Investigating the Role of Secreted EspD Protein in Mycobacterium tuberculosis Infected Human Macrophages
Zoe Sereggela 1,2, Marlene Snider 1, Ze Long Lim 1, Jeffrey Chen 1,2
1 Vaccine and Infectious Disease Organization-International Vaccine Centre (VIDO-InterVac), University of Saskatchewan, Saskatoon Canada
2 Department of Biochemistry, Microbiology & Immunology, University of Saskatchewan, Saskatoon Canada
Mycobacterium tuberculosis (M. tb) is able to persist within macrophages by utilizing its type VII ESX-1 (ESAT-6 system 1) protein secretion system to subvert host-defense mechanisms. ESX-1 is encoded by genes in two separate loci, the esx-1 locus and the espACD tri-gene cluster. EspD, encoded by the third gene on the espACD cluster, had previously been found to be necessary within the mycobacterial cell for the stability and secretion of several ESX-1 effectors. However, the function of secreted EspD within the host-cell remains unknown. This question was addressed here by comparing different M. tb Erdman strains including ones that produce single amino acid variants of the EspD protein. A transposon insertion into espA stops expression of the entire espACD operon and leads to a strain unable to secrete several ESX-1 effectors. In this study, we use this espA transposon strain transformed with a vector control (M. tb espA:: Tn + pMD31), a knockout of espD alone (espA::Tn + pMDespACDSTOP) and strains capable of producing WT, W19R, and R92A EspD variants (espA::Tn + pMDespACDWT, espA::Tn + pMDespACDW19R, and espA::Tn + pMDespACDR92A) for characterization. All strains exhibited similar growth rates. While M. tb espA::Tn + pMDespACDWT, pMDespACDW19R, and pMDespACDR92A were all capable of secreting the key virulence effector protein EsxA, only M. tb espA::Tn + pMDespACDWT and pMDespACDR92A could secrete EspD. Interestingly, M. tb espA::Tn + pMDespACDW19R appeared to be less cytotoxic to THP-1 macrophages and induced reduced amounts of the pro-inflammatory cytokines IL1b and TNFa. These results suggest that secreted EspD is involved in the induction of pro-inflammatory response. However, the role of these effectors inside the M. tb cell may be more complex than previously appreciated, especially with respect to the M. tb espA::Tn + pMDespACDSTOP strain.