Elucidating the regulon of a fur-like protein in Mycobacterium avium subsp. paratuberculosis (MAP)

Authors: SHOYAMA, FERNANDA - Michigan State University; JANETANAKIT, TAVEESAK - Chulalongkorn University; Bannantine, John; BARLETTA, RAUL - University Of Nebraska; SREEVATSAN, SRINAND - Michigan State University

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/18/2019
Publication Date: 4/23/2020
Citation: Shoyama, F.M., Janetanakit, T., Bannantine, J.P., Barletta, R., Sreevatsan, S. 2020. Elucidating the regulon of a fur-like protein in Mycobacterium avium subsp. paratuberculosis (MAP). Frontiers in Microbiology. 11:598. https://doi.org/10.3389/fmicb.2020.00598.
DOI: https://doi.org/10.3389/fmicb.2020.00598

Interpretive Summary: Iron plays a huge role in pathogenesis as the host makes extreme attempts to limit available iron that the pathogen can use. In this study, we examined counter measures the pathogen uses to collect iron from the host. Our previous studies enabled us to key in on a single M. avium subspecies paratuberculosis (Map) gene that regulates iron levels in this bacteria. This gene expresses a protein, called Fur (Ferric iron Uptake Regulator) that binds to other Map genes involved in iron uptake for metabolism. We used a technique called Chip-Seq to identify these genes. From this analysis we could identify the consensus DNA sequence that Fur binds to. Thus we were able to identify an important regulatory network used by Map, the agent that causes John's disease.

Technical Abstract: Intrabacterial iron concentration is tightly regulated to maintain cell viability. Iron plays important roles in electron transport, nucleic acid synthesis and oxidative stress. A Mycobacterium avium subsp. paratuberculosis (MAP)- specific genomic island carries a putative metal transport operon that includes MAP3773c, which encodes a Fur-like protein. Although well characterized as a global regulator of iron homeostasis in multiple bacteria, the function of Fur (ferric uptake regulator) in MAP is unknown as this organism also carries, IdeR (iron dependent regulator), a native iron regulatory protein specific to mycobacteria. Computational analysis using PRODORIC identified 23 different pathways involved in respiration, metabolism and virulence that were likely regulated by MAP3773c. Thus, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was performed to confirm the putative regulon of MAP3773c (Fur-like protein) in MAP. ChIP-Seq revealed enriched binding to 58 regions by Fur under iron replete and deplete conditions, located mostly within open reading frames (ORF). Four ChIP peaks were identified in genes that are directly related to iron regulation: MAP2961c (siderophore-interacting protein), MAP3638c (hemophore-like protein), MAP3736c (Fur box) and MAP3776c (ABC transporter). Fur box consensus sequence was identified, and binding specificity and dependence on Mn2+ availability was confirmed by a chemiluminescent electrophoresis mobility shift assay (EMSA). The results confirmed that MAP3773c is a Fur ortholog that recognizes a 19-bp DNA sequence motif (Fur box) and it is involved in metal homeostasis. This work provides a regulatory network of MAP Fur binding sites during iron replete and deplete conditions, highlighting unique properties of Fur regulon in MAP.