ABSTRACT 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.

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.

Endolysins are bacteriophage-encoded enzymatic proteins that have great potential to treat multidrug-resistant bacterial infections. Bacteriophage endolysins Cpl-1 and ClyJ-3 have shown promising antimicrobial activity against Streptococcus pneumoniae, which causes pneumonia in humans. This is the first study to investigate the feasibility of spray-dried endolysins Cpl-1 and ClyJ-3 with excipients to produce inhalable powders. The two endolysins were individually tested with leucine and sugar (lactose or trehalose) for spray drying method followed by characterization of biological and physico-chemical properties. A complete loss of ClyJ-3 bioactivity was observed after atomization of the liquid feed solution（before the drying process）, while Cpl-1 maintained its bioactivity in the spray-dried powders. Cpl-1 formulations containing leucine with lactose or trehalose showed promising physico-chemical properties (particle size, crystallinity, hygroscopicity, etc.) and aerosol performances (fine particle fraction values above 65%). The results indicated that endolysin Cpl-1 can be formulated as spray dried powders suitable for inhaled delivery to the lungs for the potential treatment of pulmonary infections.

ABSTRACT Borrelia burgdorferi, the pathogen of Lyme disease, encodes many conserved proteins of unknown structure or function, including ones that serve essential roles in microbial infectivity. One such protein is BB0238, which folds into a two-domain protein, as we have determined by X-ray crystallography and AlphaFold analysis. The N-terminal domain begins with a helix-turn-helix motif (HTH), previously referred to as a tetratricopeptide repeat (TPR) motif, known to mediate protein-protein interactions. The fold of the C-terminal domain has been seen in proteins with a range of unrelated activities and thus does not infer function. In addition to its previously known binding partner BB0323, another essential borrelial virulence determinant, we show that BB0238 also binds a second protein, BB0108, a borrelial ortholog of the chaperone protein SurA and the peptidyl-prolyl cis / trans isomerase protein PrsA. An in vitro enzymatic assay confirmed the catalytic activity. We also determined the crystal structure of the catalytic domain of BB0108, which revealed the parvulin-type organization of the key catalytic residues. We show that BB0238 influences the proteolytic processing of BB0323, although the TPR/HTH motif is not involved in the process. Instead, we show that the motif stabilizes BB0238 in the host environment and facilitates tick-to-mouse pathogen transmission by aiding spirochete evasion of early host cellular immunity. Taken together, these studies highlight the biological significance of BB0238 and its interactions with multiple B. burgdorferi proteins essential for microbial infection. 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.

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-N 335 reveals a trimeric protein comprising four domains. The baseplate anchor domain (AD) contains an intertwined triple-stranded β-helix. The ensuing XD1, XD2 and XD3 β-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-N 335 and of domain deletion proteins showed how TSP4-N 335 promotes the formation of the TSP quaternary complex. TSP1 and TSP2 bind directly to TSP4 whereas TSP3 binding requires a pre-formed TSP4-N 335 :TSP2 complex. A 3-dimensional model of the bacteriophage CBA120 TSP complex has been developed based on the structural and ultracentrifuge information.

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.