Iron-sparing response of Mycobacterium avium subsp. paratuberculosis is strain dependent

Abstract

Background: Two 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.

Results: We first complemented M.smegmatisΔideR 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).

Conclusions: Taken 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.

Figure 1

Transcriptome and proteome comparisons: Venn diagram showing the comparison of transcripts and proteins that were differentially expressed at a fold change of 1.5 or greater in cattle or sheep MAP strains in response to iron. One third of the genes differentially expressed in response to iron were represented in both the transcriptome and the proteome.

Figure 2

Repression of non-essential iron using proteins under iron-limiting conditions by cattle MAP strain: Reporter ion regions (114 - 117 m/z) of peptide tandem mass spectrum from iTRAQ labeled peptides from MAP3698c, MAP3697c and MAP1201c are shown. Quantitation of peptides and inferred proteins are made from relative peak areas of reporter ions. Peptides obtained from cattle MAP cultures grown in iron-replete and iron-limiting medium were labeled with 114 and 115 reporter ions, respectively.. Peptides obtained from sheep MAP cultures grown in iron-replete and iron-limiting medium were labeled with 116 and 117 reporter ions, respectively. The peptide sequences and shown in the parenthesis and the red dashed line illustrates the reporter ion relative peak intensities. Cattle strain of MAP shows an iron sparing response by downregulating expression of iron using proteins.

Figure 3

Peptide quantitation of proteins expressed by C and S MAP strains under iron-replete conditions: Reporter ion regions (114 - 117 m/z) of peptide tandem mass spectrum from iTRAQ labeled peptides from the (A) 35-kDa major membrane protein (MAP2121c) and (B) BfrA, and the intergenic regions of MAP1508-1509 and MAP2566-2567c. Quantitation of peptides and inferred proteins are made from relative peak areas of reporter ions. Several unique peptides (>95% confidence) were mapped to each protein. However, only one representative peptide is shown for each protein. Peptides obtained from cattle MAP cultures grown in iron-replete and iron-limiting medium were labeled with 114 and 115 reporter ions, respectively. Peptides obtained from sheep MAP cultures grown in iron-replete and iron-limiting medium were labeled with 116 and 117 reporter ions, respectively. The peptide sequences and shown in the parenthesis and the red dashed line illustrates the reporter ion relative peak intensities. MAP2121c alone was upregulated in the sheep MAP strain under iron-replete conditions.

Figure 4

Proteins expressed by type II MAP under iron-replete conditions: Proteins upregulated in cattle MAP strain whereas downregulated in sheep strain in the presence of iron. Fold change for each target is calculated and represented as a ratio of iron-replete/iron-limitation. A negative fold change represents repression and a positive fold change indicates de-repression of that particular target gene in the presence of iron. MhuD = mycobacterial heme utilization, degrader; USP = universal stress protein; CHP = conserved hypothetical protein; MIHF = mycobacterial integration host factor; CsbD = general stress response protein

Figure 5

Iron dependent metabolic programming in cattle and sheep MAP: Under iron-replete conditions, there is upregulation of ribosomal proteins, bacterioferritin, mycobacterial heme, utilization and degrader proteins in cattle strain alone. Under iron limiting conditions, siderophore synthesis and transport genes are upregulated in both cattleI and sheep MAP strains. However, under iron limitation there is downregulation of aconitase, succinate dehydrogenases and superoxide dismutase in cattle MAP strain alone. This suggests an iron-sparing response exclusively in cattle but not sheep strain.