RESUMO
Roles for the non-coding small RNA RyhB in quorum-sensing and iron-dependent gene modulation in the human pathogen V. vulnificus were assessed in this study. Both the quorum sensing master regulator SmcR and the Fur-iron complex were observed to bind to the region upstream of the non-coding small RNA RyhB gene to repress expression, which suggests that RyhB is associated with both quorum-sensing and iron-dependent signaling in this pathogen. We found that expression of LuxS, which is responsible for the biosynthesis of autoinducer-2 (AI-2), was higher in wild type than in a ryhB-deletion isotype. RyhB binds directly to the 5'-UTR (untranslated region) of the luxS transcript to form a heteroduplex, which not only stabilizes luxS mRNA but also disrupts the secondary structure that normally obscures the translational start codon and thereby allows translation of LuxS to begin. The binding of RyhB to luxS mRNA requires the chaperone protein Hfq, which stabilizes RyhB. These results demonstrate that the small RNA RyhB is a key element associated with feedback control of AI-2 production, and that it inhibits quorum-sensing signaling in an iron-dependent manner. This study, taken together with previous studies, shows that iron availability and cell density signals are funneled to SmcR and RyhB, and that these regulators coordinate cognate signal pathways that result in the proper balance of protein expression in response to environmental conditions.
Assuntos
Genes Bacterianos/genética , Homosserina/análogos & derivados , Ferro/metabolismo , Lactonas/metabolismo , Percepção de Quorum/fisiologia , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/fisiologia , Vibrio vulnificus/genética , Vibrio vulnificus/fisiologia , Regiões 5' não Traduzidas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Liases de Carbono-Enxofre/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Homosserina/biossíntese , Homosserina/metabolismo , RNA Mensageiro , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Vibrio vulnificus/metabolismoRESUMO
PURPOSE: To evaluate the effect of S.mutans luxS gene on mixed-species biofilms communities. METHODS: Biofilms were formed by S. mutans (wild type strain, its luxS overexpression strain and luxS knockout strain) and Lactobacillus acidophilus (ATCC4356) with a ratio of 1:1 at 37â for 4 h, 14 h and 24 h. MTT assay was used to detect the quantification of the biofilms formed. The structures of biofilms were observed under confocal laser scanning microscopy after 24 h, and expression of biofilm-related genes (ftf, smu630, brpA, gbpB, gtfB, vicR, comDE and relA) was investigated by real-time PCR. Statistical analysis was performed with SPSS17.0 software package. RESULTS: The results showed that biofilm formed by S. mutans(wild type strain, its luxS overexpression strain and luxS knockout strain) and L.acidophilus after 14 h were 0.481±0.024, 0.591±0.023 and 0.279±0.019, respectively. The same findings were present after 24 h, the biofilm formed by S.mutans overexpression strain with L.acidophilus was higher than wild type strain, and the biofilm formed by knockout strain significantly decreased; but there was no significant difference at 4 h time points. CLSM images revealed that both S.mutans overexpression strain and its wild type strain tended to aggregate into distinct clusters and dense structures, whereas the luxS knockout strain appeared relatively sparse. Compared with wild type strain, all of the genes examined were upregulated in the biofilms formed by the overexpression strain, and were downregulated in the biofilms formed by the luxS mutant strain in mixed-species biofilm. CONCLUSIONS: S.mutans luxS gene can affect mixed-species biofilm formation with L.acidophilus, which provides evidences for further study.
Assuntos
Proteínas de Bactérias , Biofilmes , Liases de Carbono-Enxofre , Streptococcus mutans , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/fisiologia , Lactobacillus acidophilus , Reação em Cadeia da Polimerase em Tempo Real , Streptococcus mutans/genéticaRESUMO
OBJECTIVE: To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2. MATERIALS AND METHODS: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions. RESULTS: LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05). CONCLUSIONS: Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/fisiologia , Enterococcus faecalis/crescimento & desenvolvimento , Percepção de Quorum/fisiologia , Análise de Variância , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Contagem de Colônia Microbiana , Enterococcus faecalis/genética , Técnicas de Inativação de Genes , Microscopia Confocal , Microscopia Eletrônica de Varredura , Plasmídeos , Percepção de Quorum/genética , Reação em Cadeia da Polimerase em Tempo Real , Fatores de TempoRESUMO
Several plants of agricultural and medicinal importance utilize defense chemistry that involves deployment of highly labile, reactive, and lachrymatory organosulfur molecules. However, this chemistry is difficult to investigate because the compounds are often short-lived and prone to degradation under the conditions required for analysis by common analytical techniques. This issue has complicated efforts to study the defense chemistry of plants that exploit the use of sulfur in their defense arsenals. This work illustrates how direct analysis in real time-high resolution mass spectrometry (DART-HRMS) can be used to track organosulfur defense compound chemistry under mild conditions. Petiveria alliacea was used as a model plant that exploits the enzyme alliinase to generate induced organosulfur compounds in response to herbivory. Tracking of the organosulfur compounds it produces and quantifying them by DART-HRMS using isotopically labeled analogues revealed a feedback inhibition loop through which the activities of the alliinase are stymied shortly after their activation. The results show that the downstream thiosulfinate products petivericin (100 µM) and pyruvate (8.4 mM) inhibit alliinase activity by 60% and 29%, respectively, after 1 h, and a mixture of the two inhibited alliinase activity by 65%. By 2 h, alliinase activity in the presence of these alliinase-derived products had ceased completely. Because thiosulfinate, pyruvate, and lachrymatory sulfine compounds are produced via the same alliinase-derived sulfenic acid intermediate, the inhibition of alliinase activity by increasing concentrations of downstream products shows how production of these defense compounds is modulated in real time in response to a tissue breach. These findings provide a framework within which heretofore unexplained phenomena observed in the defense chemistry of P. alliacea, onion, garlic, and other plants can be explained, as well as an approach by which to track labile compounds and enzymatic activity by DART-HRMS.
Assuntos
Liases de Carbono-Enxofre/fisiologia , Espectrometria de Massas/métodos , Phytolaccaceae/fisiologia , Liases de Carbono-Enxofre/antagonistas & inibidores , Liases de Carbono-Enxofre/isolamento & purificação , Cisteína/análogos & derivados , Cisteína/metabolismo , Retroalimentação Fisiológica , Cinética , Phytolaccaceae/enzimologia , Raízes de Plantas/enzimologia , Raízes de Plantas/fisiologia , Ácido Pirúvico/análise , Ácido Pirúvico/metabolismo , Ácidos Sulfínicos/análise , Ácidos Sulfínicos/metabolismoRESUMO
Abstract Objective: To investigate the relation between biofilm formation ability and quorum sensing gene LuxS/AI-2. Materials and Methods: Enterococcus faecalis (E. faecalis) standard strain ATCC 29212 was used in the study. Long flanking homology polymerase chain reaction method was used to build the LuxS gene knockout strain. Sequential culture turbidity measurement and CFU counting were used to assess the proliferation ability of E. faecalis after the depletion of LuxS. 96-well plate assay was used to quantify the biofilm formation ability; CLSM was used to observe the attached bacteria areas, while scanning electron microscopy (SEM) was performed to observe biofilm microstructure conditions. Results: LuxS gene knockout strains were successfully constructed and identified. The results showed that proliferation ability of E. faecalis was not affected by the depletion of the luxS gene, and the biofilm formation ability of ΔLuxS 29212 significantly decreased (P<0.05). Conclusions: Collectively, our studies provide the LuxS gene's key role in controlling biofilm formation of E. faecalis, which presented a negative regulation, and furthermore, providing us a possible way to conquer the persistent apical periodontitis.
Assuntos
Liases de Carbono-Enxofre/fisiologia , Proteínas de Bactérias/fisiologia , Enterococcus faecalis/crescimento & desenvolvimento , Biofilmes/crescimento & desenvolvimento , Percepção de Quorum/fisiologia , Plasmídeos , Liases de Carbono-Enxofre/genética , Fatores de Tempo , Proteínas de Bactérias/genética , Microscopia Eletrônica de Varredura , Contagem de Colônia Microbiana , Análise de Variância , Enterococcus faecalis/genética , Microscopia Confocal , Percepção de Quorum/genética , Técnicas de Inativação de Genes , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Fe-S cofactors are composed of iron and inorganic sulfur in various stoichiometries. A complex assembly pathway conducts their initial synthesis and subsequent binding to recipient proteins. In this minireview, we discuss how discovery of the role of the mammalian cytosolic aconitase, known as iron regulatory protein 1 (IRP1), led to the characterization of the function of its Fe-S cluster in sensing and regulating cellular iron homeostasis. Moreover, we present an overview of recent studies that have provided insights into the mechanism of Fe-S cluster transfer to recipient Fe-S proteins.
Assuntos
Homeostase , Proteína 1 Reguladora do Ferro/fisiologia , Ferro/fisiologia , Modelos Moleculares , Animais , Apoenzimas/química , Apoenzimas/metabolismo , Liases de Carbono-Enxofre/biossíntese , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/fisiologia , Transporte de Elétrons , Regulação Enzimológica da Expressão Gênica , Proteínas de Choque Térmico HSP70/biossíntese , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Proteína 1 Reguladora do Ferro/biossíntese , Proteína 1 Reguladora do Ferro/química , Proteínas de Ligação ao Ferro/biossíntese , Proteínas de Ligação ao Ferro/química , Proteínas de Ligação ao Ferro/fisiologia , Proteínas Reguladoras de Ferro/biossíntese , Proteínas Reguladoras de Ferro/química , Proteínas Reguladoras de Ferro/fisiologia , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/fisiologia , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/química , Proteínas Mitocondriais/fisiologia , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiologia , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Elementos de Resposta , Succinato Desidrogenase/biossíntese , Succinato Desidrogenase/química , Succinato Desidrogenase/fisiologia , FrataxinaRESUMO
Capnocytophaga ochracea is present in the dental plaque biofilm of patients with periodontitis. Biofilm cells change their phenotype through quorum sensing in response to fluctuations in cell-population density. Quorum sensing is mediated by auto-inducers (AIs). AI-2 is involved in intercellular signaling, and production of its distant precursor is catalyzed by LuxS, an enzyme involved in the activated methyl cycle. Our aim was to clarify the role of LuxS in biofilm formation by C. ochracea. Two luxS-deficient mutants, TmAI2 and LKT7, were constructed from C. ochracea ATCC 27872 by homologous recombination. The mutants produced significantly less AI-2 than the wild type. The growth rates of these mutants were similar to that of the wild-type in both undiluted Tryptic soy broth and 0.5 × Tryptic soy broth. However, according to crystal violet staining, they produced significantly less biofilm than the wild type. Confocal laser scanning microscopy and scanning electron microscopy showed that the biofilm of the TmAI2 strain had a rougher structure than that of the wild type. Complementation of TmAI-2 with extrinsic AI-2 from the culture supernatant of wild-type strain did not restore biofilm formation by the TmAI2 strain, but complementation of LKT7 strain with luxS partially restored biofilm formation. These results indicate that LuxS is involved in biofilm formation by C. ochracea, and that the attenuation of biofilm formation by the mutants is likely caused by a defect in the activated methyl cycle rather than by a loss of AI-2.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes , Capnocytophaga/fisiologia , Liases de Carbono-Enxofre/fisiologia , Proteínas de Bactérias/genética , Capnocytophaga/genética , Liases de Carbono-Enxofre/genética , Recombinação Homóloga , Microscopia Confocal , Microscopia Eletrônica de Varredura , MutaçãoRESUMO
Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.
Assuntos
Liases de Carbono-Enxofre/fisiologia , Proteínas Reguladoras de Ferro/fisiologia , Doenças Mitocondriais/fisiopatologia , Sequência de Aminoácidos , Liases de Carbono-Enxofre/química , Liases de Carbono-Enxofre/metabolismo , Progressão da Doença , Células HeLa , Humanos , Proteínas Reguladoras de Ferro/química , Proteínas Reguladoras de Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Doenças Mitocondriais/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estabilidade Proteica , Homologia de Sequência de AminoácidosRESUMO
To investigate the effect of the luxS gene on the expression of virulence factors in Shiga-like toxin producing and verotoxin-producing Escherichia coli, the luxS gene from E. coli 107/86 (wild type, O139:H1:F18ab, Stx2e) was deleted. The successful deletion of luxS was confirmed by bioluminescence assays. The luxS deletion mutant exhibited changed flagella-related phenotypes, like impaired expression of flagella, decreased flagella motility, reduced biofilm formation, and reduced ability to induce pro-immunity response in host cells, which were restored after complementation with the intact luxS gene. The mutant strain also displayed attenuated production of Stx2e. This study provides new information to the crucial function of luxS in regulating Shiga-like toxin producing E. coli virulence.
Assuntos
Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Flagelos/fisiologia , Percepção de Quorum/genética , Toxinas Shiga/biossíntese , Escherichia coli Shiga Toxigênica/genética , Animais , Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/fisiologia , Chlorocebus aethiops , Flagelos/genética , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Medições Luminescentes , Toxinas Shiga/genética , Escherichia coli Shiga Toxigênica/metabolismo , Escherichia coli Shiga Toxigênica/patogenicidade , Células Vero , Virulência/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Increasing methionine in potato tubers is desirable, both to increase the availability of this limiting essential amino acid and to enhance the aroma of baked and fried potatoes. Previous attempts to elevate potato methionine content using transgenic approaches have focused on increasing methionine biosynthesis. Higher isoleucine accumulation in these transgenic tubers suggested that the potatoes compensate for increased methionine biosynthesis with enhanced catabolism via methionine gamma-lyase (MGL), thereby producing 2-ketybutyrate for isoleucine biosynthesis. In the current study, we show that potato StMGL1 encodes a functional MGL in potato tubers. In planta silencing of StMGL1 results in an increased methionine to isoleucine ratio in the free amino acid profile of potato tubers and, in some transgenic lines, elevated accumulation of free methionine. In both wild-type and transgenic tubers, the ratio of methionine to isoleucine is negatively correlated with the level of StMGL1 transcript. A three-dimensional distribution of free amino acids in potato tubers is also described.
Assuntos
Liases de Carbono-Enxofre/fisiologia , Metionina/metabolismo , Proteínas de Plantas/fisiologia , Solanum tuberosum/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Isoleucina/metabolismo , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tubérculos/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo , Alinhamento de Sequência , Solanum tuberosum/crescimento & desenvolvimentoRESUMO
Quorum sensing (QS) enables bacterial multicellularity and selective advantage for communicating populations. While genetic "switching" phenomena are a common feature, their mechanistic underpinnings have remained elusive. The interplay between circuit components and their regulation are intertwined and embedded. Observable phenotypes are complex and context dependent. We employed a combination of experimental work and mathematical models to decipher network connectivity and signal transduction in the autoinducer-2 (AI-2) quorum sensing system of E. coli. Negative and positive feedback mechanisms were examined by separating the network architecture into sub-networks. A new unreported negative feedback interaction was hypothesized and tested via a simple mathematical model. Also, the importance of the LsrR regulator and its determinant role in the E. coli QS "switch", normally masked by interfering regulatory loops, were revealed. Our simple model allowed mechanistic understanding of the interplay among regulatory sub-structures and their contributions to the overall native functioning network. This "bottom up" approach in understanding gene regulation will serve to unravel complex QS network architectures and lead to the directed coordination of emergent behaviors.
Assuntos
Simulação por Computador , Escherichia coli/fisiologia , Modelos Biológicos , Percepção de Quorum/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/fisiologia , Biologia Computacional , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/fisiologia , Retroalimentação Fisiológica , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Genes Bacterianos , Homosserina/análogos & derivados , Homosserina/fisiologia , Lactonas , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Percepção de Quorum/genética , Transdução de SinaisRESUMO
LuxS is an enzyme involved in the activated methyl cycle and the by-product autoinducer-2 (AI-2) was a quorum sensing signal in some species. In our previous study, the functional LuxS in AI-2 production was verified in the porcine respiratory pathogen Actinobacillus pleuropneumoniae. Enhanced biofilm formation and reduced virulence were observed in the luxS mutant. To comprehensively understand the luxS function, in this study, the transcriptional profiles were compared between the A. pleuropneumoniae luxS mutant and its parental strain in four different growth phases using microarray. Many genes associated with infection were differentially expressed. The biofilm formation genes pgaABC in the luxS mutant were up-regulated in early exponential phase, while 9 genes associated with adhesion were down-regulated in late exponential phase. A group of genes involved in iron acquisition and metabolism were regulated in four growth phases. Phenotypic investigations using luxS mutant and both genetic and chemical (AI-2) complementation on these virulence traits were performed. The results demonstrated that the luxS mutant showed enhanced biofilm formation and reduced adhesion ability and these effects were not due to lack of AI-2. But AI-2 could increase biofilm formation and adhesion of A. pleuropneumoniae independent of LuxS. Growth under iron restricted condition could be controlled by LuxS through AI-2 production. These results revealed pleiotropic roles of LuxS and AI-2 on A. pleuropneumoniae virulence traits.
Assuntos
Actinobacillus pleuropneumoniae/fisiologia , Proteínas de Bactérias/fisiologia , Biofilmes/crescimento & desenvolvimento , Liases de Carbono-Enxofre/fisiologia , Homosserina/análogos & derivados , Ferro/metabolismo , Actinobacillus pleuropneumoniae/genética , Actinobacillus pleuropneumoniae/metabolismo , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Regulação Bacteriana da Expressão Gênica , Homosserina/genética , Homosserina/fisiologia , Lactonas , Mutação , Percepção de Quorum , Transcrição GênicaRESUMO
Despite the significant role of S-ribosylhomocysteinase (LuxS) in the activated methyl cycle pathway and quorum sensing, the connectivity between luxS and other cellular functions remains incomplete. Herein, we show that luxS deletion significantly increases swimming motility and flagella synthesis in Escherichia coli K12 using motility, transcriptome, and scanning electron microscopy assays. Further, based on the transcriptome and network component analyses, and known regulatory relations, we propose a conceptual genetic regulatory network underlying the increased flagella synthesis in response to luxS deletion.
Assuntos
Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/fisiologia , Escherichia coli K12/fisiologia , Flagelos/fisiologia , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Escherichia coli K12/genética , Flagelos/genética , Deleção de Genes , Regulação Bacteriana da Expressão GênicaRESUMO
Mature dental biofilms consist of towering microcolonies in which the resident bacterial cells interact with one another and exchange messages in the form of signalling molecules and metabolites. These structures have been compared with the bustling office blocks and apartment buildings of busy cities. Social and communication networks are the lifeblood of large communities, and there is mounting evidence that mutually beneficial interactions between microbial cells are essential to the development of biofilms in the oral cavity. This review discusses the mutualistic partnerships that form between oral bacteria, and the contribution of interspecies communication to the formation of mixed microbial communities.
Assuntos
Biofilmes , Placa Dentária/microbiologia , Interações Microbianas , Aderência Bacteriana , Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/fisiologia , Proteínas de Ligação a DNA/fisiologia , Homosserina/análogos & derivados , Homosserina/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Lactonas , Percepção de Quorum/fisiologia , Saliva/microbiologiaRESUMO
The enteric pathogen Salmonella enterica serovar Typhimurium uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 requires the luxS gene for its synthesis. The regulation of global gene expression in Salmonella Typhimurium by luxS/AI-2 is currently not known; therefore, the focus of this study was to elucidate the global gene expression patterns in Salmonella Typhimurium as regulated by luxS/AI-2. The genes controlled by luxS/AI-2 were identified using microarrays with RNA samples from wild-type (WT) Salmonella Typhimurium and its isogenic DeltaluxS mutant, in two growth conditions (presence and absence of glucose) at mid-log and early stationary phases. The results indicate that luxS/AI-2 has very different effects in Salmonella Typhimurium depending on the stage of cell growth and the levels of glucose. Genes with p < or = 0.05 were considered to be significantly expressed differentially between WT and DeltaluxS mutant. In the mid-log phase of growth, AI-2 activity was higher (1500-fold) in the presence of glucose than in its absence (450-fold). There was differential gene expression of 13 genes between the WT and its isogenic DeltaluxS mutant in the presence of glucose and 547 genes in its absence. In early stationary phase, AI-2 activity was higher (650-fold) in the presence of glucose than in its absence (1.5-fold). In the presence of glucose, 16 genes were differentially expressed, and in its absence, 60 genes were differentially expressed. Our microarray study indicates that both luxS and AI-2 could play a vital role in several cellular processes including metabolism, biofilm formation, transcription, translation, transport, and binding proteins, signal transduction, and regulatory functions in addition to previously identified functions. Phenotypic analysis of DeltaluxS mutant confirmed the microarray results and revealed that luxS did not influence growth but played a role in the biofilm formation and motility.
Assuntos
Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/fisiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Homosserina/análogos & derivados , Salmonella typhimurium/genética , Proteínas de Bactérias/genética , Biofilmes , Bioensaio , Transporte Biológico/genética , Liases de Carbono-Enxofre/genética , Ciclo Celular , Quimiotaxia/genética , Fímbrias Bacterianas/genética , Flagelos/genética , Glucose/administração & dosagem , Homosserina/análise , Homosserina/fisiologia , Lactonas/análise , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Biossíntese de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salmonella typhimurium/crescimento & desenvolvimento , Salmonella typhimurium/fisiologia , Transdução de Sinais/genética , Transcrição GênicaRESUMO
Sulfur-containing amino acids (SAAs) are essential components in many biological processes and ubiquitously distributed to all organisms. Both biosynthetic and catabolic pathways of SAAs are heterogeneous among organisms and between developmental stages, and regulated by the environmental changes. Limited lineage of organisms ranging from archaea to plants, but not human, possess a unique enzyme methionine gamma-lyase (MGL, EC 4.4.1.11) to directly degrade SAA to alpha-keto acids, ammonia, and volatile thiols. The reaction mechanisms and the physiological roles of this enzyme are partially demonstrated by the enzymological analyzes, structure determination, isotopic labeling of the intermediate metabolites, and functional analyzes of deficient mutants. MGL has been exploited as a drug target for the infectious diseases caused by parasitic protozoa and anaerobic periodontal bacteria. In addition, MGL has been utilized to develop therapeutic interventions of various cancers, by introducing recombinant proteins to deplete methionine essential for the growth of cancer cells. In this review, we discuss the current understanding of enzymological properties, putative physiological roles, and therapeutic applications of MGL.
Assuntos
Liases de Carbono-Enxofre/fisiologia , Liases de Carbono-Enxofre/uso terapêutico , Alcenos/farmacologia , Sequência de Aminoácidos , Anaerobiose , Animais , Antineoplásicos/uso terapêutico , Apoptose , Arabidopsis/enzimologia , Benzofuranos/farmacologia , Liases de Carbono-Enxofre/antagonistas & inibidores , Entamoeba histolytica/enzimologia , Humanos , Metionina/análogos & derivados , Metionina/farmacologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Doenças Periodontais/tratamento farmacológico , Infecções por Protozoários/tratamento farmacológico , Trichomonas vaginalis/enzimologiaRESUMO
Bacterial species can communicate by producing and sensing small autoinducer molecules by a process known as quorum sensing. Salmonella enterica produces autoinducer 2 (AI-2) via the luxS synthase gene, which is used by some bacterial pathogens to coordinate virulence gene expression with population density. We investigated whether the luxS gene might affect the ability of Salmonella enterica serovar Typhimurium to invade epithelial cells. No differences were found between the wild-type strain of S. Typhimurium, SL1344, and its isogenic luxS mutant with respect to the number and morphology of the membrane ruffles induced or their ability to invade epithelial cells. The dynamics of the ruffling process were also similar in the wild-type strain (SL1344) and the luxS mutant. Furthermore, comparing the Salmonella pathogenicity island 1 (SPI-1) type 3 secretion profiles of wild-type SL1344 and the luxS mutant by Western blotting and measuring the expression of a single-copy green fluorescent protein fusion to the prgH (an essential SPI-1 gene) promoter indicated that SPI-1 expression and activity are similar in the wild-type SL1344 and luxS mutant. Genetic deletion of luxS did not alter the virulence of S. Typhimurium in the mouse model, and therefore, it appears that luxS does not play a significant role in regulating invasion of Salmonella in vitro or in vivo.
Assuntos
Proteínas de Bactérias/fisiologia , Liases de Carbono-Enxofre/fisiologia , Células Epiteliais/microbiologia , Percepção de Quorum/fisiologia , Salmonella enterica/metabolismo , Actinas/metabolismo , Animais , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Linhagem Celular , Cães , Feminino , Citometria de Fluxo , Células HeLa , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Contraste de Fase , Percepção de Quorum/genética , Salmonella enterica/genéticaRESUMO
Bacteria utilize quorum-sensing communication to organize their behavior by monitoring the concentration of bacterial signals, referred to as autoinducers (AIs). The widespread detection of AI-2 signals and its enzymatic synthase (LuxS) in bacteria suggests that AI-2 is an inter- and intraspecies communication signal. We have previously shown that antibiotic susceptibility is affected by AI-2 signaling in Streptococcus anginosus. Since chronic infections involve persistent biofilms resilient to antibiotic treatment, we explored the role of AI-2/LuxS in Streptococcus intermedius biofilm formation and cell viability when the organism was exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. The S. intermedius wild type (WT) and its isogenic luxS mutant, strain SI006, were exposed to sub-MICs of ampicillin, ciprofloxacin, or tetracycline. Biofilms were formed on polystyrene discs in microtiter plates. To assess planktonic cell viability, the ATP microbial viability assay was performed and the numbers of CFU were determined. For complementation assays, the AI-2 precursor dihydroxy pentanedione (DPD) was used as a supplement for SI006. Relative luxS expression was quantified by real-time PCR. The sub-MICs of all three antibiotics increased biofilm formation in S. intermedius WT. However, biofilm formation by SI006 was either unaffected or reduced (P < or = 0.05). Bacterial viability tests of biofilm and planktonic cell cultures indicated that SI006 was more susceptible to antibiotics than the WT. DPD complemented the luxS mutant phenotype. Real-time PCR revealed modest yet significant changes in luxS expression in the presence of antibiotic concentrations that increased biofilm formation. In conclusion, in S. intermedius, AI-2/LuxS was involved in antibiotic susceptibility and increased biofilm formation at sub-MICs of antibiotic.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/fisiologia , Biofilmes/efeitos dos fármacos , Liases de Carbono-Enxofre/fisiologia , Homosserina/análogos & derivados , Streptococcus intermedius/efeitos dos fármacos , Ampicilina/farmacologia , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Ciprofloxacina/farmacologia , Homosserina/metabolismo , Homosserina/fisiologia , Lactonas/metabolismo , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase , Streptococcus intermedius/genética , Streptococcus intermedius/metabolismo , Streptococcus intermedius/ultraestrutura , Tetraciclina/farmacologiaRESUMO
Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.
Assuntos
Proteínas de Bactérias/fisiologia , Biofilmes , Liases de Carbono-Enxofre/fisiologia , Haemophilus influenzae/fisiologia , Lipopolissacarídeos/análise , Animais , Chinchila , Haemophilus influenzae/química , Homosserina/análogos & derivados , Homosserina/fisiologia , Lactonas , Otite Média/microbiologia , Fosforilcolina/análise , Sequências Repetitivas de Ácido NucleicoRESUMO
The fungus Candida albicans colonizes human oral cavity surfaces in conjunction with a complex microflora. C. albicans SC5314 formed biofilms on saliva-coated surfaces that in early stages of development consisted of approximately 30% hyphal forms. In mixed biofilms with the oral bacterium Streptococcus gordonii DL1, hyphal development by C. albicans was enhanced so that biofilms consisted of approximately 60% hyphal forms. Cell-cell contact between S. gordonii and C. albicans involved Streptococcus cell wall-anchored proteins SspA and SspB (antigen I/II family polypeptides). Repression of C. albicans hyphal filament and biofilm production by the quorum-sensing molecule farnesol was relieved by S. gordonii. The ability of a luxS mutant of S. gordonii deficient in production of autoinducer 2 to induce C. albicans hyphal formation was reduced, and this mutant suppressed farnesol inhibition of hyphal formation less effectively. Coincubation of the two microbial species led to activation of C. albicans mitogen-activated protein kinase Cek1p, inhibition of Mkc1p activation by H(2)O(2), and enhanced activation of Hog1p by farnesol, which were direct effects of streptococci on morphogenetic signaling. These results suggest that interactions between C. albicans and S. gordonii involve physical (adherence) and chemical (diffusible) signals that influence the development of biofilm communities. Thus, bacteria may play a significant role in modulating Candida carriage and infection processes in the oral cavity.