The detection of environmental autoinducing peptide quorum-sensing genes from an uncultured Clostridium sp in landfill leachate reactor biomass

Aims: To elucidate whether a dominant uncultured clostridial (Clostridium thermocellum-like) species in an environmental sample (landfill leachate), possesses an autoinducing peptide (AIP) quorum-sensing (QS) gene, although it may not be functional. Methods and Results: A modified AIP accessory gene regulator (agr)C PCR protocol was performed on extracted DNA from a landfill leachate sample (also characterized by 16S rRNA gene cloning) and the PCR products were cloned, sequenced and phylogenetically analysed. It appeared that two agrC gene phylotypes existed, most closely related to the C. thermocellum agrC gene, differing by only 1 bp. Conclusions: It is possible to specifically identify and characterize the agrC AIP QS gene from uncultured Firmicutes (C. thermocellum-like) bacteria derived from environmental (landfill leachate) sample. Significance and Impact of the Study: This is the first successful attempt at identifying AIP QS genes from a cellulolytic environment (landfill). The agrC gene was identified as being most closely related to the C. thermocellum agrC gene, the same bacterium identified as being dominant, according to 16S rRNA gene cloning and subsequently fluorescence in situ hybridization analyses, in the same biomass.

Anti-quorum sensing agents from south Florida medicinal plants and their attenuation of Pseudomonas aeruginosa pathogenicity

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus . Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.

Role of Pseudomonas aeruginosa PA4157in Quorum Sensing and Iron Transport Regulation

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is a pnmary contributing factor responsible for the morbidity and mortality in patients with cystic fibrosis. One of the trademarks of P. aeruginosa is its ability to resist antibiotics. P. aeruginosa does so in part through the LysR-type transcription factor, AmpR. To identify additional members of the AmpR regulon, a new algorithm called iterative enhancement of motifs was used to identify putative AmpR binding sites upstream of open reading frames in the P. aeruginosa genome. The surprising primary hit of this analysis was the promoter of an uncharacterized open reading frame, P A 415 7. P A 415 7 is located upstream ofthefep operon, which is known to be involved in iron acquisition. PA4157 shares high homology to the IclR family of transcriptional regulators which are known to regulate quorum sensing (QS), an elaborate cell-cell communication signaling system that uses quoromones. We postulated two hypotheses: 1) AmpR regulation of QS genes is mediated by PA4157, and 2) PA4157 may be involved in iron acquisition. To address the role of P A 415 7 we generated an in-frame chromosomal deletion of P A 415 7 in P. aeruginosa PA01 (PA0 PA4157). We compared PA0 PA4157 with its parent strain P A0 1 for its ability to produce quoromones using Chromobacterium violaceum as an indicator strain and LasA proteases using Staphylococcus aureus. We also tested its role in virulence using a Caenorhabditis elegans killing assay. Growth in iron-deficient media was also examined to determine if P A4157 has a potential role in iron uptake regulation. Our preliminary results suggest that P A 415 7 is not involved in quorum sensing regulation but does seem to exert a negative regulatory effect on iron uptake in P. aeruginosa P A0 1.

Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: Co-existence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia

Background Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novelN-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest. Results By using a basal growth medium containing N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) as the sole source of carbon and nitrogen, the ginger rhizosphere associated bacteria were enriched for strains with AHL-degrading capabilities. Three isolates belonging to the generaAcinetobacter (GG2), Burkholderia (GG4) and Klebsiella (Se14) were identified and selected for further study. Strains GG2 and Se14 exhibited the broadest spectrum of AHL-degrading activities via lactonolysis while GG4 reduced 3-oxo-AHLs to the corresponding 3-hydroxy compounds. In GG2 and GG4, QQ was found to co-exist with AHL-dependent QS and GG2 was shown to inactivate both self-generated and exogenously supplied AHLs. GG2, GG4 and Se14 were each able to attenuate virulence factor production in both human and plant pathogens. Conclusions Collectively our data show that ginger rhizosphere bacteria which make and degrade a wide range of AHLs are likely to play a collective role in determining the QS-dependent phenotype of a polymicrobial community.

Characterization of the Anti-Quorum Sensing Activity Exhibited by Marine Macro algae of South Florida

Quorum sensing is a communication mechanism employed by many bacteria. The bacteria secrete signal molecules known as acyl homoseriene lactones (AHLs) that cue to population size/density. Bacteria can be alerted of this optimum population by the concentration of these signal molecules. When the concentration of AHLs exceed a threshold valve, they enter the bacterial cell and causes the transcription of genes encoding virulence factors necessary for their colonization and survival. The marine algae Delise a pulchra, found off the coast of Australia is thought to produce compounds that inhibit the activity of the AHLs. The algae employ these compounds, known as furanones, as an anti-fouling agent. We postulated that marine algae of South Florida might contain similar activity; we screened 30 different algal species and found 22 species had the activity. Algal extracts were made from Halimeda incrassata using hexane, chloroform, ethyl acetate and methanol as solvents. The extracts were assayed for anti-quorum sensing activity. The results showed many of the South Florida green algae to possess anti-quorum sensing activity, however extracts of H incrassata did not show quorum sensing inhibition.

Anti-Quorum Sensing Agents from South Florida Medicinal Plants and their Attenuation of Pseudomonas Aeruginosa Pathogenicity

With the difficulty in treating recalcitrant infections and the growing resistance to antibiotics, new therapeutic modalities are becoming increasingly necessary. The interruption of bacterial quorum sensing (QS), or cell-cell communication is known to attenuate virulence, while limiting selective pressure toward resistance. This study initiates an ethnobotanically-directed search for QS inhibiting agents in south Florida medicinal plants. Fifty plants were screened for anti-QS activity using two biomonitor strains, Chromobacterium violaceum and Agrobacterium tumefaciens. Of these plants, six showed QS inhibition: Conocarpus erectus L. (Combretaceae), Chamaecyce hypericifolia (L.) Millsp. (Euphorbiaceae), Callistemon viminalis (Sol.ex Gaertn.) G. Don (Myrtaceae), Bucida burceras L. (Combretaceae), Tetrazygia bicolor (Mill.) Cogn. (Melastomataceae), and Quercus virginiana Mill. (Fagaceae). These plants were further examined for their effects on the QS system and virulence of Pseudomonas aeruginosa, an intractable opportunistic pathogen responsible for morbidity and mortality in the immunocompromised patient. C. erectus, B. buceras, and C. viminalis were found to significantly inhibit multiple virulence factors and biofilm formation in this organism. Each plant presented a distinct profile of effect on QS genes and signaling molecules, suggesting varying modes of action. Virulence attenuation was observed with marginal reduction of bacterial growth, suggesting quorum quenching mechanisms unrelated to static or cidal effects. Extracts of these plants were also investigated for their effects on P. aeruginosa killing of the nematode Caenorhabditis elegans. Results were evaluated in both toxin-based and infection-based assays with P. aeruginosa strains PA01 and PA14. Overall nematode mortality was reduced 50-90%. There was no indication of host toxicity, suggesting the potential for further development as anti-infectives. Using low-pressure chromatography and HPLC, two stereoisomeric ellagitannins, vescalagin and castalagin were isolated from an aqueous extract of C. erectus. Structures were confirmed via mass spectrometry and NMR spectroscopy. Both ellagitannins were shown to decrease signal production, QS gene expression, and virulence factor production in P. aeruginosa. This study introduces a potentially new therapeutic direction for the treatment of bacterial infections. In addition, this is the first report of vescalagin and castalagin being isolated from C. erectus, and the first report of ellagitannin activity on the QS system.

Diffusible Signal Factor (DSF)-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control

Cell-to-cell signals of the Diffusible Signal Factor (DSF) family are cis-2-unsaturated fatty acids of differing chain length and branching pattern. DSF signalling has been described in diverse bacteria to include plant and human pathogens where it acts to regulate functions such as biofilm formation, antibiotic tolerance and the production of virulence factors. DSF family signals can also participate in interspecies signalling with other bacteria and interkingdom signaling such as with the yeast Candida albicans. Interference with DSF signalling may afford new opportunities for the control of bacterial disease. Such strategies will depend in part on detailed knowledge of the molecular mechanisms underlying the processes of signal synthesis, perception and turnover. Here, I review both recent progress in understanding DSF signalling at the molecular level and prospects for translating this knowledge into approaches for disease control.

Non-thermal Plasma Exposure Rapidly Attenuates Bacterial AHL-Dependent Quorum Sensing and Virulence.

The antimicrobial activity of atmospheric pressure non-thermal plasma has been exhaustively characterised, however elucidation of the interactions between biomolecules produced and utilised by bacteria and short plasma exposures are required for optimisation and clinical translation of cold plasma technology. This study characterizes the effects of non-thermal plasma exposure on acyl homoserine lactone (AHL)-dependent quorum sensing (QS). Plasma exposure of AHLs reduced the ability of such molecules to elicit a QS response in bacterial reporter strains in a dose-dependent manner. Short exposures (30-60 s) produce of a series of secondary compounds capable of eliciting a QS response, followed by the complete loss of AHL-dependent signalling following longer exposures. UPLC-MS analysis confirmed the time-dependent degradation of AHL molecules and their conversion into a series of by-products. FT-IR analysis of plasma-exposed AHLs highlighted the appearance of an OH group. In vivo assessment of the exposure of AHLs to plasma was examined using a standard in vivo model. Lettuce leaves injected with the rhlI/lasI mutant PAO-MW1 alongside plasma treated N-butyryl-homoserine lactone and n-(3-oxo-dodecanoyl)-homoserine lactone, exhibited marked attenuation of virulence. This study highlights the capacity of atmospheric pressure non-thermal plasma to modify and degrade AHL autoinducers thereby attenuating QS-dependent virulence in P. aeruginosa.

Quorum Sensing and Microbial Interactions in Coral Black Band Disease and Coral-Associated Bacteria

The black band disease (BBD) microbial consortium often causes mortality of reef-building corals. Microbial chemical interactions (i.e., quorum sensing (QS) and antimicrobial production) may be involved in the BBD disease process. Culture filtrates (CFs) from over 150 bacterial isolates from BBD and the surface mucopolysaccharide layer (SML) of healthy and diseased corals were screened for acyl homoserine lactone (AHL) and Autoinducer-2 (AI-2) QS signals using bacterial reporter strains. AHLs were detected in all BBD mat samples and nine CFs. More than half of the CFs (~55%) tested positive for AI-2. Approximately 27% of growth challenges conducted among 19 isolates showed significant growth inhibition. These findings demonstrate that QS is actively occurring within the BBD microbial mat and that culturable bacteria from BBD and the coral SML are able to produce QS signals and antimicrobial compounds. This is the first study to identify AHL production in association with active coral disease.

New weapons to fight old enemies: novel strategies for the (bio)control of bacterial biofilms in the food industry

Biofilms are microbial communities characterized by their adhesion to solid surfaces and the production of a matrix of exopolymeric substances, consisting of polysaccharides, proteins, DNA and lipids, which surround the microorganisms lending structural integrity and a unique biochemical profile to the biofilm. Biofilm formation enhances the ability of the producer/s to persist in a given environment. Pathogenic and spoilage bacterial species capable of forming biofilms are a significant problem for the healthcare and food industries, as their biofilm-forming ability protects them from common cleaning processes and allows them to remain in the environment post-sanitation. In the food industry, persistent bacteria colonize the inside of mixing tanks, vats and tubing, compromising food safety and quality. Strategies to overcome bacterial persistence through inhibition of biofilm formation or removal of mature biofilms are therefore necessary. Current biofilm control strategies employed in the food industry (cleaning and disinfection, material selection and surface preconditioning, plasma treatment, ultrasonication, etc.), although effective to a certain point, fall short of biofilm control. Efforts have been explored, mainly with a view to their application in pharmaceutical and healthcare settings, which focus on targeting molecular determinants regulating biofilm formation. Their application to the food industry would greatly aid efforts to eradicate undesirable bacteria from food processing environments and, ultimately, from food products. These approaches, in contrast to bactericidal approaches, exert less selective pressure which in turn would reduce the likelihood of resistance development. A particularly interesting strategy targets quorum sensing systems, which regulate gene expression in response to fluctuations in cell-population density governing essential cellular processes including biofilm formation. This review article discusses the problems associated with bacterial biofilms in the food industry and summarizes the recent strategies explored to inhibit biofilm formation, with special focus on those targeting quorum sensing.

INTEGRATED THRESHOLD-ACTIVATED FEEDBACK MICROSYSTEM FOR REAL-TIME CHARACTERIZATION, SENSING AND TREATMENT OF BACTERIAL BIOFILMS

Biofilms are the primary cause of clinical bacterial infections and are impervious to typical amounts of antibiotics, necessitating very high doses for treatment. Therefore, it is highly desirable to develop new alternate methods of treatment that can complement or replace existing approaches using significantly lower doses of antibiotics. Current standards for studying biofilms are based on end-point studies that are invasive and destroy the biofilm during characterization. This dissertation presents the development of a novel real-time sensing and treatment technology to aid in the non-invasive characterization, monitoring and treatment of bacterial biofilms. The technology is demonstrated through the use of a high-throughput bifurcation based microfluidic reactor that enables simulation of flow conditions similar to indwelling medical devices. The integrated microsystem developed in this work incorporates the advantages of previous in vitro platforms while attempting to overcome some of their limitations. Biofilm formation is extremely sensitive to various growth parameters that cause large variability in biofilms between repeated experiments. In this work we investigate the use of microfluidic bifurcations for the reduction in biofilm growth variance. The microfluidic flow cell designed here spatially sections a single biofilm into multiple channels using microfluidic flow bifurcation. Biofilms grown in the bifurcated device were evaluated and verified for reduced biofilm growth variance using standard techniques like confocal microscopy. This uniformity in biofilm growth allows for reliable comparison and evaluation of new treatments with integrated controls on a single device. Biofilm partitioning was demonstrated using the bifurcation device by exposing three of the four channels to various treatments. We studied a novel bacterial biofilm treatment independent of traditional antibiotics using only small molecule inhibitors of bacterial quorum sensing (analogs) in combination with low electric fields. Studies using the bifurcation-based microfluidic flow cell integrated with real-time transduction methods and macro-scale end-point testing of the combination treatment showed a significant decrease in biomass compared to the untreated controls and well-known treatments such as antibiotics. To understand the possible mechanism of action of electric field-based treatments, fundamental treatment efficacy studies focusing on the effect of the energy of the applied electrical signal were performed. It was shown that the total energy and not the type of the applied electrical signal affects the effectiveness of the treatment. The linear dependence of the treatment efficacy on the applied electrical energy was also demonstrated. The integrated bifurcation-based microfluidic platform is the first microsystem that enables biofilm growth with reduced variance, as well as continuous real-time threshold-activated feedback monitoring and treatment using low electric fields. The sensors detect biofilm growth by monitoring the change in impedance across the interdigitated electrodes. Using the measured impedance change and user inputs provided through a convenient and simple graphical interface, a custom-built MATLAB control module intelligently switches the system into and out of treatment mode. Using this self-governing microsystem, in situ biofilm treatment based on the principles of the bioelectric effect was demonstrated by exposing two of the channels of the integrated bifurcation device to low doses of antibiotics.

Peroxisome proliferator-activated receptors mediate host cell proinflammatory responses to Pseudomonas aeruginosa autoinducer.

The pathogenic bacterium Pseudomonas aeruginosa utilizes the 3-oxododecanoyl homoserine lactone (3OC(12)-HSL) autoinducer as a signaling molecule to coordinate the expression of virulence genes through quorum sensing. 3OC(12)-HSL also affects responses in host cells, including the upregulation of genes encoding inflammatory cytokines. This proinflammatory response may exacerbate underlying disease during P. aeruginosa infections. The specific mechanism(s) through which 3OC(12)-HSL influences host responses is unclear, and no mammalian receptors for 3OC(12)-HSL have been identified to date. Here, we report that 3OC(12)-HSL increases mRNA levels for a common panel of proinflammatory genes in murine fibroblasts and human lung epithelial cells. To identify putative 3OC(12)-HSL receptors, we examined the expression patterns of a panel of nuclear hormone receptors in these two cell lines and determined that both peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) and PPARgamma were expressed. 3OC(12)-HSL functioned as an agonist of PPARbeta/delta transcriptional activity and an antagonist of PPARgamma transcriptional activity and inhibited the DNA binding ability of PPARgamma. The proinflammatory effect of 3OC(12)-HSL in lung epithelial cells was blocked by the PPARgamma agonist rosiglitazone, suggesting that 3OC(12)-HSL and rosiglitazone are mutually antagonistic negative and positive regulators of PPARgamma activity, respectively. These data identify PPARbeta/delta and PPARgamma as putative mammalian 3OC(12)-HSL receptors and suggest that PPARgamma agonists may be employed as anti-inflammatory therapeutics for P. aeruginosa infections.

Positive control of swarming, rhamnolipid synthesis, and lipase production by the posttranscriptional RsmA/RsmZ system in Pseudomonas aeruginosa PAO1.

In Pseudomonas aeruginosa, the small RNA-binding, regulatory protein RsmA is a negative control element in the formation of several extracellular products (e.g., pyocyanin, hydrogen cyanide, PA-IL lectin) as well as in the production of N-acylhomoserine lactone quorum-sensing signal molecules. RsmA was found to control positively the ability to swarm and to produce extracellular rhamnolipids and lipase, i.e., functions contributing to niche colonization by P. aeruginosa. An rsmA null mutant was entirely devoid of swarming but produced detectable amounts of rhamnolipids, suggesting that factors in addition to rhamnolipids influence the swarming ability of P. aeruginosa. A small regulatory RNA, rsmZ, which antagonized the effects of RsmA, was identified in P. aeruginosa. Expression of the rsmZ gene was dependent on both the global regulator GacA and RsmA, increased with cell density, and was subject to negative autoregulation. Overexpression of rsmZ and a null mutation in rsmA resulted in quantitatively similar, negative or positive effects on target genes, in agreement with a model that postulates titration of RsmA protein by RsmZ RNA.

Three small RNAs jointly ensure secondary metabolism and biocontrol in Pseudomonas fluorescens CHA0.

In many Gram-negative bacteria, the GacS/GacA two-component system positively controls the expression of extracellular products or storage compounds. In the plant-beneficial rhizosphere bacterium Pseudomonas fluorescens CHA0, the GacS/GacA system is essential for the production of antibiotic compounds and hence for biological control of root-pathogenic fungi. The small (119-nt) RNA RsmX discovered in this study, together with RsmY and RsmZ, forms a triad of GacA-dependent small RNAs, which sequester the RNA-binding proteins RsmA and RsmE and thereby antagonize translational repression exerted by these proteins in strain CHA0. This small RNA triad was found to be both necessary and sufficient for posttranscriptional derepression of biocontrol factors and for protection of cucumber from Pythium ultimum. The same three small RNAs also positively regulated swarming motility and the synthesis of a quorum-sensing signal, which is unrelated to N-acyl-homoserine lactones, and which autoinduces the Gac/Rsm cascade. Expression of RsmX and RsmY increased in parallel throughout cell growth, whereas RsmZ was produced during the late growth phase. This differential expression is assumed to facilitate fine tuning of GacS/A-controlled cell population density-dependent regulation in P. fluorescens.

Identificação de bactérias do filoplano de citros capazes de interromper o quórum sensing em Xanthomonas citri subsp. citri

Pós-graduação em Microbiologia Agropecuária - FCAV