Bacterial pathogens resistant to multiple antibiotics are an increasing global public health problem. In human tuberculosis (TB) caused by acid-fast bacteria known as the Mycobacterium tuberculosis complex (MTBC), the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains is threatening to make one of humankind’s most important infectious diseases incurable. MDR-TB is caused by MTBC strains resistant to at least isoniazid (INH) and rifampicin (RIF), which are the two most important first-line drugs used to treat TB. XDR-TB is caused by MDR strains with additional resistance to second-line agents. Both MDR-TB and XDR-TB are associated with high mortality, particularly in HIV co-infected patients. Although the mutations and mechanisms conferring resistance to the different anti-TB drugs have been well studied, the forces driving the emergence and spread of MDR-TB are much less understood. Mathematical models predict that the relative fitness of MDR strains compared to drug-susceptible strains is a key determinant of the future epidemics of MDR-TB. We and other have shown using competitive growth experiments that i) the relative fitness of drug-resistant strains differs depending on the specific resistance-conferring mutation(s), ii) different resistance mutations interact epistatically, and iii) the initial fitness cost of drug resistance can be mitigated through compensatory evolution. Moreover, the MTBC comprises different phylogenetic lineages, some of which have been associated with increased drug resistance, suggesting that the natural genetic diversity among strains influences to pathway to resistance. Taken together, these experimental data suggest that drug-resistant strains of MTBC are highly heterogeneous. Indeed, the clinical prevalence of MDR-TB differs widely across regions, and the World Health Organization (WHO) has defined many geographical hotspots. Although extrinsic factors such as the quality of the local TB control program are important determinants of MDR-TB, there is increasing evidence that intrinsic bacterial factors influence the emergence and transmission potential (i.e. in clinico fitness) of drug-resistant MTBC. Newly acquired drug resistance-conferring mutations can be conceptualized as “internal” perturbations of the baseline metabolic profile of MTBC, which is further modulated by the acquisition of compensatory mutations. Both of these occur on the context of “external” perturbationsin form of exposure to anti-TB drug(s). Finally, pre-existing phylogenetic diversity in MTBC represents a kind of “natural” perturbation which interacts with the novel “internal” and “external” perturbations. Most of the experimental work on the subject has been focusing on differential growth characteristics. However, little is known on the effect of these perturbations on the molecular phenotype of MTBC defined based on the differential transcriptome, proteome, metabolome and lipidome. Here we hypothesise that i) external and internal perturbations as defined above will be reflected in the molecular phenotype of the MTBC, and ii) particular in vitro molecular phenotypic profiles are predictive of a high in clinicofitness of MDR MTBC strains in patient populations. We will test these hypotheses by addressing the following Objectives divided into 5 Subprojects:
Define and model the molecular phenotypic space across the phylogenomic diversity of the human-associated MTBC (irrespective of drug resistance; i.e. determine the effect of “natural” perturbations).
Measure the impact of drug resistance and compensatory mutations on the MTBC phenotype (i.e. determine the effect of “internal” perturbations), building on the metabolic model(s) established for drug-susceptible MTBC (Subproject 1).
Measure the effect of exposure to anti-TB drugs (both existing and novel) on the MTBC phenotype in presence and absence of drug resistance mutations (i.e. determine the effect of “external” perturbations).
Develop a phylogenomic-based model of transmission to differentiate between highly transmissible MDR and unsuccessful MDR strains (i.e. determine differential in clinico Darwinian fitness of MDR strains).
Define a metabolic profile that is predictive of high in clinico fitness in MDR MTBC (i.e. linking the combined effects of natural, internal, and external perturbations on the MTBC metabolome with the in clinico Darwinian fitness of MDR strains).
We will use a combination of detailed population genomic analyses using existing collections of whole-genome sequences of drug-susceptible and drug-resistant clinical strains of MTBC, and detailed transcriptional, proteomic, metabolomic, and lipidomic profiling of a subset of rationally selected strains both in presence and absence of the relevant drug(s). These data will then be integrated into modified metabolic models starting with the existing models that have been developed by others. Moreover, we will construct phylodynamic models of TB transmission based on whole-genome sequences that will be generated through this consortium. The latter will serve as the gold standard for validating our metabolic models proposed to be predictive of successful transmission. Although all 5 subprojects are highly integrated, each will stand on its own and generate valuable new datasets and insights into the biology of drug-resistant MTBC. TbX will also generate much added value. Specifically, the hotspots of MDR-TB defined by WHO include Georgia, where up to 70% of TB cases are resistant to at least one drug. Prof. Gagneux, the PI of this consortium has recently been awarded an ERC Starting Grant to study the evolution and epidemiology of MDR-TB in Georgia. Through this ERC project, the TbX consortium will get access to a nation-wide collection of MTBC strains collected over 5 years. TbX will also have access to a series of defined drug-resistant mutants with and without compensatory mutations which are being generated through the ERC project. Moreover, Prof. Aebersold’s and Prof. Sauer’s groups are currently members of an EU-funded consortium on system biology of TB (SysteMtb), which is focusing on the laboratory strain H37Rv and the BCG vaccine strain. The various protocols developed during SysteMtb will serve as an excellent basis for this new and complementary consortium. TbX also teamed up with a recent ETHZ spin-off company based in Basel (BioVersys), which is developing new strategies to circumvent drug resistance in the MTBC. Hence, TbX, through its focus on relevant MTBC clinical strains will both benefit from and generate added value for existing projects as well as ongoing efforts in TB drug development.