Functional correlation of bacterial LuxS with their quaternary associations: interface analysis of the structure networks

Background The genome of a wide variety of prokaryotes contains the luxS gene homologue, which encodes for the protein S-ribosylhomocysteinelyase (LuxS). This protein is responsible for the production of the quorum sensing molecule, AI-2 and has been implicated in a variety of functions such as flagellar motility, metabolic regulation, toxin production and even in pathogenicity. A high structural similarity is present in the LuxS structures determined from a few species. In this study, we have modelled the structures from several other species and have investigated their dimer interfaces. We have attempted to correlate the interface features of LuxS with the phenotypic nature of the organisms. Results The protein structure networks (PSN) are constructed and graph theoretical analysis is performed on the structures obtained from X-ray crystallography and on the modelled ones. The interfaces, which are known to contain the active site, are characterized from the PSNs of these homodimeric proteins. The key features presented by the protein interfaces are investigated for the classification of the proteins in relation to their function. From our analysis, structural interface motifs are identified for each class in our dataset, which showed distinctly different pattern at the interface of LuxS for the probiotics and some extremophiles. Our analysis also reveals potential sites of mutation and geometric patterns at the interface that was not evident from conventional sequence alignment studies. Conclusion The structure network approach employed in this study for the analysis of dimeric interfaces in LuxS has brought out certain structural details at the side-chain interaction level, which were elusive from the conventional structure comparison methods. The results from this study provide a better understanding of the relation between the luxS gene and its functional role in the prokaryotes. This study also makes it possible to explore the potential direction towards the design of inhibitors of LuxS and thus towards a wide range of antimicrobials.

Elucidation of the conformational free energy landscape in H.pylori LuxS and its implications to catalysis

Background: One of the major challenges in understanding enzyme catalysis is to identify the different conformations and their populations at detailed molecular level in response to ligand binding/environment. A detail description of the ligand induced conformational changes provides meaningful insights into the mechanism of action of enzymes and thus its function. Results: In this study, we have explored the ligand induced conformational changes in H. pylori LuxS and the associated mechanistic features. LuxS, a dimeric protein, produces the precursor (4,5-dihydroxy-2,3-pentanedione) for autoinducer-2 production which is a signalling molecule for bacterial quorum sensing. We have performed molecular dynamics simulations on H. pylori LuxS in its various ligand bound forms and analyzed the simulation trajectories using various techniques including the structure network analysis, free energy evaluation and water dynamics at the active site. The results bring out the mechanistic details such as co operativity and asymmetry between the two subunits, subtle changes in the conformation as a response to the binding of active and inactive forms of ligands and the population distribution of different conformations in equilibrium. These investigations have enabled us to probe the free energy landscape and identify the corresponding conformations in terms of network parameters. In addition, we have also elucidated the variations in the dynamics of water co-ordination to the Zn2+ ion in LuxS and its relation to the rigidity at the active sites. Conclusions: In this article, we provide details of a novel method for the identification of conformational changes in the different ligand bound states of the protein, evaluation of ligand-induced free energy changes and the biological relevance of our results in the context of LuxS structure-function. The methodology outlined here is highly generalized to illuminate the linkage between structure and function in any protein of known structure.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Attenuation of Edwardsiella tarda Virulence by Small Peptides That Interfere with LuxS/Autoinducer Type 2 Quorum Sensing

Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Regulation of the Edwardsiella tarda Hemolysin Gene and luxS by EthR

Edwardsiella tarda is a pathogen with a broad host range that includes human and animals. The E. tarda hemolysin (Eth) system, which comprises EthA and EthB, is a noted virulence element that is widely distributed in pathogenic isolates of E. tarda. Previous study has shown that the expression of ethB is regulated by iron, which suggests the possibility that the ferric uptake regulator (Fur) is involved in the regulation of ethB. The work presented in this report supports the previous findings and demonstrates that ethB expression was decreased under conditions when the E. tarda Fur (Fur(Et)) was overproduced, and enhanced when Fur(Et) was inactivated. We also identified a second ethB regulator, EthR, which is a transcription regulator of the GntR family. EthR represses ethB expression by direct interaction with the ethB promoter region. In addition to ethB, EthR also modulates, but positively, luxS expression and AI-2 production by binding to the luxS promoter region. The expression of ethR itself is subject to negative autoregulation; interference with this regulation by overexpressing ethR during the process of infection caused (i) drastic changes in ethB and luxS expressions, (ii) vitiation in the tissue dissemination and survival ability of the bacterium, and (iii) significant attenuation of the overall bacterial virulence. These results not only provide new insights into the regulation mechanisms of the Eth hemolysin and LuxS/AI-2 quorum sensing systems but also highlight the importance of these systems in bacterial virulence.

迟缓爱德华氏菌(Edwardsiella tarda) LuxS/AI-2群体感应系统研究

从患病牙鲆中分离出迟缓爱德华氏菌株TX1，经报告菌株检测发现TX1有AI-2活性。用梯度PCR和Genome walking的方法克隆了TX1 luxS基因，将luxS基因在大肠杆菌DH5α中表达，证明其具有功能活性。在TX1中，luxS的表达与AI-2的活性基本是一致的，二者均受生长时期和生长条件的调节，即在glucose存在的条件下luxS表达和AI-2活性升高，而在高温条件下luxS表达和AI-2活性降低。glucose对AI-2活性以及luxS表达的影响经过荧光定量PCR，启动子活性检测，AI-2活性检测以及凝胶滞缓等一系列的实验证实是由cAMP-CRP复合物介导的，该复合物可以通过与luxS启动子相互作用而抑制luxS的表达。RNA干扰表明，TX1中luxS表达被干扰以后，对细菌产生了多方面的影响，包括：(1) 降低AI-2水平；(2) 降低细菌的生长能力；(3) 降低Ⅲ型分泌系统相关基因的表达水平以及生物膜的形成能力；(4) 减弱细菌毒力。外源AI-2的添加可以回复Ⅲ型分泌系统相关基因的表达水平以及生物膜的形成，但是并不能修复生长状况，表明LuxS在TX1中具有双重功能，即参与细胞代谢以及群体感应信号传导。基于LuxS/AI-2群体感应系统对细菌毒力的重要性，设计并筛选了一个该系统的阻遏因子5411。Pull-down实验证明5411可以和LuxS特异性结合。研究表明5411在TX1中表达导致细菌毒力显著下降。将5411克隆至牙鲆共生菌FP3中，发现5411可以被分泌到胞外并能被TX1吸收。将表达5411的共生菌导入牙鲆，发现其能够有效阻遏TX1对牙鲆的侵染。 这些结果表明：(1) TX1中AI-2的活性受控于LuxS，而后者则受生长时期和生长条件的调控；(2) luxS的正常表达对于细菌的正常生长和侵染是必需的；(3) LuxS/AI-2群体感应系统调控Ⅲ型分泌系统相关毒力因子的表达；(4) 通过阻遏LuxS/AI-2群体感应系统来抑制病原菌侵染是一种具有潜力的新型病害防控方法。

In Helicobacter pylori auto-inducer-2, but not LuxS/MccAB catalysed reverse transsulphuration, regulates motility through modulation of flagellar gene transcription

BACKGROUND: LuxS may function as a metabolic enzyme or as the synthase of a quorum sensing signalling molecule, auto-inducer-2 (AI-2); hence, the mechanism underlying phenotypic changes upon luxS inactivation is not always clear. In Helicobacter pylori, we have recently shown that, rather than functioning in recycling methionine as in most bacteria, LuxS (along with newly-characterised MccA and MccB), synthesises cysteine via reverse transsulphuration. In this study, we investigated whether and how LuxS controls motility of H. pylori, specifically if it has its effects via luxS-required cysteine metabolism or via AI-2 synthesis only.

RESULTS: We report that disruption of luxS renders H. pylori non-motile in soft agar and by microscopy, whereas disruption of mccAHp or mccBHp (other genes in the cysteine provision pathway) does not, implying that the lost phenotype is not due to disrupted cysteine provision. The motility defect of the DeltaluxSHp mutant was complemented genetically by luxSHp and also by addition of in vitro synthesised AI-2 or 4, 5-dihydroxy-2, 3-pentanedione (DPD, the precursor of AI-2). In contrast, exogenously added cysteine could not restore motility to the DeltaluxSHp mutant, confirming that AI-2 synthesis, but not the metabolic effect of LuxS was important. Microscopy showed reduced number and length of flagella in the DeltaluxSHp mutant. Immunoblotting identified decreased levels of FlaA and FlgE but not FlaB in the DeltaluxSHp mutant, and RT-PCR showed that the expression of flaA, flgE, motA, motB, flhA and fliI but not flaB was reduced. Addition of DPD but not cysteine to the DeltaluxSHp mutant restored flagellar gene transcription, and the number and length of flagella.

CONCLUSIONS: Our data show that as well as being a metabolic enzyme, H. pylori LuxS has an alternative role in regulation of motility by modulating flagellar transcripts and flagellar biosynthesis through production of the signalling molecule AI-2.

Analysis of the effect of copper on the virulence of a pathogenic Edwardsiella tarda strain

Aim: To investigate the effect of copper on the virulence of Edwardsiella tarda. Methods and Results: The pathogenic Edw. tarda strain TX5 was cultured under copper-stressed conditions and examined for any potential alteration in capacities that are associated with pathogenicity. The results showed that compared to untreated TX5, Cu-treated TX5 exhibits reduced planktonic and biofilm growth, an impaired ability to adhere to host mucus, modulation of host immune response, and dissemination in host blood and liver. Consistent with these observations, the overall bacterial virulence of Cu-treated TX5 is significantly attenuated. SDS-PAGE analyses of whole cell protein production showed that Cu-treated TX5 differs from the untreated TX5 in its production of at least one protein. Quantitative real time reverse transcriptase PCR analyses showed that copper treatment decreased the expression of virulence-associated genes encoding components of the type III and type VI secretion systems, the Eth haemolysin system, and the LuxS/AI-2 quorum-sensing system. Conclusions: Prolonged exposure to copper has multiple effects on TX5 and results in significant attenuation of bacterial virulence. Significance and Impact of the Study: The results of this study demonstrate that copper treatment has a broad and profound effect on the virulence-associated capacities of TX5, which is exerted at least in part at the transcription level. These findings provide new insights to the antimicrobial mechanism of copper.

Biomolecular Mechanisms of Pseudomonas aeruginosa and Escherichia coli Biofilm Formation

Pseudomonas aeruginosa and Escherichia coli are the most prevalent Gram-negative biofilm forming medical device associated pathogens, particularly with respect to catheter associated urinary tract infections. In a similar manner to Gram-positive bacteria, Gram-negative biofilm formation is fundamentally determined by a series of steps outlined more fully in this review, namely adhesion, cellular aggregation, and the production of an extracellular polymeric matrix. More specifically this review will explore the biosynthesis and role of pili and flagella in Gram-negative adhesion and accumulation on surfaces in Pseudomonas aeruginosa and Escherichia coli. The process of biofilm maturation is compared and contrasted in both species, namely the production of the exopolysaccharides via the polysaccharide synthesis locus (Psl), pellicle Formation (Pel) and alginic acid synthesis in Pseudomonas aeruginosa, and UDP-4-amino-4-deoxy-l-arabinose and colonic acid synthesis in Escherichia coli. An emphasis is placed on the importance of the LuxR homologue sdiA; the luxS/autoinducer-II; an autoinducer-III/epinephrine/norepinephrine and indole mediated Quorum sensing systems in enabling Gram-negative bacteria to adapt to their environments. The majority of Gram-negative biofilms consist of polysaccharides of a simple sugar structure (either homo- or heteropolysaccharides) that provide an optimum environment for the survival and maturation of bacteria, allowing them to display increased resistance to antibiotics and predation.

Bacterial inter-species communication mediated by the autoinducer-2 signal

Dissertation presented to obtain the Ph.D degree in Biology by Universidade Nova de Lisboa, Instituto de Tecnologia Química e Biológica, Instituto Gulbenkian de Ciência.

Production of biofilm by Listeria monocytogenes in different materials and temperatures

Listeria monocytogenes, considered as one of the most important foodborne pathogens, is easily found on surfaces, particularly in the form of a biofilm. Biofilms are aggregates of cells that facilitate the persistence of these pathogens in food processing environments conferring resistance to the processes of cleaning and may cause contamination of food during processing, thus, representing a danger to public health. Little is known about the dynamics of the formation and regulation of biofilm production in L.monocytogenes, but several authors reported that the luxS gene may be a precursor in this process. In addition, the product of the inlA gene is responsible for facilitating the entry of the microorganism into epithelial cells that express the receptor E-cadherin, also participates in surface attachment. Thus, 32 strains of L.monocytogenes isolated from different foods (milk and vegetables) and from food processing environments were analyzed for the presence of these genes and their ability to form biofilms on three different surfaces often used in the food industry and retail (polystyrene, glass and stainless steel) at different temperatures (4, 20 and 30°C). All strains had the ilnA gene and 25 out of 32 strains (78.1%) were positive for the presence of the luxS gene, but all strains produced biofilm in at least one of the temperatures and materials tested. This suggests that genes in addition to luxS may participate in this process, but were not the decisive factors for biofilm formation. The bacteria adhered better to hydrophilic surfaces (stainless steel and glass) than to hydrophobic ones (polystyrene), since at 20°C for 24h, 30 (93.8%) and 26 (81.3%) produced biofilm in stainless steel and glass, respectively, and just 2 (6.2%) in polystyrene. The incubation time seemed to be an important factor in the process of biofilm formation, mainly at 35°C for 48h, because the results showed a decrease from 30 (93.8%) to 20 (62.5%) and from 27 (84.4%) to 12 (37.5%), on stainless steel and glass, respectively, although this was not significant (. p=0.3847). We conclude that L.monocytogenes is capable of forming biofilm on different surfaces independent of temperature, but the surface composition may be important factor for a faster development of biofilm. © 2013 Elsevier Ltd.