Biology subjects

Rock, C. O.

Publications and source records attributed to Rock, C. O..

7 recordsLinked to original sources

bioRxiv2025

Isoleucine binding and regulation of Escherichia coli and Staphylococcus aureus threonine dehydratase (IlvA)

In Staphylococcus aureus, the branched-chain amino acid biosynthetic pathway provides essential intermediates for membrane biosynthesis. Threonine deaminase (IlvA) is the first enzyme in the pathway, and isoleucine feedback-regulates the enzyme in Escherichia coli. These studies on E. coli IlvA (EcIlvA) introduced the concept of allosteric regulation. To investigate the regulation of S. aureus IlvA (SaIlvA), we first conducted additional studies on EcIlvA. The previously determined crystal structure of EcIlvA revealed a tetrameric assembly of protomers, each with catalytic and regulatory domains, but the structural basis of isoleucine regulation was not characterized. Here, we present the crystal structure of the EcIlvA regulatory domain bound to isoleucine, which reveals the isoleucine binding site and conformational changes that initiate at Phe352 and propagate 23 [A] across the domain. This suggests an allosteric pathway that extends to the active site of the adjacent protomer, mediating regulation across the protomer-protomer interface. The EcIlvA(F352A) mutant binds isoleucine but is feedback-resistant due to the absence of the initiating Phe352. In contrast, SaIlvA is not feedback-regulated by isoleucine and does not bind it. The structure of the SaIlvA regulatory domain reveals a different organization that lacks the isoleucine binding site. Other potential allosteric inhibitors of SaIlvA, including phospholipid intermediates, do not affect enzyme activity. We propose that the absence of feedback inhibition in SaIlvA is due to its role in membrane biosynthesis. These findings enhance our understanding of IlvAs allosteric regulation and offer opportunities for engineering feedback-resistant IlvA variants for biotechnological use.

microbiology↗

bioRxiv2024

The Bacillus subtilis cell envelope stress-inducible ytpAB operon modulates membrane properties and contributes to bacitracin resistance

Antibiotics that inhibit peptidoglycan synthesis trigger the activation of both specific and general protective responses. {sigma}M responds to diverse antibiotics that inhibit cell wall synthesis. Here, we demonstrate that cell wall inhibiting drugs, such as bacitracin and cefuroxime, induce the {sigma}M-dependent ytpAB operon. YtpA is a predicted hydrolase previously proposed to generate the putative lysophospholipid antibiotic bacilysocin (lysophosphatidylglycerol), and YtpB is the branchpoint enzyme for the synthesis of membrane-localized C35 terpenoids. Using targeted lipidomics we reveal that YtpA is not required for the production of lysophosphatidylglycerol. Nevertheless, ytpA was critical for growth in a mutant strain defective for homeoviscous adaptation due to a lack of genes for the synthesis of branched chain fatty acids and the Des phospholipid desaturase. Consistently, overexpression of ytpA increased membrane fluidity as monitored by fluorescence anisotropy. The ytpA gene contributes to bacitracin resistance in mutants additionally lacking the bceAB or bcrC genes, which directly mediate bacitracin resistance. These epistatic interactions support a model in which {sigma}M-dependent induction of the ytpAB operon helps cells tolerate bacitracin stress, either by facilitating the flipping of the undecaprenyl-phosphate carrier lipid or by impacting the assembly or function of membrane-associated complexes proteins involved in cell wall homeostasis. ImportancePeptidoglycan synthesis inhibitors include some of our most important antibiotics. In Bacillus subtilis, peptidoglycan synthesis inhibitors induce the {sigma}M regulon, which is critical for intrinsic antibiotic resistance. The {sigma}M-dependent ytpAB operon encodes a predicted hydrolase (YtpA) and the enzyme that initiates the synthesis of C35 terpenoids (YtpB). Our results suggest that YtpA is critical in cells defective in homeoviscous adaptation. Further, we find that YtpA functions cooperatively with the BceAB and BcrC proteins in conferring intrinsic resistance to bacitracin, a peptide antibiotic that binds tightly to the UPP lipid carrier that sustains peptidoglycan synthesis.

microbiology↗

bioRxiv2023

Vaginal Lactobacillus fatty acid response mechanisms reveal a novel strategy for bacterial vaginosis treatment

Bacterial vaginosis (BV), a common syndrome characterized by Lactobacillus-deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by Lactobacillus iners instead of Lactobacillus crispatus, which has more beneficial health associations. Strategies to promote L. crispatus and inhibit L. iners are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit L. iners and enhance L. crispatus growth. These phenotypes require OA-inducible genes conserved in L. crispatus and related species, including an oleate hydratase (ohyA) and putative fatty acid efflux pump (farE). FarE mediates OA resistance, while OhyA is robustly active in the human vaginal microbiota and sequesters OA in a derivative form that only ohyA-harboring organisms can exploit. Finally, OA promotes L. crispatus dominance more effectively than antibiotics in an in vitro model of BV, suggesting a novel approach for treatment.

microbiology↗

bioRxiv2022

Metabolic adaptations underpin resistance to histone acetyltransferase inhibition

Histone acetyltransferases (HAT) catalyze the acylation of lysine side chains and are implicated in diverse human cancers as both oncogenes and non-oncogene dependencies1. Acetyl-CoA-competitive HAT inhibitors have garnered attention as potential cancer therapeutics and the first clinical trial for this class is ongoing (NCT04606446). Despite broad enthusiasm for these targets, notably including CBP/p300 and KAT6A/B2-5, the potential mechanisms of therapeutic response and evolved drug resistance remain poorly understood. Using comparative transcriptional genomics, we found that the direct gene regulatory consequences of CBP/p300 HAT inhibition are indistinguishable in models of intrinsically hypersensitive and insensitive acute myeloid leukemia (AML). We therefore modelled acquired drug resistance using a forward genetic selection and identified dysregulation of coenzyme A (CoA) metabolism as a facile driver of resistance to HAT inhibitors. Specifically, drug resistance selected for mutations in PANK3, a pantothenate kinase that controls the rate limiting step in CoA biosynthesis6. These mutations prevent negative feedback inhibition, resulting in drastically elevated concentrations of intracellular acetyl-CoA, which directly outcompetes drug-target engagement. This not only impacts the activity of structurally diverse CBP/p300 HAT inhibitors, but also agents related to an investigational KAT6A/B inhibitor that is currently in Phase-1 clinical trials. We further validated these results using a genome-scale CRISPR/Cas9 loss-of-function genetic modifier screen, which identified additional gene-drug interactions between HAT inhibitors and the CoA biosynthetic pathway. Top hits from the screen included the phosphatase, PANK4, which negatively regulates CoA production and therefore suppresses sensitivity to HAT inhibition upon knockout7, as well as the pantothenate transporter, SLC5A68, which enhances sensitivity. Altogether, this work uncovers CoA plasticity as an unexpected but potentially class-wide liability of anti-cancer HAT inhibitors and will therefore buoy future efforts to optimize the efficacy of this new form of targeted therapy.

molecular biology↗

bioRxiv2022

A genome-wide atlas of antibiotic susceptibility targets and pathways to tolerance.

Detailed knowledge on how bacteria evade antibiotics and eventually develop resistance could open avenues for novel therapeutics and diagnostics. It is thereby key to develop a comprehensive genome-wide understanding of how bacteria process antibiotic stress, and how modulation of the involved processes affects their ability to overcome said stress. Here we undertake a comprehensive genetic analysis of how the major human pathogen Streptococcus pneumoniae responds to 20 antibiotics. We built a genome-wide atlas of drug susceptibility determinants and generate a genetic interaction network that connects cellular processes and genes of unknown function, which we show can be used as therapeutic targets. Pathway analysis reveals a genome-wide "tolerome", defined by cellular processes that can make a bacterium less susceptible, and often tolerant, in an antibiotic specific manner. Importantly, modulation of these processes confers fitness benefits during active infections under antibiotic selection. Moreover, screening of sequenced clinical isolates demonstrates that mutations in tolerome genes readily evolve and are frequently associated with resistant strains, indicating such mutations may be an important harbinger for the emergence of antibiotic resistance.

microbiology↗

bioRxiv2021

Structural transitions permitting ligand entry and exit in bacterial fatty acid binding proteins.

Fatty acid (FA) transfer proteins extract FA from membranes and sequester their ligand to facilitate its movement through the cytosol. While detailed views of soluble protein-FA complexes are available, how FA exchange occurs at the membrane has remained unknown. Staphylococcus aureus FakB1 is a prototypical bacterial FA transfer protein that binds palmitate within a narrow, buried tunnel. Here, we determine the conformational change from this closed state to an open state that engages the phospholipid bilayer. Upon membrane binding, a dynamic loop in FakB1 that covers the FA binding site disengages and folds into an amphipathic helix. This helix inserts below the phosphate plane of the bilayer to create a diffusion channel for the FA to exchange between the protein and the membrane. The structure of the bilayer-associated conformation of FakB1 has local similarities with mammalian FA binding proteins and provides a general conceptual framework for how these proteins interact with the membrane to promote lipid transfer.

biophysics↗

bioRxiv2021

Competence-associated peptide BriC alters fatty acid biosynthesis in Streptococcus pneumoniae

Membrane lipid homeostasis is required for bacteria to survive in a spectrum of host environments. This homeostasis is achieved by regulation of fatty acid chain length and of the ratio of saturated to unsaturated fatty acids. In the pathogen Streptococcus pneumoniae, fatty acid biosynthesis is encoded by a cluster of fatty acid biosynthesis (fab) genes (FASII locus) whose expression is controlled by the FabT repressor. Encoded immediately downstream of the FASII locus is BriC, a competence-induced, cell-cell communication peptide that promotes biofilm development as well as nasopharyngeal colonization in a murine model of pneumococcal carriage. Here, we demonstrate that briC is co-transcribed with genes of the fab gene cluster and that a reduction of briC levels, caused by decoupling its transcription from fab gene cluster, negatively impacts biofilm development. BriC elevates fabT transcription, which is predicted to alter the balance of saturated and unsaturated fatty acids produced by the pathway. We find that briC inactivation results in a decreased production of unsaturated fatty acids that impact the membrane properties by decreasing the abundance of di-unsaturated phosphatidylglycerol molecular species. We propose that the link between BriC, FabT and phospholipid composition contributes to the ability of S. pneumoniae to alter membrane homeostasis in response to the production of a quorum-sensing peptide. IMPORTANCEAdaptation of bacteria to their host environment is a key component of colonization and pathogenesis. As an essential component of bacterial membranes, fatty acid composition contributes to host adaptation. Similarly, so does cell-cell communication, which serves as a mechanism for population levels responses. While much is known about the pathways that control the biosynthesis of fatty acids, many questions remain regarding regulation of these pathways and consequently the factors that impacts the balance between saturated and unsaturated fatty acids. We find that BriC, a cell-cell communication peptide implicated in biofilm regulation and colonization, is both influenced by a fatty acid biosynthesis pathway and impacts this same pathway. This study identified a link between cell-cell communication, fatty acid composition, and biofilms and, in doing so, suggests that these pathways are integrated into the networks that control pneumococcal colonization and host adaptation.

microbiology↗