Biology subjects

Papavinasasundaram, K.

Publications and source records attributed to Papavinasasundaram, K..

7 recordsLinked to original sources

bioRxiv2025

VadK, a Non-Canonical Kinase that Regulates the Methylcitrate Cycle and is Essential for Mycobacterium tuberculosis Virulence

The evolution of new enzymatic functions is constrained and guided by the architecture of an organisms metabolic and regulatory networks as well as by environmental constraints. Here, we identify a previously uncharacterized kinase that has evolved from pyruvate phosphate dikinase (PPDK). Through biochemical and systems-level analyses, we show that this enzyme, encoded by Rv1127c in Mycobacterium tuberculosis (Mtb), has diverged from its ancestral role in central carbon metabolism to function as a histidine kinase in pathogenic mycobacteria and related species. We designate this enzyme Virulence Associated DiKinase (VadK), reflecting its ability to autophosphorylate and role in coordinating metabolism and virulence. VadK is essential for the utilization of exogenous carbon sources critical for survival within the host and is required for Mtb pathogenicity in murine models of tuberculosis. Furthermore, VadK interacts with key enzymes of the methylcitrate cycle, and 13C-metabolic flux analysis indicates that it fine-tunes flux through this pathway, with elevated flux proving growth limiting. Together, these findings identify VadK as a previously unrecognized regulatory kinase that integrates metabolic control with virulence in Mtb, revealing a new facet of metabolic regulation in bacterial pathogenesis and a potential target for therapeutic intervention.

microbiology↗

bioRxiv2024

Chemical genetic interactions elucidate pathways controlling tuberculosis antibiotic efficacy during infection

Successful tuberculosis therapy requires treatment with an unwieldy multidrug combination for several months. Thus, there is a growing need to identify novel genetic vulnerabilities that can be leveraged to develop new, more effective antitubercular drugs. Consequently, recent efforts to optimize TB therapy have exploited Mtb chemical genetics to identify pathways influencing antibiotic efficacy, novel mechanisms of antibiotic action, and new targets for TB drug discovery. However, the influence of the complex host environment on these interactions remains largely unknown, leaving the therapeutic potential of the identified targets unclear. In this study, we leveraged a library of conditional mutants targeting 467 essential Mtb genes to characterize the chemical-genetic interactions (CGIs) with TB drugs directly in the mouse infection model. We found that these in vivo CGIs differ significantly from those identified in vitro. Both drug-specific and drug-agnostic effects were identified, and many were preserved during treatment with a multidrug combination, suggesting numerous strategies for enhancing therapy. This work also elucidated the complex effects of pyrazinamide (PZA), a drug that relies on aspects of the infection environment for efficacy. Specifically, our work supports the importance of coenzyme A synthesis inhibition during infection, as well as the antagonistic effect of iron limitation on PZA activity. In addition, we found that inhibition of thiamine and purine synthesis increases PZA efficacy, suggesting novel therapeutically exploitable metabolic dependencies. Our findings present a map of the unique in vivo CGIs, characterizing the mechanism of PZA activity in vivo and identifying novel targets for TB drug development. SignificanceThe inevitable rise of multi-drug-resistant tuberculosis underscores the urgent need for new TB drugs and novel drug targets while prioritizing synergistic drug combinations. Chemical-genetic interaction (CGI) studies have delineated bacterial pathways influencing antibiotic efficacy and uncovered druggable pathways that synergize with TB drugs. However, most studies are conducted in vitro, limiting our understanding of how the host environment influences drug-mutant interactions. Using an inducible mutant library targeting essential Mtb genes to characterize CGIs during infection, this study reveals that CGIs are both drug-specific and drug-agnostic and differ significantly from those observed in vitro. Synergistic CGIs comprised distinct metabolic pathways mediating antibiotic efficacy, revealing novel drug mechanisms of action, and defining potential drug targets that would synergize with frontline antitubercular drugs.

microbiology↗

bioRxiv2022

High bacillary burden and the ESX-1 type VII secretion system promote MHC class I presentation to CD8 T-cells during Mycobacterium tuberculosis infection

Mycobacterium tuberculosis (Mtb) subverts host defenses to persist in macrophages despite immune pressure. CD4 T-cells can recognize macrophages infected with a single bacillus in vitro. Under identical conditions, CD8 T-cells inefficiently recognize infected macrophages and fail to restrict Mtb growth, although they can inhibit Mtb growth during high burden intracellular infection. We show that high intracellular Mtb numbers cause macrophage death, leading other macrophages to scavenge cellular debris and cross-present the TB10.4 antigen to CD8 T-cells. Presentation by infected macrophages requires Mtb to have a functional ESX-1 type VII secretion system. These data indicate that phagosomal membrane damage and cell death promote class I MHC presentation of the immunodominant antigen TB10.4 by macrophages. Although this mode of antigen-presentation stimulates cytokine production that we presume would be host beneficial; killing of uninfected cells could worsen immunopathology. We suggest that shifting the focus of CD8 T-cell recognition to uninfected macrophages would limit the interaction of CD8 T-cells with infected macrophages and impair CD8 T-cell mediated resolution of tuberculosis.

immunology↗

bioRxiv2020

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice

The outcome of an encounter with Mycobacterium tuberculosis depends on the pathogens ability to adapt to the variable immune pressures exerted by the host. Understanding this interplay has proven difficult, largely because experimentally tractable animal models do not recapitulate the heterogeneity of tuberculosis disease. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to associate bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and produce qualitatively distinct immune states. Global analysis of Mtb mutant fitness across the CC panel revealed that many virulence pathways are only in specific host microenvironments, identifying the large fraction of the pathogens genome that has been maintained to ensure fitness in a diverse population. Both immunological and bacterial traits were associated with genetic variants distributed across the mouse genome, identifying the specific host-pathogen genetic interactions that influence pathogenesis.

genetics↗

bioRxiv2020

Host immunity increases Mycobacterium tuberculosis reliance on cytochrome bd oxidase

In order to sustain a persistent infection, Mycobacterium tuberculosis (Mtb) must adapt to a changing environment that is shaped by the developing immune response. This necessity to adapt is evident in the flexibility of many aspects of Mtb metabolism, including a respiratory chain that consists of two distinct terminal cytochrome oxidase complexes. Under the conditions tested thus far, the bc1/aa3 complex appears to play a dominant role, while the alternative bd oxidase is largely redundant. However, presence of two terminal oxidases in this obligate pathogen implies that respiratory requirements might change during infection. We report that the cytochrome bd oxidase is specifically required for resisting the adaptive immune response. While the bd oxidase was dispensable for growth in resting macrophages and the establishment of infection in mice, this complex was necessary for optimal fitness after the initiation of adaptive immunity. This requirement was dependent on lymphocyte-derived interferon gamma (IFN{gamma}), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that{Delta} cydA mutants were hypersusceptible to the low pH encountered in IFN{gamma}-activated macrophages. Unlike wild type Mtb, cytochrome bd-deficient bacteria were unable to sustain a maximal oxygen consumption rate (OCR) at low pH, indicating that the remaining cytochrome bc1/aa3 complex is preferentially inhibited under acidic conditions. Consistent with this model, the potency of the cytochrome bc1/aa3 inhibitor, Q203, is dramatically enhanced at low pH. This work identifies a critical interaction between host immunity and pathogen respiration that influences both the progression of the infection and the efficacy of potential new TB drugs. Author SummaryTuberculosis, caused by Mycobacterium tuberculosis (Mtb) is a serious global health problem that is responsible for over one million deaths annually, more than any other single infectious agent. In the host, Mtb can adapt to a wide variety of immunological and environmental pressures which is integral to its success as a pathogen. Accordingly, the respiratory capacity of Mtb is flexible. The electron transport chain of Mtb has two terminal oxidases, the cytochrome bc1/aa3 super complex and cytochrome bd, that contribute to the proton motive force and subsequent production of energy in the form of ATP. The bc1/aa3 super complex is required for optimal growth during infection but the role of cytochrome bd is unclear. Here we report that the cytochrome bd oxidase is required for resisting the adaptive immune response, in particular, acidification of the phagosome induced by lymphocyte-derived IFN{gamma}. We found that the cytochrome bd oxidase is specifically required under acidic conditions, where the bc1/aa3 complex is preferentially inhibited. Additionally, we show that acidic conditions increased the potency of Q203, a cytochrome bc1/aa3 inhibitor and candidate tuberculosis therapy. This work defines a new link between the host immune response and the respiratory requirements of Mtb that affects the potency of a potential new therapeutic.

microbiology↗

bioRxiv2019

Phosphorylation on PstP controls cell wall metabolism and antibiotic tolerance in Mycobacterium smegmatis

Mycobacterium tuberculosis and its relatives, like many bacteria, have dynamic cell walls that respond to environmental stresses. Modulation of cell wall metabolism in stress is thought to be responsible for decreased permeability and increased tolerance to antibiotics. The signaling systems that control cell wall metabolism under stress, however, are poorly understood. Here, we examine the cell wall regulatory function of a key cell wall regulator, the Serine Threonine Phosphatase PstP, in the model organism Mycobacterium smegmatis. We show that the peptidoglycan regulator CwlM is a substrate of PstP. We find that a phospho-mimetic mutation, pstP T171E, slows growth, mis-regulates both mycolic acid and peptidoglycan metabolism in different conditions, and interferes with antibiotic tolerance. These data suggest that phosphorylation on PstP affects its activity against various substrates and is important in the transition between growth and stasis. ImportanceRegulation of cell wall assembly is essential for bacterial survival and contributes to pathogenesis and antibiotic tolerance in mycobacteria, including pathogens such as Mycobacterium tuberculosis. However, little is known about how the cell wall is regulated in stress. We describe a pathway of cell wall modulation in Mycobacterium smegmatis through the only essential Ser/Thr phosphatase, PstP. We showed that phosphorylation on PstP is important in regulating peptidoglycan metabolism in the transition to stasis and mycolic acid metabolism in growth. This regulation also affects antibiotic tolerance in growth and stasis. This work helps us to better understand the phosphorylation-mediated cell wall regulation circuitry in Mycobacteria.

microbiology↗

bioRxiv2018

ORBIT: a new paradigm for geneticengineering of mycobacterial chromosomes

Current methods for genome engineering in mycobacteria rely on relatively inefficient recombination systems that require the laborious construction of a long double-stranded DNA substrate for each desired modification. We combined two efficient recombination systems to produce a versatile method for high-throughput chromosomal engineering that obviates the need for the preparation of double-stranded DNA recombination substrates. A synthetic \"targeting oligonucleotide\" is incorporated into the chromosome via homologous recombination mediated by the phage Che9c RecT annelase. This oligo contains a site-specific recombination site for the directional Bxb1 integrase (Int), which allows the simultaneous integration of a \"payload plasmid\" that contains a cognate recombination site and selectable marker. The targeting oligo and payload plasmid are co-transformed into a RecT- and Int-expressing strain, and drug-resistant homologous recombinants are selected in a single step. A library of reusable target-independent payload plasmids is available to generate knockouts and promoter replacements, or to fuse the C-terminal-encoding regions of target genes with tags of various functionalities. This new system is called ORBIT (Oligo-mediated Recombineering followed by Bxb1 Integrase Targeting) and is ideally suited for the creation of libraries consisting of large numbers of deletions, insertions or fusions in a target bacterium. We demonstrate the utility of ORBIT by the construction of insertions or deletions in over 100 genes in M. tuberculosis and M. smegmatis. The report describes the first genetic engineering technique for making selectable chromosomal fusions and deletions that does not require the construction of target- or modification-specific double-stranded DNA recombination substrates.

genetics↗