The Periplasmic Disulfide Oxidoreductase DsbA Contributes to Haemophilus influenzae Pathogenesis

Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, 55 Lake Ave., N., S6-242, Worcester, MA 01655, USA.
Infection and immunity (Impact Factor: 3.73). 05/2008; 76(4):1498-508. DOI: 10.1128/IAI.01378-07
Source: PubMed


Haemophilus influenzae is an obligate human pathogen that persistently colonizes the nasopharynx and causes disease when it invades the bloodstream,
lungs, or middle ear. Proteins that mediate critical interactions with the host during invasive disease are likely to be secreted.
Many secreted proteins require addition of disulfide bonds by the DsbA disulfide oxidoreductase for activity or stability.
In this study, we evaluated the role in H. influenzae pathogenesis of DsbA, as well as HbpA, a substrate of DsbA. Mutants of H. influenzae Rd and type b strain Eagan having nonpolar deletions of dsbA were attenuated for bacteremia in animal models, and complemented strains exhibited virulence equivalent to that of the parental
strains. Comparison of predicted secreted proteins in H. influenzae to known DsbA substrates in other species revealed several proteins that could contribute to the role of dsbA in virulence. One candidate, the heme transport protein, HbpA, was examined because of the importance of exogenous heme for
aerobic growth of H. influenzae. The presence of a dsbA-dependent disulfide bond in HbpA was verified by an alkylation protection assay, and HbpA was less abundant in a dsbA mutant. The hbpA mutant exhibited reduced bacteremia in the mouse model, and complementation restored its in vivo phenotype to that of the
parental strain. These results indicate that dsbA is required in vivo and that HbpA and additional DsbA-dependent factors are likely to participate in H. influenzae pathogenesis.

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Available from: Charles V Rosadini, Aug 26, 2014
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    • "This analysis utilized an H. influenzae Rd strain with a high level of genetic competence for DNA uptake to allow high-density transposon mutagenesis. Although Rd lacks some of the virulence factors present in NTHi isolates, it exhibits infection properties in animal models similar to those of clinical isolates and serves as a useful model for many aspect of H. influenzae biology and pathogenesis (Weiser et al., 1995; Daines et al., 2003; Wong et al., 2007; Rosadini et al., 2008). With refinements in understanding natural transformation mechanisms in H. influenzae or by scaling up mutagenesis procedures, it is likely that large mutant libraries suitable for the HITS procedure could be generated in NTHi isolates. "
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    ABSTRACT: Haemophilus influenzae is a Gram-negative bacterium that has no identified natural niche outside of the human host. It primarily colonizes the nasopharyngeal mucosa in an asymptomatic mode, but has the ability to disseminate to other anatomical sites to cause otitis media, upper, and lower respiratory tract infections, septicemia, and meningitis. To persist in diverse environments the bacterium must exploit and utilize the nutrients and other resources available in these sites for optimal growth/survival. Recent evidence suggests that regulatory factors that direct such adaptations also control virulence determinants required to resist and evade immune clearance mechanisms. In this review, we describe the recent application of whole-genome approaches that together provide insight into distinct survival mechanisms of H. influenzae in the context of different sites of pathogenesis.
    Frontiers in Cellular and Infection Microbiology 03/2012; 2:23. DOI:10.3389/fcimb.2012.00023 · 3.72 Impact Factor
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    • "Additional copper responsive oxidative defense enzymes detected by whole cell proteomics analysis includes a plasmid encoded DSBA oxidoreductase (Krad4572), a member of the thioredoxin family that catalyzes disulfide bonding and proper folding of secreted proteins. This periplasmic protein is involved in the pathogenesis of Haemophilus influenzae [68], toxin production in Vibrio cholerae [69], and intestinal colonization of adherent-invasive E. coli (AIEC) in Crohn's disease [70]. Its potential role in this Gram positive actinobacterium is unknown but most likely involved in oxidative stress response. "
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    ABSTRACT: Copper is a highly reactive, toxic metal; consequently, transport of this metal within the cell is tightly regulated. Intriguingly, the actinobacterium Kineococcus radiotolerans has been shown to not only accumulate soluble copper to high levels within the cytoplasm, but the phenotype also correlated with enhanced cell growth during chronic exposure to ionizing radiation. This study offers a first glimpse into the physiological and proteomic responses of K. radiotolerans to copper at increasing concentration and distinct growth phases. Aerobic growth rates and biomass yields were similar over a range of Cu(II) concentrations (0-1.5 mM) in complex medium. Copper uptake coincided with active cell growth and intracellular accumulation was positively correlated with Cu(II) concentration in the growth medium (R(2)=0.7). Approximately 40% of protein coding ORFs on the K. radiotolerans genome were differentially expressed in response to the copper treatments imposed. Copper accumulation coincided with increased abundance of proteins involved in oxidative stress and defense, DNA stabilization and repair, and protein turnover. Interestingly, the specific activity of superoxide dismutase was repressed by low to moderate concentrations of copper during exponential growth, and activity was unresponsive to perturbation with paraquot. The biochemical response pathways invoked by sub-lethal copper concentrations are exceptionally complex; though integral cellular functions are preserved, in part, through the coordination of defense enzymes, chaperones, antioxidants and protective osmolytes that likely help maintain cellular redox. This study extends our understanding of the ecology and physiology of this unique actinobacterium that could potentially inspire new biotechnologies in metal recovery and sequestration, and environmental restoration.
    PLoS ONE 08/2010; 5(8):e12427. DOI:10.1371/journal.pone.0012427 · 3.23 Impact Factor
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    • "In pathogenic Vibrio cholerae, the DsbA homolog (TcpG) is responsible for the folding, maturation and secretion of virulence factors [27]. The importance of DsbA for virulence has been demonstrated in a variety of organisms [28], [29], [30], [31], [32], [33], [34]. Compensation for this deletion by the other copy of DsbA is likely for some functions, but in Neisseria meningitides which has three DsbA homologs, they vary in functional activity in complementation assays [35]. "
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    ABSTRACT: Rickettsia peacockii, also known as the East Side Agent, is a non-pathogenic obligate intracellular bacterium found as an endosymbiont in Dermacentor andersoni ticks in the western USA and Canada. Its presence in ticks is correlated with reduced prevalence of Rickettsia rickettsii, the agent of Rocky Mountain Spotted Fever. It has been proposed that a virulent SFG rickettsia underwent changes to become the East Side Agent. We determined the genome sequence of R. peacockii and provide a comparison to a closely related virulent R. rickettsii. The presence of 42 chromosomal copies of the ISRpe1 transposon in the genome of R. peacockii is associated with a lack of synteny with the genome of R. rickettsii and numerous deletions via recombination between transposon copies. The plasmid contains a number of genes from distantly related organisms, such as part of the glycosylation island of Pseudomonas aeruginosa. Genes deleted or mutated in R. peacockii which may relate to loss of virulence include those coding for an ankyrin repeat containing protein, DsbA, RickA, protease II, OmpA, ScaI, and a putative phosphoethanolamine transferase. The gene coding for the ankyrin repeat containing protein is especially implicated as it is mutated in R. rickettsii strain Iowa, which has attenuated virulence. Presence of numerous copies of the ISRpe1 transposon, likely acquired by lateral transfer from a Cardinium species, are associated with extensive genomic reorganization and deletions. The deletion and mutation of genes possibly involved in loss of virulence have been identified by this genomic comparison. It also illustrates that the introduction of a transposon into the genome can have varied effects; either correlating with an increase in pathogenicity as in Francisella tularensis or a loss of pathogenicity as in R. peacockii and the recombination enabled by multiple transposon copies can cause significant deletions in some genomes while not in others.
    PLoS ONE 12/2009; 4(12):e8361. DOI:10.1371/journal.pone.0008361 · 3.23 Impact Factor
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