Recombinant Depolymerase Research Articles

Recombinant TP-84 Bacteriophage Glycosylase-Depolymerase Confers Activity against Thermostable Geobacillus stearothermophilus via Capsule Degradation.

The TP-84 bacteriophage, which infects Geobacillus stearothermophilus strain 10 (G. stearothermophilus), has a genome size of 47.7 kilobase pairs (kbps) and contains 81 predicted protein-coding ORFs. One of these, TP84_26 encodes a putative tail fiber protein possessing capsule depolymerase activity. In this study, we cloned the TP84_26 gene into a high-expression Escherichia coli (E. coli) system, modified its N-terminus with His-tag, expressed both the wild type gene and His-tagged variant, purified the recombinant depolymerase variants, and further evaluated their properties. We developed a direct enzymatic assay for the depolymerase activity toward G. stearothermophilus capsules. The recombinant TP84_26 protein variants effectively degraded the existing bacterial capsules and inhibited the formation of new ones. Our results provide insights into the novel TP84_26 depolymerase with specific activity against thermostable G. stearothermophilus and its role in the TP-84 life cycle. The identification and characterization of novel depolymerases, such as TP84_26, hold promise for innovative strategies to combat bacterial infections and improve various industrial processes.

Effect of a Depolymerase Encoded by Phage168 on a Carbapenem-Resistant Klebsiella pneumoniae and Its Biofilm.

Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are becoming increasingly common within clinical settings, requiring the development of alternative therapies. In this study, we isolated, characterized, and sequenced the genome of a CRKP phage, Phage168. The total genomic DNA of Phage168 was 40,222 bp in length, encoding 49 predicted proteins. Among these proteins, Dep40, the gene product of ORF40, is a putative tail fiber protein that exhibits depolymerase activity based on the result of bioinformatics analyses. In vitro, we confirmed that the molecular weight of the Phage168 depolymerase protein was about 110 kDa, the concentration of the produced phage 168 depolymerase protein was quantified as being 1.2 mg/mL, and the depolymerase activity was still detectable after the dilution of 1.2 µg/mL. This recombinant depolymerase exhibited enzyme activity during the depolymerization of the formed CRKP biofilms. We also found that depolymerase, when combined with polymyxin B, was able to enhance the bactericidal effect of polymyxin B on CRKP strains by disrupting their biofilm. When recombinant depolymerase was used in combination with human serum, it enhanced the sensitivity of the CRKP strain UA168 to human serum, and the synergistic bactericidal effect reached the strongest level when the ratio of depolymerase to human serum was 3:1. Our results indicated that depolymerase encoded by Phage168 may be a promising strategy for combating infections caused by drug-resistant CRKP formed within the biofilm.

Friunavirus Phage-Encoded Depolymerases Specific to Different Capsular Types of Acinetobacter baumannii.

Acinetobacter baumannii is a critical priority nosocomial pathogen that produces a variety of capsular polysaccharides (CPSs), the primary receptors for specific depolymerase-carrying phages. In this study, the tailspike depolymerases (TSDs) encoded in genomes of six novel Friunaviruses, APK09, APK14, APK16, APK86, APK127v, APK128, and one previously described Friunavirus phage, APK37.1, were characterized. For all TSDs, the mechanism of specific cleavage of corresponding A. baumannii capsular polysaccharides (CPSs) was established. The structures of oligosaccharide fragments derived from K9, K14, K16, K37/K3-v1, K86, K127, and K128 CPSs degradation by the recombinant depolymerases have been determined. The crystal structures of three of the studied TSDs were obtained. A significant reduction in mortality of Galleria mellonella larvae infected with A. baumannii of K9 capsular type was shown in the example of recombinant TSD APK09_gp48. The data obtained will provide a better understanding of the interaction of phage-bacterial host systems and will contribute to the formation of principles of rational usage of lytic phages and phage-derived enzymes as antibacterial agents.

Isolation and Characterization of the First Zobellviridae Family Bacteriophage Infecting Klebsiella pneumoniae.

In order to address the upcoming crisis in the treatment of Klebsiella pneumoniae infections, caused by an increasing proportion of resistant isolates, new approaches to antimicrobial therapy must be developed. One approach would be to use (bacterio)phages and/or phage derivatives for therapy. In this study, we present a description of the first K. pneumoniae phage from the Zobellviridae family. The vB_KpnP_Klyazma podovirus, which forms translucent halos around the plaques, was isolated from river water. The phage genome is composed of 82 open reading frames, which are divided into two clusters located on opposite strands. Phylogenetic analysis revealed that the phage belongs to the Zobellviridae family, although its identity with the closest member of this family was not higher than 5%. The bacteriophage demonstrated lytic activity against all (n = 11) K. pneumoniae strains with the KL20 capsule type, but only the host strain was lysed effectively. The receptor-binding protein of the phage was identified as a polysaccharide depolymerase with a pectate lyase domain. The recombinant depolymerase protein showed concentration-dependent activity against all strains with the KL20 capsule type. The ability of a recombinant depolymerase to cleave bacterial capsular polysaccharides regardless of a phage's ability to successfully infect a particular strain holds promise for the possibility of using depolymerases in antimicrobial therapy, even though they only make bacteria sensitive to environmental factors, rather than killing them directly.

Phage-Derived Depolymerase as an Antibiotic Adjuvant Against Multidrug-Resistant Acinetobacter baumannii.

Bacteriophage-encoded depolymerases are responsible for degrading capsular polysaccharides (CPS), lipopolysaccharides (LPS), and exopolysaccharides (EPS) of the host bacteria during phage invasion. They have been considered as promising antivirulence agents in controlling bacterial infections, including those caused by multidrug-resistant (MDR) bacteria. This feature inspires hope of utilizing these enzymes to disarm the polysaccharide capsules of the bacterial cells, which then strengthens the action of antibiotics. Here we have identified, cloned, and expressed a depolymerase Dpo71 from a bacteriophage specific for the gram-negative bacterium Acinetobacter baumannii in a heterologous host Escherichia coli. Dpo71 sensitizes the MDR A. baumannii to the host immune attack, and also acts as an adjuvant to assist or boost the action of antibiotics, for example colistin. Specifically, Dpo71 at 10 μg/ml enables a complete bacterial eradication by human serum at 50% volume ratio. A mechanistic study shows that the enhanced bactericidal effect of colistin is attributed to the improved outer membrane destabilization capacity and binding rate to bacteria after stripping off the bacterial capsule by Dpo71. Dpo71 inhibits biofilm formation and disrupts the pre-formed biofilm. Combination of Dpo71 could significantly enhance the antibiofilm activity of colistin and improve the survival rate of A. baumannii infected Galleria mellonella. Dpo71 retains the strain-specificity of the parent phage from which Dpo71 is derived: the phage-sensitive A. baumannii strains respond to Dpo71 treatment, whereas the phage-insensitive strains do not. In summary, our work demonstrates the feasibility of using recombinant depolymerases as an antibiotic adjuvant to supplement the development of new antibacterials and to battle against MDR pathogens.

Novel Klebsiella pneumoniae K23-Specific Bacteriophages From Different Families: Similarity of Depolymerases and Their Therapeutic Potential.

Antibiotic resistance is a major public health concern in many countries worldwide. The rapid spread of multidrug-resistant (MDR) bacteria is the main driving force for the development of novel non-antibiotic antimicrobials as a therapeutic alternative. Here, we isolated and characterized three virulent bacteriophages that specifically infect and lyse MDR Klebsiella pneumoniae with K23 capsule type. The phages belonged to the Autographiviridae (vB_KpnP_Dlv622) and Myoviridae (vB_KpnM_Seu621, KpS8) families and contained highly similar receptor-binding proteins (RBPs) with polysaccharide depolymerase enzymatic activity. Based on phylogenetic analysis, a similar pattern was also noted for five other groups of depolymerases, specific against capsule types K1, K30/K69, K57, K63, and KN2. The resulting recombinant depolymerases Dep622 (phage vB_KpnP_Dlv622) and DepS8 (phage KpS8) demonstrated narrow specificity against K. pneumoniae with capsule type K23 and were able to protect Galleria mellonella larvae in a model infection with a K. pneumoniae multidrug-resistant strain. These findings expand our knowledge of the diversity of phage depolymerases and provide further evidence that bacteriophages and phage polysaccharide depolymerases represent a promising tool for antimicrobial therapy.

Identification of a phage-derived depolymerase specific for KL64 capsule of Klebsiella pneumoniae and its anti-biofilm effect.

The increasing prevalence of Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to global health. Phages and phage-derived enzymes gained increasing attention for controling CRKP infections. In this study, a lytic phage P510 infecting KL64 type K. pneumoniae was isolated and characterized. Whole genome analysis and electron microscopy analysis showed that phage P510 belonged to genus Przondovirus, family Autographiviridae, the order Caudovirales. The tail fiber protein of the phage was predicted to encode capsule depolymerase. Further analysis demonstrated that recombinant depolymerase P510dep had polysaccharide-degrading activity against KL64-types capsule of K. pneumoniae, and its lysis spectrum matched to host range of phage P510. Wealso demonstrated that the recombinant depolymerase was able to significantly inhibit biofilm formation. The discovery of the phage-derived depolymerase lays the foundation for controlling the spread of CRKPs.

Specific Interaction of Novel Friunavirus Phages Encoding Tailspike Depolymerases with Corresponding Acinetobacter baumannii Capsular Types.

Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organisation refers it to its «critical priority» category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-depolymerasing enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89 and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae The linear double-stranded DNA phage genomes of 41,105-42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K-units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that cleave specifically the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K-units.IMPORTANCE Acinetobacter baumannii, a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K-types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidate to the cocktail designed to control A. baumannii with different K-types.

Advantages and limitations of microtiter biofilm assays in the model of antibiofilm activity of Klebsiella phage KP34 and its depolymerase

One of the potential antibiofilm strategies is to use lytic phages and phage-derived polysaccharide depolymerases. The idea is to uncover bacteria embedded in the biofilm matrix making them accessible and vulnerable to antibacterials and the immune system. Here we present the antibiofilm efficiency of lytic phage KP34 equipped with virion-associated capsule degrading enzyme (depolymerase) and its recombinant depolymerase KP34p57, depolymerase-non-bearing phage KP15, and ciprofloxacin, separately and in combination, using a multidrug-resistant K. pneumoniae biofilm model. The most effective antibiofilm agents were (1) phage KP34 alone or in combination with ciprofloxacin/phage KP15, and (2) depolymerase KP34p57 with phage KP15 and ciprofloxacin. Secondly, applying the commonly used biofilm microtiter assays: (1) colony count, (2) LIVE/DEAD BacLight Bacterial Viability Kit, and (3) crystal violet (CV) biofilm staining, we unravelled the main advantages and limitations of the above methods in antibiofilm testing. The diverse mode of action of selected antimicrobials strongly influenced obtained results, including a false positive enlargement of biofilm mass (CV staining) while applying polysaccharide degrading agents. We suggest that to get a proper picture of antimicrobials’ effectiveness, multiple examination methods should be used and the results must be read considering the principle of each technique and the antibacterial mechanism.

Functional Analysis and Antivirulence Properties of a New Depolymerase from a Myovirus That Infects Acinetobacter baumannii Capsule K45.

Acinetobacter baumannii is an important pathogen causative of health care-associated infections and is able to rapidly develop resistance to all known antibiotics, including colistin. As an alternative therapeutic agent, we have isolated a novel myovirus (vB_AbaM_B9) which specifically infects and makes lysis from without in strains of the K45 and K30 capsule types, respectively. Phage B9 has a genome of 93,641 bp and encodes 167 predicted proteins, of which 29 were identified by mass spectrometry. This phage holds a capsule depolymerase (B9gp69) able to digest extracted exopolysaccharides of both K30 and K45 strains and remains active in a wide range of pH values (5 to 9), ionic strengths (0 to 500 mM), and temperatures (20 to 80°C). B9gp69 was demonstrated to be nontoxic in a cell line model of the human lung and to make the K45 strain fully susceptible to serum killing in vitro Contrary to the case with phage, no resistance development was observed by bacteria targeted with the B9gp69. Therefore, capsular depolymerases may represent attractive antimicrobial agents against A. baumannii infections.IMPORTANCE Currently, phage therapy has revived interest for controlling hard-to-treat bacterial infections. Acinetobacter baumannii is an emerging Gram-negative pathogen able to cause a variety of nosocomial infections. Additionally, this species is becoming more resistant to several classes of antibiotics. Here we describe the isolation of a novel lytic myophage B9 and its recombinant depolymerase. While the phage can be a promising alternative antibacterial agent, its success in the market will ultimately depend on new regulatory frameworks and general public acceptance. We therefore characterized the phage-encoded depolymerase, which is a natural enzyme that can be more easily managed and used. To our knowledge, the therapeutic potential of phage depolymerase against A. baumannii is still unknown. We show for the first time that the K45 capsule type is an important virulence factor of A. baumannii and that capsule removal via the recombinant depolymerase activity helps the host immune system to combat the bacterial infection.

Identification of three podoviruses infecting Klebsiella encoding capsule depolymerases that digest specific capsular types.

Summary Klebsiella pneumoniae is an important human pathogen causing opportunistic nosocomial and community‐acquired infections. A major public health concern regarding K. pneumoniae is the increasing incidence of multidrug‐resistant strains. Here, we isolated three novel Klebsiella bacteriophages, KN1‐1, KN3‐1 and KN4‐1, which infect KN1, KN3 and K56, and KN4 types respectively. We determined their genome sequences and conducted a comparative analysis that revealed a variable region containing capsule depolymerase‐encoding genes. Recombinant depolymerase proteins were produced, and their enzymatic activity and specificity were evaluated. We identified four capsule depolymerases in these phages that could only digest the capsule types of their respective hosts. Our results demonstrate that the activities of these capsule depolymerases were correlated with the host range of each phage; thus, the capsule depolymerases are host specificity determinants. By generating a capsule mutant, we demonstrate that capsule was essential for phage adsorption and infection. Further, capsule depolymerases can enhance bacterial susceptibility to serum killing. The discovery of these phages and depolymerases lays the foundation for the typing of KN1, KN3, KN4 and K56 Klebsiella and could be useful alternative therapeutics for the treatment of K. pneumoniae infections.

Therapeutic Application of Bacteriophage PHB02 and Its Putative Depolymerase Against Pasteurella multocida Capsular Type A in Mice.

Phage PHB02 specifically infects Pasteurella multocida capsular serogroup A strains. In this study, we found that capsule deletion mutants were not lysed by PHB02, suggesting that the capsule of P. multocida serogroup A strains might be the primary receptor. Based on sequence analysis, a gene encoding a phage-associated putative depolymerase was identified. The corresponding recombinant depolymerase demonstrated specific activity against capsular serogroup A strains but did not strip capsule deletion mutants. In vivo experiments showed that PHB02 was retained at detectable levels in the liver, spleen, kidneys, lung, and blood, at 24 h post-administration in mice. Depolymerase plus serum significantly reduced the number of viable wild-type P. multocida strain HB03 cells (3.5–4.5 log decrease in colony-forming units). Moreover, treatment with phage or purified depolymerase resulted in significantly increased survival of mice infected with P. multocida HB03, and an absence of increase of eosinophils and basophils or other pathological changes when compared with the control group. These results show that phage PHB02 and its putative depolymerase represent a novel strategy for controlling P. multocida serogroup A strains.

Comparative genome analysis of novel Podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1, K2, and K57 capsular types

Hypermucoviscous (HV) strains of capsular types K1, K2 and K57 are the most virulent representatives of the Klebsiella pneumoniae species. Eight novel bacteriophages lytic for HV K. pneumoniae were isolated and characterized. Three bacteriophages, KpV41, KpV475, and KpV71 were found to have a lytic activity against mainly K. pneumoniae of capsular type K1. Two phages, KpV74, and KpV763 were lytic for K2 capsular type K. pneumoniae, and the phage KpV767 was specific to K57-type K. pneumoniae only. Two more phages, KpV766, and KpV48 had no capsular specificity. The phage genomes consist of a linear double-stranded DNA of 40,395–44,623bp including direct terminal repeats of 180–246 bp. The G + C contents are 52.3–54.2 % that is slightly lower than that of genomes of K. pneumoniae strains being used for phage propagation. According to the genome structures, sequence similarity and phylogenetic data, the phages are classified within the genus Kp32virus and Kp34virus of subfamily Autographivirinae, family Podoviridae. In the phage genomes, genes encoding proteins with putative motifs of polysaccharide depolymerase were identified. Depolymerase genes of phages KpV71 and KpV74 lytic for hypermucoviscous K. pneumoniae of K1 and K2 capsular type, respectively, were cloned and expressed in Escherichia coli, and the recombinant gene products were purified. The specificity and polysaccharide-degrading activity of the recombinant depolymerases were demonstrated.

Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis.

Purification of poly(3-hydroxybutyrate) depolymerase (EC 3.1.1.75) from Paucimonas lemoignei is complicated because the bacterium produces several isoenzymes which are difficult to separate from each other. The phaZ5 gene of P. lemoignei encoding extracellular poly(3-hydroxybutyrate) depolymerase A was functionally expressed from the constitutive P43 promoter of pWB980 in a multiple protease-negative mutant of Bacillus subtilis (strain WB800) and secreted to the culture medium. The depolymerase (apparent M(r), 42 kDa; 1.9 mg purified protein per liter culture) was purified from cell-free culture fluid to homogenity by applying only one chromatography step in comparison to at least two necessary steps if poly(3-hydroxybutyrate) depolymerases are purified from P. lemoignei. The recombinant depolymerase lacked any carbohydrate content in contrast to the glycosylated depolymerase of the wild-type. Glycosylation was not essential for activity but enhanced the thermal stability of the enzyme at high temperature. Overexpression of poly(3-hydroxybutyrate) depolymerase in B. subtilis is more efficient than in Escherichia coli.

Production of PHA depolymerase A (PhaZ5) from Paucimonas lemoignei in Bacillus subtilis

Purification of poly(3-hydroxybutyrate) depolymerase (EC 3.1.1.75) from Paucimonas lemoignei is complicated because the bacterium produces several isoenzymes which are difficult to separate from each other. The phaZ5 gene of P. lemoignei encoding extracellular poly(3-hydroxybutyrate) depolymerase A was functionally expressed from the constitutive P43 promoter of pWB980 in a multiple protease-negative mutant of Bacillus subtilis (strain WB800) and secreted to the culture medium. The depolymerase (apparent M r, 42 kDa; 1.9 mg purified protein per liter culture) was purified from cell-free culture fluid to homogenity by applying only one chromatography step in comparison to at least two necessary steps if poly(3-hydroxybutyrate) depolymerases are purified from P. lemoignei. The recombinant depolymerase lacked any carbohydrate content in contrast to the glycosylated depolymerase of the wild-type. Glycosylation was not essential for activity but enhanced the thermal stability of the enzyme at high temperature. Overexpression of poly(3-hydroxybutyrate) depolymerase in B. subtilis is more efficient than in Escherichia coli.