CsrA-Mediated Translational Activation of the hmsE mRNA Enhances HmsD-Dependent C-di-GMP-Enabled Biofilm Production in Yersinia pestis.

The plague bacterium, Yersinia pestis, forms a biofilm-mediated blockage in the flea foregut that enhances its transmission by fleabite. Biofilm formation is positively controlled by cyclic di-GMP (c-di-GMP), which is synthesized by the diguanylate cyclases (DGC), HmsD and HmsT. While HmsD primarily promotes biofilm-mediated blockage of fleas, HmsT plays a more minor role in this process. HmsD is a component of the HmsCDE tripartite signaling system. HmsC and HmsE posttranslationally inhibit or activate HmsD, respectively. HmsT-dependent c-di-GMP levels and biofilm formation are positively regulated by the RNA-binding protein CsrA. In this study we determined whether CsrA positively regulates HmsD-dependent biofilm formation through interactions with the hmsE mRNA. Gel mobility shift assays determined that CsrA binds specifically to the hmsE transcript. RNase T1 footprint assays identified a single CsrA binding site and CsrA-induced structural changes in the hmsE leader region. Translational activation of the hmsE mRNA was confirmed in vivo using plasmid-encoded inducible translational fusion reporters and by HmsE protein expression studies. Furthermore, mutation of the CsrA binding site in the hmsE transcript significantly reduced HmsD-dependent biofilm formation. These results suggest that CsrA binding leads to structural changes in the hmsE mRNA that enhance its translation to enable increased HmsD-dependent biofilm formation. Given the requisite function of HmsD in biofilm-mediated flea blockage, this CsrA-dependent increase in HmsD activity underscores that complex and conditionally defined modulation of c-di-GMP synthesis within the flea gut is required for Y. pestis transmission. IMPORTANCE Mutations enhancing c-di-GMP biosynthesis drove the evolution of Y. pestis to flea-borne transmissibility. c-di-GMP-dependent biofilm-mediated blockage of the flea foregut enables regurgitative transmission of Y. pestis by fleabite. The Y. pestis diguanylate cyclases (DGC), HmsT and HmsD, which synthesize c-di-GMP, play significant roles in transmission. Several regulatory proteins involved in environmental sensing, as well as signal transduction and response regulation, tightly control DGC function. An example is CsrA, a global posttranscriptional regulator that modulates carbon metabolism and biofilm formation. CsrA integrates alternative carbon usage metabolism cues to activate c-di-GMP biosynthesis through HmsT. Here, we demonstrated that CsrA additionally activates hmsE translation to promote c-di-GMP biosynthesis through HmsD. This emphasizes that a highly evolved regulatory network controls c-di-GMP synthesis and Y. pestis transmission.

A novel subtype of sporadic Creutzfeldt-Jakob disease with PRNP codon 129MM genotype and PrP plaques.

The presence of amyloid kuru plaques is a pathological hallmark of sporadic Creutzfeldt-Jakob disease (sCJD) of the MV2K subtype. Recently, PrP plaques (p) have been described in the white matter of a small group of CJD (p-CJD) cases with the 129MM genotype and carrying resPrPD type 1 (T1). Despite the different histopathological phenotype, the gel mobility and molecular features of p-CJD resPrPD T1 mimic those of sCJDMM1, the most common human prion disease. Here, we describe the clinical features, histopathology, and molecular properties of two distinct PrP plaque phenotypes affecting the gray matter (pGM) or the white matter (pWM) of sCJD cases with the PrP 129MM genotype (sCJDMM). Prevalence of pGM- and pWM-CJD proved comparable and was estimated to be ~ 0.6% among sporadic prion diseases and ~ 1.1% among the sCJDMM group. Mean age at onset (61 and 68 years) and disease duration (~ 7 months) of pWM- and pGM-CJD did not differ significantly. PrP plaques were mostly confined to the cerebellar cortex in pGM-CJD, but were ubiquitous in pWM-CJD. Typing of resPrPD T1 showed an unglycosylated fragment of ~ 20 kDa (T120) in pGM-CJD and sCJDMM1 patients, while a doublet of ~ 21-20 kDa (T121-20) was a molecular signature of pWM-CJD in subcortical regions. In addition, conformational characteristics of pWM-CJD resPrPD T1 differed from those of pGM-CJD and sCJDMM1. Inoculation of pWM-CJD and sCJDMM1 brain extracts to transgenic mice expressing human PrP reproduced the histotype with PrP plaques only in mice challenged with pWM-CJD. Furthermore, T120 of pWM-CJD, but not T121, was propagated in mice. These data suggest that T121 and T120 of pWM-CJD, and T120 of sCJDMM1 are distinct prion strains. Further studies are required to shed light on the etiology of p-CJD cases, particularly those of T120 of the novel pGM-CJD subtype.

CsrA Enhances Cyclic-di-GMP Biosynthesis and Yersinia pestis Biofilm Blockage of the Flea Foregut by Alleviating Hfq-Dependent Repression of the hmsT mRNA.

Plague-causing Yersinia pestis is transmitted through regurgitation when it forms a biofilm-mediated blockage in the foregut of its flea vector. This biofilm is composed of an extracellular polysaccharide substance (EPS) produced when cyclic-di-GMP (c-di-GMP) levels are elevated. The Y. pestis diguanylate cyclase enzymes HmsD and HmsT synthesize c-di-GMP. HmsD is required for biofilm blockage formation but contributes minimally to in vitro biofilms. HmsT, however, is necessary for in vitro biofilms and contributes to intermediate rates of biofilm blockage. C-di-GMP synthesis is regulated at the transcriptional and posttranscriptional levels. In this, the global RNA chaperone, Hfq, posttranscriptionally represses hmsT mRNA translation. How c-di-GMP levels and biofilm blockage formation is modulated by nutritional stimuli encountered in the flea gut is unknown. Here, the RNA-binding regulator protein CsrA, which controls c-di-GMP-mediated biofilm formation and central carbon metabolism responses in many Gammaproteobacteria, was assessed for its role in Y. pestis biofilm formation. We determined that CsrA was required for markedly greater c-di-GMP and EPS levels when Y. pestis was cultivated on alternative sugars implicated in flea biofilm blockage metabolism. Our assays, composed of mobility shifts, quantification of mRNA translation, stability, and abundance, and epistasis analyses of a csrA hfq double mutant strain substantiated that CsrA represses hfq mRNA translation, thereby alleviating Hfq-dependent repression of hmsT mRNA translation. Additionally, a csrA mutant exhibited intermediately reduced biofilm blockage rates, resembling an hmsT mutant. Hence, we reveal CsrA-mediated control of c-di-GMP synthesis in Y. pestis as a tiered, posttranscriptional regulatory process that enhances biofilm blockage-mediated transmission from fleas. IMPORTANCE Yersinia pestis, the bacterial agent of bubonic plague, produces a c-di-GMP-dependent biofilm-mediated blockage of the flea vector foregut to facilitate its transmission by flea bite. However, the intricate molecular regulatory processes that underlie c-di-GMP-dependent biofilm formation and thus, biofilm-mediated blockage in response to the nutritional environment of the flea are largely undefined. This study provides a novel mechanistic understanding of how CsrA transduces alternative sugar metabolism cues to induce c-di-GMP-dependent biofilm formation required for efficient Y. pestis regurgitative transmission through biofilm-mediated flea foregut blockage. The Y. pestis-flea interaction represents a unique, biologically relevant, in vivo perspective on the role of CsrA in biofilm regulation.

Co-infection Assay to Determine Yersinia pestis Competitive Fitness in Fleas.

Co-infection refers to the simultaneous infection of a host by multiple pathogenic organisms. Experimental co-infection studies using a mutant and its isogenic wild type have proven to be profoundly sensitive to analysis of pathogen factor mutation-associated fitness effects in in vivo models of infectious disease. Here we discuss the use of such co-infection experiments in studying the interaction between Yersinia pestis and its flea vector to more sensitively determine the critical bacterial determinants for Y. pestis survival, adaptation, and transmission from fleas. This chapter comprises two main sections, the first detailing how to infect fleas with mutant and wild type Y. pestis strains, and secondly how to process infected fleas and specifically quantify distinct Y. pestis strain burdens per flea. The Y. pestis competitive fitness co-infection model in fleas is insightful in evaluating the consequence of a mutation which may not be obvious in single-strain flea infections where there is less selective pressure.