Iron Acquisition in Mycobacterium avium subsp. paratuberculosis

Abstract

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.

Importance: 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.

FIG 1

M. avium subsp. paratuberculosis in vitro “dependence” on mycobactin. (A) ⁵⁵FeCl₃ uptake assay in MAP-cattle (MAPc), MAP-sheep (MAPs), and several other M. avium subspecies. (B) ⁵⁵FeCl₃ uptake assay in MAP-cattle in the presence or absence of exogenous mycobactin J. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) MAP-cattle growth assay in the presence or absence of exogenous mycobactin J on 7H10 agar. Tenfold serial dilutions of wild-type M. avium subsp. paratuberculosis K10 grown in 7H9 without mycobactin J supplementation were spotted onto 7H10 with or without mycobactin J. The plates were incubated at 37°C for 6 weeks and then photographed.

FIG 2

Investigation of mycobactin production and biosynthetic pathway. (A) TLC of mycobactins extracted from various mycobacterial cultures labeled with 7-¹⁴C salicylic acid. The bracketed region includes labeled species present in M. smegmatis, M. tuberculosis, and M. avium species but not M. avium subsp. paratuberculosis. S, secreted (medium); C, cell associated; MAA, M. avium subsp. avium; MI, M. intracellulare; MAH, M. avium subsp. hominissuis. (B) TLC of mycobactins extracted from M. intracellulare, MAP-cattle K10, and MAP-sheep S6756 and P465. The bracketed region includes labeled species present in M. intracellulare species but not M. avium subsp. paratuberculosis. S, secreted (medium); C, cell associated. (C) Organization of the mbt clusters in M. avium subsp. hominissuis, MAP-cattle (MAPc), and MAP-sheep (MAPs). Striped arrows represent protein products with less than 80% amino acid similarity.

FIG 3

Characterization of LSP^P15. (A) ⁵⁵FeCl₃ uptake assay in wild-type M. avium subsp. paratuberculosis and MAP3776c::Tn. **, P < 0.01; ***, P < 0.001. (B) ICP-MS quantification of intracellular metal contents (Fe:Mg) of strains grown in the presence of 0%, 0.01%, and 1.0% FAC for 48 h. Data are presented as means ± standard deviations of the results of two individual experiments, each performed in triplicate. *, P < 0.05 (compared with wild-type M. avium subsp. paratuberculosis intracellular Fe:Mg ratio). (C) Transcriptional analysis of LSP^P15 genes in wild-type M. avium subsp. paratuberculosis and MAP3776c::Tn, quantified by qRT-PCR. Data shown are normalized to the expression of sigA, an endogenous housekeeping gene. The arrow indicates the transposon insertion. (D) Detection of cotranscription by amplifying the junction of each gene pair by PCR. Wild-type cDNA was used as a template; water and RNA served as negative controls. Lanes corresponding to reactions performed with each set of primers are separated by 100-bp DNA ladders (Thermo Scientific).

FIG 4

Functional consequences of LSP^P15 overexpression. (A) ⁵⁵FeCl₃ uptake assay in wild-type M. avium subsp. paratuberculosis and MAP::MAP3775-2c. *, P < 0.05; ***, P < 0.001. (B) ICP-MS quantification of intracellular metal contents (Fe:Mg) of wild-type M. avium subsp. paratuberculosis and MAP::MAP3775-2c grown in 7H9 supplemented with 0%, 0.1%, or 1.0% FAC after 48 h. Data are presented as means ± standard deviations of the results of two individual experiments, each performed in triplicate. *, P < 0.05 (compared with wild-type M. avium subsp. paratuberculosis intracellular Fe:Mg ratio).