Biology subjects

Ojha, A. K.

Publications and source records attributed to Ojha, A. K..

7 recordsLinked to original sources

bioRxiv2026

Visualizing zinc-starved slow-replicating Mycobacterium tuberculosis in neutrophil-rich lung lesions

The months-long treatment for tuberculosis is partially attributed to a minor Mycobacterium tuberculosis (Mtb) subpopulation exhibiting phenotypic drug resistance, although origins of these bacilli in hosts remain unknown. Previously, we observed that mycobacteria respond to growth-restrictive limitations of zinc by accumulating Mrf, and inducing Mpy recruitment to the decoding center of the ribosome, thereby hibernating the ribosome in an inactive state. Mrf accumulation - regulated at both transcriptional and post-translational levels by Zur and Clp, respectively, in a zinc-dependent manner - facilitates the formation of stable Mpy-70S ribosome complex in zinc-starved cells. Here, using a translational fusion of Mrf with a fluorescent-reporter protein we visualized zinc-starved mycobacteria with a physiology conducive for Mpy-dependent ribosome hibernation. About half of zinc-starved mycobacterial population expressed microscopically detectable levels of the reporter and exhibited Mpy-dependent slowdown of growth and isoniazid tolerance in vitro. In chronic, high-burden mouse lung infections, ~3% of Mtb bacilli expressed the reporter, and the frequency increased to ~35% upon isoniazid treatment. Transcriptomics of mouse lungs suggests an important role of S100A8/A9 and ZIP2 in neutrophils in zinc mobilization during Mtb infections. Moreover, depletion of neutrophils, or loss of S100A9 caused significant attenuation of zinc starvation response in Mtb. Altogether, the study offers a new approach for visualizing and manipulating slow-replicating Mtb in host tissues.

microbiology↗

bioRxiv2024

A Novel Actinobacteria-Specific Ribosome Hibernation Factor in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), persists within its host for long periods. Treatment of TB requires prolonged administration of antibiotics, some of which target Mtb ribosomes, the protein synthesizing machine of the cell. We determined high-resolution cryo-EM structures of Mtb ribosomes obtained from biofilms under zinc-depleted conditions that are expected to induce ribosome hibernation. In these structures, in addition to finding the Mtb homolog of the hibernation promoting factor Mpy that binds to the decoding center in the small ribosomal subunit, we discovered an actinobacteria-specific protein, previously of unknown function, bound in the nascent polypeptide-exit tunnel (NPET) within the large ribosomal subunit. We refer to this NPET-bound protein as a actinobacteria-specific ribosome tunnel occlusion factor (RTOF). RTOF is found with vacant or E-tRNA-bound 70S ribosomes, but not in actively translating Mtb ribosomes, suggesting that its binding coincides with ribosome hibernation. RTOF makes strong interactions through the entire stretch of NPET extending up to the peptidyl-transferase center (PTC), such that it would block the passage of nascent polypeptide chain. The highly conserved FRRKSG motif of RTOF also interferes with accommodation of the formyl-methionyl moiety of initiator fMet-tRNAfMet in the conformation required for translation initiation. A molar excess of mRNA and initiator fMet-tRNAfMet displaces RTOF by triggering a significant alteration to the conformation of FRRKSG motif, which suggests how RTOF-bound hibernating ribosomes can be reactivated under optimal cellular conditions. The presence of RTOF would interfere with the binding of PTC-targeting drugs, such as macrolides, lincosamides, and oxazolidines, thereby maintaining a drug-free pool of hibernating Mtb ribosomes.

microbiology↗

bioRxiv2024

Uncovering organizing principles of protein-protein interaction networks

This study explores the intricate world of protein-protein interaction (PPI) networks and the critical role of hub proteins within them. PPIs, mediated by various biomolecular forces, orchestrate diverse cellular processes through transient or stable interactions. The study delves into the unique ability of hubs to connect with numerous partners within the network. This research investigates the network properties and focuses on hub-centric questions. This study explores information-carrying capacity, power laws in protein circuits, connectivity profiles of hub proteins, and network expansion or shrinkage during evolution. The study validates the power law for connectivity, identifies a marginal network compression in evolution, unveils hub connectivity patterns, and encourages the exploration of the correlation between protein structure, connectivity, and function. These findings offer valuable insights into network design principles and the critical role of hub proteins in cellular function and evolution.

systems biology↗

bioRxiv2023

Fatty acid metabolism in neutrophils promotes lung damage and bacterial replication during tuberculosis.

Mycobacterium tuberculosis (Mtb) infection triggers a significant influx of neutrophils to the lungs, which is linked to tuberculosis (TB) severity. The mechanism by which Mtb infection induces neutrophillic inflammation remains unclear. Using a clinically relevant and hypervirulent Mtb strain from the W-Beijing family, HN878, we found that genes related to both glycolysis and fatty acid metabolism are upregulated in the lung neutrophils of susceptible mice. Similar effects in gene expression were observed in rabbits, and humans with pulmonary TB compared to healthy controls. Inhibiting glycolysis with 2-deoxy D-glucose (2-DG) exacerbated disease pathology, while fatty acid oxidation (FAO) inhibitor Etomoxir (ETO) improved outcomes by reducing weight loss, immunopathology, and bacterial replication within neutrophils in genetically susceptible mice. Notably, ETO reduced neutrophil production in the bone marrow and their recruitment to the lungs. ETO specifically restrained the recruitment of Ly6Glow/dim immature neutrophil population, which is elevated during disease progression and harbors the bulk of bacilli. In a transwell setup, we demonstrated that ETO dose-dependently inhibited neutrophil chemotaxis towards infected macrophages. In summary, our research highlights the crucial role of fatty acid metabolism in regulating neutrophilic inflammation during TB and provides a rationale for targeting immunometabolism of neutrophils for potential TB treatment.

immunology↗

bioRxiv2022

Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homologue (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in non-starved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12-stalk base, suggesting that the aminoacylation status of A-site tRNA is surveyed during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle. SignificanceBacteria persist under nutrient starvation by activating RelA/SpoT homologue (Rsh), which synthesizes a growth regulating alarmone, ppGpp. Rsh is activated specifically upon recognizing a translation elongation complex with deacylated tRNA at the A-site. It is however unclear how Rsh identifies such a complex in vivo. We show here that conditions inducing ribosome hibernation in mycobacteria cause loss of intracellular Rsh, implying that association with translating ribosomes is necessary for intracellular stability of Rsh. Using structural analysis of Rsh-bound 70S translation initiation complex, we propose here that mycobacterial Rsh identifies a Rsh-activating ribosomal complex by constitutively surveying the ribosome entering the translation cycle at the early elongation stage.

molecular biology↗

bioRxiv2022

Neutrophils reprograms the bone marrow to impair T-cell immunity during tuberculosis

Mycobacterium tuberculosis (Mtb) infection induces persistent influx of neutrophils that associates with poor bacterial control and clinical outcome from tuberculosis (TB). Although implicated in TB pathology, the mechanism by which these cells contribute to pathogenesis is poorly understood. Using Cell-DIVE multiplexed immunofluorescence imaging and spatial analysis of inflammatory TB lesions, we demonstrated that persistent neutrophil infiltration affects the spatiotemporal organization of T-lymphocytes and impairs their function. Instead of directly suppressing T-cells, neutrophils produce granulocyte colony stimulating factor (CSF3/G-CSF) that collaborates with type I interferon (IFN-I) to promote a granulocyte-skewed hematopoiesis impacting T-lymphocyte production. Importantly, neutrophil-intrinsic IFN-I receptor 1 (IFNAR1) is both necessary and sufficient to promote pathologic granulopoiesis. Finally, inhibition of IFNAR1-signaling alone mitigates immunopathogenesis by restoring hematopoietic equilibrium. Collectively, our work uncovers a potential immunevasion strategy by which virulent Mtb strains induce IFN-I to generate pathogen-permissive neutrophils that produce G-CSF and sustain pathogenic hematopoiesis to impair T-cell immunity during TB.

immunology↗

bioRxiv2019

Adaptation of Mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance

Mycobacterium tuberculosis (Mtb) spontaneously grows at the air-medium interface forming pellicle biofilms, which harbor more drug tolerant persisters than planktonic cultures. The underlying basis for increased persisters in Mtb biofilms is unknown. Using a Tn-seq approach, we show here that multiple genes that are necessary for fitness of Mtb cells within biofilms, but not in planktonic cultures, are also important for their tolerance to a diverse set of stressors and antibiotics. Thus, development of Mtb biofilms appears to be associated with population enrichment, in which endogenous stresses presumably generated by challenging growth conditions within biofilm architecture select for cells that maintain tolerance to exogenous stresses including antibiotic exposure. We further observed that the intrinsic drug tolerance of constituent cells of biofilms determines the frequency of persisters: morphologically indistinguishable monoculture biofilms of a {Delta}pstC2A1 mutant hypersensitive to rifampicin harbor [~]20-fold fewer persisters than wild-type. These findings together allow us to propose that the selection of elite cells during biofilm development significantly contributes to the persister frequency. Furthermore, probing the possibility that the population enrichment is an outcome of unique environment within biofilms, we demonstrate biofilm-specific induction in the synthesis of isonitrile lipopeptides (INLP). Mutation analysis indicates that INLP is necessary for the architecture development of Mtb biofilms. In summary, the study offers an insight into persistence of Mtb biofilms under antibiotic exposure, while identifying INLP as a biomarker for further investigation of this phenomenon.\n\nSIGNIFICANCEThe tuberculosis (TB) pathogen Mycobacterium tuberculosis (Mtb) is one of the deadliest bacterial pathogens known to mankind, and TB treatment is inefficient. A lengthy chemotherapy for TB is attributed to a small subpopulation of Mtb bacilli exhibiting phenotypic tolerance to antibiotics. Drugs targeting these persisters are expected to shorten TB chemotherapy, but their development is dependent on in vitro growth models that reproducibly generate high frequency of persisters. Biofilms of Mtb are a suitable model for understanding the origin of persisters. Here, we provide an explanation for the elevated persister frequency in Mtb biofilms. We also identify isonitrile lipopetides as a biomarker of Mtb biofilms. These findings will facilitate further advancements of our efforts to identify and target Mtb persisters.

microbiology↗