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1.
Microbiol Spectr ; 10(1): e0229521, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35107319

RESUMO

Bacteriophages represent a promising option for the treatment of Clostridioides difficile (formerly Clostridium difficile) infection (CDI), which at present relies on conventional antibiotic therapy. The specificity of bacteriophages should prevent dysbiosis of the colonic microbiota associated with antibiotic treatment of CDI. While numerous phages have been isolated, none have been characterized with broad host range activity toward PCR ribotype (RT) 078 strains, despite their relevance to medicine and agriculture. In this study, we isolated four novel C. difficile myoviruses: ΦCD08011, ΦCD418, ΦCD1801, and ΦCD2301. Their characterization revealed that each was comparable with other C. difficile phages described in the literature, with the exception of ΦCD1801, which exhibited broad host range activity toward RT 078, infecting 15/16 (93.8%) of the isolates tested. In order for wild-type phages to be exploited in the effective treatment of CDI, an optimal phage cocktail must be assembled that provides broad coverage against all C. difficile RTs. We conducted experiments to support previous findings suggesting that SlpA, a constituent of the C. difficile surface layer (S-layer) is the likely phage receptor. Through interpretation of phage-binding assays, our data suggested that ΦCD1801 could bind to an RT 012 strain only in the presence of a plasmid-borne S-layer cassette corresponding to the slpA allele found in RT 078. Armed with this information, efforts should be directed toward the isolation of phages with broad host range activity toward defined S-layer cassette types, which could form the basis of an effective phage cocktail for the treatment of CDI. IMPORTANCE Research into phage therapy has seen a resurgence in recent years owing to growing concerns regarding antimicrobial resistance. Phage research for potential therapy against Clostridioides difficile infection (CDI) is in its infancy, where an optimal "one size fits all" phage cocktail is yet to be derived. The pursuit thus far has aimed to find phages with the broadest possible host range. However, for C. difficile strains belonging to certain PCR ribotypes (RTs), in particular RT 078, phages with broad host range activity are yet to be discovered. In this study, we isolate four novel myoviruses, including ΦCD1801, which exerts the broadest host range activity toward RT 078 reported in the literature. Through the application of ΦCD1801 to phage-binding assays, we provide data to support the prior notion that SlpA represents the likely phage receptor on the bacterial cell surface. Our finding directs research attention toward the isolation of phages with activity toward strains possessing defined S-layer cassette types.


Assuntos
Proteínas de Bactérias/metabolismo , Receptores de Bacteriófagos/metabolismo , Bacteriófagos/fisiologia , Clostridioides difficile/metabolismo , Clostridioides difficile/virologia , Especificidade de Hospedeiro , Proteínas de Bactérias/genética , Receptores de Bacteriófagos/genética , Bacteriófagos/classificação , Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Clostridioides difficile/genética , Infecções por Clostridium/microbiologia , Infecções por Clostridium/terapia , Humanos , Terapia por Fagos , Filogenia , Ribotipagem
2.
ACS Infect Dis ; 7(11): 3077-3087, 2021 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-34618422

RESUMO

Klebsiella pneumoniae is a Gram-negative bacterium that has become one of the leading causes of life-threatening healthcare-associated infections (HAIs), including pneumonia and sepsis. Moreover, due to its increasingly antibiotic resistance, K. pneumoniae has been declared a global top priority concern. The problem of K. pneumoniae infections is due, in part, to the inability to detect this pathogen rapidly and accurately and thus to treat patients within the early stages of infections. The success in bacterial detection is greatly dictated by the biorecognition molecule used, with the current diagnostic tools relying on expensive probes often lacking specificity and/or sensitivity. (Bacterio)phage receptor-binding proteins (RBPs) are responsible for the recognition and adsorption of phages to specific bacterial host receptors and thus present high potential as biorecognition molecules. In this study, we report the identification and characterization of a novel RBP from the K. pneumoniae phage KpnM6E1 that presents high specificity against the target bacteria and high sensitivity (80%) to recognize K. pneumoniae strains. Moreover, adsorption studies validated the role of gp86 in the attachment to bacterial receptors, as it highly inhibits (86%) phage adsorption to its Klebsiella host. Overall, in this study, we unravel the role and potential of a novel Klebsiella phage RBP as a powerful tool to be used coupled with analytical techniques or biosensing platforms for the diagnosis of K. pneumoniae infections.


Assuntos
Receptores de Bacteriófagos , Infecções por Klebsiella , Proteínas de Transporte , Humanos , Klebsiella , Klebsiella pneumoniae
3.
Int J Biol Sci ; 17(13): 3573-3582, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34512166

RESUMO

Emergence of antibiotic resistance presents a major setback to global health, and shortage of antibiotic pipelines has created an urgent need for development of alternative therapeutic strategies. Bacteriophage (phage) therapy is considered as a potential approach for treatment of the increasing number of antibiotic-resistant pathogens. Phage-antibiotic synergy (PAS) refers to sublethal concentrations of certain antibiotics that enhance release of progeny phages from bacterial cells. A combination of phages and antibiotics is a promising strategy to reduce the dose of antibiotics and the development of antibiotic resistance during treatment. In this review, we highlight the state-of-the-art advancements of PAS studies, including the analysis of bacterial-killing enhancement, bacterial resistance reduction, and anti-biofilm effect, at both in vitro and in vivo levels. A comprehensive review of the genetic and molecular mechanisms of phage antibiotic synergy is provided, and synthetic biology approaches used to engineer phages, and design novel therapies and diagnostic tools are discussed. In addition, the role of engineered phages in reducing pathogenicity of bacteria is explored.


Assuntos
Antibacterianos/uso terapêutico , Terapia por Fagos , Animais , Receptores de Bacteriófagos/genética , Terapia Combinada , Farmacorresistência Bacteriana , Humanos
4.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2614-2622, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472282

RESUMO

Bacteriophages bind to the bacteria receptor through the receptor binding proteins (RBPs), a process that requires the involvement of complex atomic structures and conformational changes. In response to bacteriophage infection, bacteria have developed a variety of resistance mechanisms, while bacteriophages have also evolved multiple antagonistic mechanisms to escape host resistance. The exploration of the "adsorption-anti adsorption-escape process" between bacteriophages and bacteria helps us better understand the co-evolution process of bacteriophages and bacteria, which is important for the development of phage therapeutic technologies and phage-based biotechnologies. This review summarizes the bacteriophage adsorption related proteins, how bacteriophages escape host resistance based on the RBP alternations, and the recent progress of RBP-related biotechnologies.


Assuntos
Receptores de Bacteriófagos , Bacteriófagos , Bactérias , Bacteriófagos/genética , Proteínas de Transporte , Ligação Proteica
5.
Viruses ; 13(8)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34452328

RESUMO

Bacteriophage receptor binding proteins (RBPs) are employed by viruses to recognize specific surface structures on bacterial host cells. Recombinant RBPs have been utilized for detection of several pathogens, typically as fusions with reporter enzymes or fluorescent proteins. Identification of Bacillus anthracis, the etiological agent of anthrax, can be difficult because of the bacterium's close relationship with other species of the Bacillus cereussensu lato group. Here, we facilitated the identification of B. anthracis using two implementations of enzyme-linked phage receptor binding protein assays (ELPRA). We developed a single-tube centrifugation assay simplifying the rapid analysis of suspect colonies. A second assay enables identification of suspect colonies from mixed overgrown solid (agar) media derived from the complex matrix soil. Thus, these tests identified vegetative cells of B. anthracis with little processing time and may support or confirm pathogen detection by molecular methods such as polymerase chain reaction.


Assuntos
Antraz/microbiologia , Bacillus anthracis/isolamento & purificação , Proteínas de Bactérias/química , Técnicas Bacteriológicas/métodos , Receptores de Bacteriófagos/química , Medições Luminescentes/métodos , Fagos Bacilares/genética , Fagos Bacilares/fisiologia , Bacillus anthracis/genética , Bacillus anthracis/metabolismo , Bacillus anthracis/virologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas/instrumentação , Receptores de Bacteriófagos/genética , Receptores de Bacteriófagos/metabolismo , Genes Reporter , Humanos , Luciferases/química , Luciferases/genética , Luciferases/metabolismo , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microbiologia do Solo
6.
Viruses ; 13(8)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34452334

RESUMO

Escherichia coli O157:H7 is a global concern that causes serious diseases, such as hemolytic uremic syndrome and bloody diarrhea. To control E. coli O157:H7 in food, a novel siphophage, BECP10, that targets the O157 serotype was isolated and characterized. Unlike other E. coli phages, BECP10 can only infect E. coli O157 strains, and thus, did not infect other strains. The 48 kbp genome of BECP10 contained 76 open reading frames (ORFs), including 33 putative functional ORFs. The phage did not contain lysogeny-related modules or toxin-associated genes, suggesting that the phage might be strictly lytic. The tail spike protein (TSP) sequence had very low homology with the reported T1-like phages, indicating that TSP might be related to this unique host spectrum. The specific O-antigen residue of E. coli O157:H7 may be a key factor for phage infection by adsorption and receptor identification. The phage exhibited strong antibacterial activity against E. coli O157:H7 over a broad pH range and showed little development of phage-insensitive mutants. The phage sustained viability on the burger patties and reduced E. coli O157:H7 to a non-detectable level without the emergence of resistant cells at low temperatures for five days. Therefore, phage BECP10 might be a good biocontrol agent for E. coli O157:H7-contaminated food matrices.


Assuntos
Receptores de Bacteriófagos/metabolismo , Bacteriófagos/fisiologia , Escherichia coli O157/virologia , Antígenos O/metabolismo , Receptores de Bacteriófagos/genética , Bacteriófagos/genética , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/metabolismo , Contaminação de Alimentos/prevenção & controle , Microbiologia de Alimentos , Genoma Viral , Antígenos O/genética , Ligação Viral
7.
PLoS Comput Biol ; 17(6): e1009067, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34125841

RESUMO

Campylobacter jejuni (C. jejuni) causes gastroenteritis following the consumption of contaminated poultry meat, resulting in a large health and economic burden worldwide. Phage therapy is a promising technique for eradicating C. jejuni from poultry flocks and chicken carcasses. However, C. jejuni can resist infections by some phages through stochastic, phase-variable ON/OFF switching of the phage receptors mediated by simple sequence repeats (SSR). While selection strength and exposure time influence the evolution of SSR-mediated phase variation (PV), phages offer a more complex evolutionary environment as phage replication depends on having a permissive host organism. Here, we build and explore several continuous culture bacteria-phage computational models, each analysing different phase-variable scenarios calibrated to the experimental SSR rates of C. jejuni loci and replication parameters for the F336 phage. We simulate the evolution of PV rates via the adaptive dynamics framework for varying levels of selective pressures that act on the phage-resistant state. Our results indicate that growth reducing counter-selection on a single PV locus results in the stable maintenance of the phage, while compensatory selection between bacterial states affects the evolutionary stable mutation rates (i.e. very high and very low mutation rates are evolutionarily disadvantageous), whereas, in the absence of either selective pressure the evolution of PV rates results in mutation rates below the basal values. Contrastingly, a biologically-relevant model with two phase-variable loci resulted in phage extinction and locking of the bacteria into a phage-resistant state suggesting that another counter-selective pressure is required, instance, the use of a distinct phage whose receptor is an F336-phage-resistant state. We conclude that a delicate balance between counter-selection and phage-attack can result in both the evolution of phase-variable phage receptors and persistence of PV-receptor-specific phage.


Assuntos
Receptores de Bacteriófagos/genética , Infecções por Campylobacter/terapia , Campylobacter jejuni/genética , Campylobacter jejuni/virologia , Terapia por Fagos , Animais , Receptores de Bacteriófagos/fisiologia , Infecções por Campylobacter/microbiologia , Infecções por Campylobacter/virologia , Biologia Computacional , Simulação por Computador , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Humanos , Interações Microbianas/genética , Interações Microbianas/fisiologia , Repetições de Microssatélites , Modelos Biológicos , Mutação , Terapia por Fagos/métodos , Terapia por Fagos/estatística & dados numéricos
8.
J Virol ; 95(6)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33408174

RESUMO

Bacteriophage VP1 is a typing phage used for the phage subtyping of Vibrio cholerae O1 biotype El Tor, but the molecular mechanisms of its receptor recognition and the resistance of its host to infection are mostly unknown. In this study, we aimed to identify the host receptor and its role in resistance in natural VP1-resistant strains. Generating spontaneous resistance mutations and genome sequencing mutant strains found the polyQ protein VcpQ, which carries 46 glutamine residues in its Q-rich region, to be responsible for infection by VP1. VcpQ is a membrane protein and possibly forms homotrimers. VP1 adsorbed to V. cholerae through VcpQ. Sequence comparisons showed that 72% of natural VP1-resistant strains have fewer glutamines in the VcpQ Q-rich stretch than VP1-sensitive strains. This difference did not affect the membrane location and oligomer of VcpQ but abrogated VP1 adsorption. These mutant VcpQs did not recover VP1 infection sensitivity in a V. cholerae strain with vcpQ deleted. Our study revealed that the polyQ protein VcpQ is responsible for the binding of VP1 during its infection of V. cholerae and that glutamine residue reduction in VcpQ affects VP1 adsorption to likely be the main cause of VP1 resistance in natural resistant strains. The physiological functions of this polyQ protein in bacteria need further clarification; however, mutations in the polyQ stretch may endow V. cholerae with phage resistance and enhance survival against VP1 or related phages.IMPORTANCE Receptor recognition and binding by bacteriophage are the first step for its infection of bacterial cells. In this study, we found the Vibrio cholerae subtyping phage VP1 uses a polyQ protein named VcpQ (V. cholerae polyQ protein) as the receptor for VP1 infection. Our study reveals the receptor's recognition of phage VP1 during its adsorption and the VP1 resistance mechanism of the wild resistant V. cholerae strains bearing the mutagenesis in the receptor VcpQ. These mutations may confer the survival advantage on these resistant strains in the environment containing VP1 or its similar phages.


Assuntos
Receptores de Bacteriófagos/metabolismo , Bacteriófagos/fisiologia , Peptídeos/metabolismo , Vibrio cholerae/virologia , Sequência de Aminoácidos , Receptores de Bacteriófagos/química , Receptores de Bacteriófagos/genética , Glutamina , Humanos , Mutação , Peptídeos/química , Peptídeos/genética , Multimerização Proteica , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Ligação Viral
9.
Biotechnol Bioeng ; 117(11): 3286-3298, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32658303

RESUMO

Healthcare-associated infections (HCAIs) affect hundreds of millions of patients, representing a significant burden for public health. They are usually associated to multidrug resistant bacteria, which increases their incidence and severity. Bloodstream infections are among the most frequent and life-threatening HCAIs, with Enterococcus and Staphylococcus among the most common isolated pathogens. The correct and fast identification of the etiological agents is crucial for clinical decision-making, allowing to rapidly select the appropriate antimicrobial and to prevent from overuse and misuse of antibiotics and the consequent increase in antimicrobial resistance. Conventional culture methods are still the gold standard to identify these pathogens, however, are time-consuming and may lead to erroneous diagnosis, which compromises an efficient treatment. (Bacterio)phage receptor binding proteins (RBPs) are the structures responsible for the high specificity conferred to phages against bacteria and thus are very attractive biorecognition elements with high potential for specific detection and identification of pathogens. Taking into account all these facts, we have designed and developed a new, fast, accurate, reliable and unskilled diagnostic method based on newly identified phage RBPs and spectrofluorometric techniques that allows the multiplex detection of Enterococcus and Staphylococcus in blood samples in less than 1.5 hr after an enrichment step.


Assuntos
Bacteriemia , Bacteriófagos/genética , Enterococcus , Proteínas Recombinantes de Fusão , Staphylococcus , Proteínas Virais , Animais , Bacteriemia/sangue , Bacteriemia/diagnóstico , Receptores de Bacteriófagos/química , Receptores de Bacteriófagos/metabolismo , Enterococcus/química , Enterococcus/metabolismo , Cavalos , Limite de Detecção , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Staphylococcus/química , Staphylococcus/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
10.
Environ Microbiol ; 22(8): 3126-3142, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32363756

RESUMO

Environmental monitoring of bacteria using phage-based biosensors has been widely developed for many different species. However, there are only a few available methods to detect specific bacteriophages in raw environmental samples. In this work, we developed a simple and efficient assay to rapidly monitor the phage content of a given sample. The assay is based on the bistable expression of the Salmonella enterica opvAB operon. Under regular growth conditions, opvAB is only expressed by a small fraction of the bacterial subpopulation. In the OpvABON subpopulation, synthesis of the OpvA and OpvB products shortens the O-antigen and confers resistance to phages that use LPS as a receptor. As a consequence, the OpvABON subpopulation is selected in the presence of such phages. Using an opvAB::gfp fusion, we could monitor LPS-binding phages in various media, including raw water samples. To enlarge our phage-biosensor panoply, we also developed biosensors able to detect LPS, as well as protein-binding coliphages. Moreover, the combination of these tools allowed to identify the bacterial receptor triggering phage infection. The epigenetic opvAB::gfp biosensor thus comes in different flavours to detect a wide range of bacteriophages and identify the type of receptor they recognize.


Assuntos
Bacteriófagos/isolamento & purificação , Técnicas Biossensoriais/métodos , Monitoramento Ambiental/métodos , Epigênese Genética , Proteínas da Membrana Bacteriana Externa/genética , Receptores de Bacteriófagos/análise , Proteínas de Escherichia coli/genética , Antígenos O , Óperon , Salmonella enterica/genética
11.
FEMS Microbiol Lett ; 367(2)2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-32009155

RESUMO

Many Escherichia coli phages have been sequenced, but in most cases their sequences alone do not suffice to predict their host specificity. Analysis of phage resistant E. coli K-12 mutants have uncovered a certain set of outer membrane proteins and polysaccharides as receptors. In this review, a compilation of E. coli K12 phage receptors is provided and their functional characterization, often driven by studies on phage resistant mutants, is discussed in the historical context. While great progress has been made in this field thus far, several proteins in the outer membrane still await characterization as phage receptors.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Receptores de Bacteriófagos/metabolismo , Colífagos/fisiologia , Escherichia coli K12/metabolismo , Escherichia coli K12/virologia , Proteínas da Membrana Bacteriana Externa/genética , Receptores de Bacteriófagos/genética , Colífagos/genética , Escherichia coli K12/genética
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