Phenotypic Profiling of Mycobacterium tuberculosis EspA Point Mutants Reveals that Blockage of ESAT-6 and CFP-10 Secretion In Vitro Does Not Always Correlate with Attenuation of Virulence

Abstract

bThe EspA protein of Mycobacterium tuberculosis is essential for the type VII ESX-1 protein secretion apparatus, which delivers the principal virulence factors ESAT-6 and CFP-10. In this study, site-directed mutagenesis of EspA was performed to elucidate its influence on the ESX-1 system. Replacing Trp 55 (W55) or Gly 57 (G57) residues in the putative W-X-G motif of EspA with arginines impaired ESAT-6 and CFP-10 secretion in vitro and attenuated M. tuberculosis. Replacing the Phe 50 (F50) and Lys 62 (K62) residues, which flank the W-X-G motif, with arginine and alanine, respectively, destabilized EspA, abolished ESAT-6 and CFP-10 secretion in vitro, and attenuated M. tuberculosis. Likewise, replacing the Phe 5 (F5) and Lys 41 (K41) residues with arginine and alanine, respectively, also destabilized EspA and blocked ESAT-6 and CFP-10 secretion in vitro. However, these two particular mutations did not attenuate M. tuberculosis in cellular models of infection or during acute infection in mice. We have thus identified amino acid residues in EspA that are important for facilitating ESAT-6 and CFP-10 secretion and virulence. However, our data also indicate for the first time that blockage of M. tuberculosis ESAT-6 and CFP-10 secretion in vitro and attenuation are mutually exclusive.

DisconnectingIn VitroESX-1 Secretion from Mycobacterial Virulence

“…Chen et al report for the first time a scenario where ESX-1 secretion appears to be disrupted in vitro but does not result in attenuation of M. tuberculosis in various infection models (1). The authors focused on EspA, a known ESX-1 substrate.…”

“…The report by Chen et al (1) calls into question whether the in vitro secretion assay is a reliable indicator of ESX-1 function within a host. There are two commonly used ways to induce ESX-1 secretion in vitro.…”

“…As suggested by Chen et al (1), the disconnect between in vitro ESX-1 secretion phenotypes and virulence could simply be explained by the limits of protein detection by Western blot analysis. The EspA variants (EspAF5R and EspAK41A) may be produced at "sub-Western" levels.…”

“…Low levels of ESAT-6 and CFP-10 may be sufficient to mediate virulence in a host cell. Alternatively, Chen et al suggest that the instability of the EspAF5R and EspAK41A proteins in vitro may be stabilized in a host cell, allowing increased ESX-1 secretion in vivo (1).…”

Interplay of human macrophages and Mycobacterium tuberculosis phenotypes

“…Some evidence however is questioning the central role of secreted ESAT6 for Mtb virulence, as for example blocked ESAT6 and CFP10 secretion due to a point mutation in EspA, an ESX-1 substrate co-secreted with the ESAT6-CFP10 dimer, did not reduce virulence in macrophages and in vivo, constituting the unique case of disconnection of ESAT6 secretion and virulence [411]. Failure to detect secreted ESAT6 could be due to lower levels of secretion and bacilli being cultured in detergent-free medium, leading to surface retention of ESX-1 substrates.…”