RESUMO
Aeromonas salmonicida is a Gram-negative bacterium that can infect a wide host range of fish populations, including salmonids and non-salmonids as well as freshwater and marine life. Some strains of A. salmonicida cause the disease furunculosis, which can cause lethargy, intestinal inflammation, ulcers, haemorrhaging and death. The infection is spread through fish-to-fish contact, and the presence of infection can have devastating effects on cultivated fish populations. The purpose of this study was to explore the ability of non-A-layer and A-layer A. salmonicida strains to incorporate polyunsaturated fatty acids (PUFAs) into their lipid profile and test the phenotypic effects thereof. Lipids were extracted from PUFA-exposed cultures and analysed for lipid modification by thin-layer chromatography and ultraperformance liquid chromatography-mass spectrometry, showing A. salmonicida, regardless of A-layer, capable of incorporating all seven of the PUFAs studied. Phenotypic effects were determined through the use of assays that tested for biofilm formation, membrane permeability and cyclic peptide susceptibility. Temperature-dependent effects on biofilm formation were observed, and PUFA exposure showed significant (p < .001) increases in membrane permeability as tested by the uptake of the hydrophobic compounds crystal violet and ethidium bromide. Additionally, some PUFAs elicited modest protection and vulnerability against the membrane-targeting cyclic peptides polymyxin B (PMB) and colistin. The diverse, strain-specific responses to exogenous PUFAs may allude to evolved adaptive strategies that enhance survival, persistence and virulence of non-pathogenic and pathogenic members of bacteria that oscillate between environmental and fish host niches.
Assuntos
Aeromonas salmonicida , Doenças dos Peixes , Animais , Peptídeos Antimicrobianos , Fosfolipídeos , Ácidos Graxos InsaturadosRESUMO
BACKGROUND: The utilization of exogenous fatty acids by Gram-negative bacteria has been linked to many cellular processes, including fatty acid oxidation for metabolic gain, assimilation into membrane phospholipids, and control of phenotypes associated with virulence. The expanded fatty acid handling capabilities have been demonstrated in several bacteria of medical importance; however, a survey of the polyunsaturated fatty acid responses in the model organism Escherichia coli has not been performed. The current study examined the impacts of exogenous fatty acids on E. coli. RESULTS: All PUFAs elicited higher overall growth, with several fatty acids supporting growth as sole carbon sources. Most PUFAs were incorporated into membrane phospholipids as determined by Ultra performance liquid chromatography-mass spectrometry, whereas membrane permeability was variably affected as measured by two separate dye uptake assays. Biofilm formation, swimming motility and antimicrobial peptide resistance were altered in the presence of PUFAs, with arachidonic and docosahexaenoic acids eliciting strong alteration to these phenotypes. CONCLUSIONS: The findings herein add E. coli to the growing list of Gram-negative bacteria with broader capabilities for utilizing and responding to exogenous fatty acids. Understanding bacterial responses to PUFAs may lead to microbial behavioral control regimens for disease prevention.
Assuntos
Biofilmes/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Ácidos Graxos Insaturados/farmacologia , Fosfolipídeos/classificação , Ampicilina/farmacologia , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Ácido Araquidônico/farmacologia , Biofilmes/crescimento & desenvolvimento , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Colistina/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/química , Escherichia coli/crescimento & desenvolvimento , Movimento/efeitos dos fármacos , Movimento/fisiologia , Fenótipo , Fosfolipídeos/química , Fosfolipídeos/isolamento & purificação , Polimixina B/farmacologia , VirulênciaRESUMO
BACKGROUND: Pseudomonas aeruginosa, a common opportunistic pathogen, is known to cause infections in a variety of compromised human tissues. An emerging mechanism for microbial survival is the incorporation of exogenous fatty acids to alter the cell's membrane phospholipid profile. With these findings, we show that exogenous fatty acid exposure leads to changes in bacterial membrane phospholipid structure, membrane permeability, virulence phenotypes and consequent stress responses that may influence survival and persistence of Pseudomonas aeruginosa. RESULTS: Thin-layer chromatography and ultra performance liquid chromatography / ESI-mass spectrometry indicated alteration of bacterial phospholipid profiles following growth in the presence of polyunsaturated fatty acids (PUFAs) (ranging in carbon length and unsaturation). The exogenously supplied fatty acids were incorporated into the major bacterial phospholipids phosphatidylethanolamine and phosphatidylglycerol. The incorporation of fatty acids increased membrane permeability as judged by both accumulation and exclusion of ethidium bromide. Individual fatty acids were identified as modifying resistance to the cyclic peptide antibiotics polymyxin B and colistin, but not the beta-lactam imipenem. Biofilm formation was increased by several PUFAs and significant fluctuations in swimming motility were observed. CONCLUSIONS: Our results emphasize the relevance and complexity of exogenous fatty acids in the membrane physiology and pathobiology of a medically important pathogen. P. aeruginosa exhibits versatility with regard to utilization of and response to exogenous fatty acids, perhaps revealing potential strategies for prevention and control of infection.
Assuntos
Membrana Celular/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fosfolipídeos/química , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidade , Membrana Celular/química , Permeabilidade da Membrana Celular , Humanos , Fosfolipídeos/metabolismo , Pseudomonas aeruginosa/genética , VirulênciaRESUMO
Acinetobacter baumannii is a ubiquitous multidrug-resistant bacteria that is found on a variety of surfaces, including skin, hair and soil. During the past decade, A. baumannii has emerged as a significant cause of nosocomial infections in the United States. Recent studies have highlighted the ability of some bacteria to utilize a wide variety of fatty acids as a membrane remodelling strategy. Considering this, we hypothesized that fatty acids may have an effect on the emerging pathogen A. baumannii. Thin-layer chromatography indicated structural alterations to major phospholipids. Liquid chromatography/mass spectrometry confirmed the assimilation of numerous exogenous polyunsaturated fatty acids (PUFAs) into the phospholipid species of A. baumannii. The incorporation of fatty acids affected several bacterial phenotypes, including membrane permeability, biofilm formation, surface motility and antimicrobial peptide resistance.
Assuntos
Acinetobacter baumannii/fisiologia , Translocação Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , Permeabilidade da Membrana Celular/fisiologia , Farmacorresistência Bacteriana/fisiologia , Ácidos Graxos Insaturados/metabolismo , Fosfolipídeos/metabolismo , Acinetobacter baumannii/química , Acinetobacter baumannii/efeitos dos fármacos , Adaptação Fisiológica , Antibacterianos/farmacologia , Cromatografia Líquida de Alta Pressão , Cromatografia em Camada Fina , Colistina/farmacologia , Testes de Sensibilidade Microbiana , Fosfolipídeos/análise , Fosfolipídeos/classificação , Polimixina B/farmacologia , Espectrometria de Massas em TandemRESUMO
The pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, and V. vulnificus) represent a constant threat to human health, causing foodborne and skin wound infections as a result of ingestion of or exposure to contaminated water and seafood. Recent studies have highlighted Vibrio's ability to acquire fatty acids from environmental sources and assimilate them into cell membranes. The possession and conservation of such machinery provokes consideration of fatty acids as important factors in the pathogenic lifestyle of Vibrio species. The findings here link exogenous fatty acid exposure to changes in bacterial membrane phospholipid structure, permeability, phenotypes associated with virulence, and consequent stress responses that may impact survival and persistence of pathogenic Vibrio species. Polyunsaturated fatty acids (PUFAs) (ranging in carbon length and unsaturation) supplied in growth medium were assimilated into bacterial phospholipids, as determined by thin-layer chromatography and liquid chromatography-mass spectrometry. The incorporation of fatty acids variably affected membrane permeability, as judged by uptake of the hydrophobic compound crystal violet. For each species, certain fatty acids were identified as affecting resistance to antimicrobial peptide treatment. Significant fluctuations were observed with regard to both motility and biofilm formation following growth in the presence of individual PUFAs. Our results illustrate the important and complex roles of exogenous fatty acids in the membrane physiology and virulence of a bacterial genus that inhabits aquatic and host environments containing an abundance of diverse fatty acids.IMPORTANCE Bacterial responses to fatty acids include, but are not limited to, degradation for metabolic gain, modification of membrane lipids, alteration of protein function, and regulation of gene expression. Vibrio species exhibit significant diversity with regard to the machinery known to participate in the uptake and incorporation of fatty acids into their membranes. Both aquatic and host niches occupied by Vibrio are rife with various free fatty acids and fatty acid-containing lipids. The roles of fatty acids in the environmental survival and pathogenesis of bacteria have begun to emerge and are expected to expand significantly. The current study demonstrates the responsiveness of V. cholerae, V. parahaemolyticus, and V. vulnificus to exogenous PUFAs. In addition to phospholipid remodeling, PUFA assimilation impacts membrane permeability, motility, biofilm formation, and resistance to polymyxin B.
Assuntos
Membrana Celular/metabolismo , Ácidos Graxos Insaturados/metabolismo , Vibrio cholerae/metabolismo , Vibrio parahaemolyticus/metabolismo , Vibrio vulnificus/metabolismo , Membrana Celular/química , Cromatografia em Camada Fina , Ácidos Graxos Insaturados/química , Humanos , Espectrometria de Massas , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Vibrioses/microbiologia , Vibrio cholerae/química , Vibrio cholerae/genética , Vibrio cholerae/patogenicidade , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidade , Vibrio vulnificus/química , Vibrio vulnificus/genética , Vibrio vulnificus/patogenicidade , VirulênciaRESUMO
Despite its highly inflammatory nature, LPS is a molecule with remarkable therapeutic potential. Lipid A is a glycolipid that serves as the hydrophobic anchor of LPS and constitutes a potent ligand of the Toll-like receptor (TLR)4/myeloid differentiation factor 2 receptor of the innate immune system. A less toxic mixture of monophosphorylated lipid A species (MPL) recently became the first new Food and Drug Administration-approved adjuvant in over 70 y. Whereas wild-type Escherichia coli LPS provokes strong inflammatory MyD88 (myeloid differentiation primary response gene 88)-mediated TLR4 signaling, MPL preferentially induces less inflammatory TRIF (TIR-domain-containing adaptor-inducing IFN-ß)-mediated responses. Here, we developed a system for combinatorial structural diversification of E. coli lipid A, yielding a spectrum of bioactive variants that display distinct TLR4 agonist activities and cytokine induction. Mice immunized with engineered lipid A/antigen emulsions exhibited robust IgG titers, indicating the efficacy of these molecules as adjuvants. This approach demonstrates how combinatorial engineering of lipid A can be exploited to generate a spectrum of immunostimulatory molecules for vaccine and therapeutics development.
Assuntos
Imunidade Inata , Lipídeo A/imunologia , Imunidade Adaptativa , Animais , Sequência de Bases , Técnicas de Química Combinatória , Citocinas/metabolismo , DNA Bacteriano/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/imunologia , Biblioteca Gênica , Genes Bacterianos , Engenharia Genética/métodos , Humanos , Imunidade Inata/genética , Lipídeo A/química , Lipídeo A/genética , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Monócitos/imunologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome. To facilitate interpretation and assignment of the resulting UVPD mass spectra of peptide anions, the MassMatrix database search algorithm was modified to allow automated analysis of negative polarity MS/MS spectra. The new UVPD algorithms were developed based on the MassMatrix database search engine by adding specific fragmentation pathways for UVPD. The new UVPD fragmentation pathways in MassMatrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS(1) and MS(2) data acquired on an Orbitrap mass spectrometer for complex Halobacterium and HeLa proteome samples. Negative mode UVPD led to the identification of 3663 and 2350 peptides for the Halo and HeLa tryptic digests, respectively, corresponding to 655 and 645 peptides that were unique when compared with electron transfer dissociation (ETD), higher energy collision-induced dissociation, and collision-induced dissociation results for the same digests analyzed in the positive mode. In sum, 805 and 619 proteins were identified via UVPD for the Halobacterium and HeLa samples, respectively, with 49 and 50 unique proteins identified in contrast to the more conventional MS/MS methods. The algorithm also features automated charge determination for low mass accuracy data, precursor filtering (including intact charge-reduced peaks), and the ability to combine both positive and negative MS/MS spectra into a single search, and it is freely open to the public. The accuracy and specificity of the MassMatrix UVPD search algorithm was also assessed for low resolution, low mass accuracy data on a linear ion trap. Analysis of a known mixture of three mitogen-activated kinases yielded similar sequence coverage percentages for UVPD of peptide anions versus conventional collision-induced dissociation of peptide cations, and when these methods were combined into a single search, an increase of up to 13% sequence coverage was observed for the kinases. The ability to sequence peptide anions and cations in alternating scans in the same chromatographic run was also demonstrated. Because ETD has a significant bias toward identifying highly basic peptides, negative UVPD was used to improve the identification of the more acidic peptides in conjunction with positive ETD for the more basic species. In this case, tryptic peptides from the cytosolic section of HeLa cells were analyzed by polarity switching nanoLC-MS/MS utilizing ETD for cation sequencing and UVPD for anion sequencing. Relative to searching using ETD alone, positive/negative polarity switching significantly improved sequence coverages across identified proteins, resulting in a 33% increase in unique peptide identifications and more than twice the number of peptide spectral matches.
Assuntos
Cromatografia Líquida/métodos , Bases de Dados de Proteínas , Ensaios de Triagem em Larga Escala , Proteoma/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Raios Ultravioleta , Algoritmos , Ânions , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Halobacterium/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Peptídeos/metabolismo , Proteoma/química , Curva ROC , Reprodutibilidade dos Testes , Análise de Sequência de ProteínaRESUMO
Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms associated with this phenotypic distinction have remained a mystery for 50 y. Both gram-negative and gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the gram-positive system responsible for D-alanylation of teichoic acids. Such machinery was not thought to be used by gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between gram-positive and gram-negative organisms.
Assuntos
Glicina/metabolismo , Bactérias Gram-Negativas/metabolismo , Bactérias Gram-Positivas/metabolismo , Lipopolissacarídeos/metabolismo , Vibrio cholerae/metabolismo , Sequência de Aminoácidos , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Glicina/química , Bactérias Gram-Negativas/química , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/química , Bactérias Gram-Positivas/genética , Lipídeo A/química , Lipídeo A/metabolismo , Lipopolissacarídeos/química , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Polimixina B/farmacologia , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vibrio cholerae/química , Vibrio cholerae/genéticaRESUMO
Campylobacter jejuni is a natural commensal of the avian intestinal tract. However, the bacterium is also the leading cause of acute bacterial diarrhea worldwide and is implicated in development of Guillain-Barré syndrome. Like many bacterial pathogens, C. jejuni assembles complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work in C. jejuni identified a gene encoding a novel phosphoethanolamine (pEtN) transferase, EptC (Cj0256), that plays a promiscuous role in modifying the flagellar rod protein, FlgG; the lipid A domain of lipooligosaccharide (LOS); and several N-linked glycans. In this work, we report that EptC catalyzes the addition of pEtN to the first heptose sugar of the inner core oligosaccharide of LOS, a fourth enzymatic target. We also examine the role pEtN modification plays in circumventing detection and/or killing by host defenses. Specifically, we show that modification of C. jejuni lipid A with pEtN results in increased recognition by the human Toll-like receptor 4-myeloid differentiation factor 2 (hTLR4-MD2) complex, along with providing resistance to relevant mammalian and avian antimicrobial peptides (i.e., defensins). We also confirm the inability of aberrant forms of LOS to activate Toll-like receptor 2 (TLR2). Most exciting, we demonstrate that strains lacking eptC show decreased commensal colonization of chick ceca and reduced colonization of BALB/cByJ mice compared to wild-type strains. Our results indicate that modification of surface structures with pEtN by EptC is key to its ability to promote commensalism in an avian host and to survive in the mammalian gastrointestinal environment.
Assuntos
Infecções por Campylobacter/metabolismo , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/fisiologia , Etanolaminofosfotransferase/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Aves/genética , Aves/metabolismo , Aves/microbiologia , Infecções por Campylobacter/genética , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Campylobacter jejuni/patogenicidade , Linhagem Celular , Proteínas de Escherichia coli , Etanolaminofosfotransferase/genética , Etanolaminas/metabolismo , Células HEK293 , Interações Hospedeiro-Patógeno , Humanos , Lipídeo A/genética , Lipídeo A/metabolismo , Lipopolissacarídeos/genética , Lipopolissacarídeos/metabolismo , Antígeno 96 de Linfócito/genética , Antígeno 96 de Linfócito/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Camundongos , Camundongos Endogâmicos BALB C , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Fenótipo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Virulência/genéticaRESUMO
Campylobacter jejuni is a major cause of bacterial diarrheal disease worldwide. The organism is characterized by a diversity of polysaccharide structures, including a polysaccharide capsule. Most C. jejuni capsules are known to be decorated nonstoichiometrically with methyl phosphoramidate (MeOPN). The capsule of C. jejuni 81-176 has been shown to be required for serum resistance, but here we show that an encapsulated mutant lacking the MeOPN modification, an mpnC mutant, was equally as sensitive to serum killing as the nonencapsulated mutant. A nonencapsulated mutant, a kpsM mutant, exhibited significantly reduced colonization compared to that of wild-type 81-176 in a mouse intestinal colonization model, and the mpnC mutant showed an intermediate level of colonization. Both mutants were associated with higher levels of interleukin 17 (IL-17) expression from lamina propria CD4(+) cells than from cells from animals infected with 81-176. In addition, reduced levels of Toll-like receptor 4 (TLR4) and TLR2 activation were observed following in vitro stimulation of human reporter cell lines with the kpsM and mpnC mutants compared to those with wild-type 81-176. The data suggest that the capsule polysaccharide of C. jejuni and the MeOPN modification modulate the host immune response.
Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter jejuni/fisiologia , Polissacarídeos Bacterianos/fisiologia , Animais , Citocinas/genética , Citocinas/metabolismo , Regulação da Expressão Gênica/imunologia , Células HEK293 , Humanos , Camundongos , Mutação , NF-kappa B/genética , NF-kappa B/metabolismo , Transdução de Sinais , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMO
Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4'-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4'-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4'-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4'-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, ß-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.
Assuntos
Membrana Celular/metabolismo , Mucosa Gástrica/microbiologia , Helicobacter pylori/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Imunidade Inata/fisiologia , Lipídeo A/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/imunologia , Mucosa Gástrica/imunologia , Células HEK293 , Humanos , Lipídeo A/genética , Lipídeo A/imunologia , Camundongos , Camundongos Knockout , Mutação , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/imunologia , Monoéster Fosfórico Hidrolases/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismoRESUMO
The Gram-negative bacteria Vibrio cholerae poses significant public health concerns by causing an acute intestinal infection afflicting millions of people each year. V. cholerae motility, as well as virulence factor expression and outer membrane protein production, has been shown to be affected by bile. The current study examines the effects of bile on V. cholerae phospholipids. Bile exposure caused significant alterations to the phospholipid profile of V. cholerae but not of other enteric pathogens. These changes consisted of a quantitative increase and migratory difference in cardiolipin, decreases in phosphatidylglycerol and phosphatidylethanolamine, and the dramatic appearance of an unknown phospholipid determined to be lyso-phosphatidylethanolamine. Major components of bile were not responsible for the observed changes, but long-chain polyunsaturated fatty acids, which are minor components of bile, were shown to be incorporated into phospholipids of V. cholerae. Although the bile-induced phospholipid profile was independent of the V. cholerae virulence cascade, we identified another relevant environment in which V. cholerae assimilates unique fatty acids into its membrane phospholipids - marine sediment. Our results suggest that Vibrio species possess unique machinery conferring the ability to take up a wider range of exogenous fatty acids than other enteric bacteria.
Assuntos
Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Interações Hospedeiro-Patógeno , Vibrio cholerae/citologia , Microbiologia da Água , Bile/metabolismo , Ácidos e Sais Biliares/metabolismo , Bilirrubina/metabolismo , Escherichia coli/metabolismo , Ácidos Graxos Insaturados/metabolismo , Sedimentos Geológicos/química , Lecitinas/metabolismo , Fosfolipídeos/química , Fosfolipídeos/isolamento & purificação , Salmonella enterica/metabolismo , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , Vibrio cholerae/crescimento & desenvolvimento , Vibrio cholerae/metabolismo , Vibrio cholerae/patogenicidade , VirulênciaRESUMO
Similar to most Gram-negative bacteria, the outer leaflet of the outer membrane of Vibrio cholerae is comprised of lipopolysaccharide. Previous reports have proposed that V. cholerae serogroups O1 and O139 synthesize structurally different lipid A domains, which anchor lipopolysaccharide within the outer membrane. In the current study, intact lipid A species of V. cholerae O1 and O139 were analysed by mass spectrometry. We demonstrate that V. cholerae serogroups associated with human disease synthesize a similar asymmetrical hexa-acylated lipid A species, bearing a myristate (C14:0) and 3-hydroxylaurate (3-OH C12:0) at the 2'- and 3'-positions respectively. A previous report from our laboratory characterized the V. cholerae LpxL homologue Vc0213, which transfers a C14:0 to the 2'-position of the glucosamine disaccharide. Our current findings identify V. cholerae Vc0212 as a novel lipid A secondary hydroxy-acyltransferase, termed LpxN, responsible for transferring the 3-hydroxylaurate (3-OH C12:0) to the V. cholerae lipid A domain. Importantly, the presence of a 3-hydroxyl group on the 3'-linked secondary acyl chain was found to promote antimicrobial peptide resistance in V. cholerae; however, this functional group was not required for activation of the innate immune response.
Assuntos
Aciltransferases/imunologia , Membrana Celular/imunologia , Imunidade Inata , Lipídeo A/biossíntese , Lipopolissacarídeos/imunologia , Vibrio cholerae/imunologia , Membrana Celular/ultraestrutura , Cólera/imunologia , Cólera/microbiologia , Farmacorresistência Bacteriana , Células HEK293 , Humanos , Lipídeo A/química , Lipídeo A/imunologia , Espectrometria de Massas , Antígenos O/análise , Antígenos O/biossíntese , Antígenos O/genética , Polimixina B/farmacologia , Vibrio cholerae/enzimologiaRESUMO
Vibrio alginolyticus and Vibrio (Aliivibrio) fischeri are Gram-negative bacteria found globally in marine environments. During the past decade, studies have shown that certain Gram-negative bacteria, including Vibrio species (cholerae, parahaemolyticus, and vulnificus) are capable of using exogenous polyunsaturated fatty acids (PUFAs) to modify the phospholipids of their membrane. Moreover, exposure to exogenous PUFAs has been shown to affect certain phenotypes that are important factors of virulence. The purpose of this study was to investigate whether V. alginolyticus and V. fischeri are capable of responding to exogenous PUFAs by remodeling their membrane phospholipids and/or altering behaviors associated with virulence. Thin-layer chromatography (TLC) analyses and ultra-performance liquid chromatography-electrospray ionization mass spectrometry (UPLC/ESI-MS) confirmed incorporation of all PUFAs into membrane phosphatidylglycerol and phosphatidylethanolamine. Several growth phenotypes were identified when individual fatty acids were supplied in minimal media and as sole carbon sources. Interestingly, several PUFAs acids inhibited growth of V. fischeri. Significant alterations to membrane permeability were observed depending on fatty acid supplemented. Strikingly, arachidonic acid (20:4) reduced membrane permeability by approximately 35% in both V. alginolyticus and V. fischeri. Biofilm assays indicated that fatty acid influence was dependent on media composition and temperature. All fatty acids caused decreased swimming motility in V. alginolyticus, while only linoleic acid (18:2) significantly increased swimming motility in V. fischeri. In summary, exogenous fatty acids cause a variety of changes in V. alginolyticus and V. fischeri, thus adding these bacteria to a growing list of Gram-negatives that exhibit versatility in fatty acid utilization and highlighting the potential for environmental PUFAs to influence phenotypes associated with planktonic, beneficial, and pathogenic associations.
Assuntos
Aliivibrio fischeri/fisiologia , Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Ácidos Graxos Insaturados/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfatidilgliceróis/metabolismo , Vibrio alginolyticus/fisiologia , Organismos Aquáticos/fisiologia , Biofilmes , Fenótipo , Vibrioses/microbiologia , Virulência/efeitos dos fármacosRESUMO
Soft tissue mobilization and massage requiring lotions or creams are commonly used interventions in outpatient rehabilitation clinics. For at least 50 years hand creams used in healthcare settings have been found to be contaminated by bacteria. The purpose of this study was to determine the current state of bacterial contamination of lotions used in clinics and to determine the efficacy of lotion preservatives to kill bacteria. Unopened containers of lotions were studied, along with 81 lotion containers used in 22 outpatient clinics in southeast Tennessee and northwest Georgia. Three sites on each container were sampled using sterile swabs. At a microbiology lab, bacterial growth media was inoculated and incubated. Of the 81 containers sampled, 16 supported bacterial growth (19.8%). Container threads displayed the highest contamination compared with other container locations (p < 0.01). No bacteria were found in unopened lotion containers, although when challenged with live bacterial cultures lotion preservatives did not kill bacteria tested. Enrichment cultures using lotions studied here supported the growth of several bacterial species. These findings suggest the need for standardized protocols to help reduce potential healthcare-associated infections due to use of lotions. Improved efficacy of preservatives added to lotions should be a priority.
Assuntos
Contaminação de Medicamentos/estatística & dados numéricos , Creme para a Pele , Instituições de Assistência Ambulatorial/estatística & dados numéricos , Humanos , Terapia de Tecidos MolesRESUMO
Klebsiella pneumoniae represents a major threat to human health due to a combination of its nosocomial emergence and a propensity for acquiring antibiotic resistance. Dissemination of the bacteria from its native intestinal location creates severe, complicated infections that are particularly problematic in healthcare settings. Thus, there is an urgency for identifying novel treatment regimens as the incidence of highly antibiotic-resistant bacteria rises. Recent findings have highlighted the ability of some Gram-negative bacteria to utilize exogenous fatty acids in ways that modify membrane phospholipids and influence virulence phenotypes, such as biofilm formation and antibiotic resistance. This study explores the ability of K. pneumoniae to assimilate and respond to exogenous fatty acids. The combination of thin-layer chromatography liquid chromatography-mass spectrometry confirmed adoption of numerous exogenous polyunsaturated fatty acids (PUFAs) into the phospholipid species of K. pneumoniae. Membrane permeability was variably affected as determined by two dye uptake assays. Furthermore, the availability of many PUFAs lowered the MICs to the antimicrobial peptides polymyxin B and colistin. Biofilm formation was significantly affected depending upon the supplemented fatty acid.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Membrana Celular/química , Ácidos Graxos Insaturados/metabolismo , Klebsiella pneumoniae/efeitos dos fármacos , Fosfolipídeos/análise , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Cromatografia Líquida , Cromatografia em Camada Fina , Klebsiella pneumoniae/química , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/fisiologia , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Permeabilidade/efeitos dos fármacosRESUMO
Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.
Assuntos
Antígenos de Bactérias/metabolismo , Chlamydia trachomatis/imunologia , Retículo Endoplasmático/química , Células Epiteliais/metabolismo , Apresentação de Antígeno , Linhagem Celular Tumoral , Chlamydia trachomatis/ultraestrutura , Células Epiteliais/ultraestrutura , Humanos , Lipopolissacarídeos/análise , Microscopia ImunoeletrônicaRESUMO
Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.
Assuntos
Antígenos de Bactérias/imunologia , Infecções por Chlamydia/imunologia , Chlamydia trachomatis/imunologia , Chlamydia trachomatis/ultraestrutura , Vesículas Citoplasmáticas/ultraestrutura , Sequência de Aminoácidos , Antibacterianos/farmacologia , Azitromicina/farmacologia , Proteínas de Bactérias/metabolismo , Linhagem Celular , Chaperonina 60/metabolismo , Infecções por Chlamydia/tratamento farmacológico , Infecções por Chlamydia/microbiologia , Vesículas Citoplasmáticas/imunologia , Endométrio/citologia , Endométrio/imunologia , Endométrio/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Complexo de Golgi/imunologia , Complexo de Golgi/ultraestrutura , Humanos , Corpos de Inclusão/efeitos dos fármacos , Corpos de Inclusão/imunologia , Corpos de Inclusão/microbiologia , Corpos de Inclusão/ultraestrutura , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Microscopia Imunoeletrônica , Dados de Sequência MolecularRESUMO
UNLABELLED: Previous work from our laboratory showed that the Gram-negative aquatic pathogen Vibrio cholerae can take up a much wider repertoire of fatty acids than other Gram-negative organisms. The current work elaborated on the ability of V. cholerae to exploit an even more diverse pool of lipid nutrients from its environment. We have demonstrated that the bacterium can use lysophosphatidylcholine as a metabolite for growth. Using a combination of thin-layer chromatography and mass spectrometry, we also showed that lysophosphatidylcholine-derived fatty acid moieties can be used for remodeling the V. cholerae membrane architecture. Furthermore, we have identified a lysophospholipase, VolA (Vibrio outer membrane lysophospholipase A), required for these activities. The enzyme is well conserved in Vibrio species, is coexpressed with the outer membrane fatty acid transporter FadL, is one of very few surface-exposed lipoprotein enzymes to be identified in Gram-negative bacteria and the first instance of a surface lipoprotein phospholipase. We propose a model whereby the bacterium efficiently couples the liberation of fatty acid from lysophosphatidylcholine to its subsequent metabolic uptake. An expanded ability to scavenge diverse environmental lipids at the bacterial surface increases overall bacterial fitness and promotes homeoviscous adaptation through membrane remodeling. IMPORTANCE: Our understanding of how bacteria utilize environmental lipid sources has been limited to lipids such as fatty acids and cholesterol. This narrow scope may be attributed to both the intricate nature of lipid uptake mechanisms and the diversity of lipid substrates encountered within an ecological niche. By examining the ability of the pathogen Vibrio cholerae to utilize exogenous lipids, we uncovered a surface-exposed lipoprotein (VolA) that is required for processing the prevalent host lipid lysophosphatidylcholine. VolA functions as a lipase liberating a fatty acid from exogenous lysophospholipids. The freed fatty acid is then transported into the cell, serving as a carbon source, or shunted into phospholipid synthesis for membrane assembly. A limited number of surface-exposed lipoproteins have been found in Gram-negative organisms, and few have enzymatic function. This work highlights the ability of bacteria to exploit exogenous lipids for both maintenance of the membrane and carbon source acquisition.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Metabolismo dos Lipídeos , Lipoproteínas/metabolismo , Lisofosfolipase/metabolismo , Vibrio cholerae/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Cromatografia em Camada Fina , Metabolismo Energético , Humanos , Lipoproteínas/genética , Lisofosfolipase/genética , Espectrometria de Massas , Vibrio cholerae/química , Vibrio cholerae/enzimologia , Vibrio cholerae/crescimento & desenvolvimentoRESUMO
Chlamydial 60-kDa heat shock proteins (cHsp60s) are known to play a prominent role in the immunopathogenesis of disease. It is also known that several stress-inducing growth conditions, such as heat, iron deprivation, or exposure to gamma interferon, result in the development of persistent chlamydial forms that often exhibit enhanced expression of cHsp60. We have shown previously that the expression of cHsp60 is greatly enhanced in Chlamydia trachomatis serovar E propagated in an iron-deficient medium. The objective of this work was to determine which single cHsp60 or combination of the three cHsp60 homologs encoded by this organism responds to iron limitation. Using monospecific polyclonal peptide antisera that recognize only cHsp60-1, cHsp60-2, or cHsp60-3, we found that expression of cHsp60-2 is responsive to iron deprivation. Overall, our studies suggest that the expression of cHsp60 homologs differs among the mechanisms currently known to induce persistence.