Biology subjects

Davis, B. M.

Publications and source records attributed to Davis, B. M..

11 recordsLinked to original sources

bioRxiv2026

Disrupting aberrant EGFR catalytic trimers reverses T790M gefitinib resistance

Epidermal growth factor receptor (EGFR) mutations drive up to 50% of non-small-cell lung cancers (NSCLC). Although tyrosine kinase inhibitors provide substantial clinical benefit, remissions are prematurely terminated by the inevitable acquisition of on-target resistance. Beyond structural changes that alter ATP-pocket affinity, EGFR oligomerization drives this resistance, though the underlying mechanisms remain unclear. Here we show that progressive secondary and tertiary resistant NSCLC EGFR-mutants form ligand-free cell surface oligomers that contain catalytic trimers instead of the canonical dimers found within these oligomers in wild-type and gefitinib-sensitive EGFR-mutants. Genetically disrupting these pathological trimers into dimers via a single-point mutation rewires downstream signaling, decelerates tumor progression, and reverses gefitinib resistance in vivo. Conversely, genetic engineering of dimers into trimers reinstates normal tumor growth. These findings reveal a structural vulnerability specific to these refractory variants, demonstrating that targeting intra-oligomer interactions can overcome resistance, and providing a blueprint for protein-protein interface modulation strategies in NSCLC.

cancer biology↗

bioRxiv2025

Integration of Multiomic and Multi-phenotypic Data Identifies Biological Pathways Associated with Physical Fitness

Unraveling the complex associations between human phenotypes and molecular pathways can pave the way to improved health and performance, but faces a fundamental challenge: the measurable genes, proteins, and metabolites vastly outnumber the participants in even the largest studies, yielding spurious correlations. To address this imbalance, we have developed a bioinformatic framework and computational approach ("PhenoMol") to discover biological drivers of phenotypic characteristics that integrates all available phenotypic data predictive of outcomes and reduces multi-omic data dimensionality by generating "expression circuits" via graph theory constrained by prior biological knowledge of molecular interactions. We applied PhenoMol to analyze causal patterns and predict elite physical performance in a healthy cohort with deep physiological, physical, behavioral, cognitive, and molecular characterization. PhenoMol outperforms regression models based on equivalent analytic methodologies that do not employ network biology for dimensionality reduction. The PhenoMol software is provided for future studies.

bioinformatics↗

bioRxiv2024

RhoA Allosterically Activates Phospholipase Cε via its EF Hands

Phospholipase C{varepsilon} (PLC{varepsilon}) cleaves phosphatidylinositol lipids to increase intracellular Ca2+ and activate protein kinase C (PKC) in response to stimulation of cell surface receptors. PLC{varepsilon} is activated via direct binding of small GTPases at the cytoplasmic leaflets of cellular membranes. In the cardiovascular system, the RhoA GTPase regulates PLC{varepsilon} to initiate a pathway that protects against ischemia/reperfusion injuries, but the underlying molecular mechanism is not known. We present here the cryo-electron microscopy (cryo-EM) reconstruction of RhoA bound to PLC{varepsilon}, showing that the G protein binds a unique insertion within the PLC{varepsilon} EF hands. Deletion of or mutations to this PLC{varepsilon} insertion decrease RhoA-dependent activation without impacting regulation by other G proteins. Together, our data support a model wherein RhoA binding to PLC{varepsilon} allosterically activates the lipase and increases its interactions with the membrane, resulting in maximum activity and cardiomyocyte survival.

biochemistry↗

bioRxiv2023

The T766M-EGFR lung cancer mutation promotes tumor growth by exploiting newfound assembly mechanisms in ligand-free EGFR oligomers

Epidermal growth factor receptor (EGFR) is central to cell growth in physiology and pathophysiologies, including non-small cell lung cancer (NSCLC). EGFR has been successfully targeted with tyrosine kinase inhibitor generations, but the missense secondary T766M mutation is a common cause of resistance. Overcoming this therapeutic challenge has been hindered by poor understanding of how T766M dysregulates EGFR function leading to tumor progression. Here we show that T766M amplifies tumor growth in vivo by exploiting newly discovered oligomer assembly mechanisms employed by wild type (WT)-EGFR to maintain ligand-independent basal phosphorylation. These mechanisms, also shared by drug-resistant exon 20 EGFR insertions, reveal tumor growth promoting functions for hitherto orphan transmembrane and kinase interfaces and for the ectodomain tethered conformation of EGFR. Placing our findings into the context of a ligand-free oligomer structure model, we provide a framework for future drug discovery directed at tackling EGFR mutations in cancer by disabling oligomer-assembling interactions.

cancer biology↗

bioRxiv2023

Abnormal enteric nervous system and motor activity in the ganglionic proximal bowel of Hirschsprung's disease

Hirschsprungs disease (HSCR) is a congenital defect in which the enteric nervous system (ENS) does not develop in the distal bowel, requiring surgical removal of the portions of bowel without ENS ganglia ( aganglionic) and reattachment of the normal proximal bowel with ENS ganglia. Unfortunately, many HSCR patients have persistent dysmotility (e.g., constipation, incontinence) and enterocolitis after surgery, suggesting that the remaining bowel is not normal despite having ENS ganglia. Anatomical and neurochemical alterations have been observed in the ENS-innervated proximal bowel from HSCR patients and mice, but no studies have recorded ENS activity to define the circuit mechanisms underlying post-surgical HSCR dysfunction. Here, we generated a HSCR mouse model with a genetically-encoded calcium indicator to map the ENS connectome in the proximal colon. We identified abnormal spontaneous and synaptic ENS activity in proximal colons from GCaMP-Ednrb-/- mice with HSCR that corresponded to motor dysfunction. Many HSCR-associated defects were also observed in GCaMP-Ednrb+/- mice, despite complete ENS innervation. Results suggest that functional abnormalities in the ENS-innervated bowel contribute to post-surgical bowel complications in HSCR patients, and HSCR-related mutations that do not cause aganglionosis may cause chronic colon dysfunction in patients without a HSCR diagnosis.

physiology↗

bioRxiv2020

Unique molecular characteristics of visceral afferents arising from different levels of the neuraxis: location of afferent somata predicts function and stimulus detection modalities

Visceral organs receive neural innervation from sensory ganglia located adjacent to multiple levels of the brainstem and spinal cord. Here we examined whether molecular profiling could be used to identify functional clusters of colon afferents from thoracolumbar (TL), lumbosacral (LS), and nodose ganglia (NG) in the mouse. Profiling of TL and LS bladder afferents was also done. Visceral afferents were back-labeled using retrograde tracers injected into proximal and distal regions of colon or bladder, followed by single cell RT-qPCR and analysis via an automated hierarchical clustering method. Genes were chosen for assay (32 for bladder; 48 for colon) based on their established role in stimulus detection, regulation of sensitivity/function or neuroimmune interaction. A total of 132 colon afferents (from NG, TL and LS) and 128 bladder afferents (from TL and LS) were analyzed. Retrograde labeling from the colon showed NG and TL afferents innervate proximal and distal regions of the colon whereas 98% of LS afferents only project to distal regions. There were clusters of colon and bladder afferents, defined by mRNA profiling, that localized to either TL or LS ganglia. Mixed TL/LS clustering also was found. In addition, transcriptionally, NG colon afferents were almost completely segregated from colon DRG (TL or LS) neurons. These results indicate that populations of primary visceral afferents are functionally "tuned" to detect and interact with the internal environment and that information from all levels is integrated at higher (CNS) levels, not only for regulation of homeostatic functions, but for conscious visceral sensations including pain. Significance StatementVisceral organs are innervated by sensory neurons whose cell bodies are located in multiple ganglia associated with the brainstem and spinal cord. For the colon, this overlapping innervation is proposed to facilitate visceral sensation and homeostasis, where sensation and pain is mediated by spinal afferents and fear and anxiety (the affective aspects of visceral pain) are the domain of nodose afferents. Transcriptomic analysis performed here reveals that genes implicated in both homeostatic regulation and pain are found in afferents across all ganglia types, suggesting that conscious sensation and homeostatic regulation is the result of convergence, and not segregation, of sensory input.

neuroscience↗

bioRxiv2020

Optogenetic inhibition of colon epithelium reduces hypersensitivity in a mouse model of inflammatory bowel disease

Visceral pain is a prevalent symptom of inflammatory bowel disease (IBD) that can be difficult to treat. Pain and hypersensitivity are mediated by extrinsic primary afferent neurons (ExPANs) that innervate the colon. Recent studies indicate that the colon epithelium contributes to initiating ExPAN firing and nociceptive responses. Based on these findings we hypothesized that the epithelium contributes to inflammation-induced hypersensitivity. A key prediction of this hypothesis is that inhibition of the epithelium would attenuate nociceptive signaling and inflammatory hypersensitivity. To test this hypothesis, the inhibitory yellow light activated protein archaerhodopsin was targeted to the intestinal epithelium (villin-Arch) or the ExPANs (TRPV1-Arch) that innervate the colon. Visceral sensitivity was assessed by measuring the visceromotor response (VMR) to colorectal distension (CRD), with and without yellow light illumination of the colon lumen. Inhibition of the colon epithelium in healthy villin-Arch mice significantly diminished the CRD-induced VMR. Direct inhibition of ExPANs during CRD using TRPV1-Arch mice showed that ExPAN and epithelial inhibition were similarly effective in reducing the VMR to CRD. We then investigated the effect of epithelial and ExPAN inhibition in the dextran sulfate sodium (DSS) model of inflammatory bowel disease (IBD). Inhibition of the colon epithelium significantly decreased DSS-induced hypersensitivity and was comparable to inhibition of ExPANS. Together these results reveal the potential of targeting the colon epithelium for treatment of pain.

neuroscience↗

bioRxiv2019

A New Suite of Allelic Exchange Vectors for the Scarless Modification of Proteobacterial Genomes

Despite the advent of new techniques for genetic engineering of bacteria, allelic exchange through homologous recombination remains an important tool for genetic analysis. Currently, sacB-based vector systems are often used for allelic exchange, but counter-selection escape, which prevents isolation of cells with the desired mutation, limits its utility. To circumvent this limitation, we engineered a series of \"pTOX\" allelic exchange vectors. Each plasmid encodes one of a set of inducible toxins, chosen for their potential utility in a wide range of medically important Proteobacteria. A codon-optimized rhaS transcriptional activator with a strong synthetic ribosome binding site enables tight toxin induction even in organisms lacking an endogenous rhamnose regulon. Expression of the blue amilCP or magenta tsPurple non-fluorescent chromoproteins facilitates monitoring of successful single- and double-crossover events using these vectors. The versatility of these vectors was demonstrated by deleting genes in Serratia marcescens, Escherichia coli O157:H7, Enterobacter cloacae, and Shigella flexneri. Finally, pTOX was used to characterize the impact of disruption of all combinations of the 3 orthologous S. marcescens peptidoglycan amidohydrolases on chromosomal ampC beta-lactamase activity and corresponding beta-lactam antibiotic resistance. Mutation of multiple amidohydrolases was necessary for high level ampC derepression and beta-lactam resistance. These data suggest why beta-lactam resistance may emerge during treatment less frequently in S. marcescens than in other AmpC-producing pathogens like E. cloacae. Collectively, our findings suggest that the pTOX vectors should be broadly useful for genetic engineering of Gram-negative bacteria.\n\nImportanceTargeted modification of bacterial genomes is critical for genetic analyses of microorganisms. Allelic exchange is a technique that relies on homologous recombination to substitute native loci for engineered sequences. However, current allelic exchange vectors often enable only weak selection for successful homologous recombination. We developed a suite of new allelic exchange vectors, pTOX, which were validated in several medically important Proteobacteria. They encode visible non-fluorescent chromoproteins that enable easy identification of colonies bearing integrated vector, and permit stringent selection for the second step of homologous recombination, yielding modified loci. We demonstrate the utility of these vectors by using them to investigate the effect of inactivation of Serratia marcescens peptidoglycan amidohydrolases on beta-lactam antibiotic resistance.

microbiology↗

bioRxiv2019

A Vibrio cholerae BolA-like protein is required for proper cell shape and cell envelope integrity

BolA family proteins are conserved in gram-negative bacteria and many eukaryotes. While diverse cellular phenotypes have been linked to this protein family, the molecular pathways through which these proteins mediate their effects are not well-described. Here, we investigated the role of BolA family proteins in Vibrio cholerae, the cholera pathogen. Like Escherichia coli, V. cholerae encodes two BolA proteins, BolA and IbaG. However, in marked contrast to E. coli, where bolA is linked to cell shape and ibaG is not, in V. cholerae, bolA mutants lack morphological defects, whereas ibaG proved critical for the generation and/or maintenance of the pathogens morphology. Notably, the bizarre-shaped, multi-polar, elongated and wide cells that predominated in exponential phase {Delta}ibaG V. cholerae cultures were not observed in stationary phase cultures. The V. cholerae {Delta}ibaG mutant exhibited increased sensitivity to cell envelope stressors, including cell wall acting antibiotics and bile, and was defective in intestinal colonization. {Delta}ibaG V. cholerae had reduced peptidoglycan and lipid II and altered outer membrane lipids, likely contributing to the mutants morphological defects and sensitivity to envelope stressors. Transposon-insertion sequencing analysis of ibaGs genetic interactions suggested that ibaG is involved in several processes involved in the generation and homeostasis of the cell envelope. Furthermore, co-purification studies revealed that IbaG interacts with proteins containing iron-sulfur clusters or involved in their assembly. Collectively, our findings suggest that V. cholerae IbaG controls cell morphology and cell envelope integrity through its role in biogenesis or trafficking of iron-sulfur cluster proteins.\n\nImportanceBolA-like proteins are conserved across prokaryotes and eukaryotes. These proteins have been linked to a variety of phenotypes, but the pathways and mechanisms through which they act have not been extensively characterized. Here, we unraveled the role of the BolA-like protein IbaG in the cholera pathogen Vibrio cholerae. The absence of IbaG was associated with dramatic changes in cell morphology, sensitivity to envelope stressors, and intestinal colonization defects. IbaG was found to be required for biogenesis of several components of the V. cholerae cell envelope and to interact with numerous iron-sulfur cluster containing proteins and factors involved in their assembly. Thus, our findings suggest that IbaG governs V. cholerae cell shape and cell envelope homeostasis through its effects on iron-sulfur proteins and associated pathways. The diversity of processes involving iron-sulfur containing proteins is likely a factor underlying the range of phenotypes associated with BolA family proteins.

microbiology↗

bioRxiv2019

Transposon-insertion sequencing screens unveil requirements for EHEC growth and intestinal colonization

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to identify genes required for EHEC and commensal E. coli K-12 growth in vitro and for EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHECs but not to K-12s growth in vitro, suggesting that gene acquisition during EHEC evolution has heightened the pathogens reliance on certain metabolic processes that are dispensable for K-12. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth in vivo. Both a refined version of an existing analytic framework as well as PCA-based analysis were used to compensate for the effects of the infection bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion apparatus is required for growth in vivo and revealed that only a few effectors are critical for in vivo fitness. Numerous mutants not previously associated with EHEC survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative in vivo fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, including tatABC, oxyR, envC, acrAB, and cvpA, promote EHEC resistance to host-derived stresses encountered in vivo. cvpA, which is also required for intestinal growth of several other enteric pathogens, proved to be required for EHEC, Vibrio cholerae and Vibrio parahaemolyticus resistance to the bile salt deoxycholate. Collectively, our findings provide a comprehensive framework for understanding EHEC growth in the intestine. Author SummaryEnterohemorrhagic E. coli (EHEC) are important food-borne pathogens that infect the colon. We created a highly saturated EHEC transposon library and used transposon insertion sequencing to identify the genes required for EHEC growth in vitro and in vivo in the infant rabbit colon. We found that there is a large infection bottleneck in the rabbit model of intestinal colonization, and refined two analytic approaches to facilitate rigorous identification of new EHEC genes that promote fitness in vivo. Besides the known type III secretion system, more than 200 additional genes were found to contribute to EHEC survival and/or growth within the intestine. The requirement for some of these new in vivo fitness factors was confirmed, and their contributions to infection were investigated. This set of genes should be of considerable value for future studies elucidating the processes that enable the pathogen to proliferate in vivo and for design of new therapeutics.

microbiology↗

bioRxiv2018

CRISPR screen reveals that EHEC’s T3SS and Shiga toxin rely on shared host factors for infection

Enterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors - a type III secretion system (T3SS) and Shiga toxins (Stx) - that are required for the pathogen to colonize the intestine and cause diarrheal disease. Here, we carried out a genome-wide CRISPR/Cas9 loss-of-function screen to identify host loci that facilitate EHEC infection of intestinal epithelial cells. Many of the guide RNAs identified targeted loci known to be associated with sphingolipid biosynthesis, particularly for production of globotriaosylceramide (Gb3), the Stx receptor. Two loci (TM9SF2 and LAPTM4A) with largely unknown functions were also targeted. Mutations in these loci not only rescued cells from Stx-mediated cell death, but also prevented cytotoxicity associated with the EHEC T3SS. These mutations interfered with early events associated with T3SS and Stx pathogenicity, markedly reducing entry of T3SS effectors into host cells and binding of Stx. The convergence of Stx and T3SS onto overlapping host targets provides guidance for design of new host-directed therapeutic agents to counter EHEC infection.\n\nImportanceEnterohemorrhagic Escherichia coli (EHEC) has two critical virulence factors - a type III secretion system (T3SS) and Shiga toxins (Stx) - that are required for colonizing the intestine and causing diarrheal disease. We screened a genome-wide collection of CRISPR mutants derived from intestinal epithelial cells and identified mutants with enhanced survival following EHEC infection. Many had mutations that disrupted synthesis of a subset of lipids (sphingolipids) that includes the Stx receptor globotriaosylceramide (Gb3), and hence protect against Stx intoxication. Unexpectedly, we found that sphingolipids also mediate early events associated with T3SS pathogenicity. Since antibiotics are contraindicated for the treatment of EHEC, therapeutics targeting sphingolipid biosynthesis are a promising alternative, as they could provide protection against both of the pathogens key virulence factors.

microbiology↗