Investigating the Function of Secreted EspD from Mycobacterium tuberculosis

Abstract

Mycobacterium tuberculosis (M. tb) is able to persist within macrophages by utilizing its type VII ESX-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 operon. EspD, encoded by the third gene on the espACD operon, had previously been found to be necessary within the mycobacterial cell for the stability and secretion of several ESX-1 proteins. However, the function of secreted EspD within the host-cell remains unknown. This question was addressed here through studies using M. tb strains expressing variant EspD proteins and through the characterization of recombinant EspD protein expressed and purified from Escherichia coli (E. coli). To generate these M. tb strains, an espA transposon insertion mutant (M. tb espA::Tn), which is unable to express the entire espACD operon, was used. M. tb espA::Tn was transformed with an empty plasmid vector (M. tb espA::Tn pMD31) or plasmid vectors encoding the espACD genes containing either wild-type espD (M. tb espA::Tn pMDespACD_WT), espD containing a premature stop codon (M. tb espA::Tn pMDespACD_STOP), or espD containing single point mutations. While all strains exhibited similar growth rates, M. tb espA::Tn pMD31 and M. tb espA::Tn pMDespACD_STOP were deficient in both EsxA and EspD secretion. M. tb espA::Tn pMDespACD_W19R was capable of secreting EsxA, but not EspD. All other strains expressing variants of EspD were able to secrete both EspD and EsxA to some level. During infections of THP-1 human macrophages, M. tb espA::Tn pMDespACD_W19R appeared less cytotoxic and induced reduced production of the pro-inflammatory cytokines IL1B and TNFa. These results strongly suggest that secreted EspD is involved in the induction of pro-inflammatory response. M. tb espA::Tn pMDespACD_STOP also appeared less cytotoxic to THP-1 cells, but in contrast induced an increased production of IL1B and TNFa. This is a novel observation indicating that ESX-1 mediated cell death in macrophages does not necessarily correspond to an increased pro-inflammatory response. Additionally, purified recombinant EspD appears to be capable of forming a large oligomeric structure and appears to bind specifically to several eukaryotic phospholipids, including PI4P. It is unknown if this binding occurs in vivo or if this binding underlies the inflammatory response induced by secreted EspD during macrophage infections.