Cysteine, Histidine-dependent Amidohydrolase/peptidase Domain Research Articles

Rapid Screening and Comparison of Chimeric Lysins for Antibacterial Activity against Staphylococcus aureus Strains.

Chimeric lysins composed of various combinations of cell wall-lysing (enzymatic) and cell-wall-binding (CWB) domains of endolysins, autolysins, and bacteriocins have been developed as alternatives to or adjuvants of conventional antibiotics. The screening of multiple chimeric lysin candidates for activity via E. coli expression is not cost effective, and we previously reported on a simple cell-free expression system as an alternative. In this study, we sufficiently improved upon this cell-free expression system for use in screening activity via a turbidity reduction test, which is more appropriate than a colony reduction test when applied in multiple screening. Using the improved protocol, we screened and compared the antibacterial activity of chimeric lysin candidates and verified the relatively strong activity associated with the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain of secretory antigen SsaA-like protein (ALS2). ALS2 expressed in E. coli showed two major bands, and the smaller one (subprotein) was shown to be expressed by an innate downstream promoter and start codon (ATG). The introduction of synonymous mutations in the promoter resulted in clearly reduced expression of the subprotein, whereas missense mutations in the start codon abolished antibacterial activity as well as subprotein production. Interestingly, most of the S. aureus strains responsible for bovine mastitis were susceptible to ALS2, but those from human and chicken were less susceptible. Thus, the simple and rapid screening method can be applied to select functional chimeric lysins and define mutations affecting antibacterial activity, and ALS2 may be useful in itself and as a lead molecule to control bovine mastitis.

Genome Sequence of the Siphoviridae Staphylococcus aureus Phage vB_SauS_BaqSau1.

Here, we report the genome sequence of a Siphoviridae phage named vB_SauS_BaqSau1 (BaqSau1), infecting Staphylococcus aureus Phage BaqSau1 was isolated from a sewage water treatment plant in Sahagún, Córdoba, Colombia. It has a double-stranded DNA (dsDNA) genome of 44,384 bp with 67 predicted genes, including a lysin containing a CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain.

Cell-Wall Hydrolases as Antimicrobials against Staphylococcus Species: Focus on Sle1.

Some staphylococcal species are opportunistic pathogens of humans and/or animals with Staphylococcus epidermidis as one of the most important. It causes a broad spectrum of diseases in humans and animals. This species is able to form biofilms and has developed antibiotic resistance, which has motivated research on new antibacterial agents. Cell-wall hydrolases (CWHs) can constitute a potential alternative. Following a hijacking strategy, we inventoried the CWHs of S. epidermidis. The lytic potential of representative CWHs that could be turned against staphylococci was explored by turbidity assays which revealed that cell wall glycosidases were not efficient, while cell wall amidases and cell wall peptidases were able to lyse S. epidermidis. Sle1, which is encoded by chromosomal gene and composed of three anchoring LysM domains and a C-terminal CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain, was one of the most active CWHs. The phylogeny of Sle1 revealed seven clusters mostly identified among staphylococci. Sle1 was able to lyse several staphylococcal species, including Staphylococcus aureus, both in planktonic and sessile forms, but not Micrococcus.

Characterization of the Lytic Capability of a LysK-Like Endolysin, Lys-phiSA012, Derived from a Polyvalent Staphylococcus aureus Bacteriophage

Antibiotic-resistant bacteria (ARB) have spread widely and rapidly, with their increased occurrence corresponding with the increased use of antibiotics. Infections caused by Staphylococcus aureus have a considerable negative impact on human and livestock health. Bacteriophages and their peptidoglycan hydrolytic enzymes (endolysins) have received significant attention as novel approaches against ARB, including S. aureus. In the present study, we purified an endolysin, Lys-phiSA012, which harbors a cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain, an amidase domain, and a SH3b cell wall binding domain, derived from a polyvalent S. aureus bacteriophage which we reported previously. We demonstrate that Lys-phiSA012 exhibits high lytic activity towards staphylococcal strains, including methicillin-resistant S. aureus (MRSA). Analysis of deletion mutants showed that only mutants possessing the CHAP and SH3b domains could lyse S. aureus, indicating that lytic activity of the CHAP domain depended on the SH3b domain. The presence of at least 1 mM Ca2+ and 100 µM Zn2+ enhanced the lytic activity of Lys-phiSA012 in a turbidity reduction assay. Furthermore, a minimum inhibitory concentration (MIC) assay showed that the addition of Lys-phiSA012 decreased the MIC of oxacillin. Our results suggest that endolysins are a promising approach for replacing current antimicrobial agents and may contribute to the proper use of antibiotics, leading to the reduction of ARB.

Characterization of a novel endolysin LysSA11 and its utility as a potent biocontrol agent against Staphylococcus aureus on food and utensils

Here we show that the LysSA11 endolysin, derived from the virulent Staphylococcus aureus phage SA11, has lytic activity against staphylococcal strains. Bioinformatics analysis revealed an enzymatically active CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain at the N-terminus of LysSA11 that showed amidase activity. A novel cell wall binding domain (CBD) in the C-terminus could bind to a broad spectrum of staphylococcal cells. The bactericidal activity of LysSA11 was determined in food and utensils artificially contaminated with methicillin-resistant S. aureus (MRSA). The amounts of MRSA bacteria in milk and on ham were significantly reduced by 1.44-log CFU/mL and 3.12-log CFU/cm3, respectively, within 15 min at refrigeration temperature (4 °C) and by 2.02-log CFU/mL and 3.37-log CFU/cm2, respectively, within 15 min at room temperature (25 °C). Moreover, a polypropylene plastic cutting board and a stainless steel knife artificially contaminated with approximately 4-log CFU/cm2 of MRSA also showed complete bacterial elimination after a 30-min treatment with 1.35 μM of LysSA11. The data presented here strongly suggest that the novel CBD-containing staphylococcal endolysin LysSA11 can be used both as a food antimicrobial and as a practical sanitizer for utensils.

Increasing the stability of the bacteriophage endolysin PlyC using rationale-based FoldX computational modeling.

Endolysins are bacteriophage-derived peptidoglycan hydrolases that represent an emerging class of proteinaceous therapeutics. While the streptococcal endolysin PlyC has been validated in vitro and in vivo for its therapeutic efficacy, the inherent thermosusceptible structure of the enzyme correlates to transient long-term stability, thereby hindering the feasibility of developing the enzyme as an antimicrobial. Here, we thermostabilized the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain of the PlyCA catalytic subunit of PlyC using a FoldX-driven computational protein engineering approach. Using a combination of FoldX and Rosetta algorithms, as well as visual inspection, a final list of PlyC point mutant candidates with predicted stabilizing ΔΔG values was assembled and thermally characterized. Five of the eight point mutations were found experimentally to be destabilizing, a result most likely attributable to computationally modeling a complex and dynamic nine-subunit holoenzyme with a corresponding 3.3-Å X-ray crystal structure. However, one of the mutants, PlyC (PlyCA) T406R, was shown experimentally to increase the thermal denaturation temperature by ∼2.2°C and kinetic stability 16-fold over wild type. This mutation is expected to introduce a thermally advantageous hydrogen bond between the Q106 side chain of the N-terminal glycosyl hydrolase domain and the R406 side chain of the C-terminal CHAP domain.

Characterization of the Modular Design of the Autolysin/Adhesin Aaa from Staphylococcus Aureus

Background Staphylococcus aureus is a frequent cause of serious and life-threatening infections, such as endocarditis, osteomyelitis, pneumonia, and sepsis. Its adherence to various host structures is crucial for the establishment of diseases. Adherence may be mediated by a variety of adhesins, among them the autolysin/adhesins Atl and Aaa. Aaa is composed of three N-terminal repeated sequences homologous to a lysin motif (LysM) that can confer cell wall attachment and a C-terminally located cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain having bacteriolytic activity in many proteins.Methodology/Principal FindingsHere, we show by surface plasmon resonance that the LysM domain binds to fibrinogen, fibronectin, and vitronectin respresenting a novel adhesive function for this domain. Moreover, we demonstrated that the CHAP domain not only mediates the bacteriolytic activity, but also adherence to fibrinogen, fibronectin, and vitronectin, thus demonstrating for the first time an adhesive function for this domain. Adherence of an S. aureus aaa mutant and the complemented aaa mutant is slightly decreased and increased, respectively, to vitronectin, but not to fibrinogen and fibronectin, which might at least in part result from an increased expression of atl in the aaa mutant. Furthermore, an S. aureus atl mutant that showed enhanced adherence to fibrinogen, fibronectin, and endothelial cells also demonstrated increased aaa expression and production of Aaa. Thus, the redundant functions of Aaa and Atl might at least in part be interchangeable. Lastly, RT-PCR and zymographic analysis revealed that aaa is negatively regulated by the global virulence gene regulators agr and SarA.Conclusions/SignificanceWe identified novel functions for two widely distributed protein domains, LysM and CHAP, i.e. the adherence to the extracellular matrix proteins fibrinogen, fibronectin, and vitronectin. The adhesive properties of Aaa might promote S. aureus colonization of host extracellular matrix and tissue, suggesting a role for Aaa in the pathogenesis of S. aureus infections.

Enhanced Staphylolytic Activity of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA88 HydH5 Virion-Associated Peptidoglycan Hydrolase: Fusions, Deletions, and Synergy with LysH5

Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.

A novel bacteriophage Tail-Associated Muralytic Enzyme (TAME) from Phage K and its development into a potent antistaphylococcal protein

BackgroundStaphylococcus aureus is a major cause of nosocomial and community-acquired infections. However, the rapid emergence of antibiotic resistance limits the choice of therapeutic options for treating infections caused by this organism. Muralytic enzymes from bacteriophages have recently gained attention for their potential as antibacterial agents against antibiotic-resistant gram-positive organisms. Phage K is a polyvalent virulent phage of the Myoviridae family that is active against many Staphylococcus species.ResultsWe identified a phage K gene, designated orf56, as encoding the phage tail-associated muralytic enzyme (TAME). The gene product (ORF56) contains a C-terminal domain corresponding to cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), which demonstrated muralytic activity on a staphylococcal cell wall substrate and was lethal to S. aureus cells. We constructed N-terminal truncated forms of ORF56 and arrived at a 16-kDa protein (Lys16) that retained antistaphylococcal activity. We then generated a chimeric gene construct encoding Lys16 and a staphylococcal cell wall-binding SH3b domain. This chimeric protein (P128) showed potent antistaphylococcal activity on global clinical isolates of S. aureus including methicillin-resistant strains. In addition, P128 was effective in decolonizing rat nares of S. aureus USA300 in an experimental model.ConclusionsWe identified a phage K gene that encodes a protein associated with the phage tail structure. The muralytic activity of the phage K TAME was localized to the C-terminal CHAP domain. This potent antistaphylococcal TAME was combined with an efficient Staphylococcus-specific cell-wall targeting domain SH3b, resulting in the chimeric protein P128. This protein shows bactericidal activity against globally prevalent antibiotic resistant clinical isolates of S. aureus and against the genus Staphylococcus in general. In vivo, P128 was efficacious against methicillin-resistant S. aureus in a rat nasal colonization model.

Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of Staphylococcus aureusbacteriophage vB_SauS-phiIPLA88

BackgroundStaphylococcus 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.ResultsS. 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.ConclusionsThe virion-associated PG hydrolase HydH5 has lytic activity against S. aureus, which makes it attractive as antimicrobial for food biopreservation and anti-staphylococcal therapy.

LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells

LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d-alanine of the stem peptide and glycine of the cross-bridge peptide, and N-acetylmuramoyl-l-alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK.

Structural elucidation of the Cys‐His‐Glu‐Asn proteolytic relay in the secreted CHAP domain enzyme from the human pathogen Staphylococcus saprophyticus

Cysteine peptidases (CP) are ubiquitous enzymes that play fundamental roles in many cellular metabolic pathways.1 In mammalian cells they are highly regulated in apoptotic pathways related to cancer and other severe disorders.2,3 In bacterial cell division, cell-growth and lysis peptidases are employed in the cleavage of peptidoglycans (autolysin).4,5 As secreted antigens and toxins, CPs are key to virulence in Gram-positive pathogens,6 and used to attack competing species in bacterial warfare.7 Viral genomes encode forms of the enzyme for the purpose of capsid trimming during maturation.8 CPs have been classified in clans based on their evolutionary relationships,9 in particular, the CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain (pfam: PF05257, MEROPS ID: C51) is classified as an endopeptidase and part of the CA clan of peptidases (CL0125). This 27-member superfamily includes synthetase/amidases, peptidases, viral proteinases, the NPLC/P60 families and other papain-related families.10 Pfam 22.0 lists 602 members for the CHAP domain family, including 475 from the bacterial kingdom (396 in the firmicutes phylum without any known 3D structure), 39 from eukaryota, and 81 from viruses and phage (predominantly involving Gram-positive phage). CHAP domains are often associated with other peptidases, bifunctional glutathionyl spermidine (GSP) amidases (type 2 and 3), choline binding, and with SH3 and/or Von Willebrand (VWA) domains to form multidomain systems that act cooperatively as versatile machineries for murein septum processing while anchored to the cell surface. Deletion of the CHAP-containing cse gene in Streptococcus thermophilus results in impaired separation of cells during mitosis, demonstrating its involvement in cell division.11 The staphylococcal phage ϕ11 hydrolase, a CHAP domain-containing enzyme, exhibits D-alanyl-glycyl endopeptidase and N-acetylmuramyl-L-alanyl amidase activity.12 In this note we present the solution NMR structure of CHAP domain encoded by gene SSP0609 of Staphylococcus saprophyticus [SWISS-PROT ID: Q49ZM2_STAS1; NESG target ID: SyR11]. S. saprophyticus, one of the three major human Gram-positive pathogens, possesses anchoring fimbriae that help colonize the urinary tract resulting in infection.13 Bacterial antigens are known virulence agents7, and SSP0609 is a secreted antigen with potential roles in the onset of S. saprophyticus infection. The protein comprises a type-I signal peptide in the N-terminal region (res. 1 - 47) and a globular CHAP domain in the C-terminal region (res. 48 - 155). The SSP0609 CHAP domain was found to have a highly-conserved Cys – His – Glu – Asn proteolytic relay active site.

The Cell Lysis Activity of the Streptococcus agalactiae Bacteriophage B30 Endolysin Relies on the Cysteine, Histidine-Dependent Amidohydrolase/Peptidase Domain

The Streptococcus agalactiae bacteriophage B30 endolysin contains three domains: cysteine, histidine-dependent amidohydrolase/peptidase (CHAP), Acm glycosidase, and the SH3b cell wall binding domain. Truncations and point mutations indicated that the Acm domain requires the SH3b domain for activity, while the CHAP domain is responsible for nearly all the cell lysis activity.

Skl, a novel choline-binding N-acetylmuramoyl- l-alanine amidase of Streptococcus mitis SK137 containing a CHAP domain

The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N-acetylmuramoyl- l-alanine amidase (EC 3.5.1.28), showing optimum activity at 30 °C and at a pH of 6.5. Skl is a unique member of the choline-binding family of proteins since it contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP-containing choline-binding proteins.

The bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30

A group B streptococcal (GBS) bacteriophage lysin gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme, calculated to have a molecular mass of 49 677 Da, lysed GBS cells. The susceptibility of GBS cells to lysis by the enzyme depended upon the growth stage at which they were harvested, with early exponential phase cells most sensitive. Calcium ions enhanced the activity of the enzyme. The enzyme also lysed other beta-haemolytic streptococci, including groups A, C, E and G streptococci, but not common oral streptococci, including Streptococcus mutans. The generation of both reducing activity and N-terminal alanine residues during lysis indicated that the lysin is a bifunctional enzyme, possessing both glycosidase and endopeptidase activities. This is consistent with the presence of two conserved sequence domains, an Acm (acetylmuramidase) domain associated with lysozyme activity, and a CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain associated with endopeptidase activity. Site-directed mutagenesis of conserved cysteine and histidine residues in the CHAP domain and conserved aspartate and glutamate residues in the Acm domain confirmed their importance for lysozyme and endopeptidase activity respectively.

Defective cell wall synthesis in Streptococcus pneumoniae R6 depleted for the essential PcsB putative murein hydrolase or the VicR (YycF) response regulator

PcsB is a protein of unknown function(s) that influences the cell morphology of several pathogenic species of streptococcus. PcsB contains a CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain found in bacterial murein hydrolases; however, direct links between steps in cell wall biosynthesis and PcsB function(s) have not been demonstrated. We show here that pcsB is essential in the human respiratory pathogen, Streptococcus pneumoniae, that depletion of PcsB is bacteriostatic and that alanine substitutions in the conserved cysteine and histidine residues of the CHAP domain appear to be lethal. We stained wild-type parent and mutant bacteria deficient in expression of PcsB with fluorescent vancomycin and DAPI to determine patterns of cell wall synthesis and nucleoid segregation respectively. The wild-type parent strain exhibited ordered, simultaneous septal and equatorial cell wall synthesis. In contrast, reduced expression of PcsB resulted in formation of long chains of cells in which peptidoglycan synthesis occurred at nearly every division septum and cell equator. Severe depletion of PcsB led to abnormal, uncontrolled cell wall synthesis at misplaced septa and around large cells. Together, these physiological properties are consistent with a role for PcsB as a murein hydrolase that balances the extent of cell wall synthesis in S. pneumoniae. Finally, we show that the defects in morphology and cell wall synthesis that result from depletion of PcsB strongly resemble those caused by depletion of the essential VicRK two component regulatory system (TCS). This result and the essentiality of pcsB support the hypothesis that the essentiality of the VicRK TCS results from its positive regulation of PcsB expression.

The CHAP domain: a large family of amidases including GSP amidase and peptidoglycan hydrolases

Cleavage of peptidoglycan plays an important role in bacterial cell division, cell growth and cell lysis. Here, we reveal that several known peptidoglycan amidases fall into a family, which includes many proteins of previously unknown function. The family includes two different peptidoglycan cleavage activities: l-muramoyl- l-alanine amidase and d-alanyl-glycyl endopeptidase activity. The family includes the amidase portion of the bifunctional glutathionylspermidine synthase/amidase enzyme from bacteria and pathogenic trypanosomes. The glutathionylspermidine synthase is thought to be a key component of the alternative pathway in trypanosomes for protection from oxygen-radical damage and has been proposed as a potential drug target. The CHAP (cysteine, histidine-dependent amidohydrolases/peptidases) domain is often found in association with other domains that cleave peptidoglycan. The large number of multifunctional hydrolases suggests that they might act in a cooperative manner to cleave specialized substrates.