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Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific.

ABSTRACT: Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes.

SUBMITTER: Castellanos E

PROVIDER: S-EPMC2708469 | biostudies-literature | 2009 Jul

REPOSITORIES: biostudies-literature

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Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific.

Castellanos Elena E Aranaz Alicia A de Juan Lucia L Alvarez Julio J Rodríguez Sabrina S Romero Beatriz B Bezos Javier J Stevenson Karen K Mateos Ana A Domínguez Lucas L

Journal of clinical microbiology 20090513 7

Insertion sequence IS900 is used as a target for the identification of Mycobacterium avium subsp. paratuberculosis. Previous reports have revealed single nucleotide polymorphisms within IS900. This study, which analyzed the IS900 sequences of a panel of isolates representing M. avium subsp. paratuberculosis strain types I, II, and III, revealed conserved type-specific polymorphisms that could be utilized as a tool for diagnostic and epidemiological purposes. ...[more]

PMID: 19439536

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Project description:Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease or paratuberculosis, a chronic infection affecting domestic ruminants worldwide. Despite sporadic reports of MAP occurrence in non-ruminants, information on the risk factors predisposing for infection is still scarce and evidence of transmission paths linking the livestock-wildlife-environment interfaces also remains lacking. In this study, we predicted that environmental, host-related, land use and human driven disturbance factors would modulate carnivore exposure to MAP. To test these hypotheses, we performed a retrospective survey, based on microbiological and molecular methods, in mainland Portugal including five sympatric species from the Herpestidae, Canidae, Viverridae, and Mustelidae families (n = 202) and examined 16 variables as putative predictors of MAP occurrence. Molecular evidence of MAP using IS900 as proxy was demonstrated in 7.43% (95%CI: 4.55-11.9) of surveyed carnivores, the highest proportions being registered for red fox (Vulpes vulpes) (10%; 95%CI: 4.0-23) and Egyptian mongoose (Herpestes ichneumon) (6.0%; 95%CI: 3.2-11). We demonstrate that important species of the Mediterranean carnivore guild, such as stone marten (Martes foina) and common genet (Genetta genetta), may also be exposed to MAP, being this the first time that occurrence in genet is reported. The high proportion of DNA-positive specimens, concurrent with the apparent lack of gastro-enteric lesions and molecular confirmation of IS900 in feces, argue for the presence of subclinical carriers that occasionally shed bacteria, potentially aiding as source of infection to susceptible species and possibly contributing for environmental contamination. Achievement of MAP isolation would prove beyond any doubt that MAP is present in this wildlife population. Ecological modelling results suggested that the probability of MAP infection using IS900 as proxy in mongoose is positively associated with higher altitude and temperature stability, as well as with lower annual rainfall. Density of livestock farms was found not to be a significant predictor, which may indicate that the livestock-wildlife interface is probably not important as an infection route for mongoose.

Project description:BackgroundTwo genotypically and microbiologically distinct strains of Mycobacterium avium subsp. paratuberculosis (MAP) exist - S and C MAP strains that primarily infect sheep and cattle, respectively. Concentration of iron in the cultivation medium has been suggested as one contributing factor for the observed microbiologic differences. We recently demonstrated that S strains have defective iron storage systems, leading us to propose that these strains might experience iron toxicity when excess iron is provided in the medium. To test this hypothesis, we carried out transcriptional and proteomic profiling of these MAP strains under iron-replete or -deplete conditions.ResultsWe first complemented M.smegmatis?ideR with IdeR of C MAP or that derived from S MAP and compared their transcription profiles using M. smegmatis mc(2)155 microarrays. In the presence of iron, sIdeR repressed expression of bfrA and MAP2073c, a ferritin domain containing protein suggesting that transcriptional control of iron storage may be defective in S strain. We next performed transcriptional and proteomic profiling of the two strain types of MAP under iron-deplete and -replete conditions. Under iron-replete conditions, C strain upregulated iron storage (BfrA), virulence associated (Esx-5 and antigen85 complex), and ribosomal proteins. In striking contrast, S strain downregulated these proteins under iron-replete conditions.. iTRAQ (isobaric tag for relative and absolute quantitation) based protein quantitation resulted in the identification of four unannotated proteins. Two of these were upregulated by a C MAP strain in response to iron supplementation. The iron-sparing response to iron limitation was unique to the C strain as evidenced by repression of non-essential iron utilization enzymes (aconitase and succinate dehydrogenase) and upregulation of proteins of essential function (iron transport, [Fe-S] cluster biogenesis and cell division).ConclusionsTaken together, our study revealed that C and S strains of MAP utilize divergent metabolic pathways to accommodate in vitro iron stress. The knowledge of the metabolic pathways these divergent responses play a role in are important to 1) advance our ability to culture the two different strains of MAP efficiently, 2) aid in diagnosis and control of Johne's disease, and 3) advance our understanding of MAP virulence.

Project description:Mycobacterium avium subsp. paratuberculosis is a host-adapted pathogen that evolved from the environmental bacterium M. avium subsp. hominissuis through gene loss and gene acquisition. Growth of M. avium subsp. paratuberculosis in the laboratory is enhanced by supplementation of the media with the iron-binding siderophore mycobactin J. Here we examined the production of mycobactins by related organisms and searched for an alternative iron uptake system in M. avium subsp. paratuberculosis. Through thin-layer chromatography and radiolabeled iron-uptake studies, we showed that M. avium subsp. paratuberculosis is impaired for both mycobactin synthesis and iron acquisition. Consistent with these observations, we identified several mutations, including deletions, in M. avium subsp. paratuberculosis genes coding for mycobactin synthesis. Using a transposon-mediated mutagenesis screen conditional on growth without myobactin, we identified a potential mycobactin-independent iron uptake system on a M. avium subsp. paratuberculosis-specific genomic island, LSP(P)15. We obtained a transposon (Tn) mutant with a disruption in the LSP(P)15 gene MAP3776c for targeted study. The mutant manifests increased iron uptake as well as intracellular iron content, with genes downstream of the transposon insertion (MAP3775c to MAP3772c [MAP3775-2c]) upregulated as the result of a polar effect. As an independent confirmation, we observed the same iron uptake phenotypes by overexpressing MAP3775-2c in wild-type M. avium subsp. paratuberculosis. These data indicate that the horizontally acquired LSP(P)15 genes contribute to iron acquisition by M. avium subsp. paratuberculosis, potentially allowing the subsequent loss of siderophore production by this pathogen.Many microbes are able to scavenge iron from their surroundings by producing iron-chelating siderophores. One exception is Mycobacterium avium subsp. paratuberculosis, a fastidious, slow-growing animal pathogen whose growth needs to be supported by exogenous mycobacterial siderophore (mycobactin) in the laboratory. Data presented here demonstrate that, compared to other closely related M. avium subspecies, mycobactin production and iron uptake are different in M. avium subsp. paratuberculosis, and these phenotypes may be caused by numerous deletions in its mycobactin biosynthesis pathway. Using a genomic approach, supplemented by targeted genetic and biochemical studies, we identified that LSP(P)15, a horizontally acquired genomic island, may encode an alternative iron uptake system. These findings shed light on the potential physiological consequence of horizontal gene transfer in M. avium subsp. paratuberculosis evolution.

Dataset Information

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific.

Publications

Single nucleotide polymorphisms in the IS900 sequence of Mycobacterium avium subsp. paratuberculosis are strain type specific.

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