Fermentation Practices Select for Thermostable Endolysins in Phages.

Endolysins are produced by (bacterio)phages and play a crucial role in degrading the bacterial cell wall and the subsequent release of new phage progeny. These lytic enzymes exhibit a remarkable diversity, often occurring in a multimodular form that combines different catalytic and cell wall-binding domains, even in phages infecting the same species. Yet, our current understanding lacks insight into how environmental factors and ecological niches may have influenced the evolution of these enzymes. In this study, we focused on phages infecting Streptococcus thermophilus, as this bacterial species has a well-defined and narrow ecological niche, namely, dairy fermentation. Among the endolysins found in phages targeting this species, we observed limited diversity, with a singular structural type dominating in most of identified S. thermophilus phages. Within this prevailing endolysin type, we discovered a novel and highly conserved calcium-binding motif. This motif proved to be crucial for the stability and activity of the enzyme at elevated temperatures. Ultimately, we demonstrated its positive selection within the host's environmental conditions, particularly under the temperature profiles encountered in the production of yogurt, mozzarella, and hard cheeses that rely on S. thermophilus.

Dairy phages escape CRISPR defence of Streptococcus thermophilus via the anti-CRISPR AcrIIA3.

Bacterial community collapse due to phage infection is a major risk in cheese making processes. As virulent phages are ubiquitous and diverse in milk fermentation factories, the use of phage-resistant lactic acid bacteria (LAB) is essential to obtain high-quality fermented dairy products. The LAB species Streptococcus thermophilus contains two type II-A CRISPR-Cas systems (CRISPR1 and CRISPR3) that can effectively protect against phage infection. However, virulent streptococcal phages carrying anti-CRISPR proteins (ACR) that block the activity of CRISPR-Cas systems have emerged in yogurt and cheese environments. For example, phages carrying AcrIIA5 can impede both CRISPR1 and CRISPR3 systems, while AcrIIA6 stops only CRISPR1. Here, we explore the activity and diversity of a third streptococcal phage anti-CRISPR protein, namely AcrIIA3. We were able to demonstrate that AcrIIA3 is efficiently active against the CRISPR3-Cas system of S. thermophilus. We used AlphaFold2 to infer the structure of AcrIIA3 and we predicted that this new family of functional ACR in virulent streptococcal phages has a new α-helical fold, with no previously identified structural homologs. Because ACR proteins are being explored as modulators in genome editing applications, we also tested AcrIIA3 against SpCas9. We found that AcrIIA3 could block SpCas9 in bacteria but not in human cells. Understanding the diversity and functioning of anti-defence mechanisms will be of importance in the design of long-term stable starter cultures.

Characterization of a (p)ppApp Synthetase Belonging to a New Family of Polymorphic Toxin Associated with Temperate Phages.

Polymorphic toxins (PTs) are a broad family of toxins involved in interbacterial competition and pathogenesis. PTs are modular proteins that are comprised of a conserved N-terminal domain responsible for its transport, and a variable C-terminal domain bearing toxic activity. Although the mode of transport has yet to be elucidated, a new family of putative PTs containing an N-terminal MuF domain, resembling the Mu coliphage F protein, was identified in prophage genetic elements. The C-terminal toxin domains of these MuF PTs are predicted to bear nuclease, metallopeptidase, ADP-ribosyl transferase and RelA_SpoT activities. In this study, we characterized the MuF-RelA_SpoT toxin associated with the temperate phage of Streptococcus pneumoniae SPNA45. We show that the RelA_SpoT domain has (p)ppApp synthetase activity, which is bactericidal under our experimental conditions. We further determine that the two genes located downstream encode two immunity proteins, one binding to and inactivating the toxin and the other detoxifying the cell via a pppApp hydrolase activity. Finally, based on protein sequence alignments, we propose a signature for (p)ppApp synthetases that distinguishes them from (p)ppGpp synthetases.

Bacteriophage-host interactions in Streptococcus thermophilus and their impact on co-evolutionary processes.

Bacteriophages (or phages) represent a persistent threat to the success and reliability of food fermentation processes. Recent reports of phages that infect Streptococcus thermophilus have highlighted the diversification of phages of this species. Phages of S. thermophilus typically exhibit a narrow range, a feature that is suggestive of diverse receptor moieties being presented on the cell surface of the host. Cell wall polysaccharides, including rhamnose-glucose polysaccharides and exopolysaccharides have been implicated as being involved in the initial interactions with several phages of this species. Following internalization of the phage genome, the host presents several defences, including CRISPR-Cas and restriction and modification systems to limit phage proliferation. This review provides a current and holistic view of the interactions of phages and their S. thermophilus host cells and how this has influenced the diversity and evolution of both entities.

Investigation of CRISPR-Independent Phage Resistance Mechanisms Reveals a Role for FtsH in Phage Adsorption to Streptococcus thermophilus.

Prokaryotes are under constant pressure from phage infection and thus have evolved multiple means of defense or evasion. While CRISPR-Cas constitutes a robust immune system and appears to be the predominant means of survival for Streptococcus thermophilus when facing lytic phage infection, other forms of phage resistance coexist in this species. Here, we show that S. thermophilus strains with deleted CRISPR-Cas loci can still give rise to phage-resistant clones following lytic phage challenge. Notably, non-CRISPR phage-resistant survivors had multiple mutations which would truncate or recode a membrane-anchored host protease, FtsH. Phage adsorption was dramatically reduced in FtsH mutants, implicating this protein in phage attachment. Phages were isolated which could bypass FtsH-based resistance through mutations predicted to alter tape measure protein translation. Together, these results identify key components in phage propagation that are subject to mutation in the molecular arms race between phage and host cell. IMPORTANCE Streptococcus thermophilus is an important organism for production of cultured dairy foods, but it is susceptible to lytic phages which can lead to failed products. Consequently, mechanisms for phage resistance are an active area of research. One such mechanism is CRISPR-Cas, and S. thermophilus is a model organism for the study of this form of adaptive immunity. Here, we expand on known mechanisms with our finding that spontaneous mutations in ftsH, a gene encoding a membrane-anchored protease, protected against phage infection by disrupting phage adsorption. In turn, mutations in phage tail protein genes allowed phages to overcome ftsH-based resistance. Our results identified components in phage propagation that are subject to mutation in the molecular arms race between phage and host.

Streptococcus suis prophage lysin as a new strategy for combating streptococci-induced mastitis and Streptococcus suis infection.

OBJECTIVES: The genus Streptococcus contains species of important zoonotic pathogens such as those that cause bovine mastitis. Unfortunately, many Streptococcus species have developed antibiotic resistance. Phage lysins are considered promising alternatives to antibiotics because it is difficult for bacteria to develop lysin resistance. However, there remains a lack of phage lysin resources for the treatment of streptococci-induced mastitis. METHODS: We identified the prophage lysin Lys0859 from the genome of the Streptococcus suis SS0859 strain. Lys0859 was subsequently characterized to determine its host range, MIC, bactericidal activity in milk, and ability to clear biofilms in vitro. Finally, to determine the effects of Lys0859 on the treatment of both bovine mastitis and S. suis infection in vivo, we established models of Streptococcus agalactiae ATCC 13813-induced mastitis and S. suis serotype 2 SC19 systemic infection. RESULTS: Our results demonstrate that Lys0859 possesses broad-spectrum lytic activity against Streptococcus and Staphylococcus species isolated from animals with bovine mastitis and 15 serotypes of S. suis isolated from swine. Intramammary and intramuscular injection of Lys0859 reduced the number of bacteria in mammary tissue by 3.75 and 1.45 logs compared with the PBS group, respectively. Furthermore, 100 µg/mouse of Lys0859 administered intraperitoneally at 1 h post-infection protected 83.3% (5/6) of mice from a lethal dose of S. suis infection. CONCLUSIONS: Overall, our results enhance the understanding and development of new strategies to combat both streptococci-induced mastitis and S. suis infection.

Implications of oral streptococcal bacteriophages in autism spectrum disorder.

Growing evidence suggests altered oral and gut microbiota in autism spectrum disorder (ASD), but little is known about the alterations and roles of phages, especially within the oral microbiota in ASD subjects. We enrolled ASD (n = 26) and neurotypical subjects (n = 26) with their oral hygiene controlled, and the metagenomes of both oral and fecal samples (n = 104) are shotgun-sequenced and compared. We observe extensive and diverse oral phageome comparable to that of the gut, and clear signals of mouth-to-gut phage strain transfer within individuals. However, the overall phageomes of the two sites are widely different and show even less similarity in the oral communities between ASD and control subjects. The ASD oral phageome exhibits significantly reduced abundance and alpha diversity, but the Streptococcal phages there are atypically enriched, often dominating the community. The over-representation of Streptococcal phages is accompanied by enriched oral Streptococcal virulence factors and Streptococcus bacteria, all exhibiting a positive correlation with the severity of ASD clinical manifestations. These changes are not observed in the parallel sampling of the gut flora, suggesting a previously unknown oral-specific association between the excessive Streptococcal phage enrichment and ASD pathogenesis. The findings provide new evidence for the independent microbiome-mouth-brain connection, deepen our understanding of how the growth dynamics of bacteriophages and oral microbiota contribute to ASD, and point to novel effective therapeutics.

Streptococcus thermophilus Phages in Whey Derivatives: From Problem to Application in the Dairy Industry.

Fifteen samples of whey protein concentrate (WPC) were tested against 37 commercial Streptococcus thermophilus strains to detect infective bacteriophages. Seventy-three diverse phages were isolated from 12 samples, characterized by using DNA restriction patterns and host range analyses. Sixty-two of them were classified as cos, two as pac, and nine as 5093, according to PCR multiplex assays. Phage concentration was greater than 104 PFU/g for 25.3% of isolated phages. Seven phages showed an unusual wide host range, being able to infect a high number of the tested strains. Regarding thermal resistance, pac phages were the most sensitive, followed by cos phages, those classified as 5093 being the most resistant. Treatments at 85 °C for 5 min in TMG buffer were necessary to completely inactivate all phages. Results demonstrated that the use, without control, of these whey derivatives as additives in dairy fermentations could be a threat because of the potential phage infection of starter strains. In this sense, these phages constitute a pool of new isolates used to improve the phage resistance of starter cultures applied today in the fermentative industry.

Brussowvirus SW13 Requires a Cell Surface-Associated Polysaccharide To Recognize Its Streptococcus thermophilus Host.

Four bacteriophage-insensitive mutants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following challenge with Brussowvirus SW13. The BIMs displayed an altered sedimentation phenotype. Whole-genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family 2 glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated rhamnose-glucose polymer (Rgp) biosynthesis locus (designated the rgp gene cluster here). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis polysaccharide pellicle (PSP) initiator WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescence labeling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide that is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. IMPORTANCE Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and, in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process, which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements that specify the surface receptor(s) is apparent. Our research identifies a single gene that is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

Prophage integration into CRISPR loci enables evasion of antiviral immunity in Streptococcus pyogenes.

CRISPR loci are composed of short DNA repeats separated by sequences, known as spacers, that match the genomes of invaders such as phages and plasmids. Spacers are transcribed and processed to generate RNA guides used by CRISPR-associated nucleases to recognize and destroy the complementary nucleic acids of invaders. To counteract this defence, phages can produce small proteins that inhibit these nucleases, termed anti-CRISPRs (Acrs). Here we demonstrate that the ΦAP1.1 temperate phage utilizes an alternative approach to antagonize the type II-A CRISPR response in Streptococcus pyogenes. Immediately after infection, this phage expresses a small anti-CRISPR protein, AcrIIA23, that prevents Cas9 function, allowing ΦAP1.1 to integrate into the direct repeats of the CRISPR locus, neutralizing immunity. However, acrIIA23 is not transcribed during lysogeny and phage integration/excision cycles can result in the deletion and/or transduction of spacers, enabling a complex modulation of the type II-A CRISPR immune response. A bioinformatic search identified prophages integrated not only in the CRISPR repeats, but also the cas genes, of diverse bacterial species, suggesting that prophage disruption of the CRISPR-cas locus is a recurrent mechanism to counteract immunity.

Novel Virulent Bacteriophage ΦSG005, Which Infects Streptococcus gordonii, Forms a Distinct Clade among Streptococcus Viruses.

Bacteriophages are viruses that specifically infect bacteria and are classified as either virulent phages or temperate phages. Despite virulent phages being promising antimicrobial agents due to their bactericidal effects, the implementation of phage therapy depends on the availability of virulent phages against target bacteria. Notably, virulent phages of Streptococcus gordonii, which resides in the oral cavity and is an opportunistic pathogen that can cause periodontitis and endocarditis have previously never been found. We thus attempted to isolate virulent phages against S. gordonii. In the present study, we report for the first time a virulent bacteriophage against S. gordonii, ΦSG005, discovered from drainage water. ΦSG005 is composed of a short, non-contractile tail and a long head, revealing Podoviridae characteristics via electron microscopic analysis. In turbidity reduction assays, ΦSG005 showed efficient bactericidal effects on S. gordonii. Whole-genome sequencing showed that the virus has a DNA genome of 16,127 bp with 21 coding sequences. We identified no prophage-related elements such as integrase in the ΦSG005 genome, demonstrating that the virus is a virulent phage. Phylogenetic analysis indicated that ΦSG005 forms a distinct clade among the streptococcus viruses and is positioned next to streptococcus virus C1. Molecular characterization revealed the presence of an anti-CRISPR (Acr) IIA5-like protein in the ΦSG005 genome. These findings facilitate our understanding of streptococcus viruses and advance the development of phage therapy against S. gordonii infection.

Inhibition of Fibrinolysis by Streptococcal Phage LysinSM1.

Expression of bacteriophage lysinSM1 by Streptococcus oralis strain SF100 is thought to be important for the pathogenesis of infective endocarditis, due to its ability to mediate bacterial binding to fibrinogen. To better define the lysinSM1 binding site on fibrinogen Aα, and to investigate the impact of binding on fibrinolysis, we examined the interaction of lysinSM1 with a series of recombinant fibrinogen Aα variants. These studies revealed that lysinSM1 binds the C-terminal region of fibrinogen Aα spanned by amino acid residues 534 to 610, with an affinity of equilibrium dissociation constant (KD) of 3.23 × 10-5 M. This binding site overlaps the known binding site for plasminogen, an inactive precursor of plasmin, which is a key protease responsible for degrading fibrin polymers. When tested in vitro, lysinSM1 competitively inhibited plasminogen binding to the αC region of fibrinogen Aα. It also inhibited plasminogen-mediated fibrinolysis, as measured by thromboelastography (TEG). These results indicate that lysinSM1 is a bi-functional virulence factor for streptococci, serving as both an adhesin and a plasminogen inhibitor. Thus, lysinSM1 may facilitate the attachment of bacteria to fibrinogen on the surface of damaged cardiac valves and may also inhibit plasminogen-mediated lysis of infected thrombi (vegetations) on valve surfaces. IMPORTANCE The interaction of streptococci with human fibrinogen and platelets on damaged endocardium is a central event in the pathogenesis of infective endocarditis. Streptococcus oralis can bind platelets via the interaction of bacteriophage lysinSM1 with fibrinogen on the platelet surface, and this process has been associated with increased virulence in an animal model of endocarditis. We now report that lysinSM1 binds to the αC region of the human fibrinogen Aα chain. This interaction blocks plasminogen binding to fibrinogen and inhibits fibrinolysis. In vivo, this inhibition could prevent the lysis of infected vegetations, thereby promoting bacterial persistence and virulence.

Isolation and Characterization of Streptococcus mutans Phage as a Possible Treatment Agent for Caries.

Streptococcus mutans is a key bacterium in dental caries, one of the most prevalent chronic infectious diseases. Conventional treatment fails to specifically target the pathogenic bacteria, while tending to eradicate commensal bacteria. Thus, caries remains one of the most common and challenging diseases. Phage therapy, which involves the use of bacterial viruses as anti-bacterial agents, has been gaining interest worldwide. Nevertheless, to date, only a few phages have been isolated against S. mutans. In this study, we describe the isolation and characterization of a new S. mutans phage, termed SMHBZ8, from hundreds of human saliva samples that were collected, filtered, and screened. The SMHBZ8 genome was sequenced and analyzed, visualized by TEM, and its antibacterial properties were evaluated in various states. In addition, we tested the lytic efficacy of SMHBZ8 against S. mutans in a human cariogenic dentin model. The isolation and characterization of SMHBZ8 may be the first step towards developing a potential phage therapy for dental caries.

Survival Strategies of Streptococcus pyogenes in Response to Phage Infection.

Bacteriophages exert strong evolutionary pressure on their microbial hosts. In their lytic lifecycle, complete bacterial subpopulations are utilized as hosts for bacteriophage replication. However, during their lysogenic lifecycle, bacteriophages can integrate into the host chromosome and alter the host's genomic make-up, possibly resulting in evolutionary important adjustments. Not surprisingly, bacteria have evolved sophisticated immune systems to protect against phage infection. Streptococcus pyogenes isolates are frequently lysogenic and their prophages have been shown to be major contributors to the virulence of this pathogen. Most S. pyogenes phage research has focused on genomic prophages in relation to virulence, but little is known about the defensive arsenal of S. pyogenes against lytic phage infection. Here, we characterized Phage A1, an S. pyogenes bacteriophage, and investigated several mechanisms that S. pyogenes utilizes to protect itself against phage predation. We show that Phage A1 belongs to the Siphoviridae family and contains a circular double-stranded DNA genome that follows a modular organization described for other streptococcal phages. After infection, the Phage A1 genome can be detected in isolated S. pyogenes survivor strains, which enables the survival of the bacterial host and Phage A1 resistance. Furthermore, we demonstrate that the type II-A CRISPR-Cas system of S. pyogenes acquires new spacers upon phage infection, which are increasingly detectable in the absence of a capsule. Lastly, we show that S. pyogenes produces membrane vesicles that bind to phages, thereby limiting the pool of phages available for infection. Altogether, this work provides novel insight into survival strategies employed by S. pyogenes to combat phage predation.

The multidomain architecture of a bacteriophage endolysin enables intramolecular synergism and regulation of bacterial lysis.

Endolysins are peptidoglycan hydrolases produced at the end of the bacteriophage (phage) replication cycle to lyse the host cell. Endolysins in Gram-positive phages come in a variety of multimodular forms that combine different catalytic and cell wall binding domains. However, the reason why phages adopt endolysins with such complex multidomain architecture is not well understood. In this study, we used the Streptococcus dysgalactiae phage endolysin PlySK1249 as a model to investigate the role of multidomain architecture in phage-induced bacterial lysis and lysis regulation. PlySK1249 consists of an amidase (Ami) domain that lyses bacterial cells, a nonbacteriolytic endopeptidase (CHAP) domain that acts as a dechaining enzyme, and a central LysM cell wall binding domain. We observed that the Ami and CHAP domains synergized for peptidoglycan digestion and bacteriolysis in the native enzyme or when expressed individually and reunified. The CHAP endopeptidase resolved complex polymers of stem-peptides to dimers and helped the Ami domain to digest peptidoglycan to completion. We also found that PlySK1249 was subject to proteolytic cleavage by host cell wall proteases both in vitro and after phage induction. Cleavage disconnected the different domains by hydrolyzing their linker regions, thus hindering their bacteriolytic cooperation and possibly modulating the lytic activity of the enzyme. PlySK1249 cleavage by cell-wall-associated proteases may represent another example of phage adaptation toward the use of existing bacterial regulation mechanism for their own advantage. In addition, understanding more thoroughly the multidomain interplay of PlySK1249 broadens our knowledge on the ideal architecture of therapeutic antibacterial endolysins.

Deep-ABPpred: identifying antibacterial peptides in protein sequences using bidirectional LSTM with word2vec.

The overuse of antibiotics has led to emergence of antimicrobial resistance, and as a result, antibacterial peptides (ABPs) are receiving significant attention as an alternative. Identification of effective ABPs in lab from natural sources is a cost-intensive and time-consuming process. Therefore, there is a need for the development of in silico models, which can identify novel ABPs in protein sequences for chemical synthesis and testing. In this study, we propose a deep learning classifier named Deep-ABPpred that can identify ABPs in protein sequences. We developed Deep-ABPpred using bidirectional long short-term memory algorithm with amino acid level features from word2vec. The results show that Deep-ABPpred outperforms other state-of-the-art ABP classifiers on both test and independent datasets. Our proposed model achieved the precision of approximately 97 and 94% on test dataset and independent dataset, respectively. The high precision suggests applicability of Deep-ABPpred in proposing novel ABPs for synthesis and experimentation. By utilizing Deep-ABPpred, we identified ABPs in the tail protein sequences of Streptococcus bacteriophages, chemically synthesized identified peptides in lab and tested their activity in vitro. These ABPs showed potent antibacterial activity against selected Gram-positive and Gram-negative bacteria, which confirms the capability of Deep-ABPpred in identifying novel ABPs in protein sequences. Based on the proposed approach, an online prediction server is also developed, which is freely accessible at https://abppred.anvil.app/. This web server takes the protein sequence as input and provides ABPs with high probability (>0.95) as output.

Dairy lactococcal and streptococcal phage-host interactions: an industrial perspective in an evolving phage landscape.

Almost a century has elapsed since the discovery of bacteriophages (phages), and 85 years have passed since the emergence of evidence that phages can infect starter cultures, thereby impacting dairy fermentations. Soon afterward, research efforts were undertaken to investigate phage interactions regarding starter strains. Investigations into phage biology and morphology and phage-host relationships have been aimed at mitigating the negative impact phages have on the fermented dairy industry. From the viewpoint of a supplier of dairy starter cultures, this review examines the composition of an industrial phage collection, providing insight into the development of starter strains and cultures and the evolution of phages in the industry. Research advances in the diversity of phages and structural bases for phage-host recognition and an overview of the perpetual arms race between phage virulence and host defense are presented, with a perspective toward the development of improved phage-resistant starter culture systems.

A Choline-Recognizing Monomeric Lysin, ClyJ-3m, Shows Elevated Activity against Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading pathogen for bacterial pneumonia, which can be treated with bacteriophage lysins harboring a conserved choline binding module (CBM). Such lysins regularly function as choline-recognizing dimers. Previously, we reported a pneumococcus-specific lysin ClyJ comprising the binding domain from the putative endolysin gp20 from the Streptococcus phage SPSL1 and the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain from the PlyC lysin. A variant of ClyJ with a shortened linker, i.e., ClyJ-3, shows improved activity and reduced cytotoxicity. Resembling typical CBM-containing lysins, ClyJ-3 dimerized upon binding with choline. Herein, we further report a choline-recognizing variant of ClyJ-3, i.e., ClyJ-3m, constructed by deleting its C-terminal tail. Biochemical characterization showed that ClyJ-3m remains a monomer after it binds to choline yet exhibits improved bactericidal activity against multiple pneumococcal strains with different serotypes. In an S. pneumoniae-infected bacteremia model, a single intraperitoneal administration of 2.32 µg/mouse of ClyJ-3m showed 70% protection, while only 20% of mice survived in the group receiving an equal dose of ClyJ-3 (P < 0.05). A pharmacokinetic analysis following single intravenously doses of 0.29 and 1.16 mg/kg of ClyJ-3 or ClyJ-3m in BALB/c mice revealed that ClyJ-3m shows a similar half-life but less clearance and a greater area under curve than ClyJ-3. Taken together, the choline-recognizing monomer ClyJ-3m exhibited enhanced bactericidal activity and improved pharmacokinetic proprieties compared to those of its parental ClyJ-3 lysin. Our study also provides a new way for rational design and programmed engineering of lysins targeting S. pneumoniae.

Characterization of a Type II-A CRISPR-Cas System in Streptococcus mutans.

Streptococcus mutans and its virulent phages are important members of the human oral microbiota. S. mutans is also the primary causal agent of dental caries. To survive in this ecological niche, S. mutans must encode phage defense mechanisms, which include CRISPR-Cas systems. Here, we describe the CRISPR-Cas type II-A system of S. mutans strain P42S, which was found to display natural adaptation and interference activity in response to phage infection and plasmid transformation. Newly acquired spacers were integrated both at the 5' end of the CRISPR locus and ectopically. In comparisons of the cas genes of P42S to those of other strains of S. mutans, cas1, cas2, and csn2 appear to be highly conserved within the species. However, more diversity was observed with cas9 While the nuclease domains of S. mutans Cas9 (SmCas9) are conserved, the C terminus of the protein, including the protospacer adjacent motif (PAM) recognition domain, is less conserved. In support of these findings, we experimentally demonstrated that the PAMs associated with SmCas9 of strain P42S are NAA and NGAA. These PAMs are different from those previously reported for the CRISPR-Cas system of the model strain S. mutans UA159. This study illustrates the diversity of CRISPR-Cas type II-A systems that can be found within the same bacterial species.IMPORTANCE CRISPR-Cas is one of the mechanisms used by bacteria to defend against viral predation. Increasing our knowledge of the biology and diversity of CRISPR-Cas systems will also improve our understanding of virus-bacterium interactions. As CRISPR-Cas systems acquiring novel immunities under laboratory conditions are rare, Streptococcus mutans strain P42S provides an alternative model to study the adaptation step, which is still the least understood step in CRISPR-Cas biology. Furthermore, the availability of a natural Cas9 protein recognizing an AT-rich PAM opens up new avenues for genome editing purposes.

Host range, morphological and genomic characterisation of bacteriophages with activity against clinical Streptococcus agalactiae isolates.

Streptococcus agalactiae or Group B Streptococcus (GBS) is a leading cause of sepsis in neonates. As a preventative measure prophylactic antibiotic administration is common in pregnant women colonised with GBS, but antibiotic-resistance and adverse effects on neonatal microbiomes may result. Use of bacteriophages (phages) is one option for targeted therapy. To this end, four phages (LF1 -LF4) were isolated from wastewater. They displayed lytic activity in vitro against S. agalactiae isolates collected from pregnant women and neonates, with 190/246 isolates (77.2%) and 10/10 (100%) isolates susceptible to at least one phage, respectively. Phage genomes ranged from 32,205-44,768 bp and all phages were members of the Siphoviridae family. High nucleotide identity (99.9%) was observed between LF1 and LF4, which were closely related to a putative prophage of S. agalactiae. The genome organisation of LF2 differed, and it showed similarity to a different S. agalactiae prophage, while LF3 was more closely related to a Streptococcus pyogenes phage. Lysogenic gene presence (integrase, repressor and regulatory modules), was suggestive of temperate phages. In a therapeutic context, temperate phages are not ideal candidates, however, the broad host range activity of these phages observed on clinical isolates in vitro is promising for future therapeutic approaches including bioengineered phage or lysin applications.