# P-1698. Geovirulence Factors Unique to M.bovis May Contribute to bTB Outcomes in the Mediterranean

## TL;DR

This study identifies a mutation in the mbtB gene of Mycobacterium bovis that may influence bovine tuberculosis outcomes and zoonotic transmission in the Mediterranean.

## Contribution

The study reveals that the R584C mutation in mbtB is prevalent in Mediterranean isolates and may affect bacterial fitness and zoonotic transmission.

## Key findings

- The R584C mutation in mbtB is present in over 90% of analyzed M. bovis isolates.
- The mutation may alter local binding interactions or enzymatic function in the mycobactin synthesis pathway.
- The high prevalence of the mutation suggests selective pressure related to iron acquisition in bovine hosts.

## Abstract

Zoonotic tuberculosis (zTB), primarily caused by Mycobacterium bovis, requires a One Health approach integrating human, animal, and environmental health. To support this, we surveyed bovine tuberculosis (bTB) in Moroccan slaughterhouses and explored links to the broader Mediterranean region. Our goal was to identify virulence factors (VFs) unique to regional isolates, highlighting potential geographic variation in bTB outbreaks and their implications for human zTB transmission.Wild Type and R584C mbtB ProteinsChimera alignment of mbtB WT and R854C (most probable conformation) mutant proteinsSTRING Pathway Analysis of mbtBAnalysis of mbtB associated proteins and pathways, implicating further downstream effector molecules and upstream cofactors.

Wild Type and R584C mbtB Proteins

Chimera alignment of mbtB WT and R854C (most probable conformation) mutant proteins

STRING Pathway Analysis of mbtB

Analysis of mbtB associated proteins and pathways, implicating further downstream effector molecules and upstream cofactors.

SNP calling was conducted using vSNP and R, with genome annotation and comparative analysis performed in Geneious. Chimera was used for proteomic visualization and structural modeling of predicted virulence factors.Top VF SNP HitsTop ten SNPs across the sample pool, demonstrating that mbtB R584C is present in 171 of the analyzed isolates. Other SNPs include regulatory proteins, and canonical VFs.

Top VF SNP Hits

Top ten SNPs across the sample pool, demonstrating that mbtB R584C is present in 171 of the analyzed isolates. Other SNPs include regulatory proteins, and canonical VFs.

The mbtB locus encodes a protein essential for the iron-scavenging siderophore complex, mycobactin. Structural modeling of the R584C variant, identified in over 90% of isolates, revealed no global conformational changes (RMSD = 1.108 Å across 840 pruned atom pairs), though localized structural perturbations were noted (RMSD = 40.970 Å across 1400 pairs). R584C maps to regions with NRPS TlmIV-like adenylation, acetyl-CoA synthetase, amino acid adenylation, and AMP-binding motifs, which are involved in substrate activation and cofactor binding. This suggests the R584C substitution could alter local binding interactions or enzymatic function.

The identification of mbtB, a core component of the mycobactin synthesis pathway, as a top mutational hotspot suggests selective pressure on iron acquisition systems in Mediterranean cattle populations. This may reflect an iron-limited microenvironment in bovine hosts, where efficient siderophore-mediated iron uptake is essential for M. bovis survival and dissemination. The high prevalence of the R584C mutation raises the possibility of a gain- or loss-of-function variant that influences bacterial fitness, with implications for zoonotic transmission and the development of strain-specific diagnostics or therapeutics. Our findings underscore the critical role of mbtB as a canonical virulence factor in M. bovis and its potential impact on bTB outcomes and zTB transmission to humans

All Authors: No reported disclosures