Biology subjects

Srinivas, P.

Publications and source records attributed to Srinivas, P..

9 recordsLinked to original sources

bioRxiv2026

Orally available designed miniproteins inhibit enterotoxigenic Bacteroides fragilis pathology by blocking toxin receptor binding

Toxigenic bacterial infections in the gut are a significant contributor to the global burden of disease. Advanced tools for protein and live biotherapeutic engineering offer potentially transformative strategies for treating such diseases, while avoiding the collateral effects of traditional antibacterials. Here we used de novo protein design to identify inhibitors of the metzincin family protease Bacteroides fragilis toxin (BFT). These inhibitors, which bind distal to the active site, interfere with toxin-mediated E-cadherin cleavage and downstream proinflammatory signaling by blocking claudin-4 receptor binding. We tested the inhibitors as disulfide-stabilized variants administered directly to the cecum or in drinking water, as well as through in situ secretion by an engineered live biotherapeutic. Across these delivery modalities, the inhibitors successfully neutralized the toxin and effectively prevented BFT-associated gut pathology, including tumor formation. These results highlight the potential of de novo designed proteins as precise, non-antibiotic interventions to mitigate bacterial toxin-driven disease in the gut.

microbiology↗

bioRxiv2025

Chronic Dietary Exposure to Methylparaben and Ethyl paraben Induces Developmental, Biochemical, and Behavioural Toxicity in Drosophila melanogaster

Parabens, particularly methylparaben (MP) and ethylparaben (EP), are extensively used preservatives in cosmetics, foods, and pharmaceuticals. Although considered safe at low concentrations, recent evidence questions their biological inertness under chronic exposure. This study evaluated the developmental, biochemical, and behavioral effects of continuous dietary MP and EP exposure in Drosophila melanogaster, an established in vivo model for toxicological screening. Flies were chronically exposed to MP (0.5-2%) or EP (0.5-1.5%) throughout development and adulthood. Developmental timing, lifespan, oxidative-stress markers (MDA, FRAP, total protein), and locomotor performance (negative geotaxis in adults, crawling in larvae) were quantified. Paraben exposure significantly delayed development ([~]15% increase in eclosion time), reduced median lifespan (up to 50% decrease at 2% MP), and elevated oxidative damage ({uparrow}MDA, {downarrow}FRAP) in a dose-dependent manner. Protein content declined more rapidly with age, suggesting oxidative degradation or proteolysis. Both adult climbing and larval crawling performances were impaired, linking biochemical stress to neuromuscular dysfunction. MP produced stronger oxidative and behavioral effects than EP. Feeding controls confirmed that observed deficits were not due to nutritional differences. Chronic MP and EP exposure induces systemic toxicity in D. melanogaster, integrating endocrine disruption and redox imbalance as plausible mechanisms. Given conserved stress and hormonal pathways, these findings reinforce the need to re-evaluate low-dose paraben safety limits and highlight Drosophila as a rapid, ethically viable platform for screening environmental preservatives and safer substitutes.

neuroscience↗

bioRxiv2025

Recognition of unique cell surface glycopolymers by diversified lectin domains specifies umbrella toxin targeting

The bacterial cell envelope provides structural integrity and is an essential conduit through which the organism interacts with its environment. However, the molecular structures of its constituents can also be exploited as receptors, permitting threats such as toxins and phage access to the cell. We previously demonstrated that Streptomyces coelicolor secretes an umbrella toxin particle that acts in a highly selective manner to inhibit the hyphal growth of competing Streptomyces strains. Here, we identify the receptor of umbrella toxins as teichuronic acid (TUA) oligosaccharides anchored to the cell surface through linkage to wall teichoic acids (WTA). We show that the carbohydrate portion of this previously undescribed hybrid TUA-WTA molecule is variable across species and that targeting specificity derives from its selective recognition by diversified lectin domains associated with umbrella particles. A cryo-EM structure of a lectin-TUA complex reveals the molecular basis for umbrella toxin cell targeting and, in conjunction with bioinformatic analyses, provides insights into a molecular arms race that we posit drives diversification of umbrella particles and the chemical composition of Streptomyces cell envelopes.

microbiology↗

bioRxiv2024

Distant Ribose 2'-O-Methylation of 23S rRNA Helix 69 Pre-Orders the Capreomycin Drug Binding Pocket at the Ribosome Subunit Interface

Loss of ribosomal RNA (rRNA) modifications incorporated by the intrinsic methyltransferase TlyA results in reduced sensitivity to tuberactinomycin antibiotics such as capreomycin. However, the mechanism by which rRNA methylation alters drug binding, particularly at the distant but functionally more important site in 23S rRNA Helix 69 (H69), is currently unknown. We determined high-resolution cryo-electron microscopy structures of the Mycolicibacterium smegmatis 70S ribosome with or without the two ribose 2-O-methyl modifications incorporated by TlyA. In the unmodified ribosome, the tip of H69 adopts a more compact conformation, positioning two key nucleotides (A2137 and C2138) such that interactions with capreomycin would be lost and the binding pocket partially occluded. In contrast, methylation of 23S rRNA nucleotide C2144 results in conformational changes that propagate from the site of modification to the H69 tip, resulting in its movement away from h44, a more favorable positioning of C2138 and adoption of a more open conformation to enable capreomycin binding. Methylation of h44 also results in structural rearrangements at the H69-h44 interface that further support antibiotic binding. These structures thus reveal the effect and regulation of distant rRNA methylation on ribosome-targeting antibiotic binding.

microbiology↗

bioRxiv2023

Umbrella toxin particles produced by Streptomyces block mycelial growth of competing species

The Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive. The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species. Here we show that in addition to small molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-EM structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, leading to our naming them umbrella particles. S. coelicolor encodes three umbrella particles with distinct toxin and lectin composition, and supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, S. griseus, we find inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative mycelial growth. Our data show that Streptomyces umbrella particles mediate competition between vegetative mycelia of related species, a function distinct from small molecule antibiotics, which are produced at the onset of reproductive growth and act broadly. Sequence analyses suggest this role of umbrella particles extends beyond Streptomyces, as we find umbrella loci in nearly one-thousand species across Actinobacteria.

microbiology↗

bioRxiv2023

30S subunit recognition and G1405 modification by the aminoglycoside-resistance 16S ribosomal RNA methyltransferase RmtC

Acquired ribosomal RNA (rRNA) methylation has emerged as a significant mechanism of aminoglycoside resistance in pathogenic bacterial infections. Modification of a single nucleotide in the ribosome decoding center by the aminoglycoside-resistance 16S rRNA (m7G1405) methyltransferases effectively blocks the action of all 4,6-deoxystreptamine ring-containing aminoglycosides, including the latest generation of drugs. To define the molecular basis of 30S subunit recognition and G1405 modification by these enzymes, we used a S-adenosyl-L-methionine (SAM) analog to trap the complex in a post-catalytic state to enable determination of an overall 3.0 [A] cryo-electron microscopy structure of the m7G1405 methyltransferase RmtC bound to the mature Escherichia coli 30S ribosomal subunit. This structure, together with functional analyses of RmtC variants, identifies the RmtC N-terminal domain as critical for recognition and docking of the enzyme on a conserved 16S rRNA tertiary surface adjacent to G1405 in 16S rRNA helix 44 (h44). To access the G1405 N7 position for modification, a collection of residues across one surface of RmtC, including a loop that undergoes a disorder to order transition upon 30S subunit binding, induces significant distortion of h44. This distortion flips G1405 into the enzyme active site where it is positioned for modification by two almost universally conserved RmtC residues. These studies expand our understanding of ribosome recognition by rRNA modification enzymes and present a more complete structural basis for future development of strategies to inhibit m7G1405 modification to re-sensitize bacterial pathogens to aminoglycosides. SignificanceIncreasing prevalence of bacterial antibiotic resistance threatens our ability to treat bacterial infections and with it, many other facets of modern healthcare. For the ribosome-targeting aminoglycoside antibiotics, diverse pathogenic bacteria have acquired ribosomal RNA (rRNA) methyltransferase enzymes that confer exceptionally high-level resistance through site-specific modification of the drug binding site. Here, we define the molecular basis for ribosomal substrate recognition and modification by an enzyme (RmtC) representing the most clinically prevalent methyltransferase family. Specifically, RmtC exploits a conserved rRNA surface for binding and induces significant disruption of the rRNA structure to capture the target nucleotide for modification via a "base flipping" mechanism. These insights also present a platform for methyltransferase inhibitor development to extend usefulness of aminoglycoside antibiotics.

biochemistry↗

bioRxiv2022

BRCA1 Hypermethylation In Sporadic Breast Cancers: Discovering A Novel Pathway To Tumorigenesis Via Coordinate NBR2 Deregulation And TNBC Transformation

Women with a family history of mutations in the Breast cancer susceptibility gene, BRCA1 will have an increased risk of developing breast neoplasms. However, majority of the breast cancers are sporadic where BRCA1 mutations are very rare. Instead, 5-65% of sporadic cases manifest BRCA1 promoter hypermethylation and 30-40% of such cases develop into Triple Negative Breast Cancers. Even then, the molecular mechanism of BRCA1 hypermethylation mediated breast tumorigenesis has remained an enigma till date. Here, we present a novel tumorigenesis pathway for breast cancers that engenders from BRCA1 hypermethylation by generating site-specific methylations in the BRCA1 promoter using a modified version of CRISPR technology. We report that induction of site-specific methylation on BRCA1 promoter effectuates a downregulation in BRCA1 expression via alteration in the balance between its alternate transcripts {beta} and . Induced BRCA1 hypermethylation is also responsible for the attenuation of a long noncoding RNA, NBR2 (Neighbour of BRCA1 gene 2), which is transcribed through the bidirectional BRCA1 promoter in the reverse direction. Downregulation of NBR2 activates a feedback loop by leading to further downregulation of BRCA1 which is more evident under glucose starvation conditions and is associated with impaired DNA damage repair. BRCA1 hypermethylation also results in significant overexpression of {beta}-hCG (human chorionic gonadotrophin), which was found to be associated with highly aggressive and drug-resistant forms of BRCA1 mutated breast cancers invitro & in vivo in our previous study. Further, we report a change in the hormone receptor levels as the tumor progresses which demonstrates how BRCA1 deficient cells modulate their expression of ER- and ER-{beta} to promote their proliferation in early stages of tumor development and at later stages, transform to a basal tumor subtype by shedding down the expression of ER- & PR. Interestingly, we also discovered that modulation of ER- expression upon BRCA1 hypermethylation is responsible for the alteration in BRCA1 transcript ratio. Finally, in in vivo mouse studies, BRCA1 hypermethylated tumors were found to be much larger, aggressive and invasive as compared to wildtype, BRCA1 and NBR2 knockdown tumors with downregulation of ER- and PR; which explains the most probable reason behind high relapse rates in BRCA1 hypermethylated tumors. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/490082v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1c81155org.highwire.dtl.DTLVardef@1ea1fdcorg.highwire.dtl.DTLVardef@1d1b5e9org.highwire.dtl.DTLVardef@fddd22_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

50S subunit recognition and modification by the Mycobacterium tuberculosis ribosomal RNA methyltransferase TlyA

Changes in bacterial ribosomal RNA methylation status can alter the activity of diverse groups of ribosome-targeting antibiotics. These modifications are typically incorporated by a single methyltransferase that acts on one nucleotide target and rRNA methylation directly prevents drug binding, thereby conferring drug resistance. Loss of intrinsic methylation can also result in antibiotic resistance. For example, Mycobacterium tuberculosis becomes sensitized to tuberactinomycin antibiotics, such as capreomycin and viomycin, due to the action of the intrinsic methyltransferase TlyA. TlyA is unique among antibiotic resistance-associated methyltransferases as it has dual 16S and 23S rRNA substrate specificity and can incorporate cytidine-2-O-methylations within two structurally distinct contexts. Here, we report the structure of a mycobacterial 50S subunit-TlyA complex trapped in a post-catalytic state with a S-adenosyl-L-methionine analog using single-particle cryogenic electron microscopy. Together with complementary functional analyses, this structure reveals critical roles in 23S rRNA substrate recognition for conserved residues across an interaction surface that spans both TlyA domains. These interactions position the TlyA active site over the target nucleotide C2144 which is flipped from 23S Helix 69 in a process stabilized by stacking of TlyA residue Phe157 on the adjacent A2143. Base flipping may thus be a common strategy among rRNA methyltransferase enzymes even in cases where the target site is accessible without such structural reorganization. Finally, functional studies with 30S subunit suggest that the same TlyA interaction surface is employed to recognize this second substrate, but with distinct dependencies on essential conserved residues. Significance StatementThe bacterial ribosome is an important target for antibiotics used to treat infection. However, resistance to these essential drugs can arise through changes in ribosomal RNA (rRNA) modification patterns through the action of intrinsic or acquired rRNA methyltransferase enzymes. How these antibiotic resistance-associated enzymes recognize their ribosomal targets for site-specific modification is currently not well defined. Here, we uncover the molecular basis for large ribosomal (50S) subunit substrate recognition and modification by the Mycobacterium tuberculosis methyltransferase TlyA, necessary for optimal activity of the antitubercular drug capreomycin. From this work, recognition of complex rRNA structures distant from the site of modification and "flipping" of the target nucleotide base both emerge as general themes in ribosome recognition for bacterial rRNA modifying enzymes.

biochemistry↗

bioRxiv2020

Ribosome rescue inhibitors clear Neisseria gonorrhoeae in vivo using a new mechanism

The trans-translation pathway for rescuing stalled ribosomes is conserved and essential in bacterial pathogens but has no mammalian homolog, making it an ideal target for new antibiotics. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, resulting in broad-spectrum antibiotic activity. Optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles produced MBX-4132, which cleared multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Cryo-EM studies of non-stop ribosomes showed that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein L27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. One Sentence SummaryRibosome rescue inhibitors reveal a new conformation of the ribosome and kill drug-resistant Neisseria gonorrhoeae in vivo.

microbiology↗