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.
The 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.
Global Health Institute, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Jeffrey M
JM
Chen
Ming
M
Zhang
Jan
J
Rybniker
Laetitia
L
Basterra
Neeraj
N
Dhar
Anna D
AD
Tischler
Florence
F
Pojer
Stewart T
ST
Cole
D019943:Amino Acid Substitution; D000818:Animals; D000942:Antigens, Bacterial; D001426:Bacterial Proteins / Q000378:metabolism*; D002460:Cell Line; D004195:Disease Models, Animal; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000472:pathogenicity*; D014376:Tuberculosis / Q000382:microbiology / Q000473:pathology; D014774:Virulence
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; C117940:CFP-10 protein, Mycobacterium tuberculosis; C093276:ESAT-6 protein, Mycobacterium tuberculosis; D026901:Membrane Transport Proteins; D050505:Mutant Proteins