Tyloxapol inhibits ESX-1 secretion in Mycobacterium marinum

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 M. tuberculosis in the 1950s. In the 1980s, microscopy studies suggested that tyloxapol impacted the interaction between M. tuberculosis 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. Mycobacterium marinum 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 M. marinum 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 M. marinum 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 M. marinum, 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 Mycobacterium tuberculosis, is one of the world's deadliest diseases. We lack a clear understanding of how M. tuberculosis and related mycobacterial species cause disease. In the 1950s, it was reported that treating M. tuberculosis-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 M. tuberculosis 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 M. marinum, 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 M. tuberculosis 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.