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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.

Haemophilus influenzae is subdivided into encapsulated strains, which express different serotypes of capsular polysaccharide (designated types a-f), and nonencapsulated strains, which are designated nontypeable H. influenzae (NTHi) (1). Since the [...]

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.

Copper- and zinc-containing superoxide dismutases ([Cu,Zn]-SODs) are generally considered almost exclusively eukaryotic enzymes, protecting the cytosol and extracellular compartments of higher organisms from damage by oxygen free-radicals. The recent description of a few examples of bacterial forms of the enzyme, located in the periplasm of different Gram-negative micro-organisms, prompted a re-evaluation of this general perception. A PCR-based approach has been developed and used successfully to identify bacterial genes encoding [Cu,Zn]-SOD in a wide range of important human and animal pathogens - members of the Haemaphilus, Actinobacillus and Pasteurella (HAP) group, and Neisseria meningitidis. Comparison of [Cu,Zn]-SOD peptide sequences found in Haemophilus ducreyi, Actinobacillus pleuropneumoniae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, and N. meningitidis with previously described bacterial proteins and examples of eukaryotic [Cu,Zn]-SOD has shown that the bacterial proteins constitute a distinct family apparently widely separated in evolutionary terms from the eukaryotic examples. The widespread occurrence of [Cu,Zn]-SOD in the periplasm of bacterial pathogens, appropriately located to dismute exogenously derived superoxide radical anions, suggests that this enzyme may play a role in the interactive biology of organisms with their hosts and so contribute to their capacity to cause disease.

Copper and zinc containing superoxide dismutases (Cu,Zn-SOD) which protect against oxygen toxicity are common in eukaryotes but are rarely presentin prokaryotes. A gene coding for Cu,Zn SOD was recently found in H. influenza type b and H. parainfluenza cells. Southern blotting techniques were used to investigate the presence of a similar gene among other members of the Haemophilus species. The presence of Cu, Zn-SOD activity in several Haemophilusspecies prove that the prevalence of this metalloenzyme among prokaryotes is greatly underestimated.

Abstract Background Many bacteria can take up DNA, but the evolutionary history and function of natural competence and transformation remain obscure. The sporadic distribution of competence suggests it is frequently lost and/or gained, but this has not been examined in an explicitly phylogenetic context. Additional insight may come from the sequence specificity of uptake by species such as Haemophilus influenzae, where a 9 bp uptake signal sequence (USS) repeat is both highly overrepresented in the genome and needed for efficient DNA uptake. We used the distribution of competence genes and DNA uptake specificity in H. influenzae's family, the Pasteurellaceae, to examine the ancestry of competence. Results A phylogeny of the Pasteurellaceae based on 12 protein coding genes from species with sequenced genomes shows two strongly supported subclades: the Hin subclade (H. influenzae, Actinobacillus actinomycetemcomitans, Pasteurella multocida, Mannheimia succiniciproducens, and H. somnus), and the Apl subclade (A. pleuropneumoniae, M. haemolytica, and H. ducreyi). All species contained homologues of all known H. influenzae competence genes, consistent with an ancestral origin of competence. Competence gene defects were identified in three species (H. somnus, H. ducreyi and M. haemolytica); each appeared to be of recent origin. The assumption that USS arise by mutation rather than copying was first confirmed using alignments of H. influenzae proteins with distant homologues. Abundant USS-like repeats were found in all eight Pasteurellacean genomes; the repeat consensuses of species in the Hin subclade were identical to that of H. influenzae (AAGTGCGGT), whereas members of the Apl subclade shared the consensus ACAAGCGGT. All species' USSs had the strong consensus and flanking AT-rich repeats of H. influenzae USSs. DNA uptake and competition experiments demonstrated that the Apl-type repeat is a true USS distinct from the Hin-type USS: A. pleuropneumoniae preferentially takes up DNA fragments containing the Apl-type USS over both H. influenzae and unrelated DNAs, and H. influenzae prefers its own USS over the Apl type. Conclusion Competence and DNA uptake specificity are ancestral properties of the Pasteurellaceae, with divergent USSs and uptake specificity distinguishing only the two major subclades. The conservation of most competence genes over the ~350 million year history of the family suggests that lineages that lose competence may be evolutionary dead ends.

Background and aims: The etiology of necrotizing enterocolitis (NEC) is unclear and postulated as being multifactorial. It has been suggested that one causative factor is the transfusion of packed red blood cells (PRBCs) leading to the disease entity commonly referred to as transfusion-associated NEC (TANEC). TANEC has been reported in North America but its incidence has not been formally investigated in the UK. Our aims were to identify the incidence of NEC and TANEC in tertiary-level UK neonatal units and to describe characteristics of TANEC cases. Materials and methods: Using strict case definitions for NEC and TANEC, we undertook a retrospective review to estimate the incidence of TANEC cases occurring in four UK tertiary-level centers during a 38-month period. Results: Of 8007 consecutive neonatal admissions of all gestations to the four centers, 68 babies went on to develop NEC and all affected infants were of very low birth weight (VLBW); 34 of these had previously received a transfusion of PRBCs but did not fit the diagnostic criteria for TANEC, whereas 15 (22%) of the 68 babies with NEC qualified as TANEC cases. UK cases occurred at an earlier postnatal age than cases reported in multiple large North American series and were of a lower birth weight. Conclusions: We have confirmed the presence of TANEC in the UK VLBW neonatal population. Its incidence lies within the wide range described in previous reports of this phenomenon globally, though with some local variation in characteristics. Further work is needed to clarify causation, pathophysiology, and possible mechanisms of prevention of TANEC. [ABSTRACT FROM AUTHOR]