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.

Computational models in the service of X-ray and cryo-electron microscopy structure determination.

Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulties. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a posteriori analysis showed that, in some cases, models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X-ray crystallography, backbone tracing and sequence positioning in a cryo-electron microscopy structure, and correction of local features. The results suggest that, in future, there will be greatly increased synergy between computational and experimental approaches to structure determination.

Antibiofilm Activities of a Novel Chimeolysin against Streptococcus mutans under Physiological and Cariogenic Conditions.

Streptococcus mutans often survives as a biofilm on the tooth surface and contributes to the development of dental caries. We investigated the efficacy of ClyR, an engineered chimeolysin, against S. mutans biofilms under physiological and cariogenic conditions. Susceptibility tests showed that ClyR was active against all clinical S. mutans isolates tested as well as S. mutans biofilms that displayed resistance to penicillin. The S. mutans biofilms that formed on hydroxyapatite discs under physiological sugar conditions and cariogenic conditions were reduced ∼2 logs and 3 logs after treatment with 100 µg/ml ClyR, respectively. In comparison, only a 1-log reduction was observed in the chlorhexidine gluconate (ChX)-treated group, and no killing effect was observed in the NaF-treated group. A mouse dental colonization model showed that repeated use of ClyR for 3 weeks (5 µg/day) reduced the number of colonized S. mutans cells in the dental plaques significantly (P < 0.05) and had no harmful effects on the mice. Furthermore, toxicity was not noted at concentrations exceeding those used for the in vitro and in vivo studies, and ClyR-specific antibodies could not be detected in mouse saliva after repeated use of ClyR in the oral cavity. Our data collectively demonstrate that ClyR is active against S. mutans biofilms both in vitro and in vivo, thus representing a preventative or therapeutic agent for use against dental caries.

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.

Catheter-associated urinary tract infection by Pseudomonas aeruginosa is mediated by exopolysaccharide-independent biofilms.

Pseudomonas aeruginosa is an opportunistic human pathogen that is especially adept at forming surface-associated biofilms. P. aeruginosa causes catheter-associated urinary tract infections (CAUTIs) through biofilm formation on the surface of indwelling catheters. P. aeruginosa encodes three extracellular polysaccharides, PEL, PSL, and alginate, and utilizes the PEL and PSL polysaccharides to form biofilms in vitro; however, the requirement of these polysaccharides during in vivo infections is not well understood. Here we show in a murine model of CAUTI that PAO1, a strain harboring pel, psl, and alg genes, and PA14, a strain harboring pel and alg genes, form biofilms on the implanted catheters. To determine the requirement of exopolysaccharide during in vivo biofilm infections, we tested isogenic mutants lacking the pel, psl, and alg operons and showed that PA14 mutants lacking these operons can successfully form biofilms on catheters in the CAUTI model. To determine the host factor(s) that induces the ΔpelD mutant to form biofilm, we tested mouse, human, and artificial urine and show that urine can induce biofilm formation by the PA14 ΔpelD mutant. By testing the major constituents of urine, we show that urea can induce a pel-, psl-, and alg-independent biofilm. These pel-, psl-, and alg-independent biofilms are mediated by the release of extracellular DNA. Treatment of biofilms formed in urea with DNase I reduced the biofilm, indicating that extracellular DNA supports biofilm formation. Our results indicate that the opportunistic pathogen P. aeruginosa utilizes a distinct program to form biofilms that are independent of exopolysaccharides during CAUTI.

Understanding the Molecular Basis for Homodimer Formation of the Pneumococcal Endolysin Cpl-1.

The rise of multi-drug-resistant bacteria that cannot be treated with traditional antibiotics has prompted the search for alternatives to combat bacterial infections. Endolysins, which are bacteriophage-derived peptidoglycan hydrolases, are attractive tools in this fight. Several studies have already demonstrated the efficacy of endolysins in targeting bacterial infections. Endolysins encoded by bacteriophages that infect Gram-positive bacteria typically possess an N-terminal catalytic domain and a C-terminal cell-wall binding domain (CWBD). In this study, we have uncovered the molecular mechanisms that underlie formation of a homodimer of Cpl-1, an endolysin that targets Streptococcus pneumoniae. Here, we use site-directed mutagenesis, analytical size exclusion chromatography, and analytical ultracentrifugation to disprove a previous suggestion that three residues at the N-terminus of the CWBD are involved in the formation of a Cpl-1 dimer in the presence of choline in solution. We conclusively show that the C-terminal tail region of Cpl-1 is involved in formation of the dimer. Alanine scanning mutagenesis generated various tail mutant constructs that allowed identification of key residues that mediate Cpl-1 dimer formation. Finally, our results allowed identification of a consensus sequence (FxxEPDGLIT) required for choline-dependent dimer formation─a sequence that occurs frequently in pneumococcal autolysins and endolysins. These findings shed light on the mechanisms of Cpl-1 and related enzymes and can be used to inform future engineering efforts for their therapeutic development against S. pneumoniae.

A unique borrelial protein facilitates microbial immune evasion.

IMPORTANCE: Lyme disease is a major tick-borne infection caused by a bacterial pathogen called Borrelia burgdorferi, which is transmitted by ticks and affects hundreds of thousands of people every year. These bacterial pathogens are distinct from other genera of microbes because of their distinct features and ability to transmit a multi-system infection to a range of vertebrates, including humans. Progress in understanding the infection biology of Lyme disease, and thus advancements towards its prevention, are hindered by an incomplete understanding of the microbiology of B. burgdorferi, partly due to the occurrence of many unique borrelial proteins that are structurally unrelated to proteins of known functions yet are indispensable for pathogen survival. We herein report the use of diverse technologies to examine the structure and function of a unique B. burgdorferi protein, annotated as BB0238-an essential virulence determinant. We show that the protein is structurally organized into two distinct domains, is involved in multiplex protein-protein interactions, and facilitates tick-to-mouse pathogen transmission by aiding microbial evasion of early host cellular immunity. We believe that our findings will further enrich our understanding of the microbiology of B. burgdorferi, potentially impacting the future development of novel prevention strategies against a widespread tick-transmitted infection.

Structure of Escherichia coli O157:H7 bacteriophage CBA120 tailspike protein 4 baseplate anchor and tailspike assembly domains (TSP4-N).

Four tailspike proteins (TSP1-4) of Escherichia coli O157:H7 bacteriophage CBA120 enable infection of multiple hosts. They form a branched complex that attaches to the tail baseplate. Each TSP recognizes a different lipopolysaccharide on the membrane of a different bacterial host. The 335 N-terminal residues of TSP4 promote the assembly of the TSP complex and anchor it to the tail baseplate. The crystal structure of TSP4-N335 reveals a trimeric protein comprising four domains. The baseplate anchor domain (AD) contains an intertwined triple-stranded ß-helix. The ensuing XD1, XD2 and XD3 ß-sheet containing domains mediate the binding of TSP1-3 to TSP4. Each of the XD domains adopts the same fold as the respective XD domains of bacteriophage T4 gp10 baseplate protein, known to engage in protein-protein interactions via its XD2 and XD3 domains. The structural similarity suggests that XD2 and XD3 of TSP4 also function in protein-protein interactions. Analytical ultracentrifugation analyses of TSP4-N335 and of domain deletion proteins showed how TSP4-N335 promotes the formation of the TSP quaternary complex. TSP1 and TSP2 bind directly to TSP4 whereas TSP3 binding requires a pre-formed TSP4-N335:TSP2 complex. A 3-dimensional model of the bacteriophage CBA120 TSP complex has been developed based on the structural and ultracentrifuge information.

Application of bacteriophage-derived endolysins to combat streptococcal disease: current state and perspectives.

The decline in new antibiotic candidates combined with an increase in antibiotic-resistance necessitates development of alternative antimicrobials. Bacteriophage-encoded endolysins (lysins) are a class of peptidoglycan hydrolases that have been proposed to fill this antimicrobial void. The past 20 years has seen a dramatic expansion of studies on endolysin discovery, structure/function, engineering, immunogenicity, toxicity/safety, and efficacy in animal models. These collective efforts have led to current human clinical trials on at least three different endolysins that are antimicrobial toward staphylococcal species. It can be anticipated that endolysins targeting streptococcal species may be next in line for translational development. Notably, streptococcal diseases largely manifest at accessible mucous membranes, which should be beneficial for protein therapeutics. Additionally, there are a number of well-identified streptococcal diseases in both humans and animals that are associated with a single species, further favoring a targeted endolysin therapeutic.

Characterization of the Bacteriophage-Derived Endolysins PlySs2 and PlySs9 with In Vitro Lytic Activity against Bovine Mastitis Streptococcus uberis.

Bovine mastitis, an infection of the cow's mammary gland, is frequently caused by Streptococcus uberis and causes major economic losses in the dairy industry. The intramammary administration of antibiotics currently remains the predominant preventive and therapeutic measure. These antimicrobial compounds, of which some are considered critical in human health care, are frequently applied as dry therapy resulting in their consistent overuse. Therefore, the use of antibiotics in the dairy sector is being questioned. We here identified two endolysins, i.e., PlySs2 and PlySs9, respectively derived from Streptococcus suis serotype-2 and -9 prophages, with lytic activity against S. uberis in an in vitro setting. Both endolysins gave clear lysis zones in spot-on-plate assays and caused a reduction of the optical density in a turbidity reduction assay. In depth characterization identified PlySs9 as the more potent endolysin over PlySs2 with a lower MIC value and about one additional log of killing. PlySs2 and PlySs9 were challenged to a panel of subclinical and clinical S. uberis milk isolates and were both able to lyse all strains tested. Molecular dissection of these endolysins in catalytic and cell wall binding subdomains resulted in major loss of killing and binding activity, respectively. Taken together, we here propose PlySs2 and PlySs9 as candidate compounds to the current antimicrobial arsenal known against bovine mastitis-causing S. uberis as future add-on or replacement strategy to the currently used intramammary antibiotics.

Structure and function of bacteriophage CBA120 ORF211 (TSP2), the determinant of phage specificity towards E. coli O157:H7.

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.

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.

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.

Complete Genome Sequence of Klebsiella pneumoniae Phages SopranoGao, MezzoGao, and AltoGao.

The Klebsiella pneumoniae phages SopranoGao, MezzoGao, and AltoGao were isolated from the Seneca Wastewater Treatment Plant in Germantown, MD. The following reports the complete genome sequence of these bacteriophages and describes their major features.

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.

A chimeolysin with extended-spectrum streptococcal host range found by an induced lysis-based rapid screening method.

The increasing emergence of multi-drug resistant streptococci poses a serious threat to public health worldwide. Bacteriophage lysins are promising alternatives to antibiotics; however, their narrow lytic spectrum restricted to closely related species is a central shortcoming to their translational development. Here, we describe an efficient method for rapid screening of engineered chimeric lysins and report a unique "chimeolysin", ClyR, with robust activity and an extended-spectrum streptococcal host range against most streptococcal species, including S. pyogenes, S. agalactiae, S. dysgalactiae, S. equi, S. mutans, S. pneumoniae, S. suis and S. uberis, as well as representative enterococcal and staphylococcal species (including MRSA and VISA). ClyR is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen as well as the first lysin that kills all four of the bovine mastitis-causing pathogens. This study demonstrates the success of the screening method resulting in a powerful lysin with potential for treating most streptococcal associated infections.