John Simon Kroll

Bacterial [Cu,Zn]-superoxide dismutase : phylogenetically distinct from the eukaryotic enzyme, and not so rare after all !

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 Haemophilus, 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.

Two TonB Systems in Actinobacillus pleuropneumoniae: Their Roles in Iron Acquisition and Virulence

ABSTRACT Iron acquisition in vivo by Actinobacillus pleuropneumoniae depends upon a functional TonB system. Tonpitak et al. (W. Tonpitak, S. Thiede, W. Oswald, N. Baltes, and G.-F. Gerlach, Infect. Immun. 68:1164-1170, 2000) have described one such system, associated with tbpBA encoding the transferrin receptor, and here we report a second, termed tonB2. This gene cluster (exbB2-exbD2-tonB2) is highly homologous to those in other Pasteurellaceae, unlike the earlier system described (now termed tonB1), suggesting that it is the indigenous system for this organism. Both tonB2 and tonB1 are upregulated upon iron restriction. TonB2, but not TonB1, was found to be essential for growth in vitro when the sole source of iron was hemin, porcine hemoglobin, or ferrichrome. In the case of iron provided as iron-loaded porcine transferrin, neither tonB mutant was viable. The tonB1 phenotype could be explained by a polar effect of the mutation on transcription of downstream tbp genes. We propose that TonB2 is crucial for the acquisition of iron provided in this form, interacting with accessory proteins of the TonB1 system that have been demonstrated to be necessary by Tonpitak et al. TonB2 appears to play a much more important role in A. pleuropneumoniae virulence than TonB1. In an acute porcine infection model, the tonB2 mutant was found to be highly attenuated, while the tonB1 mutant was not. We hypothesize that acquisition of the tonB1-tbp gene cluster confers a biological advantage through its capacity to utilize transferrin-iron but that TonB1 itself plays little or no part in this process.

Multiplex PCR That Can Distinguish between Immunologically Cross- Reactive Serovar 3, 6, and 8 Actinobacillus pleuropneumoniae Strains

ABSTRACT 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.

[Cu, Zn]-superoxide dismutase mutants of the swine pathogen Actinobacillus pleuropneumoniae are unattenuated in infections of the natural host

ABSTRACT Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, contains a periplasmic Cu- and Zn-cofactored superoxide dismutase ([Cu,Zn]-SOD, or SodC) which has the potential, realized in other pathogens, to promote bacterial survival during infection by dismutating host-defense-derived superoxide. Here we describe the construction of a site-specific, [Cu,Zn]-SOD-deficientA. pleuropneumoniae serotype 1 mutant and show that although the mutant is highly sensitive to the microbicidal action of superoxide in vitro, it remains fully virulent in experimental pulmonary infection in pigs.

Molecular and genetic characterization of superoxide dismutase in Haemophilus influenzae type b

Oxygen free radicals present a serious potential threat to microbial survival, through their ability to inflict Indiscriminate damage on proteins and DNA. Superoxide dismutase (SOD, EC 1.15.1.1), among other oxygen‐metabolizing enzymes, is essential to prevent these toxic molecules from accumulating in the bacterial cytosol during aerobic metabolism. The gene sodA, encoding manganese‐containing SOD ([Mn]‐SOD), has been cloned from a virulent strain of Haemophilus influenzae type b using degenerate oligonucleotides encoding regions of the gene conserved across different bacterial species. The gene product has been identified as [Mn]‐SOD by its similarity at key amino acid residues to known examples of the enzyme, by expression of enzymatically active protein from cloned DNA expressed in Escherichia coli, and by demonstration that an in‐frame deletion in the gene abolishes this activity. In contrast to the situation in E. coli, this [Mn]‐SOD is the only active SOD detected in H. influenzae. In further contrast to E. coli, [Mn]‐SOD gene expression in H. influenzae has been found to be only partially repressed under anaerobic conditions. When expressed in E. coli the gene is regulated by Fur and Fnr, and the promoter region, identified experimentally, has been found to contain nucleotide sequence motifs similar to the Fur‐ and Fnr‐binding sequences of E. coli, suggesting the involvement of analogues of these aerobiosis‐ responsive activators in H. influenzae gene expression.

Prevalence of Actinobacillus pleuropneumoniae serovars in England and Wales

Actinobacillus pleuropneumoniae is the causative agent of porcine pleuropneumonia, an economically important endemic bacterial disease of pigs worldwide ([Bosse and others 2002][1]). Epidemiologically, serotyping is the gold standard method, with A pleuropneumoniae being classified into 16

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 organisms 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.

The neonatal gastrointestinal microbiota: the foundation of future health?

There is a developing appreciation of the abundance and diversity of the trillions of micro-organisms that live on and within us,1 ,2 and how they influence human health and disease.3 Outnumbering human cells in our bodies by 10:1, and their genes outnumbering ours by 100:1,4 bacteria in the gastrointestinal (GI) tract have the potential to significantly modulate human metabolism.5 Previous studies of the microbiota (all the microbes in a given environment) have been hampered by the difficulties of culturing complex samples containing fastidious or unculturable organisms, resulting in inaccurate depictions of microbial communities. Microbiota studies have now progressed to identifying organisms by their DNA sequence. A particular focus has been on the bacterial component, exploiting sequence variation in the ubiquitous 16S rRNA gene. By careful choice of primers hybridising to highly conserved domains within 16S rRNA ,6 a sufficiently large and variable region of this gene can be amplified and sequenced for organisms to be identified at least at genus level, without the bias arising from the need for culture. Utilising next-generation sequencing, which allows millions of sequencing reactions to be performed in parallel, we can determine in quantitative fashion, as never before, the composition of complex bacterial communities,7 such as exist in faecal samples, a pragmatic surrogate for the microbiota of the GI tract mucosal surface.8 Two large international initiatives, The Human Microbiome Project (HMP)9 and Metagenomics of the Human Intestinal Tract (MetaHIT),10 have used this approach to characterise the adult human microbiota at different body sites in health and disease. There is a burgeoning interest in the neonatal GI microbiota, and its association with diseases of prematurity,11 normal child development4 and future health.12 Our group is conducting a longitudinal prospective study assessing the importance of …

Antibiotic resistance potential of the healthy preterm infant gut microbiome

Background Few studies have investigated the gut microbiome of infants, fewer still preterm infants. In this study we sought to quantify and interrogate the resistome within a cohort of premature infants using shotgun metagenomic sequencing. We describe the gut microbiomes from preterm but healthy infants, characterising the taxonomic diversity identified and frequency of antibiotic resistance genes detected. Results Dominant clinically important species identified within the microbiomes included C. perfringens, K. pneumoniae and members of the Staphylococci and Enterobacter genera. Screening at the gene level we identified an average of 13 antimicrobial resistance genes per preterm infant, ranging across eight different antibiotic classes, including aminoglycosides and fluoroquinolones. Some antibiotic resistance genes were associated with clinically relevant bacteria, including the identification of mecA and high levels of Staphylococci within some infants. We were able to demonstrate that in a third of the infants the S. aureus identified was unrelated using MLST or metagenome assembly, but low abundance prevented such analysis within the remaining samples. Conclusions We found that the healthy preterm infant gut microbiomes in this study harboured a significant diversity of antibiotic resistance genes. This broad picture of resistances and the wider taxonomic diversity identified raises further caution to the use of antibiotics without consideration of the resident microbial communities.

Population-based analysis of Actinobacillus pleuropneumoniae ApxIVA for use as a DIVA antigen

APXIVA is an RTX toxin of Actinobacillus pleuropneumoniae that is a candidate antigen to differentiate infected from vaccinated animals (DIVA). Insertion of ISApl1 into the apxIVA gene is known to compromise an APXIVA-based DIVA approach, as is potentially a TGG to TGA mutation in the apxIVA gene. ISApl1 was found in 63/349 (18.1%) A. pleuropneumoniae isolates from England and Wales including serovars 2, 3, 6-8 and 12. No ISApl1 insertions into apxIVA were found. Only two serovar 3 isolates contained the TGG to TGA mutation. We conclude that an ApxIVA-based DIVA approach would potentially be viable in England and Wales.