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1.
Biomed Res Int ; 2021: 4134713, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34540993

RESUMO

Previous work stated that Khaya senegalensis, Anacardium ouest L., Pterocarpus erinaceus, Diospyros mespiliformis, Ocimum gratissimum, Manihot esculenta, Vernonia amygdalina Delile, and Daniellia oliveri have a great potential for the fight against infectious diarrhea. However, data on their antibacterial activity on strains of bacteria responsible for infectious diarrhea are not available. This study is aimed at elucidating the mechanism of action of the antibacterial effect of these plants on some bacterial strains responsible for diarrheal infections. The design of the study included first evaluating the degree of sensitivity of Salmonella typhimurium 14028, Escherichia coli ATCC 25922, Shigella spp., and Salmonella spp. strains to aqueous and hydroethanolic extracts of each plant, followed by the determination of minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and antibiotic power (Pa). This screening was completed with the evaluation of the possible mode of action of the extracts by testing the membrane permeability of these bacterial strains. The data collected indicate that the bacterial strains tested were sensitive to the extracts to varying degrees, except Cassia sieberiana DC and Pseudocedrela kotschyi extracts. For the active extracts, inhibition diameters ranged from 18.33 mm to 7 mm. With the exception of Escherichia coli, all strains were sensitive to the aqueous and hydroethanolic extracts of Anacardium occidentale. MICs vary between 3.37 and 25 mg/ml. Membrane permeability test data show that all active extracts affect the bacterial strains tested by attacking the stability of their outer membrane. For all active extracts, the high percentage of membrane destabilization of the bacteria is significantly (p < 0.05) better than that of cefixime used as a reference. Thus, it appears that these extracts can destroy Gram-negative bacteria and increase the fluidity and permeability of their cytoplasmic membrane. The knowledge of the mechanism of action of these extracts is an interesting contribution to the fundamental knowledge on the alternative that medicinal plants represent to antibiotics. These extracts can be used in the management of infectious diarrhea.


Assuntos
Membrana Externa Bacteriana/efeitos dos fármacos , Disenteria/tratamento farmacológico , Extratos Vegetais/farmacologia , África Ocidental , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Membrana Externa Bacteriana/metabolismo , Diarreia/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Humanos , Medicina Tradicional Africana/métodos , Testes de Sensibilidade Microbiana , Plantas Medicinais , Salmonella/efeitos dos fármacos , Shigella/efeitos dos fármacos
2.
Science ; 373(6552)2021 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-34437126

RESUMO

Activation of cell-autonomous defense by the immune cytokine interferon-γ (IFN-γ) is critical to the control of life-threatening infections in humans. IFN-γ induces the expression of hundreds of host proteins in all nucleated cells and tissues, yet many of these proteins remain uncharacterized. We screened 19,050 human genes by CRISPR-Cas9 mutagenesis and identified IFN-γ-induced apolipoprotein L3 (APOL3) as a potent bactericidal agent protecting multiple non-immune barrier cell types against infection. Canonical apolipoproteins typically solubilize mammalian lipids for extracellular transport; APOL3 instead targeted cytosol-invasive bacteria to dissolve their anionic membranes into human-bacterial lipoprotein nanodiscs detected by native mass spectrometry and visualized by single-particle cryo-electron microscopy. Thus, humans have harnessed the detergent-like properties of extracellular apolipoproteins to fashion an intracellular lysin, thereby endowing resident nonimmune cells with a mechanism to achieve sterilizing immunity.


Assuntos
Apolipoproteínas L/metabolismo , Membrana Celular/metabolismo , Citosol/microbiologia , Bactérias Gram-Negativas/fisiologia , Interferon gama/imunologia , Apolipoproteínas L/química , Apolipoproteínas L/genética , Membrana Externa Bacteriana/metabolismo , Bacteriólise , Sistemas CRISPR-Cas , Membrana Celular/química , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular , Células Cultivadas , Detergentes/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Edição de Genes , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/patogenicidade , Bactérias Gram-Negativas/ultraestrutura , Humanos , Imunidade Inata , Lipoproteínas/química , Viabilidade Microbiana , Antígenos O/metabolismo , Domínios Proteicos , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Salmonella typhimurium/fisiologia , Salmonella typhimurium/ultraestrutura , Solubilidade
3.
Int J Mol Sci ; 22(11)2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-34205995

RESUMO

The increasing spread of multidrug-resistant pathogenic bacteria is one of the major threats to public health worldwide. Bacteria can acquire antibiotic resistance and virulence genes through horizontal gene transfer (HGT). A novel horizontal gene transfer mechanism mediated by outer membrane vesicles (OMVs) has been recently identified. OMVs are rounded nanostructures released during their growth by Gram-negative bacteria. Biologically active toxins and virulence factors are often entrapped within these vesicles that behave as molecular carriers. Recently, OMVs have been reported to contain DNA molecules, but little is known about the vesicle packaging, release, and transfer mechanisms. The present review highlights the role of OMVs in HGT processes in Gram-negative bacteria.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Membrana Externa Bacteriana/metabolismo , Técnicas de Transferência de Genes , Transferência Genética Horizontal/genética , Bactérias/genética , Bactérias/patogenicidade , Humanos , Fatores de Virulência/genética , Fatores de Virulência/metabolismo
4.
Appl Environ Microbiol ; 87(17): e0056721, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34132592

RESUMO

Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli (ExPEC), causes colibacillosis in chickens and is reportedly associated with urinary tract infections and meningitis in humans. Development of resistance is a major limitation of current ExPEC antibiotic therapy. New antibacterials that can circumvent resistance problem such as antimicrobial peptides (AMPs) are critically needed. Here, we evaluated the efficacy of Lactobacillus rhamnosus GG (LGG)-derived peptides against APEC and uncovered their potential antibacterial targets. Three peptides (NPSRQERR [P1], PDENK [P2], and VHTAPK [P3]) displayed inhibitory activity against APEC. These peptides were effective against APEC in biofilm and chicken macrophage HD11 cells. Treatment with these peptides reduced the cecum colonization (0.5 to 1.3 log) of APEC in chickens. Microbiota analysis revealed two peptides (P1 and P2) decreased Enterobacteriaceae abundance with minimal impact on overall cecal microbiota of chickens. Bacterial cytological profiling showed peptides disrupt APEC membranes either by causing membrane shedding, rupturing, or flaccidity. Furthermore, gene expression analysis revealed that peptides downregulated the expression of ompC (>13.0-fold), ompF (>11.3-fold), and mlaA (>4.9-fold), genes responsible for the maintenance of outer membrane (OM) lipid asymmetry. Consistently, immunoblot analysis also showed decreased levels of OmpC and MlaA proteins in APEC treated with peptides. Alanine scanning studies revealed residues crucial (P1, N, E, R and P; P2, D and E; P3, T, P, and K) for their activity. Overall, our study identified peptides with a new antibacterial target that can be developed to control APEC infections in chickens, thereby curtailing poultry-originated human ExPEC infections. IMPORTANCE Avian pathogenic Escherichia coli (APEC) is a subgroup of extraintestinal pathogenic E. coli (ExPEC) and considered a foodborne zoonotic pathogen transmitted through consumption of contaminated poultry products. APEC shares genetic similarities with human ExPECs, including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC). Our study identified Lactobacillus rhamnosus GG (LGG)-derived peptides (P1 [NPSRQERR], P2 [PDENK], and P3 [VHTAPK]) effective in reducing APEC infection in chickens. Antimicrobial peptides (AMPs) are regarded as ideal candidates for antibacterial development because of their low propensity for resistance development and ability to kill resistant bacteria. Mechanistic studies showed peptides disrupt the APEC membrane by affecting the MlaA-OmpC/F system responsible for the maintenance of outer membrane (OM) lipid asymmetry, a promising new druggable target to overcome resistance problems in Gram-negative bacteria. Altogether, these peptides can provide a valuable approach for development of novel anti-ExPEC therapies, including APEC, human ExPECs, and other related Gram-negative pathogens. Furthermore, effective control of APEC infections in chickens can curb poultry-originated ExPEC infections in humans.


Assuntos
Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/metabolismo , Escherichia coli Extraintestinal Patogênica/efeitos dos fármacos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Porinas/metabolismo , Doenças das Aves Domésticas/microbiologia , Animais , Membrana Externa Bacteriana/efeitos dos fármacos , Membrana Externa Bacteriana/metabolismo , Biofilmes/efeitos dos fármacos , Galinhas/microbiologia , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/crescimento & desenvolvimento , Escherichia coli Extraintestinal Patogênica/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas de Transferência de Fosfolipídeos/genética , Porinas/genética , Doenças das Aves Domésticas/tratamento farmacológico
5.
Int J Mol Sci ; 22(9)2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-34062919

RESUMO

Extracellular vesicles (EVs) are cell-derived vesicles important in intercellular communication that play an essential role in host-pathogen interactions, spreading pathogen-derived as well as host-derived molecules during infection. Pathogens can induce changes in the composition of EVs derived from the infected cells and use them to manipulate their microenvironment and, for instance, modulate innate and adaptive inflammatory immune responses, both in a stimulatory or suppressive manner. Gastric cancer is one of the leading causes of cancer-related deaths worldwide and infection with Helicobacter pylori (H. pylori) is considered the main risk factor for developing this disease, which is characterized by a strong inflammatory component. EVs released by host cells infected with H. pylori contribute significantly to inflammation, and in doing so promote the development of disease. Additionally, H. pylori liberates vesicles, called outer membrane vesicles (H. pylori-OMVs), which contribute to atrophia and cell transformation in the gastric epithelium. In this review, the participation of both EVs from cells infected with H. pylori and H. pylori-OMVs associated with the development of gastric cancer will be discussed. By deciphering which functions of these external vesicles during H. pylori infection benefit the host or the pathogen, novel treatment strategies may become available to prevent disease.


Assuntos
Vesículas Extracelulares/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori/metabolismo , Gastropatias/metabolismo , Membrana Externa Bacteriana/metabolismo , Progressão da Doença , Vesículas Extracelulares/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Humanos , Gastropatias/microbiologia , Gastropatias/patologia
6.
ACS Chem Biol ; 16(6): 1079-1089, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34032403

RESUMO

Lysobacter are new biocontrol agents known for their prolific production of lytic enzymes and bioactive metabolites. L. enzymogenes is a predator of fungi and produces several structurally distinct antimicrobial compounds, such as the antifungal HSAF (heat stable antifungal factor) and analogs. The mechanism by which L. enzymogenes interacts with fungal prey is not well understood. Here, we found that the production of HSAF and analogs in L. enzymogenes OH11 was significantly induced in media supplemented with ground fungal mycelia or chitin. In the OH11 genome, we identified a gene (LeLPMO10A) that was annotated to encode a chitin-binding protein. The stimulation of HSAF and analogs by chitin was diminished when LeLPMO10A was deleted. We expressed the gene in E. coli and demonstrated that purified LeLPMO10A oxidatively cleaved chitin into oligomeric products, including 1,5 δ-lactones and aldonic acids. The results revealed that LeLPMO10A encodes a lytic polysaccharide monooxygenase, which has not been reported in Lysobacter. The metabolite analysis, antifungal assay, and proteomic analysis showed that the antifungal compounds and the chitin-cleaving LeLPMO10A are colocalized in outer membrane vesicles. The enzymatic products that resulted from in vitro LeLPMO10A-cleaved chitin also significantly induced HSAF and analogs in OH11. Scanning electron microscopic analysis indicated that spherical vesicles were formed outside of OH11 cells, and fewer OH11 cells were observed to attach to fungal hyphae when LeLPMO10A was deleted. Together, the study revealed a previously uncharacterized synergistic strategy utilized by the predatory Lysobacter during interaction with fungal prey.


Assuntos
Antifúngicos/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de Bactérias/metabolismo , Agentes de Controle Biológico/metabolismo , Lysobacter/fisiologia , Oxigenases de Função Mista/metabolismo , Quitina/metabolismo , Fungos/fisiologia , Controle Biológico de Vetores , Polissacarídeos/metabolismo
7.
J Med Chem ; 64(11): 7630-7645, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34009979

RESUMO

A class of quinazolone thiazoles was identified as new structural scaffolds for potential antibacterial conquerors to tackle dreadful resistance. Some prepared compounds exhibited favorable bacteriostatic efficiencies on tested bacteria, and the most representative 5j featuring the 4-trifluoromethylphenyl group possessed superior performances against Escherichia coli and Pseudomonas aeruginosa to norfloxacin. Further studies revealed that 5j with inappreciable hemolysis could hinder the formation of bacterial biofilms and trigger reactive oxygen species generation, which could take responsibility for emerging low resistance. Subsequent paralleled exploration discovered that 5j not only disintegrated outer and inner membranes to induce leakage of cytoplasmic contents but also broke the metabolism by suppressing dehydrogenase. Meanwhile, derivative 5j could intercalate into DNA to exert powerful antibacterial properties. Moreover, compound 5j gave synergistic effects against some Gram-negative bacteria in combination with norfloxacin. These findings indicated that this novel structural type of quinazolone thiazoles showed therapeutic foreground in struggling with Gram-negative bacterial infections.


Assuntos
Antibacterianos/farmacologia , Azóis/química , Bactérias Gram-Negativas/efeitos dos fármacos , Tiazóis/química , Antibacterianos/química , Antibacterianos/metabolismo , Membrana Externa Bacteriana/efeitos dos fármacos , Membrana Externa Bacteriana/metabolismo , Sítios de Ligação , Ligação Competitiva , Biofilmes/efeitos dos fármacos , Cristalografia por Raios X , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/fisiologia , Bactérias Gram-Positivas/efeitos dos fármacos , L-Lactato Desidrogenase/antagonistas & inibidores , L-Lactato Desidrogenase/metabolismo , Testes de Sensibilidade Microbiana , Conformação Molecular , Simulação de Acoplamento Molecular , Pseudomonas aeruginosa/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Tiazóis/metabolismo , Tiazóis/farmacologia
8.
Appl Environ Microbiol ; 87(13): e0314420, 2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-33893117

RESUMO

Recent work with Methylorubrum extorquens AM1 identified intracellular, cytoplasmic lanthanide storage in an organism that harnesses these metals for its metabolism. Here, we describe the extracellular and intracellular accumulation of lanthanides in the Beijerinckiaceae bacterium RH AL1, a newly isolated and recently characterized methylotroph. Using ultrathin-section transmission electron microscopy (TEM), freeze fracture TEM (FFTEM), and energy-dispersive X-ray spectroscopy, we demonstrated that strain RH AL1 accumulates lanthanides extracellularly at outer membrane vesicles (OMVs) and stores them in the periplasm. High-resolution elemental analyses of biomass samples revealed that strain RH AL1 can accumulate ions of different lanthanide species, with a preference for heavier lanthanides. Its methanol oxidation machinery is supposedly adapted to light lanthanides, and their selective uptake is mediated by dedicated uptake mechanisms. Based on transcriptome sequencing (RNA-seq) analysis, these presumably include the previously characterized TonB-ABC transport system encoded by the lut cluster but potentially also a type VI secretion system. A high level of constitutive expression of genes coding for lanthanide-dependent enzymes suggested that strain RH AL1 maintains a stable transcript pool to flexibly respond to changing lanthanide availability. Genes coding for lanthanide-dependent enzymes are broadly distributed taxonomically. Our results support the hypothesis that central aspects of lanthanide-dependent metabolism partially differ between the various taxa. IMPORTANCE Although multiple pieces of evidence have been added to the puzzle of lanthanide-dependent metabolism, we are still far from understanding the physiological role of lanthanides. Given how widespread lanthanide-dependent enzymes are, only limited information is available with respect to how lanthanides are taken up and stored in an organism. Our research complements work with commonly studied model organisms and showed the localized storage of lanthanides in the periplasm. This storage occurred at comparably low concentrations. Strain RH AL1 is able to accumulate lanthanide ions extracellularly and to selectively utilize lighter lanthanides. The Beijerinckiaceae bacterium RH AL1 might be an attractive target for developing biorecovery strategies to obtain these economically highly demanded metals in environmentally friendly ways.


Assuntos
Beijerinckiaceae/metabolismo , Lantânio/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas de Bactérias/genética , Beijerinckiaceae/genética , Beijerinckiaceae/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Metanol/metabolismo , Microscopia Eletrônica de Transmissão , Periplasma/metabolismo
9.
Int J Mol Sci ; 22(8)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917862

RESUMO

Pneumonia due to respiratory infection with most prominently bacteria, but also viruses, fungi, or parasites is the leading cause of death worldwide among all infectious disease in both adults and infants. The introduction of modern antibiotic treatment regimens and vaccine strategies has helped to lower the burden of bacterial pneumonia, yet due to the unavailability or refusal of vaccines and antimicrobials in parts of the global population, the rise of multidrug resistant pathogens, and high fatality rates even in patients treated with appropriate antibiotics pneumonia remains a global threat. As such, a better understanding of pathogen virulence on the one, and the development of innovative vaccine strategies on the other hand are once again in dire need in the perennial fight of men against microbes. Recent data show that the secretome of bacteria consists not only of soluble mediators of virulence but also to a significant proportion of extracellular vesicles-lipid bilayer-delimited particles that form integral mediators of intercellular communication. Extracellular vesicles are released from cells of all kinds of organisms, including both Gram-negative and Gram-positive bacteria in which case they are commonly termed outer membrane vesicles (OMVs) and membrane vesicles (MVs), respectively. (O)MVs can trigger inflammatory responses to specific pathogens including S. pneumonia, P. aeruginosa, and L. pneumophila and as such, mediate bacterial virulence in pneumonia by challenging the host respiratory epithelium and cellular and humoral immunity. In parallel, however, (O)MVs have recently emerged as auspicious vaccine candidates due to their natural antigenicity and favorable biochemical properties. First studies highlight the efficacy of such vaccines in animal models exposed to (O)MVs from B. pertussis, S. pneumoniae, A. baumannii, and K. pneumoniae. An advanced and balanced recognition of both the detrimental effects of (O)MVs and their immunogenic potential could pave the way to novel treatment strategies in pneumonia and effective preventive approaches.


Assuntos
Bactérias/metabolismo , Membrana Externa Bacteriana/metabolismo , Vesículas Extracelulares/metabolismo , Pneumonia Bacteriana/microbiologia , Imunidade Adaptativa , Animais , Antígenos de Bactérias/imunologia , Bactérias/imunologia , Membrana Externa Bacteriana/imunologia , Vacinas Bacterianas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Pneumonia Bacteriana/imunologia , Pneumonia Bacteriana/prevenção & controle , Mucosa Respiratória/imunologia , Mucosa Respiratória/microbiologia , Infecções Respiratórias/imunologia , Infecções Respiratórias/microbiologia , Infecções Respiratórias/prevenção & controle , Virulência
10.
J Microbiol Biotechnol ; 31(5): 645-658, 2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-33879642

RESUMO

Porins are essential for the viability of Gram-negative bacteria. They ensure the uptake of nutrients, can be involved in the maintenance of outer membrane integrity and define the antibiotic or drug resistance of organisms. The function and structure of porins in proteobacteria is well described, while their function in photoautotrophic cyanobacteria has not been systematically explored. We compared the domain architecture of nine putative porins in the filamentous cyanobacterium Anabaena sp. PCC 7120 and analyzed the seven candidates with predicted OprB-domain. Single recombinant mutants of the seven genes were created and their growth capacity under different conditions was analyzed. Most of the putative porins seem to be involved in the transport of salt and copper, as respective mutants were resistant to elevated concentrations of these substances. In turn, only the mutant of alr2231 was less sensitive to elevated zinc concentrations, while mutants of alr0834, alr4741 and all4499 were resistant to high manganese concentrations. Notably the mutant of alr4550 shows a high sensitivity against harmful compounds, which is indicative for a function related to the maintenance of outer membrane integrity. Moreover, the mutant of all5191 exhibited a phenotype which suggests either a higher nitrate demand or an inefficient nitrogen fixation. The dependency of porin membrane insertion on Omp85 proteins was tested exemplarily for Alr4550, and an enhanced aggregation of Alr4550 was observed in two omp85 mutants. The comparative analysis of porin mutants suggests that the proteins in parts perform distinct functions related to envelope integrity and solute uptake.


Assuntos
Anabaena/metabolismo , Porinas/genética , Anabaena/genética , Anabaena/crescimento & desenvolvimento , Antibacterianos/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Metais/metabolismo , Mutação , Nitrogênio/metabolismo , Fenótipo , Porinas/metabolismo , Sais/metabolismo , Estresse Fisiológico/genética
11.
Microb Cell Fact ; 20(1): 73, 2021 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-33743682

RESUMO

Escherichia coli is generally used as model bacteria to define microbial cell factories for many products and to investigate regulation mechanisms. E. coli exhibits phospholipids, lipopolysaccharides, colanic acid, flagella and type I fimbriae on the outer membrane which is a self-protective barrier and closely related to cellular morphology, growth, phenotypes and stress adaptation. However, these outer membrane associated molecules could also lead to potential contamination and insecurity for fermentation products and consume lots of nutrients and energy sources. Therefore, understanding critical insights of these membrane associated molecules is necessary for building better microbial producers. Here the biosynthesis, function, influences, and current membrane engineering applications of these outer membrane associated molecules were reviewed from the perspective of synthetic biology, and the potential and effective engineering strategies on the outer membrane to improve fermentation features for microbial cell factories were suggested.


Assuntos
Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Engenharia Celular/métodos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Escherichia coli/química , Escherichia coli/citologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fermentação , Biologia Sintética/métodos
12.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33741623

RESUMO

Extracellular electron transfer (EET) is an important biological process in microbial physiology as found in dissimilatory metal oxidation/reduction and interspecies electron transfer in syntrophy in natural environments. EET also plays a critical role in microorganisms relevant to environmental biotechnology in metal-contaminated areas, metal corrosion, bioelectrochemical systems, and anaerobic digesters. Geobacter species exist in a diversity of natural and artificial environments. One of the outstanding features of Geobacter species is the capability of direct EET with solid electron donors and acceptors, including metals, electrodes, and other cells. Therefore, Geobacter species are pivotal in environmental biogeochemical cycles and biotechnology applications. Geobacter sulfurreducens, a representative Geobacter species, has been studied for direct EET as a model microorganism. G. sulfurreducens employs electrically conductive pili (e-pili) and c-type cytochromes for the direct EET. The biological function and electronics applications of the e-pili have been reviewed recently, and this review focuses on the cytochromes. Geobacter species have an unusually large number of cytochromes encoded in their genomes. Unlike most other microorganisms, Geobacter species localize multiple cytochromes in each subcellular fraction, outer membrane, periplasm, and inner membrane, as well as in the extracellular space, and differentially utilize these cytochromes for EET with various electron donors and acceptors. Some of the cytochromes are functionally redundant. Thus, the EET in Geobacter is complicated. Geobacter coordinates the cytochromes with other cellular components in the elaborate EET system to flourish in the environment.


Assuntos
Citocromos/metabolismo , Geobacter/metabolismo , Membrana Externa Bacteriana/metabolismo , Transporte de Elétrons , Membranas Intracelulares/metabolismo , Periplasma/metabolismo
13.
Chem Rev ; 121(9): 5158-5192, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33724823

RESUMO

Despite tremendous successes in the field of antibiotic discovery seen in the previous century, infectious diseases have remained a leading cause of death. More specifically, pathogenic Gram-negative bacteria have become a global threat due to their extraordinary ability to acquire resistance against any clinically available antibiotic, thus urging for the discovery of novel antibacterial agents. One major challenge is to design new antibiotics molecules able to rapidly penetrate Gram-negative bacteria in order to achieve a lethal intracellular drug accumulation. Protein channels in the outer membrane are known to form an entry route for many antibiotics into bacterial cells. Up until today, there has been a lack of simple experimental techniques to measure the antibiotic uptake and the local concentration in subcellular compartments. Hence, rules for translocation directly into the various Gram-negative bacteria via the outer membrane or via channels have remained elusive, hindering the design of new or the improvement of existing antibiotics. In this review, we will discuss the recent progress, both experimentally as well as computationally, in understanding the structure-function relationship of outer-membrane channels of Gram-negative pathogens, mainly focusing on the transport of antibiotics.


Assuntos
Antibacterianos/metabolismo , Membrana Externa Bacteriana/metabolismo , Bactérias Gram-Negativas/metabolismo , Porinas/metabolismo , Antibacterianos/química , Membrana Externa Bacteriana/química , Membrana Celular/química , Membrana Celular/metabolismo , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Modelos Moleculares , Porinas/química
14.
Chem Rev ; 121(9): 5417-5478, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33761243

RESUMO

Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis because they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realized that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition, and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilization of petroleum.


Assuntos
Antibacterianos/metabolismo , Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Membrana Externa Bacteriana/química , Membrana Externa Bacteriana/metabolismo , Resistência Microbiana a Medicamentos , Humanos , Proteínas de Membrana Transportadoras/química
15.
Mol Cell ; 81(9): 2000-2012.e3, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33705710

RESUMO

The ß-barrel assembly machine (BAM) integrates ß-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. An essential BAM subunit (BamA) catalyzes integration by promoting the formation of a hybrid-barrel intermediate state between its own ß-barrel domain and that of its client proteins. Here we show that in addition to catalyzing the integration of ß-barrel proteins, BamA functions as a polypeptide export channel. In vivo structural mapping via intermolecular disulfide crosslinking showed that the extracellular "passenger" domain of a member of the "autotransporter" superfamily of virulence factors traverses the OM through the BamA ß-barrel lumen. Furthermore, we demonstrate that a highly conserved residue within autotransporter ß-barrels is required to position the passenger inside BamA to initiate translocation and that during translocation, the passenger stabilizes the hybrid-barrel state. Our results not only establish a new function for BamA but also unify the divergent functions of BamA and other "Omp85" superfamily transporters.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Membrana Externa Bacteriana/metabolismo , Escherichia coli K12/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Transporte Biológico , Sequência Conservada , Escherichia coli K12/genética , Proteínas de Escherichia coli/genética , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , Triptofano
16.
mBio ; 12(2)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33758090

RESUMO

Cobamides are cobalt-containing cyclic tetrapyrroles used by cells from all domains of life but only produced de novo by some bacteria and archaea. The "late steps" of the adenosylcobamide biosynthetic pathway are responsible for the assembly of the nucleotide loop and are required during de novo synthesis and precursor salvaging. These steps are characterized by activation of the corrin ring and lower ligand base, condensation of the activated precursors to adenosylcobamide phosphate, and removal of the phosphate, yielding a complete adenosylcobamide molecule. The condensation of the activated corrin ring and lower ligand base is performed by an integral membrane protein, cobamide (5' phosphate) synthase (CobS), and represents an important convergence of two pathways necessary for nucleotide loop assembly. Interestingly, membrane association of this penultimate step is conserved among all cobamide producers, yet the physiological relevance of this association is not known. Here, we present the purification and biochemical characterization of the CobS enzyme of the enterobacterium Salmonella enterica subsp. enterica serovar Typhimurium strain LT2, investigate its association with liposomes, and quantify the effect of the lipid bilayer on its enzymatic activity and substrate affinity. We report a purification scheme that yields pure CobS protein, allowing in vitro functional analysis. Additionally, we report a method for liposome reconstitution of CobS, allowing for physiologically relevant studies of this inner membrane protein in a phospholipid bilayer. In vitro and in vivo data reported here expand our understanding of CobS and the implications of membrane-associated adenosylcobamide biosynthesis.IMPORTANCE Salmonella is a human pathogen of worldwide importance, and coenzyme B12 is critical for the pathogenic lifestyle of this bacterium. The importance of the work reported here lies on the improvements to the methodology used to isolate cobamide synthase, a polytopic integral membrane protein that catalyzes the penultimate step of coenzyme B12 biosynthesis. This advance is an important step in the analysis of the proposed multienzyme complex responsible for the assembly of the nucleotide loop during de novo coenzyme B12 biosynthesis and for the assimilation of incomplete corrinoids from the environment. We proposed that cobamide synthase is likely localized to the cell membrane of every coenzyme B12-producing bacterium and archaeum sequenced to date. The new knowledge of cobamide synthase advances our understanding of the functionality of the enzyme in the context of the lipid bilayer and sets the foundation for the functional-structural analysis of the aforementioned multienzyme complex.


Assuntos
Amida Sintases/genética , Membrana Externa Bacteriana/metabolismo , Cobamidas/biossíntese , Salmonella/enzimologia , Salmonella/genética , Amida Sintases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Lipossomos/metabolismo , Salmonella/metabolismo
17.
J Bacteriol ; 203(10)2021 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-33685973

RESUMO

Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host.IMPORTANCE Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.


Assuntos
Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos/metabolismo , Lipopolissacarídeos/metabolismo , Porphyromonas gingivalis/metabolismo , Membrana Externa Bacteriana/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/genética , Sistemas de Secreção Bacterianos/genética , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/genética , Mutação , Pigmentação/genética , Porphyromonas gingivalis/genética
18.
Chem Rev ; 121(9): 5597-5631, 2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-33596653

RESUMO

Cell envelope plays a dual role in the life of bacteria by simultaneously protecting it from a hostile environment and facilitating access to beneficial molecules. At the heart of this ability lie the restrictive properties of the cellular membrane augmented by efflux transporters, which preclude intracellular penetration of most molecules except with the help of specialized uptake mediators. Recently, kinetic properties of the cell envelope came into focus driven on one hand by the urgent need in new antibiotics and, on the other hand, by experimental and theoretical advances in studies of transmembrane transport. A notable result from these studies is the development of a kinetic formalism that integrates the Michaelis-Menten behavior of individual transporters with transmembrane diffusion and offers a quantitative basis for the analysis of intracellular penetration of bioactive compounds. This review surveys key experimental and computational approaches to the investigation of transport by individual translocators and in whole cells, summarizes key findings from these studies and outlines implications for antibiotic discovery. Special emphasis is placed on Gram-negative bacteria, whose envelope contains two separate membranes. This feature sets these organisms apart from Gram-positive bacteria and eukaryotic cells by providing them with full benefits of the synergy between slow transmembrane diffusion and active efflux.


Assuntos
Bactérias Gram-Negativas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Membrana Externa Bacteriana/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/efeitos dos fármacos , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Modelos Biológicos , Simulação de Dinâmica Molecular
19.
Acc Chem Res ; 54(4): 930-939, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33539084

RESUMO

Antibiotics are miracle drugs that can cure infectious bacterial diseases. However, their utility is challenged by antibiotic-resistant bacteria emerging in clinics and straining modern medicine and our ways of life. Certain bacteria such as Gram-negative (Gram(-)) and Mycobacteriales species are intrinsically resistant to most clinical antibiotics and can further gain multidrug resistance through mutations and plasmid acquisition. These species stand out by the presence of an additional external lipidic membrane, the outer membrane (OM), that is composed of unique glycolipids. Although formidable, the OM is a passive permeability barrier that can reduce penetration of antibiotics but cannot affect intracellular steady-state concentrations of drugs. The two-membrane envelopes are further reinforced by active efflux transporters that expel antibiotics from cells against their concentration gradients. The major mechanism of antibiotic resistance in Gram(-) pathogens is the active efflux of drugs, which acts synergistically with the low permeability barrier of the OM and other mutational and plasmid-borne mechanisms of antibiotic resistance.The synergy between active efflux and slow uptake offers Gram(-) bacteria an impressive degree of protection from potentially harmful chemicals, but it is also their Achilles heel. Kinetic studies have revealed that even small changes in the efficiency of either of the two factors can have dramatic effects on drug penetration into the cell. In line with these expectations, two major approaches to overcome this antibiotic resistance mechanism are currently being explored: (1) facilitation of antibiotic penetration across the outer membranes and (2) avoidance and inhibition of clinically relevant multidrug efflux pumps. Herein we summarize the progress in the latter approach with a focus on efflux pumps from the resistance-nodulation-division (RND) superfamily. The ability to export various substrates across the OM at the expense of the proton-motive force acting on the inner membrane and the engagement of accessory proteins for their functions are the major mechanistic advantages of these pumps. Both the RND transporters and their accessory proteins are being targeted in the discovery of efflux pump inhibitors, which in combination with antibiotics can potentiate antibacterial activities. We discuss intriguing relationships between substrates and inhibitors of efflux pumps, as these two types of ligands face similar barriers and binding sites in the transporters and accessory proteins and both types of activities often occur with the same chemical scaffold. Several distinct chemical classes of efflux inhibitors have been discovered that are as structurally diverse as the substrates of efflux pumps. Recent mechanistic insights, both empirical and computational, have led to the identification of features that distinguish OM permeators and efflux pump avoiders as well as efflux inhibitors from substrates. These findings suggest a path forward for optimizing the OM permeation and efflux-inhibitory activities in antibiotics and other chemically diverse compounds.


Assuntos
Antibacterianos/química , Proteínas de Membrana Transportadoras/metabolismo , Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Membrana Externa Bacteriana/metabolismo , Fluoroquinolonas/química , Fluoroquinolonas/metabolismo , Fluoroquinolonas/farmacologia , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/efeitos dos fármacos , Proteínas de Membrana Transportadoras/química , Testes de Sensibilidade Microbiana
20.
J Appl Microbiol ; 130(5): 1611-1629, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33025668

RESUMO

AIM: To test whether engineered nanopores on the outer membrane (OM) of Escherichia coli can increase expression of heterologous proteins by making additional nutrients available to the host. METHODS AND RESULTS: Outer membrane nanopores were generated by expressing recombinant Vibrio parahaemolyticus CsgG (rVpCsgG), which spontaneously assembles into a pore-forming channel on the OM, allowing spontaneous diffusion of small chemical entities from the exterior. Protein expression was probed using a reporter protein, sfGFP, expressed on a second compatible plasmid. OM pore formation was shown by acquired erythromycin sensitivity in cells transformed with rVpCsgG, influx of propidium iodide as well as by surface localization of recombinant CsgG by immunogold-labeled transmission electron microscopy. Expression of recombinant CsgG showed increased growth and also enhanced expression of sfGFP in minimal medium and is due to both enhanced transcription as well as translation. Similar enhancement of expression was also observed for a number of different proteins of different origin, sizes and nature. CONCLUSIONS: Our findings clearly demonstrate that engineered nanopores on the OM of E. coli enhance expression of different heterologous proteins in minimal medium. SIGNIFICANCE AND IMPACT OF THE STUDY: Vibrio parahaemolyticus CsgG ß-nanopore mediated co-expression strategy to improve recombinant protein expression is fully compatible with other methods of protein expression enhancement, and therefore can be a useful tool in biotechnology particularly for whole-cell bio-transformations for production of secondary metabolite.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Porinas/metabolismo , Vibrio parahaemolyticus/genética , Membrana Externa Bacteriana/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genes Bacterianos , Nanoporos , Porinas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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