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826800.9973Sustained coevolution of phage Lambda and Escherichia coli involves inner- as well as outer-membrane defences and counter-defences. Bacteria often evolve resistance to phage through the loss or modification of cell surface receptors. In Escherichia coli and phage λ, such resistance can catalyze a coevolutionary arms race focused on host and phage structures that interact at the outer membrane. Here, we analyse another facet of this arms race involving interactions at the inner membrane, whereby E. coli evolves mutations in mannose permease-encoding genes manY and manZ that impair λ's ability to eject its DNA into the cytoplasm. We show that these man mutants arose concurrently with the arms race at the outer membrane. We tested the hypothesis that λ evolved an additional counter-defence that allowed them to infect bacteria with deleted man genes. The deletions severely impaired the ancestral λ, but some evolved phage grew well on the deletion mutants, indicating that they regained infectivity by evolving the ability to infect hosts independently of the mannose permease. This coevolutionary arms race fulfils the model of an inverse gene-for-gene infection network. Taken together, the interactions at both the outer and inner membranes reveal that coevolutionary arms races can be richer and more complex than is often appreciated.202134032565
826710.9971Why put up with immunity when there is resistance: an excursion into the population and evolutionary dynamics of restriction-modification and CRISPR-Cas. Bacteria can readily generate mutations that prevent bacteriophage (phage) adsorption and thus make bacteria resistant to infections with these viruses. Nevertheless, the majority of bacteria carry complex innate and/or adaptive immune systems: restriction-modification (RM) and CRISPR-Cas, respectively. Both RM and CRISPR-Cas are commonly assumed to have evolved and be maintained to protect bacteria from succumbing to infections with lytic phage. Using mathematical models and computer simulations, we explore the conditions under which selection mediated by lytic phage will favour such complex innate and adaptive immune systems, as opposed to simple envelope resistance. The results of our analysis suggest that when populations of bacteria are confronted with lytic phage: (i) In the absence of immunity, resistance to even multiple bacteriophage species with independent receptors can evolve readily. (ii) RM immunity can benefit bacteria by preventing phage from invading established bacterial populations and particularly so when there are multiple bacteriophage species adsorbing to different receptors. (iii) Whether CRISPR-Cas immunity will prevail over envelope resistance depends critically on the number of steps in the coevolutionary arms race between the bacteria-acquiring spacers and the phage-generating CRISPR-escape mutants. We discuss the implications of these results in the context of the evolution and maintenance of RM and CRISPR-Cas and highlight fundamental questions that remain unanswered. This article is part of a discussion meeting issue 'The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems'.201930905282
836320.9970Hundreds of antimicrobial peptides create a selective barrier for insect gut symbionts. The spatial organization of gut microbiota is crucial for the functioning of the gut ecosystem, although the mechanisms that organize gut bacterial communities in microhabitats are only partially understood. The gut of the insect Riptortus pedestris has a characteristic microbiota biogeography with a multispecies community in the anterior midgut and a monospecific bacterial population in the posterior midgut. We show that the posterior midgut region produces massively hundreds of specific antimicrobial peptides (AMPs), the Crypt-specific Cysteine-Rich peptides (CCRs) that have membrane-damaging antimicrobial activity against diverse bacteria but posterior midgut symbionts have elevated resistance. We determined by transposon-sequencing the genetic repertoire in the symbiont Caballeronia insecticola to manage CCR stress, identifying different independent pathways, including AMP-resistance pathways unrelated to known membrane homeostasis functions as well as cell envelope functions. Mutants in the corresponding genes have reduced capacity to colonize the posterior midgut, demonstrating that CCRs create a selective barrier and resistance is crucial in gut symbionts. Moreover, once established in the gut, the bacteria differentiate into a CCR-sensitive state, suggesting a second function of the CCR peptide arsenal in protecting the gut epithelia or mediating metabolic exchanges between the host and the gut symbionts. Our study highlights the evolution of an extreme diverse AMP family that likely contributes to establish and control the gut microbiota.202438865264
936430.9970Predictable properties of fitness landscapes induced by adaptational tradeoffs. Fitness effects of mutations depend on environmental parameters. For example, mutations that increase fitness of bacteria at high antibiotic concentration often decrease fitness in the absence of antibiotic, exemplifying a tradeoff between adaptation to environmental extremes. We develop a mathematical model for fitness landscapes generated by such tradeoffs, based on experiments that determine the antibiotic dose-response curves of Escherichia coli strains, and previous observations on antibiotic resistance mutations. Our model generates a succession of landscapes with predictable properties as antibiotic concentration is varied. The landscape is nearly smooth at low and high concentrations, but the tradeoff induces a high ruggedness at intermediate antibiotic concentrations. Despite this high ruggedness, however, all the fitness maxima in the landscapes are evolutionarily accessible from the wild type. This implies that selection for antibiotic resistance in multiple mutational steps is relatively facile despite the complexity of the underlying landscape.202032423531
826440.9969Anti-CRISPR Phages Cooperate to Overcome CRISPR-Cas Immunity. Some phages encode anti-CRISPR (acr) genes, which antagonize bacterial CRISPR-Cas immune systems by binding components of its machinery, but it is less clear how deployment of these acr genes impacts phage replication and epidemiology. Here, we demonstrate that bacteria with CRISPR-Cas resistance are still partially immune to Acr-encoding phage. As a consequence, Acr-phages often need to cooperate in order to overcome CRISPR resistance, with a first phage blocking the host CRISPR-Cas immune system to allow a second Acr-phage to successfully replicate. This cooperation leads to epidemiological tipping points in which the initial density of Acr-phage tips the balance from phage extinction to a phage epidemic. Furthermore, both higher levels of CRISPR-Cas immunity and weaker Acr activities shift the tipping points toward higher initial phage densities. Collectively, these data help elucidate how interactions between phage-encoded immune suppressors and the CRISPR systems they target shape bacteria-phage population dynamics.201830033365
937650.9969Historical Contingency Drives Compensatory Evolution and Rare Reversal of Phage Resistance. Bacteria and lytic viruses (phages) engage in highly dynamic coevolutionary interactions over time, yet we have little idea of how transient selection by phages might shape the future evolutionary trajectories of their host populations. To explore this question, we generated genetically diverse phage-resistant mutants of the bacterium Pseudomonas syringae. We subjected the panel of mutants to prolonged experimental evolution in the absence of phages. Some populations re-evolved phage sensitivity, whereas others acquired compensatory mutations that reduced the costs of resistance without altering resistance levels. To ask whether these outcomes were driven by the initial genetic mechanisms of resistance, we next evolved independent replicates of each individual mutant in the absence of phages. We found a strong signature of historical contingency: some mutations were highly reversible across replicate populations, whereas others were highly entrenched. Through whole-genome sequencing of bacteria over time, we also found that populations with the same resistance gene acquired more parallel sets of mutations than populations with different resistance genes, suggesting that compensatory adaptation is also contingent on how resistance initially evolved. Our study identifies an evolutionary ratchet in bacteria-phage coevolution and may explain previous observations that resistance persists over time in some bacterial populations but is lost in others. We add to a growing body of work describing the key role of phages in the ecological and evolutionary dynamics of their host communities. Beyond this specific trait, our study provides a new insight into the genetic architecture of historical contingency, a crucial component of interpreting and predicting evolution.202235994371
826560.9968Mathematical modelling of CRISPR-Cas system effects on biofilm formation. Clustered regularly interspaced short palindromic repeats (CRISPR), linked with CRISPR associated (Cas) genes, can confer adaptive immunity to bacteria, against bacteriophage infections. Thus from a therapeutic standpoint, CRISPR immunity increases biofilm resistance to phage therapy. Recently, however, CRISPR-Cas genes have been implicated in reducing biofilm formation in lysogenized cells. Thus CRISPR immunity can have complex effects on phage-host-lysogen interactions, particularly in a biofilm. In this contribution, we develop and analyse a series of dynamical systems to elucidate and disentangle these interactions. Two competition models are used to study the effects of lysogens (first model) and CRISPR-immune bacteria (second model) in the biofilm. In the third model, the effect of delivering lysogens to a CRISPR-immune biofilm is investigated. Using standard analyses of equilibria, stability and bifurcations, our models predict that lysogens may be able to displace CRISPR-immune bacteria in a biofilm, and thus suggest strategies to eliminate phage-resistant biofilms.201728426329
917370.9968Bacterial defences: mechanisms, evolution and antimicrobial resistance. Throughout their evolutionary history, bacteria have faced diverse threats from other microorganisms, including competing bacteria, bacteriophages and predators. In response to these threats, they have evolved sophisticated defence mechanisms that today also protect bacteria against antibiotics and other therapies. In this Review, we explore the protective strategies of bacteria, including the mechanisms, evolution and clinical implications of these ancient defences. We also review the countermeasures that attackers have evolved to overcome bacterial defences. We argue that understanding how bacteria defend themselves in nature is important for the development of new therapies and for minimizing resistance evolution.202337095190
937780.9968Experimental Evolution of the TolC-Receptor Phage U136B Functionally Identifies a Tail Fiber Protein Involved in Adsorption through Strong Parallel Adaptation. Bacteriophages have received recent attention for their therapeutic potential to treat antibiotic-resistant bacterial infections. One particular idea in phage therapy is to use phages that not only directly kill their bacterial hosts but also rely on particular bacterial receptors, such as proteins involved in virulence or antibiotic resistance. In such cases, the evolution of phage resistance would correspond to the loss of those receptors, an approach termed evolutionary steering. We previously found that during experimental evolution, phage U136B can exert selection pressure on Escherichia coli to lose or modify its receptor, the antibiotic efflux protein TolC, often resulting in reduced antibiotic resistance. However, for TolC-reliant phages like U136B to be used therapeutically, we also need to study their own evolutionary potential. Understanding phage evolution is critical for the development of improved phage therapies as well as the tracking of phage populations during infection. Here, we characterized phage U136B evolution in 10 replicate experimental populations. We quantified phage dynamics that resulted in five surviving phage populations at the end of the 10-day experiment. We found that phages from all five surviving populations had evolved higher rates of adsorption on either ancestral or coevolved E. coli hosts. Using whole-genome and whole-population sequencing, we established that these higher rates of adsorption were associated with parallel molecular evolution in phage tail protein genes. These findings will be useful in future studies to predict how key phage genotypes and phenotypes influence phage efficacy and survival despite the evolution of host resistance. IMPORTANCE Antibiotic resistance is a persistent problem in health care and a factor that may help maintain bacterial diversity in natural environments. Bacteriophages ("phages") are viruses that specifically infect bacteria. We previously discovered and characterized a phage called U136B, which infects bacteria through TolC. TolC is an antibiotic resistance protein that helps bacteria pump antibiotics out of the cell. Over short timescales, phage U136B can be used to evolutionarily "steer" bacterial populations to lose or modify the TolC protein, sometimes reducing antibiotic resistance. In this study, we investigate whether U136B itself evolves to better infect bacterial cells. We discovered that the phage can readily evolve specific mutations that increase its infection rate. This work will be useful for understanding how phages can be used to treat bacterial infections.202337191555
917590.9968Fitness Trade-Offs Resulting from Bacteriophage Resistance Potentiate Synergistic Antibacterial Strategies. Bacteria that cause life-threatening infections in humans are becoming increasingly difficult to treat. In some instances, this is due to intrinsic and acquired antibiotic resistance, indicating that new therapeutic approaches are needed to combat bacterial pathogens. There is renewed interest in utilizing viruses of bacteria known as bacteriophages (phages) as potential antibacterial therapeutics. However, critics suggest that similar to antibiotics, the development of phage-resistant bacteria will halt clinical phage therapy. Although the emergence of phage-resistant bacteria is likely inevitable, there is a growing body of literature showing that phage selective pressure promotes mutations in bacteria that allow them to subvert phage infection, but with a cost to their fitness. Such fitness trade-offs include reduced virulence, resensitization to antibiotics, and colonization defects. Resistance to phage nucleic acid entry, primarily via cell surface modifications, compromises bacterial fitness during antibiotic and host immune system pressure. In this minireview, we explore the mechanisms behind phage resistance in bacterial pathogens and the physiological consequences of acquiring phage resistance phenotypes. With this knowledge, it may be possible to use phages to alter bacterial populations, making them more tractable to current therapeutic strategies.202032094257
9241100.9968Evolutionary Mechanisms Shaping the Maintenance of Antibiotic Resistance. Antibiotics target essential cellular functions but bacteria can become resistant by acquiring either exogenous resistance genes or chromosomal mutations. Resistance mutations typically occur in genes encoding essential functions; these mutations are therefore generally detrimental in the absence of drugs. However, bacteria can reduce this handicap by acquiring additional mutations, known as compensatory mutations. Genetic interactions (epistasis) either with the background or between resistances (in multiresistant bacteria) dramatically affect the fitness cost of antibiotic resistance and its compensation, therefore shaping dissemination of antibiotic resistance mutations. This Review summarizes current knowledge on the evolutionary mechanisms influencing maintenance of resistance mediated by chromosomal mutations, focusing on their fitness cost, compensatory evolution, epistasis, and the effect of the environment on these processes.201829439838
9176110.9968Evolutionary Dynamics between Phages and Bacteria as a Possible Approach for Designing Effective Phage Therapies against Antibiotic-Resistant Bacteria. With the increasing global threat of antibiotic resistance, there is an urgent need to develop new effective therapies to tackle antibiotic-resistant bacterial infections. Bacteriophage therapy is considered as a possible alternative over antibiotics to treat antibiotic-resistant bacteria. However, bacteria can evolve resistance towards bacteriophages through antiphage defense mechanisms, which is a major limitation of phage therapy. The antiphage mechanisms target the phage life cycle, including adsorption, the injection of DNA, synthesis, the assembly of phage particles, and the release of progeny virions. The non-specific bacterial defense mechanisms include adsorption inhibition, superinfection exclusion, restriction-modification, and abortive infection systems. The antiphage defense mechanism includes a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system. At the same time, phages can execute a counterstrategy against antiphage defense mechanisms. However, the antibiotic susceptibility and antibiotic resistance in bacteriophage-resistant bacteria still remain unclear in terms of evolutionary trade-offs and trade-ups between phages and bacteria. Since phage resistance has been a major barrier in phage therapy, the trade-offs can be a possible approach to design effective bacteriophage-mediated intervention strategies. Specifically, the trade-offs between phage resistance and antibiotic resistance can be used as therapeutic models for promoting antibiotic susceptibility and reducing virulence traits, known as bacteriophage steering or evolutionary medicine. Therefore, this review highlights the synergistic application of bacteriophages and antibiotics in association with the pleiotropic trade-offs of bacteriophage resistance.202235884169
8266120.9967Remarkable Mechanisms in Microbes to Resist Phage Infections. Bacteriophages (phages) specifically infect bacteria and are the most abundant biological entities on Earth. The constant exposure to phage infection imposes a strong selective pressure on bacteria to develop viral resistance strategies that promote prokaryotic survival. Thus, this parasite-host relationship results in an evolutionary arms race of adaptation and counteradaptation between the interacting partners. The evolutionary outcome is a spectrum of remarkable strategies used by the bacteria and phages as they attempt to coexist. These approaches include adsorption inhibition, injection blocking, abortive infection, toxin-antitoxin, and CRISPR-Cas systems. In this review, we highlight the diverse and complementary antiphage systems in bacteria, as well as the evasion mechanisms used by phages to escape these resistance strategies.201426958724
8234130.9967Contradictory roles for antibody and complement in the interaction of Brucella abortus with its host. The ability of serum complement to kill bacteria has been linked to host resistance to Gram-negative bacteria. A mechanism for killing extracellular organisms during early invasion, following release from infected phagocytic cells, or during bacteremia would contribute to a host's ability to resist disease. In fact, the ability of serum complement to kill bacteria has been linked to disease resistance. Brucella abortus are Gram-negative intracellular pathogens. Resistance to these bacteria involves the coordinated activities of the cellular and humoral immune systems. The existence of serum-resistant forms of B. abortus has been established, and it has been shown that these bacteria can resist the killing action of complement even in the presence of specific antibody. Antibody is usually necessary for complement-mediated killing of smooth (virulent) forms of Gram-negative bacteria. An anomolous situation exists with some isolates of smooth B. abortus. Sera containing high titers of specific antibody do not support killing unless they are diluted. In the bovine, this phenomenon is associated with IgG1 and IgG2 antibodies. This finding may account for the lack of positive correlation between antibody levels and resistance to disease, which has led, perhaps wrongly, to the idea that antibody and complement are not important in resistance to brucellosis. Available evidence suggests that antibody may have contradictory roles in the interactions between a host and bacteria. Avirulent (rough) forms of the organism would be rapidly killed by complement shortly after invasion, but serum-resistant smooth forms of the organism would survive and invade resident phagocytic cells. During the process of invasion and phagocytosis, the bacteria would initiate an immune response. With time, some B. abortus organisms would be released from infected phagocytic cells. In the early stages of this process, the bacteria would encounter IgM antibody and low concentrations of IgG antibody. These would cause complement-mediated killing, and infection would be restricted to resident phagocytic cells. However, the immune response to B. abortus antigens would be intensified, and IgG antibody levels would increase. High concentrations of antibody do no support complement-mediated killing of extracellular B. abortus, but the bacteria would be opsonized by antibody and complement component fragments. This would lead to increased phagocytosis of extracellular B. abortus as they appear, and concomitant extension of disease. Because of high levels of antibody would block complement-mediated killing of B. abortus, resistance to disease at this point would be dependent on cell-mediated immunity.19958845060
8362140.9967Lifestyle evolution in symbiotic bacteria: insights from genomics. Bacteria that live only in eukaryotic cells and tissues, including chronic pathogens and mutualistic bacteriocyte associates, often possess a distinctive set of genomic traits, including reduced genome size, biased nucleotide base composition and fast polypeptide evolution. These phylogenetically diverse bacteria have lost certain functional categories of genes, including DNA repair genes, which affect mutational patterns. However, pathogens and mutualistic symbionts retain loci that underlie their unique interaction types, such as genes enabling nutrient provisioning by mutualistic bacteria-inhabiting animals. Recent genomic studies suggest that many of these bacteria are irreversibly specialized, precluding shifts between pathogenesis and mutualism.200010884696
777150.9967Multiantibiotic resistance caused by active drug extrusion in Pseudomonas aeruginosa and other gram-negative bacteria. All living organisms have been exposed to noxious compounds throughout their long evolutionary history and those surviving have evolved to fabricate devices that detoxicate and extrude these life threatening substances. It is likely, therefore, that all viable organisms, from bacteria to mammals, are equipped with active extrusion machinery. When bacteria are attacked by antibiotics, they use these tactics to combat the drugs and to develop resistance. Drugs extrusion machinery in Gram-negative bacteria is complex, consisting of the inner membrane transporter which acts as an energy-dependent extrusion pump; a binding protein which presumably connect both membranes; and the outer membrane exit channel. The extrusion pump assemblies are often encoded by chromosomal genes and might be expressed by mutation(s) or induced in the presence of drug(s).19979353746
8239160.9967Surviving bacterial sibling rivalry: inducible and reversible phenotypic switching in Paenibacillus dendritiformis. Natural habitats vary in available nutrients and room for bacteria to grow, but successful colonization can lead to overcrowding and stress. Here we show that competing sibling colonies of Paenibacillus dendritiformis bacteria survive overcrowding by switching between two distinct vegetative phenotypes, motile rods and immotile cocci. Growing colonies of the rod-shaped bacteria produce a toxic protein, Slf, which kills cells of encroaching sibling colonies. However, sublethal concentrations of Slf induce some of the rods to switch to Slf-resistant cocci, which have distinct metabolic and resistance profiles, including resistance to cell wall antibiotics. Unlike dormant spores of P. dendritiformis, the cocci replicate. If cocci encounter conditions that favor rods, they secrete a signaling molecule that induces a switch to rods. Thus, in contrast to persister cells, P. dendritiformis bacteria adapt to changing environmental conditions by inducible and reversible phenotypic switching. IMPORTANCE: In favorable environments, species may face space and nutrient limits due to overcrowding. Bacteria provide an excellent model for analyzing principles underlying overcrowding and regulation of density in nature, since their population dynamics can be easily and accurately assessed under controlled conditions. We describe a newly discovered mechanism for survival of a bacterial population during overcrowding. When competing with sibling colonies, Paenibacillus dendritiformis produces a lethal protein (Slf) that kills cells at the interface of encroaching colonies. Slf also induces a small proportion of the cells to switch from motile, rod-shaped cells to nonmotile, Slf-resistant, vegetative cocci. When crowding is reduced and nutrients are no longer limiting, the bacteria produce a signal that induces cocci to switch back to motile rods, allowing the population to spread. Genes encoding components of this phenotypic switching pathway are widespread among bacterial species, suggesting that this survival mechanism is not unique to P. dendritiformis.201121628502
9736170.9967Coevolutionary phage training leads to greater bacterial suppression and delays the evolution of phage resistance. The evolution of antibiotic-resistant bacteria threatens to become the leading cause of worldwide mortality. This crisis has renewed interest in the practice of phage therapy. Yet, bacteria's capacity to evolve resistance may debilitate this therapy as well. To combat the evolution of phage resistance and improve treatment outcomes, many suggest leveraging phages' ability to counter resistance by evolving phages on target hosts before using them in therapy (phage training). We found that in vitro, λtrn, a phage trained for 28 d, suppressed bacteria ∼1,000-fold for three to eight times longer than its untrained ancestor. Prolonged suppression was due to a delay in the evolution of resistance caused by several factors. Mutations that confer resistance to λtrn are ∼100× less common, and while the target bacterium can evolve complete resistance to the untrained phage in a single step, multiple mutations are required to evolve complete resistance to λtrn. Mutations that confer resistance to λtrn are more costly than mutations for untrained phage resistance. Furthermore, when resistance does evolve, λtrn is better able to suppress these forms of resistance. One way that λtrn improved was through recombination with a gene in a defunct prophage in the host genome, which doubled phage fitness. This transfer of information from the host genome is an unexpected but highly efficient mode of training phage. Lastly, we found that many other independently trained λ phages were able to suppress bacterial populations, supporting the important role training could play during phage therapeutic development.202134083444
8287180.9967Characterizing the Mechanism of Action of an Ancient Antimicrobial, Manuka Honey, against Pseudomonas aeruginosa Using Modern Transcriptomics. Manuka honey has broad-spectrum antimicrobial activity, and unlike traditional antibiotics, resistance to its killing effects has not been reported. However, its mechanism of action remains unclear. Here, we investigated the mechanism of action of manuka honey and its key antibacterial components using a transcriptomic approach in a model organism, Pseudomonas aeruginosa We show that no single component of honey can account for its total antimicrobial action, and that honey affects the expression of genes in the SOS response, oxidative damage, and quorum sensing. Manuka honey uniquely affects genes involved in the explosive cell lysis process and in maintaining the electron transport chain, causing protons to leak across membranes and collapsing the proton motive force, and it induces membrane depolarization and permeabilization in P. aeruginosa These data indicate that the activity of manuka honey comes from multiple mechanisms of action that do not engender bacterial resistance.IMPORTANCE The threat of antimicrobial resistance to human health has prompted interest in complex, natural products with antimicrobial activity. Honey has been an effective topical wound treatment throughout history, predominantly due to its broad-spectrum antimicrobial activity. Unlike traditional antibiotics, honey-resistant bacteria have not been reported; however, honey remains underutilized in the clinic in part due to a lack of understanding of its mechanism of action. Here, we demonstrate that honey affects multiple processes in bacteria, and this is not explained by its major antibacterial components. Honey also uniquely affects bacterial membranes, and this can be exploited for combination therapy with antibiotics that are otherwise ineffective on their own. We argue that honey should be included as part of the current array of wound treatments due to its effective antibacterial activity that does not promote resistance in bacteria.202032606022
9584190.9967Using bacteria to express and display anti-parasite molecules in mosquitoes: current and future strategies. Vector-borne diseases impose enormous health and economical burdens throughout the world. Unfortunately, as insecticide and drug resistance spread, these burdens will increase unless new control measures are developed. Genetically modifying vectors to be incapable of transmitting parasites is one possible control strategy and much progress has been made towards this goal. Numerous effector molecules have been identified that interfere with parasite development in its insect vectors, and techniques for transforming the vectors with genes encoding these molecules have been established. While the ability to generate refractory vectors is close at hand, a mechanism for replacing a wild vector population with a refractory one remains elusive. This review examines the feasibility of using bacteria to deliver the anti-parasitic effector molecules to wild vector populations. The first half briefly examines paratransgenic approaches currently being tested in both the triatomine bug and tsetse fly. The second half explores the possibility of using midgut bacteria to control malaria transmission by Anopheles mosquitoes.200515894187