Abstract The life cycle of Mycobacterium tuberculosis (Mtb) is complex, encompassing an acute phase, during which the pathogen replicates exponentially; a chronic phase, when bacterial burden is stably maintained, and a latent paucibacillary state that can reactivate. Chronic tuberculosis (TB) is associated with the development of tissue-damaging immunopathology and can promote disease transmission. It has been estimated that approximately 1/4 of the world's population are infected with Mtb, and a significant proportion of these individuals harbor latent bacilli that can reactivate to cause diseases. Unraveling the mechanisms that regulate Mtb growth in an infected host in the different phases of infection is paramount to understanding TB pathogenesis. It is generally thought that certain host environmental conditions (e.g., hypoxia, nitrosative stress, starvation) can promote the establishment of a latent infection. However, the precise mechanisms that regulate TB latency are incompletely defined. Mtb Rv2623, which is among the most upregulated genes in the dormancy regulon, encodes a universal stress protein (USP) that can regulate bacillary growth both in vivo and in vitro. A deletion mutant ?Rv2623 is hypervirulent in susceptible mice and Guinea pigs, and in the latter, it is defective in establishing a chronic persistent infection. In vitro, overexpression of Rv2623 in mycobacteria retards growth in recipient cells; and Mtb ?Rv2623 exits from the non-replicative phase of the hypoxia-induced Wayne latency model more expeditiously than wild-type (WT) Mtb upon transfer into O2-sufficient media. These results provide evidence that Rv2623 regulates Mtb growth, including possibly during the latent/reactivation phase of infection. We showed that Rv2623 interacts with the FHA domain-containing Mtb Rv1747, a putative exporter of lipooligosaccharides. The FHA domain is a signaling protein module that mediates a wide variety of biological processes via phosphorylation-dependent mechanisms. We further showed that the Rv2623-Rv1747 interaction is mediated through binding of the FHAI domain of Rv1747 with a phosphothreonine (at position 237)-containing motif of Rv2623, and that the T237 residue is essential for mediating the growth-regulatory attribute of Rv2623. In contrast to the hypervirulent ?Rv2623, ?Rv1747 is attenuated for growth in vivo. And while the hypervirulent ?Rv2623 expresses enhanced levels of the immunoregulatory phosphatidyl-myo- inositol mannosides (PIMs) relative to WT Mtb, the hypovirulent ?Rv1747 is a hypo-producer of PIMs. In addition, we showed that Rv1747-overexpressing strains hyperproduce PIMs. The correlation of Rv1747's expression levels and Mtb cell wall PIMs amounts suggests that Rv1747 may function as an exporter of Mtb cell wall biogenesis intermediates. This, together with the opposing PIMs phenotype and in vivo growth phenotype of ?Rv2623 and ?Rv1747, has led us to hypothesize that Rv2623 negatively regulates the functional activity of Rv1747 to modulate the levels of Mtb cell wall PIMs, which immunoregulatory properties can alter Mtb-host interactions, thereby influencing the in vivo fate of the tubercle bacillus. We will use biochemical, genetics, and immunological approaches, in conjunction with animal modeling and integrative bioinformatics and computational data analysis, to rigorously test this hypothesis. Finally, accumulating knowledge derived from functional and structural analysis of Rv1747, and the discovery of the relationship between Rv1747 expression and PIM levels, will enable the generation of a set of isogenic Mtb mutants expressing graded levels of PIMs, which can be used to stringently probe the significance of these immunoregulatory glycoplids in influencing the in vivo fate of Mtb. The proposed studies should illuminate how the Rv2623-Rv1747-PIM pathway regulates in vivo Mtb growth. The data generated may help gain insight into the function of Rv1747 in modulating the cell wall PIM levels, the roles of PIMs in impacting the fate of the tubercle bacillus in an infected host, Mtb cell wall biogenesis, and potentially the mechanisms that regulate TB latency and reactivation.
|Effective start/end date||1/16/20 → 12/31/21|
- Infectious Diseases
- Pulmonary and Respiratory Medicine
- Cell Biology
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