A third of the world's population is infected with Mycobacterium tuberculosis (Mtb). Tubercle bacilli can remain inactive in lung lesions only to emerge decades later to seed new outbreaks of tuberculosis. In addition, tuberculosis is one of the most difficult bacterial infections to treat and continues to cause more deaths than any other bacterial infection. Bacilli exist in replicating and non-replicating states in a range of microenvironments that vary in oxygen concentration and nutrient availability. The bacilli that survive during latent infection likely exist in a non-replicating state and antimicrobials, effectie against actively growing bacteria, are often not effective against non-replicating bacteria. Population heterogeneity, the presence of more than one phenotypic variant in a clonal population, provides bacteria diverse mechanisms to endure environmental challenges. We have demonstrated that Mtb exists in two semi-stable states that appear to be epigenetically controlled. When mycobacteria were grown as a dense population, either by pellicle or settled growth, the vast majority of the population took on characteristics of a form termed pellicle. If bacilli were passaged starting with a solitary bacillus or just a few cells, however, the ensuing population shifted to a form termed solitary. One of the most striking contrasts between the solitary- and pellicle-prepared Mtb was the solitary form adapted to hypoxic and anaerobic conditions by maintaining high transcriptional activity, while the pellicle form failed to adjust t hypoxia and became truly dormant under anaerobic conditions. We hypothesize that populations of Mtb contain at least two phenotypic variants that allow for divergent responses to imbalances in proton homeostasis, particularly resulting from hypoxia. To test this hypothesis we will determine the differential abilities of the solitary and pellicle variants in adjusting to conditions that exert pressure on internal pH homeostasis. We will also identify regulatory elements that control the shift between the variants. Pellicle growth has historically been used to increase or maintain Mtb virulence during in vitro growth. Therefore, we will investigate if the pellicle-passaged variant is more virulent than the solitary form by using a mouse model of TB that reproduces hypoxic necrotic granulomas.
A third of the world's population is infected with Mycobacterium tuberculosis. Tuberculosis is one of the most difficult bacterial infections to treat and continues to cause more deaths than any other bacterial infection. The bacteria that persist during latent infection and antibacterial treatment are likely in non-replicating states with low levels of metabolic activity resulting from decreased aerobic respiration. Our research is designed to uncover the heterogeneity within a population that allows for drastically different approaches used by M. tuberculosis to adapt to non-replicating states.
