The Role of Tetraspanins in Pseudomonas aeruginosa Adherence to Human Cells

Infection of host tissue is commonly initiated by adherence of pathogenic bacteria, such as Pseudomonas aeruginosa, to cell surface membrane proteins, sugars or lipids. One class of proteins known to play a major role in cellular processes, such as adhesion are tetraspanins, specifically CD9, CD81 and CD151. Prevention of adhesion could provide a new therapeutic strategy for treating infectious diseases. This work aims to investigate the potential of using recombinant tetraspanin extracellular domains (EC2), anti-tetraspanin antibodies and CD9 EC2-derived peptides as anti-adhesives in treating P. aeruginosa infections. Also, to create a new higher throughput method to measure bacterial adhesion using flow cytometry. Expression profiles of different tetraspanins in human epithelial cell lines, A549, HEC1B and keratinocyte HaCaT cells were visualised using immunofluorescent microscopy and quantified using flow cytometry. Monolayers of adherent cells were pre-treated with the tetraspanin reagents (EC2: GST recombinant proteins, anti-tetraspanin antibodies and CD9 EC2-derived peptides), before infection with GFP-labelled P. aeruginosa (PAO1) for 60 minutes. Bacterial adhesion was determined by counting the number of bacteria per infected cells with fluorescence microscopy. Analysis revealed the anti-adhesive properties of these tetraspanins (anti-tetraspanin antibodies and recombinant proteins). In conclusion, a flow cytometry method was developed but was found to be too un sensitive to measure changes in bacterial adhesion. Moreover, anti-tetraspanin antibodies, recombinant proteins and CD9 EC2- derived peptides may have the potential to inhibit adherence of P. aeruginosa. This work has identified a role for tetraspanins in bacteria-host cell adhesion, and the possible use of tetraspanin reagents as therapeutics for treating P. aeruginosa infection

A New Class of Antibiotic That Prevents Drug Tolerance, Persistence, and Resistance By Controlling Emergence of Phenotypes.

The fight against bacterial infections and innovations in antibiotic therapy has never halted throughout human history. However, bacteria have evolved smartly and resistance against practically all conventional antibiotics has been developed. Furthermore, bacteria can form biofilms, which are surface-attached multicellular colonies. Biofilms shield bacteria against antibiotics and makes it harder to entirely eradicate infections . Under antibiotics stress, bacteria evolve into different phenotypes like hyper motile, hyper adherent and hyper virulent which are tolerant and persistent to antibiotic treatment. The use of antibiotic therapy to combat such resilient bacterial phenotypes is extremely tough. As a result, the scientific community is always looking for novel ways to defeat these resilient phenotypes. It important to notice that all antibiotic induce tolerant and persistent phenotypes and there is no direct remedy for eradication of these phenotypes. On other hands, resistance to one antibiotic can be tackled by alternative antibiotic usage.Here, we demonstrate a class of small molecules that inhibits a wide range of phenotypes of Pseudomonas aeruginosa and enables the antibiotics to kill tolerant bacteria and to prevent formation of new persistent bacteria. We identified two proteins, type IV pili and lectin LecA as receptors for our molecules by different methods, including a new label-free assay based on bacterial motility sensing the environment, chemical inhibition of bacteriophage adsorption on pili appendages of bacteria, and fluorescence polarization. Structure-activity relationship studies reveal a molecule that inhibits only pili appendage, and a class of chimeric ligands that inhibit both LecA and pili, with important structural elements of the ligand identified for each protein. This selective control of two proteins makes the correlation between the protein receptor to their controlled phenotypes. Inhibiting LecA results in reducing biofilm formation, eliminating small colony variants, and correlates with killing tolerant bacteria and preventing the development of new persistent bacteria. Inhibiting pili appendages impedes the swarming and twitching motilities, and pyocyanin and elastase production. Because these phenotypes are controlled by a broad range of signaling pathways, this approach simultaneously control the multiple signaling mechanisms, by which bacteria elude antibiotic treatments. Glycolipid, ganglio-N-tetraosylceramide (asialo-GM1), on the mammalian cells are known to be recognized by type IV pili of Pseudomonas aeruginosa. In this work, we show that asialo-GM1 can also be recognized by Lectin A (LecA), another adhesin protein of the P. aeruginosa, by a fluorescent polarization assay, a label-free bacterial motility enabled binding assay, and bacterial mutant studies. On hydrated semi-solid gel surfaces, asialo-GM1 enables swarming and twitching motilities, while on solid surfaces facilitates the bacterial adherence of P. aeruginosa. These results indicate that asialo-GM1 can modulate bioactivities, adherence, and motilities, that are controlled by opposite signaling pathways. We demonstrate that when a solution of pilin monomers or LecA proteins are spread on hydrated gel surfaces, the asialo-GM1 mediated swarming motility is inhibited. Treatment of artificial liposomes containing asialo-GM1 as a component of lipid bilayer with pilin monomers or LecA proteins caused transient leakage of encapsulated dye from liposomes. These results suggest that pili and LecA proteins not only bind to asialo-GM1 but can also cause asialo-GM1 mediated leakage. We also show that both pili and LecA mutants of P. aeruginosa adhere to asialo-GM1 coated solid surfaces, and that a class of synthetic ligands for pili and LecA inhibits both pili and LecA-mediated adherence of P. aeruginosa on asialo-GM1-coated surfaces. The inspired by adjuvant molecule\u27s control over different P. aeruginosa phenotypes, we here designed new class of antibiotics which showed broad spectrum activity against different bacteria. This class of antibiotics inhibits pili and lecA and hence control multiple phenotypes in P. aeruginosa. This antibiotic also did not show emergence any tolerant population. We also did not develop any resistance against this class of antibiotics after repeated exposure. Silver therapy is most commonly used against burn wounds to treat P. aeruginosa and S. aureus infections. These bacteria develop cooperative resistance against silver with the help of pyocyanin, a virulence factor of P. aeruginosa. Here, we showed Farnesol as adjuvant molecule with silver therapy against P. aeruginosa and S. aureus. Farnesol eradicates resistance against silver by inhibition of pyocyanin. Farnesol also potentiated silver against dual biofilms of P. aeruginosa and S. aureus

Chimera Ligand for Pili and Lectin A Protein Controls Antibiotic-Promoted Biofilm Formation, Swarming Motility, Tolerance and Persister Formation by Pseudomonas aeruginosa

Throughout the human history, the fight against bacterial infections had never stopped but the remedies for bacterial infections were often insufficient and for many infectious diseases, there was no treatment available. The revolution in antimicrobial infection therapy began with the discovery of penicillin by Alexander Fleming in 1928. However, since the first introduction of antibiotics, bacteria over time have evolved sophisticated resistant strains against almost all the available antibiotics which cause selection pressure on the bacteria to evolve their genetic makeup and develop resistance against such agents. Furthermore, bacteria can form surface attached multicellular communities known as biofilms. Bacteria residing within biofilms are protected by biofilms which renders the bacteria more difficult to eliminate because of the low permeability of antibiotics through outer membranes. Combating such resistant bacteria is an extremely difficult task if using antibiotics alone. Hence scientific community continuously seeks new strategies to overpower these resistant bacteria. The focus of the research work presented here is to develop a class of chimera ligands that can bind to both pili and LecA protein of Pseudomonas aeruginosa to inhibit both swarming motility and biofilm formation. The potential adjuvant agents of these chimera ligands that can increase the effectiveness of antibiotics were demonstrated. In addition, the ability of our adjuvant molecules to eliminate drug-tolerant bacteria and to reduce persisters, in combination with antibiotics was demonstrated. The binding property of chimera ligands was demonstrated by competitive fluorescence polarization assay (LecA) and by adding a functional group to a ligand that can covalently attach to the receptor protein only when the physical ligand-receptor binding takes place (Pili). In addition, the effect of externally added pili on the swarming motility of Pseudomonas aeruginosa was tested to support the mechanistic study of the pili as the receptor (or one of the receptors) that will bind to rhamnolipids and our synthetic agents, and upon binding, causing the bacterial activities. For quantification of polysaccharides, two efficient detection and quantification methods that make use of the negative charges of the alginate polymer and do not involve degradation of the targeted polysaccharide were described. Both approaches provide efficient methods for monitoring alginate production by mucoid Pseudomonas aeruginosa. The effect of a class of synthetic analogs of rhamnolipids at controlling (promoting and inhibiting) the biofilm formation activities of a non-rhamnolipid-producing strain – rhlA – of Pseudomonas aeruginosa was demonstrated. The bioactive synthetic analogs of rhamnolipids promote biofilm formation by rhlA mutant at low concentrations but inhibit the biofilm formation at high concentrations. To explore the internal structures formed by the biofilms, the wild-type biofilms formed with substantial topography (hills and valleys) when the sample is under shaking conditions were observed by confocal microscope. Using this observation as a comparison, the effect of synthetic analogs of rhamnolipids on promoting structured (porous) biofilm of rhlA mutant, at intermediate concentrations between the low ones that promoted biofilm formation and the high ones that inhibited biofilm formation was demonstrated. This study suggests a potential chemical signaling approach to control multiple bacterial activities

Controlling Microbial Multicellular Behaviors With Saccharide Derivatives

Microbial multicellular behaviors like biofilm formation and swarming motility are known to increase their tolerance against antimicrobials. From microbial standpoint, nonmicrobicidal agents that do not impede growth are tolerable and therefore, there is a lower propensity to develop resistance against such agents as compared to microbicidal ones (antibiotics). This study describes a new antibiofilm approach of using nonmicrobicidal saccharide derivatives for controlling the multicellular behaviors of gram-negative bacteria, Pseudomonas aeruginosa and fungus, Candida albicans. Pseudomonas aeruginosa is known to secrete rhamnolipids, a class of biosurfactants that plays an important role in maintaining the architecture of its biofilm and promoting its swarming motility. Here we show the ability of certain synthetic nonmicrobicidal disaccharide derivatives (DSDs) to mimic the biofunctions of rhamnolipids. The rhlA mutant of P. aeruginosa is incapable of synthesizing rhamnolipids and also unable to swarm on semi-solid agar gel. When the natural ligands, rhamnolipids were externally added into the semi-solid agar gel in a concentration dependent manner, the swarming of the rhlA mutant reactivated at lower concentrations (10 μM) and then at relatively higher concentrations (15 μM), the swarming reactivation was reversed. When some active synthetic DSDs were tested on the rhlA mutant, the bacterial swarming first reactivated and then the activation reversed at higher DSD concentrations, similar to the effect of externally added rhamnolipids. Previously, a known bacterial signalling molecule has been shown to exhibit a similar concentration dependent activation and then activation reversal for light simulation by Vibrio fischeri. Some DSDs having disaccharide stereochemistries (cellobiose or maltose) and a bulky aliphatic tail (3, 7, 11-trimethyl-dodecanyl) caused swarming reactivation of the rhlA mutant at concentrations lower than that caused by the externally added rhamnolipids. The synthetic nonmicrobicidal DSDs were also very effective at inhibiting the adhesion of P. aeruginosa to polystyrene surface, and at inhibiting the bacterial biofilm formation. These DSDs were also potent dispersers of pre-formed biofilm of P. aeruginosa. The potent antibiofilm (inhibition and dispersion) activities were observed for those DSDs that possessed a disaccharide (cellobiose or maltose) stereochemistry and a bulky aliphatic chain such as 3, 7, 11-trimethyl-dodecanyl. These potent DSDs had half-maximal inhibitory concentrations for biofilm inhibition (IC50) and dispersion (DC50) comparable to those of known potent antibiofilm agents against P. aeruginosa. Gene-reporting assays indicate that the mechanism of action of such DSDs is not via the known las or rhl quorum sensing systems of P. aeruginosa but that the adhesin potein, pilin maybe a likely target of such molecules. Biofilms formed under natural settings are usually formed by both bacteria and fungus that co-reside in the same microenvironment. Therefore, agents that can prevent mixed biofilms are desirable from a therapeutic standpoint. Despite being nonmicrobicidal to both fungal blastospores and hyphae, the synthetic DSDs were able to inhibit the biofilm formation of fungus Candida albicans. Microscopic evaluation showed that most DSDs did not prevent the blastospores-to-hyphae morphogenesis. The DSDs were effective at inhibiting biofilm formation of Candida albicans when applied within five minutes of seeding the test surface with fungal cells. Using a surface based assay it was shown that one DSD dramatically reduced the surface adhesion of Candida albicans hyphae. The antibiofilm activity of such DSDs against Candida albicans is probably due to their ability to prevent hyphae surface adhesion

Antimicrobial efficacy against Pseudomonas aeruginosa biofilm formation in a three-dimensional lung epithelial model and the influence of fetal bovine serum

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS

ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY OF SELECTED MEDICINAL PLANTS AGAINST HUMAN ORAL PATHOGENS

Objective: The aim of the study was focused on determining the phytochemicals, antibacterial, antiadherence, antifungal and antioxidant activities of Glycyrrhiza glabra, Matricaria chamomilla and Eclipta alba and also their mechanism of action towards human oral pathogens.Methods: Qualitative analysis and quantitative estimation of phenols and flavonoids were performed in methanolic extracts. Antibacterial, anti adherence, antifungal assays were performed by plate assays. Antioxidant assays were done by ABTS and DPPH methods. SEM, TEM and flow cytometry analysis were executed to find out the mechanism of action of plant extract.Results:  The total phenol contents were 0.85, 1.24, 0.64 GAE/g and the total flavonoid contents were 356, 231.34 and 88 µg QE/mg for G. glabra, M. chamomilla and E. alba respectively.Matricaria chamomilla possesses highest antioxidant activity (DPPH and ABTS assays) among the all extracts tested. E. alba showed a highest zone of inhibition against S. aureus (21.6 mm) whereas G. glabra and M. chamomilla revealed the better result of 21 mm and 19.8 mm respectively against S. mutans. Glycyrrhiza glabra showed antifungal activity against Candida parapsilosis whereas Pseudomonas aeruginosa, Candida krusei, Candida tropicalis and Candida albicans showed resistance towards all the extracts tested. The MIC, MBC and antiadherence tests were also performed. Sorbitol assay confirmed that G. glabra has no impact on the fungal cell wall. To confirm the mode of action SEM, TEM and flow cytometric analysis were performed which showed the cell elongation and damage in cytoplasmic membrane resulting in oozing of cellular constituents. Conclusion: This work concluded that all the plant extracts showed potent activities among the various tests. Oral care product can be developed if the active constituents responsible for the activities were analysed.Â

Chemical Control and Understanding of Horizontal Gene Transfers, Drug-Resistance Development, and Filament and Biofilm Formation

Biofilms formed by microbes on surfaces are the sources for persistent infectious diseases and environmental problems. The mechanism and details of how antibiotics promote biofilm formation is largely unknown. For instance, it not clear what stages of biofilm growth are promoted to proceed faster than without antibiotics, what phenotypes of bacteria form in an antibiotic-promoted biofilm, and what different biofilm compositions and structures are caused by the presence of antibiotics. Among other effects, antibiotics can cause bacteria to form filaments of living bacteria. Here, we conduct a real-time study of the adherence of bacteria and antibiotic-induced filamentous bacteria on surfaces and characterize the kinetics of surface adherence of these two forms of bacteria. Studying the effect of different surfaces on promoting filamentous bacteria’s adherence on surfaces, we characterize an unexpected correlation between the stage of bacterial growth and the formation and growth of filamentous bacteria on surfaces. Based on these results, we outline the lifestyle of filamentous bacteria and a mechanism by which antibiotics promote biofilm formation. The drug resistance of bacteria is becoming more severe since antibiotics are first discovered. The development of drug-resistance among bacteria involves spreading and drug-resistant gene “horizontally” between bacteria. These horizontal gene transfers impact the gene composition and evolution of the bacteria, and facilitate the transfer of antibiotic resistance genes. At the molecular level, all three major mechanisms of HGTs, transformation, conjugation and transduction, involve type IV pili appendages on bacterial surfaces. Here, the three mechanisms of horizontal gene transfer are studied. Saturated farnesol derivatives show inhibition effect on tetracycline-enhanced plasmid transformation, ciprofloxacin-enhances PAPI-1 transduction, and PAPI-1 conjugation. Further study of ciprofloxacin-resistance development in P. aeruginosa is conducted by using serial passage assay. The inhibited development of MIC of ciprofloxacin suggest that saturated farnesol derivative inhibits the development of resistance to ciprofloxacin in P. aeruginosa. This thesis also describes the mechanism of pili inhibition by saturated farnesol derivatives. The pili-mediated bacteriophage adsorption is studied, and the results demonstrate that saturated farnesol derivative could cause pili retraction, leading to inhibition of bacteriophage adsorption. More evidence was obtained from other colleagues in Dr. Luk’s lab to support the small molecules binding to pili. The MALDI-MS done by Hewen Zheng suggests that saturated farnesol derivative covalently binds to pili. Hewen also performed the alkaline buffer extraction experiment to demonstrate that PAO1 culturing with saturated farnesol derivative resulted in decrease in pili protein expression. Together with the bacteriophage adsorption results, we conclude that saturated farnesol derivative binding to pili cause pili retraction

Development and validation of a high-content screening assay for inhibitors of enteropathogenic E. coli adhesion

Enteropathogenic E. coli (EPEC) causes intestinal infections leading to severe diarrhea. EPEC attaches to the host cell causing lesions to the intestinal epithelium coupled with the effacement of microvilli. In the process, actin accumulates into a pedestal-like structure under bacterial microcolonies. We designed an automated fluorescence microscopy-based screening method for discovering compounds capable of inhibiting EPEC adhesion and virulence using aurodox, a type three secretion system (T3SS) inhibitor, as a positive control. The screening assay employs an EPEC strain (2348/69) expressing a fluorescent protein and actin staining for monitoring the bacteria and their pedestals respectively, analyzing these with a custom image analysis pipeline. The assay allows for the discovery of compounds capable of preventing the formation of pathogenic actin rearrangements. These compounds may be interfering with virulence-related molecular pathways relevant for developing antivirulence leads.Peer reviewe