Regulation of Alginate Production of Pseudomonas Aeruginosa Pseudomonas aeruginosa

Pseudomonas aeruginosa (P.a.) is a major threat to the health and well being of a person living with cystic fibrosis (CF). P.a. is capable of conversion to mucoidy or alginate overproducing phenotype. Mucoidy facilitates the chronic infection of P.a. infection in the CF lung. Mutations in the anti-sigma factor mucA, result in activation of the sigma factor AlgU which promotes expression of alginate through the alginate biosynthetic operon. However, P.a. can produce alginate independent of mucA mutation via a mechanism known as regulated proteolysis. Proteases AlgW and MucP can degrade MucA to liberate and activate AlgU. In this dissertation, two pathways to alginate production will be characterized through investigation of two negative regulators of alginate production: KinB and MucD. KinB is a histidine kinase which controls alginate production through two transcription factors: AlgB and RpoN along with the MucA protease AlgW. In the absence of KinB, the degradation rate of MucA is elevated which results in alginate production. MucD is a periplasmic protease that is presumed to degrade protein signals in the periplasm. It is hypothesized that if MucD does not degrade certain protein signals then they will accumulate and activate alginate production. Here, it is presented that in the absence of MucD, the intramembrane protease MucP activates degradation of MucA. To further characterize MucD, the roles of the PDZ domains in suppression of alginate production were probed. PDZ domains are conserved protein domains that function in protein to protein interaction. The PDZ domains of MucD were found to be dispensable for suppression of alginate production but required for the stability of MucD. When regulated proteolysis is activated, AlgU promotes expression of many genes. One such gene is LptF or Lipotoxin F. Here it is shown that in the absence of kinB, LptF is highly expressed. LptF was shown to be an outer membrane protein, but it is not involved in production of alginate. However, LptF mediates adhesion to lung epithelia and resistance to reactive oxygen species. P.a. is capable of activating AlgU through MucA proteolysis by several mechanisms that are characterized in this dissertation. Through elucidation of these pathways, we will be more prepared to combat alginate production and P.a. infection of the CF lung

Characterization of Vinyl Chloride Degrading Bacteria From a Serial Anaerobic-Aerobic Chloroethene Bioreactor

Chloroethenes are among the most common groundwater contaminants in the United States. In anoxic groundwater, chloroethenes can be reduced to vinyl chloride (VC), a known carcinogen. The goal of this research was to characterize bacteria that could mineralize VC from a microbial community within a serial aerobic-anaerobic chloroethene bioreactor. Isolations for organisms capable of VC degradation were performed. The ability to degrade VC was first indicated by accumulation of chloride. Gas chromatography assay showed that VC was degraded in cultures capable of chloride accumulation. The epoxyalkane:coenzyme M transferase (EaCoMT) gene which codes for an enzyme previously shown to function in VC mineralization, was observed in several mixed VC degrading cultures. Pure cultures isolated on nutrient rich media from EaCoMT positive cultures did not retain ability to mineralize VC. A confirmed VC degrading pure culture without the EaCoMT gene was isolated but the species identity could not be ascertained

Intranasal Peptide-Based FpvA-KLH Conjugate Vaccine Protects Mice From Pseudomonas aeruginosa Acute Murine Pneumonia

Pseudomonas aeruginosa is an opportunistic pathogen causing acute and chronic respiratory infections associated with morbidity and mortality, especially in patients with cystic fibrosis. Vaccination against P. aeruginosa before colonization may be a solution against these infections and improve the quality of life of at-risk patients. To develop a vaccine against P. aeruginosa, we formulated a novel peptide-based P. aeruginosa subunit vaccine based on the extracellular regions of one of its major siderophore receptors, FpvA. We evaluated the effectiveness and immunogenicity of the FpvA peptides conjugated to keyhole limpet hemocyanin (KLH) with the adjuvant curdlan in a murine vaccination and challenge model. Immunization with the FpvA-KLH vaccine decreased the bacterial burden and lung edema after P. aeruginosa challenge. Vaccination with FpvA-KLH lead to antigen-specific IgG and IgM antibodies in sera, and IgA antibodies in lung supernatant. FpvA-KLH immunized mice had an increase in recruitment of CD11b+ dendritic cells as well as resident memory CD4+ T cells in the lungs compared to non-vaccinated challenged mice. Splenocytes isolated from vaccinated animals showed that the FpvA-KLH vaccine with the adjuvant curdlan induces antigen-specific IL-17 production and leads to a Th17 type of immune response. These results indicate that the intranasal FpvA-KLH conjugate vaccine can elicit both mucosal and systemic immune responses. These observations suggest that the intranasal peptide-based FpvA-KLH conjugate vaccine with curdlan is a potential vaccine candidate against P. aeruginosa pneumonia

An extracytoplasmic function sigma factor-dependent periplasmic glutathione peroxidase is involved in oxidative stress response of Shewanella oneidensis

AbstractBackgroundBacteria use alternative sigma factors (σs) to regulate condition-specific gene expression for survival and Shewanella harbors multiple ECF (extracytoplasmic function) σ genes and cognate anti-sigma factor genes. Here we comparatively analyzed two of the rpoE-like operons in the strain MR-1: rpoE-rseA-rseB-rseC and rpoE2-chrR.ResultsRpoE was important for bacterial growth at low and high temperatures, in the minimal medium, and high salinity. The degP/htrA orthologue, required for growth of Escherichia coli and Pseudomonas aeruginosa at high temperature, is absent in Shewanella, while the degQ gene is RpoE-regulated and is required for bacterial growth at high temperature. RpoE2 was essential for the optimal growth in oxidative stress conditions because the rpoE2 mutant was sensitive to hydrogen peroxide and paraquat. The operon encoding a ferrochelatase paralogue (HemH2) and a periplasmic glutathione peroxidase (PgpD) was identified as RpoE2-dependent. PgpD exhibited higher activities and played a more important role in the oxidative stress responses than the cytoplasmic glutathione peroxidase CgpD under tested conditions. The rpoE2-chrR operon and the identified regulon genes, including pgpD and hemH2, are coincidently absent in several psychrophilic and/or deep-sea Shewanella strains.ConclusionIn S. oneidensis MR-1, the RpoE-dependent degQ gene is required for optimal growth under high temperature. The rpoE2 and RpoE2-dependent pgpD gene encoding a periplasmic glutathione peroxidase are involved in oxidative stress responses. But rpoE2 is not required for bacterial growth at low temperature and it even affected bacterial growth under salt stress, indicating that there is a tradeoff between the salt resistance and RpoE2-mediated oxidative stress responses

Cyclic di-GMP Regulates the Type III Secretion System and Virulence in Bordetella bronchiseptica

The second messenger cyclic di-GMP (c-di-GMP) is a ubiquitous molecule in bacteria that regulates diverse phenotypes. Among them, motility and biofilm formation are the most studied. Furthermore, c-di-GMP has been suggested to regulate virulence factors, making it important for pathogenesis. Previously, we reported that c-di-GMP regulates biofilm formation and swimming motility in Bordetella bronchiseptica. Here, we present a multi-omics approach for the study of B. bronchiseptica strains expressing different cytoplasmic c-di-GMP levels, including transcriptome sequencing (RNA-seq) and shotgun proteomics with label-free quantification. We detected 64 proteins significantly up- or downregulated in either low or high c-di-GMP levels and 358 genes differentially expressed between strains with high c-di-GMP levels and the wild-type strain. Among them, we found genes for stress-related proteins, genes for nitrogen metabolism enzymes, phage-related genes, and virulence factor genes. Interestingly, we observed that a virulence factor like the type III secretion system (TTSS) was regulated by c-di-GMP. B. bronchiseptica with high c-di-GMP levels showed significantly lower levels of TTSS components like Bsp22, BopN, and Bcr4. These findings were confirmed by independent methods, such as quantitative reverse transcription- PCR (q-RT-PCR) and Western blotting. Higher intracellular levels of c-di-GMP correlated with an impaired capacity to induce cytotoxicity in a eukaryotic cell in vitro and with attenuated virulence in a murine model. This work presents data that support the role that the second messenger c-di-GMP plays in the pathogenesis of Bordetella.Instituto de Biotecnología y Biología Molecula

Highlights of the 12th International Bordetella Symposium

To commemorate the 100th anniversary of the Nobel prize being awarded to Jules Bordet, the discoverer of Bordetella pertussis, the 12th International Bordetella Symposium was held from 9 to 12 April 2019 at the Université Libre de Bruxelles, where Jules Bordet studied and was Professor of Microbiology. The symposium attracted more than 300 Bordetella experts from 34 countries. They discussed the latest epidemiologic data and clinical aspects of pertussis, Bordetella biology and pathogenesis, immunology and vaccine development, and genomics and evolution. Advanced technological and methodological tools provided novel insights into the genomic diversity of Bordetella and a better understanding of pertussis disease and vaccine performance. New molecular approaches revealed previously unrecognized complexity of virulence gene regulation. Innovative insights into the immune responses to infection by Bordetella resulted in the development of new vaccine candidates. Such discoveries will aid in the design of more effective approaches to control pertussis and other Bordetella-related diseases.SCOPUS: ar.jinfo:eu-repo/semantics/publishe