RESUMO
The isolation and identification of pathogenic bacteria from a variety of samples are critical for controlling bacterial infection-related health problems. The conventional methods, such as plate counting and polymerase chain reaction-based approaches, tend to be time-consuming and reliant on specific instruments, severely limiting the effective identification of these pathogens. In this study, we employed the specificity of the cell wall-binding (CBD) domain of the Staphylococcus aureus bacteriophage 80 alpha (80α) endolysin towards the host bacteria for isolation. Amidase 3-CBD conjugated magnetic beads successfully isolated as few as 1 × 102 CFU/mL of S. aureus cells from milk, blood, and saliva. The cell wall hydrolyzing activity of 80α endolysin promoted the genomic DNA extraction efficiency by 12.7 folds on average, compared to the commercial bacterial genomic DNA extraction kit. Then, recombinase polymerase amplification (RPA) was exploited to amplify the nuc gene of S. aureus from the extracted DNA at 37 °C for 30 min. The RPA product activated Cas12a endonuclease activity to cleave fluorescently labeled ssDNA probes. We then converted the generated signal into a fluorescent readout, detectable by either the naked eye or a portable, self-assembled instrument with ultrasensitivity. The entire procedure, from isolation to identification, can be completed within 2 h. The simplicity and sensitivity of the method developed in this study make it of great application value in S. aureus detection, especially in areas with limited resource supply.
Assuntos
Técnicas Biossensoriais , Endopeptidases , Staphylococcus aureus , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/virologia , Técnicas Biossensoriais/métodos , Endopeptidases/química , Endopeptidases/isolamento & purificação , Endopeptidases/genética , Bacteriófagos/química , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Humanos , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/química , Fagos de Staphylococcus/isolamento & purificação , Animais , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções Estafilocócicas/microbiologia , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Nuclease do Micrococo/química , Nuclease do Micrococo/metabolismo , Nuclease do Micrococo/genética , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
Human activities such as agriculturalization and domestication have led to the emergence of many new pathogens via host-switching events between humans, domesticated and wild animals. Staphylococcus aureus is a multi-host opportunistic pathogen with a global healthcare and economic burden. Recently, it was discovered that laboratory and wild rodents can be colonised and infected with S. aureus, but the origins and zoonotic potential of rodent S. aureus is unknown. In order to trace their evolutionary history, we employed a dataset of 1249 S. aureus genome sequences including 393 of isolates from rodents and other small mammals (including newly determined sequences for 305 isolates from 7 countries). Among laboratory mouse populations, we identified multiple widespread rodent-specific S. aureus clones that likely originated in humans. Phylogeographic analysis of the most common murine lineage CC88 suggests that it emerged in the 1980s in laboratory mouse facilities most likely in North America, from where it spread to institutions around the world, via the distribution of mice for research. In contrast, wild rodents (mice, voles, squirrels) were colonized with a unique complement of S. aureus lineages that are widely disseminated across Europe. In order to investigate the molecular basis for S. aureus adaptation to rodent hosts, genome-wide association analysis was carried out revealing a unique complement of bacteriophages associated with a rodent host ecology. Of note, we identified novel prophages and pathogenicity islands in rodent-derived S. aureus that conferred the potential for coagulation of rodent plasma, a key phenotype of abscess formation and persistence. Our findings highlight the remarkable capacity of S. aureus to expand into new host populations, driven by the acquisition of genes promoting survival in new host-species.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Animais , Staphylococcus aureus/genética , Staphylococcus aureus/virologia , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Camundongos , Roedores/microbiologia , Roedores/virologia , Bacteriófagos/genética , Humanos , Filogenia , Genoma Bacteriano , Fagos de Staphylococcus/genéticaRESUMO
OBJECTIVE: This study aimed to introduce a lytic bacteriophage against Staphylococcus saprophyticus from wastewater in Gorgan, northern Iran. RESULTS: The vB_SsapS-46 phage was isolated from urban wastewater and formed round and clear plaques on bacterial culture. It was visualized by electron microscopy and had a large head (approximately 106 nm) and a long tail (approximately 150 nm), indicating that it belongs to the Siphoviridae family. The host range of vB_SsapS-46 was determined using a spot test on 35 S. saprophyticus clinical isolates, and it was able to lyse 12 of the 35 clinical isolates (34%). Finally, the relationship between phage sensitivity and adherence genes was assessed, revealing no significant correlation between phage sensitivity and the frequency of adherence genes. The vB_SsapS-46 phage can be used alone or in a mixture in future studies to control urinary tract infections caused by this bacterium, especially in the elimination of drug-resistant pathogens.
Assuntos
Fagos de Staphylococcus , Staphylococcus saprophyticus , Staphylococcus saprophyticus/virologia , Staphylococcus saprophyticus/genética , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/isolamento & purificação , Fagos de Staphylococcus/ultraestrutura , Fagos de Staphylococcus/fisiologia , Siphoviridae/genética , Siphoviridae/isolamento & purificação , Siphoviridae/ultraestrutura , Irã (Geográfico) , Águas Residuárias/microbiologia , Águas Residuárias/virologia , Especificidade de Hospedeiro , Humanos , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologiaRESUMO
In nature, bacteria often survive in a stationary state with low metabolic activity. Phages use the metabolic machinery of the host cell to replicate, and, therefore, their efficacy against non-dividing cells is usually limited. Nevertheless, it was previously shown that the Staphylococcus epidermidis phage SEP1 has the remarkable capacity to actively replicate in stationary-phase cells, reducing their numbers. Here, we studied for the first time the transcriptomic profiles of both exponential and stationary cells infected with SEP1 phage using RNA-seq to gain a better understanding of this rare phenomenon. We showed that SEP1 successfully takes over the transcriptional apparatus of both exponential and stationary cells. Infection was, however, delayed in stationary cells, with genes within the gp142-gp154 module putatively implicated in host takeover. S. epidermidis responded to SEP1 infection by upregulating three genes involved in a DNA modification system, with this being observed already 5 min after infection in exponential cells and later in stationary cells. In stationary cells, a significant number of genes involved in translation and RNA metabolic and biosynthetic processes were upregulated after 15 and 30 min of SEP1 infection in comparison with the uninfected control, showing that SEP1 activates metabolic and biosynthetic pathways necessary to its successful replication.IMPORTANCEMost phage-host interaction studies are performed with exponentially growing cells. However, this cell state is not representative of what happens in natural environments. Additionally, most phages fail to replicate in stationary cells. The Staphylococcus epidermidis phage SEP1 is one of the few phages reported to date to be able to infect stationary cells. Here, we unveiled the interaction of SEP1 with its host in both exponential and stationary states of growth at the transcriptomic level. The findings of this study provide valuable insights for a better implementation of phage therapy since phages able to infect stationary cells could be more efficient in the treatment of recalcitrant infections.
Assuntos
Fagos de Staphylococcus , Staphylococcus epidermidis , Staphylococcus epidermidis/virologia , Staphylococcus epidermidis/metabolismo , Staphylococcus epidermidis/genética , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/metabolismo , Replicação Viral , Transcriptoma , Regulação Bacteriana da Expressão GênicaRESUMO
Nowadays finding the new antimicrobials is necessary due to the emerging of multidrug resistant strains. The present study aimed to isolate and characterize bacteriophages against S. aureus. Strains Huma and Simurgh were the two podovirus morphology phages which isolated and then characterized. Huma and Simurgh had a genome size of 16,853 and 17,245 bp, respectively and both were Rosenblumvirus with G + C content of 29%. No lysogeny-related genes, nor virulence genes were identified in their genomes. They were lytic only against two out of four S. aureus strains. They also were able to inhibit S. aureus for 8 h in-vitro. Both showed a rapid adsorption. Huma and Simurgh had the latent period of 80 and 60 m and the burst sizes of 45 and 40 PFU/ml and also, they showed very low cell toxicity of 1.23%-1.79% on HT-29 cells, respectively. Thus, they can be considered potential candidates for biocontrol applications.
Assuntos
Genoma Viral , Fagos de Staphylococcus , Staphylococcus aureus , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/fisiologia , Fagos de Staphylococcus/isolamento & purificação , Staphylococcus aureus/virologia , Staphylococcus aureus/genética , Humanos , Composição de Bases , Podoviridae/genética , Podoviridae/isolamento & purificação , Podoviridae/classificação , Podoviridae/fisiologia , Células HT29 , Tamanho do GenomaRESUMO
Non-aureus staphylococci (NAS) are an essential group of bacteria causing antimicrobial resistant intramammary infections in livestock, particularly dairy cows. Therefore, bacteriophages emerge as a potent bactericidal agent for NAS mastitis. This study aimed to obtain NAS-specific bacteriophages using bacterial strains isolated from cows with mastitis, subsequently evaluating their morphological, genomic, and lytic characteristics. Four distinct NAS bacteriophages were recovered from sewage or the environment of Chinese dairy farms; PT1-1, PT94, and PT1-9 were isolated using Staphylococcus chromogenes and PT1-4 using Staphylococcus gallinarum. Both PT1-1 (24/54, 44â¯%) and PT94 (28/54, 52â¯%) had broader lysis than PT1-4 (3/54, 6â¯%) and PT1-9 (10/54, 19â¯%), but PT1-4 and PT1-9 achieved cross-species lysis. All bacteriophages had a short latency period and good environmental tolerance, including surviving at pH=4-10 and at 30-60â. Except for PT1-9, all bacteriophages had excellent bactericidal efficacy within 5â¯h of co-culture with host bacteria in vitro at various multiplicity of infection (MOIs). Based on whole genome sequencing, average nucleotide identity (ANI) analysis of PT1-1 and PT94 can be classified as the same species, consistent with whole-genome synteny analysis. Although motifs shared by the 4 bacteriophages differed little from those of other bacteriophages, a phylogenetic tree based on functional proteins indicated their novelty. Moreover, based on whole genome comparisons, we inferred that cross-species lysis of bacteriophage may be related to the presence of "phage tail fiber." In conclusion 4 novel NAS bacteriophages were isolated; they had good biological properties and unique genomes, with potential for NAS mastitis therapy.
Assuntos
Genoma Viral , Mastite Bovina , Esgotos , Staphylococcus , Esgotos/virologia , Esgotos/microbiologia , Animais , Staphylococcus/virologia , Staphylococcus/efeitos dos fármacos , Staphylococcus/genética , Bovinos , Feminino , Mastite Bovina/microbiologia , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/fisiologia , Fagos de Staphylococcus/classificação , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Bacteriófagos/classificação , Bacteriófagos/fisiologia , Infecções Estafilocócicas/veterinária , Infecções Estafilocócicas/microbiologia , Filogenia , Genômica , Sequenciamento Completo do GenomaRESUMO
Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors that remodel host metabolism will provide valuable information for target development for antimicrobials. Here, we perform a comprehensive screen for early-gene products of ΦNM1 that inhibit cell growth in Staphylococcus aureus. A small membrane protein, Gp11, with inhibitory effects on S. aureus cell division was identified. A bacterial two-hybrid library containing 345 essential S. aureus genes was constructed to screen for targets of Gp11, and Gp11 was found to interact with MurG and DivIC. Defects in cell growth and division caused by Gp11 were dependent on MurG and DivIC, which was further confirmed using CRISPRi hypersensitivity assay. Gp11 interacts with MurG, the protein essential for cell wall formation, by inhibiting the production of lipid II to regulate peptidoglycan (PG) biosynthesis on the cell membrane. Gp11 also interacts with cell division protein DivIC, an essential part of the division machinery necessary for septal cell wall assembly, to disrupt the recruitment of division protein FtsW. Mutations in Gp11 result in loss of its ability to cause growth defects, whereas infection with phage in which the gp11 gene has been deleted showed a significant increase in lipid II production in S. aureus. Together, our findings reveal that a phage early-gene product interacts with essential host proteins to disrupt PG biosynthesis and block S. aureus cell division, suggesting a potential pathway for the development of therapeutic approaches to treat pathogenic bacterial infections. IMPORTANCE: Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant Staphylococcus aureus infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in S. aureus, and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in S. aureus. Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.
Assuntos
Divisão Celular , Peptidoglicano , Fagos de Staphylococcus , Staphylococcus aureus , Proteínas Virais , Staphylococcus aureus/virologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Peptidoglicano/metabolismo , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Parede Celular/virologia , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genéticaRESUMO
The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) emphasises the urgent need for novel antimicrobial agents as alternatives to antibiotics. Bacteriophage therapy is one of the most promising antimicrobial strategies. Here, we isolated and comprehensively characterized a novel Staphylococcus phage, vB_SauM_VL10 (VL10), from urban sewage. The VL10 genome displays 141,746 bp of linear double-stranded DNA, containing 193 open reading frames and lacking tRNA, virulence, or antibiotic resistance genes. Phylogenetic analysis categorizes VL10 as a novel species within the Silviavirus genus, Twortvirinae subfamily. VL10 exhibits lytic behaviour characterized by efficient adsorption, a short latent period, and substantial burst size, with environmental stability. It demonstrates lytic activity against 79.06% of tested S. aureus strains, highlighting its species specificity. Additionally, VL10 effectively targets MRSA biofilms, reducing biomass and viable cells. In MRSA-infected G. mellonella larvae, VL10 enhances survival rates, supporting its potential for phage therapy applications. Moreover, the emergence of VL10-resistant S. aureus strains associated with fitness trade-offs, including reduced growth, biofilm formation, and virulence. Altogether, these findings emphasize VL10 as a promising candidate for developing therapeutic agents against MRSA infections, providing insights into phage biology and resistance dynamics.
Assuntos
Biofilmes , Genoma Viral , Staphylococcus aureus Resistente à Meticilina , Filogenia , Fagos de Staphylococcus , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/virologia , Fagos de Staphylococcus/genética , Biofilmes/efeitos dos fármacos , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/terapia , Infecções Estafilocócicas/tratamento farmacológico , Terapia por Fagos , Esgotos/microbiologia , Esgotos/virologia , Animais , Humanos , Antibacterianos/farmacologiaRESUMO
Dormant prophages protect lysogenic cells by expressing diverse immune systems, which must avoid targeting their cognate prophages upon activation. Here we report that multiple Staphylococcus aureus prophages encode Tha (tail-activated, HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain-containing anti-phage system), a defence system activated by structural tail proteins of incoming phages. We demonstrate the function of two Tha systems, Tha-1 and Tha-2, activated by distinct tail proteins. Interestingly, Tha systems can also block reproduction of the induced tha-positive prophages. To prevent autoimmunity after prophage induction, these systems are inhibited by the product of a small overlapping antisense gene previously believed to encode an excisionase. This genetic organization, conserved in S. aureus prophages, allows Tha systems to protect prophages and their bacterial hosts against phage predation and to be turned off during prophage induction, balancing immunity and autoimmunity. Our results show that the fine regulation of these processes is essential for the correct development of prophages' life cycle.
Assuntos
Prófagos , Staphylococcus aureus , Prófagos/genética , Staphylococcus aureus/virologia , Staphylococcus aureus/imunologia , Autoimunidade , Lisogenia , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/imunologia , Fagos de Staphylococcus/fisiologia , Proteínas da Cauda Viral/genética , Proteínas da Cauda Viral/metabolismo , Bacteriófagos/genética , Bacteriófagos/imunologia , Bacteriófagos/fisiologiaRESUMO
The human skin microbiome comprises diverse populations that differ temporally between body sites and individuals. The virome is a less studied component of the skin microbiome and the study of bacteriophages is required to increase knowledge of the modulation and stability of bacterial communities. Staphylococcus species are among the most abundant colonisers of skin and are associated with both health and disease yet the bacteriophages infecting the most abundant species on skin are less well studied. Here, we report the isolation and genome sequencing of 40 bacteriophages from human skin swabs that infect coagulase-negative Staphylococcus (CoNS) species, which extends our knowledge of phage diversity. Six genetic clusters of phages were identified with two clusters representing novel phages, one of which we characterise and name Alsa phage. We identified that Alsa phages have a greater ability to infect the species S. hominis that was otherwise infected less than other CoNS species by the isolated phages, indicating an undescribed barrier to phage infection that could be in part due to numerous restriction-modification systems. The extended diversity of Staphylococcus phages here enables further research to define their contribution to skin microbiome research and the mechanisms that limit phage infection.
Assuntos
Bacteriófagos , Humanos , Bacteriófagos/genética , Coagulase/genética , Genoma Viral , Pele/microbiologia , Fagos de Staphylococcus/genética , Staphylococcus/genéticaRESUMO
Although bacteriophage-based biosensors are promising tools for rapid, convenient, and sensitive detection of Staphylococcus aureus in food products, the effect of biosensors using temperate phages as biorecognition elements to detect viable S. aureus isolates remains unclear. In this study, three temperate S. aureus phages were isolated and their biological features (one-step growth, host range, pH stability, temperature stability, and adsorption rate) were evaluated as the biological element. The selected phage SapYZUs8 was immobilized on the nanozyme Cu-MOF via electrostatic interactions to generate SapYZUs8@Cu-MOF, and its detection performance in real food (skim milk and pork) was then evaluated. Compared with phages SapYZUm7 and SapYZUs16, phage SapYZUs8 exhibited a broader host range, greater pH stability (3-12), and a better absorption rate (92 %, 8 min) suitable for S. aureus detection, which is likely the result of the DNA replication (DNA helicase) and phage tail protein genes in the SapYZUs8 genome. Therefore, phage SapYZUs8 was fixed on Cu-MOF to generate SapYZUs8@Cu-MOF, which exhibited good sensitivity and specificity for rapid colourimetric detection of viable S. aureus. The method took <0.5 h, and the detection limit was 1.09 × 102 CFU/mL. In addition, SapYZUs8@Cu-MOF was successfully employed for the colourimetric detection of S. aureus in food samples without interference from different food additives, NaCl concentrations, or pH values. With these benefits, it allows rapid visual assessment of S. aureus levels.
Assuntos
Bacteriófagos , Infecções Estafilocócicas , Humanos , Staphylococcus aureus , Colorimetria , Alimentos , Fagos de Staphylococcus/genéticaRESUMO
The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.
Assuntos
Infecções Estafilocócicas , Staphylococcus epidermidis , Humanos , Staphylococcus epidermidis/genética , Staphylococcus epidermidis/metabolismo , Staphylococcus aureus/genética , Coagulase/metabolismo , Glucose/metabolismo , Ácidos Teicoicos/metabolismo , Staphylococcus/metabolismo , Fagos de Staphylococcus/genética , DNA/metabolismo , Parede Celular/metabolismo , Infecções Estafilocócicas/metabolismoRESUMO
Staphylococcus epidermidis is a common member of the human skin and nose microbiomes and a frequent cause of invasive infections. Transducing phages accomplish the horizontal transfer of resistance and virulence genes by mispackaging of mobile-genetic elements, contributing to severe, therapy-refractory S. epidermidis infections. Lytic phages on the other hand can be interesting candidates for new anti-S. epidermidis phage therapies. Despite the importance of phages, we are only beginning to unravel S. epidermidis phage interactions. Recent studies shed new light on S. epidermidis phage diversity, host range, and receptor specificities. Modulation of cell wall teichoic acids, the major phage receptor structures, along with other phage defense mechanisms, are crucial determinants for S. epidermidis susceptibility to different phage groups.
Assuntos
Terapia por Fagos , Infecções Estafilocócicas , Humanos , Staphylococcus epidermidis/genética , Fagos de Staphylococcus/genética , Especificidade de Hospedeiro , Virulência , Infecções Estafilocócicas/terapiaRESUMO
Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific "helper" bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal itself, produced by overexpression, and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.
Assuntos
Ilhas Genômicas , Fagos de Staphylococcus , Staphylococcus aureus , Humanos , Proteínas do Capsídeo/química , Microscopia Crioeletrônica , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/virologia , Fagos de Staphylococcus/genética , Fatores de Virulência/genética , Transdução Genética , Empacotamento do DNA , Conformação de Ácido NucleicoRESUMO
Cyclic oligonucleotide-based antiphage signalling systems (CBASS) protect prokaryotes from viral (phage) attack through the production of cyclic oligonucleotides, which activate effector proteins that trigger the death of the infected host1,2. How bacterial cyclases recognize phage infection is not known. Here we show that staphylococcal phages produce a structured RNA transcribed from the terminase subunit genes, termed CBASS-activating bacteriophage RNA (cabRNA), which binds to a positively charged surface of the CdnE03 cyclase and promotes the synthesis of the cyclic dinucleotide cGAMP to activate the CBASS immune response. Phages that escape the CBASS defence harbour mutations that lead to the generation of a longer form of the cabRNA that cannot activate CdnE03. As the mammalian cyclase OAS1 also binds viral double-stranded RNA during the interferon response, our results reveal a conserved mechanism for the activation of innate antiviral defence pathways.
Assuntos
Bactérias , Nucleotidiltransferases , RNA Viral , Fagos de Staphylococcus , Animais , 2',5'-Oligoadenilato Sintetase/metabolismo , Bactérias/enzimologia , Bactérias/imunologia , Evolução Molecular , Imunidade Inata , Nucleotidiltransferases/metabolismo , Oligonucleotídeos/imunologia , Oligonucleotídeos/metabolismo , RNA Viral/imunologia , RNA Viral/metabolismo , Transdução de Sinais/imunologia , Fagos de Staphylococcus/genética , Fagos de Staphylococcus/imunologiaRESUMO
Staphylococcus aureus is one of the most relevant mastitis pathogens in dairy cattle, and the acquisition of antimicrobial resistance genes presents a significant health issue in both veterinary and human fields. Among the different strategies to tackle S. aureus infection in livestock, bacteriophages have been thoroughly investigated in the last decades; however, few specimens of the so-called jumbo phages capable of infecting S. aureus have been described. Herein, we report the biological, genomic, and structural proteomic features of the jumbo phage vB_SauM-UFV_DC4 (DC4). DC4 exhibited a remarkable killing activity against S. aureus isolated from the veterinary environment and stability at alkaline conditions (pH 4 to 12). The complete genome of DC4 is 263,185 bp (GC content: 25%), encodes 263 predicted CDSs (80% without an assigned function), 1 tRNA (Phe-tRNA), multisubunit RNA polymerase, and an RNA-dependent DNA polymerase. Moreover, comparative analysis revealed that DC4 can be considered a new viral species belonging to a new genus DC4 and showed a similar set of lytic proteins and depolymerase activity with closely related jumbo phages. The characterization of a new S. aureus jumbo phage increases our understanding of the diversity of this group and provides insights into the biotechnological potential of these viruses. KEY POINTS: ⢠vB_SauM-UFV_DC4 is a new viral species belonging to a new genus within the class Caudoviricetes. ⢠vB_SauM-UFV_DC4 carries a set of RNA polymerase subunits and an RNA-directed DNA polymerase. ⢠vB_SauM-UFV_DC4 and closely related jumbo phages showed a similar set of lytic proteins.
Assuntos
Bacteriófagos , Fagos de Staphylococcus , Animais , Bovinos , Feminino , Humanos , Fagos de Staphylococcus/genética , Staphylococcus aureus/genética , Proteômica , Genoma Viral , Genômica , Bacteriófagos/genética , RNA Polimerases Dirigidas por DNA/genética , RNA de TransferênciaRESUMO
The issue of antibiotic resistance in healthcare worldwide has led to a pressing need to explore and develop alternative approaches to combat infectious diseases. Among these methods, phage therapy has emerged as a potential solution to tackle this growing challenge. Virulent phages of the Herelleviridae family, known for their ability to cause lysis of Staphylococcus aureus, a clinically significant pathogen frequently associated with multidrug resistance, have proven to be one of the most effective viruses utilized in phage therapy. In order to utilize phages for therapeutic purposes effectively, a thorough investigation into their physiology and mechanisms of action on infected cells is essential. The use of omics technologies, particularly total RNA sequencing, is a promising approach for analyzing the interaction between phages and their hosts, allowing for the assessment of both the behavior of the phage during infection and the cell's response. This review aims to provide a comprehensive overview of the physiology of the Herelleviridae family, utilizing existing analyses of their total phage transcriptomes. Additionally, it sheds light on the changes that occur in the metabolism of S. aureus when infected with virulent bacteriophages, contributing to a deeper understanding of the phage-host interaction.
Assuntos
Bacteriófagos , Caudovirales , Terapia por Fagos , Infecções Estafilocócicas , Humanos , Staphylococcus aureus/genética , Bacteriófagos/genética , Fagos de Staphylococcus/genética , Infecções Estafilocócicas/terapiaRESUMO
S. epidermidis is an important opportunistic pathogen causing chronic prosthetic joint infections associated with biofilm growth. Increased tolerance to antibiotic therapy often requires prolonged treatment or revision surgery. Phage therapy is currently used as compassionate use therapy and continues to be evaluated for its viability as adjunctive therapy to antibiotic treatment or as an alternative treatment for infections caused by S. epidermidis to prevent relapses. In the present study, we report the isolation and in vitro characterization of three novel lytic S. epidermidis phages. Their genome content analysis indicated the absence of antibiotic resistance genes and virulence factors. Detailed investigation of the phage preparation indicated the absence of any prophage-related contamination and demonstrated the importance of selecting appropriate hosts for phage development from the outset. The isolated phages infect a high proportion of clinically relevant S. epidermidis strains and several other coagulase-negative species growing both in planktonic culture and as a biofilm. Clinical strains differing in their biofilm phenotype and antibiotic resistance profile were selected to further identify possible mechanisms behind increased tolerance to isolated phages.
Assuntos
Bacteriófagos , Terapia por Fagos , Infecções Estafilocócicas , Humanos , Bacteriófagos/genética , Staphylococcus epidermidis , Antibacterianos/farmacologia , Biofilmes , Fagos de Staphylococcus/genéticaRESUMO
The study of bacteriophages is experiencing a resurgence with the increasing development of antimicrobial resistance in Staphylococcus aureus. Nonetheless, the genetic features of highly efficient lytic S. aureus phage remain to be explored. In this study, two lytic S. aureus phages, SapYZU11 and SapYZU15, were isolated from sewage samples from Yangzhou, China. The phage morphology, one-step growth, host spectrum and lytic activity of these phages were examined, and their whole-genome sequences were analysed and compared with 280 published genomes of staphylococcal phages. The structural organisation and genetic contents of SapYZU11 and SapYZU15 were investigated. The Podoviridae phage SapYZU11 and Herelleviridae phage SapYZU15 effectively lysed all of the 53 S. aureus strains isolated from various sources. However, SapYZU15 exhibited a shorter latent period, larger burst size and stronger bactericidal ability with an anti-bacterial rate of approximately 99.9999% for 24 h. Phylogenetic analysis revealed that Herelleviridae phages formed the most ancestral clades and the S. aureus Podoviridae phages were clustered in the staphylococcal Siphoviridae phage clade. Moreover, phages in different morphology families contain distinct types of genes associated with host cell lysis, DNA packaging and lysogeny. Notably, SapYZU15 harboured 13 DNA metabolism-related genes, 5 lysin genes, 1 holin gene and 1 DNA packaging gene. The data suggest that S. aureus Podoviridae and Siphoviridae phages originated from staphylococcal Herelleviridae phages, and the module exchange of S. aureus phages occurred in the same morphology family. Moreover, the extraordinary lytic capacity of SapYZU15 was likely due to the presence of speciï¬c genes associated with DNA replication, DNA packaging and the lytic cycle.
Assuntos
Bacteriófagos , Siphoviridae , Infecções Estafilocócicas , Humanos , Staphylococcus aureus/genética , Esgotos , Filogenia , Infecções Estafilocócicas/microbiologia , Fagos de Staphylococcus/genéticaRESUMO
Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.