Quinolone trafficking via outer membrane vesicles in Pseudomonas aeruginosa

textPseudomonas aeruginosa is a Gram-negative opportunistic pathogen often infecting the lungs of individuals with the heritable genetic disease cystic fibrosis and the peritoneum of those undergoing continuous peritoneal dialysis. Often these infections are not caused by colonization with P. aeruginosa alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of P. aeruginosa in vivo, and less is known about the impact of coinfecting bacteria on P. aeruginosa pathogenesis and physiology. In this dissertation I used a rat dialysis membrane peritoneal model to evaluate the in vivo transcriptome of P. aeruginosa in monoculture and in coculture with Staphylococcus aureus. Monoculture results indicate that approximately 5% of all P. aeruginosa genes are differentially regulated during growth in vivo. Included in this analysis are genes important for iron acquisition and growth in lowoxygen environments. The presence of S. aureus caused decreased transcription of P. aeruginosa iron-regulated genes during in vivo coculture, indicating that the presence of S. aureus increases usable iron for P. aeruginosa in the environment. This lysis was shown to be dependent on antimicrobial quinolones produced by P. aeruginosa. I demonstrate that these quinolones are present in outer membrane vesicles (MVs). Not only were these quinolones present in MVs, but the quorum sensing molecule; 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal; PQS) was also packaged into MVs and was necessary for MV formation. These findings illustrate that a prokaryote possesses a signal trafficking system with features common to those used by higher organisms and outlines a novel mechanism for delivery of a signal critical for coordinating group behaviors in P. aeruginosa. Although MVs are involved in important processes besides signaling, the molecular mechanism is unknown. To provide insight into the molecular mechanism of MV formation, I examined the interaction of PQS with bacterial lipids. In this work, I demonstrated that PQS interacts strongly with the acyl chains and 4’-phosphate of bacterial lipopolysaccharide. The results of my studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signaling functions. Finally, I propose a model of PQSmediated MV formation where PQS interacts with specific outer membrane components to allow the necessary curvature for MV formation.Microbiolog

The conserved mosaic prophage protein paratox inhibits the natural competence regulator ComR in Streptococcus

Abstract Horizontal gene transfer is an important means of bacterial evolution. This includes natural genetic transformation, where bacterial cells become “competent” and DNA is acquired from the extracellular environment. Natural competence in many species of Streptococcus, is regulated by quorum sensing via the ComRS receptor-signal pair. The ComR-XIP (mature ComS peptide) complex induces expression of the alternative sigma factor SigX, which targets RNA polymerase to CIN-box promoters to activate genes involved in DNA uptake and recombination. In addition, the widely distributed Streptococcus prophage gene paratox (prx) also contains a CIN-box, and here we demonstrate it to be transcriptionally activated by XIP. In vitro experiments demonstrate that Prx binds ComR directly and prevents the ComR-XIP complex from interacting with DNA. Mutations of prx in vivo caused increased expression of the late competence gene ssb when induced with XIP as compared to wild-type, and Prx orthologues are able to inhibit ComR activation by XIP in a reporter strain which lacks an endogenous prx. Additionally, an X-ray crystal structure of Prx reveals a unique fold that implies a novel molecular mechanism to inhibit ComR. Overall, our results suggest Prx functions to inhibit the acquisition of new DNA by Streptococcus

Streptococcus pyogenes biofilm growth in vitro and in vivo and its role in colonization, virulence, and genetic exchange

BACKGROUND: Group A streptococcus (GAS) commonly colonizes the oropharynx and nonintact skin. However, colonization has been little studied and the role of biofilm formation is unclear, as biofilm experiments to date have not been conducted under conditions that mimic the host environment.METHODS: In this study we grew GAS biofilms on human keratinocytes under various environmental conditions and used this model to evaluate colonization, invasive disease and natural transformation.RESULTS: GAS grown on epithelial cells, but not biofilms grown on abiotic surfaces, produced biofilms with characteristics similar to in vivo colonization. These biofilm bacteria showed a 100-fold higher bacterial burden of nasal-associated lymphoid tissue in mice than broth-grown bacteria, and were not virulent during septic infection, which was attributed in part to down-regulation of genes typically involved in localized and invasive disease. We also showed for the first time that GAS were naturally transformable when grown in biofilms and during colonization of NALT in vivo.CONCLUSIONS: These findings provide novel model systems to study biofilm formation of GAS in vitro and in vivo, suggest an important role for biofilm formation during GAS colonization, and provide an explanation for the known genome diversity within the GAS population

The extracellular DNA lattice of bacterial biofilms is structurally related to Holliday junction recombination intermediates

Extracellular DNA (eDNA) is a critical component of the extracellular matrix of bacterial biofilms that protects the resident bacteria from environmental hazards which includes imparting significantly greater resistance to antibiotics and host immune effectors. eDNA is organized into a lattice-like structure, stabilized by the DNABII family of proteins, known to have high affinity and specificity for HJs. Accordingly, we demonstrated that the branched eDNA structures present within the biofilms formed by NTHI in the middle ear of the chinchilla in an experimental otitis media model, and in sputum samples that contain multiple mixed bacterial species and were recovered from cystic fibrosis (CF) patients possess a HJ-like configuration. Next, we showed that the prototypic E. coli HJ-specific DNA-binding protein RuvA could be functionally exchanged for DNABII proteins in the stabilization of biofilms formed by three diverse human pathogens, UPEC, NTHI and Staphylococcus epidermidis. Importantly, while replacement of DNABII proteins within the NTHI biofilm matrix with RuvA was shown to retain similar mechanical properties when compared to the control NTHI biofilm structure, we also demonstrated that biofilm eDNA matrices stabilized by RuvA could be subsequently undermined upon addition of the HJ resolvase complex, RuvABC, which resulted in significant biofilm disruption. Collectively, our data suggested that nature has recapitulated a functional equivalent of the HJ recombination intermediate to maintain the structural integrity of bacterial biofilms

A Biochemical Characterization of the DNA Binding Activity of the Response Regulator VicR from <i>Streptococcus mutans</i>

<div><p>Two-component systems (TCSs) are ubiquitous among bacteria and are among the most elegant and effective sensing systems in nature. They allow for efficient adaptive responses to rapidly changing environmental conditions. In this study, we investigated the biochemical characteristics of the <i>Streptococcus mutans</i> protein VicR, an essential response regulator that is part of the VicRK TCS. We dissected the DNA binding requirements of the recognition sequences for VicR in its phosphorylated and unphosphorylated forms. In doing so, we were able to make predictions for the expansion of the VicR regulon within <i>S. mutans</i>. With the ever increasing number of bacteria that are rapidly becoming resistant to even the antibiotics of last resort, TCSs such as the VicRK provide promising targets for a new class of antimicrobials.</p></div

DNaseI footprinting assays of VicR with the 7^th position mutants of <i>gcrR</i> and <i>plsX</i> promoter regions.

<p>(A) DNaseI footprint analysis of <i>gcrR</i> 7^th position mutants. VicR at increasing concentrations was incubated with labeled probe. (B) DNaseI footprint analysis of <i>plsX</i> 77 bp 7^th position mutants. VicR at increasing concentrations was incubated with labeled probe. The S indicates that the substrate was incubated in the absence of VicR. The solid bars to the left of the autoradiograph indicate the match to the WalR consensus. The arrows indicate the bases that are referentially protected in the 7^th position mutants.</p

WebLogo alignment of high affinity VicR targets with a 5 base pair spacer.

<p>The output of the WebLogo software is featured above. The hexameric half sites are indicated by the black bars. The stars indicate the conserved adenines immediately following the half sites. The sequences that were used for the sequence alignment are below the Weblogo. The hexameric sites are highlighted within the sequences.</p