Cation Regulation And Proteomic Analysis Of Phase Variation And Biofilm Formation In The Human Pathogen Vibrio Vulnificus

Vibrio vulnificus, a Gram-negative halophile which is ubiquitously present in the marine environments, can cause diseases like gastroenteritis, septicemia and wound infections in susceptible human beings. Production of capsular polysaccharide results in an opaque phenotype, which is pathogenic to humans. Loss or reduction of capsule results in translucent colonies composed of cells that are nonpathogenic or have diminished virulence, respectively. The opaque and the translucent phenotypes, upon production of an exopolysaccharide, give rise to a dry, wrinkled rugose form that can form copious biofilms. These three phenotypes can spontaneously switch from one form to another in a process known as phase variation. In this study, we have sought to identify environmental factors and genetic mechanisms that affect phase variation and biofilm formation in V. vulnificus. Elements such as Ba2+, Mn2+, and Sr2+, were tested to determine their effect on V. vulnificus phase variation and we found that manganese at micromolar to millimolar concentrations, induces V.vulnificus polysaccharide phase variation from opaque to translucent or rugose forms in this species. Since calcium was previously shown to induce significant phase variation in different V.vulnificus strains, we have also attempted to compare the effects of equimolar addition of manganese and calcium on phase variation of different V.vulnificus strains. Here, we have also attempted to better understand the process of V. vulnificus rugose colony formation. Our study has identified 28 proteins that include translational elongation factors, various metabolic enzymes, proteases, sugar binding proteins, amino acid transporters, polar flagellins and an uncharacterized protein that are differentially expressed in the biofilm proficient rugose phase variant compared to the parent isogenic opaque phase variant. One protein of particular interest, MalE (VVA0397 gene product), a maltose binding periplasmic protein, was identified as being expressed at higher amounts, and concomitantly showed higher transcript levels in the rugose variant compared to the opaque variant. Interestingly, generation of a targeted mutant of the malE gene did not knock out rugosity, indicating that the malE (VVA0397) gene is not required for rugosity in V.vulnificus. However, its up-regulation at both the transcriptional and translational levels suggests that MalE protein is involved in rugose colony formation in a way that remains to be determined

Genetic analysis and prevalence studies of the brp exopolysaccharide locus of Vibrio vulnificus

Phase variation in the Gram-negative human pathogen Vibrio vulnificus involves three colonial morphotypes- smooth opaque colonies due to production of capsular polysaccharide (CPS), smooth translucent colonies as the result of little or no CPS expression, and rugose colonies due to production of a separate extracellular polysaccharide (EPS), which greatly enhances biofilm formation. Previously, it was shown that the brp locus, which consists of nine genes arranged as an operon, is up-regulated in rugose strains in a c-di-GMP-dependent manner, and that plasmid insertions into the locus resulted in loss of rugosity and efficient biofilm production. Here, we have used non-polar mutagenesis to assess the involvement of individual brp genes in production of EPS and related phenotypes. Inactivation of genes predicted to be involved in various stages of EPS biosynthesis eliminated both the rugose colonial appearance and production of EPS, while knockout of a predicted flippase function involved in EPS transport resulted in a dry, lightly striated phenotype, which was associated with a reduction of brp-encoded EPS on the cell surface. All brp mutants retained the reduced motility characteristic of rugose strains. Lastly, we provide evidence that the brp locus is highly prevalent among strains of V. vulnificus. © 2014 Garrison-Schilling et al

Alterations in the steroid hormone receptor co-chaperone FKBPL are associated with male infertility: a case-control study

RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are.Abstract Background Male infertility is a common cause of reproductive failure in humans. In mice, targeted deletions of the genes coding for FKBP6 or FKBP52, members of the FK506 binding protein family, can result in male infertility. In the case of FKBP52, this reflects an important role in potentiating Androgen Receptor (AR) signalling in the prostate and accessory glands, but not the testis. In infertile men, no mutations of FKBP52 or FKBP6 have been found so far, but the gene for FKBP-like (FKBPL) maps to chromosome 6p21.3, an area linked to azoospermia in a group of Japanese patients. Methods To determine whether mutations in FKBPL could contribute to the azoospermic phenotype, we examined expression in mouse and human tissues by RNA array blot, RT-PCR and immunohistochemistry and sequenced the complete gene from two azoospermic patient cohorts and matching control groups. FKBPL-AR interaction was assayed using reporter constructs in vitro. Results FKBPL is strongly expressed in mouse testis, with expression upregulated at puberty. The protein is expressed in human testis in a pattern similar to FKBP52 and also enhanced AR transcriptional activity in reporter assays. We examined sixty patients from the Japanese patient group and found one inactivating mutation and one coding change, as well as a number of non-coding changes, all absent in fifty-six controls. A second, Irish patient cohort of thirty showed another two coding changes not present in thirty proven fertile controls. Conclusions Our results describe the first alterations in the gene for FKBPL in azoospermic patients and indicate a potential role in AR-mediated signalling in the testis.Published versio

Capsular polysaccharide production and serum survival of Vibrio vulnificus are dependent on antitermination control by RfaH

© 2016 Federation of European Biochemical Societies The human pathogen Vibrio vulnificus undergoes phase variation among colonial morphotypes, including a virulent opaque form which produces capsular polysaccharide (CPS) and a translucent phenotype that produces little or no CPS and is attenuated. Here, we found that a V. vulnificus mutant defective for RfaH antitermination control showed a diminished capacity to undergo phase variation and displayed significantly reduced distal gene expression within the Group I CPS operon. Moreover, the rfaH mutant produced negligible CPS and was highly sensitive to killing by normal human serum, results which indicate that RfaH is likely essential for virulence in this bacterium

Implementation of Bayesian Inference Technique to Address Data Limited Problems in Acology: A Case study with Peary Caribou in Canadian Arctic Archipelago

In the present era, rates of decline in species’ abundance provide some of the most compelling evidence of biodiversity loss rates globally. To address the problem of biodiversity loss, a critical piece of knowledge is the understanding of species interactions with their environment, because environmental variables are generally better predictors of population integrity than intrinsic biological traits. Peary caribou (Rangifer tarandus pearyi), the smallest of all caribou subspecies, are endemic to the Canadian Arctic Archipelago (CAA) and a characteristic example of species at risk. Climate change can affect their habitat availability, as well as the makeup of the entire Arctic ecosystem. Logistical and financial constraints in the CAA often compromise the frequency and the spatial extent of Peary caribou surveys, and therefore inconsistent sampling, errors in measurements, or faults in data acquisition encumber the robust assessment of their population status. To remedy such data gaps in surveys and, improve the robustness of any modelling exercise, I first developed a regression-based imputation framework to reconstruct the Peary caribou time series. The model was able to capture more than 65% of the variability in the dataset. To date, little work has been done to evaluate the net impact of changes from the climate on Peary caribou population dynamics, as it has been argued that the net balance of limited forage accessibility due to severe weather conditions relative to that of increased forage biomass due to prolonged growing season will depend on local climate, floral abundance and composition, and landscape characteristics. Using a two-pronged modelling approach, I characterized the year-to-year variability of the habitat conditions across the CAA, using meteorological variables, landscape features, and resource competition. My dissertation also introduced a spatially explicit modelling framework to examine the strength and nature of the relationships of snow density and vegetation with Peary caribou populations. My dissertation concludes by identifying critical augmentations of the available scientific knowledge that necessitate to design the optimal management actions of Peary caribou populations across the Canadian Arctic Archipelago.Ph.D

Quantitative chemical imaging in atom probe tomography

Atom Probe Tomography (APT) resolves atoms in real space and detects their chemical identity. In the realm of microscopic techniques, it has its unique place with sub nanometer spatial resolution and high chemical sensitivity. In this thesis, data driven techniques have been developed to identify nano scale chemical features from reconstructed atomic data obtained from APT experiments. Main drawbacks in present day methods, to detect nano scale features, are use of input parameters involving heuristics on part of user. Techniques developed in this work use the APT data to determine the input parameters, thereby making the process more quantitative. In particular, techniques have been developed to select optimal voxel size to calculate the concentration profile, and to select the concentration threshold using ideas from computational topology. Voxel size is selected using an error minimization technique. A framework to quantify and visualize spatial uncertainty in isosurface has also been developed. Approaches developed in this work are generic in nature and can be applied on any APT data. In this work, results have been shown for Ni based superalloy data.</p

An aggressive live range splitting and coalescing framework for efficient registrar allocation

M.S.Committee Chair: Santosh Pand