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2.
J Microbiol Biotechnol ; 29(12): 1916-1924, 2019 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-31635440

RESUMO

Outbreaks of staphylococcal food poisoning (SFP) causing serious human diseases and economic losses have been reported globally. Furthermore, the spread of Staphylococcus aureus with increased resistance to multiple antimicrobial agents has become a major concern in the food industries and medicine. Here, we isolated an endolysin LysSAP8, as one of the peptidoglycan hydrolases, derived from the bacteriophage SAP8 infecting S. aureus. This endolysin was tagged with a 6×His at the C-terminal of the target protein and purified using affinity chromatography. LysSAP8 demonstrated lytic activity against a broad spectrum of bacteria, which included a majority of the staphylococcal strains tested in this study as well as the methicillin-resistant S. aureus (MRSA); however, no such activity was observed against other gram-positive or gram-negative bacteria. Additionally, LysSAP8 could maintain bactericidal activity until 0.1 nM working concentration and after heat treatment at 37°C for 30 min. The ability of LysSAP8 to lyse cells under varying conditions of temperature (4-43°C), pH (3-9), and NaCl concentrations (0-1,000 mM), and divalent metal ions (Ca2+, Co2+, Cu2+, Mg2+, Mn2+, Hg2+, and Zn2+) was examined. At the optimized condition, LysSAP8 could disrupt approximately 3.46 log CFU/ml of the planktonic cells in their exponential phase of growth within 30 min. In this study, we have suggested that LysSAP8 could be a potent alternative as a biocontrol agent that can be used to combat MRSA.


Assuntos
N-Acetil-Muramil-L-Alanina Amidase/farmacologia , Fagos de Staphylococcus/enzimologia , Staphylococcus aureus/efeitos dos fármacos , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Endopeptidases/farmacologia , Estabilidade Enzimática , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , N-Acetil-Muramil-L-Alanina Amidase/classificação , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/isolamento & purificação , Filogenia , Fagos de Staphylococcus/genética
3.
Int J Biol Macromol ; 138: 881-889, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31356938

RESUMO

Quantitative measurement of cell lysis against a given microbial strain is essential to calculate the antimicrobial potency of protein/peptide/nanomaterial based formulations. Fluorescence spectroscopy based measurements offer precise quantification of a process via selected flurophore emission profile. In this context, we elucidate a reliable and robust green fluorescent protein (GFP) based fluorescence spectroscopy protocol to evaluate the antimicrobial activity of proteins. The technique is based on the fact that the intensity of the GFP emission released from cells correlates with cell lysis and henceforth the antimicrobial potential of the chosen agent. The technique was demonstrated with two different families of bacteriophage endolysins (T7 and T4 endolysins) using GFP expressing E. coli cells. The GFP based method allowed the absolute quantification of T4 and T7 endolysins cell lysis characteristics at different pH, salt concentrations, and metal ions. The results obtained from GFP based fluorimetric assay were substantiated with turbidimetric assay and fluorescence microscopy. This fluorimetric method in conjugation with different GFP expressing microbial strains and antimicrobial agents can be efficiently applied as a quantification technique to precisely measure cell lysis.


Assuntos
Escherichia coli/metabolismo , Fluorometria/métodos , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Análise Espectral
4.
Microbiology ; 165(9): 1013-1023, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31264955

RESUMO

Mycobacteriophages are viruses that infect and kill mycobacteria. The peptidoglycan hydrolase, lysin A (LysA), coded by one of the most potent mycobacteriophages, D29, carries two catalytic domains at its N-terminus and a cell wall-binding domain at its C-terminus. Here, we have explored the importance of the centrally located lysozyme-like catalytic domain (LD) of LysA in phage physiology. We had previously identified an R198A substitution that causes inactivation of the LD when it is present alone on a polypeptide. Here, we show that upon incorporation of the same mutation (i.e. R350A) in full-length LysA, the protein demonstrates substantially reduced activity in vitro, even in the presence of the N-terminal catalytic domain, and has less efficient mycobacterial cell lysis ability when it is expressed in Mycobacterium smegmatis. These data suggest that an active LD is required for the full-length protein to function optimally. Moreover, a mutant D29 phage harbouring this substitution (D29R350A) in its LysA protein shows significantly delayed host M. smegmatis lysis. However, the mutant phage demonstrates an increase in burst size and plaque diameter. Taken together, our data show the importance of an intact LD region in D29 LysA PG hydrolase, and indicate an evolutionary advantage over other phages that lack such a domain in their endolysins.


Assuntos
Endopeptidases/genética , Micobacteriófagos , Mycobacterium smegmatis/virologia , N-Acetil-Muramil-L-Alanina Amidase/genética , Domínio Catalítico/genética , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/metabolismo , Mutação , Micobacteriófagos/genética , Micobacteriófagos/crescimento & desenvolvimento , Micobacteriófagos/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/metabolismo
5.
Vet Microbiol ; 234: 92-100, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213278

RESUMO

Streptococcus suis (SS) is a major pathogen in the swine industry, and also an important zoonotic agent for humans. The novel SS cell surface protein, AtlASS, comprising the special GW module and N-acetylmuramidases domain, was designated as a putative autolysin. Indeed, the atlASS deletion mutant almost completely lost its activity in Triton X-100 induced bacterial autolysis, while the wild-type and CΔatlASS strains showed significant decrease, to less than 20% of the initial OD600 values. Unexpectedly, both immunofluorescence and immunogold electron microscopy confirmed that AtlASS is mainly located in the cell division septum, suggesting autolytic activity in peptidoglycan hydrolysis may be required for cell separation, thus modulating and truncating bacterial chain length. The biofilm capacity of the AtlASS mutation was reduced ˜ 40%, as compared to the wild-type strain. The ΔatlASS strain also attenuated bacterial adherence in human brain microvessel endothelial cells (HBMECs). Furthermore, we confirmed that AtlASS has fibrinogen/fibronectin binding capacities. In mouse infection model, the AtlASS inactivation also significantly attenuated bacterial virulence and proliferation in vivo. In conclusion, these results indicate that AtlASS autolysin modulates bacterial chain length, and contributes to the full virulence of SS during infection.


Assuntos
Divisão Celular , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Streptococcus suis/química , Streptococcus suis/patogenicidade , Animais , Autólise , Biofilmes/crescimento & desenvolvimento , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , N-Acetil-Muramil-L-Alanina Amidase/genética , Organismos Livres de Patógenos Específicos , Virulência
6.
Gene ; 704: 25-30, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980942

RESUMO

Yersinia enterocolitica strains produce two chromosomal ß­lactamases, BlaA - a constitutively produced penicillinase, and BlaB - an inducible "AmpC-type" cephalosporinase. As in other members of Enterobacteriaceae, expression of ampC in Y. enterocolitica is regulated by the genes - ampR and ampD. The ampR encodes a transcriptional regulator which represses the expression of ampC and, ampD encodes a cytoplasmic N­acetyl­anhydromuramyl­l­alanine amidase which participates in recycling of peptidoglycan. Exposure of bacteria to antibiotics like imipenem and cefoxitin results in generation and accumulation of large quantities of muropeptides in cytoplasm which is beyond the recycling capability of AmpD. These muropeptides bind to AmpR, converting it into an activator of ampC expression (ampC de-repression). Earlier studies from our laboratory indicated that instead of BlaB, Y. enterocolitica biotype 1A strains produced a "BlaB-like" enzyme which was non-heterogeneous and showed a differential expression when induced with imipenem. The detection of "BlaB-like" cephalosporinase which was also induced differentially in Y. enterocolitica biotype 1A strains presented an opportunity to discern newer mechanisms, if any, which may underlie inducible expression of "AmpC-type" cephalosporinases. Thus, the objective of the present study was to understand the role of ampR and ampD in regulating differential expression of "BlaB-like" cephalosporinases in biotype 1A strains. Analysis of promoters and amino acid sequences of AmpR revealed that these were conserved in all strains of biotype 1A. Analysis of AmpD amino acid sequences revealed that five variants of AmpD were present which did not contribute to hyper-inducible production of "BlaB-like" enzyme. In-silico prediction of the mRNA secondary structures of ampD revealed significant differences, which might have affected the rate of translation of ampD and accumulation of un-recycled muropeptides inside the cell leading to hyper production of "BlaB-like" cephalosporinases in some Y. enterocolitica biotype 1A strains. The findings provide newer insights to our understanding of the mechanisms underlying regulation of expression of "AmpC-type" ß­lactamases.


Assuntos
Proteínas de Bactérias/genética , Cefalosporinase/genética , N-Acetil-Muramil-L-Alanina Amidase/genética , Yersinia enterocolitica/genética , Clonagem Molecular , Indução Enzimática/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Homologia de Sequência , Yersinia enterocolitica/classificação , Yersinia enterocolitica/enzimologia , beta-Lactamases/genética
7.
Proc Natl Acad Sci U S A ; 116(16): 7825-7830, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30940749

RESUMO

Bacteria are surrounded by a protective exoskeleton, peptidoglycan (PG), a cross-linked mesh-like macromolecule consisting of glycan strands interlinked by short peptides. Because PG completely encases the cytoplasmic membrane, cleavage of peptide cross-links is a prerequisite to make space for incorporation of nascent glycan strands for its successful expansion during cell growth. In most bacteria, the peptides consist of l-alanine, d-glutamate, meso-diaminopimelic acid (mDAP) and d-alanine (d-Ala) with cross-links occurring either between d-Ala and mDAP or two mDAP residues. In Escherichia coli, the d-Ala-mDAP cross-links whose cleavage by specialized endopeptidases is crucial for expansion of PG predominate. However, a small proportion of mDAP-mDAP cross-links also exist, yet their role in the context of PG expansion or the hydrolase(s) capable of catalyzing their cleavage is not known. Here, we identified an ORF of unknown function, YcbK (renamed MepK), as an mDAP-mDAP cross-link cleaving endopeptidase working in conjunction with other elongation-specific endopeptidases to make space for efficient incorporation of nascent PG strands into the sacculus. E. coli mutants lacking mepK and another d-Ala-mDAP-specific endopeptidase (mepS) were synthetic sick, and the defects were abrogated by lack of l,d-transpeptidases, enzymes catalyzing the formation of mDAP cross-links. Purified MepK was able to cleave the mDAP cross-links of soluble muropeptides and of intact PG sacculi. Overall, this study describes a PG hydrolytic enzyme with a hitherto unknown substrate specificity that contributes to expansion of the PG sacculus, emphasizing the fundamental importance of cross-link cleavage in bacterial peptidoglycan synthesis.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Proteínas de Bactérias , Parede Celular , N-Acetil-Muramil-L-Alanina Amidase , Aminoácidos/química , Aminoácidos/metabolismo , Bactérias/química , Bactérias/citologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Parede Celular/química , Parede Celular/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Especificidade por Substrato
8.
Infect Genet Evol ; 69: 211-215, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30710654

RESUMO

Inducible 'AmpC-type' chromosomal cephalosporinases have been reported to be differentially expressed in different biotypes of Yersinia entercolocolitica. AmpD amidases are key regulators of the expression of ampC genes in Y. entercolocolitica as their inactivation results in hyper production of AmpC. To understand the differences in regulation of ampC expression in different biotypes of Y. enterocolitica, characteristics of ampD homologs were studied in strains of Y. enterocolitica belonging to five biotypes namely 1A, 1B, 2, 3 and 4. Our results indicated that the mechanisms which regulate expression of ampC might differ in different biotypes. While a three-step regulation mechanism seemed to be functional in biotypes 2, 3 and 4, a two-step regulation mechanism using other AmiD like proteins might be functional in biotypes 1A and 1B. The existence of ampD homolog(s)-mediated expression of ampC in other members of the family Enterobacteriaceae may provide further credence to our findings.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , N-Acetil-Muramil-L-Alanina Amidase/genética , Yersiniose/microbiologia , Yersinia enterocolitica/classificação , Yersinia enterocolitica/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Genes Bacterianos , Genótipo , Humanos , Modelos Moleculares , Tipagem de Sequências Multilocus , N-Acetil-Muramil-L-Alanina Amidase/química , Filogenia , Conformação Proteica , beta-Lactamases/genética
9.
Adv Virus Res ; 103: 33-70, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30635077

RESUMO

The first steps in phage lysis involve a temporally controlled permeabilization of the cytoplasmic membrane followed by enzymatic degradation of the peptidoglycan. For Caudovirales of Gram-negative hosts, there are two different systems: the holin-endolysin and pinholin-SAR endolysin pathways. In the former, lysis is initiated when the holin forms micron-scale holes in the inner membrane, releasing active endolysin into the periplasm to degrade the peptidoglycan. In the latter, lysis begins when the pinholin causes depolarization of the membrane, which activates the secreted SAR endolysin. Historically, the disruption of the first two barriers of the cell envelope was thought to be necessary and sufficient for lysis of Gram-negative hosts. However, recently a third functional class of lysis proteins, the spanins, has been shown to be required for outer membrane disruption. Spanins are so named because they form a protein bridge that connects both membranes. Most phages produce a two-component spanin complex, composed of an outer membrane lipoprotein (o-spanin) and an inner membrane protein (i-spanin) with a predominantly coiled-coil periplasmic domain. Some phages have a different type of spanin which spans the periplasm as a single molecule, by virtue of an N-terminal lipoprotein signal and a C-terminal transmembrane domain. Evidence is reviewed supporting a model in which the spanins function by fusing the inner membrane and outer membrane. Moreover, it is proposed that spanin function is inhibited by the meshwork of the peptidoglycan, thus coupling the spanin step to the first two steps mediated by the holin and endolysin.


Assuntos
Bacteriólise/fisiologia , Bacteriófagos/fisiologia , Bactérias Gram-Negativas/virologia , Proteínas Virais/genética , Bacteriófagos/genética , Parede Celular/metabolismo , Parede Celular/virologia , DNA/genética , DNA/metabolismo , Evolução Molecular , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Fusão de Membrana/fisiologia , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Transdução de Sinais/genética , Proteínas Virais/metabolismo
10.
Immunol Lett ; 212: 125-131, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30496765

RESUMO

Staphylococcus aureus is a leading infectious cause of life-threatening diseases in human beings, with no effective vaccine available to date against this bacterium. Treatment of methicillin-resistant S. aureus (MRSA) infections has become increasingly difficult because of the emergence of multidrug-resistant isolates. Immunotherapy represents a potential approach to prevent S. aureus-related infections. Autolysin is one of the virulence factors, which controls the growth, cell lysis, daughter-cell separation, and biofilm formation. Our study focused on passive immunization against MRSA infection. Herein, rabbit polyclonal IgG was produced following the preparation of r-autolysin. Specificity of IgG against r-autolysin was investigated by ELISA and western blotting assays. IgG fraction was prepared using sulfate ammonium precipitation, and the ability of antiserum to promote phagocytosis of bacteria was assessed by opsonophagocytosis assay. Then, passive immunization of mice was carried out with polyclonal IgG fraction and, mice were sacrificed three days after challenge and their kidneys, liver, and spleen were collected. Results exhibited that the passive immunization with rabbit polyclonal anti-IgG fraction tremendously improved survival rates of mice challenged by S. aureus as well as vancomycin treatment compared with the negative control groups. In addition, a remarkable decrease in bacterial numbers was observed in mice treated with rabbit polyclonal anti-IgG. Importantly, our findings demonstrated that passive immunotherapy and antibiotic therapy lead to decreased histopathological damage in mice infected by S. aureus as compared with control groups. Our results suggested that the passive immunization may result in the introduction of excellent strategies to control infections caused by MRSA, like antibiotic therapy.


Assuntos
Antibacterianos/uso terapêutico , Imunização Passiva/métodos , Imunoglobulina G/administração & dosagem , Staphylococcus aureus Resistente à Meticilina/imunologia , N-Acetil-Muramil-L-Alanina Amidase/imunologia , Infecções Estafilocócicas/terapia , Animais , Proteínas de Bactérias/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina G/imunologia , Staphylococcus aureus Resistente à Meticilina/genética , Camundongos , N-Acetil-Muramil-L-Alanina Amidase/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Resultado do Tratamento , Fatores de Virulência/imunologia
11.
Int J Med Microbiol ; 309(1): 73-83, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30563740

RESUMO

Tuberculosis is a highly infectious disease and of high incidence in low-income countries that is caused by Mycobacterium tuberculosis (M. tuberculosis). M. tuberculosis can form biofilms in vitro and in vivo, and the cells in the biofilm can survive at high concentrations of antibiotics. CwlM is a peptidoglycan hydrolase (amidase) and can hydrolyze bacterial cell walls, and the effects of CwlM on autolysis and biofilms is worthy of in-depth study. In this study, we successfully constructed an in vitro biofilm model of M. tuberculosis and Mycobacterium smegmatis (M. smegmatis). Reverse transcription followed by real-time quantitative PCR (qPCR) revealed that the expression of cwlM in M. tuberculosis and M. smegmatis was significantly up-regulated during the middle stage of biofilm formation. Treatment with recombinant CwlM enhanced the autolytic ability of M. tuberculosis and M. smegmatis and reduced the formation of their biofilms. As M. smegmatis is a model bacterium of M. tuberculosis, we built the M. smegmatis cwlM-deletion strain MSΔ6935, whose autolytic ability, biofilm production, and eDNA and eRNA content were determined to be lower than those of its parental strain. In conclusion, the cwlM gene plays a key regulatory role in biofilm formation in M. tuberculosis and M. smegmatis. This study provided a theoretical basis for using peptidoglycan hydrolase as a target for the inhibition of biofilms.


Assuntos
Bacteriólise/genética , Biofilmes/crescimento & desenvolvimento , Mycobacterium smegmatis/genética , Mycobacterium tuberculosis/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Parede Celular/metabolismo , Humanos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Tuberculose/microbiologia
12.
mBio ; 9(6)2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30538181

RESUMO

The human eukaryotic pathogen Trichomonas vaginalis causes trichomoniasis, a prevalent sexually transmitted infection. This extracellular protozoan is intimately associated with the human vaginal mucosa and microbiota, but key aspects of the complex interactions between the parasite and the vaginal bacteria remain elusive. We report that T. vaginalis has acquired, by lateral gene transfer from bacteria, genes encoding peptidoglycan hydrolases of the NlpC/P60 family. Two of the T. vaginalis enzymes were active against bacterial peptidoglycan, retaining the active-site fold and specificity as dl-endopeptidases. The endogenous NlpC/P60 genes are transcriptionally upregulated in T. vaginalis in the presence of bacteria. The overexpression of an exogenous copy enables the parasite to outcompete bacteria from mixed cultures, consistent with the biochemical activity of the enzyme. Our study results highlight the relevance of the interactions of this eukaryotic pathogen with bacteria, a poorly understood aspect of the biology of this important human parasite.IMPORTANCE Trichomonas vaginalis is a parasitic protozoan of the human urogenital tract that causes trichomoniasis, a very common sexually transmitted disease. Despite residing extracellularly and in close association with the vaginal bacteria (i.e., the microbiota), very little is known about the nature of the parasite-bacterium interactions. Our study showed that this parasite had acquired genes from bacteria which retained their original function. They produce active enzymes capable of degrading peptidoglycan, a unique polymer of the bacterial cell envelope, helping the parasite to outcompete bacteria in mixed cultures. This study was the first to show that a laterally acquired group of genes enables a eukaryotic mucosal pathogen to control bacterial population. We highlight the importance of understanding the interactions between pathogens and microbiota, as the outcomes of these interactions are increasingly understood to have important implications on health and disease.


Assuntos
Antibiose , Bactérias/efeitos dos fármacos , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/fisiologia , Feminino , Regulação da Expressão Gênica , Humanos , N-Acetil-Muramil-L-Alanina Amidase/genética , Trichomonas vaginalis/genética , Vagina/microbiologia , Vagina/parasitologia
13.
J Antimicrob Chemother ; 73(11): 2960-2968, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30124902

RESUMO

Background: Acinetobacter baumannii is intrinsically resistant to fosfomycin; however, the mechanisms underlying this resistance are poorly understood. Objectives: To identify and characterize genes that contribute to intrinsic fosfomycin resistance in A. baumannii. Methods: More than 9000 individual transposon mutants of the A. baumannii ATCC 17978 strain (fosfomycin MIC ≥1024 mg/L) were screened to identify mutations conferring increased susceptibility to fosfomycin. In-frame deletion mutants were constructed for the identified genes and their susceptibility to fosfomycin was characterized by MIC determination and growth in the presence of fosfomycin. The effects of these mutations on membrane permeability and peptidoglycan integrity were characterized. Susceptibilities to 21 antibiotics were determined for the mutant strains. Results: Screening of the transposon library identified mutants in the ampD and anmK genes, both encoding enzymes of the peptidoglycan recycling pathway, that demonstrated increased susceptibility to fosfomycin. MIC values for in-frame deletion mutants were ≥42-fold (ampD) and ≥8-fold (anmK) lower than those for the parental strain, and growth of the mutant strains in the presence of 32 mg/L fosfomycin was significantly reduced. Neither mutation resulted in increased cell permeability; however, the ampD mutant demonstrated decreased peptidoglycan integrity. Susceptibility to 21 antibiotics was minimally affected by mutations in ampD and anmK. Conclusions: This study demonstrates that AmpD and AnmK of the peptidoglycan recycling pathway contribute to intrinsic fosfomycin resistance in A. baumannii, indicating that inhibitors of these enzymes could be used in combination with fosfomycin as a novel treatment approach for MDR A. baumannii.


Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Fosfomicina/farmacologia , Peptidoglicano/metabolismo , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Parede Celular/efeitos dos fármacos , Elementos de DNA Transponíveis , Testes de Sensibilidade Microbiana , Mutação , N-Acetil-Muramil-L-Alanina Amidase/genética , Peptidoglicano/genética
14.
Viruses ; 10(8)2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30110929

RESUMO

Mycobacteriophages are viruses that specifically infect mycobacteria, which ultimately culminate in host cell death. Dedicated enzymes targeting the complex mycobacterial cell envelope arrangement have been identified in mycobacteriophage genomes, thus being potential candidates as antibacterial agents. These comprise lipolytic enzymes that target the mycolic acid-containing outer membrane and peptidoglycan hydrolases responsive to the atypical mycobacterial peptidoglycan layer. In the recent years, a remarkable progress has been made, particularly on the comprehension of the mechanisms of bacteriophage lysis proteins activity and regulation. Notwithstanding, information about mycobacteriophages lysis strategies is limited and is mainly represented by the studies performed with mycobacteriophage Ms6. Since mycobacteriophages target a specific group of bacteria, which include Mycobacterium tuberculosis responsible for one of the leading causes of death worldwide, exploitation of the use of these lytic enzymes demands a special attention, as they may be an alternative to tackle multidrug resistant tuberculosis. This review focuses on the current knowledge of the function of lysis proteins encoded by mycobacteriophages and their potential applications, which may contribute to increasing the effectiveness of antimycobacterial therapy.


Assuntos
Membrana Celular/química , Parede Celular/química , Lisogenia , Micobacteriófagos/genética , Mycobacterium tuberculosis/virologia , Proteínas Virais/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Endopeptidases/química , Endopeptidases/genética , Endopeptidases/metabolismo , Expressão Gênica , Hidrólise , Lipase/química , Lipase/genética , Lipase/metabolismo , Micobacteriófagos/enzimologia , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/química , Peptidoglicano/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo
15.
J Bacteriol ; 200(20)2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30082459

RESUMO

The ESAT-6-like secretion system (ESS) of Staphylococcus aureus is assembled in the bacterial membrane from core components that promote the secretion of WXG-like proteins (EsxA, EsxB, EsxC, and EsxD) and the EssD effector. Genes encoding the ESS secretion machinery components, effector, and WXG-like proteins are located in the ess locus. Here, we identify essH, a heretofore uncharacterized gene of the ess locus, whose product is secreted via an N-terminal signal peptide into the extracellular medium of staphylococcal cultures. EssH exhibits two peptidoglycan hydrolase activities, cleaving the pentaglycine cross bridge and the amide bond of N-acetylmuramyl-l-alanine, thereby separating glycan chains and wall peptides with cleaved cross bridges. Unlike other peptidoglycan hydrolases, EssH does not promote the lysis of staphylococci. EssH residues Cys199 and His254, which are conserved in other CHAP domain enzymes, are required for peptidoglycan hydrolase activity and for S. aureus ESS secretion. These data suggest that EssH and its murein hydrolase activity are required for protein secretion by the ESS pathway.IMPORTANCE Gene clusters encoding WXG-like proteins and FtsK/SpoIIIE-like P loop ATPases in Firmicutes encode type 7b secretion systems (T7bSS) for the transport of select protein substrates. The Staphylococcus aureus T7bSS assembles in the bacterial membrane and promotes the secretion of WXG-like proteins and effectors. The mechanisms whereby staphylococci extend the T7SS across the bacterial cell wall envelope are not known. Here, we show that staphylococci secrete EssH to cleave their peptidoglycan, thereby enabling T7bSS transport of proteins across the bacterial cell wall envelope.


Assuntos
Proteínas de Bactérias/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Staphylococcus aureus/enzimologia , Sistemas de Secreção Tipo VII/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Família Multigênica , Mutação , N-Acetil-Muramil-L-Alanina Amidase/genética , Transporte Proteico , Staphylococcus aureus/genética , Sistemas de Secreção Tipo VII/genética
16.
Mol Microbiol ; 110(1): 114-127, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30039535

RESUMO

Chronic infection with Helicobacter pylori can lead to the development of gastric ulcers and stomach cancers. The helical cell shape of H. pylori promotes stomach colonization. Screens for loss of helical shape have identified several periplasmic peptidoglycan (PG) hydrolases and non-enzymatic putative scaffolding proteins, including Csd5. Both over and under expression of the PG hydrolases perturb helical shape, but the mechanism used to coordinate and localize their enzymatic activities is not known. Using immunoprecipitation and mass spectrometry we identified Csd5 interactions with cytosolic proteins CcmA, a bactofilin required for helical shape, and MurF, a PG precursor synthase, as well as the inner membrane spanning ATP synthase. A combination of Csd5 domain deletions, point mutations, and transmembrane domain chimeras revealed that the N-terminal transmembrane domain promotes MurF, CcmA, and ATP synthase interactions, while the C-terminal SH3 domain mediates PG binding. We conclude that Csd5 promotes helical shape as part of a membrane associated, multi-protein shape complex that includes interactions with the periplasmic cell wall, a PG precursor synthesis enzyme, the bacterial cytoskeleton, and ATP synthase.


Assuntos
Parede Celular/metabolismo , Citoesqueleto/metabolismo , Helicobacter pylori/citologia , Helicobacter pylori/enzimologia , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptídeo Sintases/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Deleção de Genes , Helicobacter pylori/genética , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/química , N-Acetil-Muramil-L-Alanina Amidase/genética , Peptídeo Sintases/química , Peptídeo Sintases/genética , Periplasma/metabolismo , Análise de Sequência de Proteína
17.
mBio ; 9(4)2018 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-30018106

RESUMO

Carboxy-terminal processing proteases (CTPs) occur in all three domains of life. In bacteria, some of them have been associated with virulence. However, the precise roles of bacterial CTPs are poorly understood, and few direct proteolytic substrates have been identified. One bacterial CTP is the CtpA protease of Pseudomonas aeruginosa, which is required for type III secretion system (T3SS) function and for virulence in a mouse model of acute pneumonia. Here, we have investigated the function of CtpA in P. aeruginosa and identified some of the proteins it cleaves. We discovered that CtpA forms a complex with a previously uncharacterized protein, which we have named LbcA (lipoprotein binding partner of CtpA). LbcA is required for CtpA activity in vivo and promotes its activity in vitro We have also identified four proteolytic substrates of CtpA, all of which are uncharacterized proteins predicted to cleave the peptide cross-links within peptidoglycan. Consistent with this, a ctpA null mutant was found to have fewer peptidoglycan cross-links than the wild type and grew slowly in salt-free medium. Intriguingly, the accumulation of just one of the CtpA substrates was required for some ΔctpA mutant phenotypes, including the defective T3SS. We propose that LbcA-CtpA is a proteolytic complex in the P. aeruginosa cell envelope, which controls the activity of several peptidoglycan cross-link hydrolases by degrading them. Furthermore, based on these and other findings, we suggest that many bacterial CTPs might be similarly controlled by partner proteins as part of a widespread mechanism to control peptidoglycan hydrolase activity.IMPORTANCE Bacterial carboxy-terminal processing proteases (CTPs) are widely conserved and have been associated with the virulence of several species. However, their roles are poorly understood, and few direct substrates have been identified in any species. Pseudomonas aeruginosa is an important human pathogen in which one CTP, known as CtpA, is required for type III secretion system function and for virulence. This work provides an important advance by showing that CtpA works with a previously uncharacterized binding partner to degrade four substrates. These substrates are all predicted to hydrolyze peptidoglycan cross-links, suggesting that the CtpA complex is an important control mechanism for peptidoglycan hydrolysis. This is likely to emerge as a widespread mechanism used by diverse bacteria to control some of their peptidoglycan hydrolases. This is significant, given the links between CTPs and virulence in several pathogens and the importance of peptidoglycan remodeling to almost all bacterial cells.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/enzimologia , Endopeptidases/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/genética , Parede Celular/metabolismo , Endopeptidases/genética , Modelos Biológicos , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/isolamento & purificação , Peptidoglicano/química , Ligação Proteica , Proteólise , Pseudomonas aeruginosa/crescimento & desenvolvimento , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo
18.
Viruses ; 10(7)2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30029517

RESUMO

The antibiotic-resistant bacterium Paenibacillus larvae is the causative agent of American foulbrood (AFB), currently the most destructive bacterial disease in honeybees. Phages that infect P. larvae were isolated as early as the 1950s, but it is only in recent years that P. larvae phage genomes have been sequenced and annotated. In this study we analyze the genomes of all 48 currently sequenced P. larvae phage genomes and classify them into four clusters and a singleton. The majority of P. larvae phage genomes are in the 38⁻45 kbp range and use the cohesive ends (cos) DNA-packaging strategy, while a minority have genomes in the 50⁻55 kbp range that use the direct terminal repeat (DTR) DNA-packaging strategy. The DTR phages form a distinct cluster, while the cos phages form three clusters and a singleton. Putative functions were identified for about half of all phage proteins. Structural and assembly proteins are located at the front of the genome and tend to be conserved within clusters, whereas regulatory and replication proteins are located in the middle and rear of the genome and are not conserved, even within clusters. All P. larvae phage genomes contain a conserved N-acetylmuramoyl-l-alanine amidase that serves as an endolysin.


Assuntos
Bacteriófagos/classificação , Bacteriófagos/genética , Genoma Viral , Paenibacillus larvae/virologia , Animais , Bacteriófagos/isolamento & purificação , Abelhas , Proteínas do Capsídeo/genética , Genômica , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Filogenia , Análise de Sequência de DNA
19.
Trends Biotechnol ; 36(9): 966-984, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29778530

RESUMO

Bacteriophages encode many distinct proteins for the successful infection of a bacterial host. Each protein plays a specific role in the phage replication cycle, from host recognition, through takeover of the host machinery, and up to cell lysis for progeny release. As the roles of these proteins are being revealed, more biotechnological applications can be anticipated. Phage-encoded proteins are now being explored for the control, detection, and typing of bacteria; as vehicles for drug delivery; and for vaccine development. In this review, we discuss how engineering approaches can be used to improve the natural properties of these proteins and set forth the most innovative applications that demonstrate the unlimited biotechnological potential held by phage-encoded proteins.


Assuntos
Bacteriófagos/química , Técnicas Biossensoriais/métodos , Biotecnologia/métodos , Sistemas de Liberação de Medicamentos/métodos , Proteoma/química , Biologia Sintética/métodos , Proteínas Virais/química , Bactérias/virologia , Bacteriófagos/genética , Bacteriófagos/metabolismo , Técnicas Biossensoriais/instrumentação , Peptídeos Penetradores de Células/genética , Peptídeos Penetradores de Células/metabolismo , Endopeptidases/genética , Endopeptidases/metabolismo , Glicoconjugados/síntese química , Glicoconjugados/imunologia , Humanos , Lisogenia/genética , Interações Microbianas/genética , N-Acetil-Muramil-L-Alanina Amidase/genética , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Proteoma/metabolismo , Proteoma/farmacologia , Vacinas/biossíntese , Proteínas Virais/metabolismo , Proteínas Virais/farmacologia , Replicação Viral
20.
Biochim Biophys Acta Gen Subj ; 1862(7): 1635-1643, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29674124

RESUMO

The type VI secretion system (T6SS) is considered as one of the key competition strategies by injecting toxic effectors for intestinal pathogens to acquire optimal colonization in host gut, a microenviroment with high-density polymicrobial community where bacteria compete for niches and resources. Enterotoxigenic Escherichia coli (ETEC), a major cause of infectious diarrhea in human and animals, widely encode T6SS clusters in their genomes. In this report, we first identified VT1, a novel amidase effector in ETEC, significantly hydrolyzed D-lactyl-L-Ala crosslinks between N-acetylmuramoyl and L-Ala in peptidoglycan. Further study showed that the VT1/VTI1 effector/immunity pair is encoded within a typical vgrG island, and plays a critical role for the successful establishment of ETEC in host gut. Numerous putative effectors with diverse toxin domains were found by retrieving vgrG islands in pathogenic E. coli, and designated as VT modules. Therein, VT5, a lysozyme-like effector widely encoded in ETEC, was confirmed to effectively kill adjacent cells, suggesting that VT toxin modules may be critical for pathogenic E. coli to seize a significantly competitive advantage for optimal intestinal colonization. To expand our analyses for large-scale search of VT antibacterial effectors based on vgrG island, >200 predicted effectors from 20 bacterial species were found and classified into 11 predicted toxins. This work reports a new retrieval strategy for screening T6SS effectors, and provides an example how pathogenic bacteria antagonize and displace commensal microbiome to successfully colonize in the host niches through a T6SS-dependent manner.


Assuntos
Escherichia coli Enterotoxigênica/genética , Proteínas de Escherichia coli/genética , N-Acetil-Muramil-L-Alanina Amidase/genética , Sistemas de Secreção Tipo VI/genética , Animais , Antibiose , Transporte Biológico , Parede Celular/metabolismo , Escherichia coli Enterotoxigênica/metabolismo , Proteínas de Escherichia coli/metabolismo , Microbioma Gastrointestinal , Bactérias Gram-Negativas/genética , Bactérias Gram-Negativas/metabolismo , Camundongos , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Domínios Proteicos
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