Cells consist of molecular modules which perform vital biological functions. Cellular modules are key units of adaptive evolution because organismal fitness depends on their performance. Theory shows that in rapidly evolving populations, such as those of many microbes, adaptation is driven primarily by common beneficial mutations with large effects, while other mutations behave as if they are effectively neutral. As a consequence, if a module can be improved only by rare and/or weak beneficial mutations, its adaptive evolution would stall. However, such evolutionary stalling has not been empirically demonstrated, and it is unclear to what extent stalling may limit the power of natural selection to improve modules. Here we empirically characterize how natural selection improves the translation machinery (TM), an essential cellular module. We experimentally evolved populations of Escherichia coli with genetically perturbed TMs for 1,000 generations. Populations with severe TM defects initially adapted via mutations in the TM, but TM adaptation stalled within about 300 generations. We estimate that the genetic load in our populations incurred by residual TM defects ranges from 0.5 to 19%. Finally, we found evidence that both epistasis and the depletion of the pool of beneficial mutations contributed to evolutionary stalling. Our results suggest that cellular modules may not be fully optimized by natural selection despite the availability of adaptive mutations.

Candida albicans (CA), a commensal and opportunistic eukaryotic organism, frequently inhabits the gastrointestinal (GI) tract and causes life-threatening infections. Antibiotic-induced gut dysbiosis is a major risk factor for increased CA colonization and dissemination from the GI tract. We identified a significant increase of taurocholic acid (TCA), a major bile acid in antibiotic-treated mice susceptible to CA infection. In vivo findings indicate that administration of TCA through drinking water is sufficient to induce colonization and dissemination of CA in wild-type and immunosuppressed mice. Treatment with TCA significantly reduced mRNA expression of immune genes ang4 and Cxcr3 in the colon. In addition, TCA significantly decreased the relative abundance of three culturable species of commensal bacteria, Turicibacter sanguinis, Lactobacillus johnsonii, and Clostridium celatum, in both cecal contents and mucosal scrapings from the colon. Taken together, our results indicate that TCA promotes fungal colonization and dissemination of CA from the GI tract by controlling the host defense system and intestinal microbiota that play a critical role in regulating CA in the intestine.

Morbidity and mortality attributed to Clostridium difficile infection (CDI) have increased over the past 20 years. Currently, antibiotics are the only US FDA-approved treatment for primary C. difficile infection, and these are, ironically, associated with disease relapse and the threat of burgeoning drug resistance. We previously showed that non-toxin virulence factors play key roles in CDI, and that colonization factors are critical for disease. Specifically, a C. difficile adhesin, Surface Layer Protein A (SlpA) is a major contributor to host cell attachment. In this work, we engineered Syn-LAB 2.0 and Syn-LAB 2.1, two synthetic biologic agents derived from lactic acid bacteria, to stably and constitutively express a host-cell binding fragment of the C. difficile adhesin SlpA on their cell-surface. Both agents harbor conditional suicide plasmids expressing a codon-optimized chimera of the lactic acid bacterium’s cell-wall anchoring surface-protein domain, fused to the conserved, highly adherent, host-cell-binding domain of C. difficile SlpA. Both agents also incorporate engineered biocontrol, obviating the need for any antibiotic selection. Syn-LAB 2.0 and Syn-LAB 2.1 possess positive biophysical and in vivo properties compared with their parental antecedents in that they robustly and constitutively display the SlpA chimera on their cell surface, potentiate human intestinal epithelial barrier function in vitro, are safe, tolerable and palatable to Golden Syrian hamsters and neonatal piglets at high daily doses, and are detectable in animal feces within 24 h of dosing, confirming robust colonization. In combination, the engineered strains also delay (in fixed doses) or prevent (when continuously administered) death of infected hamsters upon challenge with high doses of virulent C. difficile. Finally, fixed-dose Syn-LAB ameliorates diarrhea in a non-lethal model of neonatal piglet enteritis. Taken together, our findings suggest that the two synthetic biologics may be effectively employed as non-antibiotic interventions for CDI.

Background & AimsThe diarrheagenic pathogen, enteropathogenic Escherichia coli (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss.MethodsDSG2 abundance and localization was monitored via immunoblotting and immunofluorescence, respectively. Junctional perturbations were visualized by electron microscopy, and cell–cell adhesion was assessed using dispase assays. EspH alanine-scan mutants as well as pharmacologic agents were used to evaluate impacts on desmosomal alterations. EPEC-mediated DSG2 loss, and its impact on bacterial colonization in vivo, was assessed using a murine model.ResultsThe secreted virulence protein EspH mediates EPEC-induced DSG2 degradation, and contributes to desmosomal perturbation, loss of cell junction integrity, and barrier disruption in infected IECs. EspH sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatase signaling; EspH mutants impaired for Rho guanine nucleotide exchange factor interaction failed to inhibit RhoA or deplete DSG2. Cytotoxic necrotizing factor 1, which locks Rho guanosine triphosphatase in the active state, jasplakinolide, a molecule that promotes actin polymerization, and the lysosomal inhibitor bafilomycin A, respectively, rescued infected cells from EPEC-induced DSG2 loss. Wild-type EPEC, but not an espH-deficient strain, colonizes mouse intestines robustly, widens paracellular junctions, and induces DSG2 re-localization in vivo.ConclusionsOur studies define the mechanism and consequences of EPEC-induced desmosomal alterations in IECs. These perturbations contribute to the colonization and virulence of EPEC, and likely related pathogens.

10Biological organisms are modular. Theory predicts that natural selection would steadily improve 11 modules towards their performance optima up to the margin of effective neutrality. This classical 12 theory may break down for populations evolving in the clonal interference regime because 13 natural selection may focus on some modules while adaptation of others stalls. Such evolutionary 14 stalling has not been observed and it is unclear whether it limits the power of natural selection to 15 optimize module performance. To empirically characterize evolutionary stalling, we evolved 16 populations of Escherichia coli with genetically perturbed translation machineries (TMs). We 17show that populations with different suboptimal TMs embark on statistically distinct trajectories 18 of TM optimization. Yet, before TMs approach the margin of effective neutrality, the focus of 19 natural selection shifts to other cellular modules, and TM optimization stalls. Our results suggest 20 that module optimization within an organism may take much longer than suggested by classical 21 theory.