Tyloxapol inhibits ESX-1 secretion in <i>Mycobacterium marinum</i>

Mycobacteria have a hydrophobic cell envelope that makes uniform growth in liquid culture challenging. Non-ionic detergents, including Tween-80 and tyloxapol, are commonly added to media when culturing mycobacterial species in the laboratory. Tyloxapol was reported to exhibit anti-tuberculous activity during animal infection with <i>M. tuberculosis</i> in the 1950s. In the 1980s, microscopy studies suggested that tyloxapol impacted the interaction between <i>M. tuberculosis</i> and the phagosomal membrane, preventing mycobacterial access to the cytoplasm. It is now known that the ESX-1 Type VII secretion system mediates the interaction between pathogenic mycobacteria and the phagosomal membrane. <i>Mycobacterium marinum</i> is a pathogenic mycobacterial species that has been widely used to understand the molecular mechanisms and host responses to the ESX-1 system. The hemolytic activity of <i>M. marinum</i> allows the study of ESX-1 lytic activity outside of the context of a host cell. We found that tyloxapol inhibits the hemolytic activity of <i>M. marinum</i> in a concentration-dependent manner. We discovered the mechanism underlying the inhibition of hemolysis is the differential inhibition of the production and secretion of ESX-1 substrates. Proteomics analysis indicates that tyloxapol specifically inhibited the secretion of a subset of ESX-1 substrates in addition to specific PE/PPE/PEGRS proteins, but not the secretion of proteins secreted by the Sec, ESX-3, or ESX-5 secretion systems. Our findings directly impact how the field interprets data from studies where <i>M. marinum</i>, and potentially other mycobacterial species, were grown in tyloxapol. Our findings may explain the original observations linking tyloxapol to anti-tuberculosis activity.IMPORTANCETuberculosis, which is caused by <i>Mycobacterium tuberculosis</i>, is one of the world's deadliest diseases. We lack a clear understanding of how <i>M. tuberculosis</i> and related mycobacterial species cause disease. In the 1950s, it was reported that treating <i>M. tuberculosis</i>-infected animals with tyloxapol improved the survival and, in some cases, protected the animals from death. Tyloxapol is a detergent that is commonly added to mycobacterial cultures to promote dispersed growth in the laboratory. Later studies suggested that tyloxapol altered the interaction between <i>M. tuberculosis</i> and the phagosomal membrane during macrophage infection. The ability to escape the phagosome is essential for mycobacteria to cause disease and is mediated by the ESX-1 Type VII protein secretion system. Using <i>M. marinum</i>, a well-established model for understanding the molecular mechanisms of ESX-1 secretion, we show that tyloxapol used at more than 100-fold less than what is commonly used to grow mycobacteria in the lab inhibits ESX-1 secretion. Our findings have widespread implications on how we interpret our findings as a field and may explain why tyloxapol impacted <i>M. tuberculosis</i> infection of both animals and macrophages. Our study also indicates that tyloxapol can be used as a tool to understand the molecular mechanisms of ESX-1 protein secretion.