Loss of the ESX-5 secretion locus in Mycobacterium tuberculosis reshapes the mycomembrane and enhances ESX-1 substrate secretion

Abstract

The ESX-5 secretion system, uniquely found in slow-growing mycobacteria, is predicted to secrete over 150 proteins across the inner membrane of Mycobacterium tuberculosis (M.tb). Although many of these substrates are believed to promote M.tb virulence, most remain poorly characterized. Here, we use a complete locus deletion strain of ESX-5 in M.tb to examine the molecular changes caused by a broad loss in ESX-5 secretory substrates. We confirmed the selective loss of PE/PPE proteins secreted by ESX-5 into both the culture filtrate (CF) and outer mycomembrane (OMM) fractions of the M.tb {Delta}esx5 mutant. In examining other ESX systems, we found that ESX-1 substrate levels were increased in both the CF and OMM fractions of the {Delta}esx5 mutant. Conversely, the ESX-3 locus was transcriptionally repressed upon ESX-5 deletion. We noted that the {Delta}esx5 mutant had altered morphology in the form of wrinkled distortions of the bacterial surface. Likewise, we identified increased susceptibility of the {Delta}esx5 mutant to a variety of large (molecular weight >550 g/mol) antimicrobial compounds, suggesting that an intact ESX-5 system is required for M.tb to exclude such molecules. Our findings suggest that removing the ESX-5 system from M.tb fundamentally alters the properties of the mycobacterial OMM and impacts the expression and secretion activity of other ESX systems. Significance StatementMycobacterium tuberculosis (M.tb) uses the ESX-5 secretion system to export numerous proteins that shape host-pathogen interactions. Here, we found that deleting ESX-5 from M.tb not only prevented the secretion of many ESX-5 substrates but also impacted other ESX systems. The M.tb {Delta}esx5 mutant had increased ESX-1 substrate secretion but reduced ESX-3 expression. In addition, the M.tb {Delta}esx5 mutant displayed altered cell surface morphology and increased vulnerability to large antibiotic drugs, suggesting a critical role for ESX-5 for maintaining outer membrane integrity. These findings highlight ESX-5 as a central modulator of secretion and cell envelope composition with implications for drug targeting and vaccine development.