Tim Bull

Characterization of IS900 loci in mycobacterium avium subsp. paratuberculosis and development of multiplex PCR typing

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes chronic inflammation of the intestine in many animals, including primates, and is implicated in Crohns disease in humans. It differs from other members of the M. avium complex in having 14-18 copies of IS900 inserted into conserved loci in its genome. In the present study, genomic DNA flanking 14 of these insertions was characterized and homologues in the Mycobacterium tuberculosis and M. avium subsp. avium genomes were identified. These included regions encoding a sigma factor (sigJ) at locus 3, a nitrate reductase (nirA) at locus 4, a transcription regulator (tetR) and polyketide synthase at locus 6, and a 6-O-methylguanine methyltransferase at locus 9. In addition, locus numbers were assigned to 9 of 15 RFLP bands previously described. IS900 insertion at 7 of the 14 characterized loci was into the RBS of a gene substituting an RBS encoded by IS900 sited two bases closer to the initiation codon. IS900 insertion at five loci interrupted an ORF at the target site, one of which encoded a homologue of the immunodominant mycobacterial DesA1 protein. Eleven of eighty-one M. avium subsp. paratuberculosis isolates lacked the insertion site at locus 6 together with flanking genomic DNA. This region was also absent from seven reference strains of M. avium subsp. avium, from one M. avium subsp. silvaticum and from six other mycobacterial species. A multiplex PCR of IS900 loci (MPIL) typing method was developed which was able to discriminate 10 different types of M. avium subsp. paratuberculosis from the panel of 81 isolates with consistent differences between those of bovine and ovine origin. Nine MPIL types corresponded with a single PstI/Bst:EII RFLP type, suggesting that this method may be applicable to typing of M. avium subsp. paratuberculosis directly from a sample without the need for culture. The remaining MPIL type corresponded with seven PstI/BstEII RFLP types. Further resolution of these may come from sequencing the remaining four uncharacterized IS900 loci.

Mycobacterium avium Subspecies paratuberculosis Infection in Cases of Irritable Bowel Syndrome and Comparison with Crohn's Disease and Johne's Disease: Common Neural and Immune Pathogenicities

ABSTRACT Mycobacterium avium subsp. paratuberculosis causes Johnes disease, a systemic infection and chronic inflammation of the intestine that affects many species, including primates. Infection is widespread in livestock, and human populations are exposed. Johnes disease is associated with immune dysregulation, with involvement of the enteric nervous system overlapping with features of irritable bowel syndrome in humans. The present study was designed to look for an association between Mycobacterium avium subsp. paratuberculosis infection and irritable bowel syndrome. Mucosal biopsy specimens from the ileum and the ascending and descending colon were obtained from patients with irritable bowel syndrome attending the University of Sassari, Sassari, Sardinia, Italy. Crohns disease and healthy control groups were also included. Mycobacterium avium subsp. paratuberculosis was detected by IS900 PCR with amplicon sequencing. Data on the potential risk factors for human exposure to these pathogens and on isolates from Sardinian dairy sheep were also obtained. Mycobacterium avium subsp. paratuberculosis was detected in 15 of 20 (75%) patients with irritable bowel syndrome, 3 of 20 (15%) healthy controls, and 20 of 23 (87%) people with Crohns disease (P = 0.0003 for irritable bowel syndrome patients versus healthy controls and P = 0.0000 for Crohns disease patients versus healthy controls). One subject in each group had a conserved single-nucleotide polymorphism at position 247 of IS900 that was also found in isolates from seven of eight dairy sheep. There was a significant association (P = 0.0018) between Mycobacterium avium subsp. paratuberculosis infection and the consumption of hand-made cheese. Mycobacterium avium subsp. paratuberculosis is a candidate pathogen in the causation of a proportion of cases of irritable bowel syndrome as well as in Crohns disease.

Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex

Mycobacterial interspersed repetitive units (MIRU) comprise short tandem repeat structures found at multiple loci throughout the Mycobacterium tuberculosis genome and have been used for typing these pathogens. We have identified MIRU at 18 conserved loci throughout the common portions of the Mycobacterium avium subspecies paratuberculosis (MAP) and M. avium subspecies avium (MAA) genomes. Six of these loci were found to differ between MAA and MAP in the number of tandem repeat motifs occurring at each MIRU locus. Locus specific PCR at 4 of these loci segregated MAP into two major groups, which could be differentiated from ovine-pigmented strains of MAP and the MAP vaccine strain 316F. The same PCR differentiated MAA into five MIRU profiles. PCR at either MIRU locus 1 or MIRU locus 4 distinguished between MAP and all other M. avium complex (MAC) tested. PCR at both loci 1 and 4 also distinguished MAP from Mycobacterium intracellulare. MIRU typing may provide an additional simple and rapid procedure for the differentiation between MAP and other MAC.

Replication and Long-Term Persistence of Bovine and Human Strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

ABSTRACT Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johnes disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohns disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.

Exploring the zoonotic potential of Mycobacterium avium subspecies paratuberculosis through comparative genomics

A comparative genomics approach was utilised to compare the genomes of Mycobacterium avium subspecies paratuberculosis (MAP) isolated from early onset paediatric Crohns disease (CD) patients as well as Johnes diseased animals. Draft genome sequences were produced for MAP isolates derived from four CD patients, one ulcerative colitis (UC) patient, and two non-inflammatory bowel disease (IBD) control individuals using Illumina sequencing, complemented by comparative genome hybridisation (CGH). MAP isolates derived from two bovine and one ovine host were also subjected to whole genome sequencing and CGH. All seven human derived MAP isolates were highly genetically similar and clustered together with one bovine type isolate following phylogenetic analysis. Three other sequenced isolates (including the reference bovine derived isolate K10) were genetically distinct. The human isolates contained two large tandem duplications, the organisations of which were confirmed by PCR. Designated vGI-17 and vGI-18 these duplications spanned 63 and 109 open reading frames, respectively. PCR screening of over 30 additional MAP isolates (3 human derived, 27 animal derived and one environmental isolate) confirmed that vGI-17 and vGI-18 are common across many isolates. Quantitative real-time PCR of vGI-17 demonstrated that the proportion of cells containing the vGI-17 duplication varied between 0.01 to 15% amongst isolates with human isolates containing a higher proportion of vGI-17 compared to most animal isolates. These findings suggest these duplications are transient genomic rearrangements. We hypothesise that the over-representation of vGI-17 in human derived MAP strains may enhance their ability to infect or persist within a human host by increasing genome redundancy and conferring crude regulation of protein expression across biologically important regions.

Recently recognized mycobacteria of clinical significance

In 1885. 3 years after Koch’s discovery of the tubercle bacillus, mycobacteria other than 1M. tubemdosis were found in human secretions. Occasional cases of nontuberculous infection were subsequently described over the next seven decades but it was not until 1948, with the discovery of ,M~coDacteriun~ ulcer-am, that these organisms were recognized as important human pathogens. In 1949. a case of disseminated Mycobacteriurn intracellulare was described and followed, over the next 5 years. by reports of cases of infection caused by M. fortuitmm, M. marinzm, M. kansasii, ICI. aviun? and M. chelonae. In the 1980s with the advent of the AIDS era the situation changed dramatically. There was almost an exponential increase in the incidence of disseminated nontuberculous infection, and reports of, what were previously thought to be. non-pathogenic mycobacteria causing disease in humans. At this time not only was there increased awareness of any mycobacteria being a potential pathogen but also laboratory methods for identification were being improved and the use of molecular techniques for identification, drug susceptibility and epidemiology developed. The mere presence of non-tuberculous mycobacteria, however, does not necessarily indicate the presence of disease and when such mycobacteria are recovered from non-sterile anatomical sites such as the respiratory tract or gut, it is important to try and distinguish infection from colonization from contamination. In order to address this issue. clinical diagnostic criteria have been developed,l but it is important to note that they may not be wholly applicable to immunodeficient patients. In the last 30 years several new important mycobacterial species of clinical significance have been recognized. Some such as M. genevensae cause disease almost exclusively in immunosuppressed patients whereas others such as M. n~almoel?se can cause disease in the immunocompetent host. These recently discovered mycobacteria will be reviewed here.

Use of Bioinformatics to Predict a Function for the GS Element in Mycobacterium avium Subspecies paratuberculosis

Mycobacterium avium subsp. paratuberculosis (MAP) is a member of the Mycobacterium avium complex (MAC) and causes the inflammatory bowel disease, Johne’s disease, in livestock. MAP has also been implicated as the causative agent of a similar disease, Crohn’s disease, in humans. One of three major genetic differences between MAP and non-pathogenic MAC is the 6,496-bp GS element. Based on the output from freely available protein sequence and structural bioinformatics tools, and the close homology of GS genes with the ser2 region of the closely related Mycobacterium avium subsp. avium (MAA), we predict that GS encoded enzymes are involved in the biosynthesis of GDP-fucose, and the addition to, and modification of fucose on, the oligosaccharide moiety of GPL. GPL is a major constituent of the cell wall of the MAC and has immunomodulatory properties. Therefore, the enzymes involved in its synthesis may provide novel drug targets against MAP and other pathogenic MAC members.

Persistent survival of luminescent mycobacterium avium subspecies paratuberculosis in peripheral blood monocytes from both crohn's disease and healthy controls is associated with differential cytokine response

Introduction Abnormal intestinal macrophage function which could allow persistence of candidate bacterial triggering agents such as E coli and Mycobacterium avium subspecies paratuberculosis (MAP), has been implicated in the pathogenesis of Crohns disease (CD).1 Prolonged survival of E coli in CD-derived peripheral blood monocytes (PBM) has been demonstrated,2 however current methods of determining MAP infection are difficult and time-consuming. The application of a luminescent MAP strain in a novel rapid technique may improve survival assays.3 Aims Determine the sensitivity and accuracy of a method to quantify luminescent MAP and develop an intracellular survival assay. Monitor cytokine profiles induced during MAP infection in CD and healthy controls (HC). Methods Quantitative real-time PCR (qRT PCR) was used to calibrate a standard curve of luminescence from MAP ‘Lux’ 19 698 strain. Monocytes from CD and HC were isolated and infected with MAP lux 19 698 (multiplicity of infection 4:1) for up to 4 weeks. Luminescence was recorded at intervals to determine killing activity compared to medium only controls. Intracellular uptake was visualised using an anti-MAP antibody and confocal microscopy. Cytokine release (TNFα, IL10, IL6 and IL23) from PBM was measured using ELISA pre and 24 h post infection. Results A close linear relationship (R2= 0.9999) was seen between luminescence and number of MAP organisms by qRT PCR. Sensitivities were also similar. Monocytes from 8 CD patients and 7 HC infected with MAP lux 19 698 assayed over a mean of 15 days (range 8 to 27) showed some killing during the experiment time course. However a majority of MAP survived intracellularly (visualised by confocal microscopy) in all samples and the degree of killing was not significantly different between CD and HC. Cytokine profiling showed only a proportion of all subjects (CD or HC) PBM responded to MAP infection. Of these responders, there was a difference in cytokine profiles between CD and HC and in particular there was a strong trend to greater IL6 response in CD. Conclusion This study provides initial validation for a luminescent MAP intracellular survival assay. Consistent, persistent survival of MAP was demonstrated in both CD and controls. Alterations in cytokine responses to MAP infection between CD and normals suggest that this agent is perceived and processed differently by CD monocytes.

Use of bioinformatics to model the structure of the cell surface fucosyl transferasegsd on Mycobacterium avium subspecies paratuberculosis (MAP) and develop specific anti-peptide antibodies for the immunolocalization of these pathogens

Use of bioinformatics to model the structure of the cell surface fucosyl transferasegsd on Mycobacterium avium subspecies paratuberculosis (MAP) and develop specific anti-peptide antibodies for the immunolocalization of these pathogens