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1.
Antimicrob Agents Chemother ; 64(2)2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31767724

RESUMO

Streptococcus pneumoniae is a leading human pathogen uniquely characterized by choline moieties on the bacterial surface. Our previous work reported a pneumococcus-specific chimeric lysin, ClyJ, which combines the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) enzymatically active domain (EAD) from the PlyC lysin and the cell wall binding domain (CBD) from the phage SPSL1 lysin, which imparts choline binding specificity. Here, we demonstrate that the lytic activity of ClyJ can be further improved by editing the linker sequence adjoining the EAD and CBD. Keeping the net charge of the linker constant, we constructed three ClyJ variants containing different lengths of linker sequence. Circular dichroism showed that linker editing has only minor effects on the folding of the EAD and CBD. However, thermodynamic examination combined with biochemical analysis demonstrated that one variant, ClyJ-3, with the shortest linker, displayed improved thermal stability and bactericidal activity, as well as reduced cytotoxicity. In a pneumococcal mouse infection model, ClyJ-3 showed significant protective efficacy compared to that of the ClyJ parental lysin or the Cpl-1 lysin, with 100% survival at a single ClyJ-3 intraperitoneal dose of 100 µg/mouse. Moreover, a ClyJ-3 dose of 2 µg/mouse had the same efficacy as a ClyJ dose of 40 µg/mouse, suggesting a 20-fold improvement in vivo Taking these results together, the present study not only describes a promising pneumococcal lysin with improved potency, i.e., ClyJ-3, but also implies for the first time that the linker sequence plays an important role in determining the activity of a chimeric lysin, providing insight for future lysin engineering studies.

2.
Antibiotics (Basel) ; 8(3)2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31546935

RESUMO

Bacillus cereus, a Gram-positive bacterium, is an agent of food poisoning. B. cereus is closely related to Bacillus anthracis, a deadly pathogen for humans, and Bacillus thuringenesis, an insect pathogen. Due to the growing prevalence of antibiotic resistance in bacteria, alternative antimicrobials are needed. One such alternative is peptidoglycan hydrolase enzymes, which can lyse Gram-positive bacteria when exposed externally. A bioinformatic search for bacteriolytic enzymes led to the discovery of a gene encoding an endolysin-like endopeptidase, LysBC17, which was then cloned from the genome of B. cereus strain Bc17. This gene is also present in the B. cereus ATCC 14579 genome. The gene for LysBC17 encodes a protein of 281 amino acids. Recombinant LysBC17 was expressed and purified from E. coli. Optimal lytic activity against B. cereus occurred between pH 7.0 and 8.0, and in the absence of NaCl. The LysBC17 enzyme had lytic activity against strains of B. cereus, B. anthracis, and other Bacillus species.

3.
Sci Rep ; 9(1): 7349, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089181

RESUMO

Bacteriophage tailspike proteins mediate virion absorption through reversible primary receptor binding, followed by lipopolysaccharide or exopolysaccharide degradation. The Escherichia coli O157:H7 bacteriophage CBA120 genome encodes four distinct tailspike proteins, annotated as ORFs 210 through 213. Previously, we reported the crystal structure of ORF210 (TSP1). Here we describe the crystal structure of ORF212 (TSP3) determined at 1.85 Å resolution. As observed with other tailspike proteins, TSP3 assembles into a trimer. Each subunit of TSP3 has an N-terminal head domain that is structurally similar to that of TSP1, consistent with their high amino acid sequence identity. In contrast, despite sharing a ß-helix fold, the overall structure of the C-terminal catalytic domain of TSP3 is quite different when compared to TSP1. The TSP3 structure suggests that the glycosidase active site resides in a cleft at the interface between two adjacent subunits where three acidic residues, Glu362 and Asp383 on one subunit, and Asp426 on a second subunit, are located in close proximity. Comparing the glycosidase activity of wild-type TSP3 to various point mutants revealed that catalysis requires the carboxyl groups of Glu362 and Asp426, and not of Asp383, confirming the enzyme employs two carboxyl groups to degrade lippopolysaccharide using an acid/base mechanism.

4.
Antibiotics (Basel) ; 8(2)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31142020

RESUMO

Bacteriophage endolysins, enzymes that degrade the bacterial peptidoglycan (PG), have gained an increasing interest as alternative antimicrobial agents, due to their ability to kill antibiotic resistant pathogens efficiently when applied externally as purified proteins. Typical endolysins derived from bacteriophage that infect Gram-positive hosts consist of an N-terminal enzymatically-active domain (EAD) that cleaves covalent bonds in the PG, and a C-terminal cell-binding domain (CBD) that recognizes specific ligands on the surface of the PG. Although CBDs are usually essential for the EADs to access the PG substrate, some EADs possess activity in the absence of CBDs, and a few even display better activity profiles or an extended host spectrum than the full-length endolysin. A current hypothesis suggests a net positive charge on the EAD enables it to reach the negatively charged bacterial surface via ionic interactions in the absence of a CBD. Here, we used the PlyC CHAP domain as a model EAD to further test the hypothesis. We mutated negatively charged surface amino acids of the CHAP domain that are not involved in structured regions to neutral or positively charged amino acids in order to increase the net charge from -3 to a range from +1 to +7. The seven mutant candidates were successfully expressed and purified as soluble proteins. Contrary to the current hypothesis, none of the mutants were more active than wild-type CHAP. Analysis of electrostatic surface potential implies that the surface charge distribution may affect the activity of a positively charged EAD. Thus, we suggest that while charge should continue to be considered for future engineering efforts, it should not be the sole focus of such engineering efforts.

5.
Appl Environ Microbiol ; 85(9)2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30850428

RESUMO

Lytic bacteriophages (or phages) drive bacterial mortality by elaborating exquisite abilities to bind, breach, and destroy bacterial cell membranes and subjugate critical bacterial cell functions. These antimicrobial activities make phages ideal candidates to serve as, or provide sources of, biological control measures for bacterial pathogens. In this study, we isolated the Myoviridae phage vB_BanS_Bcp1 (here referred to as Bcp1) from landfill soil, using a Bacillus anthracis host. The antimicrobial activities of both Bcp1 and its encoded endolysin, PlyB, were examined across different B. cereus sensu lato group species, including B. cereus sensu stricto, Bacillus thuringiensis, and Bacillus anthracis, with pathogenic potential in humans and multiple different uses in biotechnological applications. The Bcp1 phage infected only a subset (11 to 66%) of each B. cereus sensu lato species group tested. In contrast, functional analysis of purified PlyB revealed a potent bacteriolytic activity against all B. cereus sensu lato isolates tested (n = 79). PlyB was, furthermore, active across broad temperature, pH, and salt ranges, refractory to the development of resistance, bactericidal as a single agent, and synergistic with a second endolysin, PlyG. To confirm the potential for PlyB as an antimicrobial agent, we demonstrated the efficacy of a single intravenous treatment with PlyB alone or combination with PlyG in a murine model of lethal B. anthracis infection. Overall, our findings show exciting potential for the Bcp1 bacteriophage and the PlyB endolysin as potential new additions to the antimicrobial armamentarium.IMPORTANCE Organisms of the Bacillus cereus sensu lato lineage are ubiquitous in the environment and are responsible for toxin-mediated infections ranging from severe food poisoning (B. cereus sensu stricto) to anthrax (Bacillus anthracis). The increasing incidence of many of these infections, combined with the specter of antibiotic resistance, has created a need for novel antimicrobials with potent activity, including bacteriophages (or phages) and phage-encoded products (i.e., endolysins). In this study, we describe a broadly infective phage, Bcp1, and its encoded endolysin, PlyB, which exhibited a rapidly bacteriolytic effect against all B. cereus sensu lato isolates tested with no evidence of evolving resistance. Importantly, PlyB was highly efficacious in a mouse model of lethal bacteremia with B. anthracis Both the Bcp1 phage and the PlyB endolysin represent novel mechanisms of action compared to antibiotics, with potential applications to address the evolving problem of antimicrobial resistance.

6.
Artigo em Inglês | MEDLINE | ID: mdl-30642930

RESUMO

Streptococcus pneumoniae is one of the leading pathogens that cause a variety of mucosal and invasive infections. With the increased emergence of multidrug-resistant S. pneumoniae, new antimicrobials with mechanisms of action different from conventional antibiotics are urgently needed. In this study, we identified a putative lysin (gp20) encoded by the Streptococcus phage SPSL1 using the LytA autolysin as a template. Molecular dissection of gp20 revealed a binding domain (GPB) containing choline-binding repeats (CBRs) that are high specificity for S. pneumoniae By fusing GPB to the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) catalytic domain of the PlyC lysin, we constructed a novel chimeric lysin, ClyJ, with improved activity to the pneumococcal Cpl-1 lysin. No resistance was observed in S. pneumoniae strains after exposure to incrementally doubling concentrations of ClyJ for 8 continuous days in vitro In a mouse bacteremia model using penicillin G as a control, a single intraperitoneal injection of ClyJ improved the survival rate of lethal S. pneumoniae-infected mice in a dose-dependent manner. Given its high lytic activity and safety profile, ClyJ may represent a promising alternative to combat pneumococcal infections.

7.
Viruses ; 10(11)2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30445722

RESUMO

Bacteriophage-derived endolysins have gained increasing attention as potent antimicrobial agents and numerous publications document the in vivo efficacy of these enzymes in various rodent models. However, little has been documented about their safety and toxicity profiles. Here, we present preclinical safety and toxicity data for two pneumococcal endolysins, Pal and Cpl-1. Microarray, and gene profiling was performed on human macrophages and pharyngeal cells exposed to 0.5 µM of each endolysin for six hours and no change in gene expression was noted. Likewise, in mice injected with 15 mg/kg of each endolysin, no physical or behavioral changes were noted, pro-inflammatory cytokine levels remained constant, and there were no significant changes in the fecal microbiome. Neither endolysin caused complement activation via the classic pathway, the alternative pathway, or the mannose-binding lectin pathway. In cellular response assays, IgG levels in mice exposed to Pal or Cpl-1 gradually increased for the first 30 days post exposure, but IgE levels never rose above baseline, suggesting that hypersensitivity or allergic reaction is unlikely. Collectively, the safety and toxicity profiles of Pal and Cpl-1 support further preclinical studies.


Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/efeitos adversos , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Endopeptidases/administração & dosagem , Endopeptidases/efeitos adversos , Fagos de Streptococcus/enzimologia , Animais , Antibacterianos/imunologia , Anticorpos Antivirais/sangue , Endopeptidases/imunologia , Endopeptidases/toxicidade , Células Epiteliais/efeitos dos fármacos , Perfilação da Expressão Gênica , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Macrófagos/efeitos dos fármacos , Camundongos
8.
Viruses ; 10(5)2018 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-29883383

RESUMO

Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0⁻8.0), over a broad temperature range (4⁻55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.


Assuntos
Fagos Bacilares/enzimologia , Bacillus/virologia , Endopeptidases/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Bacillus/efeitos dos fármacos , Fagos Bacilares/classificação , Fagos Bacilares/genética , Domínio Catalítico , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/farmacologia , Estabilidade Enzimática , Especificidade de Hospedeiro , Modelos Moleculares , Peptidoglicano/metabolismo , Filogenia , Ligação Proteica , Homologia de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/farmacologia
9.
Cell Microbiol ; 20(9): e12855, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29749010

RESUMO

One of the Borrelia burgdorferi virulence determinants, annotated as Lmp1, is a surface-exposed, conserved, and potential multi-domain protein involved in various functions in spirochete infectivity. Lmp1 contributes to host-pathogen interactions and evasion of host adaptive immunity by spirochetes. Here, we show that in diverse B. burgdorferi species, Lmp1 exists as distinct, region-specific, and lower molecular mass polypeptides encompassing 1 or more domains, including independent N-terminal and middle regions and a combined middle and C-terminal region. These polypeptides originate from complex posttranslational maturation events, partly supported by a periplasmic serine protease termed as BbHtrA. Although spirochete persistence in mice is independently supported by domain-specific Lmp1 polypeptides, transmission of B. burgdorferi from ticks to mammals requires essential contributions from both N-terminal and middle regions. Interference with the functions of Lmp1 domains or their complex posttranslational maturation events may aid in development of novel therapeutic strategies to combat infection and transmission of pathogens.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/fisiologia , Proteínas de Membrana/metabolismo , Viabilidade Microbiana , Processamento de Proteína Pós-Traducional , Serina Proteases/metabolismo , Fatores de Virulência/metabolismo , Animais , Camundongos , Proteólise , Carrapatos
10.
Genome Announc ; 5(45)2017 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-29122857

RESUMO

The Klebsiella pneumoniae phages SopranoGao, MezzoGao, and AltoGao were isolated from the Seneca Wastewater Treatment Plant in Germantown, MD. The following reports the complete genome sequence of these bacteriophages and describes their major features.

11.
Antimicrob Agents Chemother ; 60(12): 7436-7443, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27736755

RESUMO

Streptococcus mutans often survives as a biofilm on the tooth surface and contributes to the development of dental caries. We investigated the efficacy of ClyR, an engineered chimeolysin, against S. mutans biofilms under physiological and cariogenic conditions. Susceptibility tests showed that ClyR was active against all clinical S. mutans isolates tested as well as S. mutans biofilms that displayed resistance to penicillin. The S. mutans biofilms that formed on hydroxyapatite discs under physiological sugar conditions and cariogenic conditions were reduced ∼2 logs and 3 logs after treatment with 100 µg/ml ClyR, respectively. In comparison, only a 1-log reduction was observed in the chlorhexidine gluconate (ChX)-treated group, and no killing effect was observed in the NaF-treated group. A mouse dental colonization model showed that repeated use of ClyR for 3 weeks (5 µg/day) reduced the number of colonized S. mutans cells in the dental plaques significantly (P < 0.05) and had no harmful effects on the mice. Furthermore, toxicity was not noted at concentrations exceeding those used for the in vitro and in vivo studies, and ClyR-specific antibodies could not be detected in mouse saliva after repeated use of ClyR in the oral cavity. Our data collectively demonstrate that ClyR is active against S. mutans biofilms both in vitro and in vivo, thus representing a preventative or therapeutic agent for use against dental caries.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Cárie Dentária/tratamento farmacológico , Placa Dentária/tratamento farmacológico , N-Acetil-Muramil-L-Alanina Amidase/farmacologia , Streptococcus mutans/efeitos dos fármacos , Proteínas Virais/farmacologia , Animais , Antibacterianos/biossíntese , Antibacterianos/química , Bacteriófagos/química , Bacteriófagos/enzimologia , Biofilmes/crescimento & desenvolvimento , Células CHO , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Clorexidina/análogos & derivados , Clorexidina/farmacologia , Cricetulus , Cárie Dentária/microbiologia , Placa Dentária/microbiologia , Modelos Animais de Doenças , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , N-Acetil-Muramil-L-Alanina Amidase/biossíntese , N-Acetil-Muramil-L-Alanina Amidase/genética , Penicilinas/farmacologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Saliva/química , Fluoreto de Sódio/farmacologia , Streptococcus mutans/crescimento & desenvolvimento , Proteínas Virais/biossíntese , Proteínas Virais/genética
12.
Sci Rep ; 6: 25063, 2016 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-27121552

RESUMO

Multi-drug resistant bacteria are a persistent problem in modern health care, food safety and animal health. There is a need for new antimicrobials to replace over used conventional antibiotics. Here we describe engineered triple-acting staphylolytic peptidoglycan hydrolases wherein three unique antimicrobial activities from two parental proteins are combined into a single fusion protein. This effectively reduces the incidence of resistant strain development. The fusion protein reduced colonization by Staphylococcus aureus in a rat nasal colonization model, surpassing the efficacy of either parental protein. Modification of a triple-acting lytic construct with a protein transduction domain significantly enhanced both biofilm eradication and the ability to kill intracellular S. aureus as demonstrated in cultured mammary epithelial cells and in a mouse model of staphylococcal mastitis. Interestingly, the protein transduction domain was not necessary for reducing the intracellular pathogens in cultured osteoblasts or in two mouse models of osteomyelitis, highlighting the vagaries of exactly how protein transduction domains facilitate protein uptake. Bacterial cell wall degrading enzyme antimicrobials can be engineered to enhance their value as potent therapeutics.


Assuntos
Antibacterianos/metabolismo , Antibacterianos/uso terapêutico , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/prevenção & controle , Staphylococcus aureus/efeitos dos fármacos , Animais , Portador Sadio/prevenção & controle , Células Cultivadas , Modelos Animais de Doenças , Humanos , Mastite/tratamento farmacológico , Camundongos , N-Acetil-Muramil-L-Alanina Amidase/genética , Osteomielite/tratamento farmacológico , Ratos , Proteínas Recombinantes de Fusão/genética , Resultado do Tratamento
13.
Elife ; 52016 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-26978792

RESUMO

PlyC, a bacteriophage-encoded endolysin, lyses Streptococcus pyogenes (Spy) on contact. Here, we demonstrate that PlyC is a potent agent for controlling intracellular Spy that often underlies refractory infections. We show that the PlyC holoenzyme, mediated by its PlyCB subunit, crosses epithelial cell membranes and clears intracellular Spy in a dose-dependent manner. Quantitative studies using model membranes establish that PlyCB interacts strongly with phosphatidylserine (PS), whereas its interaction with other lipids is weak, suggesting specificity for PS as its cellular receptor. Neutron reflection further substantiates that PlyC penetrates bilayers above a PS threshold concentration. Crystallography and docking studies identify key residues that mediate PlyCB-PS interactions, which are validated by site-directed mutagenesis. This is the first report that a native endolysin can traverse epithelial membranes, thus substantiating the potential of PlyC as an antimicrobial for Spy in the extracellular and intracellular milieu and as a scaffold for engineering other functionalities.


Assuntos
Endopeptidases/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Fagos de Streptococcus/enzimologia , Streptococcus pyogenes/efeitos dos fármacos , Membrana Celular/metabolismo , Cristalografia por Raios X , Análise Mutacional de DNA , Endopeptidases/química , Endopeptidases/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Fosfatidilserinas/metabolismo , Transporte Proteico
14.
Cell Microbiol ; 18(1): 97-110, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26247174

RESUMO

Borrelia burgdorferi surface-located membrane protein 1, also known as Lmp1, has been shown to play critical roles in pathogen evasion of host-acquired immune defences, thereby facilitating persistent infection. Lmp1 possesses three regions representing potentially discrete domains: Lmp1N, Lmp1M and Lmp1C. Because of its insignificant homology to known proteins, how Lmp1 or its specific regions contribute to microbial biology and infection remains enigmatic. Here, we show that distinct from Lmp1N and Lmp1C, Lmp1M is composed of at least 70% alpha helices and completely lacks recognizable beta sheets. The region binds to host glycosaminoglycan chondroitin-6-sulfate molecules and facilitates mammalian cell attachment, suggesting an adhesin function of Lmp1M. Phenotypic analysis of the Lmp1-deficient mutant engineered to produce Lmp1M on the microbial surface suggests that Lmp1M can independently support B. burgdorferi infectivity in murine hosts. Further exploration of functions of Lmp1 distinct regions will shed new light on the intriguing biology and infectivity of spirochetes and help develop novel interventions to combat Lyme disease.


Assuntos
Proteínas de Bactérias/metabolismo , Borrelia burgdorferi/fisiologia , Sulfatos de Condroitina/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Animais , Aderência Bacteriana , Camundongos , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
15.
Sci Rep ; 5: 17257, 2015 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-26607832

RESUMO

The increasing emergence of multi-drug resistant streptococci poses a serious threat to public health worldwide. Bacteriophage lysins are promising alternatives to antibiotics; however, their narrow lytic spectrum restricted to closely related species is a central shortcoming to their translational development. Here, we describe an efficient method for rapid screening of engineered chimeric lysins and report a unique "chimeolysin", ClyR, with robust activity and an extended-spectrum streptococcal host range against most streptococcal species, including S. pyogenes, S. agalactiae, S. dysgalactiae, S. equi, S. mutans, S. pneumoniae, S. suis and S. uberis, as well as representative enterococcal and staphylococcal species (including MRSA and VISA). ClyR is the first lysin that demonstrates activity against the dominant dental caries-causing pathogen as well as the first lysin that kills all four of the bovine mastitis-causing pathogens. This study demonstrates the success of the screening method resulting in a powerful lysin with potential for treating most streptococcal associated infections.


Assuntos
Bacteriólise/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Especificidade de Hospedeiro/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Streptococcus/fisiologia , Animais , Bovinos , Modelos Animais de Doenças , Escherichia coli , Feminino , Camundongos Endogâmicos BALB C , Leite/química , Infecções Estreptocócicas , Streptococcus/efeitos dos fármacos
16.
Infect Immun ; 83(11): 4293-303, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26283338

RESUMO

Invasive M1T1 group A Streptococcus (GAS) can have a mutation in the regulatory system CovRS, and this mutation can render strains hypervirulent. Interestingly, via mechanisms that are not well understood, the host innate immune system's neutrophils select spontaneous M1T1 GAS CovRS hypervirulent mutants, thereby enhancing the pathogen's ability to evade immune killing. It has been reported that the DNase Sda1 is critical for the resistance of M1T1 strain 5448 to killing in human blood and provides pressure for in vivo selection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion of sda1 or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion of sda1 in strain 5448 resulted in Δsda1 mutants with (5448 Δsda1(M+) strain) and without (5448 Δsda1(M-) strain) M protein production. The 5448 Δsda1(M+) strain accumulated CovRS mutations in vivo and resisted killing in the bloodstream, whereas the 5448 Δsda1(M-) strain lost in vivo selection of CovRS mutations and was sensitive to killing. The deletion of emm and a spontaneous Mga mutation in MGAS2221 reduced and prevented in vivo selection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical for in vivo selection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to the in vivo selection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Desoxirribonuclease I/imunologia , Imunidade Inata , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Repressoras/genética , Infecções Estreptocócicas/imunologia , Infecções Estreptocócicas/microbiologia , Streptococcus pyogenes/imunologia , Animais , Desoxirribonuclease I/genética , Histidina Quinase , Humanos , Evasão da Resposta Imune , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regulon , Proteínas Repressoras/imunologia , Streptococcus pyogenes/enzimologia , Streptococcus pyogenes/genética
17.
Protein Eng Des Sel ; 28(4): 85-92, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25740429

RESUMO

Endolysins are bacteriophage-derived peptidoglycan hydrolases that represent an emerging class of proteinaceous therapeutics. While the streptococcal endolysin PlyC has been validated in vitro and in vivo for its therapeutic efficacy, the inherent thermosusceptible structure of the enzyme correlates to transient long-term stability, thereby hindering the feasibility of developing the enzyme as an antimicrobial. Here, we thermostabilized the cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain of the PlyCA catalytic subunit of PlyC using a FoldX-driven computational protein engineering approach. Using a combination of FoldX and Rosetta algorithms, as well as visual inspection, a final list of PlyC point mutant candidates with predicted stabilizing ΔΔG values was assembled and thermally characterized. Five of the eight point mutations were found experimentally to be destabilizing, a result most likely attributable to computationally modeling a complex and dynamic nine-subunit holoenzyme with a corresponding 3.3-Å X-ray crystal structure. However, one of the mutants, PlyC (PlyCA) T406R, was shown experimentally to increase the thermal denaturation temperature by ∼2.2°C and kinetic stability 16-fold over wild type. This mutation is expected to introduce a thermally advantageous hydrogen bond between the Q106 side chain of the N-terminal glycosyl hydrolase domain and the R406 side chain of the C-terminal CHAP domain.


Assuntos
Bacteriófagos/química , Endopeptidases/química , Engenharia de Proteínas , Proteínas Virais/química , Algoritmos , Amidoidrolases/química , Amidoidrolases/genética , Bacteriófagos/genética , Biologia Computacional , Cristalografia por Raios X , Endopeptidases/genética , Estabilidade Enzimática/genética , Histidina/química , Ligações de Hidrogênio , Cinética , Mutação Puntual
18.
J Antimicrob Chemother ; 70(5): 1453-65, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25630640

RESUMO

OBJECTIVES: In the light of increasing drug resistance in Staphylococcus aureus, bacteriophage endolysins [peptidoglycan hydrolases (PGHs)] have been suggested as promising antimicrobial agents. The aim of this study was to determine the antimicrobial activity of nine enzymes representing unique homology groups within a diverse class of staphylococcal PGHs. METHODS: PGHs were recombinantly expressed, purified and tested for staphylolytic activity in multiple in vitro assays (zymogram, turbidity reduction assay and plate lysis) and against a comprehensive set of strains (S. aureus and CoNS). PGH cut sites in the staphylococcal peptidoglycan were determined by biochemical assays (Park-Johnson and Ghuysen procedures) and MS analysis. The enzymes were tested for their ability to eradicate static S. aureus biofilms and compared for their efficacy against systemic MRSA infection in a mouse model. RESULTS: Despite similar modular architectures and unexpectedly conserved cleavage sites in the peptidoglycan (conferred by evolutionarily divergent catalytic domains), the enzymes displayed varying degrees of in vitro lytic activity against numerous staphylococcal strains, including cell surface mutants and drug-resistant strains, and proved effective against static biofilms. In a mouse model of systemic MRSA infection, six PGHs provided 100% protection from death, with animals being free of clinical signs at the end of the experiment. CONCLUSIONS: Our results corroborate the high potential of PGHs for treatment of S. aureus infections and reveal unique antimicrobial and biochemical properties of the different enzymes, suggesting a high diversity of potential applications despite highly conserved peptidoglycan target sites.


Assuntos
Antibacterianos/uso terapêutico , Bacteriófagos/enzimologia , Terapia Biológica/métodos , Endopeptidases/uso terapêutico , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Infecções Estafilocócicas/tratamento farmacológico , Animais , Bacteriemia/tratamento farmacológico , Bacteriemia/microbiologia , Parede Celular/efeitos dos fármacos , Modelos Animais de Doenças , Endopeptidases/genética , Endopeptidases/metabolismo , Feminino , Hidrólise , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Peptidoglicano/efeitos dos fármacos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Infecções Estafilocócicas/microbiologia , Análise de Sobrevida , Resultado do Tratamento
19.
Virology ; 477: 125-132, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25432575

RESUMO

Endolysins are lytic enzymes encoded by bacteriophage that represent an emerging class of protein therapeutics. Considering macromolecular thermoresistance correlates with shelf life, PlyG, a Bacillus anthracis endolysin, was thermally characterized to further evaluate its therapeutic potential. Results from a biophysical thermal analysis revealed full-length PlyG and its isolated domains comprised thermal denaturation temperatures exceeding 63°C. In the absence of reducing agent, PlyG was determined to be kinetically unstable, a finding hypothesized to be attributable to the chemical oxidation of cysteine and/or methionine residues. The presence of reducing agent kinetically stabilized the endolysin, with PlyG retaining at least ~50% residual lytic activity after being heated at temperatures up to 80°C and remaining enzymatically functional after being boiled. Furthermore, the endolysin had a kinetic half-life at 50°C and 55°C of 35 and 5.5h, respectively. PlyG represents a thermostable proteinaceous antibacterial with subsequent prolonged therapeutic shelf life expectancy.


Assuntos
Bacillus anthracis/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/efeitos da radiação , Proteínas Virais/química , Proteínas Virais/efeitos da radiação , Antibacterianos/química , Antibacterianos/efeitos da radiação , Estabilidade Enzimática , Cinética , Desnaturação Proteica/efeitos da radiação , Estabilidade Proteica , Temperatura Ambiente
20.
Appl Microbiol Biotechnol ; 99(2): 741-52, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25038926

RESUMO

The increasing rate of resistance of pathogenic bacteria, such as Staphylococcus aureus, to classical antibiotics has driven research toward identification of other means to fight infectious disease. One particularly viable option is the use of bacteriophage-encoded peptidoglycan hydrolases, called endolysins or enzybiotics. These enzymes lyse the bacterial cell wall upon direct contact, are not inhibited by traditional antibiotic resistance mechanisms, and have already shown great promise in the areas of food safety, human health, and veterinary science. We have identified and characterized an endolysin, PlyGRCS, which displays dose-dependent antimicrobial activity against both planktonic and biofilm S. aureus, including methicillin-resistant S. aureus (MRSA). The spectrum of lytic activity for this enzyme includes all S. aureus and Staphylococcus epidermidis strains tested, but not other Gram-positive pathogens. The contributions of the PlyGRCS putative catalytic and cell wall binding domains were investigated through deletion analysis. The cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) catalytic domain displayed activity by itself, though reduced, indicating the necessity of the binding domain for full activity. In contrast, the SH3_5 binding domain lacked activity but was shown to interact directly with the staphylococcal cell wall via fluorescent microscopy. Site-directed mutagenesis studies determined that the active site residues in the CHAP catalytic domain were C29 and H92, and its catalytic functionality required calcium as a co-factor. Finally, biochemical assays coupled with mass spectrometry analysis determined that PlyGRCS displays both N-acetylmuramoyl-L-alanine amidase and D-alanyl-glycyl endopeptidase hydrolytic activities despite possessing only a single catalytic domain. These results indicate that PlyGRCS has the potential to become a revolutionary therapeutic option to combat bacterial infections.


Assuntos
Bacteriófagos/enzimologia , Endopeptidases/metabolismo , Staphylococcus aureus Resistente à Meticilina/virologia , Bacteriófagos/genética , Biofilmes , Domínio Catalítico , Parede Celular/química , Dicroísmo Circular , Clonagem Molecular , Cisteína/química , Endopeptidases/genética , Histidina/química , Mutagênese Sítio-Dirigida , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Staphylococcus epidermidis/virologia
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