Gene Expression Profiles of Pathogenic Mycobacteria Reveal Espj as a Novel Substrate and Whib6 as a Transcriptional Regulator of Esx-1

5 SUMMARY The mycobacterial type VII secretion system ESX-1 is responsible for secretion of a number of proteins that play an important role during infection of the host. Regulation of expression of secreted proteins is often essential to establish a successful infection. Using transcriptome sequencing (RNA-seq), we found that abrogation of ESX-1 function in M. marinum leads to a pronounced increase in gene expression levels of the espA operon during infection of macrophages, suggesting an important role in ESX-1-mediated virulence during the early phase of infection. In addition, we found that disruption of ESX-1-mediated protein secretion leads to a specific down-regulation of substrates, but not the structural components of the transport machinery, in both M. marinum and M. tuberculosis during growth in culture medium. Based on these gene expression data, we could identify EspJ as a novel ESX-1 substrate that is cleaved upon transport. We established that down-regulation of ESX-1 substrates is the result of a regulatory process, which is influenced by the transcriptional regulatory gene whib6, located adjacent to the esx-1 locus. In addition, overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Together, these data show that ESX-1 substrates are regulated independently from the structural components of the transport machinery, both during infection and as a result of active secretion.