Regulation of the Staphylococcal Superantigen-Like Protein 1 Gene of Community-Associated Methicillin-Resistant Staphylococcus aureus in Murine Abscesses.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) causes substantial skin and soft tissue infections annually in the United States and expresses numerous virulence factors, including a family of toxins known as the staphylococcal superantigen-like (SSL) proteins. Many of the SSL protein structures have been determined and implicated in immune system avoidance, but the full scope that these proteins play in different infection contexts remains unknown and continues to warrant investigation. Analysis of ssl gene regulation may provide valuable information related to the function of these proteins. To determine the transcriptional regulation of the ssl1 gene of CA-MRSA strain MW2, an ssl1 promoter::lux fusion was constructed and transformed into S.aureus strains RN6390 and Newman. Resulting strains were grown in a defined minimal medium (DSM) broth and nutrient-rich brain-heart infusion (BHI) broth and expression was determined by luminescence. Transcription of ssl1 was up-regulated and occurred earlier during growth in DSM broth compared to BHI broth suggesting expression is regulated by nutrient availability. RN6390 and Newman strains containing the ssl1::lux fusion were also used to analyze regulation in vivo using a mouse abscess model of infection. A marked increase in ssl1 transcription occurred early during infection, suggesting SSL1 is important during early stages of infection, perhaps to avoid the immune system.

Suppressor mutations reveal an NtrC-like response regulator, NmpR, for modulation of Type-IV Pili-dependent motility in Myxococcus xanthus.

Two-component signaling systems (TCS) regulate bacterial responses to environmental signals through the process of protein phosphorylation. Specifically, sensor histidine kinases (SK) recognize signals and propagate the response via phosphorylation of a cognate response regulator (RR) that functions to initiate transcription of specific genes. Signaling within a single TCS is remarkably specific and cross-talk between TCS is limited. However, regulation of the flow of information through complex signaling networks that include closely related TCS remains largely unknown. Additionally, many bacteria utilize multi-component signaling networks which provide additional genetic and biochemical interactions that must be regulated for signaling fidelity, input and output specificity, and phosphorylation kinetics. Here we describe the characterization of an NtrC-like RR that participates in regulation of Type-IV pilus-dependent motility of Myxococcus xanthus and is thus named NmpR, NtrC Modulator of Pili Regulator. A complex multi-component signaling system including NmpR was revealed by suppressor mutations that restored motility to cells lacking PilR, an evolutionarily conserved RR required for expression of pilA encoding the major Type-IV pilus monomer found in many bacterial species. The system contains at least four signaling proteins: a SK with a protoglobin sensor domain (NmpU), a hybrid SK (NmpS), a phospho-sink protein (NmpT), and an NtrC-like RR (NmpR). We demonstrate that ΔpilR bypass suppressor mutations affect regulation of the NmpRSTU multi-component system, such that NmpR activation is capable of restoring expression of pilA in the absence of PilR. Our findings indicate that pilus gene expression in M. xanthus is regulated by an extended network of TCS which interact to refine control of pilus function.

Molecular Mechanisms of Signaling in Myxococcus xanthus Development.

Myxococcus xanthus is an environmental bacterium that displays a complex life cycle that includes motility, predation, multicellular fruiting body development, and sporulation. Given the elaborate fruiting body development of this bacterial species, M. xanthus has served as a model organism for the study of multicellular development of bacteria, and a remarkable number of genes have been identified that contribute to the regulation of this highly dynamic process. Included among these developmental factors is a robust repertoire of signaling proteins, which have arisen from extensive gene duplication in M. xanthus and related species. In this review, we explore several aspects of the molecular mechanisms of signaling in M. xanthus development. This includes mechanisms of kin selection, single-cell sensing of nutrient depletion and the stringent response, the production of and response to extracellular population cues, and the contribution of several two-component signaling systems regulating developmental transcriptional programs. Collectively, these signaling mechanisms function to tightly regulate the sensing of nutrient depletion, the aggregation of populations of cells, and the temporal and spatial formation of complex fruiting bodies and sporulation of M. xanthus.

Type IV-pili dependent motility is co-regulated by PilSR and PilS2R2 two-component systems via distinct pathways in Myxococcus xanthus.

Myxococcus xanthus is an environmental bacterium with two forms of motility. One type, known as social motility, is dependent on extension and retraction of Type-IV pili (T4P) and production of extracellular polysaccharides (EPS). Several signaling systems have been linked to regulation of T4P-dependent motility. In particular, expression of the pilin subunit pilA requires the PilSR two-component signaling system (TCS). A second TCS, PilS2R2, encoded within the same locus that encodes PilSR, has also been linked to M. xanthus T4P-dependent motility. We demonstrate that PilSR and PilS2R2 regulate M. xanthus T4P-dependent motility through distinct pathways. Consistent with known roles of PilSR, our results indicate that the primary function of PilSR is to regulate expression of pilA. In contrast, PilS2 and PilR2 have little to no affect on PilA protein levels. However, deletion of pilR2 resulted in a reduction of assembled pili, significant decreases in EPS production and loss of T4P-dependent motility. Furthermore, the pilR2 mutation led to increased production of outer membrane vesicles (OMV). Collectively, we propose that PilS2R2 is required for proper assembly of T4P and regulation of OMV production, and hypothesize that production of these vesicles is related to M. xanthus motility.

Rv2744c Is a PspA Ortholog That Regulates Lipid Droplet Homeostasis and Nonreplicating Persistence in Mycobacterium tuberculosis.

UNLABELLED: Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant cause of morbidity and mortality worldwide, despite the availability of a live attenuated vaccine and anti-TB antibiotics. The vast majority of individuals infected with M. tuberculosis develop an asymptomatic latent infection in which the bacterium survives within host-generated granulomatous lesions in a physiologically altered metabolic state of nonreplicating persistence. The granuloma represents an adverse environment, as M. tuberculosis is exposed to various stressors capable of disrupting the essential constituents of the bacterium. In Gram-negative and Gram-positive bacteria, resistance to cell envelope stressors that perturb the plasma membrane is mediated in part by proteins comprising the phage shock protein (Psp) system. PspA is an important component of the Psp system; in the presence of envelope stress, PspA localizes to the inner face of the plasma membrane, homo-oligomerizes to form a large scaffold-like complex, and helps maintain plasma membrane integrity to prevent a loss of proton motive force. M. tuberculosis and other members of the Mycobacterium genus are thought to encode a minimal functional unit of the Psp system, including an ortholog of PspA. Here, we show that Rv2744c possesses structural and physical characteristics that are consistent with its designation as a PspA family member. However, although Rv2744c is upregulated under conditions of cell envelope stress, loss of Rv2744c does not alter resistance to cell envelope stressors. Furthermore, Rv2744c localizes to the surface of lipid droplets in Mycobacterium spp. and regulates lipid droplet number, size, and M. tuberculosis persistence during anaerobically induced dormancy. Collectively, our results indicate that Rv2744c is a bona fide ortholog of PspA that may function in a novel role to regulate lipid droplet homeostasis and nonreplicating persistence (NRP) in M. tuberculosis IMPORTANCE: Mycobacterium tuberculosis is the causative agent of tuberculosis, a disease associated with significant morbidity and mortality worldwide. M. tuberculosis is capable of establishing lifelong asymptomatic infections in susceptible individuals and reactivating during periods of immune suppression to cause active disease. The determinants that are important for persistent infection of M. tuberculosis or for reactivation of this organism from latency are poorly understood. In this study, we describe our initial characterizations of Rv2744c, an ortholog of phage shock protein A (PspA) that regulates the homeostasis of lipid bodies and nonreplicating persistence in M. tuberculosis This function of PspA in M. tuberculosis is novel and suggests that PspA may represent a unique bacterial target upon which to base therapeutic interventions against this organism.

Bacteriocin production augments niche competition by enterococci in the mammalian gastrointestinal tract.

Enterococcus faecalis is both a common commensal of the human gastrointestinal tract and a leading cause of hospital-acquired infections. Systemic infections with multidrug-resistant enterococci occur subsequent to gastrointestinal colonization. Preventing colonization by multidrug-resistant E. faecalis could therefore be a valuable approach towards limiting infection. However, little is known about the mechanisms E. faecalis uses to colonize and compete for stable gastrointestinal niches. Pheromone-responsive conjugative plasmids encoding bacteriocins are common among enterococcal strains and could modulate niche competition among enterococci or between enterococci and the intestinal microbiota. We developed a model of colonization of the mouse gut with E. faecalis, without disrupting the microbiota, to evaluate the role of the conjugative plasmid pPD1 expressing bacteriocin 21 (ref. 4) in enterococcal colonization. Here we show that E. faecalis harbouring pPD1 replaces indigenous enterococci and outcompetes E. faecalis lacking pPD1. Furthermore, in the intestine, pPD1 is transferred to other E. faecalis strains by conjugation, enhancing their survival. Colonization with an E. faecalis strain carrying a conjugation-defective pPD1 mutant subsequently resulted in clearance of vancomycin-resistant enterococci, without plasmid transfer. Therefore, bacteriocin expression by commensal bacteria can influence niche competition in the gastrointestinal tract, and bacteriocins, delivered by commensals that occupy a precise intestinal bacterial niche, may be an effective therapeutic approach to specifically eliminate intestinal colonization by multidrug-resistant bacteria, without profound disruption of the indigenous microbiota.

The MprB extracytoplasmic domain negatively regulates activation of the Mycobacterium tuberculosis MprAB two-component system.

Mycobacterium tuberculosis is an acid-fast pathogen of humans and the etiological agent of tuberculosis (TB). It is estimated that one-third of the world's population is latently (persistently) infected with M. tuberculosis. M. tuberculosis persistence is regulated, in part, by the MprAB two-component signal transduction system, which is activated by and mediates resistance to cell envelope stress. Here we identify MprAB as part of an evolutionarily conserved cell envelope stress response network and demonstrate that MprAB-mediated signal transduction is negatively regulated by the MprB extracytoplasmic domain (ECD). In particular, we report that deregulated production of the MprB sensor kinase, or of derivatives of this protein, negatively impacts M. tuberculosis growth. The observed growth attenuation is dependent on MprAB-mediated signal transduction and is exacerbated in strains of M. tuberculosis producing an MprB variant lacking its ECD. Interestingly, full-length MprB, and the ECD of MprB specifically, immunoprecipitates the Hsp70 chaperone DnaK in vivo, while overexpression of dnaK inhibits MprAB-mediated signal transduction in M. tuberculosis grown in the absence or presence of cell envelope stress. We propose that under nonstress conditions, or under conditions in which proteins present in the extracytoplasmic space are properly folded, signaling through the MprAB system is inhibited by the MprB ECD. Following exposure to cell envelope stress, proteins present in the extracytoplasmic space become unfolded or misfolded, leading to removal of the ECD-mediated negative regulation of MprB and subsequent activation of MprAB.

MprA and DosR coregulate a Mycobacterium tuberculosis virulence operon encoding Rv1813c and Rv1812c.

Mycobacterium tuberculosis remains a significant global pathogen, causing extensive morbidity and mortality worldwide. This bacterium persists within granulomatous lesions in a poorly characterized, nonreplicating state. The two-component signal transduction systems MprAB and DosRS-DosT (DevRS-Rv2027c) are responsive to conditions likely to be present within granulomatous lesions and mediate aspects of M. tuberculosis persistence in vitro and in vivo. Here, we describe a previously uncharacterized locus, Rv1813c-Rv1812c, that is coregulated by both MprA and DosR. We demonstrate that MprA and DosR bind to adjacent and overlapping sequences within the promoter region of Rv1813c and direct transcription from an initiation site located several hundred base pairs upstream of the Rv1813 translation start site. We further show that Rv1813c and Rv1812c are cotranscribed, and that the genomic organization of this operon is specific to M. tuberculosis and Mycobacterium bovis. Although Rv1813c is not required for survival of M. tuberculosis in vitro, including under conditions in which MprAB and DosRST signaling are activated, an M. tuberculosis ΔRv1813c mutant is attenuated in the low-dose aerosol model of murine tuberculosis, where it exhibits a lower bacterial burden, delayed time to death, and decreased ability to stimulate proinflammatory cytokines interleukin-1ß (IL-1ß) and IL-12. Interestingly, overcomplementation of these phenotypes is observed in the M. tuberculosis ΔRv1813c mutant expressing both Rv1813c and Rv1812c, but not Rv1813c alone, in trans. Therefore, Rv1813c and Rv1812c may represent general stress-responsive elements that are necessary for aspects of M. tuberculosis virulence and the host immune response to infection.

Adaptation to environmental stimuli within the host: two-component signal transduction systems of Mycobacterium tuberculosis.

Pathogenic microorganisms encounter a variety of environmental stresses following infection of their respective hosts. Mycobacterium tuberculosis, the etiological agent of tuberculosis, is an unusual bacterial pathogen in that it is able to establish lifelong infections in individuals within granulomatous lesions that are formed following a productive immune response. Adaptation to this highly dynamic environment is thought to be mediated primarily through transcriptional reprogramming initiated in response to recognition of stimuli, including low-oxygen tension, nutrient depletion, reactive oxygen and nitrogen species, altered pH, toxic lipid moieties, cell wall/cell membrane-perturbing agents, and other environmental cues. To survive continued exposure to these potentially adverse factors, M. tuberculosis encodes a variety of regulatory factors, including 11 complete two-component signal transduction systems (TCSSs) and several orphaned response regulators (RRs) and sensor kinases (SKs). This report reviews our current knowledge of the TCSSs present in M. tuberculosis. In particular, we discuss the biochemical and functional characteristics of individual RRs and SKs, the environmental stimuli regulating their activation, the regulons controlled by the various TCSSs, and the known or postulated role(s) of individual TCSSs in the context of M. tuberculosis physiology and/or pathogenesis.

Components of the Rv0081-Rv0088 locus, which encodes a predicted formate hydrogenlyase complex, are coregulated by Rv0081, MprA, and DosR in Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, remains a significant cause of morbidity and mortality throughout the world despite a vaccine and cost-effective antibiotics. The success of this organism can be attributed, in part, to its ability to adapt to potentially harmful stress within the host and establish, maintain, and reactivate from long-term persistent infection within granulomatous structures. The DosRS-DosT/DevRS-Rv2027c, and MprAB two-component signal transduction systems have previously been implicated in aspects of persistent infection by M. tuberculosis and are known to be responsive to conditions likely to be found within the granuloma. Here, we describe initial characterization of a locus (Rv0081-Rv0088) encoding components of a predicted formate hydrogenylase enzyme complex that is directly regulated by DosR/DevR and MprA, and the product of the first gene in this operon, Rv0081. In particular, we demonstrate that Rv0081 negatively regulates its own expression and that of downstream genes by binding an inverted repeat element in its upstream region. In contrast, DosR/DevR and MprA positively regulate Rv0081 expression by binding to recognition sequences that either partially or completely overlap that recognized by Rv0081, respectively. Expression of Rv0081 initiates from two promoter elements; one promoter located downstream of the DosR/DevR binding site but overlapping the sequence recognized by both Rv0081 and MprA and another promoter downstream of the DosR/DevR, Rv0081, and MprA binding sites. Interestingly, Rv0081 represses Rv0081 and downstream determinants following activation of DosRS-DosT/DevRS-Rv2027c by nitric oxide, suggesting that expression of this locus is complex and subject to multiple levels of regulation. Based on this and other published information, a model is proposed detailing Rv0081-Rv0088 expression by these transcription factors within particular growth environments.

The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF) sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.