Production Of Several Virulence Factors Research Articles

Beyond the β-amino alcohols framework: identification of novel β-hydroxy pyridinium salt-decorated pterostilbene derivatives as bacterial virulence factor inhibitors.

Bacterial virulence factors are involved in various biological processes and mediate persistent bacterial infections. Focusing on virulence factors of phytopathogenic bacteria is an attractive strategy and crucial direction in pesticide discovery to prevent invasive and persistent bacterial infection. Hence, discovery and development of novel agrochemicals with high activity, low-risk, and potent anti-virulence is urgently needed to control plant bacterial diseases. A series of novel β-hydroxy pyridinium cation decorated pterostilbene derivatives were prepared and their antibacterial activities against Xanthomonas oryzae pv. oryzae (Xoo) were systematacially assessed. Among these pterostilbene derivatives, compound 4S exhibited the best antibacterial activity against Xoo in vitro, with an EC50 value of 0.28 μg mL-1. A series of biochemical assays including scanning electron microscopy, crystal violet staining, and analyses of biofilm formation, swimming motility, and related virulence factor gene expression levels demonstrated that compound 4S could function as a new anti-virulence factor inhibitor by interfering with the bacterial infection process. Furthermore, the pot experiments provided convinced evidence that compound 4S had the high control efficacy (curative activity:71.4%, protective activity: 72.6%), and could be used to effectively manage rice bacterial leaf blight in vivo. Compounds 4S is an attractive virulence factor inhibitor with potential for application in treating plant bacterial diseases by suppressing production of several virulence factors. This article is protected by copyright. All rights reserved.

Virulence Potential and Antibiotic Susceptibility of S. aureus Strains Isolated from Food Handlers.

Staphylococcus spp. are common members of the normal human flora. However, some Staphylococcus species are recognised as human pathogens due to the production of several virulence factors and enterotoxins that are particularly worrisome in food poisoning. Since many of Staphylococcal food poisoning outbreaks are typically associated with cross-contamination, the detection of S. aureus on food handlers was performed. Hand swabs from 167 food handlers were analysed for the presence of S. aureus. More than 11% of the samples were positive for S. aureus. All S. aureus strains were isolated and analysed for the presence of virulence and enterotoxin genes, namely, sea, seb, sec, sed, seg, sei, tsst-1 and pvl. The same strains were phenotypically characterised in terms of antibiotic susceptibility using the disc diffusion method and antimicrobial agents from 12 different classes. A low prevalence of antibiotic-resistant strains was found, with 55.6% of the strains being sensitive to all of the antimicrobial agents tested. However, a high prevalence of resistance to macrolides was found, with 44.4% of the strains showing resistance to erythromycin. At least one of the virulence or toxin genes was detected in 61.1% of the strains, and seg was the most prevalent toxin gene, being detected in 44.4% of the strains.

Anti-quorum sensing evaluation of methyleugenol, the principal bioactive component, from the Melaleuca bracteata leaf oil.

Quorum sensing (QS) is a cell-to-cell communication in bacteria that couples gene expression through the accumulation of signaling molecules, which finally induce the production of several virulence factors and modulate bacterial behaviors. Plants have evolved an array of quorum sensing inhibitors (QSIs) to inhibit the pathogens, of which aromatic compounds are widely recognized. The essential oil of Melaleuca bracteata was found to exhibit anti-quorum sensing activity, and its principal bioactive component, methyleugenol (ME), had been isolated in our previous study. Here, ME interfered effectively with the QS-regulated processes of toxin secretion in Chomobacterium violaceum ATCC31532, resulting in strong inhibition of QS genes, cviR, cviI, vioA-E, hmsHNR, lasA-B, pilE1-3, and hcnABC, leading to impaired virulence, including violacein production, biofilm biomass, and swarming motility. The accumulation of the signal molecule (N-hexanoyl-DL-homoserine lactone, C6-HSL) in C. violaceum declined upon treatment with ME, suggesting an inhibition effect on the C6-HSL production, and the ME was also capable of degrading the C6-HSL in vitro assay. Molecular docking technique and the consumption change of exogenous C6-HSL in C. violaceum CV026 revealed the anti-QS mechanism of ME consisted of inhibition of C6-HSL production, potentially via interaction with CviR and/or CviI protein. Collectively, the isolated ME, the principal active components of M. bracteata EO, exhibited a wide range of inhibition processes targeting C. violaceum QS system, which supports the potential anti-pathogenic use of M. bracteata EO and ME for treatment of pathogen contamination caused by bacterial pathogens.

Essential Oils Biofilm Modulation Activity and Machine Learning Analysis on Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients.

The opportunistic pathogen Pseudomonas aeruginosa is often involved in airway infections of cystic fibrosis (CF) patients. It persists in the hostile CF lung environment, inducing chronic infections due to the production of several virulence factors. In this regard, the ability to form a biofilm plays a pivotal role in CF airway colonization by P. aeruginosa. Bacterial virulence mitigation and bacterial cell adhesion hampering and/or biofilm reduced formation could represent a major target for the development of new therapeutic treatments for infection control. Essential oils (EOs) are being considered as a potential alternative in clinical settings for the prevention, treatment, and control of infections sustained by microbial biofilms. EOs are complex mixtures of different classes of organic compounds, usually used for the treatment of upper respiratory tract infections in traditional medicine. Recently, a wide series of EOs were investigated for their ability to modulate biofilm production by different pathogens comprising S. aureus, S. epidermidis, and P. aeruginosa strains. Machine learning (ML) algorithms were applied to develop classification models in order to suggest a possible antibiofilm action for each chemical component of the studied EOs. In the present study, we assessed the biofilm growth modulation exerted by 61 commercial EOs on a selected number of P. aeruginosa strains isolated from CF patients. Furthermore, ML has been used to shed light on the EO chemical components likely responsible for the positive or negative modulation of bacterial biofilm formation.

Malassezia virulence factors and their role in dermatological disorders

Malassezia is a commensal fungus that constitutes normal skin microbiota. However, in certain conditions and individuals, it may transform into a pathogenic yeast with multiple associated dermatological disorders and various clinical manifestations. This phenomenon is influenced by a unique host-agent interaction that triggers the production of several virulence factors, such as indoles, reactive oxygen species, azelaic acid, hyphae formation, and biofilm formation. This review article discusses Malassezia virulence factors that contribute to the transformation of Malassezia from commensal to pathogenic as well as their role in dermatological disorders, including pityriasis versicolor, seborrheic dermatitis, Malassezia folliculitis, atopic dermatitis, and psoriasis.

Chemical Composition and Antibacterial, Anti-biofilm and Anti-virulence Activities of Plant Extracts Against Human Pathogenic Bacteria

Background: Bacterial multi-drug resistance is one of the serious issues in the worldwide. The majority of drug resistance developed by bacteria is derived from the formation of biofilm and the production of several virulence factors. Objective: To combat the emerging drug resistance nature of bacteria, we have used some medicinal plants such as Stevia rebaudiana, Cymbopogon flexuosus, Matricaria chamomilla, Ocimum sanctum, Phyllanthus amarus and Thymus vulgaris for the extraction of active molecules that can inhibit the biofilm formation and bacterial growth. Materials & Methods: Each extracted compounds were checked for anti-bacterial, antibiofilm, and anti-virulence activities against Gram-positive and Gram-negative pathogenic bacteria. Soxhlet method of extraction was used for obtaining crude extracts. Agar disc diffusion and 96-wells microplate spectroscopic reading were used to detect the antibacterial and antibiofilm properties of extracts. Crystal violet staining was used for the detection of biofilm cells. GC-MS analysis was carried out to identify the chemical constituents present in the extracts. Results: At a concentration of 25 mg mL-1 , the highest antibacterial activities were obtained in M. chamomilla acetone extract (CHAAC) against E. coli (17.66±1.15 mm), S. Typhimurium (13.66±1.52 mm) and S. sonnei (14±1.73 mm). M. chamomilla chloroform extracted (CHACE) showed effective against S. aureus (30±2 mm) and P. aeruginosa (13.66±1.52 mm). The minimum inhibitory concentration of crude extracts derived from M. chamomilla was 0. 781mg.mL-1 against S. aureus. Furthermore, the oil extract from C. flexuosus (LEGO) exhibited significant antibacterial activity against S. aureus (at 0.781 µg mL-1 ). The potent anti-biofilm activity was observed in G. superba (KALO) against E. coli (78.6%) and S. typhimurium (79.1%), and V. negundo (VNO) against S. Typhimurium (87.3%). Furthermore, the GC-MS analysis showed the presence of active chemical constituents in the extracts which might have been effective anti-bacterial, anti-biofilm, and anti-virulence properties. Conclusion: The extracts from the medicinal plant showed antibiofilm, anti-virulence (attenuation of pyocyanin, LasA, vilacein, and swarming motility) activity towards the pathogenic bacteria. GC-MS analysis confirms the presence of an active component in the extracts. Future study is required to purify the bioactive molecules and elucidate the molecular mechanism of antibiofilm and anti-virulence properties.

Association of quorum sensing and biofilm formation with Salmonella virulence: story beyond gathering and cross-talk

Enteric fever (typhoid and paratyphoid fever) is a public health concern which contributes to mortality and morbidity all around the globe. It is caused mainly due to ingestion of contaminated food and water with a gram negative, rod-shaped, flagellated bacterium known as Salmonella enterica serotype typhi (typhoid fever) or paratyphi (paratyphoid fever). Clinical problems associated with Salmonellosis are mainly bacteraemia, gastroenteritis and enteric fever. The bacteria undergo various mechanisms to escape itself from immune reaction of the host, modulating immune response at the site of infection leading to virulence factor production and anti-microbial resistance. Biofilm is one of the adaptation mechanisms through which Salmonella survives in unfavourable conditions and thus is considered as a major threat to public health. Another property of the bacteria is "Quorum Sensing", which is a cell-cell communication and most of the pathogenic bacteria use it to coordinate the production of several virulence factors and other behaviours such as swarming and biofilm formation. Earlier, quorum sensing was believed to be just a medium for communication but, later on, its role in virulence has been studied. However, there are negligible information relating to interaction between quorum sensing and biofilm formation and how these events play crucial role in Salmonella pathogenesis. The review is a summary of updated information regarding how Salmonella uses these properties to spread more and survive better, making a challenge for clinicians and public health experts. Therefore, this review would help bring an insight regarding how biofilm formation and quorum sensing are inter-related and their role in pathogenesis and virulence of Salmonella.

Antilisterial Potential of Lactic Acid Bacteria in Eliminating Listeria monocytogenes in Host and Ready-to-Eat Food Application

Listeriosis is a severe food borne disease with a mortality rate of up to 30% caused by pathogenic Listeria monocytogenes via the production of several virulence factors including listeriolysin O (LLO), transcriptional activator (PrfA), actin (Act), internalin (Int), etc. It is a foodborne disease predominantly causing infections through consumption of contaminated food and is often associated with ready-to-eat food (RTE) and dairy products. Common medication for listeriosis such as antibiotics might cause an eagle effect and antibiotic resistance if it is overused. Therefore, exploration of the use of lactic acid bacteria (LAB) with probiotic characteristics and multiple antimicrobial properties is increasingly getting attention for their capability to treat listeriosis, vaccine development, and hurdle technologies. The antilisterial gene, a gene coding to produce antimicrobial peptide (AMP), one of the inhibitory substances found in LAB, is one of the potential key factors in listeriosis treatment, coupled with the vast array of functions and strategies; this review summarizes the various strategies by LAB against L. monocytogenes and the prospect in development of a ‘generally regarded as safe’ LAB for treatment of listeriosis.

Anti-quorum Sensing and Protective Efficacies of Naringin Against Aeromonas hydrophila Infection in Danio rerio.

It is now well known that the quorum sensing (QS) mechanism coordinates the production of several virulence factors and biofilm formation in most pathogenic microorganisms. Aeromonas hydrophila is a prime pathogen responsible for frequent outbreaks in aquaculture settings. Recent studies have also continuously reported that A. hydrophila regulates virulence factor production and biofilm formation through the QS system. In addition to the presence of antibiotic resistance genes, biofilm-mediated antibiotic resistance increases the severity of A. hydrophila infections. To control the bacterial pathogenesis and subsequent infections, targeting the QS mechanism has become one of the best alternative methods. Though very few compounds were identified as QS inhibitors against A. hydrophila, to date, the screening and identification of new and effective natural QS inhibitors is a dire necessity to control the infectious A. hydrophila. The present study endorses naringin (NA) as an anti-QS and anti-infective agent against A. hydrophila. Initially, the NA showed a concentration-dependent biofilm reduction against A. hydrophila. Furthermore, the results of microscopic analyses and quantitative virulence assays displayed the promise of NA as a potential anti-QS agent. Subsequently, the downregulation of ahh1, aerA, lip and ahyB validate the interference of NA in virulence gene expression. Furthermore, the in vivo assays were carried out in zebrafish model system to evaluate the anti-infective potential of NA. The outcome of the immersion challenge assay showed that the recovery rate of the zebrafish has substantially increased upon treatment with NA. Furthermore, the quantification of the bacterial load upon NA treatment showed a decreased level of bacterial counts in zebrafish when compared to the untreated control. Moreover, the NA treatment averts the pathogen-induced histoarchitecture damages in vital organs of zebrafish, compared to their respective controls. The current study has thus analyzed the anti-QS and anti-infective capabilities of NA and could be employed to formulate effective treatment measures against A. hydrophila infections.

Inhibition of Virulence Factors and Biofilm Formation of Acinetobacter Baumannii by Naturally-derived and Synthetic Drugs.

Acinetobacter baumannii is a gram-negative, aerobic, non-motile, and pleomorphic bacillus. A. baumannii is also a highly-infectious pathogen causing high mortality and morbidity rates in intensive care units. The discovery of novel agents against A. baumannii infections is urgently needed due to the emergence of drug-resistant A. baumannii strains and the limited number of efficacious antibiotics available for treatment. In addition to the production of several virulence factors, A. baumannii forms biofilms on the host cell surface as well. Formation of biofilms occurs through initial surface attachment, microcolony formation, biofilm maturation, and detachment stages, and is one of the major drug resistance mechanisms employed by A. baumannii. Several studies have previously reported the efficacy of naturally-derived and synthetic compounds as anti- biofilm and anti-virulence agents against A. baumannii. Here, inhibition of biofilm formation and virulence factors of A. baumannii using naturally-derived and synthetic compounds are reviewed.

The Lon-1 Protease Is Required by Borrelia burgdorferi To Infect the Mammalian Host.

Borrelia burgdorferi encodes a functional homolog of canonical Lon protease termed Lon-2. In addition, B. burgdorferi encodes a second Lon homolog called Lon-1. Recent studies suggest that Lon-1 may function differently from the prototypical Lon protease. However, the function of Lon-1 in B. burgdorferi biology remains virtually unknown. Particularly, the contribution of Lon-1 to B. burgdorferi fitness and infection remains hitherto unexplored. Herein, we show that Lon-1 plays a critical role for the infection of B. burgdorferi in a mammalian host. We found that lon-1 was highly expressed during animal infection, implying an important function of this protein in bacterial infection. We further generated a lon-1 deletion mutant and an isogenic complemented strain. Relative to that of the wild-type strain, the infectivity of the mutant was severely attenuated in a murine infection model. Our data also showed that the mutant displayed growth defects in regular BSK-II medium. Furthermore, bacterial resistance to osmotic stress was markedly reduced when lon-1 was inactivated. When exposed to tert-butyl hydroperoxide, survival of the lon-1 mutant was impaired. In addition, production of several virulence factors, such as BosR, RpoS, and OspC, was elevated in the mutant. These phenotypes were restored when the lon-1 mutation was complemented. Finally, we created a lon-1(S714A) mutant and found that this mutant failed to infect mice, suggesting that the proteolytic activity of Lon-1 is essential for bacterial infection. Taken together, these results demonstrate that Lon-1 is required by B. burgdorferi to infect animal hosts and to cope with environmental stresses.

Inhibition of biofilm and virulence properties of Pseudomonas aeruginosa by sub-inhibitory concentrations of aminoglycosides

Aminoglycosides are a commonly used class of antibiotics; however, their application has been discontinued due to the emergence of multi-drug resistance bacterial strains. In the present study, the subinhibitory concentrations (sub-MIC) of several aminoglycosides were determined and tested as an antibiofilm and for their anti-virulence properties against Pseudomonas aeruginosa PAO1, which is an opportunistic foodborne pathogen. P. aeruginosa PAO1 exhibits multiple mechanisms of resistance, including the formation of biofilm and production of several virulence factors, against aminoglycoside antibiotics. The sub-MIC of these antibiotics exhibited biofilm inhibition of P. aeruginosa in alkaline TSB (pH 7.9). Moreover, various concentrations of these aminoglycosides also eradicate the mature biofilm of P. aeruginosa. In the presence of sub-MIC of aminoglycosides, the morphological changes of P. aeruginosa were found to change from rod-shaped to the filamentous, elongated, and streptococcal forms. Similar growth conditions and sub-MIC of aminoglycosides were also found to attenuate several virulence properties of P. aeruginosa PAO1. Molecular docking studies demonstrate that these aminoglycosides possess strong binding properties with the LasR protein, which is a well-characterized quorum-sensing receptor of P. aeruginosa. The present study suggests a new approach to revitalize aminoglycosides as antibiofilm and antivirulence drugs to treat infections caused by pathogenic bacteria.

Participation of the pvd Gene Cluster in the Mechanism of Quorum Sensing and Virulence of Pseudomonas aeruginosa PAO1

ObjectiveDetermination of the molecular mechanism involved in the virulence modulation of Pseudomonas aeruginosa PAO1 involving the pvdI and pvdL genes.MethodsSupernatants of the P. aeruginosa PAO1 strain and mutants pvdI, pvdJ, and pvdL were obtained from LB liquid medium after 24 h of growth. Evaluation of virulence factors: biofilm was determined by growth of P. aeruginosa strains in 96‐well plates using violet crystal staining. Rhamnolipids production was determined by quantification of rhamnose by the orcinol method. Survival assays were carried out in Caenorhabditis elegans using supernatants of PAO1 strains.AbstractPseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals and plants. This bacterium possesses a system of perception of bacterial quorum (quorum sensing, QS), which modulates several virulence factors. The LasI/LasR QS system modifies the production of the cyclodipeptides (CDPs) cyclo(L‐Pro‐L‐Val), cyclo(L‐Pro‐L‐Phe) and cyclo(L‐Pro‐L‐Tyr). Although CDPs are molecules with bioactive properties, low elements concern in the mechanism of biosynthesis in bacteria and their role in communication with host cells. In addition, it has been described that the CDPs production in P. aeruginosa mainly depends of the multi‐modular non‐ribosomal peptide synthetases (MM‐NRPS), whose products such as the aeruginaldehyde has been involved in the bacterial QS mechanisms. In this study, was found that levels of pyocyanin (a virulence factor in P. aeruginosa), it was increased in mutant strains in the pvdJ gene, which encodes for a NPRS. The pvd gene cluster is directly related to the pioverdin synthesis, which is a fluorescent peptide‐siderophore involved in the iron acquisition, being essential in the bacterial pathogenicity on the host. Therefore, is important to elucidate the mechanism by which the CDPs of P. aeruginosa PAO1 contribute to its pathogenesis and/or virulence. Production of several virulence factors in P. aeruginosa PAO1 with different single and double mutants in pvdI, pvdJ and pvdL genes were compared. Mutants in the pvdI, pvdL and pvdL/pvdI genes decrease 50% the production of biofilm respect to the WT strain. Likewise, when evaluating rhamnolipids amount, the production of rhamnolipids was significantly increased in the single mutants pvdJ and pvdL compared with the WT strain, but not modified in the double mutant pvdJ/pvdL. Survival in the C. elegans worms indicated that the pvdJ mutant increase the death of the nematode 40% than the WT. The results suggest that the pvd gene cluster was involved in the production of CDPs in P. aeruginosa PAO1 and may to regulate the production of virulence factors (biofilm and rhamnolipids), impacting its pathogenicity on the nematode in vivo model.

The β-Lactamase Inhibitor Boronic Acid Derivative SM23 as a New Anti-Pseudomonas aeruginosa Biofilm.

Pseudomonas aeruginosa is a Gram-negative nosocomial pathogen, often causative agent of severe device-related infections, given its great capacity to form biofilm. P. aeruginosa finely regulates the expression of numerous virulence factors, including biofilm production, by Quorum Sensing (QS), a cell-to-cell communication mechanism used by many bacteria. Selective inhibition of QS-controlled pathogenicity without affecting bacterial growth may represent a novel promising strategy to overcome the well-known and widespread drug resistance of P. aeruginosa. In this study, we investigated the effects of SM23, a boronic acid derivate specifically designed as β-lactamase inhibitor, on biofilm formation and virulence factors production by P. aeruginosa. Our results indicated that SM23: (1) inhibited biofilm development and production of several virulence factors, such as pyoverdine, elastase, and pyocyanin, without affecting bacterial growth; (2) decreased the levels of 3-oxo-C12-HSL and C4-HSL, two QS-related autoinducer molecules, in line with a dampened lasR/lasI system; (3) failed to bind to bacterial cells that had been preincubated with P. aeruginosa-conditioned medium; and (4) reduced both biofilm formation and pyoverdine production by P. aeruginosa onto endotracheal tubes, as assessed by a new in vitro model closely mimicking clinical settings. Taken together, our results indicate that, besides inhibiting β-lactamase, SM23 can also act as powerful inhibitor of P. aeruginosa biofilm, suggesting that it may have a potential application in the prevention and treatment of biofilm-associated P. aeruginosa infections.

Antibiofilm and antivirulence properties of chitosan-polypyrrole nanocomposites to Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen which exhibits its property of pathogenesis due to several factors, including the formation of biofilm and production of several virulence factors. Development of resistance properties against antibiotics leads to the discovery of certain alternative strategies to combat its pathogenesis. In the present study, a highly stable, biocompatible and water soluble nanocomposites (NCs) are synthesized from chitosan (CS) and the polypyrrole (PPy). The resultant chitosan-polypyrrole nanocomposites (CS-PPy NCs) inhibit the establishment of biofilm and also eradicate the preformed matured biofilm formed by P. aeruginosa. CS-PPy NCs inhibit the hemolytic and protease activities of P. aeruginosa. The NCs significantly reduce the production of many virulence factors such as pyocyanin, pyroverdine and rhamnolipid. CS-PPy NCs also suppress the bacterial motility such as swimming and swarming. The present study showed that highly stable CS-PPy NCs act as a potent antibiofilm and antivirulence drug for the treatment of P. aeruginosa infection.

The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease.

Discovery of potent inhibitors targeting Vibrio harveyi LuxR through shape and e-pharmacophore based virtual screening and its biological evaluation.

Quorum sensing is widely recognized as an efficient mechanism in the regulation and production of several virulence factors, biofilm formation and stress responses. For this reason, quorum sensing circuit is emerging as a novel drug target for the development of anti-infective. Recently, cinnamaldehyde derivatives have been found to interfere with master quorum sensing transcriptional regulator and thereby decreasing the DNA binding ability of LuxR. However, the exact mode of cinnamaldehyde binding with LuxR and receptor interaction still remains inconclusive. In the current study, combined method of molecular docking and molecular dynamics simulations were performed to investigate the binding mode, dynamic and energy aspects of cinnamaldehyde derivatives into the binding site of LuxR. Based on the experimental and computational evidences, LuxR-3,4-dichloro-cinnamaldehyde complex was chosen for the development of e-pharmacophore model. Further, shape and e-pharmacophore based virtual screening were performed against ChemBridge database to find potent and suitable ligands for LuxR. By comparing the results of shape and e-pharmacophore based virtual screening; best 9 hit molecules were selected for further studies including ADMET prediction, molecular dynamics simulations and Prime MM-GBSA calculations. From the 9 hit molecules, the top most compound 3-(2,4-dichlorophenyl)-1-(1H-pyrrol-2-yl)-2-propen-1-one (ChemBridge-7364106) was selected for invitro assays using Vibrio harveyi. The result revealed that ChemBridge-7364106 significantly reduced the bioluminescence production in a dose dependent manner. In addition, ChemBridge-7364106 showed a significant inhibition in biofilm formation and motility in V.harveyi. The results from the study suggest that ChemBridge-7364106 could serve as an anti-quorum sensing molecule for V.harveyi.

Calcium induces tobramycin resistance in Pseudomonas aeruginosa by regulating RND efflux pumps

Pseudomonas aeruginosa is an opportunistic multidrug resistant pathogen causing severe chronic infections. Our previous studies showed that elevated calcium (Ca2+) enhances production of several virulence factors and plant infectivity of the pathogen. Here we show that Ca2+ increases resistance of P. aeruginosa PAO1 to tobramycin, antibiotic commonly used to treat Pseudomonas infections. LC–MS/MS-based comparative analysis of the membrane proteomes of P aeruginosa grown at elevated versus not added Ca2+, determined that the abundances of two RND (resistance-nodulation-cell division) efflux pumps, MexAB-OprM and MexVW-OprM, were increased in the presence of elevated Ca2+. Analysis of twelve transposon mutants with disrupted RND efflux pumps showed that six of them (mexB, muxC, mexY, mexJ, czcB, and mexE) contribute to Ca2+-induced tobramycin resistance. Transcriptional analyses by promoter activity and RT-qPCR showed that the expression of mexAB, muxABC, mexXY, mexJK, czcCBA, and mexVW is increased by elevated Ca2+. Disruption of mexJ, mexC, mexI, and triA significantly decreased Ca2+-induced plant infectivity of the pathogen. Earlier, our group showed that PAO1 maintains intracellular Ca2+ (Ca2+in) homeostasis, which mediates Ca2+ regulation of P. aeruginosa virulence, and identified four putative Ca2+ transporters involved in this process (Guragain et al., 2013). Here we show that three of these transporters (PA2435, PA2092, PA4614) play role in Ca2+-induced tobramycin resistance and one of them (PA2435) contributes to Ca2+ regulation of mexAB-oprM promoter activity. Furthermore, mexJ, czcB, and mexE contribute to the maintenance of Ca2+in homeostasis. This provides the first evidence that Ca2+in homeostasis mediates Ca2+ regulation of RND transport systems, which contribute to Ca2+-enhanced tobramycin resistance and plant infectivity in P. aeruginosa.

Discovery of new diketopiperazines inhibiting Burkholderia cenocepacia quorum sensing in vitro and in vivo.

Burkholderia cenocepacia, an opportunistic respiratory pathogen particularly relevant for cystic fibrosis patients, is difficult to eradicate due to its high level of resistance to most clinically relevant antimicrobials. Consequently, the discovery of new antimicrobials as well as molecules capable of inhibiting its virulence is mandatory. In this regard quorum sensing (QS) represents a good target for anti-virulence therapies, as it has been linked to biofilm formation and is important for the production of several virulence factors, including proteases and siderophores. Here, we report the discovery of new diketopiperazine inhibitors of the B. cenocepacia acyl homoserine lactone synthase CepI, and report their anti-virulence properties. Out of ten different compounds assayed against recombinant CepI, four were effective inhibitors, with IC50 values in the micromolar range. The best compounds interfered with protease and siderophore production, as well as with biofilm formation, and showed good in vivo activity in a Caenorhabditis elegans infection model. These molecules were also tested in human cells and showed very low toxicity. Therefore, they could be considered for in vivo combined treatments with established or novel antimicrobials, to improve the current therapeutic strategies against B. cenocepacia.

The Pseudomonas aeruginosa PAO1 Two-Component Regulator CarSR Regulates Calcium Homeostasis and Calcium-Induced Virulence Factor Production through Its Regulatory Targets CarO and CarP.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes severe, life-threatening infections in patients with cystic fibrosis (CF), endocarditis, wounds, or artificial implants. During CF pulmonary infections, P. aeruginosa often encounters environments where the levels of calcium (Ca(2+)) are elevated. Previously, we showed that P. aeruginosa responds to externally added Ca(2+) through enhanced biofilm formation, increased production of several secreted virulence factors, and by developing a transient increase in the intracellular Ca(2+) level, followed by its removal to the basal submicromolar level. However, the molecular mechanisms responsible for regulating Ca(2+)-induced virulence factor production and Ca(2+) homeostasis are not known. Here, we characterized the genome-wide transcriptional response of P. aeruginosa to elevated [Ca(2+)] in both planktonic cultures and biofilms. Among the genes induced by CaCl2 in strain PAO1 was an operon containing the two-component regulator PA2656-PA2657 (here called carS and carR), while the closely related two-component regulators phoPQ and pmrAB were repressed by CaCl2 addition. To identify the regulatory targets of CarSR, we constructed a deletion mutant of carR and performed transcriptome analysis of the mutant strain at low and high [Ca(2+)]. Among the genes regulated by CarSR in response to CaCl2 are the predicted periplasmic OB-fold protein, PA0320 (here called carO), and the inner membrane-anchored five-bladed β-propeller protein, PA0327 (here called carP). Mutations in both carO and carP affected Ca(2+) homeostasis, reducing the ability of P. aeruginosa to export excess Ca(2+). In addition, a mutation in carP had a pleotropic effect in a Ca(2+)-dependent manner, altering swarming motility, pyocyanin production, and tobramycin sensitivity. Overall, the results indicate that the two-component system CarSR is responsible for sensing high levels of external Ca(2+) and responding through its regulatory targets that modulate Ca(2+) homeostasis, surface-associated motility, and the production of the virulence factor pyocyanin. During infectious disease, Pseudomonas aeruginosa encounters environments with high calcium (Ca(2+)) concentrations, yet the cells maintain intracellular Ca(2+) at levels that are orders of magnitude less than that of the external environment. In addition, Ca(2+) signals P. aeruginosa to induce the production of several virulence factors. Compared to eukaryotes, little is known about how bacteria maintain Ca(2+) homeostasis or how Ca(2+) acts as a signal. In this study, we identified a two-component regulatory system in P. aeruginosa PAO1, termed CarRS, that is induced at elevated Ca(2+) levels. CarRS modulates Ca(2+) signaling and Ca(2+) homeostasis through its regulatory targets, CarO and CarP. The results demonstrate that P. aeruginosa uses a two-component regulatory system to sense external Ca(2+) and relays that information for Ca(2+)-dependent cellular processes.