ABSTRACT
Fusobacterium nucleatum is an opportunistic pathogenic bacterium that can be enriched in colorectal cancer tissues, affecting multiple stages of colorectal cancer development. The two-component system plays an important role in the regulation and expression of genes related to pathogenic resistance and pathogenicity. In this paper, we focused on the CarRS two-component system of F. nucleatum, and the histidine kinase protein CarS was recombinantly expressed and characterized. Several online software such as SMART, CCTOP and AlphaFold2 were used to predict the secondary and tertiary structure of the CarS protein. The results showed that CarS is a membrane protein with two transmembrane helices and contains 9 α-helices and 12 β-folds. CarS protein is composed of two domains, one is the N-terminal transmembrane domain (amino acids 1-170), the other is the C-terminal intracellular domain. The latter is composed of a signal receiving domain (histidine kinases, adenylyl cyclases, methyl-accepting proteins, prokaryotic signaling proteins, HAMP), a phosphate receptor domain (histidine kinase domain, HisKA), and a histidine kinase catalytic domain (histidine kinase-like ATPase catalytic domain, HATPase_c). Since the full-length CarS protein could not be expressed in host cells, a fusion expression vector pET-28a(+)-MBP-TEV-CarScyto was constructed based on the characteristics of secondary and tertiary structures, and overexpressed in Escherichia coli BL21-Codonplus(DE3)RIL. CarScyto-MBP protein was purified by affinity chromatography, ion-exchange chromatography, and gel filtration chromatography with a final concentration of 20 mg/ml. CarScyto-MBP protein showed both protein kinase and phosphotransferase activities, and the MBP tag had no effect on the function of CarScyto protein. The above results provide a basis for in-depth analysis of the biological function of the CarRS two-component system in F. nucleatum.
Subject(s)
Humans , Histidine Kinase/metabolism , Fusobacterium nucleatum/metabolism , Automobiles , Protein Kinases/genetics , Escherichia coli/metabolism , Colorectal NeoplasmsABSTRACT
@#Staphylococcus aureusis a major cause of hospital and community acquired infection,which can express many kinds of cell wall-related and extracellular virulence factors,causing a variety of infectious diseases. Ebh protein is involved in the adhesion,aggregation and biofilm formation of Staphylococcus aureus,and thus closely related to its strong pathogenicity. The pathogenesis of Staphylococcus aureus is controlled collaboratively by different two-component signal transduction systems and transcriptional regulation factors,among which ArlRS is the key two-component system(TCS)necessary for the adhesion,biofilm formation and virulence. At the same time,ArlRS regulates Ebh protein through global regulation factor MgrA,thus affecting the role of Staphylococcus aureus in human infectious diseases. In this paper,the regulatory mechanism was reviewed.
ABSTRACT
Carbapenem-resistant Klebsiella pneumoniae (CRKP) leads to high mortality of infected patients. How to deal with CRKP is an urgent problem in clinical practice, and it is imperative to carry out researchon carbapenem resistance mechanism of CRKP. The two-component systems (TCSs) areassociated with the development of drug resistance in a variety of bacteria, and TCSs were expected to be important therapeutic targets for CRKP. Therefore, this article reviewed the mechanisms of TCSs in the regulation of CRKP from the following several aspects: common mechanisms of carbapenem resistance of CRKP, research progress in drug resistance of TCSs, relationships between Klebsiella pneumoniae and TCSs, and so on. It may provide some research ideas for future research and the references for clinical diagnosis and treatment.
ABSTRACT
A interação entre membros do microbioma intestinal humano, células hospedeiras e patógenos invasores pode ocorrer de diversas formas, sendo uma delas através de pequenas moléculas chamadas metabólitos. A percepção e resposta efetiva de um microrganismo às diferentes condições encontradas em seu ambiente, incluindo metabólitos produzidos por outros microrganismos, são fatores importantes para sua adaptação, sobrevivência e disseminação. Os sistemas de dois componentes (TCS) permitem a percepção e resposta a mudanças ambientais, regulando a expressão de genes específicos. Nosso grupo mostrou anteriormente que um extrato orgânico de fezes humanas (EF), bem como o ácido 3,4-dimetilbenzoico (3,4-DMB), encontrado no EF, inibe a capacidade de Salmonella enterica sorovar Typhimurium de invadir células hospedeiras. O presente trabalho propôs investigar o impacto do microbioma intestinal humano, bem como de pequenas moléculas produzidas por Clostridium citroniae (membro deste microbioma) na expressão e atividade dos genes de TCS de Salmonella. Os metabólitos de EF e de culturas puras de C. citroniae foram extraídos com acetato de etila e adicionados a meio de cultura. O pH do meio foi ajustado (~ 7,4) e a solução foi esterilizada por filtragem. Salmonella foi cultivada na presença ou ausência do EF e do extrato de C. citroniae, bem como do ácido 3,4-DMB, em condições aeróbias e anaeróbias, até alcançar o meio da fase logarítmica de crescimento. O RNA foi extraído para a realização de PCR em Tempo Real utilizando iniciadores direcionados a quase todos os TCS de Salmonella. Nossos resultados mostraram que vários genes de TCS envolvidos na virulência de Salmonella (SsrAB, EnvZ-OmpR, QseCB, PhoQP, TorSR, TtrRS) foram regulados diferencialmente por esses metabólitos, tanto em condições aeróbias quanto anaeróbias. EnvZ-OmpR, PhoPQ e SsrAB estão diretamente envolvidos na regulação das Ilhas de Patogenicidade 1 e 2 de Salmonella. QseCB é crucial para a detecção de quorum em Salmonella, de hormônios hospedeiros e para a regulação da motilidade (swimming). Vários outros TCS também foram regulados, incluindo TorSR e TtrRS, envolvidos na regulação da respiração anaeróbica de N-óxido de trimetilamina (TMAO) e tetrationato, respectivamente. Esses compostos são importantes para a sobrevivência de Salmonella no ambiente anaeróbico do intestino humano. Nossos resultados de avaliação de expressão gênica global de Salmonella cultivada na presença de ácido 3,4-DMB (aerobiose e anaerobiose) bem como na presença do EF em anaerobiose, mostraram que genes condificados em SPI-1 e SPI-2, SPI-4 e alguns genes do TCS foram reprimidos, enquanto genes marR, marB e marA foram ativadas nessas condições. Adicionalmente, comparamos nossos resultados de RNAseq, de Salmonella cultivada na presença do ácido 3,4-DMB em aerobiose, com resultados disponíveis da base de dados Salmonella Compendium. Ainda, a capacidade de Salmonella de adentrar e sobreviver dentro de células fagocíticas (macrófagos RAW 264.7) parece ser afetada pelas três condições testadas neste trabalho. Nossos resultados mostram que importantes vias de sinalização da virulência de Salmonella podem ser moduladas pelos metabólitos presentes no microbioma intestinal humano e abrem caminhos para novas pesquisas sobre a sinalização intercelular microbioma-patógeno no ambiente intestinal.
The interaction between members of the human gut microbiome, host cells and invading pathogens often occurs through small molecules, also called metabolites. The perception and effective response of a microorganism to the different conditions found in its environment, including metabolites produced by other microbes, is important for its adaptation, survival and dissemination. Two-component systems (TCS) allow the perception and response to environmental changes by regulating the expression of specific genes. Our group previously showed that organic extracts of human feces (EF) as well as the specific metabolite 3,4-dimethylbenzoic acid (3,4-DMB) found within the EF, inhibit the ability of Salmonella enterica sorovar Typhimurium to invade host cells. In the present work, we investigated the impact of the human gut microbiome as well as small molecules produced by Clostridium citroniae (a member of this microbiome) on the expression and activity of Salmonella TCS genes. Metabolites (from feces or C. citroniae cultures) were extracted using ethyl acetate and added to culture medium. The pH of the medium was adjusted (~7.4), and the solution was filter sterilized. Salmonella was grown in the presence or absence of the organic extracts as well as 3,4-DMB acid under aerobic and anaerobic conditions until it reached mid-log growth. RNA was then extracted for Real-time PCR using primers targeting almost all Salmonella TCS. Our results showed that several TCS involved in Salmonella virulence (SsrAB, EnvZ-OmpR, QseCB, PhoQP, TorSR, TtrRS) were differentially regulated by these metabolites both in aerobic and anaerobic conditions. EnvZ-OmpR, PhoPQ, and SsrAB are directly involved in the regulation of Salmonella Pathogenicity Islands 1 and 2. QseCB is crucial for Salmonella =quorum sensing, sensing of host hormones and regulation of swimming motility. Several other TCS were also regulated, including TorSR and TtrRS, which are involved in the anaerobic respiration of trimethylamine N-oxide (TMAO) and tetrathionate, respectively. These compounds are important for Salmonella survival in the anaerobic environment of the human gut. Our results of the evaluation of global Salmonella gene expression grown in the presence of 3,4-DMB acid (aerobiosis and anaerobiosis) as well as in the presence of EF in anaerobiosis, showed that genes encoded in SPI-1 and SPI-2, SPI-4 and some TCS genes have been repressed, while multiple drug resistance genes, as well marR, marB and marA genes have been activated under these conditions. Besides, we compared our results of RNAseq, Salmonella was grown in the presence of 3,4-DMB acid in aerobiosis, with results available from the Salmonella Compendium database. Also, Salmonella's ability to enter and survive within phagocytic cells (macrophages RAW 264.7) appears to be affected by the three conditions tested in this work. Our results show that important Salmonella virulence signalling pathways can be modulated by the metabolites present in the human intestinal microbiome and open the way for further research on the microbiome-pathogen intercellular signalling in the intestinal environment.
Subject(s)
Humans , Salmonella enterica , Metabolome , Intestines/microbiology , Salmonella typhimurium , Aerobiosis , Virulence Factors , Genomic Islands , Feces/virology , Microbiota , Gastrointestinal Microbiome , AnaerobiosisABSTRACT
Beta-lactams are the most widely used antibiotics. One of the principle mechanisms for Gram-negative bacteria to resist β-lactams is by producing β-lactamases that degrade β-lactams. This review highlights two regulatory mechanisms for inducing β-lactamase in Gram-negative bacteria. In the ampR-ampC paradigm, the induction of β-lactamase is intimately linked to peptidoglycan recycling. AmpR, a LysR-type transcriptional regulator, plays a central role in regulating expression of β-lactamase. Recent studies found that two-component signal transduction pathway is activated by β-lactams, which in turn induces the expression of β-lactamase. Finally, we discussed the future research directions in β-lactam resistance in Gram-negative bacteria.
ABSTRACT
We designed a strategy for the sequencing and bioinformatical characterization of the 1,3-propanediol operon regulator genes from the Colombian Clostridium sp. strain IBUN13A, which is taxonomically related to Clostridium butyricum. Three genes are proposed to be involved in the operon's transcriptional activity, the dhaS and dhaA genes through a two-component system and the third gene named dhaY, which encodes a putative transcriptional regulator similar to the domains of the dhaS/A system. Phylogenetic analyses indicated that the predicted proteins had a modular structure consisting of domains homologous to different signal transduction systems, but had significant differences concerning their conserved residues, pointing to the possibility that they constitute ancestral domains. In accordance with the prediction of functions, we propose a mechanism of regulation of the proteins studied of the 1,3-propanediol operon of the native strain, as a response to the presence of glycerol in the medium, which provides valuable information on the overall regulation of the glycerol metabolism in Clostridium sp.
Se diseñó una estrategia de amplificación, secuenciación y caracterización bioinformática de los genes reguladores del operón 1,3-propanediol (1,3-PD) de la cepa nativa colombiana Clostridium sp. IBUN 13A, relacionada taxonómicamente con Clostridium butyricum. Se identificaron tres genes que pueden estar involucrados en la regulación transcripcional de dicho operón: los genes dhaS y dhaA -a través de un sistema de transducción de señales de dos componentes-y un tercer gen que se denominó dhaY, que codifica para un regulador transcripcional putativo, similar a los dominios presentes en las proteínas del sistema DhaS/A. Los análisis filogenéticos indican que las proteínas predichas presentan una estructura modular con dominios homólogos a diferentes sistemas de transducción de señales, pero muestran diferencias importantes en los residuos conservados, lo que sugiere que podrían ser estos los dominios ancestrales. La predicción de funciones postula un mecanismo de regulación de las proteínas estudiadas sobre el promotor del operón 1,3-PD de la cepa nativa como respuesta a la presencia de glicerol en el medio, lo cual aporta información importante sobre la regulación global del metabolismo del glicerol en Clostridium sp.
Nesta pesquisa foi feita uma estratégia para a amplificacäo, sequenciamento e caracterizacäo bioinformática dos genes reguladores do operon 1,3 propanodiol (1,3-PD) da cepa colombiana Clostridium sp. IBUN 13A, relacionada taxonomicamente com o Clostridium butyricum. Tèm sido identificados tres genes que podem estar envolvidos na regulacáo transcricional do operon. Os genes dhaS e dhaA por meio de um sistema de dois componentes e o terceiro gene nomeado de dhaY, que codifica para um regulador transcridonal putativo, parecido com os dominios presentes nas proteínas do sistema DhaS/A. A análise filogenètica mostra que estas proteínas apresentam uma estrutura modular com dominios homólogos a diferentes sistemas de traducáo de sinais, mas pressupöem diferencas importantes nos residuos conservados, indicando provavelmente que possam constituir os dominios ancestrais. De acordo com a predicáo de funcöes, é postulado um mecanismo de regulacáo do sistema DhaS/A, DhaY sobre o promotor do operon 1,3-DP da cepa nativa, como resposta à presenca de glicerol no meio, aportando informacöes importantes da regulacáo global do metabolismo do glicerol no Clostridium sp.
ABSTRACT
Objective:To explore the correlation between Mycobacterium tuberculosis PhoPR two-component system and the pathogenicity of different virulent MTB by analysing the expression levels difference of PhoP gene and PhoR gene in BCG ,H37Ra, H37Rv and XJ-MTB respectively.Methods:Total RNA extracted from four different virulent MTB strains and the integrity of total RNA were identified by using agarose gel electrophoresis.The expression of PhoP gene and PhoR gene were quantified by using SYBR Green I FQ-PCR.The expression levels difference of these genes were compared in different virulent MTB strains .Results: The relative expression levels of PhoP gene in between four different virulent MTB strains from high to low were XJ -MTB(9.05),H37Rv(1.00), H37Ra(0.25),BCG(0.08) respectively ,and the expression levels difference of PhoP gene were statistically significant in different virulent MTB strains ( P＜0.05 );the relative expression levels of PhoR gene in four different virulent MTB strains from high to low were XJ-MTB(5.72),H37Rv(1.00),H37Ra(0.18),BCG(0.07) respectively,and the expression levels difference of PhoR gene were sta-tistically significant in different virulent MTB strains ( P＜0.05 ).The expression levels of PhoP gene and PhoR gene at XJ-MTB were statistically significant difference compared with BCG ,H37Rv,H37Ra respectively (P＜0.05);the expression levels of PhoP gene and PhoR gene at H37Rv were statistically significant difference compared with BCG ,H37Ra respectively (P＜0.05);the expression levels of PhoP and PhoR gene at BCG were not statistically significant difference compared with H 37Ra (P>0.05).Conclusion:Significant expression levels difference of PhoP gene and PhoR gene are confirmed in different virulent MTB strains .Therefore,the Mycobacterium tuberculosis PhoPR two-component system is correlated with the pathogenicity in different virulent MTB strains.
ABSTRACT
Objective To explore the correlation between Mycobacterium tuberculosis ( MTB ) PhoPR two-component system and drug resistance of MTB clinical isolates widespread in Xinjiang region by analyzing the expression of PhoP gene and PhoR gene among different isolates .Methods Total RNA of MTB was extracted from drug-susceptible strains , the strains only resistant to a single first-line anti-TB drugs (INH, RFP, SM and EB) and multidrug-resistant (MDR) strains, respectively.The purity of total RNA was checked by agarose gel electrophoresis .The expressions of PhoP gene and PhoR gene were quantified by using SYBR Green I qRT-PCR and the differences of their gene expression in different isolates were ana-lyzed.Results Compared with the drug-susceptible strains of MTB, the expression of PhoP gene was up-regulated for about 1.48 times in MTB strains resistant to RFP (RFP-MTB) and 2.74 times in MDR strain (P<0.05).Compared with MDR strain, the expressions of PhoP gene in the isolates resistant to INH (IN-HMTB), RFP (RFP-MTB), SM (SM-MTB) and EB (EB-MTB) were down-regulated for 0.70, 0.50, 0.25 and 0.21 times respectively.The expressions of PhoR gene were down-regulated for 0.36, 0.54, 0.35 and 0.19 times, respectively (P<0.05).The expressions of PhoR gene in the isolates of INH-MTB, RFP-MTB, SM-MTB and EB-MTB were up-regulated for 6.33, 4.56, 2.34, 1.85 and 9.06 times, respectively as compared with the drug-susceptible strains (P<0.05).Conclusion Significant differences of PhoR gene and PhoP gene expressions were observed among drug-susceptible strains , INH-MTB, RFP-MTB, SM-MTB, EB-MTB and MDR strains.Therefore, the Mycobacterium tuberculosis PhoPR two-component system is asso-ciated with the drug resistance of MTB strains prevalent in Xinjiang region .
ABSTRACT
The Lactobacillus acidophilus group is a phylogenetically distinct group of closely related lactobacilli. Members of this group are considered to have probiotic properties and occupy different environmental niches. Bacteria generally sense and respond to environmental changes through two component systems (TCSs) which consist of a histidine protein kinase (HPK) and its cognate response regulator (RR). With the use of in silico techniques, the five completely sequenced L. acidophilus group genomes were scanned in order to predict TCSs. Five to nine putative TCSs encoding genes were detected in individual genomes of the L. acidophilus group. The L. acidophilus group HPKs and RRs were classified into subfamilies using the Grebe and Stock classification method. Putative TCSs were analyzed with respect to conserved domains to predict biological functions. Putative biological functions were predicted for the L. acidophilus group HPKs and RRs by comparing them with those of other microorganisms. Some of TCSs were putatively involved in a wide variety of functions which are related with probiotic ability, including tolerance to acid and bile, production of antimicrobial peptides, resistibility to the glycopeptide antibiotic vancomycin, and oxidative condition.
Subject(s)
Anti-Bacterial Agents , Base Sequence , DNA-Activated Protein Kinase , Glycopeptides , Histidine , Lactobacillus acidophilus/genetics , Lactobacillus acidophilus/isolation & purification , Peptides , Probiotics/isolation & purification , Signal Transduction , Computational Biology , Enzyme Activation , Methods , MethodsABSTRACT
The regulation of gene expression in the oral pathogen Aggregatibacter actinomycetemcomitans is still not fully elucidated. ArcAB is a two-component system which allows facultative anaerobic bacteria to sense various respiratory growth conditions and adapt their gene expression accordingly. This study investigated in A. actinomycetemcomitans the role of arcB on the regulation of biofilm formation, adhesion to saliva coated hydroxyapatite (SHA) and the hydrophobic properties of the cell. These phenotypic traits were determined for an A. actinomycetemcomitansarcB deficient type and a wild type strain. Differences in hydrophobic properties were shown at early and late exponential growth phases under microaerobic incubation and at late exponential phase under anaerobiosis. The arcB mutant formed less biofilm than the wild type strain when grown under anaerobic incubation, but displayed higher biofilm formation activity under microaerobic conditions. The adherence to SHA was significantly lower in the mutant when compared with the wild type strain. These results suggest that the transmembrane sensor kinase arcB, in A. actinomycetemcomitans, senses redox growth conditions and regulates the expression of surface components of the bacterial cell related to biofilm formation and adhesion to saliva coated surfaces.
A regulação da expressão gênica do patógeno oral Aggregatibacter actinomycetemcomitans não está completamente descrita. O sistema de dois componentes ArcAB permite que bactérias anaeróbias facultativas percebam diferenças nas condições respiratórias durante sua multiplicação e adaptem a expressão de genes à estas condições. Este estudo investigou em A. actinomycetemcomitans o papel de arcB na regulação da formação de biofilme, aderência à hidroxiapatita recoberta por saliva (SHA) e nas propriedades hidrofóbicas celulares. Estas características fenotípicas foram determinadas para uma linhagem de A. actinomycetemcomitans deficiente em arcB e para uma linhagem selvagem. Foram observadas diferenças nas propriedades hidrofóbicas entre as linhagens quando estas foram cultivadas em ambiente microaerófilo até início e final de fase exponencial e quando foram cultivadas em ambiente anaeróbio até final de fase exponencial. A linhagem arcB mutante formou menos biofilme do que a linhagem selvagem quando estas foram cultivadas sob incubação anaeróbica, porém, apresentou maior formação de biofilme quando a incubação foi realizada em condições de microaerofilia. A aderência à SHA apresentada pela linhagem mutante foi significantemente menor do que a observada pela linhagem selvagem. Estes estudos sugerem que a quinase sensora arcB, em A. actinomycetemcomitans, percebe as condições redox de multiplicação e regula a expressão de componentes de superfície bacterianos relacionados à formação de biofilme e adesão a superfícies recobertas com saliva.