Biology subjects

Babunovic, G. H.

Publications and source records attributed to Babunovic, G. H..

6 recordsLinked to original sources

bioRxiv2026

Polyketide synthase 12 is an in vivo essential source of novel mycolyl lipids in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) is a major pathogen worldwide that infects and transmits only among humans, yet nearly all in vivo virulence research occurs in non-human hosts. To overcome this central challenge in tuberculosis research, we leveraged a dataset of more than 50,000 sequenced isolates to identify Mtb genes that that are functionally preserved during natural infection, disease causation, and transmission between humans. This whole-genome ranking identified polyketide synthase 12 (pks12 ) as an exceptionally in vivo essential gene in human tuberculosis, which we validated in zebrafish and mouse models. Though Pks12 produces a mycoketide lipid in only trace amounts, pks12 deletion severely altered the host-facing surface and arabinoglycan architecture of Mtb. This amplified effect was explained through the discovery of mannosyl-{beta}-1-phosphomycoketide monomycolate (MPMMM), which is synthesized from the known Pks12 product at higher abundance by antigen 85 mycolyltransferases. Thus, we used a new host-facing genomic-metabolomic-phenotypic approach to discover the functions of an in vivo essential Mtb gene, which controls the physical structure of the Mtb-host interface.

microbiology↗

bioRxiv2026

Encoded metabolic remodeling amplifies drug resistance in Mycobacterium tuberculosis

Antibiotic pressure causes pathogens to evolve many forms of altered drug susceptibility. In addition to target or activator mutations conferring canonical drug resistance, mutations can serve as steppingstones to or enhancers of resistance. In clinical strains of Mycobacterium tuberculosis (Mtb), we find that idsA2, which encodes an isoprenyl pyrophosphate synthase involved in the synthesis of precursors for essential components of the cell wall and electron transport chain, is undergoing diversifying selection and that these mutations are associated with the acquisition of first-line antibiotic resistance. By engineering isogenic Mtb strains that express clinically prevalent variants of idsA2, we show that clinical variants increase the minimum inhibitory concentration of ethambutol and, to a lesser extent, of isoniazid. Targeted lipid analyses reveal that disrupting IdsA2 function redirects limited resources in the isoprenoid synthesis pathway, leading to increased production of decaprenylphosporyl pentose which can compete with ethambutol for binding to arabinosyltransferases. IdsA2 mutations most often occur after embB mutation and lead to a multiplicative increase in ethambutol resistance. Thus, identification of idsA2 mutations can be utilized to improve the specificity of genotypic ethambutol susceptibility testing. Together, this work defines idsA2 as an ethambutol resistance gene and demonstrates how metabolic remodeling can augment drug resistance. Author SummaryTuberculosis is the deadliest infectious disease in the world and becomes more difficult to treat with the acquisition of drug resistance. Defining the mechanisms and genetic basis of drug resistance will allow for improved screening and drug regimen optimization for treatment success. To identify previously unrecognized mechanisms of altered drug susceptibility, we have combined population genomics and experimental genetics approaches, focusing on Mycobacterium tuberculosis genes evolving in clinical strains. We identified idsA2 as a target of frequent mutations that are associated with drug resistance. Experimental data indicate that idsA2 variants decrease the susceptibility of the bacteria to multiple antibiotics through isoprenoid synthesis remodeling, with the strongest effect on ethambutol resistance. IdsA2 mutations often occur after embB mutations to multiplicatively increase ethambutol resistance. These data suggest that inclusion of idsA2 variants in drug resistance testing could improve the specificity of genotypic detection of ethambutol resistance.

microbiology↗

bioRxiv2025

Mapping unsolved lipidomes accelerates lipid discovery in major bacterial pathogens

Unlike gene-first approaches to understanding bacterial pathogenesis, molecule-forward discovery can uncover unexpected chemical diversity. Here, new lipidomic analytical methods and quality metrics defined the large scope of unknown lipids in the worlds deadliest pathogen, Mycobacterium tuberculosis (Mtb). This map allowed rapid discovery of Mtb lysyldiacylglycerol linked to the biosynthetic gene lysX, which controls in vivo infection outcomes in moth larvae, mice, guinea pigs, and here, zebrafish. A broader search for orthologous lysyltransferase domains identified the Staphylococcus aureus virulence gene mprF, where the same lipoamino acid was shown to be a previously unknown biosynthetic product. Thus, lipidomic mapping showed that the cell envelope composition of well-studied bacterial pathogens remains substantially unsolved and offers a new way to generate lists of discoverable lipids to accelerate molecular discovery.

microbiology↗

bioRxiv2022

Tuberculosis treatment failure associated with evolution of antibiotic resilience

Antibiotics are a cornerstone of medicine, placing bacterial pathogens under intense pressure to evolve new survival mechanisms. Analysis of 51,229 Mycobacterium tuberculosis (Mtb) clinical isolates identified an essential transcriptional regulator, Rv1830 (here named resR) as a frequent target of positive (adaptive) selection. resR mutants do not demonstrate canonical drug resistance or drug tolerance but instead have significantly faster recovery after drug treatment across all antibiotics and combinations tested, a phenotype which we term antibiotic resilience. ResR acts in a regulatory cascade with other growth-controlling transcriptional regulators WhiB2 and WhiA, which are also under positive selection in Mtb clinical isolates. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

microbiology↗

bioRxiv2020

GM-CSF differentiation of human monocytes stabilizes macrophage state via oxidative signaling

Macrophages are central mediators of immunity that integrate diverse signals derived from differentiation cues, tissue location, and disease. Controlling macrophage state and function is an appealing therapeutic objective across many diseases including cancer, atherosclerosis, and tuberculosis. Despite the growing appreciation for the in vivo complexity of macrophage state, existing in vitro models of human monocyte-derived macrophages have used a limited number of individual perturbations to explore the complex phenotypic space that macrophages can occupy. Here, we leverage a tiered differentiation, activation, and stimulation strategy to generate libraries of in vitro monocyte-derived macrophages and examine the in vitro state space of macrophage function using high-dimensional technologies. Our tiered experimental approach further revealed a striking relationship between GM-CSF differentiation and IL-10 production. Cells that were differentiated with GM-CSF produced very low or undetectable levels of IL-10 independent of activation or stimulation condition. To nominate candidate regulators of this IL-10 response, we leverage unbiased single-cell mRNA sequencing to identify transcriptional networks associated with GM-CSF-derived cells. Using these data, we identify oxidative signaling pathways as upregulated in GM-CSF derived cells and demonstrate that scavenging of oxidative radicals can enhance IL-10 production in these cells. Collectively, these data underscore the complexity of monocyte-derived macrophage state over time and highlight a dominant role for GM-CSF in tuning macrophage inflammatory phenotype, metabolic state, and plasticity.

immunology↗

bioRxiv2020

Illuminating host-mycobacterial interactions with functional genomic screening to inhibit mycobacterial pathogenesis

Existing antibiotics are inadequate to defeat tuberculosis (TB), a leading cause of death worldwide. We sought potential targets for host-directed therapies (HDTs) by investigating the host immune response to mycobacterial infection. We used CRISPR/Cas9-mediated high-throughput genetic screens to identify perturbations that improve the survival of human phagocytic cells infected with Mycobacterium bovis BCG (Bacillus Calmette-Guerin), as a proxy for Mycobacterium tuberculosis (Mtb). Many of these perturbations constrained the growth of intracellular mycobacteria. We identified over 100 genes associated with diverse biological pathways as potential HDT targets. We validated key components of the type I interferon and aryl hydrocarbon receptor signaling pathways that respond to the small-molecule inhibitors cerdulatinib and CH223191, respectively; these inhibitors enhanced human macrophage survival and limited the intracellular growth of Mtb. Thus, high-throughput functional genomic screens can elucidate highly complex host-pathogen interactions and serve to identify HDTs with the potential to improve TB treatment.

microbiology↗