PhoPR and tuberculosis transmission – MTB-Spread

First, we aim to provide structural information on PhoR. We deploy complementary biophysical approaches, including high-resolution X-ray crystallography and/or cryo-electron microscopy, to depict the structure of full-length and/or functional domains of PhoR. Then, we evaluate the impact of the mutations found in phoR on the activity of the PhoPR system. Using a set of recombinant isogenic TB bacilli expressing various phoPR alleles, we monitor the response of the PhoPR system either to in vitro stimuli or to microenvironments encountered during infection.
Second, we aim to evaluate the impact of the 5 selected phoPR alleles from TB bacilli with various transmission capacity on host pathogen cross-talk with a specific focus on steps related to transmission. For that, we employ a particular mouse model that reproduces upon infection by TB bacilli the severity and diversity of pulmonary lesions found in human TB patients. We also use human cells found in lung alveoli to mimic the initial encounter of TB bacilli with the host. Finally, we compare the impact of the various phoPR alleles on transmission in an artificial mouse transmission model.
Third, we aim to unravel the genetic adaptation(s), which endow(s) a specific phylogenetic lineage of TB bacilli to circumvent the PhoR mutation-mediated loss of PhoPR activity, and to enhance the expression of PhoPR-controlled functions. This objective will be addressed by evaluating the response of the PhoPR-regulon to PhoPR stimuli and the contribution of PhoR to the response. Then, we will deploy complementary strategies based on comparative genomics and genetic screens to search for gene(s) and adaptation(s) involved in the suppression.

Mycobacterium tuberculosis, the major causative agent of human tuberculosis, emerged from an environmental ancestor through the adaptation of endogenous pathways enhancing its ability to colonize the human host and transmit. However, the sequence of genetic events associated with this dramatic lifestyle change and the underlying functional consequences remain poorly understood. The closest extant relatives to this environmental ancestor are the members of the taxon Mycobacterium canettii. These rare strains are responsible for a few cases of tuberculosis but are unable to transmit among the human host. The first set of results from the partners of this consortium show that . functions regulated by PhoP are underexpressed in most M. canettii isolates I comparison to M. tuberculosis and that natural mutations in the two-component regulatory system PhoPR of some M. canettii strains impact the biosynthesis and secretion of key virulence factors of TB bacilli. Expression of a M. tuberculosis phoPR allele in M. canettii increases its virulence in several infectious models. These results therefore suggest that the selection of a specific PhoPR variant probably contributed to the evolution of the ancestor of TB bacilli into highly adapted human pathogens. Initial positioning of these mutations on a three-dimensional structural model of the PhoR protein, established by the use of artificial intelligence tools, corroborates the experimental results. Relevant data are currently being collected and an article describing the different data is currently underway to be finalized before submission for publication.

Expected results should reveal the structure of PhoR, explain why some phoPR mutations enhance/reduce transmission in humans, and describe how some strains have adapted their PhoP regulon response to compensate for the loss of PhoR activity.

on going

There is a clear need for new therapeutics in order to significantly impact the human tuberculosis (TB) incidence curve worldwide. This requires a better understanding of the key factors from the pathogen contributing to pathogenesis and transmission. TB bacilli have evolved unique capacities to cope with the host immune system and to use it for transmission. Surprisingly, almost nothing is known on the bacterial functions involved in transmission. Here, we aim to tackle this crucial problem. Some phylogenetic lineages of TB bacilli exhibit impaired capacities to transmit via aerosol in humans. Previous results strongly suggest that mutations affecting the two component regulatory system, PhoPR, contribute to this lower transmission capacity. PhoPR is made of the sensor protein PhoR and the transcriptional regulator PhoP which controls the expression of major bacterial virulence factors. All the non-synonymous mutations are located within the phoR gene. So far, the structure of PhoR is unknown and the impact of natural mutations found in PhoR on the activity of PhoPR and pathogen transmission capacity is not documented.
Here, we propose to address several important questions: i) what is the structure of PhoR? ii) How do mutations, found in phoPR alleles from certain phylogenetic lineages of TB bacilli, affect the activity of the PhoPR regulatory system, iii) What is the impact of the various phoPR alleles on host pathogen cross-talk, with a specific focus on steps important for transmission; and iv) how does a specific lineage of TB bacilli circumvent/compensate the loss of PhoR activity?
First, we will deploy complementary biophysical approaches, including high-resolution X-ray crystallography, small-angle X-ray scattering and/or cryo-electron microscopy to depict the structure of full-length and/or functional domains of PhoR. Then, we will evaluate the impact of the mutations found in phoR on the structure and activity of the PhoPR system. Using a set of recombinant isogenic TB bacilli expressing various selected phoPR alleles, from highly vs poorly transmissible isolates, we will monitor the response of the PhoPR variants either to in vitro stimuli or to microenvironments encountered during infection.
Second, we aim to evaluate the impact of selected phoPR alleles on host pathogen cross-talk with a specific focus on steps related to transmission. For that, we will employ a particular mouse model which reproduces upon infection by TB bacilli the severity and diversity of pulmonary lesions found in human TB patient. We will also use human cells found in lung alveoli to mimic the initial encounter of bacilli with the host. Finally, we will compare the impact of the various phoPR alleles on transmission in an artificial mouse transmission model.
Third, we aim to unravel the genetic adaptation(s), which endow(s) a specific phylogenetic lineage of TB bacilli to circumvent the loss of PhoPR activity, due to a mutation in PhoR, and to enhance the expression of PhoPR-controlled functions. This objective will be addressed by evaluating the response of PhoPR-regulon to PhoPR stimuli and the contribution of PhoR to the response. We will deploy complementary strategies based on comparative genomics and genetic screen to search for gene(s) and adaptation(s) involved in the suppression.

This proposal is highly innovative as it tackles the question of adaptations that favor long term association with the human host. This question is of major relevance but remains unexplored so far. This project relies on the collaboration of three internationally recognized groups with complementary expertise and multi-scale approaches going from molecular mechanisms to animal experimentation. The expected results should reveal the structure of PhoR, explain why some mutations in phoPR enhance/reduce transmission in humans, and depict how some strains adapted their PhoP-regulon response to compensate the loss of PhoR activity.