RESUMO
Streptococcus dysgalactiae infection can cause bovine mastitis and lead to huge economic losses for the dairy industry. The abuse of antibiotics has resulted in growing drug resistance of S. dysgalactiae, which causes hard-to-treat infections. Bacteriophage lysin, as a novel antibacterial agent, has great potential for application against drug-resistant gram-positive bacteria. However, few studies have been conducted on the prophage lysin of S. dysgalactiae. In this study, we mined a novel prophage lysin, named Lys1644, from a clinical S. dysgalactiae isolate by genome sequencing and bioinformatic analysis. Lys1644 was expressed and purified, and the lytic activity, antibacterial spectrum, optimal pH and temperature, lytic activity in milk in vitro, and synergistic bacteriostasis with antibiotics were assessed. The Lys1644 prophage lysin showed high bacteriolysis activity specifically on S. dysgalactiae, which resulted in CFU 100-fold reduction in milk. Moreover, Lys1644 maintained high activity over a wide pH range (pH 5-10) and a wide temperature range (4-42 °C). Synergistic bacteriostatic experiments showed that the combination of low-dose Lys1644 (50 µg/mL) with a subinhibitory concentration of aminoglycoside antibiotics (kanamycin or spectinomycin) can completely inhibit bacterial growth, suggesting that the combination of Lys1644 and antibiotics could be an effective therapeutic strategy against S. dysgalactiae infection.
Assuntos
Antibacterianos , Prófagos , Streptococcus , Streptococcus/efeitos dos fármacos , Prófagos/genética , Antibacterianos/farmacologia , Antibacterianos/química , Animais , Leite/microbiologia , Fagos de Streptococcus/genética , Bovinos , Mastite Bovina/microbiologia , Mastite Bovina/tratamento farmacológico , Concentração de Íons de Hidrogênio , Bacteriólise/efeitos dos fármacos , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/tratamento farmacológicoRESUMO
The bacteriophage protein paratox (Prx) blocks quorum sensing in its streptococcal host by directly binding the signal receptor and transcription factor ComR. This reduces the ability of Streptococcus to uptake environmental DNA and protects phage DNA from damage by recombination. Past work characterizing the Prx:ComR molecular interaction revealed that paratox adopts a well-ordered globular fold when bound to ComR. However, solution-state biophysical measurements suggested that Prx may be conformationally dynamic. To address this discrepancy, we investigated the stability and dynamic properties of Prx in solution using circular dichroism, nuclear magnetic resonance, and several fluorescence-based protein folding assays. Our work shows that under dilute buffer conditions Prx is intrinsically disordered. We also show that the addition of kosmotropic salts or protein stabilizing osmolytes induces Prx folding. However, the solute stabilized fold is different from the conformation Prx adopts when it is bound to ComR. Furthermore, we have characterized Prx folding thermodynamics and folding kinetics through steady-state fluorescence and stopped flow kinetic measurements. Our results show that Prx is a highly dynamic protein in dilute solution, folding and refolding within the 10 ms timescale. Overall, our results demonstrate that the streptococcal phage protein Prx is an intrinsically disordered protein in a two-state equilibrium with a solute-stabilized folded form. Furthermore, the solute-stabilized fold is likely the predominant form of Prx in a solute-crowded bacterial cell. Finally, our work suggests that Prx binds and inhibits ComR, and thus quorum sensing in Streptococcus, by a combination of conformational selection and induced-fit binding mechanisms.
Assuntos
Proteínas Intrinsicamente Desordenadas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Dobramento de Proteína , Proteínas Virais/química , Proteínas Virais/metabolismo , Proteínas Virais/genética , Fagos de Streptococcus/química , Fagos de Streptococcus/metabolismo , Fagos de Streptococcus/genética , Streptococcus/virologia , Streptococcus/química , Streptococcus/metabolismoRESUMO
CRISPR-Cas systems serve as adaptive immune systems in bacteria and archaea, protecting against phages and other mobile genetic elements. However, phages and archaeal viruses have developed countermeasures, employing anti-CRISPR (Acr) proteins to counteract CRISPR-Cas systems. Despite the revolutionary impact of CRISPR-Cas systems on genome editing, concerns persist regarding potential off-target effects. Therefore, understanding the structural and molecular intricacies of diverse Acrs is crucial for elucidating the fundamental mechanisms governing CRISPR-Cas regulation. In this study, we present the structure of AcrIIA28 from Streptococcus phage Javan 128 and analyze its structural and functional features to comprehend the mechanisms involved in its inhibition of Cas9. Our current study reveals that AcrIIA28 is a metalloprotein that contains Zn2+ and abolishes the cleavage activity of Cas9 only from Streptococcus pyrogen (SpyCas9) by directly interacting with the REC3 domain of SpyCas9. Furthermore, we demonstrate that the AcrIIA28 interaction prevents the target DNA from being loaded onto Cas9. These findings indicate the molecular mechanisms underlying AcrIIA28-mediated Cas9 inhibition and provide valuable insights into the ongoing evolutionary battle between bacteria and phages.
Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Fagos de Streptococcus , Streptococcus , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/química , DNA/metabolismo , DNA/genética , Edição de Genes , Metaloproteínas/metabolismo , Metaloproteínas/genética , Metaloproteínas/química , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Streptococcus/genética , Streptococcus/virologia , Fagos de Streptococcus/genética , Fagos de Streptococcus/metabolismo , Proteínas Virais/metabolismo , Proteínas Virais/genética , Proteínas Virais/química , Zinco/metabolismoRESUMO
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.
Assuntos
Bacteriófagos , Fagos de Streptococcus , Humanos , Bacteriófagos/genética , Bacteriófagos/metabolismo , Streptococcus thermophilus/genética , Streptococcus thermophilus/metabolismo , Fagos de Streptococcus/genética , Sistemas CRISPR-Cas/genética , Edição de GenesRESUMO
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.
Assuntos
Bacteriófagos , Fagos de Streptococcus , Bacteriófagos/genética , Streptococcus thermophilus/genética , Adsorção , Mutação , Peptídeo Hidrolases/genética , Sistemas CRISPR-Cas , Fagos de Streptococcus/genéticaRESUMO
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.
Assuntos
Bacteriófagos , Siphoviridae , Fagos de Streptococcus , Bacteriófagos/genética , Polissacarídeos , Fagos de Streptococcus/genética , Streptococcus thermophilus/genéticaRESUMO
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.
Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Prófagos/fisiologia , Fagos de Streptococcus/fisiologia , Streptococcus pyogenes/imunologia , Streptococcus pyogenes/virologia , Lisogenia , Plasmídeos/genética , Plasmídeos/metabolismo , Prófagos/genética , Fagos de Streptococcus/genética , Streptococcus pyogenes/genética , Integração ViralRESUMO
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.
Assuntos
Genoma Viral , Filogenia , Fagos de Streptococcus/genética , Fagos de Streptococcus/patogenicidade , Streptococcus gordonii/virologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Terapia por Fagos , Fagos de Streptococcus/classificação , Virulência , Sequenciamento Completo do GenomaRESUMO
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.
Assuntos
Fibrinogênio/metabolismo , Fibrinólise , Plasminogênio/metabolismo , Fagos de Streptococcus/fisiologia , Streptococcus/metabolismo , Sítios de Ligação , Endocardite/microbiologia , Fibrina/química , Fibrina/metabolismo , Humanos , Ligação Proteica , Streptococcus/genética , Streptococcus/patogenicidade , Streptococcus/virologia , Fagos de Streptococcus/genética , VirulênciaRESUMO
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.
Assuntos
Cárie Dentária/terapia , Terapia por Fagos , Fagos de Streptococcus/isolamento & purificação , Streptococcus mutans/virologia , Cárie Dentária/microbiologia , Cárie Dentária/virologia , Genoma Viral , Humanos , Saliva/virologia , Fagos de Streptococcus/classificação , Fagos de Streptococcus/genética , Fagos de Streptococcus/fisiologia , Streptococcus mutans/fisiologiaRESUMO
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.
Assuntos
Viabilidade Microbiana , Fagos de Streptococcus/genética , Fagos de Streptococcus/patogenicidade , Streptococcus pyogenes/fisiologia , Streptococcus pyogenes/virologia , Sistemas CRISPR-Cas , Genoma Viral , Lisogenia , Prófagos/genética , VirulênciaRESUMO
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.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Edição de Genes , Streptococcus mutans/genética , Plasmídeos/genética , Fagos de Streptococcus/genética , Fagos de Streptococcus/metabolismo , Streptococcus mutans/virologiaRESUMO
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.
Assuntos
Sepse Neonatal/terapia , Terapia por Fagos , Siphoviridae/genética , Fagos de Streptococcus/genética , Streptococcus agalactiae/virologia , DNA Viral/isolamento & purificação , Feminino , Genômica , Especificidade de Hospedeiro/genética , Humanos , Recém-Nascido , Lisogenia , Sepse Neonatal/microbiologia , Filogenia , Gravidez , Siphoviridae/isolamento & purificação , Fagos de Streptococcus/isolamento & purificação , Streptococcus agalactiae/isolamento & purificação , Streptococcus pyogenes/virologiaRESUMO
Streptococcus anginosus is an often overlooked and understudied emerging pathogen inhabiting many areas of the human body. Through our sequencing of S. anginosus strains isolated from the female bladder microbiota, we detected numerous prophage sequences. Bioinformatic analysis of these sequences identified 17 distinct groups of S. anginosus prophages. The majority of these phages exhibit no sequence homology to previously characterized temperate or virulent phage sequences, indicating an unexplored diversity of Streptococcus phages. By culturing these bacterial isolates, we confirmed that the prophages of five of these groups are capable of induction. One of these putative phages was imaged, the first such evidence of an S. anginosus virus-like particle; it exhibits morphological characteristics of siphoviruses.
Assuntos
Fagos de Streptococcus/genética , Streptococcus anginosus/genética , Streptococcus anginosus/virologia , Infecções Urinárias/microbiologia , Infecções Urinárias/virologia , Sistema Urinário/microbiologia , Sistema Urinário/virologia , Biologia Computacional/métodos , Feminino , Genoma Viral/genética , Humanos , Microbiota/genética , Filogenia , Prófagos/genéticaRESUMO
Streptococcus thermophilus is a lactic acid bacterium commonly used for the manufacture of yogurt and specialty cheeses. Virulent phages represent a major risk for milk fermentation processes worldwide, as they can inactivate the added starter bacterial cells, leading to low-quality fermented dairy products. To date, four genetically distinct groups of phages infecting S. thermophilus have been described. Here, we describe a fifth group. Phages P738 and D4446 are virulent siphophages that infect a few industrial strains of S. thermophilus The genomes of phages P738 and D4446 were sequenced and found to contain 34,037 and 33,656 bp as well as 48 and 46 open reading frames, respectively. Comparative genomic analyses revealed that the two phages are closely related to each other but display very limited similarities to other S. thermophilus phages. In fact, these two novel S. thermophilus phages share similarities with streptococcal phages of nondairy origin, suggesting that they emerged recently in the dairy environment.IMPORTANCE Despite decades of research and adapted antiphage strategies such as CRISPR-Cas systems, virulent phages are still a persistent risk for the milk fermentation industry worldwide, as they can cause manufacturing failures and alter product quality. Phages P738 and D4446 are novel virulent phages that infect the food-grade Gram-positive bacterial species Streptococcus thermophilus These two related viruses represent a fifth group of S. thermophilus phages, as they are significantly distinct from other known S. thermophilus phages. Both phages share similarities with phages infecting nondairy streptococci, suggesting their recent emergence and probable coexistence in dairy environments. These findings highlight the necessity of phage surveillance programs as the phage population evolves in response to the application of antiphage strategies.
Assuntos
Siphoviridae/classificação , Fagos de Streptococcus/classificação , Streptococcus thermophilus/virologia , Microscopia Eletrônica de Transmissão , Análise de Sequência de DNA , Siphoviridae/genética , Siphoviridae/ultraestrutura , Fagos de Streptococcus/genética , Fagos de Streptococcus/ultraestruturaRESUMO
Streptococcus thermophilus is a lactic acid bacterium widely used by the dairy industry for the manufacture of yogurt and specialty cheeses. It is also a Gram-positive bacterial model to study phage-host interactions. CRISPR-Cas systems are one of the most prevalent phage resistance mechanisms in S. thermophilus. Little information is available about other host factors involved in phage replication in this food-grade streptococcal species. We used the model strain S. thermophilus SMQ-301 and its virulent phage DT1, harboring the anti-CRISPR protein AcrIIA6, to show that a host gene coding for a methionine aminopeptidase (metAP) is necessary for phage DT1 to complete its lytic cycle. A single mutation in metAP provides S. thermophilus SMQ-301 with strong resistance against phage DT1. The mutation impedes a late step of the lytic cycle since phage adsorption, DNA replication, and protein expression were not affected. When the mutated strain was complemented with the wild-type version of the gene, the phage sensitivity phenotype was restored. When this mutation was introduced into other S. thermophilus strains it provided resistance against cos-type (Sfi21dt1virus genus) phages but replication of pac-type (Sfi11virus genus) phages was not affected. The mutation in the gene coding for the MetAP induces amino acid change in a catalytic domain conserved across many bacterial species. Introducing the same mutation in Streptococcus mutans also provided a phage resistance phenotype, suggesting the wide-ranging importance of the host methionine aminopeptidase in phage replication.
Assuntos
Aminopeptidases/genética , Mutação , Fagos de Streptococcus/fisiologia , Streptococcus thermophilus/virologia , Aminopeptidases/química , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Domínio Catalítico , Microbiologia de Alimentos , Fagos de Streptococcus/genética , Streptococcus thermophilus/enzimologia , Streptococcus thermophilus/genética , Replicação Viral , Sequenciamento Completo do GenomaRESUMO
Streptococci are one of the most important and common constituents of the host's microbiota and can colonize and live in the upper respiratory and urogenital tract of humans and animals. The CRISPR-Cas systems (i.e., clustered regularly interspaced short palindromic repeat, with CRISPR-associated proteins) found in bacteria and archaea provide sequence-based adaptive immunity against mobile genetic elements, especially in the streptococci. Here, recent research progress on CRISPR-Cas systems in the streptococci is reviewed, including their classification (mainly type I, type II, and type III), physiological function, defense mechanism (CRISPR adaptation, crRNA biogenesis, and target interference) and applications, which are useful for a better understanding of the functions of such systems. Finally, the advances that have been made in streptococci may help in the discovery of further novel CRISPR-Cas systems for use in new technologies and applications in other species.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Regulação Bacteriana da Expressão Gênica , RNA Guia de Cinetoplastídeos/genética , Fagos de Streptococcus/genética , Streptococcus/genética , Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Conjugação Genética , Edição de Genes/métodos , Transferência Genética Horizontal , Terapia Genética/métodos , Genoma Bacteriano , Humanos , Sequências Repetitivas Dispersas , Isoenzimas/genética , Isoenzimas/metabolismo , RNA Circular/genética , RNA Circular/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus/imunologia , Streptococcus/virologia , Fagos de Streptococcus/metabolismoRESUMO
Streptococcus pyogenes (Group A Streptococcus, GAS) is a strictly human bacterial pathogen. Since the mid-1980s, GAS M1T1 clone has been the most prevalent and globally disseminated serotype and is the culprit causing invasive and severe streptococcal infections, urging a better understanding of the emergence of hypervirulent M1T1 clone from an evolutionary perspective. This review highlights the molecular and evolutionary events leading to pandemic M1T1 strains, and discusses the pressure driving the genetic acquisition of novel virulence genes and the selection of hypervirulent isolates in host. By understanding the evolutionary selection and pressures that select and shape the pandemic M1T1 clone, we could potentially develop new therapeutic strategies to tackle challenges when dealing with the globally disseminated M1T1 GAS clone.
Assuntos
Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Pandemias , Infecções Estreptocócicas/epidemiologia , Streptococcus pyogenes/genética , Streptococcus pyogenes/patogenicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Clonais , Citotoxinas/genética , Citotoxinas/metabolismo , Desoxirribonuclease I/genética , Desoxirribonuclease I/metabolismo , Evolução Molecular , Exotoxinas/genética , Exotoxinas/metabolismo , Interações Hospedeiro-Patógeno/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Recombinação Genética , Seleção Genética , Infecções Estreptocócicas/microbiologia , Infecções Estreptocócicas/patologia , Fagos de Streptococcus/genética , Fagos de Streptococcus/metabolismo , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/virologia , Transdução Genética , VirulênciaRESUMO
Comparative genomics has proven useful in exploring the biodiversity of phages and understanding phage-host interactions. This knowledge is particularly useful for phages infecting Streptococcus thermophilus, as they constitute a constant threat during dairy fermentations. Here, we explore the genetic diversity of S. thermophilus phages to identify genetic determinants with a signature for host specificity, which could be linked to the bacterial receptor genotype. A comparative genomic analysis was performed on 142 S. thermophilus phage genomes, 55 of which were sequenced in this study. Effectively, 94 phages were assigned to the group cos (DT1), 36 to the group pac (O1205), six to the group 5093, and six to the group 987. The core genome-based phylogeny of phages from the two dominating groups and their receptor binding protein (RBP) phylogeny corresponded to the phage host-range. A role of RBP in host recognition was confirmed by constructing a fluorescent derivative of the RBP of phage CHPC951, followed by studying the binding of the protein to the host strain. Furthermore, the RBP phylogeny of the cos group was found to correlate with the host genotype of the exocellular polysaccharide-encoding operon. These findings provide novel insights towards developing strategies to combat phage infections in dairies.
Assuntos
Bacteriófagos/genética , Genoma Viral/genética , Especificidade de Hospedeiro/genética , Streptococcus thermophilus/genética , Genômica , Filogenia , Fagos de Streptococcus/genética , Streptococcus thermophilus/virologiaRESUMO
Streptococcus thermophilus strains are among the most widely employed starter cultures in dairy fermentations, second only to those of Lactococcus lactis. The extensive application of this species provides considerable opportunity for the proliferation of its infecting (bacterio)phages. Until recently, dairy streptococcal phages were classified into two groups (cos and pac groups), while more recently, two additional groups have been identified (5093 and 987 groups). This highlights the requirement for consistent monitoring of phage populations in the industry. Here, we report a survey of 35 samples of whey derived from 27 dairy fermentation facilities in ten countries against a panel of S. thermophilus strains. This culminated in the identification of 172 plaque isolates, which were characterized by multiplex PCR, restriction fragment length polymorphism analysis, and host range profiling. Based on this characterisation, 39 distinct isolates representing all four phage groups were selected for genome sequencing. Genetic diversity was observed among the cos isolates and correlations between receptor binding protein phylogeny and host range were also clear within this phage group. The 987 phages isolated within this study shared high levels of sequence similarity, yet displayed reduced levels of similarity to those identified in previous studies, indicating that they are subject to ongoing genetic diversification.