Biology subjects

Moore, J. P.

Publications and source records attributed to Moore, J. P..

18 recordsLinked to original sources

bioRxiv2026

Decoding epitope immunodominance in HIV Env using cryoEM and machine learning

Viral surface glycoproteins, such as the HIV envelope protein (Env), present numerous antibody (Ab) epitopes, yet immune responses consistently focus on only a subset, a phenomenon known as immunodominance. Although structural studies have provided insights into Env antigenicity, our understanding of the molecular features that govern efficient Ab engagement remains incomplete, thereby limiting the predictive and rational design of vaccines. Here, we characterized the structural determinants of epitope immunodominance in HIV Env by integrating high-resolution cryoEM-based polyclonal epitope mapping (cryoEMPEM) across different clades with quantitative analyses of epitope topology, accessibility, and physicochemical properties. More than 70 new structures were resolved to assemble a library of >100 Env-antibody complexes. These data informed the development of a surface-centric, machine-learning model to predict relative Antigen Surface Immunodominance (ASI model). Comparison of ASI-predicted epitope sites with the specificities of Env-induced antibodies showed that the model accurately identifies immunodominant regions and highlights the structural features driving immune bias. Notably, immunogens redesigned based on model predictions successfully redirected Ab responses toward a normally subdominant epitope, demonstrating the potential of strategies coupling targeted assembly of focused structural libraries with machine learning to uncover complex molecular patterns and enable design of more effective vaccine antigens.

immunology↗

bioRxiv2025

Germline-targeting HIV Envelope SOSIP immunization more frequently elicits broadly-neutralizing antibody precursor responses in infant compared to juvenile rhesus macaques

A vaccine capable of inducing broadly neutralizing antibodies (bnAbs) is essential for effective prevention against HIV in children and adolescents. Germline-targeting vaccine strategies aim to stimulate bnAb precursor B cells through carefully designed immunogens, such as the stabilized SOSIP trimers, which mimic native HIV envelope (Env) proteins while presenting key neutralizing epitopes to germline B cell receptors. Given the ability of children living with HIV to develop bnAbs earlier and at a higher frequency than adults, we compared the immunogenicity of a CD4 binding site (CD4bs) bnAb germline-targeting SOSIP trimer immunization strategy in infant (n = 5) and juvenile (n = 4) rhesus macaques (RMs). Animals received 3 doses of the germline-targeting BG505 GT1.1 immunogen, followed by 3 boosts of wild-type BG505 SOSIP, each adjuvanted with the TLR7/8 agonist, 3M-052-SE. After 1.5 years, the RMs were further boosted with a mixed clade B Env trimer nanoparticle to enhance heterologous virus neutralization responses. This germline-targeting strategy induced equivalent titers of neutralizing antibodies in both groups of RMs, yet the infants exhibited a higher magnitude of vaccine-specific IgG binding. Notably, after 3 doses of BG505 GT1.1 SOSIP, infants had higher BG505 GT1.1-specific IgD- B cells. Upon completion of the vaccine regimen, 4 of 5 infants developed a CD4bs bnAb precursor response detectable in serum compared to only 1 of 4 juveniles. Finally, administration of the mixed clade B nanoparticle was able to increase the breadth of antibody responses in 3 of 5 infants and 2 of 4 juveniles. These results suggest that immunization in early-life may enhance bnAb induction and highlight the potential for future pediatric HIV-1 vaccine strategies. Author SummaryVaccines that can generate antibody responses that have breadth and potency in neutralization can protect children and adolescents from acquiring multi-strain viruses like HIV. Increasingly, vaccine strategies have become better tailored to elicit these broadly neutralizing antibodies (bnAbs), such as the germline-targeting HIV envelope antigens, which have shown great promise. To determine if we could elicit protective antibody responses against HIV in early life, we immunized infant and juvenile monkeys using the same vaccine strategy. In this study, we found that while both infants and juveniles developed similar levels of antibodies that could neutralize HIV, the infants showed stronger antibody responses in several key areas. Moreso, infants developed the type of early antibody response that researchers believe is needed to eventually evolve into broadly neutralizing antibodies. These findings suggest that initiating HIV vaccination earlier in life may offer a better chance at generating the bnAbs needed to protect against various HIV strains. Our work highlights the potential benefits of initiating an HIV vaccine regimen in childhood.

immunology↗

bioRxiv2024

Impact of mutations in the HEADING DATE 1 gene on transcription and cell wall composition of rice

Plants utilize environmental information to modify their developmental trajectories for optimal survival and reproduction. Over a century ago, day length (photoperiod) was identified as a major factor influencing developmental transitions, particularly the shift from vegetative to reproductive growth. In rice, exposure to day lengths shorter than a critical threshold accelerates flowering, while longer days inhibit this process. This response is mediated by HEADING DATE 1 (Hd1), a zinc finger transcription factor that is central in the photoperiodic flowering network. Hd1 acts as a repressor of flowering under long days but functions as a promoter of flowering under short days. However, the transcriptional organization of this dual function is still not fully understood. In this study, we utilized RNA-Seq to analyze the transcriptome of hd1 mutants under both long and short day conditions. We identified genes involved in the phenylpropanoid pathway that are deregulated under long days in the mutant. Quantitative profiling of cell wall components and abiotic stress assays suggest that Hd1 is involved in processes considered unrelated to flowering control. This indicates that day length perception and responses are intertwined with physiological processes beyond flowering.

plant biology↗

bioRxiv2023

Germline-targeting SOSIP trimer immunization elicits precursor CD4 binding-site targeting broadly neutralizing antibodies in infant macaques.

A vaccine that can achieve protective immunity prior to sexual debut is critical to prevent the estimated 410,000 new HIV infections that occur yearly in adolescents. As children living with HIV can make broadly neutralizing antibody (bnAb) responses in plasma at a faster rate than adults, early childhood is an opportune window for implementation of a multi-dose HIV immunization strategy to elicit protective immunity prior to adolescence. Therefore, the goal of our study was to assess the ability of a B cell lineage-designed HIV envelope SOSIP to induce bnAbs in early life. Infant rhesus macaques (RMs) received either BG505 SOSIP or the germline-targeting BG505 GT1.1 SOSIP (n=5/group) with the 3M-052-SE adjuvant at 0, 6, and 12 weeks of age. All infant RMs were then boosted with the BG505 SOSIP at weeks 26, 52 and 78, mimicking a pediatric immunization schedule of multiple vaccine boosts within the first two years of life. Both immunization strategies induced durable, high magnitude binding antibodies and plasma autologous virus neutralization that primarily targeted the CD4-binding site (CD4bs) or C3/465 epitope. Notably, three BG505 GT1.1-immunized infants exhibited a plasma HIV neutralization signature reflective of VRC01-like CD4bs bnAb precursor development and heterologous virus neutralization. Finally, infant RMs developed precursor bnAb responses at a similar frequency to that of adult RMs receiving a similar immunization strategy. Thus, a multi-dose immunization regimen with bnAb lineage designed SOSIPs is a promising strategy for inducing protective HIV bnAb responses in childhood prior to adolescence when sexual HIV exposure risk begins.

immunology↗

bioRxiv2023

Fight not flight: parasites drive the bacterial evolution of resistance, not avoidance

In the face of ubiquitous threats from parasites, hosts can evolve strategies to resist infection or to altogether avoid parasitism, for instance by avoiding behavior that could expose them to parasites or by dispersing away from local parasite threats. At the microbial scale, bacteria frequently encounter viral parasites, bacteriophages. While bacteria are known to utilize a number of strategies to resist infection by phages, and can have the capacity to avoid moving towards phage-infected cells, it is unknown whether bacteria can evolve dispersal to escape from phages. In order to answer this question, we combined experimental evolution and mathematical modeling. Experimental evolution of the bacterium Pseudomonas fluorescens in environments with differing spatial distributions of the phage Phi2 revealed that the host bacteria evolved resistance depending on parasite distribution, but did not evolve dispersal to escape parasite infection. Simulations using parameterized mathematical models of bacterial growth and swimming motility showed that this is a general finding: while increased dispersal is adaptive in the absence of parasites, in the presence of parasites that fitness benefit disappears and resistance becomes adaptive, regardless of the spatial distribution of parasites. Together, these experiments suggest that parasites should rarely, if ever, drive the evolution of bacterial escape via dispersal.

evolutionary biology↗

bioRxiv2023

Sensory diversity and precise adaptation enable independent bet-hedging strategies for multiple signals at the same time

In uncertain environments, phenotypic diversity can be advantageous for survival. However, as the environmental uncertainty decreases, the relative advantage of having diverse phenotypes decreases. Here, we show how populations of E. coli integrate multiple chemical signals to adjust sensory diversity in response to changes in the prevalence of each ligand in the environment. Measuring kinase activity in single cells, we quantified the sensitivity distribution to various chemoattractants in different mixtures of background stimuli. We found that when ligands bind uncompetitively, the population tunes sensory diversity to each signal independently, decreasing diversity when the signal ambient concentration increases. However, amongst competitive ligands the population can only decrease sensory diversity one ligand at a time. Mathematical modeling suggests that sensory diversity tuning benefits E. coli populations by modulating how many cells are committed to tracking each signal proportionally as their prevalence changes.

systems biology↗

bioRxiv2021

Antibody responses to SARS-CoV-2 mRNA vaccines are detectable in saliva

Vaccines are critical for curtailing the COVID-19 pandemic (1, 2). In the USA, two highly protective mRNA vaccines are available: BNT162b2 from Pfizer/BioNTech and mRNA-1273 from Moderna (3, 4). These vaccines induce antibodies to the SARS-CoV-2 S-protein, including neutralizing antibodies (NAbs) predominantly directed against the Receptor Binding Domain (RBD) (1-4). Serum NAbs are induced at modest levels within [~]1 week of the first dose, but their titers are strongly boosted by a second dose at 3 (BNT162b2) or 4 weeks (mRNA-1273) (3, 4). SARS-CoV-2 is most commonly transmitted nasally or orally and infects cells in the mucosae of the respiratory and to some extent also the gastrointestinal tract (5). Although serum NAbs may be a correlate of protection against COVID-19, mucosal antibodies might directly prevent or limit virus acquisition by the nasal, oral and conjunctival routes (5). Whether the mRNA vaccines induce mucosal immunity has not been studied. Here, we report that antibodies to the S-protein and its RBD are present in saliva samples from mRNA-vaccinated healthcare workers (HCW). Within 1-2 weeks after their second dose, 37/37 and 8/8 recipients of the Pfizer and Moderna vaccines, respectively, had S-protein IgG antibodies in their saliva, while IgA was detected in a substantial proportion. These observations may be relevant to vaccine-mediated protection from SARS-CoV-2 infection and disease.

microbiology↗

bioRxiv2021

Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

In BriefHerein, we evaluated the immunogenicity of several BG505 SOSIP-based HIV Env immunogens in the rhesus macaque animal model using a combination of serology and biophysical approaches. We applied electron cryo-microscopy for high-resolution mapping of elicited polyclonal antibody responses, which provided detailed insights into the binding modes of the most common classes of antibodies elicited by BG505 SOSIP immunogens as well as the critical differences in immunogenicity that can occur as a consequence of engineered stabilizing mutations and partial glycan occupancy at different sites. SummaryEngineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

immunology↗

bioRxiv2020

Immunofocusing and enhancing autologous Tier-2 HIV-1 neutralization by displaying Env trimers on two-component protein nanoparticles

The HIV-1 envelope glycoprotein trimer is poorly immunogenic because it is covered by a dense glycan shield. As a result, recombinant Env glycoproteins generally elicit inadequate antibody levels that neutralize clinically-relevant, neutralization-resistant (Tier-2) HIV-1 strains. Multivalent antigen presentation on nanoparticles is an established strategy to increase vaccine-driven immune responses. However, due to nanoparticle instability in vivo, the display of non-native Env structures, and the inaccessibility of many neutralizing antibody (NAb) epitopes, the effects of nanoparticle display have been modest for Env trimers. Here, we generated two-component self-assembling protein nanoparticles presenting twenty SOSIP trimers of the clade C Tier-2 genotype 16055. An immunization study in rabbits demonstrated that these nanoparticles induced 60-fold higher autologous Tier-2 NAb titers than the corresponding SOSIP trimers. Epitope mapping revealed that nanoparticle presentation focused antibody responses to an immunodominant apical epitope. Thus, these nanoparticles are a promising platform to improve the immunogenicity of Env trimers with apex-proximate NAb epitopes.

microbiology↗

bioRxiv2020

Salicylate, Bile Acids and Extreme Acid Cause Fitness Tradeoffs for Multidrug Pumps in Escherichia coli K-12

The aspirin derivative salicylate selects against bacterial multidrug efflux pumps of Escherichia coli K-12 such as MdtEF-TolC and EmrAB-TolC, and acid stress regulators such as GadE. Salicylate uptake is driven by the transmembrane pH gradient ({Delta}pH) and the proton motive force (PMF) which drives many efflux pumps. We used flow cytometry to measure the fitness tradeoffs of salicylate, bile acids, and extreme low pH for E. coli cultured with pump deletants. The AcrAB-TolC efflux pump conferred a fitness advantage in the presence of bile acids, an efflux substrate. Without bile acids, AcrA incurred a small fitness cost. The fitness advantage with bile acids was eliminated by the PMF uncoupler CCCP. The Gad acid fitness island encodes components of MdtEF-TolC (an acid-adapted efflux pump) as well as acid regulator GadE. The fitness advantage of E. coli cocultured with a Gad deletant ({Delta}slp-gadX) was lost in the presence of salicylate. Salicylate caused an even larger fitness cost for GadE. MdtE incurred negative or neutral fitness under all media conditions, as did EmrA. But when the competition cycle included two hours at pH 2, MdtE conferred a fitness advantage. The MdtE advantage required the presence of bile acids. Thus, the MdtEF-TolC pump is useful to E. coli for transient extreme acid exposure comparable to passage through the acidic stomach. Salicylate selects against some multidrug efflux pumps, whereas bile acids selects for them; and these fitness tradeoffs are amplified by extreme acid. IMPORTANCEControl of drug resistance in gut microbial communities is a compelling problem for human health. Growth of gut bacteria is limited by host-produced acids such as bile acids, and may be modulated by plant-derived acids such as salicylic acid. Membrane-soluble organic acids can control bacterial growth by disrupting membranes, decreasing cell pH, and depleting PMF. Our flow cytometry assay measures the fitness effects of exposure to membrane-soluble organic acids, with growth cycles that may include a period of extreme acid. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump, MdtEF-TolC, which otherwise incurs a large fitness cost. Thus, organic acids and stomach acid may play important roles in controlling multidrug resistance in the gut microbiome. Therapeutic acids might be developed to limit the prevalence of multidrug resistance pumps in environmental and host-associated communities.

microbiology↗

bioRxiv2020

High-resolution mapping of the neutralizing and binding specificities of polyclonal rabbit serum elicited by HIV Env trimer immunization

Mapping the epitope specificities of polyclonal serum is critical to rational vaccine design. However, most high-resolution mapping approaches involve isolating and characterizing individual monoclonal antibodies, which incompletely defines the full polyclonal response. Here we use two complementary approaches to directly map the specificities of the neutralizing and binding antibodies of polyclonal anti-HIV-1 sera from rabbits immunized with BG505 Env SOSIP trimers. To map the neutralizing specificity, we used mutational antigenic profiling to determine how all amino-acid mutations in Env affected viral neutralization. To map the binding specificity, we used electron microscopy polyclonal epitope mapping (EMPEM) to directly visualize the Fabs in serum bound to Env trimers. Mutational antigenic profiling showed that the dominant neutralizing specificities were the C3/V5 and/or 241/289 glycan hole epitopes, which were generally only a subset of the more diverse binding specificities mapped with EMPEM. Additional differences between binding and neutralization reflected antigenicity differences between virus and soluble Env trimer. Further, mutational antigenic profiling was able to refine epitope specificity in residue-level detail directly from sera, revealing subtle differences across rabbits. Together, mutational antigenic profiling and EMPEM allow for a holistic view of the binding and neutralizing specificity of polyclonal sera and could be used to finely evaluate and guide vaccine design.

microbiology↗

bioRxiv2020

Enhancing glycan occupancy of soluble HIV-1 envelope trimers to mimic the native viral spike

SummaryThe HIV-1 envelope glycoprotein (Env) trimer is decorated with N-linked glycans, which are attached to asparagine residues in the amino acid sequon NxT/S by oligosaccharyltransferases (OST). Artificial glycan “holes” exist when a PNGS is under-occupied on recombinant Env-based vaccines, but not on their viral counterpart. Native-like SOSIP trimers, including clinical candidates, have these artificial holes in the glycan shield that induce strain-specific neutralizing antibodies (NAbs) or non-NAbs. To increase PNGS occupancy, eliminate artificial glycan holes, and mimic the glycosylation of native BG505 Env, we replaced all 12 NxS sequons on the BG505 SOSIP trimer with NxT, thereby increasing the affinity of the sequons for OST. All PNGS, except N133 and N160, were nearly fully occupied on the modified trimer. Occupancy of the N133 site could be increased by changing N133 to NxS, while occupancy of the N160 site could be restored by reverting the nearby N156 sequon to NxS. Hence, OST affinity can influence glycan occupancy when two PNGS are in close proximity. Increasing glycan occupancy should reduce off-target immune responses to artificial glycan holes on vaccine antigens.Competing Interest StatementR. W. S. and J. P. M. are listed on patents concerning stabilized Env trimers.View Full Text

biochemistry↗

bioRxiv2020

Mapping the immunogenic landscape of near-native HIV-1 envelope trimers in non-human primates

The induction of broad and potent immunity by vaccines is the key focus of research efforts aimed at protecting against HIV-1 infection. Soluble native-like HIV-1 envelope glycoproteins have shown promise as vaccine candidates as they can induce potent autologous neutralizing responses in rabbits and non-human primates. In this study, monoclonal antibodies were isolated and characterized from rhesus macaques immunized with the BG505 SOSIP.664 trimer to better understand vaccine-induced antibody responses. Our studies reveal a diverse landscape of antibodies recognizing immunodominant strain-specific epitopes and non-neutralizing neo-epitopes. Additionally, we isolated a subset of mAbs against an epitope cluster at the gp120-gp41 interface that recognize the highly conserved fusion peptide and the glycan at position 88 and have characteristics akin to several human-derived broadly neutralizing antibodies.

immunology↗

bioRxiv2020

Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens

Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Nanoparticles of different sizes and geometries can be designed and combined with appropriate antigens to fit the requirements of different immunization strategies. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to the underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. Comparing the humoral responses elicited by ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle to the corresponding soluble protein revealed that multivalent presentation increased the proportion of the overall antibody response directed against autologous neutralizing Ab epitopes present on the ConM-SOSIP trimers. Author SummaryProtein constructs based on soluble ectodomains of HIV glycoprotein (Env) trimers are the basis of many current HIV vaccine platforms. Multivalent antigen display is one strategy applied to improve the immunogenicity of different subunit vaccine candidates. Here, we describe and comprehensively evaluate a library of de novo designed, protein nanoparticles of different geometries for their ability to present trimeric Env antigens. We found three nanoparticle candidates that can stably incorporate model Env trimer on their surface while maintaining its structure and antigenicity. Immunogenicity of the designed nanoparticles is assessed in vitro and in vivo. In addition to introducing a novel set of reagents for multivalent display of Env trimers, this work provides both guiding principles and a detailed experimental roadmap for the generation, characterization, and optimization of Env-presenting, self-assembling nanoparticle immunogens.

immunology↗

bioRxiv2020

Tailored Design of Protein Nanoparticle Scaffolds for Multivalent Presentation of Viral Glycoprotein Antigens

The adaptive immune system is highly sensitive to arrayed antigens, and multivalent display of viral glycoproteins on symmetric scaffolds has been found to substantially increase the elicitation of antigen-specific antibodies. Motivated by the considerable promise of this strategy for next-generation anti-viral vaccines, we set out to design new self-assembling protein nanoparticles with geometries specifically tailored to scaffold ectodomains of different viral glycoproteins. We first designed and characterized homo-trimers from designed repeat proteins with N-terminal helices positioned to match the C termini of several viral glycoprotein trimers. Oligomers found to experimentally adopt the designed configuration were then used to generate nanoparticles with tetrahedral, octahedral, or icosahedral symmetry. Examples of all three target symmetries were experimentally validated by cryo-electron microscopy and several were assessed for their ability to display viral glycoproteins via genetic fusion. Electron microscopy and antibody binding experiments demonstrated that the designed nanoparticles display conformationally intact native-like HIV-1 Env, influenza hemagglutinin, and prefusion RSV F trimers in the predicted geometries. This work demonstrates that novel nanoparticle immunogens can be designed from the bottom up with atomic-level accuracy and provides a general strategy for precisely controlling epitope presentation and accessibility.

synthetic biology↗

bioRxiv2020

Genomic and phenotypic evolution of Escherichia coli in a novel citrate-only resource environment

Evolutionary innovations allow populations to colonize new, previously inaccessible ecological niches. We previously reported that aerobic growth on citrate (Cit+) evolved in a population of Escherichia coli during adaptation to a minimal glucose medium containing citrate (DM25). Cit+ can grow in citrate-only medium (DM0), which is a novel environment for E. coli. To study adaptation to this new niche, we evolved one set of Cit+ populations for 2,500 generations in DM0 and a control set in DM25. We identified numerous parallel mutations, many mediated by transposable elements. Several lineages evolved multi-copy amplifications containing the maeA gene, constituting up to [~]15% of the genome. We also found substantial cell death in ancestral and evolved clones. Our results demonstrate the importance of copy-number variation and transposable elements in the refinement of the Cit+ trait. However, the observed mortality suggests a persistent evolutionary mismatch between E. coli physiology and a citrate-only environment.

evolutionary biology↗

bioRxiv2019

Benzoate-Tolerant Escherichia coli Strains from Experimental Evolution Lose the Gad Regulon, Multi-Drug Efflux Pumps, and Hydrogenases

Benzoate, a partial uncoupler of the proton motive force (PMF), selects for sensitivity to chloramphenicol and tetracycline in Escherichia coli K-12. Genetic mechanisms of increased benzoate tolerance and decreased drug resistance were analyzed in strains isolated from experimental evolution with benzoate. Transcriptomes showed reversal of benzoate-dependent regulation, including the Gad regulon for acid resistance and multi-drug resistance (MDR). Benzoate-evolved strains knocked down the nitrate reductase narHJ and the acid-consuming hydrogenase Hyd-3, but upregulated OmpF and other porins that admit nutrients and antibiotics. Mutations were found affecting many genes of aromatic catabolism such as folD and add. Several candidate genes from benzoate-evolved strains had a role in benzoate tolerance. Growth with benzoate or salicylate was increased by deletion of the Gad activator ariR, or by deletion of the slp-gadX acid fitness island encoding Gad regulators and the MDR pump MdtEF. Benzoate growth was also increased by deletion of MDR component emrA, the RpoS post-transcriptional regulator cspC, or the adenosine deaminase add. Growth in chloramphenicol with benzoate was decreased by a point mutation in the RNA polymerase alpha-subunit rpoA, which appeared in one chloramphenicol-sensitive benzoate-evolved strain. Growth in chloramphenicol was also decreased by deletion of Mar activator rob, or of rfaY (lipopolysaccharide biosynthesis). Hydrogenase Hyd-3 deletion increased benzoate tolerance. Overall, long-term culture in the presence of benzoate or salicylate favors loss of MDR efflux pumps and of hydrogenases that generate a futile cycle of PMF; and favors upregulation of large-hole porins that increase uptake of fermentable nutrients and of antibiotics. IMPORTANCEBenzoate is a common food preservative, and salicylate is the active form of aspirin. In E. coli, benzoate derivatives upregulate multiple regulons that export antibiotics and other toxic products, and downregulate large outer-membrane porins that allow antibiotic influx. But benzoate or salicylate exposure causes energy stress by depleting the proton motive force. In the absence of antibiotics, long-term benzoate exposure selects against energy-spending systems such as multi-drug efflux pumps and the proton-consuming hydrogenase. Selection favors upregulation of porins that admit fermentable substrates but also allow entry of antibiotics. Thus, evolution with benzoate requires a tradeoff for antibiotic sensitivity. Benzoate and salicylate are naturally occurring plant signal molecules that may influence the evolution of microbiomes in plants and in animal digestive tracts. Effects on hydrogenase activity may be relevant to the biotechnology of hydrogen production.

microbiology↗

bioRxiv2018

CCCP experimental evolution of Escherichia coli K-12 selects for mutations that increase EmrA activity or that downregulate other PMF-driven drug efflux pumps

ABSTRACTExperimental evolution was conducted with Escherichia coli K-12 W3110 in the presence of carbonyl cyanide m-chlorophenylhydrazone (CCCP), an uncoupler of the proton motive force (PMF). Cultures were serially diluted daily 1:100 in broth medium containing 20-150 M CCCP at pH 6.5 or at pH 8.0. After 1,000 generations, all populations showed 5- to 10-fold increase in CCCP resistance. Sequenced isolates showed mutations in emrAB or in its negative repressor mprA; the EmrAB-TolC multidrug efflux pump confers resistance to CCCP and nalidixic acid. Deletion of emrA abolished the CCCP resistance of these strains. One CCCP-evolved isolate lacked emrA or mprA mutations; this strain (C-B11-1) showed mutations in drug efflux regulators cecR (ybiH) (upregulates drug pumps YbhG and YbhFSR) and gadE (upregulates drug pump mdtEF). A cecR::kanR deletion conferred partial resistance to CCCP. A later evolved descendant of the C-B11 population showed mutations in ybhR (MDR efflux). Another isolate showed acrB (MDR efflux pump). The acrB isolate was sensitive to chloramphenicol and tetracycline, which are effluxed by AcrAB. Other mutant genes in CCCP-evolved strains include adhE (alcohol dehydrogenase), rng (ribonuclease G), and cyaA (adenylate cyclase). Overall, experimental evolution revealed a CCCP fitness advantage for mutations increasing its own efflux via EmrA; and for mutations that may decrease proton-driven pumps that efflux other drugs not present (cecR, gadE, acrB, ybhR). These results are consistent with our previous report of drug sensitivity associated with evolved tolerance to a partial uncoupler (benzoate or salicylate).\n\nIMPORTANCEThe genetic responses of bacteria to depletion of proton motive force, and their effects on drug resistance, are poorly understood. Our evolution experiment reveals genetic mechanisms of adaptation to the PMF uncoupler CCCP, including selection for and against various multidrug efflux pumps. The results have implications for our understanding of the gut microbiome, which experiences high levels of organic acids that decrease PMF. Organic acid uncouplers may select against multidrug resistance in evolving populations of enteric bacteria.

microbiology↗