttranscriptional regulation of virulence factors by Hfq-dependent sRNAs in bacterial pathogens has been described. For instance, Hfq and sRNAs participate in the regulation of biofilms produced by uropathogenic E. coli, V. cholerae, and Y. pestis, as well as in the regulation of alpha-toxin synthesis in Staphylococcus aureus (23,(25)(26)(27)(28)(29). Indeed, Hfq and sRNAs have been shown to contribute to the virulence of a number of pathogens, including both Y. pestis and Y. pseudotuberculosis (20,(30)(31)(32).…”