Identification of a lineage specific zinc responsive genomic island in Mycobacterium avium ssp. paratuberculosis

Abstract

Background: Maintenance of metal homeostasis is crucial in bacterial pathogenicity as metal starvation is the most important mechanism in the nutritional immunity strategy of host cells. Thus, pathogenic bacteria have evolved sensitive metal scavenging systems to overcome this particular host defence mechanism. The ruminant pathogen Mycobacterium avium ssp. paratuberculosis (MAP) displays a unique gut tropism and causes a chronic progressive intestinal inflammation. MAP possesses eight conserved lineage specific large sequence polymorphisms (LSP), which distinguish MAP from its ancestral M. avium ssp. hominissuis or other M. avium subspecies. LSP14 and LSP15 harbour many genes proposed to be involved in metal homeostasis and have been suggested to substitute for a MAP specific, impaired mycobactin synthesis.

Results: In the present study, we found that a LSP14 located putative IrtAB-like iron transporter encoded by mptABC was induced by zinc but not by iron starvation. Heterologous reporter gene assays with the lacZ gene under control of the mptABC promoter in M. smegmatis (MSMEG) and in a MSMEG∆furB deletion mutant revealed a zinc dependent, metalloregulator FurB mediated expression of mptABC via a conserved mycobacterial FurB recognition site. Deep sequencing of RNA from MAP cultures treated with the zinc chelator TPEN revealed that 70 genes responded to zinc limitation. Remarkably, 45 of these genes were located on a large genomic island of approximately 90 kb which harboured LSP14 and LSP15. Thirty-five of these genes were predicted to be controlled by FurB, due to the presence of putative binding sites. This clustering of zinc responsive genes was exclusively found in MAP and not in other mycobacteria.

Conclusions: Our data revealed a particular genomic signature for MAP given by a unique zinc specific locus, thereby suggesting an exceptional relevance of zinc for the metabolism of MAP. MAP seems to be well adapted to maintain zinc homeostasis which might contribute to the peculiarity of MAP pathogenicity.

Figure 1

Metal dependent regulation of a M. avium ssp. paratuberculosis specific gene locus. MAPwt was grown in MB-complete to an OD₆₀₀ of 1.0 and treated with different chelating agents and supplements as described in Methods. After RNA extraction, changes in gene expression levels of mbtB (black bars), mptA (white bars) and sidA (grey bars) were analysed by qRT-PCR. (A) 200 μM 2,2-bipyridyl (DIP) for 2 h. (B) 14 mM nitrilotriacetic acid (NTA) for 24 h. (C) NTA treated cultures (14 mM, 24 h) supplemented with 1 mM ZnSO₄, FeSO₄, MgCl₂, CaCl₂, CuSO₄, CoCl₂ or MnSO₄. (D) 10 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN) for 2 h. (E) TPEN treated cultures (10 μM, 2 h) supplemented with ZnSO₄ or FeSO₄ both in a final concentration of 7.5 μM. Shown are the results of at least three independent experiments (mean ± SEM). Results were normalized to the housekeeping gene gap and are expressed as fold change compared to the untreated controls. Statistical analyses were performed using Kruskal-Wallis test (C) with *p < 0.01 and ***p < 0.0001 or Mann–Whitney test (E) with ***p < 0.0001.

Figure 2

Organisation and Zur dependent regulation of a MAP specific ABC transporter. (A) Analysis of the mptABC promoter. MAPwt was grown in MB-complete to an OD₆₀₀ of 1.0 and treated 2 h with 10 μM TPEN. Transcription start sites (TSS) were determined by 5’RACE. Depicted is the putative organisation of the mptABC promoter region [NCBI:NC_002944] (position 4158368 to 4158826). TSS and putative translation start sites (TLS) according to NCBI (NCBI) and 5’RACE results (RACE) are indicated in bold. A putative −10 promoter site is highlighted grey, putative Zur boxes and a ribosome binding site (RBS) are underlined. (B) Heterologous expression and regulation of the mptABC operon in MSMEG. MSMEG was transformed with pMP1102, cultured in MB-complete and treated with TPEN as described above. Gene expression of mptA was analysed by qRT-PCR. Bars represent the relative fold change of the treated transformant (wt+) to the untreated control (wt-) (three independent experiments, mean ± SEM). Statistical analysis was performed using Mann–Whitney test with **p < 0.005. (C) Analysis of Zur binding sites in MAP by FIMO analysis. Upper panel: consensus sequence of Mtb-Zur [10] used for FIMO. Middle panel: Zur box3 of mptA. Lower panel: mutated mptA Zur box3, black arrows indicate mutated nucleotides. (D) Zur box analysis of the mptABC operon by β-galactosidase assay. MSMEGwt was transformed with the indicated lacZ-reporter plasmids: mptA2, mptA8, mptA3 and mptA2-MUT. Strains were grown in MB-complete and treated with TPEN as described above (black bars) or left untreated (white bars). Proteins were extracted and promoter activity was analysed by β-galactosidase assay (three independent experiments, mean ± SEM). Activity was measured at a wavelength of 405 nm and related to mg protein per ml. Statistical analysis was performed by using the Kruskal-Wallis-Test with *p >0.01 and ***p >0.0001.

Figure 3

Analysis of mpt A regulation by FurB by heterologous expression in M. smegmatis ∆ fur B (MSMEG∆ fur B). (A) FurB amino acid sequences of MAP, Mtb and M. smegmatis (MSMEG) were compared using ClustalOmega multiple sequence alignment. Asterices indicate homologue amino acids, grey arrows show highly conserved functional sites, black arrows structural sites (according to [39]). (B) MSMEG∆furB was transformed with pMP1102, grown in MB-complete to an OD₆₀₀ of 1.0 and gene expression of mptA compared to MSMEG wildtype (wt) was analysed by qRT-PCR. Shown are the results of three independent experiments expressed as the relative fold change of gene expression of the ∆furB mutant to the wildtype, normalized to the housekeeping gene gap. Statistical analysis was performed using Mann–Whitney test with **p < 0.005. (C) MSMEG∆furB was transformed with pJEM15 or pJEM-mptA2, grown in MB-complete to an OD₆₀₀ of 1.0 treated with 10 μM TPEN for 2 h, proteins were extracted, concentration was determined and promoter activity of TPEN treated (black bars) and untreated cultures (white bars) was analysed by β-galactosidase assay. Results of at least three independent experiments (mean ± SEM) are shown. Activity was measured at a wavelength of 405 nm and related to mg protein per ml. Statistical analysis was performed by using the Kruskal-Wallis-Test with ***p >0.0001.

Figure 4

Organisation of a M. avium ssp. paratuberculosis specific zinc responsive genomic island (ZnGI). Depicted are the genes map3725 to map3788. Genes responsive to zinc starvation are colored either blue (no Zur box) or green (Zur box). Location of Zur boxes and genes under their control are marked by black arrows. LSP14, LSP15 and two other gene clusters are marked by white bold arrows at the bottom.