Successful host colonization by bacteria requires sensing and response to the local ionic milieu, and coordination of responses with the maintenance of ionic homeostasis in the face of changing conditions. We previously discovered that Mycobacterium tuberculosis (Mtb) responds synergistically to chloride (Cl - ) and pH, as cues to the immune status of its host. This raised the intriguing concept of abundant ions as important environmental signals, and we have now uncovered potassium (K + ) as an ion that can significantly impact colonization by Mtb. The bacterium has a unique transcriptional response to changes in environmental K + levels, with both distinct and shared regulatory mechanisms controlling Mtb response to the ionic signals of K + , Cl - , and pH. We demonstrate that intraphagosomal K + levels increase during macrophage phagosome maturation, and find using a novel fluorescent K + -responsive reporter Mtb strain that K + is not limiting during macrophage infection. Disruption of Mtb K + homeostasis by deletion of the Trk K + uptake system results in dampening of the bacterial response to pH and Cl - , and attenuation in host colonization, both in primary murine bone marrow-derived macrophages and in vivo in a murine model of Mtb infection. Our study reveals how bacterial ionic homeostasis can impact environmental ionic responses, and highlights the important role that abundant ions can play during host colonization by Mtb.

The pathogenesis of Mycobacterium tuberculosis (Mtb) is intrinsically linked to its intimate and enduring interaction with its host, and understanding Mtb-host interactions at a molecular level is critical to attempts to decrease the significant burden of tuberculosis disease. The marked heterogeneity that exists in lesion progression and outcome during Mtb infection necessitates the development of methods that enable in situ analyses of Mtb biology and host response within the spatial context of tissue structure. Fluorescent reporter Mtb strains have thus come to the forefront as an approach with broad utility for the study of the Mtb-host interface, enabling visualization of the bacteria during infection, and contributing to the discovery of several facets such as non-uniformity in microenvironments and Mtb physiology in vivo, and their relation to the host immune response or therapeutic intervention. We review here the different types of fluorescent reporters and ways in which they have been utilized in Mtb studies, and expand on how they may further be exploited in combination with novel imaging and other methodologies to illuminate key aspects of Mtb-host interactions.

Sensing and response to environmental cues, such as pH and chloride (Cl−), is critical in enabling Mycobacterium tuberculosis (Mtb) colonization of its host. Utilizing a fluorescent reporter Mtb strain in a chemical screen, we have identified compounds that dysregulate Mtb response to high Cl− levels, with a subset of the hits also inhibiting Mtb growth in host macrophages. Structure–activity relationship studies on the hit compound “C6,” or 2-(4-((2-(ethylthio)pyrimidin-5-yl)methyl)piperazin-1-yl)benzo[d]oxazole, demonstrated a correlation between compound perturbation of Mtb Cl− response and inhibition of bacterial growth in macrophages. C6 accumulated in both bacterial and host cells, and inhibited Mtb growth in cholesterol media, but not in rich media. Subsequent examination of the Cl− response of Mtb revealed an intriguing link with bacterial growth in cholesterol, with increased transcription of several Cl−-responsive genes in the simultaneous presence of cholesterol and high external Cl− concentration, versus transcript levels observed during exposure to high external Cl− concentration alone. Strikingly, oral administration of C6 was able to inhibit Mtb growth in vivo in a C3HeB/FeJ murine infection model. Our work illustrates how Mtb response to environmental cues can intersect with its metabolism and be exploited in antitubercular drug discovery.

For Mycobacterium tuberculosis (Mtb) to successfully infect a host, it must be able to adapt to changes in its microenvironment, including variations in ionic signals such as pH and chloride (Cl − ), and link these responses to its growth. Transcriptional changes are a key mechanism for Mtb environmental adaptation, and we identify here Rv0500A as a novel transcriptional regulator that links Mtb environmental response and division processes. Global transcriptional profiling revealed that Rv0500A acts as a repressor and influences the expression of genes related to division, with the magnitude of its effect modulated by pH and Cl − . Rv0500A can directly bind the promoters of several of these target genes, and we identify key residues required for its DNA-binding ability and biological effect. Overexpression of rv0500A disrupted Mtb growth morphology, resulting in filamentation that was exacerbated by high environmental Cl − levels and acidic pH. Finally, we show that perturbation of rv0500A leads to attenuation of the ability of Mtb to colonize its host in vivo. Our work highlights the important link between Mtb environmental response and growth characteristics, and uncovers a new transcription factor involved in this critical facet of Mtb biology.