RESUMO
BACKGROUND: Campylobacter is a genus of Gram-negative bacteria. It is considered one of the leading cause of diarrheal illness worldwide. Incidence of human campylobacteriosis has increased significantly during recent decades; the increase has been directly linked with the advancement made in Campylobacter detection methods. C. jejuni and C. coli are the two major human-pathogenic species that can colonize poultry and cause foodborne illness and outbreak. OBJECTIVE: This study was carried out to rapidly identify Campylobacter-like isolates recovered from raw poultry products by matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) MS and DNA sequencing of 16S and 23S ribosomal RNA (rRNA) regions. METHODS: Twenty-seven isolates of Campylobacter-like organisms were isolated from raw poultry products and cultured on blood agars for MALDI-TOF MS analysis and DNA isolation. For each isolate, one to two colonies were directly spotted on the VITEK MS analysis. Genomic DNA was extracted from overnight bacterial culture. Afterward, two-directional Sanger sequencing of rRNA gene was performed to confirm species identification on an ABI 3500xL Genetic Analyzer. RESULTS: The VITEK MS could provide species-level identification for all 27 Campylobacter-like bacterial isolates (including 13 isolates as C. jejuni and 14 isolates as C. coli). Species identification attained by the VITEK MS matched completely with the rRNA sequence characterization data. CONCLUSIONS: Based on the restricted number of target strains and agar plates, MALDI-TOF MS and rRNA sequence analysis can be used for rapid identification of C. jejuni and C. coli isolates. HIGHLIGHTS: MALDI-TOF MS and rRNA sequencing can provide species identification of C. jejuni and C. coli isolates.
Assuntos
Campylobacter coli , Campylobacter jejuni , Técnicas de Tipagem Bacteriana , Campylobacter coli/genética , Campylobacter jejuni/genética , Humanos , Produtos Avícolas , RNA Ribossômico 16S , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Sec-dependent protein translocation is an essential process in bacteria. SecA is a key component of the translocation machinery and has multiple domains that interact with various ligands. SecA acts as an ATPase motor to drive the precursor protein/peptide through the SecYEG protein translocation channels. As SecA is unique to bacteria and there is no mammalian counterpart, it is an ideal target for the development of new antimicrobials. Several reviews detail the assays for ATPase and protein translocation, as well as the search for SecA inhibitors. Recent studies have shown that, in addition to the SecA-SecYEG translocation channels, there are SecA-only channels in the lipid bilayers, which function independently from the SecYEG machinery. This mini-review focuses on recent advances on the newly developed SecA inhibitors that allow the evaluation of their potential as antimicrobial agents, as well as a fundamental understanding of mechanisms of SecA function(s). These SecA inhibitors abrogate the effects of efflux pumps in both Gram-positive and Gram-negative bacteria. We also discuss recent findings that SecA binds to ribosomes and nascent peptides, which suggest other roles of SecA. A model for the multiple roles of SecA is presented.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Canais de Translocação SEC/antagonistas & inibidores , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Antibacterianos/química , Bactérias/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Canais de Translocação SEC/genética , Canais de Translocação SEC/metabolismo , Proteínas SecARESUMO
Background: Lysinibacillus fusiformis is a Gram-positive, rod-shaped bacterium that can cause tropical ulcers, severe sepsis, and respiratory illnesses in humans. Objective: In this study, we analyzed cosmetic samples for the presence of human pathogenic microorganisms. Methods: Five unopened jars of exfoliating cream were examined initially by microbiological methods. Afterward, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS and 16S ribosomal RNA (rRNA) sequencing techniques were applied to characterize the recovered isolates. Results: Of the eight recovered Gram-positive bacterial subs, the VITEK® MS could provide genus-level identification to five subs and species-level identification to two subs (L. fusiformis with a 99.9% confidence value); one sub was unidentified. Subsequently, the deoxyriboneucleic acid sequencing of the 16S rRNA gene was done on an ABI 3500XL Genetic Analyzer for the confirmation of species identification. An analysis of sequencing data revealed a complete absence of genetic variation among the eight subs sequenced at this locus and confirmed the eight bacterial subs to be L. fusiformis, as their respective 16S rRNA sequences were identical to the available sequence in public domain (GenBank accession No. KU179364). Conclusions: Our results suggest that the VITEK MS and the 16S rRNA sequencing can be used for the identification of human pathogenic bacteria of public health importance. Highlights: We characterized eight isolates of Lysinibacillus spp. from cosmetics by MALDI-TOF MS and 16S rRNA sequence analyses.
Assuntos
Bacillaceae/isolamento & purificação , Cosméticos , DNA Bacteriano/genética , RNA Ribossômico 16S/genética , Bacillaceae/genética , Técnicas de Tipagem Bacteriana/métodos , Humanos , Análise de Sequência de DNA , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Campylobacteriosis is an infectious gastrointestinal disease caused by Campylobacter spp. In most cases, it is either underdiagnosed or underreported due to poor diagnostics and limited databases. Several DNA-based molecular diagnostic techniques, including 16S ribosomal RNA (rRNA) sequence typing, have been widely used in the species identification of Campylobacter. Nevertheless, these assays are time-consuming and require a high quality of bacterial DNA. Matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) MS is an emerging diagnostic technology that can provide the rapid identification of microorganisms by using their intact cells without extraction or purification. In this study, we analyzed 24 American Type Culture Collection reference isolates of 16 Campylobacter spp. and five unknown clinical bacterial isolates for rapid identification utilizing two commercially available MADI-TOF MS platforms, namely the bioMérieux VITEK® MS and Bruker Biotyper systems. In addition, 16S rRNA sequencing was performed to confirm the species-level identification of the unknown clinical isolates. Both MALDI-TOF MS systems identified the isolates of C. jejuni, C. coli, C. lari, and C. fetus. The results of this study suggest that the MALDI-TOF MS technique can be used in the identification of Campylobacter spp. of public health importance.
Assuntos
Técnicas de Tipagem Bacteriana/métodos , Campylobacter/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Campylobacter/genética , Infecções por Campylobacter/diagnóstico , Gatos , Galinhas , Humanos , Lagartos , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Ovinos , SuínosRESUMO
Staphylococcus spp. is considered as one of the most common human-pathogenic bacteria, causing illnesses ranging from nonthreatening skin infections to lethal diseases, including sepsis, pneumonia, bloodstream infections, and food poisoning. The emergence of methicillin-resistant Staphylococcus aureus strains has increased morbidity and mortality and resulted in a major healthcare burden worldwide. Single and multilocus sequence typing have been extensively used in the identification of Staphylococcus species. Nevertheless, these assays are relatively time-consuming and require high-quality DNA. Matrix-assisted laser desorption ionization-time-of-flight has been used recently for the rapid identification of several bacterial species. In this study, we have examined 47 Staphylococcus isolates recovered from food, environment, clinical samples, cosmetic products, and a medical device and 3 American Type Culture Collection Staphylococcus reference isolates using bioMérieux VITEK MS and VITEK 2 systems to determine isolate identity. Sequencing of the 16S ribosomal RNA gene was performed to confirm and compare the species identification data generated by VITEK 2 and VITEK MS systems. Although the VITEK 2 system could not identify one of the isolates, VITEK MS identified all 50 Staphylococcus spp. isolates tested. Results of this study clearly suggest that VITEK MS can be used in the rapid identification of Staphylococcus isolates of public health importance.
Assuntos
Técnicas Bacteriológicas/métodos , Microbiologia de Alimentos/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Staphylococcus aureus/isolamento & purificação , Cosméticos , Equipamentos e Provisões Hospitalares/microbiologia , Humanos , RNA Ribossômico 16S/genética , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/genéticaRESUMO
Campylobacter is one of the leading causes of foodborne travelers' diarrhea worldwide. Although a large number cases of campylobacteriosis go undiagnosed or unreported, it is considered as the second most common foodborne illness in the USA affecting over 1.3 million individuals every year. Of various Campylobacter species, C. jejuni, C. coli, and C. lari have been accounted for causing more than 99% of human infections. Thus, there is a need to have efficient isolation method to protect public health on food safety and monitoring the burden of campylobacteriosis. Nevertheless, it is a challenging task as the exposure of environmental stress during isolation process makes Campylobacter species less culturable. Sixteen Campylobacter spp. were used to evaluate the current protocols used in Campylobacter isolation. For optimal recovery, a range of growth media (Bolton, Columbia, Muller Hinton, CVA Campy and mCCDA), incubation temperatures, and additional supplements (including antibiotics) were tested. Blood agars without antibiotics were sufficient for the initial recovery. Afterward, the isolates could grow on agars without any supplements, and in some cases growth was observed in the presence of antibiotics. Incubation at 37 °C was found to be the optimal temperature for the recovery and the growth of most species. Additionally, a food adulteration study was also carried out by artificially contaminating three food matrices that included egg, milk, and infant cereal, with two isolates of C. jejuni and C. coli. Results of this study should provide the insight for culturing and isolation of Campylobacter from food and other sources.
Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter/crescimento & desenvolvimento , Meios de Cultura/química , Animais , Campylobacter/classificação , Campylobacter/genética , Campylobacter/isolamento & purificação , Bovinos , Meios de Cultura/metabolismo , Ovos/microbiologia , Contaminação de Alimentos/análise , Humanos , Leite/microbiologiaRESUMO
SecA is an essential protein in the major bacterial Sec-dependent translocation pathways. E. coli SecA has 901 aminoacyl residues which form multi-functional domains that interact with various ligands to impart function. In this study, we constructed and purified tethered C-terminal deletion fragments of SecA to determine the requirements for N-terminal domains interacting with lipids to provide ATPase activity, pore structure, ion channel activity, protein translocation and interactions with SecYEG-SecDFâ¢YajC. We found that the N-terminal fragment SecAN493 (SecA1-493) has low, intrinsic ATPase activity. Larger fragments have greater activity, becoming highest around N619-N632. Lipids greatly stimulated the ATPase activities of the fragments N608-N798, reaching maximal activities around N619. Three helices in amino-acyl residues SecA619-831, which includes the "Helical Scaffold" Domain (SecA619-668) are critical for pore formation, ion channel activity, and for function with SecYEG-SecDFâ¢YajC. In the presence of liposomes, N-terminal domain fragments of SecA form pore-ring structures at fragment-size N640, ion channel activity around N798, and protein translocation capability around N831. SecA domain fragments ranging in size between N643-N669 are critical for functional interactions with SecYEG-SecDFâ¢YajC. In the presence of liposomes, inactive C-terminal fragments complement smaller non-functional N-terminal fragments to form SecA-only pore structures with ion channel activity and protein translocation ability. Thus, SecA domain fragment interactions with liposomes defined critical structures and functional aspects of SecA-only channels. These data provide the mechanistic basis for SecA to form primitive, low-efficiency, SecA-only protein-conducting channels, as well as the minimal parameters for SecA to interact functionally with SecYEG-SecDFâ¢YajC to form high-efficiency channels.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Canais de Translocação SEC/metabolismo , Adenosina Trifosfatases/química , Animais , Proteínas de Bactérias/química , Escherichia coli/química , Transporte de Íons , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Transporte Proteico , Canais de Translocação SEC/química , Proteínas SecA , Especificidade por Substrato , XenopusRESUMO
The genus Bacillus is a group of gram-positive endospore-forming bacteria that can cause food poisoning and diarrheal illness in humans. A wide range of food products have been linked to foodborne outbreaks associated with these opportunistic pathogens. The U.S. Food and Drug Administration recommends (in their Bacteriological Analytical Manual) the use of Bacara or mannitol egg yolk polymyxin (MYP) agar plates and the most-probable-number (MPN) method for enumeration and confirmation of Bacillus cereus and related species isolated from foods, sporadic cases, outbreaks, and routine environmental surveillance samples. We performed a comparative analysis of two chromogenic media (Bacara and Brilliance) and two traditional media (MYP and polymyxin egg yolk mannitol bromothymol blue agar [PEMBA]) for the isolation and enumeration of 16 Bacillus species under modified growth conditions that included pH, temperature, and dilution factor. A total of 50 environmental, food, and American Type Culture Collection reference isolates from 16 distinct Bacillus species were evaluated. A food adulteration experiment also was carried out by artificially adulterating two baby food matrices with two isolates each of B. cereus and Bacillus thuringiensis . Our results clearly indicated that chromogenic plating media (Bacara and Brilliance) are better than conventional standard media (MYP and PEMBA) for the detection and enumeration of B. cereus in foods and other official regulatory samples. The comparison of the two chromogenic media also indicated that Brilliance medium to be more efficient and selective for the isolation of Bacillus.
Assuntos
Bacillus , Microbiologia de Alimentos , Ágar , Bacillus cereus/isolamento & purificação , Meios de Cultura , Contaminação de Alimentos , HumanosRESUMO
SecA is an essential component in the bacterial Sec-dependent protein translocation process. We previously showed that in addition to the ubiquitous, high-affinity SecYEG-SecDF·YajC protein translocation channel, there is a low-affinity SecA-only channel that elicits ion channel activity and promotes protein translocation. The SecA-only channels are less efficient, and like Prl suppressors, lack signal peptide specificity; they function in the absence of signal peptides. The presence of SecYEG-SecDF·YajC alters the sensitivity of ATPase inhibitor Rose Bengal. In this study, we found that the suppressor membranes are much more resistant to inhibition by Rose Bengal. Similar results have been found for a SecA-specific inhibitor. Moreover, biphasic responses of inhibition of ion current and protein translocation activities were observed for many PrlA/SecY and PrlG/SecE suppressor membranes, with a low IC50 value similar to that of the SecA-only channels and a very high IC50. However, the suppressor strains are as sensitive to the inhibitor as the parental strain, suggesting that SecA-only channels have some essential physiological function(s) in the cells that are inhibited by the specific SecA inhibitor.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Transporte Proteico/fisiologia , Rosa Bengala/administração & dosagem , Canais de Translocação SEC/antagonistas & inibidores , Canais de Translocação SEC/metabolismo , Escherichia coli/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas SecARESUMO
With the widespread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram-negative bacteria. Along this line, the identification of viable targets is a critical first step. The protein translocase SecA is commonly believed to be an excellent target for the development of broad-spectrum antimicrobials. In recent years, we developed three structural classes of SecA inhibitors that have proven to be very effective against Gram-positive bacteria. However, we have not achieved the same level of success against Gram-negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain an understanding as to why these inhibitors were not effective against Gram-negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram-positive and Gram-negative bacteria is in the additional permeability barrier posed by the outer membrane of Gram-negative bacteria. We also found that the expression of efflux pumps, which are responsible for multidrug resistance (MDR), have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor-resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA-azi-9 mutation increased the resistance, providing genetic evidence that SecA is indeed the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram-negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Canais de Translocação SEC/antagonistas & inibidores , Adenosina Trifosfatases/metabolismo , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Bactérias Gram-Negativas/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Canais de Translocação SEC/metabolismo , Proteínas SecA , Relação Estrutura-AtividadeRESUMO
SecA, a key component of the bacterial Sec-dependent secretion pathway, is an attractive target for the development of new antimicrobial agents. Through a combination of virtual screening and experimental exploration of the surrounding chemical space, we identified a hit bistriazole SecA inhibitor, SCA-21, and studied a series of analogues by systematic dissections of the core scaffold. Evaluation of these analogues allowed us to establish an initial structure-activity relationship in SecA inhibition. The best compounds in this group are potent inhibitors of SecA-dependent protein-conducting channel activity and protein translocation activity at low- to sub-micromolar concentrations. They also have minimal inhibitory concentration (MIC) values against various strains of bacteria that correlate well with the SecA and protein translocation inhibition data. These compounds are effective against methicillin-resistant Staphylococcus aureus strains with various levels of efflux pump activity, indicating the capacity of SecA inhibitors to null the effect of multidrug resistance. Results from studies of drug-affinity-responsive target stability and protein pull-down assays are consistent with SecA as a target for these compounds.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Pirimidinas/farmacologia , Triazóis/farmacologia , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Proteínas de Membrana Transportadoras/metabolismo , Staphylococcus aureus Resistente à Meticilina/enzimologia , Testes de Sensibilidade Microbiana , Estrutura Molecular , Pirimidinas/química , Canais de Translocação SEC , Proteínas SecA , Relação Estrutura-Atividade , Triazóis/químicaRESUMO
Due to the emergence and rapid spread of drug resistance in bacteria, there is an urgent need for the development of novel antimicrobials. SecA, a key component of the general bacterial secretion system required for viability and virulence, is an attractive antimicrobial target. Earlier we reported that systematical dissection of a SecA inhibitor, Rose Bengal (RB), led to the development of novel small molecule SecA inhibitors active against Escherichia coli and Bacillus subtilis. In this study, two potent RB analogs were further evaluated for activities against methicillin-resistant Staphylococcus aureus (MRSA) strains and for their mechanism of actions. These analogs showed inhibition on the ATPase activities of S. aureus SecA1 (SaSecA1) and SecA2 (SaSecA2), and inhibition of SaSecA1-dependent protein-conducting channel. Moreover, these inhibitors reduce the secretion of three toxins from S. aureus and exert potent bacteriostatic effects against three MRSA strains. Our best inhibitor SCA-50 showed potent concentration-dependent bactericidal activity against MRSA Mu50 strain and very importantly, 2-60 fold more potent inhibitory effect on MRSA Mu50 than all the commonly used antibiotics including vancomycin, which is considered the last resort option in treating MRSA-related infections. Protein pull down experiments further confirmed SaSecA1 as a target. Deletion or overexpression of NorA and MepA efflux pumps had minimal effect on the antimicrobial activities against S. aureus, indicating that the effects of SecA inhibitors were not affected by the presence of these efflux pumps. Our studies show that these small molecule analogs target SecA functions, have potent antimicrobial activities, reduce the secretion of toxins, and have the ability to overcome the effect efflux pumps, which are responsible for multi-drug resistance. Thus, targeting SecA is an attractive antimicrobial strategy against MRSA.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Anti-Infecciosos/química , Proteínas de Bactérias/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/enzimologia , Rosa Bengala/química , Adenosina Trifosfatases/metabolismo , Antibacterianos/química , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Cinética , Luz , Proteínas de Membrana Transportadoras/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Oxirredução , Estrutura Terciária de Proteína , Rosa Bengala/farmacologia , Canais de Translocação SEC , Proteínas SecA , Staphylococcus aureus/efeitos dos fármacosRESUMO
Establishing recordable channels in membranes of oocytes formed by expressing exogenous complementary DNA (cDNA) or messenger RNA (mRNA) has contributed greatly to understanding the molecular mechanisms of channel functions. Here, we report the extension of this semi-physiological system for monitoring the channel activity of preassembled membrane proteins in single cell oocytes by injecting reconstituted proteoliposomes along with substrates or regulatory molecules. We build on the observation that SecA from various bacteria forms active protein-conducting channels with injection of proteoliposomes, protein precursors, and ATP-Mg(2+). Such activity was enhanced by reconstituted SecYEG-SecDFâ¢YajC liposome complexes that could be monitored easily and efficiently, providing correlation of in vitro and intact cell functionality. In addition, inserting reconstituted gap junction Cx26 liposomes into the oocytes allowed the demonstration of intracellular/extracellular Ca(2+)-regulated hemi-channel activities. The channel activities can be detected rapidly after injection, can be monitored for various effectors, and are dependent on specific exogenous lipid compositions. This simple and effective functional system with low endogenous channel activity should have broad applications for monitoring the specific channel activities of complex interactions of purified membrane proteins with their effectors and regulatory molecules.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Conexinas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Oócitos/metabolismo , Proteolipídeos/metabolismo , Análise de Célula Única , Animais , Linhagem Celular , Conexina 26 , Insetos , Camundongos , Canais de Translocação SEC , Proteínas SecA , Xenopus laevis/metabolismoRESUMO
SecA is an essential protein possessing ATPase activity in bacterial protein translocation for which Rose Bengal (RB) is the first reported sub-micromolar inhibitor in ATPase activity and protein translocation. Here, we examined the mechanisms of inhibition on various forms of SecA ATPase by conventional enzymatic assays, and by monitoring the SecA-dependent channel activity in the semi-physiological system in cells. We build on the previous observation that SecA with liposomes form active protein-conducting channels in the oocytes. Such ion channel activity is enhanced by purified Escherichia coli SecYEG-SecDF·YajC liposome complexes. Inhibition by RB could be monitored, providing correlation of in vitro activity and intact cell functionality. In this work, we found the intrinsic SecA ATPase is inhibited by RB competitively at low ATP concentration, and non-competitively at high ATP concentrations while the translocation ATPase with precursors and SecYEG is inhibited non-competitively by RB. The Inhibition by RB on SecA channel activity in the oocytes with exogenous ATP-Mg(2+), mimicking translocation ATPase activity, is also non-competitive. The non-competitive inhibition on channel activity has also been observed with SecA from other bacteria which otherwise would be difficult to examine without the cognate precursors and membranes.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Corantes Fluorescentes/farmacologia , Rosa Bengala/farmacologia , Adenosina Trifosfatases/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Proteínas de Membrana Transportadoras/metabolismo , Transporte Proteico/efeitos dos fármacos , Canais de Translocação SEC , Proteínas SecA , XenopusRESUMO
SecA is an essential multifunctional protein for the translocation of proteins across bacterial membranes. Though SecA is known to function in the membrane, the detailed mechanism for this process remains unclear. In this study we constructed a series of SecA N-terminal deletions and identified two specific domains crucial for initial SecA/membrane interactions. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of a SecA ts mutant. However, deletions of N-terminal aminoacyl residues 23-25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity. All together, the results indicate that the N-terminal aminoacyl residues 23-25 play a critical role for SecA binding to membranes and that the N-terminal limit of SecA for activity is at the 25th amino acid.
Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Genes Bacterianos , Teste de Complementação Genética , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Proteínas Ligadas a Lipídeos/química , Proteínas Ligadas a Lipídeos/genética , Proteínas Ligadas a Lipídeos/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Membranas/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Deleção de SequênciaRESUMO
SecA is an essential ATPase in bacterial Sec-dependent protein translocation pathway, and equilibrates between monomers and dimers in solution. The question of whether SecA functions as monomers or dimers in membranes during the protein translocation is controversial. We previously constructed a tail-to-head SecAA tandem dimer, and showed it is fully functional by complementation in vivo and protein translocation in vitro, indicating that SecA can function at least as a dimer in the membrane without dissociating into monomers. In this study, we further constructed genetically a tail-to-head SecAAA trimer, which is functional in complementing a temperature-sensitive secA mutant. The purified SecAAA trimer per protomer is fully active as SecAA tandem dimers in ATPase activity, in protein translocation in vitro and in ion channel activities in the oocytes. With these functional tail-to-head trimer SecAAA and tandem SecAA, we examined their surface topology in the presence of liposomes using AFM. As expected, the soluble SecAAA without lipids are larger than SecAA. However, the ring/pore structures of SecAAA trimers were, surprisingly, almost identical to the SecA 2-monomers and SecAA dimers, raising the intriguing possibility that the SecA may exist and function as hexamer ring-structures in membranes. Cross-linking with formaldehyde showed that SecA, SecAA and SecAAA could form larger oligomers, including the hexamers. The molecular modeling simulation shows that both tail-to-head and tail-to-tail hexamers in the membranes are possible.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Proteínas de Membrana Transportadoras/química , Multimerização Proteica , Adenosina Trifosfatases/genética , Animais , Proteínas de Bactérias/genética , Reagentes de Ligações Cruzadas/química , Proteínas de Membrana Transportadoras/genética , Microscopia de Força Atômica , Oócitos , Canais de Translocação SEC , Proteínas SecA , XenopusRESUMO
Previous studies showed that Escherichia coli membranes depleted of SecYEG are capable of translocating certain precursor proteins, but not other precursors such as pPhoA, indicating a differential requirement for SecYEG. In this study, we examined the role of SecYEG in pPhoA translocation using a purified reconstituted SecA-liposomes system. We found that translocation of pPhoA, in contrast to that of pOmpA, requires the presence of purified SecYEG. A differential specificity of the SecYEG was also revealed in its interaction with SecA: EcSecYEG did not enhance SecA-mediated pOmpA translocation by purified SecA either from Pseudomonas aeruginosa or Bacillus subtilis. Neither was SecYEG required for eliciting ion channel activity, which could be opened by unfolded pPhoA or unfolded PhoA. Addition of the SecYEG complex did restore the specificity of signal peptide recognition in the ion-channel activity. We concluded that SecYEG confers specificity in interacting with protein precursors and SecAs.
Assuntos
Proteínas de Escherichia coli/metabolismo , Animais , Escherichia coli/metabolismo , Transporte Proteico , Canais de Translocação SEC , XenopusRESUMO
SecA, a key component of bacterial Sec-dependent secretion pathway, is an attractive target for exploring novel antimicrobials. Rose bengal (RB), a polyhalogenated fluorescein derivative, was found from our previous study as a potent SecA inhibitor. Here we describe the synthesis and structure-activity relationships (SAR) of 23 RB analogues that were designed by systematical dissection of RB. Evaluation of these analogues allowed us to establish an initial SAR in SecA inhibition. The antimicrobial effects of these SecA inhibitors are confirmed in experiments using E.â coli and B.â subtilis.
Assuntos
Adenosina Trifosfatases/antagonistas & inibidores , Anti-Infecciosos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/síntese química , Rosa Bengala/química , Adenosina Trifosfatases/metabolismo , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Proteínas de Bactérias/metabolismo , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/enzimologia , Proteínas de Membrana Transportadoras/metabolismo , Ligação Proteica , Rosa Bengala/metabolismo , Rosa Bengala/farmacologia , Canais de Translocação SEC , Proteínas SecA , Relação Estrutura-AtividadeRESUMO
Previous work showed that SecA alone can promote protein translocation and ion-channel activity in liposomes, and that SecYEG increases efficiency as well as signal peptide specificity. We now report that SecDF·YajC further increases translocation and ion-channel activity. These activities of reconstituted SecA-SecYEG-SecDF·YajC-liposome are almost the same as those of native membranes, indicating the transformation of reconstituted functional high-affinity protein-conducting channels from the low-affinity SecA-channels.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Adenosina Trifosfatases/química , Animais , Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Canais Iônicos/química , Lipossomos/química , Proteínas de Membrana/química , Proteínas de Membrana Transportadoras/química , Transporte Proteico , Canais de Translocação SEC , Proteínas SecA , Xenopus laevisRESUMO
We have developed a sensitive method to detect the opening of SecA-dependent, protein-conducting channels in Xenopus oocytes. In this study, we determined the ionic current activities of the SecA-dependent channel from membrane vesicles depleted of SecYEG. We found that these SecYEG-depleted membranes produced SecA-dependent ionic currents in the oocytes, as did membranes containing SecYEG. However, reconstituted membranes depleted of SecYEG required higher concentrations of SecA to elicit ionic currents like those in membranes containing SecYEG. In contrast to membranes containing SecYEG, the proofreading capacity of signal peptides was lost for those membranes lacking SecYEG. These findings are consistent with loss of signal peptide specificity in channel activity from membranes of SecY suppressor or SecY plug domain mutants. The signal peptide specificity of the reconstituted membranes, like SecA-liposomes, can be restored by the addition of SecYEG proteoliposomes. On the other hand, the channel activity efficiency of reconstituted membranes was fully restored, while SecA-liposomes could only be partially enhanced by the addition of SecYEG, indicating that, in addition to SecYEG, other membrane proteins contribute to the efficiency of channel activity. The SecA-dependent channels in membranes that lacked SecYEG also lost ion selectivity to monovalent cations but retained selective permeability to large anions. Thus, the electrophysiological evidence presented here indicates that SecYEG is not obligatory for the channel activity of Escherichia coli membranes, as previously shown for protein translocation, and that SecYEG is important for maintenance of the efficiency and specificity of SecA-dependent channels.