Haplotypic diversity in human CEACAM genes: effects on susceptibility to meningococcal disease.

Adhesion between the opacity-associated adhesin (Opa) proteins of Neisseria meningitidis and human carcino-embryonic antigen cell adhesion molecule (CEACAM) proteins is an important stage in the pathogenesis of meningococcal disease, a globally important bacterial infection. Most disease is caused by a small number of meningococcal genotypes known as hyperinvasive lineages. As these are also carried asymptomatically, acquisition of them alone cannot explain why only some hosts develop meningococcal disease. Our aim was to determine whether genetic diversity in CEACAM is associated with susceptibility to meningococcal disease. Frequency distributions of alleles, genotypes and haplotypes were compared in four CEACAM genes in 384 case samples and 190 controls. Linkage disequilibrium among polymorphic sites, haplotype structures and relationships were also analysed. A number of polymorphisms were observed in CEACAM genes but the diversity of CEACAM1, to which most Opa proteins bind, was lower, and a small number of high-frequency haplotypes were detected. Dose-dependent associations of three CEACAM haplotypes with meningococcal disease were observed, with the effect of carrying these haplotypes amplified in homozygous individuals. Two haplotypes were protective while one haplotype in CEACAM6 was associated with a twofold increase in disease susceptibility. These data imply that human CEACAM may be one determinant of human susceptibility to meningococcal disease.

Multiplex PCR that can distinguish between immunologically cross- reactive serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains.

We describe a highly sensitive and specific multiplex PCR, based on capsular loci and the species specific apxIV gene, that unequivocally differentiates serovar 3, 6, and 8 Actinobacillus pleuropneumoniae strains that are cross-reactive in conventional immunological tests.

PCR specific for Actinobacillus pleuropneumoniae serotype 3.

Serotypes 3 and 8 of Actinobacillus pleuropneumoniae, the aetiological agent of porcine pleuropneumonia, have been reported to predominate in the UK. Direct serotyping of isolates of the organism is typically determined by the immunological reactivity of rabbit serum to its surface polysaccharides, but the method has limitations, for example, cross-reactions between serotypes 3, 6 and 8. This study describes the development of a serotype 3-specific pcr, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The pcr test was evaluated on 266 strains of A pleuropneumoniae and 121 strains of other organisms, including all the major respiratory bacterial pathogens of pigs. The test was highly specific and sensitive and should be useful for differentiating strains of serotypes 3, 6 and 8, and in seroprevalence and epidemiological surveys in regions where serotype 3 is prevalent, such as the UK.

Cu,Zn superoxide dismutase structure from a microbial pathogen establishes a class with a conserved dimer interface.

Macrophages and neutrophils protect animals from microbial infection in part by issuing a burst of toxic superoxide radicals when challenged. To counteract this onslaught, many Gram-negative bacterial pathogens possess periplasmic Cu,Zn superoxide dismutases (SODs), which act on superoxide to yield molecular oxygen and hydrogen peroxide. We have solved the X-ray crystal structure of the Cu,Zn SOD from Actinobacillus pleuropneumoniae, a major porcine pathogen, by molecular replacement at 1.9 A resolution. The structure reveals that the dimeric bacterial enzymes form a structurally homologous class defined by a water-mediated dimer interface, and share with all Cu,Zn SODs the Greek-key beta-barrel subunit fold with copper and zinc ions located at the base of a deep loop-enclosed active-site channel. Our structure-based sequence alignment of the bacterial enzymes explains the monomeric nature of at least two of these, and suggests that there may be at least one additional structural class for the bacterial SODs. Two metal-mediated crystal contacts yielded our C222(1) crystals, and the geometry of these sites could be engineered into proteins recalcitrant to crystallization in their native form. This work highlights structural differences between eukaryotic and prokaryotic Cu,Zn SODs, as well as similarities and differences among prokaryotic SODs, and lays the groundwork for development of antimicrobial drugs that specifically target periplasmic Cu,Zn SODs of bacterial pathogens.

Functional and crystallographic characterization of Salmonella typhimurium Cu,Zn superoxide dismutase coded by the sodCI virulence gene.

The functional and three-dimensional structural features of Cu,Zn superoxide dismutase coded by the Salmonella typhimurium sodCI gene, have been characterized. Measurements of the catalytic rate indicate that this enzyme is the most efficient superoxide dismutase analyzed so far, a feature that may be related to the exclusive association of the sodCI gene with the most pathogenic Salmonella serotypes. The enzyme active-site copper ion is highly accessible to external probes, as indicated by quenching of the water proton relaxation rate upon addition of iodide. The shape of the electron paramagnetic resonance spectrum is dependent on the frozen or liquid state of the enzyme solution, suggesting relative flexibility of the copper ion environment. The crystal structure (R-factor 22.6%, at 2.3 A resolution) indicates that the dimeric enzyme adopts the quaternary assembly typical of prokaryotic Cu,Zn superoxide dismutases. However, when compared to the structures of the homologous enzymes from Photobacterium leiognathi and Actinobacillus pleuropneumoniae, the subunit interface of Salmonella Cu,Zn superoxide dismutase shows substitution of 11 out of 19 interface residues. As a consequence, the network of structural water molecules that fill the dimer interface cavity is structured differently from the other dimeric bacterial enzymes. The crystallographic and functional characterization of this Salmonella Cu,Zn superoxide dismutase indicates that structural variability and catalytic efficiency are higher in prokaryotic than in the eukaryotic homologous enzymes.

Palindromic Haemophilus DNA uptake sequences in presumed transcriptional terminators from H. influenzae and H. parainfluenzae.

We have found palindromic pairs of near matches to the 11-bp Haemophilus DNA uptake motif shortly after the stop codons of three Haemophilus genes. Short runs of thymidylate residues follow the stem-loop structures thus defined. This organization suggests that, in H. influenzae, the uptake motif may be preferentially incorporated into gene termination signals, as has been proposed for Neisseria gonorrhoeae.

DsbA: a protein-folding catalyst contributing to bacterial virulence.

DsbA, a periplasmic thiol:disulphide oxidoreductase, catalyses the folding of various factors, among which are virulence determinants or the components of type III secretory machinery. It is also necessary for intracellular survival and cell-to-cell spread of the intracellular pathogen Shigella flexneri.

Superoxide dismutases of pathogenic and non-pathogenic Streptococcus suis type 2 isolates.

Pathogenic and non-pathogenic isolates of Streptococcus suis type 2 were screened to determine whether differences in superoxide dismutase (SOD) synthesis could explain the observed differences in their pathogenicity and intracellular fate in macrophages. A single band of SOD activity of similar Rf value was visualised in PAGE gels in all isolates and inhibition studies suggested that the cofactor present was manganese. There was no correlation between specific SOD activity and virulence. It is unlikely, therefore, that SOD produced by S. suis type 2 mediates intracellular survival of pathogenic isolates in macrophages.

Identification of sodC encoding periplasmic [Cu,Zn]-superoxide dismutase in Salmonella.

sodC, encoding [Cu,Zn]-cofactored superoxide dismutase, once thought to be virtually confined to eukaryotes, has now been described in many Gram-negative pathogens that have their primary niche of colonization in the upper respiratory tract. Their role in host-parasite interactive biology is unknown. We here show that members of the major human and animal enteric pathogenic species Salmonella harbour a version of sodC most closely resembling that found in Brucella abortus. The enzyme it encodes is a novel candidate determinant of virulence in Salmonella, an intracellular pathogen potentially exposed to toxic oxygen free radicals within its intracellular niche.

Inactivation of DsbA alters the behaviour of Shigella flexneri towards murine and human-derived macrophage-like cells.

The mutants of Shigella flexneri, Sh4 (dsbA::kan) and Sh42 (dsbA33G), behave differently towards murine and human-derived macrophage-like cells in vitro. Sh4 was trapped in the phagocytic vacuoles of the murine J774 cells as evidenced by its colony forming units plus and minus chloroquine exposure in a gentamicin protection assay, and by light and transmission electron microscopy (TEM). Sh42, similar to the wild-type M90TS, was able to escape from the vacuoles and kill host cells presumably by inducing apoptosis. In U937 cells, unlike M90TS that was free in the cytosol, both Sh4 and Sh42 grew poorly. TEM revealed that Sh4 and Sh42 were trapped within the U937 phagocytic vacuoles. Furthermore, the two mutants induced different patterns of interleukin-1beta and tumour necrosis factor-alpha expression, which might explain why they possess different immunogenic properties in vivo.

Occurrence of [copper, zinc]-cofactored superoxide dismutase in Pasteurella haemolytica and its serotype distribution.

Fifty-two ovine strains of Pasteurella haemolytica and P. trehalosi representing serotypes 1-16 were examined for the presence of [copper, zinc]superoxide dismutase DNA sequences. This was done using a combination of polymerase chain reaction with degenerate primers based on the sequence of the [Cu,Zn]superoxide dismutase gene (sodC) in related species and Southern hybridization using a fragment of sodC from P. haemolytica A2 serotype as a probe. Both detection methods identified a fragment of the sodC gene in 9/9 strains of P. haemolytica serotype 2 examined and in 5/8 strains of serotype 7. No evidence of this gene was found in any other serotype of P. haemolytica or in any P. trehalosi serotype. Comparison of DNA sequence showed near identity between sodC from the A2 and A7 serotypes of P. haemolytica and substantial similarity (70%) to sodC previously sequenced in P. multocida, Haemophilus parainfluenzae and H. influenzae. Analysis by gel electrophoresis of the superoxide dismutase activity present in cell lysates showed that one or more superoxide dismutase is present in all serotypes. However, cyanide-inhibitable activity, corresponding to [Cu,Zn]superoxide dismutase, was detected only in those strains of serotypes A2 and A7 which showed evidence of the sodC gene fragment.

Distribution, cloning, characterisation and mutagenesis of sodC, the gene encoding copper/zinc superoxide dismutase, a potential determinant of virulence, in Haemophilus ducreyi.

The sodC gene encoding the periplasmic enzyme copper/zinc superoxide dismutase (CuZnSOD) has been cloned from Haemophilus ducreyi, the causative agent of the genital ulcer disease, chancroid. Examination of a collection of diverse strains indicates that it is present throughout the species. Cloned sodC has been expressed in E. coli and shown to encode active enzyme. Insertional mutagenesis was used to construct a non-functional version of the gene. This has been transferred into the chromosome of the parent H. ducreyi strain by electroporation and homologous recombination, in preparation for studies of the role of this enzyme in the interactive biology of the organism with its host, perhaps in protecting bacteria from superoxide radicals and their reactive progeny generated by neutrophils in the context of host defence.

A promoter probe plasmid based on green fluorescent protein : a strategy for studying meningococcal gene expression.

Many bacterial genes are regulated in an environment-responsive fashion, and from the perspective of a pathogen, the host represents just another environment. Many genes that contribute to virulence are differentially expressed in response to host environments that they encounter during colonization and invasion (1). Recognition of this has led to the development of selection or reporter systems that utilize the increased activity of promoters during growth in vivo to identify genes that are selectively expressed during infection, and, thus, may contribute to the infection process (2-5). One of these techniques, differential fluorescence induction (2,3), involves the use of a promoter-probe plasmid that utilizes a variant of green fluorescent protein (GFP) as its reporter. The technique has been used successfully to identify novel Salmonella typhimurium genes that are selectively expressed following exposure to acid environments (3) and during infection of macrophages (2). GFP reporter systems have also been used to evaluate in vivo gene expression in other organisms including Staphylococcus aureus (6), Listeria monocytogenes (7), and Mycobacterium marinum (8). This chapter describes the use of the GFP-promoter-probe plasmid, pJSK411, which is suitable for the evaluation of differential gene expression in Neisseria meningitidis (Fig.1). Fig. 1. Map of pJSK411 demonstrating restriction sites within the multiple cloning site. The binding site for the 401 US primer overlies the XhoI site and the 41 1DS primer binding site lies within the coding region of GFPmut3.

High-throughput identification of conditionally essential genes in bacteria: from STM to TSM.

Signature-tagged mutagenesis (STM) provided the first widely applicable high-throughput method for detecting conditionally essential genes in bacteria by using negative selection to screen large pools of transposon (Tn) mutants. STM requires no prior knowledge of the bacterium's genome sequence, and has been used to study a large number of Gram-positive and Gram-negative species, greatly expanding the repertoires of known virulence factors for these organisms. Originally, hybridization of radiolabelled probes to colony or dot blots was used to detect differences in populations of tagged mutants before and after growth under a selective condition. Modifications of the tag detection method involving polymerase chain reaction (PCR) amplification and visualisation by gel electrophoresis have been developed and can be automated through the use of robotics. Genetic footprinting is another negative selection technique that uses PCR amplification to detect loss of mutants from a pool. Unlike PCR-STM, this technique allows direct amplification of Tn-flanking sequences. However, it requires the bacterium's whole genome sequence in order to design specific primers for every gene of interest. More recently, a number of techniques have been described that combine the negative-selection principle of STM and genetic footprinting with the genome-wide screening power of DNA microarrays. These techniques, although also requiring whole genome sequences, use either a form of linker-mediated or semi-random PCR to amplify and label Tn-flanking regions for hybridization to microarrays. The superior sensitivity microarray detection allows greater numbers of mutants to be screened per pool, as well as determination of the coverage/distribution of insertions in the library prior to screening, two significant advantages over STM.

The genetics of encapsulation in Haemophilus influenzae.

In Haemophilus influenzae type b (Hib) strains the cap locus with very few exceptions contains an unstable direct repeat of approximately 17 kb of DNA flanking an approximately 1-kb bridge region containing the gene bexA. Each repeat contains genes necessary for polysaccharide synthesis, export, and surface expression, with BexA a critical component of the polysaccharide exporter. Only rare Hib strains have been identified in which cap lacks a direct repeat, though this is the norm for non-b serotypes. Examination of the ends of this single-copy locus shows that cap has the structure of a compound transposon: Copies of the insertion element IS1016 flank the gene cluster. This gives strains the capacity to amplify genes at cap by unequal homologous recombination. The cap duplication in Hib strains--subserving augmented production of polysaccharide--has apparently arisen in this way and become fixed in the population through deletion of one copy of bexA.

Pyridoxine for neonatal seizures: an unexpected danger.

A case of pyridoxine-dependent seizures is reported. Administration of pyridoxine to an infant after a long period of convulsions was followed by acute hypotonia. Other cases have been reported in the literature, in one of which assisted ventilation was required. A possible mechanism for this alarming outcome is discussed, and it is suggested that resuscitation facilities should be quickly available during such trials.

Type b capsular polysaccharide as a virulence factor of Haemophilus influenzae.

The capsular polysaccharide of Haemophilus influenzae serotype b [(3)-beta-D-ribose-(1-1)-ribitol-5-phosphate] is a major virulence factor and a target for serum antibodies which protect individuals against invasive infections. Studies in an experimental rat model of meningitis, using genetically defined H. influenzae transformants, provide evidence that chromosomal genes within or limited to a region (cap b) containing genes necessary for type b capsule are critical for efficient intravascular survival of H. influenzae. Within cap b there is a duplication of a 17 kb region organized as direct repeats separated by a smaller (1-2 kb) region of non-repeated DNA. Homologous recombination between the direct repeats is rec dependent and results in high-frequency loss of capsule expression and virulence.