Conserved ESX-1 Substrates EspE and EspF Are Virulence Factors That Regulate Gene Expression

Abstract

Mycobacterium tuberculosis, the cause of human tuberculosis, and Mycobacterium marinum, a non-tubercular pathogen with a broad host range, require the ESX-1 secretion system for virulence. The ESX-1 system secretes proteins which cause phagosomal lysis within the macrophage via an unknown mechanism. As reported in R.E. Bosserman et al (Proc Natl Acad Sci U S A doi:10.1073/pnas.1710167114), we recently discovered that the ESX-1 system regulates gene expression in M. marinum This finding has been confirmed in M. tuberculosis in reports by C. Sala et al (PLoS Pathog 14 (12):e1007491. doi: 10.1371/journal.ppat.1007491) and A.M. Abdallah et al (PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). We further demonstrated that a feedback control mechanism connects protein secretion to WhiB6-dependent expression of the esx-1 genes via an unknown mechanism. Here, we connect protein secretion and gene expression by showing for the first time that specific ESX-1 substrates have dual functions inside and outside the mycobacterial cell. We demonstrate that the EspE and EspF substrates negatively control esx-1 gene expression in the M. marinum cytoplasm through the conserved WhiB6 transcription factor. We found that EspE and EspF are required for virulence and promote lytic activity independently of the major EsxA and EsxB substrates. We show that the dual functions of EspE and EspF are conserved in the orthologous proteins from M. tuberculosis. Our findings support a role for EspE and EspF in virulence that is independent of the EsxA and EsxB substrates, and demonstrate that ESX-1 substrates have a conserved role in regulating gene expression.

A Small Protein but with Diverse Roles: A Review of EsxA in Mycobacterium–Host Interaction

“…EsxA mediates mycobacterial escape from the phagosome and translocation to the cytosol (here termed cytosolic translocation) through its acidic-pH-dependent membrane-permeabilizing activity (MPA) that disrupts lipid membranes at low pH ( Figure 1 ) [ 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ]. Interestingly, other mycobacterial factors are also involved in mycobacterial cytosolic translocation, and the co-dependent expression and secretion of EsxA with other factors increases the complexity of dissecting the precise roles of EsxA in mycobacterial pathogenesis [ 43 , 53 , 54 , 55 ]. Moreover, EsxA is a strong immunoregulator and interacts with multiple cellular proteins and signaling pathways [ 35 , 36 , 40 , 56 , 57 , 58 , 59 , 60 ].…”

Post-translational knockdown and post-secretional modification of EsxA determine contribution of EsxA membrane permeabilizing activity for mycobacterial intracellular survival

“…Also, deletion of other ESX-1 substrates would impact mycobacteria phagosome escape ability [ 17 , 38 ]. A recent study has reported that deletion of espE/F significantly inhibits Mm’s hemolysis, while the expression and secretion of EsxA/B were not affected [ 77 ], indicating that EsxA and EsxB are not sufficient for mycobacteria lysis-related phenotype. We also notice that deletion of espE or espF upregulates the expression and secretion of EsxA and EsxB, which provides an evidence that genetic deletion may introduce artifacts by altering expression of other genes.…”

Conserved and specialized functions of Type VII secretion systems in non-tuberculous mycobacteria

“…EspE and EspF negatively regulate the esx -1 locus through WhiB6 transcription factor (Chirakos et al ., 2020 [68], Sanchez et al ., 2020 [69]) inside cytoplasm and after secretion promote virulence and lytic activity (Chirakos et al ., 2020 [68]).…”

“…EspM negatively regulate the esx -1 locus through WhiB6 transcription factor (Chirakos et al ., 2020 [68], Sanchez et al ., 2020 [69]).…”

“…EspE, EspF and EspM have recently been demonstrated to negatively regulate esx-1 gene expression through the WhiB6 transcription factor in Mma [67–69]. EspE and EspF proteins share a dual function in Mma by negatively regulating esx-1 expression inside the cytoplasm and, after secretion, by their involvement in virulence and lytic activity unrelated to EsxA and EsxB [68]. EspH was shown to interact with EspE, while an espH Mma mutant abolished secretion of both EspE and EspF.…”