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UnlabelledBacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens.ImportanceBy protecting microbes against reactive oxygen insults, SODs aid survival of many bacteria within their hosts. Despite the ubiquity and conservation of these key enzymes, notable species-specific differences relevant to pathogenesis remain undefined. To probe mechanisms that govern the functioning of Neisseria meningitidis and Brucella abortus SODs, we used X-ray structures, enzymology, modeling, and murine infection experiments. We identified virulence determinants common to the two homologs, assembly differences, and a unique metal reservoir within meningococcal SOD that stabilizes the enzyme and may provide a safeguard against copper toxicity. The insights reported here provide a rationale and a basis for SOD-specific drug design and an extension of immunogen design to target two important pathogens that continue to pose global health threats.

For more than a century, Haemophilus influenzae biogroup aegyptius (Hae) has caused worldwide seasonal epidemics of acute, purulent conjunctivitis (1,2). In 1984, an entirely new syndrome, Brazilian purpuric fever (BPF), [...]

Clinical isolates of the porcine pathogen Actinobacillus pleuropneumoniae often form adherent colonies on agar plates due to expression of an operon, pgaABCD, encoding a poly-[beta]-1,6-N-acetyl-D-glucosamine (PGA) extracellular matrix. The adherent colony phenotype, which correlates with the ability to form biofilms on the surfaces of polystyrene plates, is lost following serial passage in broth culture, and repeated passage of the nonadherent variants on solid media does not result in reversion to the adherent colony phenotype. In order to investigate the regulation of PGA expression and biofilm formation in A. pleuropneumoniae, we screened a bank of transposon mutants of the nonadherent serovar 1 strain [S4074.sup.T] and identified mutations in two genes, rseA and hns, which resulted in the formation of the adherent colony phenotype. In other bacteria, including the Enterobacteriaceae, H-NS acts as a global gene regulator, and RseA is a negative regulator of the extracytoplasmic stress response sigma factor [[sigma].sup.E]. Transcription profiling of A. pleuropneumoniae rseA and hns mutants revealed that both [[sigma].sup.E] and H-NS independently regulate expression of the pga operon. Transcription of the pga operon is initiated from a [[sigma].sup.E] promoter site in the absence of H-NS, and upregulation of [[sigma].sup.E] is sufficient to displace H-NS, allowing transcription to proceed. In A. pleuropneumoniae, H-NS does not act as a global gene regulator but rather specifically regulates biofilm formation via repression of the pga operon. Positive regulation of the pga operon by [[sigma].sup.E] indicates that biofilm formation is part of the extracytoplasmic stress response in A. pleuropneumoniae. doi: 10.1128/JB.01513-09

Neisseria meningitidis colonizes the human nasopharynx asymptomatically, often for prolonged periods, but occasionally invades from this site to cause life-threatening infection. In the nasopharynx aggregated organisms are closely attached to the epithelial surface, in a state in which the expression of components of the bacterial envelope differs significantly from that found in organisms multiplying exponentially in liquid phase culture or in the blood. We and others have hypothesized that here they are in the biofilm state, and to explore this we have investigated biofilm formation by the serogroup B strain MC58 on an abiotic surface, in a sorbarod system. Transcriptional changes were analysed, focusing on alteration in gene expression relevant to polysaccharide capsulation, lipooligosaccharide and outer-membrane protein synthesis--all phenotypes of importance in epithelial colonization. We report downregulation of genes controlling capsulation and the production of core oligosaccharide, and upregulation of genes encoding a range of outer-membrane components, reflecting phenotypic changes that have been established to occur in the colonizing state. A limited comparison with organisms recovered from an extended period of co-cultivation with epithelial cells suggests that this model system may better mirror natural colonization than do short-term meningococcal/epithelial cell co-cultivation systems. Modelling prolonged meningococcal colonization with a sorbarod system offers insight into gene expression during this important, but experimentally relatively inaccessible, phase of human infection.

DsbA ensures the correct folding of many exported bacterial proteins by forming intramolecular disulphide bonds in the bacterial periplasm. The pathogen Neisseria meningitidis is unusual in its possession of three different dsbA genes (dsbA 1, dsbA2 and dsbA3), encoding two membrane-anchored (DsbA1 and DsbA2) and one periplasmic (DsbA3) thiol-disulphide oxidoreductase enzymes. In this study, the involvement of DsbA1 and DsbA2 in natural competence was confirmed and attributed to events in the early stages of the transformation process. Strains lacking both DsbA1 and DsbA2 were reduced in competence as a result of decreased DNA binding and uptake. Overexpression of DsbA3 could not overcome this defect, suggesting differences in substrate specificity and protein-folding abilities between the DsbA homologues. Competence in Neisseria is dependent on the expression of type IV pili, which are extruded and retracted through the outer-membrane secretin PilQ. Both DsbA1 and DsbA2 were able to specifically bind PilQ in solid-phase overlay assays. Consistent with this, deletion of both dsbA 1 and dsbA2 resulted in reduced levels of PilQ, confirming inefficient folding of PilQ, while pilus expression was apparently unaffected. The secretin PilQ is involved in DNA binding and transport as well as pilus biogenesis, and the defect in PiIQ folding resulting from the absence of DsbA1 and DsbA2 is revealed in the observed decreased DNA binding and uptake.

The genome of Neisseria meningitidis serogroup B strain MC58 contains three genes-nmb0278, nmb0294 and nmb0407--encoding putative homologues of DsbA, a periplasmic thiol disulphide oxidoreductase protein-folding catalyst of the Dsb protein family. DsbA assists the folding of periplasmic and membrane proteins in diverse organisms. While all three cloned genes complemented the DTT sensitivity of dsbA-null Escherichia coli, they showed different activities in folding specific target proteins in this background. NMB0278 protein was the most active in complementing defects in motility and alkaline phosphatase activity, while NMB0294 was the most active in folding periplasmic MalF. NMB0407 showed the weakest activity in all assays. It is extremely unusual for organisms to contain more than one chromosomal dsbA. Among the members of the genus Neisseria, only the meningococcus carries all three of these genes. Strains of Neisseria gonorrhoeae, Neisseria lactamica, Neisseria cinerea and Neisseria polysaccharea contained only homologues of nmb0278 and nmb0407, while Neisseria flava, Neisseria subflava and Neisseria flavescens carried only nmb0294. It is speculated that the versatility of the meningococcus in surviving in different colonizing and invasive disease settings may be derived in part from an enhanced potential to deploy outer-membrane proteins, a consequence of carrying an extended repertoire of protein-folding catalysts.

Previously, the authors have shown that inactivation of Shigella flexneri yihE, a gene of unknown function upstream of dsbA, which encodes a periplasmic disulphide catalyst, results in a global change of gene expression. Among the severely down-regulated genes are galETKM, suggesting that the yihE mutant, Sh54, may inefficiently produce the UDP-glucose and UDP-galactose required for LPS synthesis. This paper demonstrates that LPS synthesis in Sh54 is impaired. As a result, Sh54 is unable to polymerize host cell actin, due to aberrant localization of IcsA, or to cause keratoconjunctivitis in guinea pigs. Furthermore, Sh54 is more sensitive to some antimicrobial agents, and exhibits epithelial cytotoxicity characteristic of neither wild-type nor dsbA mutants. Supplying galETK in trans restores LPS synthesis and corrects all the defects. Hence, it is clear that the Shigella yihE gene is important not only in regulating global gene expression, as shown previously, but also in virulence through LPS synthesis via regulating the expression of the galETK operon.

Research has been conducted on the effect of the knockout mutations on the porcine pathogen's virulence. Results indicate that periplasmic copper- and zinc-cofactored superoxide dismutases are needed to protect bacterial periplasmic or membrane components.