PPE51 modulates membrane integrity in <i>Mycobacterium marinum</i>

PPE proteins, which are exclusive to mycobacteria, are substrates of type VII secretion systems, also unique to this genus. Their function has been an enigma for a long time, but has recently received attention for their association with nutrient uptake across the mycobacterial outer membrane. One of these nutrient transporters is PPE51, involved in glycerol and glucose uptake in Mycobacterium tuberculosis . We investigated the role of PPE51 proteins in nutrient uptake and membrane integrity using the model organism Mycobacterium marinum , whose genome encodes four paralogs. We created single, double, and triple mutants of ppe51 and noted that the presence of the final ppe51 gene was essential. To determine if PPE51's function is conserved in M. marinum , we investigated its role in glucose and glycerol uptake and found it to be retained across species. Further analysis of its secretion mechanism revealed that PPE51 proteins are ESX-5 substrates, one of the type VII secretion systems, in line with its proposed role in nutrient transport. Notably, and in contrast to its role in glycerol transport, the ppe51 mutants presented substantially altered membrane permeability, as observed by ethidium bromide influx and increased susceptibility to high-molecular-weight antibiotics, such as rifampicin and vancomycin. Scanning electron microscopy showed that the triple mutant has gross alterations in cell morphology. Furthermore, the triple mutant demonstrated attenuation during infection experiments in murine macrophages, likely due to loss of cell-wall integrity. In summary, we analyzed the characteristics of PPE51 in more detail, revealing a new role in membrane integrity and an indirect link with virulence. Importance The impermeable outer membrane of pathogenic mycobacteria presents a major obstacle to nutrient acquisition and antibiotic penetration. PPE51, a substrate of the ESX-5 secretion system, has previously been linked to glucose and glycerol uptake. Our study in Mycobacterium marinum reveals an unexpected additional role for PPE51 in maintaining membrane integrity. Loss of PPE51 not only impairs nutrient uptake but also causes increased membrane permeability, altered antibiotic susceptibility, and reduced virulence. These findings redefine PPE51 as more than a nutrient transporter, highlighting its broader role in cell envelope stability. This dual function has important implications for understanding how mycobacteria balance impermeability with metabolic needs and suggests new strategies to enhance antibiotic efficacy by targeting membrane-associated proteins like PPE51.