Mutation, selection, and quantitative genetic architecture of susceptibility to bacterial pathogens in C. elegans

Understanding the evolutionary and genetic underpinnings of susceptibility to pathogens is of fundamental importance across a wide swathe of biology. Much theoretical and empirical effort has focused on genetic variants of large effect, but pathogen susceptibility often appears to be a polygenic complex trait. Here we investigate the quantitative genetics of survival over 120 hours of exposure (“susceptibility”) of C. elegans to three bacterial pathogens of varying virulence, along with the non-pathogenic OP50 strain of E. coli. We compare the genetic (co)variance input by spontaneous mutations accumulated under minimal selection to the standing genetic (co)variance in a set of ∼50 wild isolates. Three conclusions emerge. First, with one exception, mutations increase susceptibility to pathogens, and susceptibility is uncorrelated with fitness in the absence of pathogens. Second, the orientation in trait space of the heritable (co)variance of wild isolates is sufficiently explained by mutation. However, pathogen susceptibility is clearly under purifying, apparently directional, selection of magnitude similar to that of competitive fitness in the MA conditions. The results provide no evidence for fitness tradeoffs between pathogen susceptibility and fitness in the absence of pathogens, nor that balancing selection is important in maintaining genetic variation for susceptibility to these bacterial pathogens.

Antibiotic Tolerance and Heteroresistance: Associated Fitness Costs and Potential in Evading Antibiotic Killing

Streptococcus pneumoniae is a prominent human pathogen that causes both invasive and non-invasive diseases, such as otitis media, pneumonia, meningitis, and bacteremia. Although it is frequently an asymptomatic colonizer of the human nasopharynx, S. pneumoniae is a major cause of morbidity and mortality in the immune compromised population, young children, and the elderly. Up until the 1970s, S. pneumoniae was susceptible to almost all antibiotics. Since then, this pathogen has gained resistance to a variety of antibiotic treatments, including beta-lactams, macrolides, and fluoroquinolones. In the first chapter, we focused on fluoroquinolone resistance in S. pneumoniae. Fluoroquinolones are one of the most frequently prescribed antibiotics, yet fluoroquinolone resistance in S. pneumoniae is still rare compared to other antibiotics resistance, such as beta-lactams. In this study, we investigated the mechanism(s) underlying this intriguing case by assessing the efficiency and fitness costs of horizontal transfer of fluoroquinolone resistance determinants. We hypothesized that the fitness tradeoffs incurred by resistance determinants would define the likelihood of such resistance to emerge in a clinical setting. Clinically relevant fluoroquinolone resistance requires both on-target mutations in topoisomerase IV parC and DNA gyrase gyrA. The wild-type S. pneumoniae TIGR4 was not readily transformed with single mutations in gyrA or parC; however, it was readily transformed with double on-target mutations in gyrA and parC. Compared to the wild type, the single on-target mutants were attenuated, whereas the double on-target mutant was virulent. This suggests that clinically relevant, high-level fluoroquinolone resistance requires the combination of several on-target mutations, which could be acquired via horizontal transfer. The combination of the extremely low probability of acquiring two or more mutations simultaneously from different target genes and the deleterious fitness tradeoffs imposed by individual on-target mutations in gyrA or parC likely result in the infrequent prevalence of fluoroquinolone resistance in S. pneumoniae. Through in vitro serial passaging, we identified a novel mutation (N291D) in the efflux pump patA that facilitated the acquisition of the on-target mutations in parC and gyrA via horizontal transfer with minimal fitness tradeoffs. We also modeled the evolution of fluoroquinolone resistance in a murine host and identified mutation(s) that arose and fixated during in vivo passaging. Interestingly, the experimentally-evolved isolates from the in vivo passaging study did not encode on-target mutations for fluoroquinolone resistance and instead displayed tolerance, which potentially facilitated the subsequent acquisition of fluoroquinolone resistance. In the next chapter, we investigated how fitness tradeoffs and horizontal transfer play a role in the emergence and spread of another mainstay of treatment of pneumococcal infection, beta-lactams- specifically, penicillin, which inhibit wall synthesis. We found that recombination with related viridans species via horizontal transfer may be preferable to de novo on-target mutations in penicillin-binding proteins in S. pneumoniae to acquire resistance more rapidly without initially losing in vivo fitness. Initial recombinants retained virulence in vivo and could readily acquire higher resistance via subsequent transformation. The final recombinants displayed tolerance to penicillin, having reduced kill kinetics compared to the wild type. This suggests that S. pneumoniae might have minimized fitness tradeoffs by developing tolerance via horizonal transfer with related viridans group streptococci, which would serve as a stepping stone for subsequent development of resistance. In the next study, we explored an underlying mechanism of antibiotic tolerance in S. pneumoniae. In our model of the evolution of antibiotic resistance, rny that encodes ribonuclease Y (RNAse Y) was a mutational hotspot across multiple antibiotics. The rny knockout mutant was fully virulent, indicating that deletion of this gene imposed minimal to no fitness tradeoffs. Disruptions in RNA degradation resulted in tolerance to several classes of antibiotics and reduced antibiotic treatment efficacy in vivo. In the final chapter, we investigated whether other phenomena that allow bacteria to withstand antibiotic killing, such as heteroresistance, can affect antibiotic treatment outcomes clinically. We found that vancomycin heteroresistance is associated with treatment failure and poor outcomes in coagulase-negative staphylococci (CoNS) from pediatric leukemia patients. Taken together, this dissertation provides insights into strategies of S. pneumoniae for striking a balance between maximizing resistance potential while minimizing fitness tradeoffs, thereby potentially contributing to the development of more-effective antibiotics for treatment of pneumococcal disease. It also provided insights into the association between heteroresistance in CoNS and clinical outcomes..

Extreme Acid Modulates Fitness Tradeoffs of Multidrug Efflux Pumps MdtEF-TolC and AcrAB-TolC in Escherichia coli K-12

Bacterial genomes encode various multidrug efflux pumps (MDR) whose specific conditions for fitness advantage are unknown. We show that the efflux pump MdtEF-TolC, in Escherichia coli , confers a fitness advantage during exposure to extreme acid (pH 2). Our flow cytometry method revealed pH-dependent fitness tradeoffs between bile acids (a major pump substrate) and salicylic acid, a membrane-permeant aromatic acid that induces a drug-resistance regulon but depletes proton motive force (PMF). The PMF drives MdtEF-TolC and related pumps such as AcrAB-TolC. Deletion of mdtE (with loss of pump MdtEF-TolC) increased the strain’s relative fitness during growth with or without salicylate or bile acids. However, when the growth cycle included a 2-h incubation at pH 2 (below the pH growth range), MdtEF-TolC conferred a fitness advantage. The fitness advantage required bile salts but was decreased by the presence of salicylate, whose uptake is amplified by acid. For comparison, AcrAB-TolC, the primary efflux pump for bile acids, conferred a PMF-dependent fitness advantage with or without acid exposure in the growth cycle. A different MDR pump, EmrAB-TolC, confered no selective benefit during growth in the presence of bile acids. Without bile acids, all three MDR pumps incurred a large fitness cost with salicylate when exposed at pH 2. These results are consistent with the increased uptake of salicylate at low pH. Overall, we showed that MdtEF-TolC is an MDR pump adapted for transient extreme-acid exposure; and that low pH amplifies the salicylate-dependent fitness cost for drug pumps. IMPORTANCE Antibiotics and other drugs that reach the gut must pass through stomach acid. Yet little is known of how extreme acid modulates the effect of drugs on gut bacteria. We find that extreme-acid exposure leads to a fitness advantage for a multidrug pump that otherwise incurs a fitness cost. At the same time, extreme acid amplifies the effect of salicylate selection against multidrug pumps. Thus, organic acids and stomach acid could play important roles in regulating multidrug resistance in the gut microbiome. Our flow cytometry assay provides a way to measure the fitness effects of extreme-acid exposure to various membrane-soluble organic acids including plant-derived nutrients and pharmaceutical agents. Therapeutic acids might be devised to control the prevalence of multidrug pumps in environmental and host-associated habitats.

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

ABSTRACTThe 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 (ΔpH) 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 (Δslp-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.

The Population Genetics of Pleiotropy, and the Evolution of Collateral Resistance and Sensitivity in Bacteria

Pleiotropic fitness tradeoffs and their opposite, buttressing pleiotropy, underlie many important phenomena in ecology and evolution. Yet, predicting whether a population adapting to one (“home”) environment will concomitantly gain or lose fitness in another (“non-home”) environment remains challenging, especially when adaptive mutations have diverse pleiotropic effects. Here, we address this problem using the concept of the joint distribution of fitness effects (JDFE), a local measurable property of the fitness landscape. We derive simple statistics of the JDFE that predict the expected slope, variance and co-variance of non-home fitness trajectories. We estimate these statistics from published data from the Escherichia coli knock-out collection in the presence of antibiotics. We find that, for some drug pairs, the average trend towards collateral sensitivity may be masked by large uncertainty, even in the absence of epistasis. We provide simple theoretically grounded guidelines for designing robust sequential drug protocols.

Diversity, host ranges, and potential drivers of speciation among the inquiline enemies of oak gall wasps

ABSTRACTAnimals that exploit an extended phenotype (e.g., residences, resources, etc.) of other animals are called inquilines. Not strictly parasites, inquilines may nevertheless possess specialized traits that adapt them to particular dimensions of the extended phenotype of their “host”. These adaptations to host traits can in turn lead to fitness tradeoffs that restrict the host range of an inquiline such that shifts to novel hosts might trigger inquiline diversification. Speciation via host shifting has been studied in many animal parasites, but we know far less about the role of host shifts in inquiline speciation. Synergus (Hymenoptera: Cynipidae: Synergini) is a speciose but taxonomically-challenging group of inquilines that feed on the tissue of galls induced by oak gall wasps (Hymenoptera: Cynipidae: Cynipini). Currently too little is known about Nearctic Synergus diversity or host associations to evaluate whether and how host use affects their diversification. Here, we report on a large collection of Synergus reared from galls of 33 oak gall wasp species in the upper Midwestern United States. We integrated DNA barcodes, morphology, ecology, and phenology to delimit putative species of Synergus and describe their host ranges. We find evidence of at least 23 Synergus species associated with the 33 gall wasp hosts. At least five previously described Synergus species are each a complex of two to five species, while three species fit no prior description. We also find strong evidence that oak tree section and host gall morphology define axes of specialization for Synergus. Without over-interpreting our singlegene tree, it is clear that the North American Synergus have experienced several transitions among gall hosts and tree habitats and that host-use is correlated with reproductive isolation, though it remains too early to tell whether shifts to new hosts are the initiators of speciation events in Synergus inquilines of oak gall wasps, or if host shifts occur after reproductive isolation has already evolved.

Fitness costs of parasites explain multiple life history tradeoffs in a wild mammal

SummaryReproduction in wild animals can divert limited resources away from immune defence, resulting in increased parasite burdens. A longstanding prediction of life history theory states that these parasites can harm the reproductive individual, reducing its subsequent fitness and producing reproduction-fitness tradeoffs. Here, we examined associations among reproductive allocation, immunity, parasitism, and subsequent fitness in a wild population of individually identified red deer (Cervus elaphus). Using path analysis, we investigated whether costs of lactation for downstream survival and fecundity were mediated by changes in strongyle nematode count and mucosal antibody levels. Lactating females exhibited increased parasite counts, which were in turn associated with substantially decreased fitness in the following year in terms of overwinter survival, fecundity, subsequent calf weight, and parturition date. This study offers observational evidence for parasite regulation of multiple life history tradeoffs, supporting the role of parasites as an important mediating factor in wild mammal populations.

Does batrachotoxin autoresistance co-evolve with toxicity in Phyllobates poison-dart frogs?

Toxicity is widespread among living organisms, and evolves as a multimodal phenotype. Part of this phenotype is the ability to avoid self-intoxication (autoresistance). Evolving toxin resistance can involve fitness tradeoffs, so autoresistance is often expected to evolve gradually and in tandem with toxicity, resulting in a correlation between the degrees of toxicity and autoresistance among toxic populations. We investigate this correlation in Phyllobates poison frogs, notorious for secreting batrachotoxin (BTX), a potent neurotoxin that targets sodium channels, using ancestral sequence reconstructions of BTX–sensing areas of the muscular voltage-gated sodium channel. Reconstructions suggest that BTX resistance arose at the root of Phyllobates, coinciding with the evolution of BTX secretion. After this event little or no further evolution of autoresistance seems to have occurred, despite large increases in toxicity throughout the history of these frogs. Our results therefore provide no evidence in favor of an evolutionary correlation between toxicity and autoresistance, which conflicts with previous work. Future research on the functional costs and benefits of mutations putatively involved in BTX resistance, as well as their prevalence in natural populations should shed light on the evolutionary mechanisms driving the relationship between toxicity and autoresistance in Phyllobates frogs.