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.

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. Here, we tested the hypothesis that drug-drug interactions vary in different media, and determined which in vitro models best predict drug interactions in the lungs. We systematically studied pair-wise antibiotic interactions in three different media, CAMHB, (a rich lab medium standard for antibiotic susceptibility testing), a urine mimetic medium (UMM), and a minimal medium of M9 salts supplemented with glucose and iron (M9Glu) with 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, PaO1 responded to drug combinations similarly when grown in all three different media, whereas Ab17978 and other Ab clinical isolates responded similarly when grown in CAMHB and M9Glu medium. By contrast, drug interactions in Kp43816, and other Kp clinical isolates poorly correlated across different media. To assess whether any of these media were predictive of antibiotic interactions against Kp in the lungs of mice, we tested three antibiotic combination pairs. In vitro measurements in M9Glu, but not rich medium or UMM, predicted in vivo outcomes. This work demonstrates that antibiotic interactions are highly variable across three Gram-negative pathogens and highlights the importance of growth medium by showing a superior correlation between in vitro interactions in a minimal growth medium and in vivo outcomes. IMPORTANCE: Drug-resistant bacterial infections are a growing concern and have only continued to increase during the SARS-CoV-2 pandemic. Though not routinely used for Gram-negative bacteria, drug combinations are sometimes used for serious infections and may become more widely used as the prevalence of extremely drug-resistant organisms increases. To date, reliable methods are not available for identifying beneficial drug combinations for a particular infection. Our study shows variability across strains in how drug interactions are impacted by growth conditions. It also demonstrates that testing drug combinations in tissue-relevant growth conditions for some strains better models what happens during infection and may better inform combination therapy selection.

Allosteric site variants affect GTP hydrolysis on Ras.

RAS 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 mutations found in human tumors, which potentially cooperate with RAF complex formation to stabilize the GTP-bound state of RAS.

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 resistance 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 the 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 osmoprotective 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.IMPORTANCE Horizontal gene transfer is a driving force in bacterial evolution, responsible for the spread of many traits, including antibiotic and heavy metal resistance. 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.

Identification, characterization and benefits of an exclusion system in an integrative and conjugative element of Bacillus subtilis.

Integrative and conjugative elements (ICEs) are mobile genetic elements that transfer from cell to cell by conjugation (like plasmids) and integrate into the chromosomes of bacterial hosts (like lysogenic phages or transposons). ICEs are prevalent in bacterial chromosomes and play a major role in bacterial evolution by promoting horizontal gene transfer. Exclusion prevents the redundant transfer of conjugative elements into host cells that already contain a copy of the element. Exclusion has been characterized mostly for conjugative elements of Gram-negative bacteria. Here, we report the identification and characterization of an exclusion mechanism in ICEBs1 from the Gram-positive bacterium Bacillus subtilis. We found that cells containing ICEBs1 inhibit the activity of the ICEBs1-encoded conjugation machinery in other cells. This inhibition (exclusion) was specific to the cognate conjugation machinery and the ICEBs1 gene yddJ was both necessary and sufficient to mediate exclusion by recipient cells. Through a mutagenesis and enrichment screen, we identified exclusion-resistant mutations in the ICEBs1 gene conG. Using genes from a heterologous but related ICE, we found that the exclusion specificity was determined by ConG and YddJ. Finally, we found that under conditions that support conjugation, exclusion provides a selective advantage to the element and its host cells.