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1.
Pol J Microbiol ; 68(4): 477-491, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31880892

RESUMO

This study explored a potential treatment against methicillin-resistant Staphylococcus aureus (MRSA) infections that combines thioridazine (TZ), an efflux pump inhibitor, and miconazole (MCZ), an autolysis inducer, with the anti-microbial drug cloxacillin (CXN). In vitro, the combination treatment of TZ and MCZ significantly reduced 4096-fold (Σ (FIC) = 0.1 - 1.25) the MIC value of CXN against S. aureus. In vivo, the combination therapy significantly relieved breast redness and swelling in mice infected with either clinical or standard strains of S. aureus. Meanwhile, the number of bacteria isolated from the MRSA135-infected mice decreased significantly (p = 0.0427 < 0.05) after the combination therapy when compared to monotherapy. Moreover, the number of bacteria isolated from the mice infected with a reference S. aureus strain also decreased significantly (p = 0.0191 < 0.05) after the combination therapy when compared to monotherapy. The pathological changes were more significant in the CXN-treated group when compared to mice treated with a combination of three drugs. In addition, we found that combination therapy reduced the release of the bacteria-stimulated cytokines such as IL-6, IFN-γ, and TNF-α. Cytokine assays in serum revealed that CXN alone induced IL-6, IFN-γ, and TNF-α in the mouse groups infected with ATCC 29213 or MRSA135, and the combination of these three drugs significantly reduced IL-6, IFN-γ, and TNF-α concentrations. Also, the levels of TNF-α and IFN-γ in mice treated with a combination of three drugs were significantly lower than in the CXN-treated group. Given the synergistic antibacterial activity of CXN, we concluded that the combination of CXN with TZ, and MCZ could be developed as a novel therapeutic strategy against S. aureus.This study explored a potential treatment against methicillin-resistant Staphylococcus aureus (MRSA) infections that combines thioridazine (TZ), an efflux pump inhibitor, and miconazole (MCZ), an autolysis inducer, with the anti-microbial drug cloxacillin (CXN). In vitro, the combination treatment of TZ and MCZ significantly reduced 4096-fold (Σ (FIC) = 0.1 ­ 1.25) the MIC value of CXN against S. aureus. In vivo, the combination therapy significantly relieved breast redness and swelling in mice infected with either clinical or standard strains of S. aureus. Meanwhile, the number of bacteria isolated from the MRSA135-infected mice decreased significantly (p = 0.0427 < 0.05) after the combination therapy when compared to monotherapy. Moreover, the number of bacteria isolated from the mice infected with a reference S. aureus strain also decreased significantly (p = 0.0191 < 0.05) after the combination therapy when compared to monotherapy. The pathological changes were more significant in the CXN-treated group when compared to mice treated with a combination of three drugs. In addition, we found that combination therapy reduced the release of the bacteria-stimulated cytokines such as IL-6, IFN-γ, and TNF-α. Cytokine assays in serum revealed that CXN alone induced IL-6, IFN-γ, and TNF-α in the mouse groups infected with ATCC 29213 or MRSA135, and the combination of these three drugs significantly reduced IL-6, IFN-γ, and TNF-α concentrations. Also, the levels of TNF-α and IFN-γ in mice treated with a combination of three drugs were significantly lower than in the CXN-treated group. Given the synergistic antibacterial activity of CXN, we concluded that the combination of CXN with TZ, and MCZ could be developed as a novel therapeutic strategy against S. aureus.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Farmacorresistência Bacteriana , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/efeitos dos fármacos , beta-Lactamas/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriólise/efeitos dos fármacos , Cloxacilina/farmacologia , Quimioterapia Combinada , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Miconazol/farmacologia , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/citologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Tioridazina/farmacologia
2.
PLoS Pathog ; 15(9): e1008044, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31518377

RESUMO

ß-lactam antibiotics interfere with cross-linking of the bacterial cell wall, but the killing mechanism of this important class of antibiotics is not fully understood. Serendipitously we found that sub-lethal doses of ß-lactams rescue growth and prevent spontaneous lysis of Staphylococcus aureus mutants lacking the widely conserved chaperone ClpX, and we reasoned that a better understanding of the clpX phenotypes could provide novel insights into the downstream effects of ß-lactam binding to the PBP targets. Super-resolution imaging revealed that clpX cells display aberrant septum synthesis, and initiate daughter cell separation prior to septum completion at 30°C, but not at 37°C, demonstrating that ClpX becomes critical for coordinating the S. aureus cell cycle as the temperature decreases. FtsZ localization and dynamics were not affected in the absence of ClpX, suggesting that ClpX affects septum formation and autolytic activation downstream of Z-ring formation. Interestingly, oxacillin antagonized the septum progression defects of clpX cells and prevented lysis of prematurely splitting clpX cells. Strikingly, inhibitors of wall teichoic acid (WTA) biosynthesis that work synergistically with ß-lactams to kill MRSA synthesis also rescued growth of the clpX mutant, as did genetic inactivation of the gene encoding the septal autolysin, Sle1. Taken together, our data support a model in which Sle1 causes premature splitting and lysis of clpX daughter cells unless Sle1-dependent lysis is antagonized by ß-lactams or by inhibiting an early step in WTA biosynthesis. The finding that ß-lactams and inhibitors of WTA biosynthesis specifically prevent lysis of a mutant with dysregulated autolytic activity lends support to the idea that PBPs and WTA biosynthesis play an important role in coordinating cell division with autolytic splitting of daughter cells, and that ß-lactams do not kill S. aureus simply by weakening the cell wall.


Assuntos
Proteínas de Bactérias/fisiologia , Endopeptidase Clp/fisiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriólise/efeitos dos fármacos , Bacteriólise/fisiologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Endopeptidase Clp/genética , Humanos , Modelos Biológicos , Mutação , Oxacilina/farmacologia , Staphylococcus aureus/genética , Ácidos Teicoicos/biossíntese , Tunicamicina/farmacologia , beta-Lactamas/farmacologia
3.
BMC Res Notes ; 12(1): 560, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31488211

RESUMO

OBJECTIVE: Our immediate objective is to determine whether infectivity of lytic podophage T3 has a relatively high persistence in the blood of a mouse, as suggested by previous data. Secondarily, we determine whether the T3 surface has changed during this mouse passage. The surface is characterized by native agarose gel electrophoresis (AGE). Beyond our current data, the long-term objective is optimization of phages chosen for therapy of all bacteremias and associated sepsis. RESULTS: We find that the persistence of T3 in mouse blood is higher by over an order of magnitude than the previously reported persistence of (1) lysogenic phages lambda and P22, and (2) lytic phage T7, a T3 relative. We explain these differences via the lysogenic character of lambda and P22, and the physical properties of T7. For the future, we propose testing a new, AGE-based strategy for rapidly screening for high-persistence, lytic, environmental podophages that have phage therapy-promoting physical properties.


Assuntos
Bacteriemia/terapia , Bacteriófago T3/fisiologia , Terapia por Fagos/métodos , Sepse/terapia , Animais , Bacteriemia/sangue , Bacteriólise , Bacteriófago T7/fisiologia , Feminino , Camundongos Endogâmicos C57BL , Sepse/sangue
4.
Can J Microbiol ; 65(12): 895-903, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31479619

RESUMO

Bacteriocins are antimicrobial peptides, produced by Gram-positive bacteria such as lactococci and staphylococci, that have limited bactericidal action against Gram-negative bacteria. The aim of this paper was to study the sensitivity of three strains of Escherichia coli to bacteriocins: nisin (as Nisaplin®) and two staphylococcal peptides (warnerin and hominin) during sucrose-induced osmotic stress. We found that all peptides in a 0.3 g·mL-1 sucrose solution significantly reduced the number of viable E. coli. The most pronounced antibacterial effect was achieved by nisin against E. coli K-12 (3 log reduction). Slightly less bactericidal effects were observed with warnerin (1 mg·mL-1) and hominin (1 mg·mL-1) in sucrose solution. The lytic activity of staphylococcal peptides was detected by decreased optical density and viable cell counts. Moreover, it was confirmed by the increased amount of DNA and protein in the medium and the morphological changes detected by atomic force microscopy after 20 h of treatment. Zymographic analysis revealed the release of lytic enzymes from E. coli cells after treatment with staphylococcal peptides and sucrose. These results indicated that the antimicrobial action of peptides can be extended to Gram-negative bacteria via combination with high concentrations of sucrose.


Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Escherichia coli/efeitos dos fármacos , Pressão Osmótica , Sacarose/farmacologia , Antibacterianos/metabolismo , Bacteriocinas/metabolismo , Bacteriólise/efeitos dos fármacos , Bactérias Gram-Positivas/metabolismo , Viabilidade Microbiana/efeitos dos fármacos
5.
Arch Virol ; 164(10): 2627-2630, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31363923

RESUMO

A lytic bacteriophage, designated Vibrio phage vB_VpP_BA6, was isolated from sewage collected in Guangzhou, China. The double-stranded DNA genome of phage BA6 is composed of 50,520 bp with a G+C content of 41.77%. It possesses 64 open reading frames relating to phage structure, packaging, host lysis, DNA metabolism, and additional functions. Three tRNAs genes (encoding Pro, Ile and Trp) were detected. Comparison of its genomic features and phylogenetic analysis revealed that phage BA6 is a novel member of the family Podoviridae. This phage may represent a potential therapeutic agent against multidrug-resistant Vibrio parahaemolyticus.


Assuntos
Bacteriófagos/genética , Bacteriófagos/isolamento & purificação , Genoma Viral , Podoviridae/genética , Podoviridae/isolamento & purificação , Vibrio parahaemolyticus/virologia , Bacteriólise , Bacteriófagos/classificação , Bacteriófagos/crescimento & desenvolvimento , Composição de Bases , China , DNA/química , DNA/genética , Fases de Leitura Aberta , Filogenia , Podoviridae/classificação , Podoviridae/crescimento & desenvolvimento , RNA de Transferência/genética , Esgotos/virologia
6.
PLoS Negl Trop Dis ; 13(8): e0007113, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31425525

RESUMO

Buruli Ulcer (BU) is a cutaneous disease caused by Mycobacterium ulcerans. The pathogenesis of this disease is closely related to the secretion of the toxin mycolactone that induces extensive destruction of the skin and soft tissues. Currently, there are no effective measures to prevent the disease and, despite availability of antibiotherapy and surgical treatments, these therapeutic options are often associated with severe side effects. Therefore, it is important to develop alternative strategies for the treatment of BU. Endolysins (lysins) are phage encoded enzymes that degrade peptidoglycan of bacterial cell walls. Over the past years, lysins have been emerging as alternative antimicrobial agents against bacterial infections. However, mycobacteria have an unusual outer membrane composed of mycolylarabinogalactan-peptidoglycan. To overcome this complex barrier, some mycobacteriophages encode a lipolytic enzyme, Lysin B (LysB). In this study, we demonstrate for the first time that recombinant LysB displays lytic activity against M. ulcerans isolates. Moreover, using a mouse model of M. ulcerans footpad infection, we show that subcutaneous treatment with LysB prevented further bacterial proliferation, associated with IFN-γ and TNF production in the draining lymph node. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease.


Assuntos
Úlcera de Buruli/tratamento farmacológico , Endopeptidases/administração & dosagem , Micobacteriófagos/enzimologia , Mycobacterium ulcerans/efeitos dos fármacos , Animais , Bacteriólise , Úlcera de Buruli/patologia , Modelos Animais de Doenças , Endopeptidases/farmacologia , Feminino , Interferon gama/análise , Linfonodos/imunologia , Camundongos Endogâmicos BALB C , Mycobacterium ulcerans/virologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/farmacologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/análise
7.
Arch Virol ; 164(10): 2637-2640, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31372754

RESUMO

A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.


Assuntos
Bacteriófagos/classificação , Bacteriófagos/isolamento & purificação , Enterobacteriaceae/virologia , Filogenia , Siphoviridae/classificação , Siphoviridae/isolamento & purificação , Bacteriólise , Bacteriófagos/genética , Genoma Viral , Microscopia Eletrônica de Transmissão , Análise de Sequência de DNA , Siphoviridae/genética , Sintenia , Proteínas Virais/genética , Vírion/ultraestrutura
8.
Future Microbiol ; 14: 949-955, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31373220

RESUMO

Aim: Bile salts promote the specific autolysis of pneumococcal cells, allowing the differentiation between Streptococcus pneumoniae and other viridans group streptococci (VGS). Material & methods: One hundred clinical VGS isolates identified by amplification and sequencing of 16S rRNA, groEL and sodA genes were analyzed with different variants of bile-solubility tests: tube testing read by naked eye; tube testing where the lysis was measured as the decrease of turbidity with a densitometer; and direct testing on blood agar plate. Results: As expected, all S. pneumoniae isolates were fully lysed in the presence of bile salts except for one isolate that partially lysate in tube testing as well as on the blood agar plate. None of the VGS were lysed by bile salts. Conclusion: Bile-solubility testing is an accurate and technically nondemanding method to discriminate between S. pneumoniae and other VGS species.


Assuntos
Técnicas Bacteriológicas/métodos , Ácidos e Sais Biliares , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/microbiologia , Streptococcus pneumoniae/isolamento & purificação , Estreptococos Viridans/isolamento & purificação , Bacteriólise/efeitos dos fármacos , Ácidos e Sais Biliares/farmacologia , Diagnóstico Diferencial , Humanos , Sensibilidade e Especificidade , Solubilidade
9.
Arch Virol ; 164(10): 2599-2603, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31278422

RESUMO

This work describes the characterization and genome annotation of a new lytic Enterococcus faecalis siphovirus, vB_EfaS_AL3 (referred to as AL3), isolated from wastewater samples collected in Liaoning Province, China. The genome of phage AL3 is composed of linear double-stranded DNA that is 40,789 bp in length with a G + C content of 34.84% and 61 putative protein-coding genes. Phylogenetic and comparative genomic analyses indicate that phage AL3 should be considered a novel phage.


Assuntos
Bacteriófagos/genética , Enterococcus faecalis/virologia , Genoma Viral , Filogenia , Análise de Sequência de DNA , Águas Residuárias/virologia , Bacteriólise , Composição de Bases , China , DNA/química , DNA/genética , DNA Viral/química , DNA Viral/genética , Microscopia Eletrônica de Transmissão , Anotação de Sequência Molecular , Ensaio de Placa Viral , Vírion/ultraestrutura
10.
J Basic Microbiol ; 59(10): 1049-1062, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31347183

RESUMO

The aim of this study was to identify and characterize the SE-P3, P16, P37, and P47 phages infecting Salmonella Enteritidis. Transmission electron microscopy analysis showed that the SE phages belonged to the Myoviridae or Siphoviridae family and had plaque sizes between 0.622 ± 0.027 and 1.630 ± 0.036 mm in diameter. sefC, pefA, spvC, sopE, and gipA virulent gene regions were absent in their genome and their calculated genome sizes were between 35.9 and 37.8 kbp. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the protein profiles of each phage were different. The SE phages had a short latent period (10-20 min), large burst size (76-356 PFU/cell), and a short burst time (25-35 min). The multiplicity of infection values and mutant frequency of the phages were 0.01-0.0001 and 10-7 , respectively. They were very infective against their host bacteria when applied at 20°C, 30°C, or 37°C and adsorbed to their host cells by 96.20-97.65% in the first 5 min of incubation, and also Ca2+ ions did not have a significant effect on their adsorption. The SE phages were resistant to wide pH ranges and high temperatures. These results indicate that the SE phages are good candidates as therapeutic and biocontrol agents against foodborne pathogenic S. Enteritidis.


Assuntos
Fagos de Salmonella/fisiologia , Salmonella enteritidis/virologia , Bacteriólise , Tamanho do Genoma , Genoma Viral , Temperatura Alta , Concentração de Íons de Hidrogênio , Taxa de Mutação , Myoviridae/classificação , Myoviridae/genética , Myoviridae/fisiologia , Myoviridae/ultraestrutura , Fagos de Salmonella/classificação , Fagos de Salmonella/genética , Fagos de Salmonella/ultraestrutura , Siphoviridae/classificação , Siphoviridae/genética , Siphoviridae/fisiologia , Siphoviridae/ultraestrutura , Especificidade da Espécie , Proteínas Virais/química , Proteínas Virais/metabolismo , Ligação Viral , Latência Viral
11.
Int J Antimicrob Agents ; 54(3): 329-337, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31229670

RESUMO

Orthopaedic implant-associated infections are a devastating complication of orthopaedic surgery with a significant impact on patients and healthcare systems. The aims of this work were to describe the patterns of antimicrobial resistance, pathogenicity and virulence of clinical bacterial isolates from orthopaedic implant-associated infections and to further isolate and characterise bacteriophages that are efficient in controlling these bacteria. Staphylococcus aureus, Enterococcus faecalis and Escherichia coli isolated from orthopaedic infections showed multiresistance patterns to the most frequently used antibiotics in clinical settings. The presence of mobile genetic elements (mecA, Tn916/Tn1545 and intl1) and virulence determinants (icaB, cna, hlb, cylLs, cylM, agg, gelE, fsr and fimA) highlighted the pathogenicity of these isolates. Moreover, the isolates belonged to clonal complexes associated with the acquisition of pathogenicity islands and antimicrobial resistance genes by recombination and horizontal gene transfer. Bacteriophages vB_SauM_LM12, vB_EfaS_LM99 and vB_EcoM_JB75 were characterised and their ability to infect clinical isolates of S. aureus, E. faecalis and E. coli, respectively, was assessed. Morphological and genomic analyses revealed that vB_EfaS_LM99 and vB_EcoM_JB75 belong to the Siphoviridae and Myoviridae families, respectively, and no genes associated with lysogeny were found. The bacteriophages showed low latent periods, high burst sizes, broad host ranges and tolerance to several environmental conditions. Moreover, they showed high efficiency and specificity to infect and reduce clinical bacteria, including methicillin-resistant S. aureus and vancomycin-resistant enterococci. Therefore, the results obtained suggest that the bacteriophages used in this work are a promising approach to control these pathogens involved in orthopaedic implant-associated infections.


Assuntos
Bacteriólise , Bacteriófagos/isolamento & purificação , Infecções por Escherichia coli/terapia , Infecções por Bactérias Gram-Positivas/terapia , Terapia por Fagos/métodos , Infecções Relacionadas à Prótese/terapia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriófagos/crescimento & desenvolvimento , Farmacorresistência Bacteriana Múltipla , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/isolamento & purificação , Enterococcus faecalis/patogenicidade , Enterococcus faecalis/virologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Escherichia coli/virologia , Feminino , Infecções por Bactérias Gram-Positivas/microbiologia , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos Ortopédicos/efeitos adversos , Infecções Relacionadas à Prótese/microbiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Staphylococcus aureus/patogenicidade , Staphylococcus aureus/virologia
12.
Cell Host Microbe ; 25(6): 803-814.e5, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-31175044

RESUMO

The human gut microbiome is comprised of densely colonizing microorganisms including bacteriophages, which are in dynamic interaction with each other and the mammalian host. To address how bacteriophages impact bacterial communities in the gut, we investigated the dynamic effects of phages on a model microbiome. Gnotobiotic mice were colonized with defined human gut commensal bacteria and subjected to predation by cognate lytic phages. We found that phage predation not only directly impacts susceptible bacteria but also leads to cascading effects on other bacterial species via interbacterial interactions. Metabolomic profiling revealed that shifts in the microbiome caused by phage predation have a direct consequence on the gut metabolome. Our work provides insight into the ecological importance of phages as modulators of bacterial colonization, and it additionally suggests the potential impact of gut phages on the mammalian host with implications for their therapeutic use to precisely modulate the microbiome.


Assuntos
Bacteriólise , Bacteriófagos/crescimento & desenvolvimento , Fezes/química , Microbioma Gastrointestinal , Metaboloma , Animais , Vida Livre de Germes , Camundongos , Interações Microbianas
14.
Cell Host Microbe ; 25(5): 746-755.e5, 2019 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-31071296

RESUMO

Temperate phages can adopt either a lytic or lysogenic lifestyle within their host bacteria. It was recently shown that Bacillus-subtilis-infecting phages of the SPbeta group utilize a peptide-based communication system called arbitrium to coordinate the lysogeny decision. The occurrence of peptide-based communication systems among phages more broadly remains to be explored. Here, we uncover a wide array of peptide-based communication systems utilized by phages for lysogeny decisions. These arbitrium-like systems show diverse peptide codes and can be detected in numerous genetically distant phage types and conjugative elements. The pathogens Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are commonly infected by arbitrium-carrying mobile elements, which often carry toxins essential for pathogenicity. Experiments with phages containing these arbitrium-like systems demonstrate their involvement in lysogeny decisions. Finally, our results suggest that the peptide-based decision is executed by an antisense RNA that controls the regulator of the lysogenic state.


Assuntos
Fagos Bacilares/crescimento & desenvolvimento , Bacillus anthracis/virologia , Bacillus cereus/virologia , Bacillus thuringiensis/virologia , Regulação Viral da Expressão Gênica , Peptídeos/metabolismo , Microbiologia do Solo , Fagos Bacilares/genética , Bacteriólise , Lisogenia , RNA não Traduzido/metabolismo
15.
Appl Microbiol Biotechnol ; 103(10): 4103-4112, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30953122

RESUMO

SpoIIID is a small, sequence-specific DNA-binding protein which can direct many genes' transcription and has an effect on spore formation in Bacillus subtilis. We investigated the role of SpoIIID in mother cell lysis in Bacillus thuringiensis. A ß-galactosidase assay based on the promoter fusions with lacZ indicated that the sigK gene was positively regulated by SpoIIID and σK negatively regulated the expression of sigE. The spoIIID mutant strain exhibited no mother cell lysis in Schaeffer's sporulation medium (SSM) but did in ½ Luria-Bertani (LB) medium. cwlC is an essential hydrolase gene for mother cell lysis. Moreover, the expression of a PcwlC-lacZ fusion in spoIIID mutant was proved to be higher in ½ LB medium than in SSM. HD (ΔspoIIID)(ΔcwlC) mutant was obtained by knocking out the cwlC gene in HD(ΔspoIIID) and displayed no mother cell lysis in both SSM and ½ LB mediums. The deletion of spoIIID decreased the crystal protein production in HD73. The expression of Porf1cry8E and P5014 promoter fusions with lacZ gene in the acrystalliferous HD-(ΔspoIIID) mutant showed similar activity to that in the acrystalliferous HD73- strain before T7 and slightly higher than that in the acrystalliferous HD73- after T7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Cry1Ac production in HD-(ΔspoIIID) directed by the Porf1cry8E and P5014 promoters was at a similar level as that in HD73 wild strain. Altogether, these results suggested that the spoIIID mutant with Porf1cry8E or P5014 promoters could be an alternative delivery system for cry gene expression with no mature spore formation and medium-dependent mother cell lysis.


Assuntos
Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriólise , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Bacillus thuringiensis/crescimento & desenvolvimento , Endotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento
16.
Elife ; 82019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30964003

RESUMO

Penicillin and related antibiotics disrupt cell wall synthesis to induce bacteriolysis. Lysis in response to these drugs requires the activity of cell wall hydrolases called autolysins, but how penicillins misactivate these deadly enzymes has long remained unclear. Here, we show that alterations in surface polymers called teichoic acids (TAs) play a key role in penicillin-induced lysis of the Gram-positive pathogen Streptococcus pneumoniae (Sp). We find that during exponential growth, Sp cells primarily produce lipid-anchored TAs called lipoteichoic acids (LTAs) that bind and sequester the major autolysin LytA. However, penicillin-treatment or prolonged stationary phase growth triggers the degradation of a key LTA synthase, causing a switch to the production of wall-anchored TAs (WTAs). This change allows LytA to associate with and degrade its cell wall substrate, thus promoting osmotic lysis. Similar changes in surface polymer assembly may underlie the mechanism of antibiotic- and/or growth phase-induced lysis for other important Gram-positive pathogens.


Assuntos
Antibacterianos/farmacologia , Bacteriólise/efeitos dos fármacos , Vias Biossintéticas/efeitos dos fármacos , Penicilinas/farmacologia , Streptococcus pneumoniae/efeitos dos fármacos , Ácidos Teicoicos/biossíntese
17.
Artigo em Inglês | MEDLINE | ID: mdl-30863725

RESUMO

Streptococcosis is recognized as a leading infectious disease in the swine industry. Streptococcus suis serotype 2 is regarded as the most virulent species, which threatens human and pig health and causes serious economic losses. In this study, multiple in vitro and in vivo effects of MP1102 on multidrug resistant S. suis was studied for the first time. MP1102 exhibited significant antibacterial activity against S. suis (minimum inhibitory concentration, MIC = 0.028-0.228 µM), rapid bacteriocidal action, a longer postantibiotic effect than ceftriaxone, and a synergistic or additive effect with lincomycin, penicillin, and ceftriaxone (FICI = 0.29-0.96). No resistant mutants appeared after 30 serial passages of S. suis in the presence of MP1102. Flow cytometric analysis and electron microscopy observations showed that MP1102 destroyed S. suis cell membrane integrity and affected S. suis cell ultrastructure and membrane morphology. Specifically, a significantly wrinkled surface, intracellular content leakage, and cell lysis were noted, establishing a cyto-basis of nonresistance to this pathogen. DNA gel retardation and circular dichroism analysis indicated that MP1102 interacted with DNA by binding to DNA and changing the DNA conformation, even leading to the disappearance of the helical structure. This result further supported the mechanistic basis of nonresistance via interaction with an intracellular target, which could serve as a means of secondary injury after MP1102 is transported across the membrane. Upon treatment with 2.5-5.0 mg/kg MP1102, the survival of mice challenged with S. suis was 83.3-100%. MP1102 decreased bacterial translocation in liver, lung, spleen, and blood; inhibited the release of interleukin-1ß and tumor necrosis factor-α; and relieved the lung, liver, and spleen from acute injury induced by S. suis. These results suggest that MP1102 is a potent novel antibacterial agent for the treatment of porcine streptococcal disease.


Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Streptococcus suis/efeitos dos fármacos , Estruturas Animais/microbiologia , Estruturas Animais/patologia , Animais , Bacteriólise/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , DNA Bacteriano/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos dos fármacos , Sorogrupo , Infecções Estreptocócicas/patologia , Streptococcus suis/classificação , Streptococcus suis/fisiologia , Streptococcus suis/ultraestrutura , Análise de Sobrevida
18.
mBio ; 10(1)2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30723128

RESUMO

Gram-negative bacteria have a tripartite cell envelope with the cytoplasmic membrane (CM), a stress-bearing peptidoglycan (PG) layer, and the asymmetric outer membrane (OM) containing lipopolysaccharide (LPS) in the outer leaflet. Cells must tightly coordinate the growth of their complex envelope to maintain cellular integrity and OM permeability barrier function. The biogenesis of PG and LPS relies on specialized macromolecular complexes that span the entire envelope. In this work, we show that Escherichia coli cells are capable of avoiding lysis when the transport of LPS to the OM is compromised, by utilizing LD-transpeptidases (LDTs) to generate 3-3 cross-links in the PG. This PG remodeling program relies mainly on the activities of the stress response LDT, LdtD, together with the major PG synthase PBP1B, its cognate activator LpoB, and the carboxypeptidase PBP6a. Our data support a model according to which these proteins cooperate to strengthen the PG in response to defective OM synthesis.IMPORTANCE In Gram-negative bacteria, the outer membrane protects the cell against many toxic molecules, and the peptidoglycan layer provides protection against osmotic challenges, allowing bacterial cells to survive in changing environments. Maintaining cell envelope integrity is therefore a question of life or death for a bacterial cell. Here we show that Escherichia coli cells activate the LD-transpeptidase LdtD to introduce 3-3 cross-links in the peptidoglycan layer when the integrity of the outer membrane is compromised, and this response is required to avoid cell lysis. This peptidoglycan remodeling program is a strategy to increase the overall robustness of the bacterial cell envelope in response to defects in the outer membrane.


Assuntos
Membrana Celular/metabolismo , Parede Celular/metabolismo , Escherichia coli/fisiologia , Viabilidade Microbiana , Peptidoglicano/metabolismo , Bacteriólise , Transporte Biológico , Proteínas de Escherichia coli/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Ligação às Penicilinas/metabolismo , Peptidoglicano Glicosiltransferase/metabolismo , Peptidil Transferases/metabolismo , D-Ala-D-Ala Carboxipeptidase Tipo Serina/metabolismo
19.
Int J Mol Sci ; 20(3)2019 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-30736446

RESUMO

Nasal carriage of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) represents both a source and a risk factor for subsequent infections. However, existing MRSA decolonization strategies and antibiotic treatment options are hampered by the duration of administration and particularly by the emergence of resistance. Moreover, beyond classical resistance mechanisms, functional resistance as the formation of the small-colony variant (SCV) phenotype may also impair the course and treatment of S. aureus infections. For the recombinant bacteriophage endolysin HY-133, rapid bactericidal and highly selective in vitro activities against MSSA and MRSA has been shown. In order to assess the in vitro efficacy of HY-133 against the SCV phenotype, minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were evaluated on clinical SCVs, their isogenic wild types, as well as on genetically derived and gentamicin-selected SCVs. For all strains and growth phases, HY-133 MIC and MBC ranged between 0.12 and 1 mg/L. Time-kill studies revealed a fast-acting bactericidal activity of HY-133 resulting in a ≥3 - log10 decrease in CFU/mL within 1 h compared to oxacillin, which required 4⁻24 h. Since the mode of action of HY-133 was independent of growth phase, resistance pattern, and phenotype, it is a promising candidate for future S. aureus decolonization strategies comprising rapid activity against phenotypic variants exhibiting functional resistance.


Assuntos
Bacteriófagos/fisiologia , Endopeptidases/genética , Staphylococcus aureus/virologia , Proteínas Virais/genética , Bacteriólise , Endopeptidases/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana , Tipagem Molecular , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Proteínas Virais/metabolismo
20.
Viruses ; 11(2)2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30699900

RESUMO

Phage lytic enzymes are enzymes produced by bacterial viruses, either as part of their virion to facilitate bacterial infection through local peptidoglycan degradation, or as soluble proteins to induce massive cell lysis at the end of the lytic replication cycle [...].


Assuntos
Bacteriófagos/enzimologia , Endopeptidases/farmacologia , Proteínas Virais/farmacologia , Anti-Infecciosos/farmacologia , Bactérias/efeitos dos fármacos , Infecções Bacterianas/tratamento farmacológico , Bacteriólise
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