Gammaherpesvirus modulation of mouse adenovirus type 1 pathogenesis

AbstractImmune function is likely to be shaped by multiple infections over time. Infection with one pathogen can confer cross-protection against heterologous pathogens. We tested the hypothesis that latent murine gammaherpesvirus 68 (γHV68) infection modulates host inflammatory responses and susceptibility to mouse adenovirus type 1 (MAV-1). Mice were infected intranasally (i.n.) with γHV68. 21 days later, they were infected i.n. with MAV-1. We assessed cytokine and chemokine expression by quantitative reverse transcriptase real-time PCR, cellular inflammation by histology, and viral loads by quantitative real-time PCR. Previous γHV68 infection led to persistently upregulated IFN-γ in lungs and spleen and persistently upregulated CCL2 and CCL5 in the lungs. Previous γHV68 infection amplified MAV-1-induced CCL5 upregulation and cellular inflammation in the lungs. Previous γHV68 infection was associated with lower MAV-1 viral loads in the spleen but not the lung. There was no significant effect of previous γHV68 on IFN-γ expression or MAV-1 viral loads when the interval between infections was increased to 44 days. In summary, previous γHV68 infection modulated lung inflammatory responses and decreased susceptibility to a heterologous virus in an organ- and time-dependent manner

A sigma factor and anti-sigma factor that control swarming motility and biofilm formation in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an environmental bacterium and opportunistic human pathogen of major clinical significance. It is the principal cause of morbidity and mortality in patients with cystic fibrosis (CF) and a leading cause of nosocomial infections. Although the organism is unicellular, P. aeruginosa exhibits two forms of multicellular behaviors when associated with a surface under the right conditions: swarming motility and biofilm formation. Swarming motility is a multicellular cooperative form of flagella-dependent surface motility, while biofilm formation produces a sessile community of bacteria enclosed by a self-produced extracellular polymeric matrix. P. aeruginosa is thought to grow as a biofilm in the lungs of CF patients and the growth of P. aeruginosa biofilms on indwelling medical devices, such as endotracheal tubes and catheters, is a significant source of nosocomial infection. By growing as a biofilm, P. aeruginosa resists clearance by the immune system and increases its resistance to antimicrobial therapy. In this thesis, I describe the characterization of a sigma factor and anti-sigma factor implicated in P. aeruginosa virulence and cell envelope stress that control the expression of a novel regulator of swarming motility and biofilm formation. In addition, I describe work done to investigate the role of a post-translational regulator of flagellar motility that has been described in other bacteria, but has not been studied extensively P. aeruginosa. In bacteria, RNA polymerase (RNAP) requires sigma factors for promoter-specific transcription initiation. sigma factors guide RNAP to promoters by recognizing conserved DNA sequences within the promoter called the -10 and -35 elements. Most bacteria encode a primary sigma factor and several alternative sigma factors, each of which recognizes different promoter -10 and -35 sequences. By modulating the activity of alternative sigma factors, bacteria can rapidly alter their transcriptional program in response to changes in growth, morphological development, and environmental conditions. The extracytoplasmic function (ECF) sigma factors are the largest and most diverse group of bacterial sigma factors. The gene encoding an ECF sigma factor is often cotranscribed with its own negative regulator, called an anti-sigma factor, which directly binds to and inhibits its partner sigma factor until the appropriate extracytoplasmic signal stimulates sigma factor release and expression of the sigma factor’s regulon. In this thesis, I describe the characterization of the P. aeruginosa ECF sigma factor PA2896 and its cognate anti-sigma factor PA2895. Using immunoprecipitation, we show that the ECF sigma factor PA2896 and RNAP co-purify in vivo. Utilizing DNA microarrays, we identify the genes that constitute the PA2895 and PA2896 regulon and infer the putative -10 and -35 consensus sequences recognized by PA2896. Genetic analysis revealed a subset of genes within the PA2896 regulon that share the putative promoter consensus sequence are positively regulated by the ECF sigma factor PA2896 and negatively regulated by the anti-sigma factor PA2895. Using a bacterial two-hybrid assay, we show that PA2895 directly interacts with PA2896. We present evidence that increased expression of the PA2896 regulon in ∆PA2895 mutants cells leads to the inhibition of swarming motility and enhanced biofilm formation. We further show that one gene in the PA2896 regulon, PA1494, is necessary and sufficient for the inhibition of swarming motility and promotion of biofilm formation. Thus we report the discovery of a system that may respond to a stress signal by activating PA2896-dependent expression of PA1494 to inhibit swarming motility and promote the formation of a protective biofilm. In many bacteria, swarming motility and biofilm formation are controlled by the second messenger c-di-GMP. In P. aeruginosa, elevated intracellular c-di-GMP generally results in the inhibition of flagellar-dependent swarming motility and enhanced biofilm formation. In some species of bacteria, c-di-GMP-mediated repression of flagellar motility is achieved by repressing the expression of the flagellar genes. However, flagellar gene expression in P. aeruginosa does not appear to be influenced by elevated c-di-GMP, suggesting c-di-GMP controls flagellar motility post transcriptionally in this bacterium. Mechanisms by which c-di-GMP controls flagellar function post translationally have been described in both Gram-negative and Gram-positive bacteria, however the mechanism in P. aeruginosa remains unclear. Gram-negative bacteria appear to utilize a “flagellar brake” to control flagellar function in response to c-di-GMP. P. aeruginosa encodes a homolog of this brake and in this thesis I present evidence that this homolog is involved in controlling flagellar function in P. aeruginosa. Together, the characterization of PA2895 and PA2896, the identification of PA1494 as a novel regulator of swarming motility and biofilm formation, and evidence of a functional flagellar brake in P. aeruginosa advance our understanding of how this bacterium controls the transition from motile cell to sessile biofilm.Medical Science

σ Factor and Anti-σ Factor That Control Swarming Motility and Biofilm Formation in Pseudomonas aeruginosa.

International audiencePseudomonas aeruginosa is capable of causing a variety of acute and chronic infections. Here, we provide evidence that sbrR (PA2895), a gene previously identified as required during chronic P. aeruginosa respiratory infection, encodes an anti-σ factor that inhibits the activity of its cognate extracytoplasmic-function σ factor, SbrI (PA2896). Bacterial two-hybrid analysis identified an N-terminal region of SbrR that interacts directly with SbrI and that was sufficient for inhibition of SbrI-dependent gene expression. We show that SbrI associates with RNA polymerase in vivo and identify the SbrIR regulon. In cells lacking SbrR, the SbrI-dependent expression of muiA was found to inhibit swarming motility and promote biofilm formation. Our findings reveal SbrR and SbrI as a novel set of regulators of swarming motility and biofilm formation in P. aeruginosa that mediate their effects through muiA, a gene not previously known to influence surface-associated behaviors in this organism.This study characterizes a σ factor/anti-σ factor system that reciprocally regulates the surface-associated behaviors of swarming motility and biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. We present evidence that SbrR is an anti-σ factor specific for its cognate σ factor, SbrI, and identify the SbrIR regulon in P. aeruginosa. We find that cells lacking SbrR are severely defective in swarming motility and exhibit enhanced biofilm formation. Moreover, we identify muiA (PA1494) as the SbrI-dependent gene responsible for mediating these effects. SbrIR have been implicated in virulence and in responding to antimicrobial and cell envelope stress. SbrIR may therefore represent a stress response system that influences the surface behaviors of P. aeruginosa during infection

The C-Terminal Repeating Units of CsgB Direct Bacterial Functional Amyloid Nucleation

Curli are functional amyloids produced by enteric bacteria. The major curli fiber subunit, CsgA, self-assembles into an amyloid fiber in vitro. The minor curli subunit protein, CsgB, is required for CsgA polymerization on the cell surface. Both CsgA and CsgB are composed of five predicted β–strand-loop-β–strand-loop repeating units that feature conserved glutamine and asparagine residues. Because of this structural homology, we proposed that CsgB might form an amyloid template that initiates CsgA polymerization on the cell surface. To test this model, we purified wild-type CsgB, and found that it self-assembled into amyloid fibers in vitro. Preformed CsgB fibers seeded CsgA polymerization as did soluble CsgB added to the surface of cells secreting soluble CsgA. To define the molecular basis of CsgB nucleation, we generated a series of mutants that removed each of the five repeating units. Each of these CsgB deletion mutants was capable of self-assembly in vitro. In vivo, membrane-localized mutants lacking the 1(st), 2(nd) or 3(rd) repeating units were able to convert CsgA into fibers. However, mutants missing either the 4(th) or 5(th) repeating units were unable to complement a csgB mutant. These mutant proteins were not localized to the outer membrane, but were instead secreted into the extracellular milieu. Synthetic CsgB peptides corresponding to repeating units 1, 2 and 4 self assembled into ordered amyloid polymers, while peptides corresponding to repeating units 3 and 5 did not, suggesting that there are redundant amyloidogenic domains in CsgB. Our results suggest a model where the rapid conversion of CsgB from unstructured protein to a β-sheet-rich amyloid template anchored to the cell surface is mediated by the C-terminal repeating units

Simulation of Oxygen Isotopes in a Global Ocean Model

Abstract: We incorporate the oxygen isotope composition of seawater δ18Ow into a global ocean model that is based on the Modular Ocean Model (MOM, version 2) of the Geophysical Fluid Dynamics Laboratory (GFDL). In a first experiment, this model is run to equilibrium to simulate the present-day ocean; in a second experiment, the oxygen isotope composition of Antarctic Surface Water (AASW) is set to a constant value to indirectly account for the effect of sea-ice. We check the depth distribution of δ18Ow against observations. Furthermore, we computed the equilibrium fractionation of the oxygen isotope composition of calcite δ18Oc from a paleotemperature equation and compared it with benthic foraminiferal δ18O. The simulated δ18Ow distribution compares fairly well with the GEOSECS data. We show that the δ18Ow values can be used to characterize different water masses. However, a warm bias of the global ocean model yields δ18Oc values that are too light by about 0.5 ‰ above 2 km depth and exhibit a false vertical gradient below 2 km depth. Our ultimate goal is to interpret the wealth of foraminiferal δ18O data in terms of water mass changes in the paleocean, e.g. at the Last Glacial Maximum (LGM). This requires the warm bias of the global ocean model to be corrected. Furthermore the model must probably be coupled to simple atmosphere and sea-ice models such that neither sea-surface salinity (SSS) nor surface δ18Ow need to be prescribed and the use of present-day δ18Ow-salinity relationships can be avoided