Burkholderia cepacia Complex Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition.

Burkholderia species, including opportunistic pathogens in the Burkholderia cepacia complex (Bcc), have genes to produce contact-dependent growth inhibition (CDI) system proteins. CDI is a phenomenon in which Gram-negative bacteria use the toxic C terminus of a polymorphic surface-exposed exoprotein, BcpA, to inhibit the growth of susceptible bacteria upon direct cell-cell contact. Production of a small immunity protein, BcpI, prevents autoinhibition. Although CDI systems appear widespread in Gram-negative bacteria, their function has been primarily examined in several model species. Here we demonstrate that genes encoding predicted CDI systems in Bcc species exhibit considerable diversity. We also show that Burkholderia multivorans, which causes pulmonary infections in patients with cystic fibrosis, expresses genes that encode two CDI systems, both of which appear distinct from the typical Burkholderia-type CDI system. Each system can mediate intrastrain interbacterial competition and contributes to bacterial adherence. Surprisingly, the immunity-protein-encoding bcpI gene of CDI system 1 could be mutated without obvious deleterious effects. We also show that nonpathogenic Burkholderia thailandensis uses CDI to control B. multivorans growth during coculture, providing one of the first examples of interspecies CDI and suggesting that CDI systems could be manipulated to develop therapeutic strategies targeting Bcc pathogens.IMPORTANCE Competition among bacteria affects microbial colonization of environmental niches and host organisms, particularly during polymicrobial infections. The Bcc is a group of environmental bacteria that can cause life-threatening opportunistic infections in patients who have cystic fibrosis or are immunocompromised. Understanding the mechanisms used by these bacterial pathogens to compete with one another may lead to the development of more effective therapies. Findings presented here demonstrate that a Bcc species, Burkholderia multivorans, produces functional CDI system proteins and that growth of this pathogen can be controlled by CDI system proteins produced by neighboring Burkholderia cells.

Discovery of Inhibitors of Burkholderia pseudomallei Methionine Aminopeptidase with Antibacterial Activity.

Evaluation of a series of MetAP inhibitors in an in vitro enzyme activity assay led to the first identification of potent molecules that show significant growth inhibition against Burkholderia pseudomallei. Nitroxoline analogs show excellent inhibition potency in the BpMetAP1 enzyme activity assay with the lowest IC50 of 30 nM, and inhibit the growth of B. pseudomallei and B. thailandensis at concentrations ≥ 31 µM.

Evidence for phenotypic bistability resulting from transcriptional interference of bvgAS in Bordetella bronchiseptica.

Bordetella species cause respiratory infections in mammals. Their master regulatory system BvgAS controls expression of at least three distinct phenotypic phases in response to environmental cues. The Bvg⁺ phase is necessary and sufficient for respiratory infection while the Bvg⁻ phase is required for survival ex vivo. We obtained large colony variants (LCVs) from the lungs of mice infected with B. bronchiseptica strain RBX9, which contains an in-frame deletion mutation in fhaB, encoding filamentous haemagglutinin. RBX9 also yielded LCVs when switched from Bvg⁻ phase conditions to Bvg⁺ phase conditions in vitro. We determined that LCVs are composed of both Bvg⁺ and Bvg⁻ phase bacteria and that they result from defective bvgAS positive autoregulation. The LCV phenotype was linked to the presence of a divergent promoter 5' to bvgAS, suggesting a previously undescribed mechanism of transcriptional interference that, in this case, leads to feedback-based bistability (FBM). Our results also indicate that a small proportion of RBX9 bacteria modulates to the Bvg⁻ phase in vivo. In addition to providing insight into transcriptional interference and FBM, our data provide an example of an in-frame deletion mutation exerting a 'polar' effect on nearby genes.

Functional characterization of Burkholderia pseudomallei trimeric autotransporters.

Burkholderia pseudomallei is a tier 1 select agent and the causative agent of melioidosis, a severe and often fatal disease with symptoms ranging from acute pneumonia and septic shock to a chronic infection characterized by abscess formation in the lungs, liver, and spleen. Autotransporters (ATs) are exoproteins belonging to the type V secretion system family, with many playing roles in pathogenesis. The genome of B. pseudomallei strain 1026b encodes nine putative trimeric AT proteins, of which only four have been described. Using a bioinformatic approach, we annotated putative domains within each trimeric AT protein, excluding the well-studied BimA protein, and found short repeated sequences unique to Burkholderia species, as well as an unexpectedly large proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence to, invasion of, and plaque formation in A549 cells. The majority of the ATs (and/or the proteins encoded downstream) contributed to adherence to and efficient invasion of A549 cells. Using a BALB/c mouse model of infection, we determined the contributions of each AT to bacterial burdens in the lungs, liver, and spleen. At 48 h postinoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model.

An improved recombination-based in vivo expression technology-like reporter system reveals differential cyaA gene activation in Bordetella species.

Bordetella pertussis and Bordetella bronchiseptica rely on the global two-component regulatory system BvgAS to control expression of distinct phenotypic phases. In the Bvg(-) phase, expression of vrg genes, including those required for motility in B. bronchiseptica, is activated and genes encoding virulence factors are not expressed. Conversely, in the Bvg(+) phase, genes encoding virulence factors are highly expressed while genes necessary for motility are repressed. Although several genetic analyses have demonstrated the importance of the Bvg(+) phase during respiratory infection, Bvg-regulated gene activation in B. bronchiseptica has not been investigated in vivo. To address this, we developed a plasmid, pGFLIP, that encodes a sensitive Flp recombinase-based fluorescent reporter system able to document gene activation both in vitro and in vivo. Using pGFLIP, we demonstrated that cyaA, considered to be a "late" Bvg(+) phase gene, is activated substantially earlier in B. bronchiseptica than B. pertussis following a switch from Bvg(-) to Bvg(+) phase conditions. We show that the altered activation of cyaA is not due to differences in the cyaA promoter or in the bvgAS alleles of B. bronchiseptica compared to B. pertussis, but appears to be species specific. Finally, we used pGFLIP to show that flaA remains repressed during infection, confirming that B. bronchiseptica does not modulate to the Bvg(-) phase in vivo.

Pseudomonas aeruginosa Psl polysaccharide reduces neutrophil phagocytosis and the oxidative response by limiting complement-mediated opsonization.

Pseudomonas aeruginosa causes chronic lung infections in the airways of cystic fibrosis (CF) patients. Psl is an extracellular polysaccharide expressed by non-mucoid P. aeruginosa strains, which are believed to be initial colonizers. We hypothesized that Psl protects P. aeruginosa from host defences within the CF lung prior to their conversion to the mucoid phenotype. We discovered that serum opsonization significantly increased the production of reactive oxygen species (ROS) by neutrophils exposed to a psl-deficient mutant, compared with wild-type (WT) and Psl overexpressing strains (Psl(++)). Psl-deficient P. aeruginosa were internalized and killed by neutrophils and macrophages more efficiently than WT and Psl(++) variants. Deposition of complement components C3, C5 and C7 was significantly higher on psl-deficient strains compared with WT and Psl(++) bacteria. In an in vivo pulmonary competition assay, there was a 4.5-fold fitness advantage for WT over psl-deficient P. aeruginosa. Together, these data show that Psl inhibits efficient opsonization, resulting in reduced neutrophil ROS production, and decreased killing by phagocytes. This provides a survival advantage in vivo. Since phagocytes are critical in early recognition and control of infection, therapies aimed at Psl could improve the quality of life for patients colonized with P. aeruginosa.

Direct evaluation of Pseudomonas aeruginosa biofilm mediators in a chronic infection model.

Biofilms contribute to Pseudomonas aeruginosa persistence in a variety of diseases, including cystic fibrosis, burn wounds, and chronic suppurative otitis media. However, few studies have directly addressed P. aeruginosa biofilms in vivo. We used a chinchilla model of otitis media, which has previously been used to study persistent Streptococcus pneumoniae and Haemophilus influenzae infections, to show that structures formed in vivo are biofilms of bacterial and host origin within a matrix that includes Psl, a P. aeruginosa biofilm polysaccharide. We evaluated three biofilm and/or virulence mediators of P. aeruginosa known to affect biofilm formation in vitro and pathogenesis in vivo--bis-(3',5')-cyclic dimeric GMP (c-di-GMP), flagella, and quorum sensing--in a chinchilla model. We show that c-di-GMP overproduction has a positive impact on bacterial persistence, while quorum sensing increases virulence. We found no difference in persistence attributed to flagella. We conclude from these studies that a chinchilla otitis media model provides a means to evaluate pathogenic mediators of P. aeruginosa and that in vitro phenotypes should be examined in multiple infection systems to fully understand their role in disease.

The Pseudomonas aeruginosa exopolysaccharide Psl facilitates surface adherence and NF-kappaB activation in A549 cells.

In order for the opportunistic Gram-negative pathogen Pseudomonas aeruginosa to cause an airway infection, the pathogen interacts with epithelial cells and the overlying mucous layer. We examined the contribution of the biofilm polysaccharide Psl to epithelial cell adherence and the impact of Psl on proinflammatory signaling by flagellin. Psl has been implicated in the initial attachment of P. aeruginosa to biotic and abiotic surfaces, but its direct role in pathogenesis has not been evaluated (L. Ma, K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak, J. Bacteriol. 188:8213-8221, 2006). Using an NF-kappaB luciferase reporter system in the human epithelial cell line A549, we show that both Psl and flagellin are necessary for full activation of NF-kappaB and production of the interleukin 8 (IL-8) chemokine. We demonstrate that Psl does not directly stimulate NF-kappaB activity, but indirectly as a result of increasing contact between bacterial cells and epithelial cells, it facilitates flagellin-mediated proinflammatory signaling. We confirm differential adherence of Psl and/or flagellin mutants by scanning electron microscopy and identify Psl-dependent membrane structures that may participate in adherence. Although we hypothesized that Psl would protect P. aeruginosa from recognition by the epithelial cell line A549, we instead observed a positive role for Psl in flagellin-mediated NF-kappaB activation, likely as a result of increasing contact between bacterial cells and epithelial cells.

Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production.

Exopolysaccharides contribute significantly to attachment and biofilm formation in the opportunisitc pathogen Pseudomonas aeruginosa. The Psl polysaccharide, which is synthesized by the polysaccharide synthesis locus (psl), is required for biofilm formation in non-mucoid strains that do not rely on alginate as the principal biofilm polysaccharide. In-frame deletion and complementation studies of individual psl genes revealed that 11 psl genes, pslACDEFGHIJKL, are required for Psl production and surface attachment. We also present the first structural analysis of the psl-dependent polysaccharide, which consists of a repeating pentasaccharide containing d-mannose, d-glucose and l-rhamnose: [See text]. In addition, we identified the sugar nucleotide precursors involved in Psl generation and demonstrated the requirement for GDP-d-mannose, UDP-d-glucose and dTDP-l-rhamnose in Psl production and surface attachment. Finally, genetic analyses revealed that wbpW restored Psl production in a pslB mutant and pslB promoted A-band LPS synthesis in a wbpW mutant, indicating functional redundancy and overlapping roles for these two enzymes. The structural and genetic data presented here provide a basis for further investigation of the Psl proteins and potential roles for Psl in the biology and pathogenesis of P. aeruginosa.

LuxS promotes biofilm maturation and persistence of nontypeable haemophilus influenzae in vivo via modulation of lipooligosaccharides on the bacterial surface.

Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.