In vitro Studies on the Sputum Phenotype of Mycobacterium tuberculosis

Two defining characteristics of Mycobacterium tuberculosis (Mtb) in sputum are gene expression and the production of lipid bodies (LBs). Previous analysis of the Mtb sputum transcriptome demonstrated that a population of persister-like bacilli predominate in sputum. LBs are intracellular structures consisting of triacylglycerol. Their formation is environmentally regulated, and occurs in response to a number of stresses, including conditions which induce a non-replicating persistence phenotype. Based upon these findings, Mtb in sputum may express a unique, transmission-adapted phenotype. Comparison of the gene expression of Mtb in sputum against Mtb exposed to in vitro stimuli demonstrated that no obvious single growth condition fully replicated the sputum transcriptome. In contrast, Mtb cultures exposed to multiple stimuli, including phosphate-buffered saline or RPMI medium, nitric oxide, cholesterol, oleic acid and static incubation had gene expression that correlated significantly to that in sputum. However, the exact combination of stresses is yet to be defined. It was demonstrated in these studies that the presence of LBs as a single factor does not influence the transmission adaptability of Mtb bacilli as tested here. An increased proportion of LB-positive cells did not confer increased survival to transmission stresses (desiccation and ultraviolet radiation). This was confirmed using two independent methods of LB induction (triacylglycerol synthase overexpression and nitric oxide exposure). Analysis of Mtb binding to macrophages also showed that an increased proportion of LB-positive cells did not result in increased bacterial binding. However, the Mtb multi-stimulus cultures showed increased macrophage binding as compared to control cultures, although this remains to be confirmed. It was concluded that Mtb gene expression in sputum occurs secondary to multiple stimuli, and that LBs may represent an epiphenomenon of the Mtb sputum phenotype as far as transmission is concerned. If in fact Mtb in sputum are transmission-adapted, other factors must contribute to this phenotype.