Investigation of the role of the TRK potassium transporter of Mycobacterium Tuberculosis in intra cellular survival

[摘要] Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis (TB), is amicrobial pathogen which has infected about one third of the world's population, with abouteight million new cases of TB reported annually of which almost two million are fatal. Thosecoinfected with human immunodeficiency virus (HIV) are most vulnerable for development ofsevere disease. The disease is acquired through the respiratory route, whereby M. tuberculosisbacilli overcome the mechanical defences of the upper airways to reach the lungs where theyinfect alveolar macrophages. Although considerable progress has been made in identifyingimmune mechanisms which confer protection against M. tuberculosis, this has not resulted inthe development of an effective vaccine, underscoring the fact that novel insights into theimmunopathogenesis of M. tuberculosis infection are necessary. In this respect it is noteworthythat almost nothing is known about the involvement of the major mycobacterial potassium (K+)transporters in microbial virulence.M. tuberculosis possesses two major K+-uptake systems, namely the Trk and Kdp systems. TheTrk seems to be functional when the extracellular K+ concentration is high, while the Kdp is aninducible back-up system. The Trk system consists of two proteins, CeoB and CeoC, which areencoded by the ceoB and ceoC genes, which have some degree of homology to the TrkA proteinof Escherichia coli. These proteins share the NAD+-binding motif, compatible with protonmotive force as the driver of cation uptake, suggesting that the M. tuberculosis Trk K+transporter may operate as a K+ and protons (H+) symporter, raising the possibility that it mayantagonize vacuolar acidification, a critical event in the eradication of this intracellularpathogen.The possible involvement of the Trk system in the virulence of M. tuberculosis has beenaddressed in the current study by investigating the intracellular survival of a trk-gene knockoutmutant of the microbial pathogen with that of the matched wild-type (WT) strain using human monocyte-derived macrophages. In addition, the cytokine profiles of macrophages infected byboth strains have also been investigated.Macrophages were prepared from isolated human blood monocytes following sequentialdifferential adherence of CD14+ monocytes. These were matured into large monocytes-derivedmacrophages co-expressing CD14+ / CD16+ following a 7 day incubation period. These cellswere then infected with either the WT (H37Rv) or trk-gene knockout strains of M. tuberculosisat a 1:10 cell: bacteria ratio and intracellular survival as well as cytokine (IL-1b, IL-6, IL8, IL-10, TNF-a) secretion profiles monitored over a 3 day period using a viable colony-countingprocedure and multiplex bead array technology, respectively.No significant differences with respect to either intracellular survival or cytokine secretionprofiles were detected following infection of human monocyte-derived macrophages with theWT or trk-gene knockout strains of M. tuberculosis. Although the observations are compatiblewith lack of involvement of the Trk system in the intracellular survival and virulence of M.tuberculosis, this study may lay groundwork for future studies which simultaneously encompassboth of the major K+ transporters of this microbial pathogen.

[发布机构] University of Pretoria