Gene replacement and quantitative mass spectrometry approaches validate guanosine monophosphate synthetase as essential for Mycobacterium tuberculosis growth

Abstract

Guanosine monophosphate synthetase (GMPS), encoded by <i>guaA</i> gene, is a key enzyme for guanine nucleotide biosynthesis in <i>Mycobacterium tuberculosis</i>. The <i>guaA</i> gene from several bacterial pathogens has been shown to be involved in virulence; however, no information about the physiological effect of direct <i>guaA</i> deletion in <i>M. tuberculosis</i> has been described so far. Here, we demonstrated that the <i>guaA</i> gene is essential for <i>M. tuberculosis</i> H37Rv growth. The lethal phenotype of <i>guaA</i> gene disruption was avoided by insertion of a copy of the ortholog gene from <i>Mycobacterium smegmatis,</i> indicating that this GMPS protein is functional in <i>M. tuberculosis</i>. Protein validation of the <i>guaA</i> essentiality observed by PCR was approached by shotgun proteomic analysis. A quantitative method was performed to evaluate protein expression levels, and to check the origin of common and unique peptides from <i>M. tuberculosis</i> and <i>M. smegmatis</i> GMPS proteins. These results validate GMPS as a molecular target for drug design against <i>M. tuberculosis</i>, and GMPS inhibitors might prove to be useful for future development of new drugs to treat human tuberculosis.