Genetic analysis of pellicle formation in Mycobacterium tuberculosis: New insights into an old foe

Abstract

The genus name Mycobacterium means fungus-like bacterium, reflecting the observation that the tubercle bacilli are capable of growing on the surface of media as mold-like pellicles. Despite this early observation, Mycobacterium tuberculosis pellicles have not been studied in detail, and neither their genetic basis nor their physiological significance well understood.;A forward genetic screen for M. tuberculosis mutants unable to form a pellicle using a transposon insertion library of M. tuberculosis mc27000, resulted in the identification of ten independent pellicle defective mutants. Using these mutants, we demonstrate that pellicle development requires cell-cell interaction at the surface level. Studies using the pks16 mutant coding for a possible fatty acid adenylate ligase, identified in this screen, and mycolic acid methyl transferase mutants of M. tuberculosis H37Rv resulted in the identification of keto-mycolic acids as a factor required for pellicle growth. These studies also indicated an overlap in the genetic requirement for pellicle growth and tolerance to rifampicin, a frontline anti-tuberculosis (TB) drug, in vitro.;Transcriptional analysis of M. tuberculosis within the pellicle demonstrated significant overlap between the pellicle stimulon and genes that were found to be differentially expressed in other dormancy models shown to confer drug tolerance. Growth within the pellicle conferred drug tolerance on a inherently drug-intolerant strain of Mtb, emphasizing the biofilm-like nature of these structures and suggesting that the pervasive physiological changes induced by growth within the pellicle confers drug-tolerance. These observations also suggested that the pellicle-biofilm can be a useful as a screen for identifying novel in vivo-active anti-TB drugs. Such a screen resulted in the identification of a novel compound that was found to be active against both drug-resistant and non-replicating M. tuberculosis and synergizes with existing anti-TB drugs.;In a mouse model of TB, M. tuberculosis H37RV strains carrying deletions in pks16 and mmaA4, two pellicle-defective mutants with alterations in their mycolic acid compositions, were found to be attenuated. Low burden infections established by these strains in mice were unable to reactivate to fulminant disease upon immune suppression.;These observations provide us with new paradigms for development of novel intervention strategies against tuberculosis.