Cell Wall Binding Research Articles

Effective removal of staphylococcal biofilms on various food contact surfaces by Staphylococcus aureus phage endolysin LysCSA13

Staphylococcal biofilms are recognized as a significant problem in the food industry because of their high resistance to antibiotics, and the use of bacteriophages or endolysins has been regarded as a promising alternative to antibiotics. In this study, LysCSA13, an endolysin from S. aureus virulent bacteriophage CSA13, was cloned and characterized. LysCSA13 consists of an enzymatic active domain and a cell wall binding domain. LysCSA13 showed strong antimicrobial activity against staphylococcal strains at pH 7.0–9.0, 4.0–37.0 °C and in the presence of Ca2+ and Mn2+. In addition, a high efficacy of LysCSA13 in removing staphylococcal biofilms was observed on various surfaces, including polystyrene, glass and stainless steel, displaying an approximately 80–90% decrease in biofilm mass. Furthermore, 300 nM of LysCSA13 effectively removed staphylococcal sessile cells formed on stainless steel and glass by 1–3 log units compared with the untreated control. Scanning electron microscopy analysis visualized the effective deformation and removal of cells embedded in the biofilm matrix. The results indicate that LysCSA13 can effectively control staphylococcal planktonic cells and biofilms regardless of the contact surface matrix and suggest the possible use of LysCSA13 as a promising biocontrol agent in various food processing environments.

Recovery of recombinant Mycobacterium tuberculosis antigens fused with cell wall-anchoring motif (LysM) from inclusion bodies using non-denaturing reagent (N-laurylsarcosine)

BackgroundThe current limitations of conventional BCG vaccines highlights the importance in developing novel and effective vaccines against tuberculosis (TB). The utilization of probiotics such as Lactobacillus plantarum for the delivery of TB antigens through in-trans surface display provides an effective and safe vaccine approach against TB. Such non-recombinant probiotic surface display strategy involves the fusion of candidate proteins with cell wall binding domain such as LysM, which enables the fusion protein to anchor the L. plantarum cell wall externally, without the need for vector genetic modification. This approach requires sufficient production of these recombinant fusion proteins in cell factory such as Escherichia coli which has been shown to be effective in heterologous protein production for decades. However, overexpression in E. coli expression system resulted in limited amount of soluble heterologous TB-LysM fusion protein, since most of it are accumulated as insoluble aggregates in inclusion bodies (IBs). Conventional methods of denaturation and renaturation for solubilizing IBs are costly, time-consuming and tedious. Thus, in this study, an alternative method for TB antigen-LysM protein solubilization from IBs based on the use of non-denaturating reagent N-lauroylsarcosine (NLS) was investigated.ResultsExpression of TB antigen-LysM fusion genes was conducted in Escherichia coli, but this resulted in IBs deposition in contrast to the expression of TB antigens only. This suggested that LysM fusion significantly altered solubility of the TB antigens produced in E. coli. The non-denaturing NLS technique was used and optimized to successfully solubilize and purify ~ 55% of the recombinant cell wall-anchoring TB antigen from the IBs. Functionality of the recovered protein was analyzed via immunofluorescence microscopy and whole cell ELISA which showed successful and stable cell wall binding to L. plantarum (up to 5 days).ConclusionThe presented NLS purification strategy enables an efficient and rapid method for obtaining higher yields of soluble cell wall-anchoring Mycobacterium tuberculosis antigens-LysM fusion proteins from IBs in E. coli.

Staphylococci phages display vast genomic diversity and evolutionary relationships

BackgroundBacteriophages are the most abundant and diverse entities in the biosphere, and this diversity is driven by constant predator–prey evolutionary dynamics and horizontal gene transfer. Phage genome sequences are under-sampled and therefore present an untapped and uncharacterized source of genetic diversity, typically characterized by highly mosaic genomes and no universal genes. To better understand the diversity and relationships among phages infecting human pathogens, we have analysed the complete genome sequences of 205 phages of Staphylococcus sp.ResultsThese are predicted to encode 20,579 proteins, which can be sorted into 2139 phamilies (phams) of related sequences; 745 of these are orphams and possess only a single gene. Based on shared gene content, these phages were grouped into four clusters (A, B, C and D), 27 subclusters (A1-A2, B1-B17, C1-C6 and D1-D2) and one singleton. However, the genomes have mosaic architectures and individual genes with common ancestors are positioned in distinct genomic contexts in different clusters. The staphylococcal Cluster B siphoviridae are predicted to be temperate, and the integration cassettes are often closely-linked to genes implicated in bacterial virulence determinants. There are four unusual endolysin organization strategies found in Staphylococcus phage genomes, with endolysins predicted to be encoded as single genes, two genes spliced, two genes adjacent and as a single gene with inter-lytic-domain secondary translational start site. Comparison of the endolysins reveals multi-domain modularity, with conservation of the SH3 cell wall binding domain.ConclusionsThis study provides a high-resolution view of staphylococcal viral genetic diversity, and insights into their gene flux patterns within and across different phage groups (cluster and subclusters) providing insights into their evolution.

Structural bases of peptidoglycan recognition by lysostaphin SH3b domain

Staphylococcus simulans lysostaphin cleaves pentaglycine cross-bridges between stem peptides in the peptidoglycan of susceptible staphylococci, including S. aureus. This enzyme consists of an N-terminal catalytic domain and a cell wall binding domain (SH3b), which anchors the protein to peptidoglycan. Although structures of SH3bs from lysostaphin are available, the binding modes of peptidoglycan to these domains are still unclear. We have solved the crystal structure of the lysostaphin SH3b domain in complex with a pentaglycine peptide representing the peptidoglycan cross-bridge. The structure identifies a groove between β1 and β2 strands as the pentaglycine binding site. The structure suggests that pentaglycine specificity of the SH3b arises partially directly by steric exclusion of Cβ atoms in the ligand and partially indirectly due to the selection of main chain conformations that are easily accessible for glycine, but not other amino acid residues. We have revealed further interactions of SH3b with the stem peptides with the support of bioinformatics tools. Based on the structural data we have attempted engineering of the domain specificity and have investigated the relevance of the introduced substitutions on the domain binding and specificity, also in the contexts of the mature lysostaphin and of its bacteriolytic activity.

Functional analysis of elicitins and identification of cell wall proteins in Phytophthora cinnamomi

Phytophthora cinnamomi Rands, a soil-borne oomycete plant pathogen, is a major threat to natural vegetation in many countries, especially Australia. The disease that it causes also has economic impacts on several agricultural and forestry systems worldwide. To investigate the virulence mechanisms of P. cinnamomi the roles of elicitins and elicitin-like proteins were examined using a susceptible model species, Lupinus angustifolius. Elicitins are 10 kDa proteins secreted by many Phytophthora species. Here, elicitins were isolated from P. cinnamomi liquid culture and separated according to their isoelectric point. A β-cinnamomin elicitin was then purified and used to develop an immunoaffinity purified antibody as a tool to examine virulence. The specificity of the β-cinnamomin antiserum was confirmed through Western-blot analysis. β-cinnamomin was found to be produced at different P. cinnamomi life stages and in inoculated plant roots by using immuno detection via confocal laser scanning microscopy. Pre-treatment of P. cinnamomi zoospores with β-cinnamomin antiserum and then inoculation of susceptible L. angustifolius roots revealed a partial loss of virulence of the pathogen. In addition, the expression of the β-cinnamomin gene at an early infection stage in susceptible L. angustifolius suggested an intrinsic role of the elicitin. Furthermore, the identification of covalently bound cell wall proteins in P. cinnamomi mycelia in this study has opened up the opportunity to investigate their role in virulence.

Lysis of Staphylococcal Cells by Modular Lysin Domains Linked via a Non-covalent Barnase-Barstar Interaction Bridge.

Bacteriophage endolysins and bacterial exolysins are capable of enzymatic degradation of the cell wall peptidoglycan layer and thus show promise as a new class of antimicrobials. Both exolysins and endolysins often consist of different modules, which are responsible for enzymatic functions and cell wall binding, respectively. Individual modules from different endo- or exolysins with different binding and enzymatic activities, can via gene fusion technology be re-combined into novel variants for investigations of arrangements of potential clinical interest. The aim of this study was to investigate if separately produced cell wall binding and enzyme modules could be assembled into a functional lysin via a non-covalent affinity interaction bridge composed of the barnase ribonuclease from Bacillus amyloliquefaciens and its cognate inhibitor barstar, known to form a stable heterodimeric complex. In a proof-of-principle study, using surface plasmon resonance, flow cytometry and turbidity reduction assays, we show that separately produced modules of a lysin cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) from Staphylococcus aureus bacteriophage K endolysin (LysK) fused to barnase and a cell wall binding Src homology 3 domain (SH3b) from the S. simulans exolysin lysostaphin fused to barstar can be non-covalently assembled into a functional lysin showing both cell wall binding and staphylolytic activity. We hypothesize that the described principle for assembly of functional lysins from separate modules through appended hetero-dimerization domains has a potential for investigations of also other combinations of enzymatically active and cell wall binding domains for desired applications.

LysPBC2, a Novel Endolysin Harboring a Bacillus cereus Spore Binding Domain.

To control the spore-forming human pathogen Bacillus cereus, we isolated and characterized a novel endolysin, LysPBC2, from a newly isolated B. cereus phage, PBC2. Compared to the narrow host range of phage PBC2, LysPBC2 showed very broad lytic activity against all Bacillus, Listeria, and Clostridium species tested. In addition to a catalytic domain and a cell wall binding domain, LysPBC2 has a spore binding domain (SBD) partially overlapping its catalytic domain, which specifically binds to B. cereus spores but not to vegetative cells of B. cereus Both immunogold electron microscopy and a binding assay indicated that the SBD binds the external region of the spore cortex layer. Several amino acid residues required for catalytic or spore binding activity of LysPBC2 were determined by mutagenesis studies. Interestingly, LysPBC2 derivatives with impaired spore binding activity showed an increased lytic activity against vegetative cells of B. cereus compared with that of wild-type LysPBC2. Further biochemical studies revealed that these LysPBC2 derivatives have lower thermal stability, suggesting a stabilizing role of SBD in LysPBC2 structure.IMPORTANCE Bacteriophages produce highly evolved lytic enzymes, called endolysins, to lyse peptidoglycan and release their progeny from bacterial cells. Due to their potent lytic activity and specificity, the use of endolysins has gained increasing attention as a natural alternative to antibiotics. Since most endolysins from Gram-positive-bacterium-infecting phages have a modular structure, understanding the function of each domain is crucial to make effective endolysin-based therapeutics. Here, we report the functional and biochemical characterization of a Bacillus cereus phage endolysin, LysPBC2, which has an unusual spore binding domain and a cell wall binding domain. A single point mutation in the spore binding domain greatly enhanced the lytic activity of endolysin at the cost of reduced thermostability. This work contributes to the understanding of the role of each domain in LysPBC2 and will provide insight for the rational design of efficient antimicrobials or diagnostic tools for controlling B. cereus.

Characteristics of Cd accumulation and distribution in two sweet potato cultivars

In this study, we compared the chemical forms and subcellular distribution of Cd in high-Cd (X16) and low-Cd (N88) sweet potato cultivars through hydroponic experiments and examined the Cd distribution in their roots by histochemical staining. The results showed that inorganic and pectate/protein-integrated Cd predominated in the leaves, and Cd concentrations were significantly higher in X16 than in N88. However, in the roots, Cd was mostly integrated with pectate and protein, and Cd concentration was higher in N88 than in X16. It was mainly stored through vacuolar sequestration and cell wall binding. In the leaves and stems, Cd concentrations in all subcellular fractions were higher in X16 than in N88; the opposite was observed in the roots. In X16, Cd was mostly accumulated in the root stele, and its Cd translocation factor was higher than that of N88. Overall, the subcellular fractions of X16 roots retained less Cd than N88 roots, and more Cd entered the root stele of X16 and subsequently moved to the shoots. The higher amounts of inorganic, water-soluble, and pectate/protein-integrated Cd with high mobility in the shoots of X16 than in N88 might facilitate Cd remobilization to other tissues, but this needs to be further studied.

Structural Basis for Cell Wall Recognition by Bacteriophage PBC5 Endolysin

Phage endolysins are hydrolytic enzymes that cleave the bacterial cell wall during the lytic cycle. We isolated the bacteriophage PBC5 against Bacillus cereus, a major foodborne pathogen, and describe the molecular interaction between endolysin LysPBC5 and the host peptidoglycan structure. LysPBC5 has an N-terminal glycoside hydrolase 25 domain, and a C-terminal cell-wall binding domain (CBD) that is critical for specific cell-wall recognition and lysis. The crystal and solution structure of CBD reveals tandem SH3b domains that are tightly engaged with each other. CBD binds to peptidoglycan in a bidentate manner via distal β-sheet motifs with pseudo two-fold symmetry, which can explain its high affinity and host specificity. CBD primarily interacts with the glycan strand of the peptidoglycan layer instead of the peptide crosslink, implicating the tertiary structure of peptidoglycan as the recognition motif of endolysins.

Crystal Structure of LysB4, an Endolysin from Bacillus cereus-Targeting Bacteriophage B4.

Endolysins are bacteriophage-derived enzymes that hydrolyze the peptidoglycan of host bacteria. Endolysins are considered to be promising tools for the control of pathogenic bacteria. LysB4 is an endolysin produced by Bacillus cereus-infecting bacteriophage B4, and consists of an N-terminal enzymatic active domain (EAD) and a C-terminal cell wall binding domain (CBD). LysB4 was discovered for the first time as an Lalanoyl-D-glutamate endopeptidase with the ability to breakdown the peptidoglycan among B. cereus-infecting phages. To understand the activity of LysB4 at the molecular level, this study determined the X-ray crystal structure of the LysB4 EAD, using the full-length LysB4 endolysin. The LysB4 EAD has an active site that is typical of LAS-type enzymes, where Zn2+ is tetrahedrally coordinated by three amino acid residues and one water molecule. Mutational studies identified essential residues that are involved in lytic activity. Based on the structural and biochemical information about LysB4, we suggest a ligand-docking model and a putative endopeptidase mechanism for the LysB4 EAD. These suggestions add insight into the molecular mechanism of the endolysin LysB4 in B. cereus-infecting phages.

Catalytic diversity and cell wall binding repeats in the phage-encoded endolysins.

Bacteriophage-encoded endolysins can recognize and bind specific bacteria, and act to cleave the glycosidic and/or amide bonds in the peptidoglycan (PG) bacterial cell wall. Cleavage of the cell wall generally results in the death of the bacteria. Their utility as bacteriolytic agents could be exploited for human and veterinary medicines as well as various biotechnological applications. As interest grows in the commercial uses of these proteins, there has been much effort to successfully employ rational design and engineering to produce endolysins with bespoke properties. In this review, we interrogate the current structural data and identify structural features that would be of benefit to engineering the activity and specificity of phage endolysins. We show that the growing body of structural data can be used to predict catalytic residues and mechanism of action from sequences of hypothetical endolysins, and probe the importance of secondary structure repeats in bacterial cell wall-binding domains.

Proof of concept in utilizing in-trans surface display system of Lactobacillus plantarum as mucosal tuberculosis vaccine via oral administration in mice

BackgroundTuberculosis is one of the most common and deadliest infectious diseases worldwide affecting almost a third of the world’s population. Although this disease is being prevented and controlled by the Bacille Calmette Guérin (BCG) vaccine, the protective efficacy is highly variable and substandard (0–80%) in adults. Therefore, novel and effective tuberculosis vaccine that can overcome the limitations from BCG vaccine need to be developed.ResultsA novel approach of utilizing an in-trans protein surface display system of Lactobacillus plantarum carrying and displaying combination of Mycobacterium tuberculosis subunit epitope antigens (Ag85B, CFP-10, ESAT-6, Rv0475 and Rv2031c) fused with LysM anchor motif designated as ACERL was constructed, cloned and expressed in Esherichia coli Rossetta expression host. Subsequently the binding capability of ACERL to the cell wall of L. plantarum was examined via the immunofluorescence microscopy and whole cell ELISA where successful attachment and consistent stability of cell wall binding up to 4 days was determined. The immunization of the developed vaccine of L. plantarum surface displaying ACERL (Lp ACERL) via the oral route was studied in mice for its immunogenicity effects. Lp ACERL immunization was able to invoke significant immune responses that favor the Th1 type cytokine response of IFN-γ, IL-12 and IL-2 as indicated by the outcome from the cytokine profiling of spleen, lung, gastrointestinal tract (GIT), and the re-stimulation of the splenocytes from the immunized mice. Co-administration of an adjuvant consisting of Lactococcus lactis secreting mouse IL-12 (LcIL-12) with Lp ACERL was also investigated. It was shown that the addition of LcIL-12 was able to further generate significant Th1 type cytokines immune responses, similar or better than that of Lp ACERL alone which can be observed from the cytokine profiling of the immunized mice’s spleen, lung and GIT.ConclusionsThis study represents a proof of concept in the development of L. plantarum as a carrier for a non-genetically modified organism (GMO) tuberculosis vaccine, which may be the strategy in the future for tuberculosis vaccine development.

Exploring Combined Effect of Abiotic (Soil Moisture) and Biotic (Sclerotium rolfsii Sacc.) Stress on Collar Rot Development in Chickpea.

Plants being sessile are under constant threat of multiple abiotic and biotic stresses within its natural habitat. A combined stress involving an abiotic and a biotic factor reportedly increases susceptibility of the plants to pathogens. The emerging threat, collar rot disease of chickpea (caused by Sclerotium rolfsii Sacc.) is reported to be influenced by soil moisture condition (SMC). Hence, we studied the influence of differential SMC viz. upper optimum (100%), optimum (80%), lower optimum (60%), and limiting (40%) soil moisture conditions on colonization and collar rot development over the course of infection in two chickpea cultivars, Annigeri (susceptible to collar rot) and ICCV 05530 (moderately resistant to collar rot). Disease incidence was found to be directly proportional to increase in soil moisture (R2 = 0.794). Maximum incidence was observed at 80% SMC, followed by 100 and 60% SMC. Expression of genes (qPCR analysis) associated with host cell wall binding (lectin) and degradation viz. endopolygalacturonase-2, endoglucosidase, and cellobiohydrolase during collar rot development in chickpea were relatively less at limiting soil moisture condition (40%) as compared to optimum soil moisture condition (80%). As compared to individual stress, the expression of defense response genes in chickpea seedlings were highly up-regulated in seedlings challenged with combined stress. Our qPCR results indicated that the expression of defense-related genes in chickpea during interaction with S. rolfsii at low SMC was primarily responsible for delayed disease reaction. Involvement of moisture and biotic stress-related genes in combined stress showed a tailored defense mechanism.

Changes in lignin biosynthesis and monomer composition in response to benzothiadiazole and root-knot nematode Meloidogyne incognita infection in tomato

Benzothiadiazole (BTH) acts as a priming agent in plant defence leading to a reduction in penetration and development of the root-knot nematode Meloidogyne incognita in susceptible tomato roots. Changes in lignin biosynthesis in the susceptible tomato cv. Roma following nematode infection and/or BTH treatment were investigated in comparison to the resistant cv. Rossol. Both untreated and BTH-treated susceptible infected roots (galls) showed an increased level of expression of lignin synthesis-related genes (PAL, C4H, HCT and F5H) at early times during infection (2–4 days post inoculation). Peroxidase (soluble and cell-wall bound, POX) enzyme activities increased after inoculation with M. incognita and the priming effect of BTH treatment was evident at later stages of infection (7 days post inoculation). As expected, the induction of PAL and POXs and lignin synthesis-related genes was faster and greater in resistant roots after infection. Histochemical analysis revealed accumulation of higher lignin levels at later infection stages in BTH-treated galls compared to untreated ones. Furthermore, the monomer composition of lignin indicated a different composition in guaiacyl (G) and syringyl (S) units in BTH-treated galls compared to untreated galls. The increase in G units made G/S ratio similar to that in the resistant genotype. Overall, lignin played a critical role in tomato defence to M. incognita in response to BTH.

Staphylococcus aureus virulence attenuation and immune clearance mediated by a phage lysin-derived protein.

New anti-infective approaches are much needed to control multi-drug-resistant (MDR) pathogens, such as methicillin-resistant Staphylococcus aureus (MRSA). Here, we found for the first time that a recombinant protein derived from the cell wall binding domain (CBD) of the bacteriophage lysin PlyV12, designated as V12CBD, could attenuate S.aureus virulence and enhance host immune defenses via multiple manners. After binding with V12CBD, S.aureus became less invasive to epithelial cells and more susceptible to macrophage killing. The expressions of multiple important virulence genes of S.aureus were reduced 2.4- to 23.4-fold as response to V12CBD More significantly, V12CBD could activate macrophages through NF-κB pathway and enhance phagocytosis against S.aureus As a result, good protections of the mice from MRSA infections were achieved in therapeutic and prophylactic models. These unique functions of V12CBD would render it a novel alternative molecule to control MDRS.aureus infections.

Antifungal Activity and Mechanism of Action of the Co(III) Coordination Complexes With Diamine Chelate Ligands Against Reference and Clinical Strains of Candida spp.

Although many antifungal agents are available in clinical treatment, increasing resistance of fungi, especially Candida species, to the available drugs requires the development of new safe and non-toxic compounds with novel modes of action as effective treatment against resistant microorganisms. Cobalt complexes are very interesting and attractive as potential candidates with antimicrobial activity. Their therapeutic uses as antiviral, antibacterial antifungal, antiparasitic, antitumour, transferrin transporters, and anti-inflammatory agents are being intensively investigated. In this study we examined the antifungal activity of Co(III) complexes with diamine chelate ligands against a broad spectrum of Candida species. Minimum inhibitory concentration was determined by the microbroth dilution method and with serial passaging assay; the synergistic antimicrobial activity of the tested complexes combined with two antifungal drugs (ketoconazole and amphotericin B) was made by checkerboard assay. The effects of Co(III) complexes on yeast cell morphology were studied by optical and transmission electron microscopy. The mode of action of Co(III) complexes on the yeast cell wall (sorbitol assay) and cell membrane (ergosterol assay) were investigated. The cytotoxic effects of the tested compounds on red blood cells and the human keratinocyte (HaCaT) cell line were also evaluated. The analyzed compounds revealed significant antifungal activity for selected strains of Candida species; [CoCl2(dap)2]Cl (1) and [CoCl2(en)2]Cl (2) were more effective than ketoconazole. Its probable mechanism of action did not involve the cell wall or ergosterol binding. However, the checkerboard assay showed, that the antifungal activity of ketoconazole increased in combination with the tested complexes of Co(III). Our results suggest that both diamine complexes with Co(III) analogs caused damage to mitochondrial membrane or the membrane of the endoplasmic reticulum. The effect was observed by transmission electron microscope. Co(III) complexes with diamine chelate ligands are non-toxic at concentrations active against Candida species. This study provides new data on potential antifungal drugs, especially against Candida species.

Class III peroxidases in cellulose deficient cultured maize cells during cell wall remodeling.

Maize (Zea mays L.) suspension-cultured cells habituated to a cellulose biosynthesis inhibitor 2,6-dichlorobenzonitrile (DCB) have a modified cell wall, in which the reduction in the cellulose content is compensated by a network of highly cross-linked feruloylated arabinoxylans and the deposition of lignin-like polymers. For both arabinoxylan cross-linking and lignin polymerization, class III peroxidases (POXs) have been demonstrated to have a prominent role. For the first time, a comparative study of POX activity and isoforms in control and cellulose-impaired cells has been addressed, also taking into account their cellular distribution in different compartments. Proteins from the spent medium (SM), soluble cellular (SC), ionically (ICW) and covalently bound cell wall protein fractions were assayed for total and specific peroxidase activity by using coniferyl and sinapyl alcohol and ferulic acid as substrates. The isoPOX profile was obtained by isoelectric focusing. POX activity was higher in DCB-habituated than in non-habituated cells in all protein fractions at all cell culture stages. For all substrates assayed, SC and ICW fractions showed higher activity at the early log growth phase than at the late log phase. However, the highest POX activity in the spent medium was found at the late log phase. According to the isoPOX profiles, the highest diversity of isoPOXs was detected in the ICW and SM protein fractions. The latter fraction contained isoPOXs with higher activity in DCB-habituated cells. Some of the isoPOXs detected could be involved in cross-linking of arabinoxylans and in the lignin-like polymer formation in DCB-habituated cells.

The Auxiliary Role of the Amidase Domain in Cell Wall Binding and Exolytic Activity of Staphylococcal Phage Endolysins.

In response to increasing concern over antibiotic-resistant Staphylococcus aureus, the development of novel antimicrobials has been called for, with bacteriophage endolysins having received considerable attention as alternatives to antibiotics. Most staphylococcal phage endolysins have a modular structure consisting of an N-terminal cysteine, histidine-dependent amidohydrolases/peptidase domain (CHAP), a central amidase domain, and a C-terminal cell wall binding domain (CBD). Despite extensive studies using truncated staphylococcal endolysins, the precise function of the amidase domain has not been determined. Here, a functional analysis of each domain of two S. aureus phage endolysins (LysSA12 and LysSA97) revealed that the CHAP domain conferred the main catalytic activity, while the central amidase domain showed no enzymatic activity in degrading the intact S. aureus cell wall. However, the amidase-lacking endolysins had reduced hydrolytic activity compared to the full-length endolysins. Comparison of the binding affinities of fusion proteins consisting of the green fluorescent protein (GFP) with CBD and GFP with the amidase domain and CBD revealed that the major function of the amidase domain was to enhance the binding affinity of CBD, resulting in higher lytic activity of endolysin. These results suggest an auxiliary binding role of the amidase domain of staphylococcal endolysins, which can be useful information for designing effective antimicrobial and diagnostic agents against S. aureus.

Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40-Bacillus Specific Endolysins.

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.

Clostridium perfringens Virulent Bacteriophage CPS2 and Its Thermostable Endolysin LysCPS2.

Clostridium perfringens is one of the most common causes of food-borne illness. The increasing prevalence of multidrug-resistant bacteria requires the development of alternatives to typical antimicrobial treatments. Here, we isolated and characterized a C. perfringens-specific virulent bacteriophage CPS2 from chicken feces. The CPS2 phage contains a 17,961 bp double-stranded DNA genome with 25 putative ORFs, and belongs to the Picovirinae, subfamily of Podoviridae. Bioinformatic analysis of the CPS2 genome revealed a putative endolysin, LysCPS2, which is homologous to the endolysin of Clostridium phage phiZP2 and phiCP7R. The enzyme showed strong lytic activity against C. perfringens with optimum conditions at pH 7.5–10, 25–65 °C, and over a broad range of NaCl concentrations. Interestingly, LysCPS2 was found to be highly thermostable, with up to 30% of its lytic activity remaining after 10 min of incubation at 95 °C. The cell wall binding domain in the C-terminal region of LysCPS2 showed a binding spectrum specific to C. perfringens strains. This is the first report to characterize highly thermostable endolysin isolated from virulent C. perfringens bacteriophage. The enzyme can be used as an alternative biocontrol and detection agent against C. perfringens.