The Fur-like regulatory protein MAP3773c modulates key metabolic pathways in Mycobacterium avium subsp. paratuberculosis under in-vitro iron starvation

Authors: THAPA, SAJANI - Michigan State University; RATHNAIAH, GOVARDHAN - University Of Nebraska; ZINNIEL, DENISE - University Of Nebraska; BARLETTA, RAUL - University Of Nebraska; Bannantine, John; HUEBNER, MARIANNE - Michigan State University; SREEVATSAN, SRINAND - Michigan State University

Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/14/2024
Publication Date: 4/18/2024
Citation: Thapa, S., Rathnaiah, G., Zinniel, D.K., Barletta, R.G., Bannantine, J.P., Huebner, M., Sreevatsan, S. 2024. The Fur-like regulatory protein MAP3773c modulates key metabolic pathways in Mycobacterium avium subsp. paratuberculosis under in-vitro iron starvation. Scientific Reports. https://doi.org/10.1038/s41598-024-59691-3.
DOI: https://doi.org/10.1038/s41598-024-59691-3

Interpretive Summary: Mycobacterium avium subsp. paratuberculosis (MAP), the bacterium causing Johne’s disease (JD), is difficult to grow in the lab; often requiring eight to sixteen weeks to produce colonies in culture. This represents a major hurdle in the diagnosis and control of JD. A significant gap in knowledge is the comprehensive understanding of the metabolic networks used by MAP to regulate iron under a variety of environmental conditions. Iron-binding regulatory and regulated effector proteins are well established as key virulence determinants and have been targeted as vaccines for multiple pathogens. Our preliminary work on MAP iron regulation has led to multiple discoveries, the most significant of which is the in-vivo identification of a fur-like gene. The function of Fur in iron regulation is well established in Gram-negative bacteria. However, environmental cues for MAP to deploy Fur and how this novel gene interacts with another essential transcription factor (either MAP2827c or IdeR) are unknown. Our studies have further shown that MAP likely employs alternate iron regulatory pathways, unlike other pathogenic mycobacteria. We showed that MAP3773c (fur) was acquired by horizontal gene transfer and encodes a functional protein for sensing extracellular signals and maintaining intracellular iron balance.

Technical Abstract: Mycobacterium avium subsp. paratuberculosis (MAP) has special micronutrient requirements for optimal growth in laboratory media. For example, MAP requires supplementation of a siderophore (mycobactin), and takes eight to sixteen weeks to produce colonies, a major hurdle in timely diagnosis. MAP carries MAP3773c, a putative metal regulator, on its genome which is absent in other mycobacteria. Homologues of this gene in enterobacteria have roles in global iron regulation and homeostasis. The role of MAP3773c in regulating intracellular iron in MAP is poorly understood. In the current study, a field isolate (K-10) and an in-frame MAP3773c deletion mutant ('MAP3773c) derived from K-10, were exposed to iron restricted conditions for 5, 30, 60, and 90 minutes. Total RNA was extracted at each time point, quality and integrity of the RNA was assessed and RNA-Seq was performed. A comparison of transcriptional profiles between K-10 and 'MAP3773c showed 425 differentially expressed genes (DEGs) at 30 minutes time post iron restriction. Functional analysis of DEGs in 'MAP3773c revealed that pantothenate biosynthesis, polysaccharide biosynthesis, sugar metabolism and n-acetyltransferase enhanced intracellular survival (eis) genes were downregulated at 30 minutes post iron starvation whereas arginine and proline metabolism, PPE, PE family genes and mammalian cell entry genes were upregulated 'MAP3773c strain at that same time point. Pathway analysis revealed that 'MAP3773c strain experiences an impairment in pantothenate (Pan) and CoA biosynthesis pathways at 30 minutes post iron starvation suggesting that the absence of these pathways likely affect overall metabolic process and cellular functions, affecting MAP survival and pathogenesis.