SP-CHAP, an endolysin with enhanced activity against biofilm pneumococci and nasopharyngeal colonization.

Streptococcus pneumoniae (Spn), a Gram-positive bacterium, is responsible for causing a wide variety of invasive infections. The emergence of multi-drug antibiotic resistance has prompted the search for antimicrobial alternatives. Phage-derived peptidoglycan hydrolases, known as endolysins, are an attractive alternative. In this study, an endolysin active against Spn, designated SP-CHAP, was cloned, produced, purified, biochemically characterized, and evaluated for its antimicrobial properties. Cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domains are widely represented in bacteriophage endolysins but have never previously been reported for pneumococcal endolysins. Here, we characterize the first pneumococcal endolysin with a CHAP catalytic domain. SP-CHAP was antimicrobial against all Spn serovars tested, including capsular and capsule-free pneumococci, and it was found to be more active than the most widely studied pneumococcal endolysin, Cpl-1, while not affecting various oral or nasal commensal organisms tested. SP-CHAP was also effective in eradicating Spn biofilms at concentrations as low as 1.56 µg/mL. In addition, a Spn mouse nasopharyngeal colonization model was employed, which showed that SP-CHAP caused a significant reduction in Spn colony-forming units, even more than Cpl-1. These results indicate that SP-CHAP may represent a promising alternative to combating Spn infections. IMPORTANCE: Considering the high rates of pneumococcal resistance reported for several antibiotics, alternatives are urgently needed. In the present study, we report a Streptococcus pneumoniae-targeting endolysin with even greater activity than Cpl-1, the most characterized pneumococcal endolysin to date. We have employed a combination of biochemical and microbiological assays to assess the stability and lytic potential of SP-CHAP and demonstrate its efficacy on pneumococcal biofilms in vitro and in an in vivo mouse model of colonization. Our findings highlight the therapeutic potential of SP-CHAP as an antibiotic alternative to treat Streptococcus pneumoniae infections.

Controlled Proteolysis of an Essential Virulence Determinant Dictates Infectivity of Lyme Disease Pathogens.

The Borrelia burgdorferi BB0323 protein undergoes a complex yet poorly defined proteolytic maturation event that generates N-terminal and C-terminal proteins with essential functions in cell growth and infection. Here, we report that a borrelial protease, B. burgdorferi high temperature requirement A protease (BbHtrA), cleaves BB0323 between asparagine (N) and leucine (L) at positions 236 and 237, while the replacement of these residues with alanine in the mutant protein prevents its cleavage, despite preserving its normal secondary structure. The N-terminal BB0323 protein binds BbHtrA, but its cleavage site mutant displays deficiency in such interaction. An isogenic borrelial mutant with NL-to-AA substitution in BB0323 (referred to as Bbbb0323NL) maintains normal growth yet is impaired for infection of mice or transmission from infected ticks. Notably, the BB0323 protein is still processed in Bbbb0323NL, albeit with lower levels of mature N-terminal BB0323 protein and multiple aberrantly processed polypeptides, which could result from nonspecific cleavages at other asparagine and leucine residues in the protein. The lack of infectivity of Bbbb0323NL is likely due to the impaired abundance or stoichiometry of a protein complex involving BB0238, another spirochete protein. Together, these studies highlight that a precise proteolytic event and a particular protein-protein interaction, involving multiple borrelial virulence determinants, are mutually inclusive and interconnected, playing essential roles in the infectivity of Lyme disease pathogens.

Linker Editing of Pneumococcal Lysin ClyJ Conveys Improved Bactericidal Activity.

Streptococcus pneumoniae is a leading human pathogen uniquely characterized by choline moieties on the bacterial surface. Our previous work reported a pneumococcus-specific chimeric lysin, ClyJ, which combines the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) enzymatically active domain (EAD) from the PlyC lysin and the cell wall binding domain (CBD) from the phage SPSL1 lysin, which imparts choline binding specificity. Here, we demonstrate that the lytic activity of ClyJ can be further improved by editing the linker sequence adjoining the EAD and CBD. Keeping the net charge of the linker constant, we constructed three ClyJ variants containing different lengths of linker sequence. Circular dichroism showed that linker editing has only minor effects on the folding of the EAD and CBD. However, thermodynamic examination combined with biochemical analysis demonstrated that one variant, ClyJ-3, with the shortest linker, displayed improved thermal stability and bactericidal activity, as well as reduced cytotoxicity. In a pneumococcal mouse infection model, ClyJ-3 showed significant protective efficacy compared to that of the ClyJ parental lysin or the Cpl-1 lysin, with 100% survival at a single ClyJ-3 intraperitoneal dose of 100 µg/mouse. Moreover, a ClyJ-3 dose of 2 µg/mouse had the same efficacy as a ClyJ dose of 40 µg/mouse, suggesting a 20-fold improvement in vivo Taking these results together, the present study not only describes a promising pneumococcal lysin with improved potency, i.e., ClyJ-3, but also implies for the first time that the linker sequence plays an important role in determining the activity of a chimeric lysin, providing insight for future lysin engineering studies.

ClyJ Is a Novel Pneumococcal Chimeric Lysin with a Cysteine- and Histidine-Dependent Amidohydrolase/Peptidase Catalytic Domain.

Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

Safety Studies of Pneumococcal Endolysins Cpl-1 and Pal.

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.

Borrelia burgdorferi surface-located Lmp1 protein processed into region-specific polypeptides that are critical for microbial persistence.

One of the Borrelia burgdorferi virulence determinants, annotated as Lmp1, is a surface-exposed, conserved, and potential multi-domain protein involved in various functions in spirochete infectivity. Lmp1 contributes to host-pathogen interactions and evasion of host adaptive immunity by spirochetes. Here, we show that in diverse B. burgdorferi species, Lmp1 exists as distinct, region-specific, and lower molecular mass polypeptides encompassing 1 or more domains, including independent N-terminal and middle regions and a combined middle and C-terminal region. These polypeptides originate from complex posttranslational maturation events, partly supported by a periplasmic serine protease termed as BbHtrA. Although spirochete persistence in mice is independently supported by domain-specific Lmp1 polypeptides, transmission of B. burgdorferi from ticks to mammals requires essential contributions from both N-terminal and middle regions. Interference with the functions of Lmp1 domains or their complex posttranslational maturation events may aid in development of novel therapeutic strategies to combat infection and transmission of pathogens.

A bacteriophage endolysin that eliminates intracellular streptococci.

PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB-PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities.

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.

Biochemical and biophysical characterization of PlyGRCS, a bacteriophage endolysin active against methicillin-resistant Staphylococcus aureus.

The increasing rate of resistance of pathogenic bacteria, such as Staphylococcus aureus, to classical antibiotics has driven research toward identification of other means to fight infectious disease. One particularly viable option is the use of bacteriophage-encoded peptidoglycan hydrolases, called endolysins or enzybiotics. These enzymes lyse the bacterial cell wall upon direct contact, are not inhibited by traditional antibiotic resistance mechanisms, and have already shown great promise in the areas of food safety, human health, and veterinary science. We have identified and characterized an endolysin, PlyGRCS, which displays dose-dependent antimicrobial activity against both planktonic and biofilm S. aureus, including methicillin-resistant S. aureus (MRSA). The spectrum of lytic activity for this enzyme includes all S. aureus and Staphylococcus epidermidis strains tested, but not other Gram-positive pathogens. The contributions of the PlyGRCS putative catalytic and cell wall binding domains were investigated through deletion analysis. The cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) catalytic domain displayed activity by itself, though reduced, indicating the necessity of the binding domain for full activity. In contrast, the SH3_5 binding domain lacked activity but was shown to interact directly with the staphylococcal cell wall via fluorescent microscopy. Site-directed mutagenesis studies determined that the active site residues in the CHAP catalytic domain were C29 and H92, and its catalytic functionality required calcium as a co-factor. Finally, biochemical assays coupled with mass spectrometry analysis determined that PlyGRCS displays both N-acetylmuramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase hydrolytic activities despite possessing only a single catalytic domain. These results indicate that PlyGRCS has the potential to become a revolutionary therapeutic option to combat bacterial infections.

Quantitative analysis of the thermal stability of the gamma phage endolysin PlyG: a biophysical and kinetic approach to assaying therapeutic potential.

Endolysins are lytic enzymes encoded by bacteriophage that represent an emerging class of protein therapeutics. Considering macromolecular thermoresistance correlates with shelf life, PlyG, a Bacillus anthracis endolysin, was thermally characterized to further evaluate its therapeutic potential. Results from a biophysical thermal analysis revealed full-length PlyG and its isolated domains comprised thermal denaturation temperatures exceeding 63°C. In the absence of reducing agent, PlyG was determined to be kinetically unstable, a finding hypothesized to be attributable to the chemical oxidation of cysteine and/or methionine residues. The presence of reducing agent kinetically stabilized the endolysin, with PlyG retaining at least ~50% residual lytic activity after being heated at temperatures up to 80°C and remaining enzymatically functional after being boiled. Furthermore, the endolysin had a kinetic half-life at 50°C and 55°C of 35 and 5.5h, respectively. PlyG represents a thermostable proteinaceous antibacterial with subsequent prolonged therapeutic shelf life expectancy.

Determining carbapenemase activity with 18O labeling and targeted mass spectrometry.

Carbapenems are broad spectrum antibiotics considered as a "last resort" medicine to treat bacterial infections. Carbapenem-hydrolyzing ß-lactamases (also called carbapenemases), however, can confer bacterial resistance and represent a serious health threat. Here, we report a novel approach using (18)O labeling and selected reaction monitoring to detect carbapenemase activity from pathogenic microorganisms in a rapid and quantitative manner. Four model bacterial strains bearing various classes of ß-lactamases were tested for their capability to hydrolyze Meropenem, an FDA-approved carbapenem drug. We were able to predict the Meropenem resistance of these bacteria on the basis of their carbapenemase activity, suggesting the great potential of our method in clinical diagnostics.

X-ray crystal structure of the streptococcal specific phage lysin PlyC.

Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria.

Cysteine proteinase SpeB from Streptococcus pyogenes - a potent modifier of immunologically important host and bacterial proteins.

Group A streptococcus (Streptococcus pyogenes) is an exclusively human pathogen that causes a wide spectrum of diseases ranging from pharyngitis, to impetigo, to toxic shock, to necrotizing fasciitis. The diversity of these disease states necessitates that S. pyogenes possess the ability to modulate both the innate and adaptive immune responses. SpeB, a cysteine proteinase, is the predominant secreted protein from S. pyogenes. Because of its relatively indiscriminant specificity, this enzyme has been shown to degrade the extracellular matrix, cytokines, chemokines, complement components, immunoglobulins, and serum protease inhibitors, to name but a few of the known substrates. Additionally, SpeB regulates other streptococcal proteins by degrading them or releasing them from the bacterial surface. Despite the wealth of literature on putative SpeB functions, there remains much controversy about this enzyme because many of reported activities would produce contradictory physiological results. Here we review all known host and bacterial protein substrates for SpeB, their cleavage sites, and discuss the role of this enzyme in streptococcal pathogenesis based on the current literature.