Biology subjects

Davis, K. P.

Publications and source records attributed to Davis, K. P..

5 recordsLinked to original sources

bioRxiv2025

Antibiotic dose-response curves can measure antibiotic activity against Mycobacterium abscessus and Mycobacterium peregrinum

Mycobacterium abscessus is a drug-resistant pathogen associated with poor clinical outcomes despite treatment with multidrug antibiotic regimens. Apart from clarithromycin, antimicrobial susceptibility testing (AST) results for Mycobacterium abscessus cannot guide antibiotic selection. AST involves measuring the minimum inhibitory concentration (MIC), which is allowed to span a four-fold range in concentration. This accepted variability of the MIC limits the clinical utility of AST. Antimicrobial dose-response curves, obtained by measuring the growth inhibition of a given organism to increasing concentrations of an antibiotic, can yield metrics of antibiotic activity that are less variable than the MIC. We used Clinical and Laboratory Standards Institute growth conditions for rapid-grower nontuberculous mycobacteria to generate 990 dose-response curves across three time points (72 hours, 96 hours, and 120 hours) for six guideline-recommended (clarithromycin, amikacin, cefoxitin, linezolid, tigecycline, and clofazimine) and five new (omadacycline, tedizolid, SPR719, SQ109, and bedaquiline) antibiotics against Mycobacterium abscessus subspecies abscessus ATCC 19977 and Mycobacterium peregrinum ATCC 700686. We established the fit of the dose-response curve (R2) as a quality control metric. Using the geometric standard deviation and median coefficient of variation, we demonstrated that the IC50 and IC75 (antibiotic concentrations corresponding to 50% and 75% growth inhibition, respectively) are less variable than the MIC. We identified time-dependent changes in dose-response curve metrics that allow the detection of inducible clarithromycin resistance with only five days of incubation. This study demonstrates the potential of dose-response curves in measuring antibiotic activity against Mycobacterium abscessus.

microbiology↗

bioRxiv2024

Massively parallel combination screen reveals small molecule sensitization of antibiotic-resistant Gram-negative ESKAPE pathogens

Antibiotic resistance, especially in multidrug-resistant ESKAPE pathogens, remains a worldwide problem. Combination antimicrobial therapies may be an important strategy to overcome resistance and broaden the spectrum of existing antibiotics. However, this strategy is limited by the ability to efficiently screen large combinatorial chemical spaces. Here, we deployed a high-throughput combinatorial screening platform, DropArray, to evaluate the interactions of over 30,000 compounds with up to 22 antibiotics and 6 strains of Gram-negative ESKAPE pathogens, totaling to over 1.3 million unique strain-antibiotic-compound combinations. In this dataset, compounds more frequently exhibited synergy with known antibiotics than single-agent activity. We identified a compound, P2-56, and developed a more potent analog, P2-56-3, which potentiated rifampin (RIF) activity against Acinetobacter baumannii and Klebsiella pneumoniae. Using phenotypic assays, we showed P2-56-3 disrupts the outer membrane of A. baumannii. To identify pathways involved in the mechanism of synergy between P2-56-3 and RIF, we performed genetic screens in A. baumannii. CRISPRi-induced partial depletion of lipooligosaccharide transport genes (lptA-D, lptFG) resulted in hypersensitivity to P2-56-3/RIF treatment, demonstrating the genetic dependency of P2-56-3 activity and RIF sensitization on lpt genes in A. baumannii. Consistent with outer membrane homeostasis being an important determinant of P2-56-3/RIF tolerance, knockout of maintenance of lipid asymmetry complex genes and overexpression of certain resistance-nodulation-division efflux pumps - a phenotype associated with multidrug-resistance - resulted in hypersensitivity to P2-56-3. These findings demonstrate the immense scale of phenotypic antibiotic combination screens using DropArray and the potential for such approaches to discover new small molecule synergies against multidrug-resistant ESKAPE strains. Significance StatementThere is an unmet need for new antibiotic therapies effective against the multidrug-resistant, Gram-negative ESKAPE pathogens. Combination therapies have the potential to overcome resistance and broaden the spectrum of existing antibiotics. In this study, we use DropArray, a massively parallel combinatorial screening tool, to assay more than 1.3 million combinations of small molecules against the Gram-negative ESKAPE pathogens, Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We discovered a synthetic small molecule potentiator, P2-56, of the antibiotic rifampin effective in A. baumannii and K. pneumoniae. We generated P2-56-3, a more potent derivative of P2-56, and found that it likely potentiates rifampin by compromising the outer membrane integrity. Our study demonstrates a high-throughput strategy for identifying antibiotic potentiators against multidrug-resistant bacteria.

microbiology↗

bioRxiv2023

Allosteric site variants affect GTP hydrolysis on RAS

AbstractRAS GTPases are proto-oncoproteins that regulate cell growth, proliferation, and differentiation in response to extracellular signals. The signaling functions of RAS, and other small GTPases, are dependent on their ability to cycle between GDP-bound and GTP-bound states. Structural analyses suggest that GTP hydrolysis catalyzed by HRAS can be regulated by an allosteric site located between helices 3, 4 and loop 7. Here we explore the relationship between intrinsic GTP hydrolysis on HRAS and the position of helix 3 and loop 7 through manipulation of the allosteric site, showing that the two sites are functionally connected. We generated several hydrophobic mutations in the allosteric site of HRAS to promote shifts in helix 3 relative to helix 4. By combining crystallography and enzymology to study these mutants, we show that closure of the allosteric site correlates with increased hydrolysis of GTP on HRAS in solution. Interestingly, binding to the RAS binding domain of RAF kinase (RAF-RBD) inhibits GTP hydrolysis in the mutants. This behavior may be representative of a cluster of poorly understood mutations that occur in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.

biochemistry↗

bioRxiv2022

Critical role of growth medium for detecting drug interactions in Gram-negative bacteria that model in vivo responses

The rise in infections caused by multidrug-resistant (MDR) bacteria has necessitated a variety of clinical approaches, including the use of antibiotic combinations. Antibiotic susceptibility is affected in part by the growth state of bacteria within various tissues. Here we tested the hypothesis that drug-drug interactions vary in different media, and hence, using a medium that reflects tissue environments will better predict in vivo outcomes. We systematically studied pair-wise antibiotic interactions in three different media (CAMHB, a urine mimetic, and a lung mimetic) using three Gram-negative ESKAPE pathogens, Acinetobacter baumannii (Ab), Klebsiella pneumoniae (Kp), and Pseudomonas aeruginosa (Pa). There were pronounced differences in responses to antibiotic combinations between the three bacterial species grown in the same medium. However, within species, Pa responded to drug combinations similarly when grown in all three different media, whereas Ab responded similarly when grown in CAMHB and a lung mimetic medium. By contrast, drug interactions in Kp were poorly correlated across three different media. To assess whether distinct media were predictive of antibiotic interactions in Kp in the lungs of mice, we developed a treatment strategy and tested three antibiotic combination pairs. Measurements obtained in vitro from lung mimetic medium, but not rich medium, predicted in vivo outcomes. This work demonstrates that antibiotic interactions are highly variable when comparing across three gram-negative pathogens and highlights the importance of growth medium by showing a superior correlation between in vitro interactions in a growth medium that resembles the tissue environment and in vivo outcomes.

microbiology↗

bioRxiv2021

Specificity and selective advantage of an exclusion system in the integrative and conjugative element ICEBs1 of Bacillus subtilis

Integrative and conjugative elements (ICEs) are mobile genetic elements capable of transferring their own and other DNA. They contribute to the spread of antibiotic resistances and other important traits for bacterial evolution. Exclusion is a mechanism used by many conjugative plasmids and a few ICEs to prevent their host cell from acquiring a second copy of the cognate element. ICEBs1 of Bacillus subtilis has an exclusion mechanism whereby the exclusion protein YddJ in a potential recipient inhibits the activity of the ICEBs1-encoded conjugation machinery in a potential donor. The target of YddJ-mediated exclusion is the conjugation protein ConG (a VirB6 homolog). Here we defined the regions of YddJ and ConG that confer exclusion specificity and determined the importance of exclusion to host cells. Using chimeras that had parts of ConG from ICEBs1 and the closely related ICEBat1 we identified a putative extracellular loop of ConG that conferred specificity for exclusion by the cognate YddJ. Using chimeras of YddJ from ICEBs1 and ICEBat1 we identified two regions in YddJ needed for exclusion specificity. We also found that YddJ-mediated exclusion reduced death of donor cells following conjugation into recipients. Donor death was dependent on the ability of transconjugants to themselves become donors and was reduced under osmo-protective conditions, indicating that death was likely due to alterations in the donor cell envelope caused by excessive conjugation. We postulate that elements that can have high frequencies of transfer likely evolved exclusion mechanisms to protect the host cells from excessive death. ImportanceHorizontal gene transfer is a driving force in bacterial evolution, responsible for the spread of many traits, including antibiotic and heavy metal resistances. Conjugation, one type of horizontal gene transfer, involves DNA transfer from donor to recipient cells through conjugation machinery and direct cell-cell contact. Exclusion mechanisms allow conjugative elements to prevent their host from acquiring additional copies of the element, and are highly specific enabling hosts to acquire heterologous elements. We defined regions of the exclusion protein and its target in the conjugation machinery that convey high specificity of exclusion. We found that exclusion protects donors from cell death during periods of high transfer. This is likely important for the element to enter new populations of cells.

microbiology↗