The Rv2623-Rv1747 interaction: regulation of the in vivo fate of M. tuberculosis

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 ΔRv2623 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 ΔRv2623 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 ΔRv2623, ΔRv1747 is attenuated for growth in vivo. And
while the hypervirulent ΔRv2623 expresses enhanced levels of the immunoregulatory phosphatidyl-myo-
inositol mannosides (PIMs) relative to WT Mtb, the hypovirulent ΔRv1747 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 ΔRv2623 and ΔRv1747, 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.

Grant Number: 5R01AI146340-05
NIH Institute/Center: NIH
Principal Investigator: John Chan