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1.
Microbiol Spectr ; 10(2): e0000222, 2022 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-35286133

RESUMO

Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis synthesizes a variety of sphingolipids, similar to its human host. Studies have shown that synthesis of these lipids (dihydroceramides [DHCs]) is involved in oxidative stress resistance, the survival of P. gingivalis during stationary phase, and immune modulation. Here, we constructed a deletion mutant of P. gingivalis strain W83 with a deletion of the gene encoding DhSphK1, a protein that shows high similarity to a eukaryotic sphingosine kinase, an enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate. Our data show that deletion of the dhSphK1 gene results in a shift in the sphingolipid composition of P. gingivalis cells; specifically, the mutant synthesizes higher levels of phosphoglycerol DHCs (PG-DHCs) than the parent strain W83. Although PG1348 shows high similarity to the eukaryotic sphingosine kinase, we discovered that the PG1348 enzyme is unique, since it preferentially phosphorylates dihydrosphingosine, not sphingosine. Besides changes in lipid composition, the W83 ΔPG1348 mutant displayed a defect in cell division, the biogenesis of outer membrane vesicles (OMVs), and the amount of K antigen capsule. Taken together, we have identified the first bacterial dihydrosphingosine kinase whose activity regulates the lipid profile of P. gingivalis and underlies a regulatory mechanism of immune modulation. IMPORTANCE Sphingoid base phosphates, such as sphingosine-1-phosphate (S1P) and dihydrosphingosine-1-phosphate (dhS1P), act as ligands for S1P receptors, and this interaction is known to play a central role in mediating angiogenesis, vascular stability and permeability, and immune cell migration to sites of inflammation. Studies suggest that a shift in ratio to higher levels of dhS1P in relation to S1P alters downstream signaling cascades due to differential binding and activation of the various S1P receptor isoforms. Specifically, higher levels of dhS1P are thought to be anti-inflammatory. Here, we report on the characterization of a novel kinase in Porphyromonas gingivalis that phosphorylates dihydrosphingosine to form dhS1P.


Assuntos
Transdução de Sinais , Esfingosina , Movimento Celular , Humanos , Esfingosina/análogos & derivados , Esfingosina/química , Esfingosina/metabolismo
2.
J Food Prot ; 82(8): 1336-1349, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31313962

RESUMO

Nontyphoidal Salmonella strains continue to be a major cause of foodborne illness globally. One intriguing approach to reducing the risk of salmonellosis is the direct ingestion of phages targeting Salmonella to enhance natural gut resilience and provide protection during foodborne disease outbreaks. We evaluated the ability of a prophylactically administered bacteriophage cocktail, the foodborne outbreak pill (FOP) targeting Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella, to resolve a Salmonella infection in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), a simulated gut platform populated by the human intestinal microbiome of healthy donors. The FOP preparation eliminated Salmonella enterica serovar Typhimurium from the colon compartment of the SHIME platform but health-associated metabolites, such as short-chain fatty acids and lactate, remained stable or increased in a donor-dependent manner. In studies of human intestinal cells, pretreatment of Salmonella Typhimurium with the FOP cocktail preserved lipopolysaccharide-stimulated signaling in a Caco-2-THP-1 Transwell system and prevented destruction of the Caco-2 monolayer by Salmonella. Adhesion and invasion of intestinal epithelial cells by Salmonella-a critical factor in Salmonella pathogenesis-was blunted when the bacteria were incubated with the FOP preparation before addition to the monolayer. The FOP phage cocktail was effective for (i) eliminating Salmonella from a simulated human gut without disturbing the indigenous microbiota and (ii) reducing the risk of invasion by Salmonella into the intestinal epithelia. These results suggest that the FOP preparation may be of value for reducing the risk of salmonellosis in humans, e.g., during foodborne disease outbreaks.


Assuntos
Bacteriófagos , Microbioma Gastrointestinal , Salmonella typhimurium , Bacteriófagos/fisiologia , Células CACO-2 , Colo/microbiologia , Citocinas/metabolismo , Humanos , Técnicas In Vitro , Salmonella typhimurium/virologia , Transdução de Sinais
3.
Appl Environ Microbiol ; 85(4)2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30552185

RESUMO

The asaccharolytic anaerobe Porphyromonas gingivalis metabolizes proteins it encounters in the periodontal pocket, including host-derived glycoproteins such as mucins and immunoglobulins. Often, these proteins are protected by a diverse array of carbohydrates tethered to the polypeptide chain via glycolytic bonds, and P. gingivalis produces enzymes capable of liberating these carbohydrates, exposing the proteinaceous core. In this study, we investigated the effect of individual monosaccharides, including galactose, l-fucose, mannose, and glucose, on the growth and physiology of P. gingivalis Of the carbohydrates tested, only galactose noticeably altered the density of the bacterial culture, and we observed that cultures grown with galactose reached significantly higher densities during stationary phase. Importantly, electron micrographs and plating of P. gingivalis in stationary phase demonstrated that the presence of galactose did not increase cell numbers; instead, the higher densities resulted from the expansion of individual cells which contained large intracellular granules. Initial attempts to characterize these granules revealed only a subtle increase in soluble carbohydrates, suggesting they are likely not composed of stored carbohydrate. Also, an analysis of major surface polysaccharides via an enzyme-linked immunosorbent assay (ELISA) did not reveal significant differences between cells grown with or without galactose. Finally, an initial investigation of the transcriptional changes elicited by galactose in late exponential phase suggested that genes important for cell shape and for the general stress response may play roles in this phenomenon. Overall, galactose, a monosaccharide commonly present on the surfaces of host proteins, substantially alters the physiology of P. gingivalis via the production of large, currently undefined, intracellular granules.IMPORTANCE Environmental perturbations are central to the ability of pathobionts, such as Porphyromonas gingivalis, to promote the development of diseased sites. In the case of periodontal disease, increased local pH, a shift to anaerobic surroundings, and the accumulation of Gram-negative anaerobes at the expense of Gram-positive cocci are known ecological fluctuations prominently associated with progression toward disease. Importantly, in contrast, the alterations to subgingival food webs in disease sites remain poorly characterized. We hypothesized that given the dramatic shift in community structure during disease, it is possible that free carbohydrates, which would typically be readily metabolized by Gram-positive cocci after cleavage from glycoproteins, may increase in concentration locally and thereby affect the physiological state of the subgingival microbiota. In this study, we explored the impact of free monosaccharides on P. gingivalis to gain deeper insight into the effect of dysbiotic conditions on the growth and physiology of this periodontal pathogen.


Assuntos
Galactose/metabolismo , Porphyromonas gingivalis/crescimento & desenvolvimento , Porphyromonas gingivalis/fisiologia , Meios de Cultura/química , Fucose/metabolismo , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Manose/metabolismo , Doenças Periodontais/microbiologia , Porphyromonas gingivalis/citologia , Porphyromonas gingivalis/genética , Transcriptoma
4.
Viruses ; 10(4)2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29671810

RESUMO

Foodborne illnesses remain a major cause of hospitalization and death worldwide despite many advances in food sanitation techniques and pathogen surveillance. Traditional antimicrobial methods, such as pasteurization, high pressure processing, irradiation, and chemical disinfectants are capable of reducing microbial populations in foods to varying degrees, but they also have considerable drawbacks, such as a large initial investment, potential damage to processing equipment due to their corrosive nature, and a deleterious impact on organoleptic qualities (and possibly the nutritional value) of foods. Perhaps most importantly, these decontamination strategies kill indiscriminately, including many—often beneficial—bacteria that are naturally present in foods. One promising technique that addresses several of these shortcomings is bacteriophage biocontrol, a green and natural method that uses lytic bacteriophages isolated from the environment to specifically target pathogenic bacteria and eliminate them from (or significantly reduce their levels in) foods. Since the initial conception of using bacteriophages on foods, a substantial number of research reports have described the use of bacteriophage biocontrol to target a variety of bacterial pathogens in various foods, ranging from ready-to-eat deli meats to fresh fruits and vegetables, and the number of commercially available products containing bacteriophages approved for use in food safety applications has also been steadily increasing. Though some challenges remain, bacteriophage biocontrol is increasingly recognized as an attractive modality in our arsenal of tools for safely and naturally eliminating pathogenic bacteria from foods.


Assuntos
Bacteriófagos/crescimento & desenvolvimento , Microbiologia de Alimentos , Inocuidade dos Alimentos/métodos , Indústria de Processamento de Alimentos/métodos , Doenças Transmitidas por Alimentos/prevenção & controle , Humanos
5.
Front Microbiol ; 7: 1919, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27965646

RESUMO

Bacteria alter the biophysical properties of their membrane lipids in response to environmental cues, such as shifts in pH or temperature. In essence, lipid composition determines membrane structure, which in turn influences many basic functions, such as transport, secretion, and signaling. Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis possesses the ability to synthesize a variety of novel membrane lipids, including species of dihydroceramides that are distinct, yet similar in structure to sphingolipids produced by the human host. The role of dihydroceramides in the physiology and pathogenic potential of the human microbiota is only beginning to be explored; yet there is increasing data indicating that these lipids play a role in human diseases, such as periodontitis and multiple sclerosis. Here, we report on the identification of a gene (PG1780) in the chromosome of P. gingivalis strain W83 encoding a putative serine palmitoyltransferase, the enzyme that catalyzes the first step in sphingolipid biosynthesis. While we were able to detect dihydroceramides in whole lipid extracts of P. gingivalis cells as well as crude preparations of outer membrane vesicles, sphingolipids were absent in the PG1780 mutant strain. Moreover, we show that the synthesis of sphingolipids plays an essential role in the long-term survival of the organism as well as its resistance to oxidative stress. Further, a PG1780 mutant displayed much lower activity of cell-associated arginine and lysine gingipains, yet slightly higher activity in the corresponding culture supernates, which we hypothesize is due to altered membrane properties and anchoring of these proteases to the cell surface. In addition, we determined that sphingolipid production is critical to the presentation of surface polysaccharides, with the mutant strain displaying less K-antigen capsule and more anionic polysaccharide (APS). Overall, we have discovered that, in addition to their role in pathogenicity, the synthesis of sphingolipids is critical to the cellular homeostasis and persistence of this important dental pathogen.

6.
Appl Environ Microbiol ; 82(15): 4821-4834, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27260355

RESUMO

UNLABELLED: The capacity to internalize and catabolize carbohydrates is essential for dental caries pathogens to persist and cause disease. The expression of many virulence-related attributes by Streptococcus mutans, an organism strongly associated with human dental caries, is influenced by the peptide signaling pathways that control genetic competence. Here, we demonstrate a relationship between the efficiency of competence signaling and carbohydrate source. A significant increase in the activity of the promoters for comX, comS, and comYA after exposure to competence-stimulating peptide (CSP) was observed in cells growing on fructose, maltose, sucrose, or trehalose as the primary carbohydrate source, compared to cells growing on glucose. However, only cells grown in the presence of trehalose or sucrose displayed a significant increase in transformation frequency. Notably, even low concentrations of these carbohydrates in the presence of excess glucose could enhance the expression of comX, encoding a sigma factor needed for competence, and the effects on competence were dependent on the cognate sugar:phosphotransferase permease for each carbohydrate. Using green fluorescent protein (GFP) reporter fusions, we observed that growth in fructose or trehalose resulted in a greater proportion of the population activating expression of comX and comS, encoding the precursor of comX-inducing peptide (XIP), after addition of CSP, than growth in glucose. Thus, the source of carbohydrate significantly impacts the stochastic behaviors that regulate subpopulation responses to CSP, which can induce competence in S. mutans IMPORTANCE: The signaling pathways that regulate development of genetic competence in Streptococcus mutans are intimately intertwined with the pathogenic potential of the organism, impacting biofilm formation, stress tolerance, and expression of known virulence determinants. Induction of the gene for the master regulator of competence, ComX, by competence-stimulating peptide (CSP) occurs in a subpopulation of cells. Here, we show that certain carbohydrates that are common in the human diet enhance the ability of CSP to activate transcription of comX and that a subset of these carbohydrates stimulates progression to the competent state. The cognate sugar:phosphotransferase permeases for each sugar are needed for these effects. Interestingly, single-cell analysis shows that the carbohydrates that increase com gene expression do so by enhancing the proportion of cells that respond to CSP. A mathematical model is developed to explain how carbohydrates modulate bistable behavior in the system via the ComRS pathway and ComX stability.


Assuntos
Carboidratos/química , Streptococcus mutans/genética , Streptococcus mutans/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Metabolismo dos Carboidratos , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fator sigma/genética , Fator sigma/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-25317251

RESUMO

The nature of the oral cavity and host behaviors has mandated that the oral microbiota evolve mechanisms for coping with environmental fluctuations, especially changes in the type and availability of carbohydrates. In the case of human dental caries, the presence of excess carbohydrates is often responsible for altering the local environment to be more favorable for species associated with the initiation and progression of disease, including Streptococcus mutans. Some of the earliest endeavors to understand how cariogenic species respond to environmental perturbations were carried out using chemostat cultivation, which provides fine control over culture conditions and bacterial behaviors. The development of genome-scale methodologies has allowed for the combination of sophisticated cultivation technologies with genome-level analysis to more thoroughly probe how bacterial pathogens respond to environmental stimuli. Recent investigations in S. mutans and other closely related streptococci have begun to reveal that carbohydrate metabolism can drastically impact pathogenic potential and highlight the important influence that nutrient acquisition has on the success of pathogens; inside and outside of the oral cavity. Collectively, research into pathogenic streptococci, which have evolved in close association with the human host, has begun to unveil the essential nature of careful orchestration of carbohydrate acquisition and catabolism to allow the organisms to persist and, when conditions allow, initiate or worsen disease.

8.
Appl Environ Microbiol ; 80(16): 5053-67, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24928869

RESUMO

Glucosamine and N-acetylglucosamine are among the most abundant sugars on the planet, and their introduction into the oral cavity via the diet and host secretions, and through bacterial biosynthesis, provides oral biofilm bacteria with a source of carbon, nitrogen, and energy. In this study, we demonstrated that the dental caries pathogen Streptococcus mutans possesses an inducible system for the metabolism of N-acetylglucosamine and glucosamine. These amino sugars are transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS), with the glucose/mannose enzyme II permease encoded by manLMN playing a dominant role. Additionally, a previously uncharacterized gene product encoded downstream of the manLMN operon, ManO, was shown to influence the efficiency of uptake and growth on N-acetylglucosamine and, to a lesser extent, glucosamine. A transcriptional regulator, designated NagR, was able to bind the promoter regions in vitro, and repress the expression in vivo, of the nagA and nagB genes, encoding N-acetylglucosamine-6-phosphate deacetylase and glucosamine-6-phosphate deaminase, respectively. The binding activity of NagR could be inhibited by glucosamine-6-phosphate in vitro. Importantly, in contrast to the case with certain other Firmicutes, the gene for de novo synthesis of glucosamine-6-phosphate in S. mutans, glmS, was also shown to be regulated by NagR, and NagR could bind the glmS promoter region in vitro. Finally, metabolism of these amino sugars by S. mutans resulted in the production of significant quantities of ammonia, which can neutralize cytoplasmic pH and increase acid tolerance, thus contributing to enhanced persistence and pathogenic potential.


Assuntos
Acetilglucosamina/metabolismo , Glucosamina/metabolismo , Streptococcus mutans/metabolismo , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Regulação Bacteriana da Expressão Gênica , Glucose/metabolismo , Óperon , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/genética , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/metabolismo , Regiões Promotoras Genéticas , Streptococcus mutans/enzimologia , Streptococcus mutans/genética
9.
Appl Environ Microbiol ; 80(3): 972-85, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24271168

RESUMO

The genetic and phenotypic responses of Streptococcus mutans, an organism that is strongly associated with the development of dental caries, to changes in carbohydrate availability were investigated. S. mutans UA159 or a derivative of UA159 lacking ManL, which is the EIIAB component (EIIAB(Man)) of a glucose/mannose permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and a dominant effector of catabolite repression, was grown in continuous culture to steady state under conditions of excess (100 mM) or limiting (10 mM) glucose. Microarrays using RNA from S. mutans UA159 revealed that 174 genes were differentially expressed in response to changes in carbohydrate availability (P < 0.001). Glucose-limited cells possessed higher PTS activity, could acidify the environment more rapidly and to a greater extent, and produced more ManL protein than cultures grown with excess glucose. Loss of ManL adversely affected carbohydrate transport and acid tolerance. Comparison of the histidine protein (HPr) in S. mutans UA159 and the manL deletion strain indicated that the differences in the behaviors of the strains were not due to major differences in HPr pools or HPr phosphorylation status. Therefore, carbohydrate availability alone can dramatically influence the expression of physiologic and biochemical pathways that contribute directly to the virulence of S. mutans, and ManL has a profound influence on this behavior.


Assuntos
Metabolismo dos Carboidratos , Streptococcus mutans/efeitos dos fármacos , Streptococcus mutans/metabolismo , Fatores de Virulência/biossíntese , Perfilação da Expressão Gênica , Redes e Vias Metabólicas/genética , Análise em Microsséries , Streptococcus mutans/patogenicidade , Virulência
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