Grant S. Hotter

Production of avirulent mutants of Mycobacterium bovis with vaccine properties by the use of illegitimate recombination and screening of stationary-phase cultures

A better tuberculosis vaccine is urgently required to control the continuing epidemic. Molecular techniques are now available to produce a better live vaccine than BCG by producing avirulent strains of the Mycobacterium tuberculosis complex with known gene deletions. In this study, 1000 illegitimate recombinants of Mycobacterium bovis were produced by illegitimate recombination with fragments of mycobacterial DNA containing a kanamycin resistance gene. Eight recombinant strains were selected on the basis of their inability to grow when stationary-phase cultures were inoculated into minimal medium. Five of these recombinants were found to be avirulent when inoculated into guinea pigs. Two of the avirulent recombinants produced vaccine efficacy comparable to BCG against an aerosol challenge in guinea pigs with M. bovis. One of these recombinants had an inactivated glnA2 gene encoding a putative glutamine synthetase. Transcriptional analysis showed that inactivation of glnA2 did not affect expression of the downstream glnE gene. The other recombinant had a block of 12 genes deleted, including the sigma factor gene sigG. Two avirulent recombinants with an inactivated pckA gene, encoding phosphoenolpyruvate carboxykinase which catalyses the first step of gluconeogenesis, induced poor protection against tuberculosis. It is clear that live avirulent strains of the M. tuberculosis complex vary widely in their ability as vaccines to protect against tuberculosis. Improved models may be required to more clearly determine the difference in protective effect between BCG and potential new tuberculosis vaccines.

Transposon Mutagenesis of Mb0100 at the ppe1-nrp Locus in Mycobacterium bovis Disrupts Phthiocerol Dimycocerosate (PDIM) and Glycosylphenol-PDIM Biosynthesis, Producing an Avirulent Strain with Vaccine Properties At Least Equal to Those of M. bovis BCG

The unusual and complex cell wall of pathogenic mycobacteria plays a major role in pathogenesis, with specific complex lipids acting as defensive, offensive, or adaptive effectors of virulence. The phthiocerol and phthiodiolone dimycocerosate esters (PDIMs) comprise one such category of virulence-enhancing lipids. Recent work in several laboratories has established that the Mycobacterium tuberculosis fadD26-mmpL7 (Rv2930-Rv2942) locus plays a major role in PDIM biosynthesis and secretion and that PDIM is required for virulence. Here we describe two independent transposon mutants (WAg533 and WAg537) of Mycobacterium bovis, both of which carry an insertion in Mb0100 (= M. tuberculosis Rv0097) to reveal a new locus involved in PDIM biosynthesis. The mutations have a polar effect on expression of the downstream genes Mb0101, Mb0102 (fadD10), Mb0103, and Mb0104 (nrp), and Mb0100 is shown to be in an operon comprising these genes and Mb0099. Reverse transcription-PCR analysis shows elevated transcription of genes in the operon upstream from the transposon insertion sites in both mutants. Both mutants have altered colony morphology and do not synthesize PDIMs or glycosylphenol-PDIM. Both mutants are avirulent in a guinea pig model of tuberculosis, and when tested as a vaccine, WAg533 conferred protective immunity against M. bovis infection at least equal to that afforded by M. bovis bacillus Calmette-Guerin.

Generation of Attenuated Mycobacterium bovis Strains by Signature-Tagged Mutagenesis for Discovery of Novel Vaccine Candidates

ABSTRACT Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex, has a particularly wide host range and causes tuberculosis in most mammals, including humans. A signature tag mutagenesis approach, which employed illegitimate recombination and infection of guinea pigs, was applied to M. bovis to discover genes important for virulence and to find potential vaccine candidates. Fifteen attenuated mutants were identified, four of which produced no lesions when inoculated separately into guinea pigs. One of these four mutants had nine deleted genes including mmpL4 and sigK and, in guinea pigs with aerosol challenge, provided protection against tuberculosis at least equal to that of M. bovis BCG. Seven mutants had mutations near the esxA (esat-6) locus, and immunoblot analysis of these confirmed the essential role of other genes at this locus in the secretion of EsxA (ESAT-6) and EsxB (CFP10). Mutations in the eight other attenuated mutants were widely spread through the chromosome and included pks1, which is naturally inactivated in clinical strains of M. tuberculosis. Many genes identified were different from those found by signature tag mutagenesis of M. tuberculosis by use of a mouse infection model and illustrate how the use of different approaches enables identification of a wider range of attenuating mutants.

Whole-Genome Comparison of Two Campylobacter jejuni Isolates of the Same Sequence Type Reveals Multiple Loci of Different Ancestral Lineage

Campylobacter jejuni ST-474 is the most important human enteric pathogen in New Zealand, and yet this genotype is rarely found elsewhere in the world. Insight into the evolution of this organism was gained by a whole genome comparison of two ST-474, flaA SVR-14 isolates and other available C. jejuni isolates and genomes. The two isolates were collected from different sources, human (H22082) and retail poultry (P110b), at the same time and from the same geographical location. Solexa sequencing of each isolate resulted in 1.659 Mb (H22082) and 1.656 Mb (P110b) of assembled sequences within 28 (H22082) and 29 (P110b) contigs. We analysed 1502 genes for which we had sequences within both ST-474 isolates and within at least one of 11 C. jejuni reference genomes. Although 94.5% of genes were identical between the two ST-474 isolates, we identified 83 genes that differed by at least one nucleotide, including 55 genes with non-synonymous substitutions. These covered 101 kb and contained 672 point differences. We inferred that 22 (3.3%) of these differences were due to mutation and 650 (96.7%) were imported via recombination. Our analysis estimated 38 recombinant breakpoints within these 83 genes, which correspond to recombination events affecting at least 19 loci regions and gives a tract length estimate of 2 kb. This includes a 12 kb region displaying non-homologous recombination in one of the ST-474 genomes, with the insertion of two genes, including ykgC, a putative oxidoreductase, and a conserved hypothetical protein of unknown function. Furthermore, our analysis indicates that the source of this recombined DNA is more likely to have come from C. jejuni strains that are more closely related to ST-474. This suggests that the rates of recombination and mutation are similar in order of magnitude, but that recombination has been much more important for generating divergence between the two ST-474 isolates.

Mycobacterium bovis lipids: virulence and vaccines.

Mycobacterium bovis is an important pathogen of both domesticated and wild animals in many countries, and improved vaccines have great potential to assist in its control and eventual eradication. One of the hallmarks of members of the Mycobacterium tuberculosis complex, which includes both M. bovis and M. tuberculosis, is their ability to synthesise an impressive array of unique and complex lipids, many of which act as defensive, offensive or adaptive effectors of virulence. For example, studies focussed on the development of rationally attenuated strains of both M. bovis and M. tuberculosis with efficacy as animal or human vaccines have shown that the phthiocerol dimycocerosates (PDIMs) and glycosylphenol-PDIM (phenolic glycolipid, PGL) are key virulence factors. The availability of the genome sequences for M. bovis and M. tuberculosis, together with mutants of these organisms carrying defects in lipid biosynthesis, and biochemical and molecular tools to dissect lipid biosynthesis pathways, has enabled developments in our understanding of the biosynthesis of PDIMs and PGL, as well as the possible roles played by PDIMs and PGL in virulence. In this article we review some of these developments, and also propose a cryptic lipid biosynthesis pathway in M. bovis and M. tuberculosis that may be involved in the production of an unrecognised, virulence-associated lipopeptide.

Binary genomotyping using lipooligosaccharide biosynthesis genes distinguishes between Campylobacter jejuni isolates within poultry-associated multilocus sequence types.

Campylobacter jejuni is a leading cause of human bacterial gastroenteritis throughout the industrialized world. We investigated whether or not differences in gene complement at the lipooligosaccharide (LOS) biosynthesis locus can identify epidemiologically useful binary genomotypes in 87 C. jejuni isolates from poultry-associated multilocus sequence types (STs) collected during the course of a sentinel surveillance study. Using a PCR-based approach, we correlated assignment of both isolate LOS locus class and binary genomotype with ST. We found that isolates within STs 45, 190, 354 and 474 displayed mosaicism in gene complement at the intra-ST level. For example, based upon their binary genomotypes, we assigned individual ST-45 isolates from human clinical cases as probably originating from either a poultry or wild-bird source. However, intra-ST mosaicism in gene complement was observed alongside broader patterns of congruence in LOS locus class and gene complement that distinguished between isolates from different STs.

Campylobacter jejuni colonization and population structure in urban populations of ducks and starlings in New Zealand

A repeated cross‐sectional study was conducted to determine the prevalence of Campylobacter spp. and the population structure of C. jejuni in European starlings and ducks cohabiting multiple public access sites in an urban area of New Zealand. The countrys geographical isolation and relatively recent history of introduction of wild bird species, including the European starling and mallard duck, create an ideal setting to explore the impact of geographical separation on the population biology of C. jejuni, as well as potential public health implications. A total of 716 starling and 720 duck fecal samples were collected and screened for C. jejuni over a 12 month period. This study combined molecular genotyping, population genetics and epidemiological modeling and revealed: (i) higher Campylobacter spp. isolation in starlings (46%) compared with ducks (30%), but similar isolation of C. jejuni in ducks (23%) and starlings (21%), (ii) significant associations between the isolation of Campylobacter spp. and host species, sampling location and time of year using logistic regression, (iii) evidence of population differentiation, as indicated by FST, and host‐genotype association with clonal complexes CC ST‐177 and CC ST‐682 associated with starlings, and clonal complexes CC ST‐1034, CC ST‐692, and CC ST‐1332 associated with ducks, and (iv) greater genetic diversity and genotype richness in ducks compared with starlings. These findings provide evidence that host‐associated genotypes, such as the starling‐associated ST‐177 and ST‐682, represent lineages that were introduced with the host species in the 19th century. The isolation of sequence types associated with human disease in New Zealand indicate that wild ducks and starlings need to be considered as a potential public health risk, particularly in urban areas.

Newly attenuated Mycobacterium bovis mutants as vaccines against bovine tuberculosis, particularly for possums

Bovine tuberculosis costs New Zealand more than

Identification of a cadmium‐induced gene in Mycobacterium bovis and Mycobacterium tuberculosis

80 million per year, mostly because extensive areas of the country are occupied by brushtail possums infected with Mycobacterium bovis. AgResearch has a major programme to produce new live tuberculosis vaccines that can be delivered to possums. Primary work involved development of molecular biological methods to enable genetic manipulation of M. bovis, including the production of random and specific mutants. Many avirulent mutants of M. bovis have been produced and their vaccine efficacy has been compared to BCG in guinea pigs. Selected mutants that perform at least as well as BCG are retested in guinea pigs using an extended vaccination protocol in which animals are pre-sensitized to environmental mycobacteria to mimic natural exposure. Ten candidate vaccines that have induced good protection in guinea pigs have been subsequently tested as vaccines in possums. While the protective efficacy of an M. bovis mutant inoculated into guinea pigs reliably indicated that some protection would be induced in possums, the most protective mutant in guinea pigs was different from that in possums. This illustrates the importance of testing in the target species as part of new vaccine development. An important outcome of this work was the identification of an operon in M. bovis whose inactivation produced an avirulent M. bovis vaccine candidate that was better than BCG in protecting possums from experimental tuberculosis. Allelic exchange methods are now being used to produce vaccine strains with multiple specific mutations to improve safety and immunological characteristics.