Endolysins (or lysins) are highly evolved enzymes produced by bacteriophage (phage for short) to digest the bacterial cell wall for phage progeny release. In Gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic-resistant bacteria found on mucosal surfaces and infected tissues. Their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable, make them ideal anti-infectives in an age of mounting resistance. Here we review the current literature showing the effectiveness of these enzymes in controlling a variety of infections.
Phage; Bacteriophage; Cell wall; Gram-positive bacteria; Infection; Lysin
Lysins are highly evolved enzymes produced by bacteriophage ( phage for short) to digest the bacterial cell wall for phage progeny release. In gram-positive bacteria, small quantities of purified recombinant lysin added externally results in immediate lysis causing log-fold death of the target bacterium. Lysins have been used successfully in a variety of animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and infected tissues. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins, and their ability to kill colonizing pathogens on mucosal surfaces, a capacity previously unavailable. Thus, lysins may be a much needed anti-infective in an age of mounting antibiotic resistance.
Phage; Bacteriophage; Cell wall; Gram-positive bacteria; Infection; Lysin; Lytic enzymes; Mucosal colonization; Pathogens; Peptidoglycan
With the increasing worldwide prevalence of antibiotic resistant bacteria, bacteriophage endolysins (lysins) represent a very promising novel alternative class of antibacterial in the fight against infectious disease. Lysins are phage-encoded peptidoglycan hydrolases which, when applied exogenously (as purified recombinant proteins) to Gram-positive bacteria, bring about rapid lysis and death of the bacterial cell. A number of studies have recently demonstrated the strong potential of these enzymes in human and veterinary medicine to control and treat pathogens on mucosal surfaces and in systemic infections. They also have potential in diagnostics and detection, bio-defence, elimination of food pathogens and control of phytopathogens. This review discusses the extensive research on recombinant bacteriophage lysins in the context of antibacterials, and looks forward to future development and potential.
lysin; endolysin; bacteriophage; pathogen; antibacterial; infection; lytic; enzyme
Purified phage lysins present an alternative to traditional antibiotics and work by hydrolyzing peptidoglycan. Phage lysins have been developed against Gram-positive pathogens such as Bacillus anthracis and Streptococcus pneumoniae, where the peptidoglycan layer is exposed on the cell surface. Addition of the lysin to a bacterial culture results in rapid death of the organism. Gram-negative bacteria are resistant to phage lysins because they contain an outer membrane that protects the peptidoglycan from degradation. We solved crystal structures of a Yersinia pestis outer membrane protein and the bacteriocin that targets it, which informed engineering of a bacterial-phage hybrid lysin that can be transported across the outer membrane to kill specific Gram-negative bacteria. This work provides a template for engineering phage lysins against a wide variety of bacterial pathogens.
Group B streptococci (GBS) are the leading cause of neonatal meningitis and sepsis worldwide. The current treatment strategy is limited to intrapartum antibiotic prophylaxis in pregnant women to prevent early-onset neonatal diseases, but considering the potential for antibiotic resistance, the risk of losing control over the disease is high. To approach this problem, we have developed a bacteriophage (phage) lytic enzyme to remove colonizing GBS. Bacteriophage muralytic enzymes, termed lysins, are highly evolved molecules designed to degrade the cell wall of host bacteria to release phage particles from the bacterial cytoplasm. Several different lysins have been developed to specifically kill bacterial pathogens both on mucosal surfaces and in blood and represent a novel approach to control infection. A lysin cloned from a phage infecting GBS was found to contain two putative catalytic domains and one putative binding domain, which is similar to the domain organization of some staphylococcal phage lysins. The lysin (named PlyGBS) was recombinantly expressed in Escherichia coli, and purified PlyGBS efficiently killed all tested GBS serotypes in vitro. In a mouse model, a single dose of PlyGBS significantly reduced bacterial colonization in both the vagina and oropharynx. As an alternative strategy for intrapartum antibiotic prophylaxis, this approach may be used to reduce vaginal GBS colonization in pregnant women before delivery or to decontaminate newborns, thus reducing the incidence of GBS-associated neonatal meningitis and sepsis.
Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology.
antimicrobial; bacteriophage; detection; endolysin; lysis; peptidoglycan hydrolase; protein engineering
The increase in antibiotic resistance world-wide revitalized the interest in the use of phage lysins to combat pathogenic bacteria. In this work, we analyzed the specific cleavage sites on the staphylococcal peptidoglycan produced by three phage lytic proteins. The investigated cell wall lytic enzymes were the endolysin LysH5 derived from the S. aureus bacteriophage vB_SauS-phi-IPLA88 (phi-IPLA88) and two fusion proteins between lysostaphin and the virion-associated peptidoglycan hydrolase HydH5 (HydH5SH3b and HydH5Lyso). We determined that all catalytic domains present in these proteins were active. Additionally, we tested for the emergence of resistant Staphylococcus aureus to any of the three phage lytic proteins constructs. Resistant S. aureus could not be identified after 10 cycles of bacterial exposure to phage lytic proteins either in liquid or plate cultures. However, a quick increase in lysostaphin resistance (up to 1000-fold in liquid culture) was observed. The lack of resistant development supports the use of phage lytic proteins as future therapeutics to treat staphylococcal infections.
The evolution of multi-antibiotic resistance in bacterial pathogens, often resulting from de novo mutations, is creating a public health crisis. Phages show promise for combating antibiotic-resistant bacteria, the efficacy of which, however, may also be limited by resistance evolution. Here, we suggest that phages may be used as supplements to antibiotics in treating initially sensitive bacteria to prevent resistance evolution, as phages are unaffected by most antibiotics and there should be little cross-resistance to antibiotics and phages. In vitro experiments using the bacterium Pseudomonas fluorescens, a lytic phage, and the antibiotic kanamycin supported this prediction: an antibiotic–phage combination dramatically decreased the chance of bacterial population survival that indicates resistance evolution, compared with antibiotic treatment alone, whereas the phage alone did not affect bacterial survival. This effect of the combined treatment in preventing resistance evolution was robust to immigration of bacteria from an untreated environment, but not to immigration from environment where the bacteria had coevolved with the phage. By contrast, an isogenic hypermutable strain constructed from the wild-type P. fluorescens evolved resistance to all treatments regardless of immigration, but typically suffered very large fitness costs. These results suggest that an antibiotic–phage combination may show promise as an antimicrobial strategy.
coevolution; fitness cost; immigration; mutator bacteria; phage therapy
Interest in phage therapy has grown over the past decade due to the rapid emergence of antibiotic resistance in bacterial pathogens. However, the use of bacteriophages for therapeutic purposes has raised concerns over the potential for immune response, rapid toxin release by the lytic action of phages, and difficulty in dose determination in clinical situations. A phage that kills the target cell but is incapable of host cell lysis would alleviate these concerns without compromising efficacy.
We developed a recombinant lysis-deficient Staphylococcus aureus phage P954, in which the endolysin gene was rendered nonfunctional by insertional inactivation. P954, a temperate phage, was lysogenized in S. aureus strain RN4220. The native endolysin gene on the prophage was replaced with an endolysin gene disrupted by the chloramphenicol acetyl transferase (cat) gene through homologous recombination using a plasmid construct. Lysogens carrying the recombinant phage were detected by growth in presence of chloramphenicol. Induction of the recombinant prophage did not result in host cell lysis, and the phage progeny were released by cell lysis with glass beads. The recombinant phage retained the endolysin-deficient genotype and formed plaques only when endolysin was supplemented. The host range of the recombinant phage was the same as that of the parent phage. To test the in vivo efficacy of the recombinant endolysin-deficient phage, immunocompromised mice were challenged with pathogenic S. aureus at a dose that results in 80% mortality (LD80). Treatment with the endolysin-deficient phage rescued mice from the fatal S. aureus infection.
A recombinant endolysin-deficient staphylococcal phage has been developed that is lethal to methicillin-resistant S. aureus without causing bacterial cell lysis. The phage was able to multiply in lytic mode utilizing a heterologous endolysin expressed from a plasmid in the propagation host. The recombinant phage effectively rescued mice from fatal S. aureus infection. To our knowledge this is the first report of a lysis-deficient staphylococcal phage.
Most bacteriophages (phages) release their progeny through the action of holins that form lesions in the cytoplasmic membrane and lysins that degrade the bacterial peptidoglycan. Although the function of each protein is well established in phages infecting Streptococcus pneumoniae, the role—if any—of the powerful bacterial autolysin LytA in virion release is currently unknown. In this study, deletions of the bacterial and phage lysins were done in lysogenic S. pneumoniae strains, allowing the evaluation of the contribution of each lytic enzyme to phage release through the monitoring of bacterial-culture lysis and phage plaque assays. In addition, we assessed membrane integrity during phage-mediated lysis using flow cytometry to evaluate the regulatory role of holins over the lytic activities. Our data show that LytA is activated at the end of the lytic cycle and that its triggering results from holin-induced membrane permeabilization. In the absence of phage lysin, LytA is able to mediate bacterial lysis and phage release, although exclusive dependence on the autolysin results in reduced virion egress and altered kinetics that may impair phage fitness. Under normal conditions, activation of bacterial LytA, together with the phage lysin, leads to greater phage progeny release. Our findings demonstrate that S. pneumoniae phages use the ubiquitous host autolysin to accomplish an optimal phage exiting strategy.
Bacteriophage endolysins (lysins) bind to a cell wall substrate and cleave peptidoglycan, resulting in hypotonic lysis of the phage-infected bacteria. When purified lysins are added externally to Gram-positive bacteria they mediate rapid death by the same mechanism. For this reason, novel therapeutic strategies have been developed using such enzybiotics. However, like other proteins introduced into mammalian organisms, they are quickly cleared from systemic circulation. PEGylation has been used successfully to increase the in vivo half-life of many biological molecules and was therefore applied to Cpl-1, a lysin specific for S. pneumoniae. Cysteine-specific PEGylation with either PEG 10K or 40K was achieved on Cpl-1 mutants, each containing an additional cysteine residue at different locations To the best of our knowledge, this is the first report of the PEGylation of bacteriophage lysin. Compared to the native enzyme, none of the PEGylated conjugates retained significant in vitro anti-pneumococcal lytic activity that would have justified further in vivo studies. Since the anti-microbial activity of the mutant enzymes used in this study was not affected by the introduction of the cysteine residue, our results implied that the presence of the PEG molecule was responsible for the inhibition. As most endolysins exhibit a similar modular structure, we believe that our work emphasizes the inability to improve the in vivo half-life of this class of enzybiotics using a cysteine-specific PEGylation strategy.
Bacteriophage; S. pneumoniae; Cpl-1; PEGylation; Endolysin; Enzybiotic
With their ability to lyse Gram-positive bacteria, phage lytic enzymes (or lysins) have received a great deal of attention as novel anti-infective agents. The number of known genes encoding these peptidoglycan hydrolases has increased markedly in recent years, due in large part to advances in DNA sequencing technology. As the genomes of more and more bacterial species/strains are sequenced, lysin-encoding open reading frames (ORFs) can be readily identified in lysogenized prophage regions. In the current study, we sought to assess lysin diversity for the medically relevant pathogen Clostridium perfringens. The sequenced genomes of nine C. perfringens strains were computationally mined for prophage lysins and lysin-like ORFs, revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase from strain ATCC 13124 (termed PlyCM) was chosen for recombinant analysis based on its dissimilarity to previously characterized C. perfringens lysins. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/salt-dependence and temperature stability. The enzyme exhibited activity at low µg/ml concentrations, a typical value for phage lysins. It was active against 23 of 24 strains of C. perfringens tested, with virtually no activity against other clostridial or nonclostridial species. Overall, PlyCM shows potential for development as an enzybiotic agent, demonstrating how expanding genomic databases can serve as rich pools for biotechnologically relevant proteins.
Lysin; Prophage; Enzybiotic; Muramidase; Clostridium perfringens
Peptidoglycan lytic enzymes (endolysins) induce bacterial host cell lysis in the late phase of the lytic bacteriophage replication cycle. Endolysins OBPgp279 (from Pseudomonas fluorescens phage OBP), PVP-SE1gp146 (Salmonella enterica serovar Enteritidis phage PVP-SE1) and 201ϕ2-1gp229 (Pseudomonas chlororaphis phage 201ϕ2-1) all possess a modular structure with an N-terminal cell wall binding domain and a C-terminal catalytic domain, a unique property for endolysins with a Gram-negative background. All three modular endolysins showed strong muralytic activity on the peptidoglycan of a broad range of Gram-negative bacteria, partly due to the presence of the cell wall binding domain. In the case of PVP-SE1gp146, this domain shows a binding affinity for Salmonella peptidoglycan that falls within the range of typical cell adhesion molecules (Kaff = 1.26×106 M−1). Remarkably, PVP-SE1gp146 turns out to be thermoresistant up to temperatures of 90°C, making it a potential candidate as antibacterial component in hurdle technology for food preservation. OBPgp279, on the other hand, is suggested to intrinsically destabilize the outer membrane of Pseudomonas species, thereby gaining access to their peptidoglycan and exerts an antibacterial activity of 1 logarithmic unit reduction. Addition of 0.5 mM EDTA significantly increases the antibacterial activity of the three modular endolysins up to 2–3 logarithmic units reduction. This research work offers perspectives towards elucidation of the structural differences explaining the unique biochemical and antibacterial properties of OBPgp279, PVP-SE1gp146 and 201ϕ2-1gp229. Furthermore, these endolysins extensively enlarge the pool of potential antibacterial compounds used against multi-drug resistant Gram-negative bacterial infections.
The emergence and increasing prevalence of multidrug-resistant bacterial pathogens emphasizes the need for new and innovative antimicrobial strategies. Lytic phages, which kill their host following amplification and release of progeny phage into the environment, may offer an alternative strategy for combating bacterial infections. In this study, however, we describe the use of a nonlytic phage to specifically target and deliver DNA encoding bactericidal proteins to bacteria. To test the concept of using phage as a lethal-agent delivery vehicle, we used the M13 phagemid system and the addiction toxins Gef and ChpBK. Phage delivery of lethal-agent phagemids reduced target bacterial numbers by several orders of magnitude in vitro and in a bacteremic mouse model of infection. Given the powerful genetic engineering tools available and the present knowledge in phage biology, this technology may have potential use in antimicrobial therapies and DNA vaccine development.
Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa l-alanoyl-d-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-l-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the SPslpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of SPslpA–ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3′-end coding sequence of SPslpA–ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511Δ(S262–K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent SPSlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L. monocytogenes cells. Moreover, transformation of pSL-PL511ΔC into L. lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.
Staphylococcus aureus is a food-borne pathogen and the most common cause of infections in hospitalized patients. The increase in the resistance of this pathogen to antibacterials has made necessary the development of new anti-staphylococcal agents. In this context, bacteriophage lytic enzymes such as endolysins and structural peptidoglycan (PG) hydrolases have received considerable attention as possible antimicrobials against gram-positive bacteria.
S. aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88) contains a virion-associated muralytic enzyme (HydH5) encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encoded by staphylococcal phages. The protein consists of 634 amino acid residues. Two putative lytic domains were identified: an N-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain (135 amino acid residues), and a C-terminal LYZ2 (lysozyme subfamily 2) domain (147 amino acid residues). These domains were also found when a predicted three-dimensional structure of HydH5 was made which provided the basis for deletion analysis. The complete HydH5 protein and truncated proteins containing only each catalytic domain were overproduced in E. coli and purified from inclusion bodies by subsequent refolding. Truncated and full-length HydH5 proteins were all able to bind and lyse S. aureus Sa9 cells as shown by binding assays, zymogram analyses and CFU reduction analysis. HydH5 demonstrated high antibiotic activity against early exponential cells, at 45°C and in the absence of divalent cations (Ca2+, Mg2+, Mn2+). Thermostability assays showed that HydH5 retained 72% of its activity after 5 min at 100°C.
The virion-associated PG hydrolase HydH5 has lytic activity against S. aureus, which makes it attractive as antimicrobial for food biopreservation and anti-staphylococcal therapy.
In most bacteriophages of gram-negative bacteria, the phage endolysin is released to its murein substrate through a lesion in the inner membrane. The lesion is brought about by a second phage-encoded lysis function. For the first time, we present evidence that the same strategy is elaborated by a phage of a gram-positive bacterium. Thus, there appears to be an evolutionarily conserved lysis pathway for most phages whether their host bacterium is gram negative or gram positive. Phage phi 29 gene 14, the product of which is required for efficient lysis of Bacillus subtilis, was cloned in Escherichia coli. Production of protein 14 in E. coli resulted in cell death, whereas production of protein 14 concomitantly with the phi 29 lysozyme or unrelated murein-degrading enzymes led to lysis, suggesting that membrane-bound protein 14 induces a nonspecific lesion in the cytoplasmic membrane.
Like most gram-positive oral bacteria, Actinomyces naeslundii is resistant to salivary lysozyme and to most other lytic enzymes. We are interested in studying the lysins of phages of this important oral bacterium as potential diagnostic and therapeutic agents. To identify the Actinomyces phage genes encoding these species-specific enzymes in Escherichia coli, we constructed a new cloning vector, pAD330, that can be used to enrich for and isolate phage holin genes, which are located adjacent to the lysin genes in most phage genomes. Cloned holin insert sequences were used to design sequencing primers to identify nearby lysin genes by using whole phage DNA as the template. From partial digestions of A. naeslundii phage Av-1 genomic DNA we were able to clone, in independent experiments, inserts that complemented the defective λ holin in pAD330, as evidenced by extensive lysis after thermal induction. The DNA sequence of the inserts in these plasmids revealed that both contained the complete lysis region of Av-1, which is comprised of two holin-like genes, designated holA and holB, and an endolysin gene, designated lysA. We were able to subclone and express these genes and determine some of the functional properties of their gene products.
Bacteriophage therapy of bacterial infections has received renewed attention owing to the increasing prevalence of antibiotic-resistant pathogens. A side effect of many antibiotics as well as of phage therapy with lytic phage is the release of cell wall components, e.g., endotoxins of gram-negative bacteria, which mediate the general pathological aspects of septicemia. Here we explored an alternative strategy by using genetically engineered nonreplicating, nonlytic phage to combat an experimental Pseudomonas aeruginosa infection. An export protein gene of the P. aeruginosa filamentous phage Pf3 was replaced with a restriction endonuclease gene. This rendered the Pf3 variant (Pf3R) nonreplicative and concomitantly prevented the release of the therapeutic agent from the target cell. The Pf3R phage efficiently killed a wild-type host in vitro, while endotoxin release was kept to a minimum. Treatment of P. aeruginosa infections of mice with Pf3R or with a replicating lytic phage resulted in comparable survival rates upon challenge with a minimal lethal dose of 3. However, the survival rate after phage therapy with Pf3R was significantly higher than that with the lytic phage upon challenge with a minimal lethal dose of 5. This higher survival rate correlated with a reduced inflammatory response elicited by Pf3R treatment relative to that with the lytic phage. Therefore, this study suggests that the increased survival rate of Pf3R-treated mice could result from reduced endotoxin release. Thus, the use of a nonreplicating modified phage for the delivery of genes encoding proteins toxic to bacterial pathogens may open up a new avenue in antimicrobial therapy.
Bacterial pathogens that colonize mucosal surfaces have acquired resistance to antimicrobials that are abundant at these sites. One of the main antimicrobials present on mucosal surfaces is lysozyme, a muramidase that hydrolyzes the peptidoglycan backbone of bacteria. Cleavage of the peptidoglycan backbone leads to bacterial cell death and lysis, which releases bacterial fragments, including peptidoglycan, at the site of infection. Peptidoglycan fragments can be recognized by host receptors and initiate an immune response that will aid in clearing infection. Many mucosal pathogens modify the peptidoglycan residues surrounding the cleavage site for lysozyme to avoid peptidoglycan degradation and the release of these proinflammatory fragments. This review will focus specifically on peptidoglycan modifications, their role in lysozyme resistance, and downstream effects on the host immune response to infection.
Parasites provide a selective pressure during the evolution of their hosts, and mediate a range of effects on ecological communities. Due to their short generation time, host-parasite interactions may also drive the virulence of opportunistic bacteria. This is especially relevant in systems where high densities of hosts and parasites on different trophic levels (e.g. vertebrate hosts, their bacterial pathogens, and virus parasitizing bacteria) co-exist. In farmed salmonid fingerlings, Flavobacterium columnare is an emerging pathogen, and phage that infect F. columnare have been isolated. However, the impact of these phage on their host bacterium is not well understood. To study this, four strains of F. columnare were exposed to three isolates of lytic phage and the development of phage resistance and changes in colony morphology were monitored. Using zebrafish (Danio rerio) as a model system, the ancestral rhizoid morphotypes were associated with a 25–100% mortality rate, whereas phage-resistant rough morphotypes that lost their virulence and gliding motility (which are key characteristics of the ancestral types), did not affect zebrafish survival. Both morphotypes maintained their colony morphologies over ten serial passages in liquid culture, except for the low-virulence strain, Os06, which changed morphology with each passage. To our knowledge, this is the first report of the effects of phage-host interactions in a commercially important fish pathogen where phage resistance directly correlates with a decline in bacterial virulence. These results suggest that phage can cause phenotypic changes in F. columnare outside the fish host, and antagonistic interactions between bacterial pathogens and their parasitic phage can favor low bacterial virulence under natural conditions. Furthermore, these results suggest that phage-based therapies can provide a disease management strategy for columnaris disease in aquaculture.
Acinetobacter baumannii is known for its ability to develop resistance to the major groups of antibiotics, form biofilms, and survive for long periods in hospital environments. The prevalence of infections caused by multidrug-resistant A. baumannii is a significant problem for the modern health care system, and application of lytic bacteriophages for controlling this pathogen may become a solution.
In this study, using atomic force microscopy (AFM) and microbiological assessment we have investigated A. baumannii bacteriophage AP22, which has been recently described. AFM has revealed the morphology of bacteriophage AP22, adsorbed on the surfaces of mica, graphite and host bacterial cells. Besides, morphological changes of bacteriophage AP22-infected A. baumannii cells were characterized at different stages of the lytic cycle, from phage adsorption to the cell lysis. The phage latent period, estimated from AFM was in good agreement with that obtained by microbiological methods (40 min). Bacteriophage AP22, whose head diameter is 62±1 nm and tail length is 88±9 nm, was shown to disperse A. baumannii aggregates and adsorb to the bacterial surface right from the first minute of their mutual incubation at 37°C.
High rate of bacteriophage AP22 specific adsorption and its ability to disperse bacterial aggregates make this phage very promising for biomedical antimicrobial applications. Complementing microbiological results with AFM data, we demonstrate an effective approach, which allows not only comparing independently obtained characteristics of the lytic cycle but also visualizing the infection process.
Functional redundancy in genomes arises from genes with overlapping functions, allowing phenotypes to persist after gene knockouts. Evolutionary redundancy or evolvability of a genome is one step removed, in that functional redundancy is absent but the genome has the potential to evolve to restore a lost phenotype. Exploring the extent to which this recovery alters gene networks can illuminate how functional gene interactions change through time. Here, the evolvability of lysis was studied in bacteriophage T7, revealing hidden functional interactions. Lysis is the destruction of host cell wall and membranes that releases progeny and is therefore essential for phage propagation. In most phages, lysis is mediated by a two-component genetic module: a muralytic enzyme that degrades the bacterial cell wall (endolysin) and a holin that permeabilizes the inner membrane to allow the endolysin access to the cell wall. T7 carries one known holin, one endolysin, and a second muralytic enzyme that plays little role in lysis by wild-type phage. If the primary endolysin is deleted, the second muralytic enzyme evolves to restore normal lysis after selection for faster growth. Here, a second level of evolutionary redundancy was revealed. When the second muralytic enzyme was prevented from adapting in a genome lacking the primary endolysin, the phage reevolved lysis de novo in the absence of any known muralytic enzymes by changes in multiple genes outside the original lysis module. This second level of redundancy proved to be evolutionarily inferior to the first, and both result in a lower fitness and slower lysis than wild-type T7. Deletion of the holin gene delayed lysis time modestly; fitness was restored by compensatory substitutions in genes that lack known roles in lysis of the wild type.
experimental evolution; lysis; T7 bacteriophage; modularity; genome evolution; adaptive evolution
The use of bacteriophages against pathogenic bacteria in health care and in the food industry is now being advocated as an alternative to the use of antibiotics. But what is the evolutionary response for a bacterial population if both antibiotics and phages are used in combination? We employ an experimental evolution approach to address these questions and exposed Pseudomonas fluorescens SBW25 and a related hypermutator strain (mutS−) to the action of the antibiotic rifampicin and the lytic bacteriophage SBW25ϕ2. We then compared the densities, growth rates, and the mutations at the rpoB locus leading to rifampicin resistance of the evolved bacterial populations. We observed that the evolutionary response of populations under different treatments varied depending on the order in which the antimicrobials were added and whether the bacterium was a hypermutator. We found that wild-type rifampicin-resistant populations involved in biofilm formation often reverted to rifampicin sensitivity when stresses were added sequentially. In contrast, when the mortality agents were added simultaneously, phage populations frequently went extinct and the bacteria evolved antibiotic resistance. However, populations of the hypermutator mutS− converged to a single genotype at the rpoB locus. Future investigation on other bacteria and using different antibiotics and bacteriophage are needed to evaluate the generality of our findings.
antibiotic resistance; bacteria; experimental evolution; hypermutator; phage therapy; Pseudomonas
PRD1 is a bacteriophage with an icosahedral outer protein layer surrounding the viral membrane, which encloses the linear double-stranded DNA genome. PRD1 infects gram-negative cells harboring a conjugative IncP plasmid. Here we studied the lytic functions of PRD1. Using infected cells and plasmid-borne lysis genes, we demonstrated that a two-component lysis system (holin-endolysin) operates to release progeny phage particles from the host cell. Monitoring of ion fluxes and the ATP content of the infected cells allowed us to build a model of the sequence of lysis-related physiological changes. A decrease in the intracellular level of ATP is the earliest indicator of cell lysis, followed by the leakage of K+ from the cytosol approximately 20 min prior to the decrease in culture turbidity. However, the K+ efflux does not immediately lead to the depolarization of the cytoplasmic membrane or leakage of the intracellular ATP. These effects are observed only ∼5 to 10 min prior to cell lysis. Similar results were obtained using cells expressing the holin and endolysin genes from plasmids.