Biology subjects

Nandicoori, V. K.

Publications and source records attributed to Nandicoori, V. K..

6 recordsLinked to original sources

bioRxiv2025

Sec and Tat mediated secretion safeguards Mycobacterium tuberculosis membrane homeostasis

Protein secretion drives Mycobacterium tuberculosis (Mtb) physiology and pathogenesis, yet a unified picture of the machinery and its role in cell membrane homeostasis is still lacking. By comprehensively curating published evidence, we assembled a systems-level map of Mtb secretion encompassing 92 components and 198 mechanistic reactions across Sec, Tat, and ESX pathways. The secretory components identified were integrated with high-throughput ChIP-Seq and transcriptome datasets to elucidate the regulation of the secretion system. Using CRISPRi, conditional depletion of SecA1 or TatA impaired growth in vitro and survival ex vivo. Quantitative secretome revealed decreased export of SecA1- and TatA-dependent substrates, with enrichment of cytosolic proteins in culture filtrates, indicating increased membrane permeability. Membrane proteomics showed depletion dependent increased metabolic/lipid-degrading proteins and decreased cell-wall/cell-process proteins, consistent with loss of membrane stability. Ultrastructural defects and increased ethidium bromide uptake confirmed impaired membrane integrity. Together, our multi-omics and functional genetics established SecA1 and TatA as essential guardians of Mtb membrane integrity which provided valuable datasets and a framework for secretion-dependent Mtb pathogenesis.

microbiology↗

bioRxiv2024

A clinical mutation in uvrA, a DNA repair gene, confers survival advantage to Mycobacterium tuberculosis in the host

DNA repair pathways play an essential role in maintaining the genomic integrity of bacteria, and a perturbation in their biological activity helps bacteria survive under duress. In drug-resistant clinical strains, we identified a Q135K mutation in the uvrA gene, a DNA repair pathway gene. To delineate the role of uvrA and the Q135K mutation, we generated the gene replacement mutant of UvrA (Rv{Delta}uvrA) in Mycobacterium tuberculosis H37Rv (Mtb-Rv). While the lack of UvrA function in Rv{Delta}uvrA could be restored upon complementation with uvrA, the uvrA-Q135K mutant identified in clinical drug-resistant strains failed to do so. This was reflected in higher mutation rates in Rv{Delta}uvrA and Rv{Delta}uvrA::uvrAQ135A, compared with wild-type Rv or Rv{Delta}uvrA::uvrA complemented strains in the presence and absence of oxidative stress. Killing kinetics experiments with anti-TB drugs showed increased survival of Rv{Delta}uvrA and Rv{Delta}uvrA::uvrAQ135K, strains compared with Rv or Rv{Delta}uvrA::uvrA. Importantly, Rv{Delta}uvrA and Rv{Delta}uvrA::uvrAQ135K showed enhanced survival in peritoneal macrophages and murine infection model of infection. Together, data suggests that acquiring Q135K mutation benefits the pathogen, which helps enhance the hosts survival adaptability. Author SummaryDNA repair mechanisms in an organism are necessary for correcting the errors generated during replication or when it is damaged/modified due to insults. As a GC organism, Mtb is highly prone to host-mediated attacks on its genome, which, if uncorrected, can impact its genome integrity. The drug-resistant clinical strains of Mtb harbor Q135K mutation in uvrA, the first enzyme in the nucleotide excision repair pathway. With the help of genetic, molecular, and murine challenge experiments, we show that the UvrA-Q135K mutation abrogates the enzymes activity, compromising the Mtb strain harboring the mutation in the oxidative and nitrosative stress. On the contrary, the mutation in UvrA imparts survival advantage in activated macrophages and murine infection models. Results presented argue that identified mutation helps in better adaptability in the host, which may include faster acquisition of drug resistance.

microbiology↗

bioRxiv2024

Structure-function relationship of PE11 esterase of Mycobacterium tuberculosis with respect to its role in virulence

The lipolytic enzymes of Mycobacterium tuberculosis play a critical role in immunomodulation and virulence. Among these proteins, PE11 which also belongs to the PE/PPE family, is the smallest ([~]10.8 kDa) and play a significant role in cell wall remodelling and virulence. PE11 is established to be an esterase, but its enzymatic and structural properties are not yet characterized. In this study, using homology modelling we deduced the putative structure which shows the presence of both -helix and {beta}-sheet structures which is in close agreement with that observed by CD spectra of the purified protein. PE11 was found to contain a GX3SX4G motif homologous to canonical GxSxG motif present in many serin hydrolases. The catalytic triad appears to be located within this motif as substitution of Serine26 and Glycine31 residues abrogated its enzymatic activity. Gel-filtration chromatography data indicate that PE11 possibly exists as dimer and tetramer showing positive cooperativity for binding its substrates. In addition, PE11 esterase activity was found to be critical for cell wall remodelling, antibiotic resistance and conferring survival advantages to M. tuberculosis. Our data suggest that PE11 can be targeted for designing potential therapeutic strategies.

microbiology↗

bioRxiv2023

Divergent downstream biosynthetic pathways are supported by L-cysteine synthases of Mycobacterium tuberculosis

Mycobacterium tuberculosiss (Mtb) autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA-seq performed under various stress conditions, we identified that genes belonging to Mtb sulfur metabolism pathways are significantly upregulated during oxidative stress. Using an integrated approach of microbial genetics, transcriptomics, metabolomics, animal experiments, chemical inhibition, and rescue studies, we investigated the biological role of non-canonical L-cysteine synthases, CysM and CysK2. While transcriptome signatures of Rv{Delta}cysM and Rv{Delta}cysK2 appear similar under regular growth conditions, we observed unique transcriptional signatures when subjected to oxidative stress. We followed pool size and labelling (34S) of key downstream metabolites, viz. mycothiol and ergothioneine, to monitor L-cysteine biosynthesis and utilization. This revealed the significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant during infection. With the help of genetic mutants and chemical inhibitors, we show that CysM and CysK2 serve as unique, attractive targets for adjunct therapy to combat mycobacterial infection.

microbiology↗

bioRxiv2022

GWAS and functional studies implicate a role for altered DNA repair in the evolution of drug resistance in Mycobacterium tuberculosis

The emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is alarming and demands in-depth knowledge for timely diagnosis. We performed genome-wide association analysis (GWAS) using 2237 clinical strains of Mtb to identify novel genetic factors that evoke drug resistance. In addition to the known direct targets, for the first time, we identified a strong association between the mutations in the DNA repair genes and the multidrug-resistant phenotype. To evaluate the impact of variants identified in the clinical samples in the evolution of drug resistance, we utilized knockouts and complemented strains in Mycobacterium smegmatis (Msm) and Mtb. Results show that variant mutations abrogated the function of MutY and UvrB. MutY variant showed enhanced survival compared with wild-type (Rv) when the Mtb strains were subjected to multiple rounds of ex vivo antibiotic stress. Notably, in an in vivo Guinea pig infection model, the MutY variant outcompeted the wild-type strain. Collectively, we show that novel variant mutations in the DNA repair genes abrogate their function and contribute to better survival under antibiotic/host stress conditions.

microbiology↗

bioRxiv2018

Selectivity among anti-σ factors by Mycobacterium tuberculosis ClpX influences intracellular levels of Extracytoplasmic Function σ factors

Extracytoplasmic Function {sigma} factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-{sigma} factor. M. tuberculosis membrane-associated anti-{sigma} factors have a small stable RNA gene A-like degron for targeted proteolysis. Interaction between the unfoldase, ClpX, and the substrate with an accessible degron initiates energy-dependent proteolysis. Four anti-{sigma} factors with a mutation in the degron provided a set of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-substrate processivity. We note that a point mutation in the degron (XXX-Ala-Ala) leads to an order of magnitude difference in the dwell time of the substrate on ClpX. Differences in ClpX/anti-{sigma} interactions were correlated with change in unfoldase activity. GFP chimeras or polypeptides of identical length with the anti-{sigma} degron also demonstrate degron-dependent variation in ClpX activity. We show that degron-dependent ClpX activity leads to differences in anti-{sigma} factor degradation thereby regulating the release of free {sigma} from the {sigma}/anti-{sigma} complex. M. tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF {sigma} factors.\n\nImportanceThe ability of Mycobacterium tuberculosis to quickly adapt to the changing environmental stimuli occurs by maintaining protein homeostasis. Extra-cytoplasmic function (ECF) {sigma} factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the {sigma} factor from the anti-{sigma} is governed by the ClpXP2P1 assembly. M. tuberculosis ECF anti-{sigma} factors have a ssrA-like degron for targeted degradation. A point mutation in the degron leads to differences in ClpX mediated proteolysis and affects the cellular abundance of ECF {sigma}-factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affecting M. tuberculosis physiology.

biochemistry↗