Anti-virulence Strategies Research Articles

Ostarine attenuates pyocyanin in Pseudomonas aeruginosa by interfering with quorum sensing systems.

Antimicrobial resistance has been an increasingly serious threat to global public health. Anti-virulence strategies are being developed to manage antibiotic resistance because they apply a lower selective pressure for antimicrobial-resistant pathogens than that created using traditional bactericides. We aimed to discover novel small molecules that can reduce the production of virulence factors in Pseudomonas aeruginosa and determine the mechanism of action underlying these effects. A clinical compound library was screened, and ostarine was identified as a potential anti-virulence agent. The effects of ostarine were studied via antimicrobial susceptibility testing, bacterial growth assays, pyocyanin quantitation assays, transcriptomic analysis, quorum sensing signal molecule quantification, and real-time PCR assays. Ostarine treatment significantly decreased the synthesis of pyocyanin without any bactericidal action. Besides, ostarine treatment did not affect the relative growth rate and cell morphology of bacteria. Treatment with ostarine interfered with quorum sensing by decreasing the transcription of genes associated with quorum sensing systems and the production of signalling molecules. The inhibition of ostarine on pyocyanin production and gene expression can be alleviated when signalling molecules were supplemented externally. Overall, ostarine may act as a novel anti-virulence agent that can attenuate P. aeruginosa pyocyanin by interfering with quorum sensing systems.

Anti-Virulence Strategy against the Honey Bee Pathogenic Bacterium Paenibacillus larvae via Small Molecule Inhibitors of the Bacterial Toxin Plx2A.

American Foulbrood, caused by Paenibacillus larvae, is the most devastating bacterial honey bee brood disease. Finding a treatment against American Foulbrood would be a huge breakthrough in the battle against the disease. Recently, small molecule inhibitors against virulence factors have been suggested as candidates for the development of anti-virulence strategies against bacterial infections. We therefore screened an in-house library of synthetic small molecules and a library of flavonoid natural products, identifying the synthetic compound M3 and two natural, plant-derived small molecules, Acacetin and Baicalein, as putative inhibitors of the recently identified P. larvae toxin Plx2A. All three inhibitors were potent in in vitro enzyme activity assays and two compounds were shown to protect insect cells against Plx2A intoxication. However, when tested in exposure bioassays with honey bee larvae, no effect on mortality could be observed for the synthetic or the plant-derived inhibitors, thus suggesting that the pathogenesis strategies of P. larvae are likely to be too complex to be disarmed in an anti-virulence strategy aimed at a single virulence factor. Our study also underscores the importance of not only testing substances in in vitro or cell culture assays, but also testing the compounds in P. larvae-infected honey bee larvae.

Screening of small molecules attenuating biofilm formation of Acinetobacter baumannii by inhibition of ompA promoter activity.

Anti-virulence therapeutic strategies are promising alternatives against drug-resistant pathogens. Outer membrane protein A (OmpA) plays a versatile role in the pathogenesis and antimicrobial resistance of Acinetobacter baumannii. Therefore, OmpA is an innovative target for anti-virulence therapy against A. baumannii. This study aimed to develop a high-throughput screening (HTS) system to discover small molecules inhibiting the ompA promoter activity of A. baumannii and screen chemical compounds using the bacterial growth-based HTS system. The ompA promoter and open reading frame of nptI fusion plasmids that controlled the expression of nptI encoding resistance to kanamycin by the ompA promoter were constructed and then transformed into A. baumannii ATCC 17978. This reporter strain was applied to screen small molecules inhibiting the ompA promoter activity in a chemical library. Of the 7,520 chemical compounds, 15 exhibited ≥ 70% growth inhibition of the report strain cultured in media containing kanamycin. Three compounds inhibited the expression of ompA and OmpA in the outer membrane of A. baumannii ATCC 17978, which subsequently reduced biofilm formation. In conclusion, our reporter strain is useful for large-scale screening of small molecules inhibiting the ompA expression in A. baumannii. Hit compounds identified by the HTS system are promising scaffolds to develop novel therapeutics against A. baumannii.

The CARB-X Portfolio of Nontraditional Antibacterial Products.

The growing prevalence of antibiotic-resistant bacterial pathogens and the lack of new medicines to treat the infections they cause remain a significant global threat. In recent years, this ongoing unmet need has encouraged more research groups to focus on the discovery and development of nontraditional antibacterial agents, ranging from anti-virulence strategies to bacteriophage and ways to modulate the microbiome. The Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) is a global nonprofit public-private partnership dedicated to accelerating antibacterial-related research. Importantly, the CARB-X portfolio supports a wide variety of novel and innovative nontraditional programs to help the global antibacterial research ecosystem understand the potential that these modalities can play in the management or prevention of serious infections. We describe here the breadth of the CARB-X pipeline of novel nontraditional products.

Antibacterial and Antibiofilm Activities of Chloroindoles Against Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a food-borne pathogen recognized as the prominent cause of seafood-borne gastroenteritis globally, necessitating novel therapeutic strategies. This study examined the antimicrobial and antivirulence properties of indole and 16 halogenated indoles on V. parahaemolyticus. Among them, 4-chloroindole, 7-chloroindole, 4-iodoindole, and 7-iodoindole effectively inhibited planktonic cell growth, biofilm formation, bacterial motility, fimbrial activity, hydrophobicity, protease activity, and indole production. Specifically, 4-chloroindole at 20 μg/mL inhibited more than 80% of biofilm formation with a minimum inhibitory concentration (MIC) of 50 μg/mL against V. parahaemolyticus and Vibrio harveyi. In contrast, 7-chloroindole inhibited biofilm formation without affecting planktonic cell growth with a MIC of 200 μg/mL. Both chlorinated indoles caused visible damage to the cell membrane, and 4-chloroindole at 100 μg/mL had a bactericidal effect on V. parahaemolyticus within 30 min treatment, which is superior to the effect of tetracycline at the same dose. The quantitative structure-activity relationship (QSAR) analyses revealed that chloro and bromo at positions 4 or 5 of the indole are essential for eradicating the growth of V. parahaemolyticus. These results suggest that halogenated indoles have potential use in antimicrobial and antivirulence strategies against Vibrio species.

Non-Native Peptides Capable of Pan-Activating the agr Quorum Sensing System across Multiple Specificity Groups of Staphylococcus epidermidis.

Staphylococcus epidermidis is a leading cause of hospital-acquired infections. Traditional antibiotics have significantly reduced efficacy against this pathogen due to its ability to form biofilms on abiotic surfaces and drug resistance. The accessory gene regulator (agr) quorum sensing system is directly involved in S. epidermidis pathogenesis. Activation of agr is achieved via binding of the autoinducing peptide (AIP) signal to the extracellular sensor domain of its cognate receptor, AgrC. Divergent evolution has given rise to four agr specificity groups in S. epidermidis defined by the unique AIP sequence used by each group (AIPs-I-IV) with observed cross-group activities. As agr agonism has been shown to reduce biofilm growth in S. epidermidis, the development of pan-group activators of the agr system is of interest as a potential antivirulence strategy. To date, no synthetic compounds have been identified that are capable of appreciably activating the agr system of more than one specificity group of S. epidermidis or, to our knowledge, of any of the other Staphylococci. Here, we report the characterization of the structure-activity relationships for agr agonism by S. epidermidis AIP-II and AIP-III and the application of these new SAR data and those previously reported for AIP-I for the design and synthesis of the first multigroup agr agonists. These non-native peptides were capable of inducing the expression of critical biofilm dispersal agents (i.e., phenol-soluble modulins) in cell culture and represent new tools to study the role of quorum sensing in S. epidermidis infections.

Combatting fungal infections through the discovery and elucidation of novel anti‐virulence strategies

Fungal pathogens are critically important threats to global health with over 300 million people affected by serious fungal diseases worldwide. Fungal pathogens have evolved sophisticated strategies, including the secretion of virulence factors to interfere with host cell functions and to perturb immune responses. Our ‘infectome’ analysis identifies previously undescribed proteins involved in fungal virulence and host immune response, representing an opportunity to elucidate molecular mechanisms of host-pathogen interplay during disease. Using state-of-the-art mass spectrometry-based proteomics we profile the total proteome and secretome of Cryptococcus neoformans wild-type (H99) under in vitro growth conditions. We also define the infectome of C. neoformans and BALB/c macrophages in single runs using high resolution mass spectrometry on a Quadrupole Orbitrap instrument. The in vitro and infectome datasets were integrated using Perseus and candidate fungal proteins of interest were prioritized based on predicted secretory roles, novelty, and abundance profiles. Deletion strains were constructed by double-joint PCR and characterized by phenotypic screening and cell death assays. Virulence-associated candidates will be evaluated in a murine infection model and further characterized by immunofluorescence and interactome analyses. Our preliminary results identify interactions between the host and pathogen critical for disease. Comprehensive profiling of infection from both host and pathogen perspectives unveils new anti-virulence strategies to combat fungal infection.

Characterization and development of new anti‐toxins targeting Clostridium difficile

Clostridium difficile is a bacterial pathogen that causes serious and potentially fatal inflammatory disease of the colon. One strategy for targeting C. difficile infection is to development treatments that target bacterial virulence. Anti-virulence strategies to treat C. difficile infection are attractive options to antibiotic therapy because the human microbiota is spared and damage to host tissue is minimized. The Tcd toxins, TcdA and TcdB, are the major virulence factors produced by C. difficile and mediate host cytotoxicity by glucosylating and inactivating Rho family GTPases using UDP-glucose as a glucosyl donor. Glucosylation of Rho GTPases leads to cell rounding and eventually cell death. We used kinetic isotope effects (KIEs) and transition state theory to solve the transition state structure of the glycosyltransferase domain of TcdB (TcdB-GTD). This permits the design of transition state analogues which are potent enzyme inhibitors. KIEs compare the chemical steps of enzymatic reaction rates with isotopically labeled substrates and unlabeled substrates and provide critical information on the catalytic mechanism, bond lengths and geometry at the transition state. KIE analysis on the glycohydrolase reaction of TcdB-GTD supported the formation of a late, dissociative glucocation-like transition state where positive charge develops on the anomeric carbon. Glucocationic transition states are best mimicked by cationic nitrogen groups at or near the anomeric carbon. Iminosugars contain a nitrogen atom in the sugar ring in place of oxygen and mimic glucocationic transition states. We tested iminosugars for inhibition of TcdB-GTD and TcdA-GTD. Isofagomine (Ki TcdB-GTD = 1.4 µM and Ki TcdA-GTD = 0.24 µM) and noeuromycin (Ki TcdB-GTD = 10.6 µM and Ki TcdA-GTD = 4.7 µM) were the most potent iminosugars identified. Isofagomine and noeuromycin were shown to exhibit an uncompetitive mechanism of inhibition suggesting that both iminosugars act by binding to the enzyme-UDP complex. Isothermal titration calorimetry experiments with TcdB-GTD supported this finding where isofagomine and noeuromycin could only bind to TcdB-GTD in the presence of UDP with Kd = 0.133 µM and Kd = 0.4 µM respectively. In addition, the X-ray crystal structure of TcdB-GTD in complex with either Isofagomine or noeuromycin with UDP revealed the formation of an ion-pair interaction between the cationic nitrogen atom of the iminosugar and the β-phosphate of UDP. Finally, we confirmed that isofagomine and noeuromycin could prevent Tcd toxin-induced cytotoxicity of IMR90 cells. Firstly, both iminosugars were able to prevent Tcd toxin-induced cell rounding as visualized by light microscopy. Secondly, Western blot analysis of Tcd toxin treated IMR90 cell lysates revealed that both isofagomine and noeuromycin could prevent glucosylation of Rac1 by Tcd toxin. Isofagomine and noeuromycin are effective in preventing Tcd toxin action. Transition state analogues against TcdA and TcdB have the potential to be used as treatments for C. difficile infections.

NirA Is an Alternative Nitrite Reductase from Pseudomonas aeruginosa with Potential as an Antivirulence Target.

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, antivirulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in strain PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we identified a Tn5 mutant with an insertion in PA4130 showing reduced levels of a number of virulence traits in vitro Construction of an isogenic mutant in this gene presented results similar to those for the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster and Caenorhabditis elegans as well as reduced toxicity in human cell lines. Mice infected with this mutant demonstrated an 80% increased survival rate in acute and agar bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulfite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this enzyme is a nitrite reductase operating in a ferredoxin-dependent manner. The preference for nitrite and production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named NirA.IMPORTANCE The emergence of widespread antimicrobial resistance has led to the need for development of novel therapeutic interventions. Antivirulence strategies are an attractive alternative to classic antimicrobial therapy; however, they require identification of new specific targets which can be exploited in drug discovery programs. The host-specific nature of P. aeruginosa virulence adds complexity to the discovery of these types of targets. Using a sequence of in vitro assays and phylogenetically diverse in vivo disease models, we have identified a PA4130 mutant with reduced production in a number of virulence traits and severe attenuation across all infection models tested. Characterization of PA4130 revealed that it is a ferredoxin-nitrite reductase and hence was named NirA. These results, together with attenuation of nirA mutants in different clinical isolates, high level conservation of its gene product in P. aeruginosa genomes, and the lack of orthologues in human genomes, make NirA an attractive antivirulence target.

An antipathogenic compound that targets the OxyR peroxide sensor in Pseudomonas aeruginosa.

IntroductionAntipathogenic or antivirulence strategy is to target a virulence pathway that is dispensable for growth, in the hope to mitigate the selection for drug resistance.Hypothesis/Gap StatmentPeroxide stress responses are one of the conserved virulence pathways in bacterial pathogens and thus good targets for antipathogenic strategy.AimThis study aims to identify a new chemical compound that targets OxyR, the peroxide sensor required for the full virulence of the opportunistic human pathogen, Pseudomonas aeruginosa .MethodologyComputer-based virtual screening under consideration of the ‘eNTRy’ rules and molecular docking were conducted on the reduced form of the OxyR regulatory domain (RD). Selected hits were validated by their ability to phenocopy the oxyR null mutant and modulate the redox cycle of OxyR.ResultsWe first isolated three robust chemical hits that inhibit OxyR without affecting prototrophic growth or viability. One (compound 1) of those affected the redox cycle of OxyR in response to H2O2 treatment, in a way to impair its function. Compound 1 displayed selective antibacterial efficacy against P. aeruginosa in Drosophila infection model, without antibacterial activity against Staphylococcus aureus .ConclusionThese results suggest that compound 1 could be an antipathogenic hit inhibiting the P. aeruginosa OxyR. More importantly, our study provides an insight into the computer-based discovery of new-paradigm selective antibacterials to treat Gram-negative bacterial infections presumably with few concerns of drug resistance.

Subinhibitory Concentrations of Biogenic Silver Nanoparticles Affect Motility and Biofilm Formation in Pseudomonas aeruginosa.

Biogenic silver nanoparticles (bio-AgNPs) are increasingly recognized as an antibiofilm and antivirulence strategy against P. aeruginosa, a bacterium that causes chronic infections in immunocompromised and cystic fibrosis patients. This study aimed to investigate the effects of subinhibitory concentrations of bio-AgNPs on motility and biofilm formation in P. aeruginosa. Bio-AgNPs were synthesized via reduction of ionic silver catalyzed by cell-free culture filtrate from Fusarium oxysporum. A total of 17 P. aeruginosa isolates and strains were evaluated for swarming, swimming, and twitching motility in the presence and absence (control) of bio-AgNPs, including 10 clinical isolates from patients with and without cystic fibrosis, 5 environmental isolates obtained from the public water supply system, and 2 reference strains (PAO1 and PA14). Isolates were identified by biochemical and molecular methods. Minimum inhibitory concentrations (MICs) were determined by the broth microdilution method. Swarming, swimming, and twitching motility assays were performed in Petri dishes. Biofilm formation capacity was assessed quantitatively by the crystal violet method. MIC values ranged from 15.62 to 62.50 µM. The results showed that subinhibitory concentrations of bio-AgNPs (½ MIC, 7.81–31.25 µM) significantly increased (p < 0.05) swarming, swimming, and twitching motility in 40.0, 40.0, and 46.7% of isolates, respectively. Subinhibitory bio-AgNP treatment enhanced (p < 0.05) biofilm formation capacity in PA14 and a cystic fibrosis isolate (P11). It is concluded that subinhibitory concentrations of bio-AgNPs increased biofilm formation and swarming, swimming, and twitching motility in PA14 and some P. aeruginosa isolates. These virulence factors are directly involved with quorum-sensing systems. Further research should investigate the effects of AgNPs on P. aeruginosa quorum sensing to help elucidate their mechanism of action at subinhibitory concentrations.

Resveratrol influences the pathogenesis of Aeromonas hydrophila by inhibiting production of aerolysin and biofilm

Aeromonas hydrophila is widely distributed in freshwater environments and aquatic products which can cause a variety of infectious diseases to aquatic, terrestrial species and human. However, the emergence of antibiotic resistant strains results in the treatment failure of antibiotics. Therefore, novel strategies need to be clarified. The pathogenesis of the bacterium is composed by the virulence factors, such as hemolytic toxins, biofilm and adhesions. Therefore, aerolysin and biofilm have been developed as targets identifying novel drugs against A. hydrophila infections. In the present study, resveratrol with little anti-A. hydrophila activity could decrease the pathogenesis of A. hydrophila by inhibiting the expression of aerolysin and biofilm formation at sub-inhibitory concentrations. Results of western-blot and qPCR assays demonstrated that RSV could decrease aerolysin and biofilm production by inhibiting aerolysin encoding gene and quorum sensing system. Furthermore, results of cell viability and experimental therapeutics assays indicated that RSV could provide a significant protection both in vitro and in vivo. Taken together, these findings provide a new approach in identifying drugs based on anti-virulence strategies. RSV is a promising candidate anti- A. hydrophila infections by decreasing the production of aerolysin and biofilm.

Bacterial virulence plays a crucial role in MRSA sepsis.

Bacterial sepsis is a major global cause of death. However, the pathophysiology of sepsis has remained poorly understood. In industrialized nations, Staphylococcus aureus represents the pathogen most commonly associated with mortality due to sepsis. Because of the alarming spread of antibiotic resistance, anti-virulence strategies are often proposed to treat staphylococcal sepsis. However, we do not yet completely understand if and how bacterial virulence contributes to sepsis, which is vital for a thorough assessment of such strategies. We here examined the role of virulence and quorum-sensing regulation in mouse and rabbit models of sepsis caused by methicillin-resistant S. aureus (MRSA). We determined that leukopenia was a predictor of disease outcome during an early critical stage of sepsis. Furthermore, in device-associated infection as the most frequent type of staphylococcal blood infection, quorum-sensing deficiency resulted in significantly higher mortality. Our findings give important guidance regarding anti-virulence drug development strategies for the treatment of staphylococcal sepsis. Moreover, they considerably add to our understanding of how bacterial sepsis develops by revealing a critical early stage of infection during which the battle between bacteria and leukocytes determines sepsis outcome. While sepsis has traditionally been attributed mainly to host factors, our study highlights a key role of the invading pathogen and its virulence mechanisms.

Tailored liposomal nanotraps for the treatment of Streptococcal infections

BackgroundStreptococcal infections are associated with life-threatening pneumonia and sepsis. The rise in antibiotic resistance calls for novel approaches to treat bacterial diseases. Anti-virulence strategies promote a natural way of pathogen clearance by eliminating the advantage provided to bacteria by their virulence factors. In contrast to antibiotics, anti-virulence agents are less likely to exert selective evolutionary pressure, which is a prerequisite for the development of drug resistance. As part of their virulence mechanism, many bacterial pathogens secrete cytolytic exotoxins (hemolysins) that destroy the host cell by destabilizing their plasma membrane. Liposomal nanotraps, mimicking plasmalemmal structures of host cells that are specifically targeted by bacterial toxins are being developed in order to neutralize-by competitive sequestration-numerous exotoxins.ResultsIn this study, the liposomal nanotrap technology is further developed to simultaneously neutralize the whole palette of cytolysins produced by Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus dysgalactiae subspecies equisimilis-pathogens that can cause life-threatening streptococcal toxic shock syndrome. We show that the mixture of liposomes containing high amounts of cholesterol and liposomes composed exclusively of choline-containing phospholipids is fully protective against the combined action of exotoxins secreted by these pathogens.ConclusionsUnravelling the universal mechanisms that define targeting of host cells by streptococcal cytolysins paves the way for a broad-spectrum anti-toxin therapy that can be applied without a diagnostic delay for the treatment of bacterial infections including those caused by antibiotic-resistant pathogens.

Antivirulence DsbA inhibitors attenuate Salmonella enterica serovar Typhimurium fitness without detectable resistance.

ABSTRACTInhibition of the DiSulfide Bond (DSB) oxidative protein folding machinery, a major facilitator of virulence in Gram‐negative bacteria, represents a promising antivirulence strategy. We previously developed small molecule inhibitors of DsbA from Escherichia coli K‐12 (EcDsbA) and showed that they attenuate virulence of Gram‐negative pathogens by directly inhibiting multiple diverse DsbA homologues. Here we tested the evolutionary robustness of DsbA inhibitors as antivirulence antimicrobials against Salmonella enterica serovar Typhimurium under pathophysiological conditions in vitro. We show that phenylthiophene DsbA inhibitors slow S. Typhimurium growth in minimal media, phenocopying S. Typhimurium isogenic dsbA null mutants. Through passaging experiments, we found that DsbA inhibitor resistance was not induced under conditions that rapidly induced resistance to ciprofloxacin, an antibiotic commonly used to treat Salmonella infections. Furthermore, no mutations were identified in the dsbA gene of inhibitor‐treated S. Typhimurium, and S. Typhimurium virulence remained susceptible to DsbA inhibitors. Our work demonstrates that under in vitro pathophysiological conditions, DsbA inhibitors can have both antivirulence and antibiotic action. Importantly, our finding that DsbA inhibitors appear to be evolutionarily robust offers promise for their further development as next‐generation antimicrobials against Gram‐negative pathogens.

Sorting out the Superbugs: Potential of Sortase A Inhibitors among Other Antimicrobial Strategies to Tackle the Problem of Antibiotic Resistance.

Rapid spread of antibiotic resistance throughout the kingdom bacteria is inevitably bringing humanity towards the “post-antibiotic” era. The emergence of so-called “superbugs”—pathogen strains that develop resistance to multiple conventional antibiotics—is urging researchers around the globe to work on the development or perfecting of alternative means of tackling the pathogenic bacteria infections. Although various conceptually different approaches are being considered, each comes with its advantages and drawbacks. While drug-resistant pathogens are undoubtedly represented by both Gram(+) and Gram(−) bacteria, possible target spectrum across the proposed alternative approaches of tackling them is variable. Numerous anti-virulence strategies aimed at reducing the pathogenicity of target bacteria rather than eliminating them are being considered among such alternative approaches. Sortase A (SrtA) is a membrane-associated cysteine protease that catalyzes a cell wall sorting reaction by which surface proteins, including virulence factors, are anchored to the bacterial cell wall of Gram(+) bacteria. Although SrtA inhibition seems perspective among the Gram-positive pathogen-targeted antivirulence strategies, it still remains less popular than other alternatives. A decrease in virulence due to inactivation of SrtA activity has been extensively studied in Staphylococcus aureus, but it has also been demonstrated in other Gram(+) species. In this manuscript, results of past studies on the discovery of novel SrtA inhibitory compounds and evaluation of their potency were summarized and commented on. Here, we discussed the rationale behind the inhibition of SrtA, raised some concerns on the comparability of the results from different studies, and touched upon the possible resistance mechanisms as a response to implementation of such therapy in practice. The goal of this article is to encourage further studies of SrtA inhibitory compounds.

Epigallocatechin gallate alters leukotoxin secretion and Aggregatibacter actinomycetemcomitans virulence

We and others have previously shown that epigallocatechin gallate (EGCg) inhibits the activity of an important virulence factor, leukotoxin (LtxA), produced by the oral bacterium Aggregatibacter actinomycetemcomitans, suggesting the potential use of this molecule as an anti-virulence strategy to treat periodontal infections. Here, we sought to better understand the effects of EGCg on toxin secretion and A. actinomycetemcomitans pathogenicity in a co-culture model. We used a quantitative immunoblot assay to determine the concentrations of LtxA in the bacterial supernatant and on the bacterial cell surface. Using a co-culture model, consisting of A. actinomycetemcomitans and THP-1 cells, we studied the impact of EGCg-mediated changes in LtxA secretion on the toxicity of A. actinomycetemcomitans. EGCg increased production of LtxA and changed the localization of secreted LtxA from the supernatant to the surface of the bacterial cells. In the co-culture model, a single low dose of EGCg did not protect host THP-1 cells from A. actinomycetemcomitans-mediated cytotoxicity, but a multiple dosing strategy had improved effects. Together, these results demonstrate that EGCg has important, but complicated, effects on toxin secretion and activity; new dosing strategies and comprehensive model systems may be required to properly develop these anti-virulence activities.

QseC Inhibition as a Novel Antivirulence Strategy for the Prevention of Acute Hepatopancreatic Necrosis Disease (AHPND)-Causing Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus resulted in great economic losses in global shrimp aquaculture. There is an urgent need for development of novel strategies to combat AHPND-causing V. parahaemolyticus (Vp AHPND), given that one of the greatest challenges currently is the widespread use of antibiotics and subsequent emergence of multidrug-resistant bacteria. Here, we proposed a broad-spectrum antivirulence approach targeting a conserved histidine kinase, QseC, which has been demonstrated to activate virulence expression in several Gram-negative pathogens. Our results showed that QseC mediated the catecholamine stimulated effects on growth and flagellar motility of Vp AHPND. Transcriptome analysis revealed that QseC was involved in the global regulation of the virulence of Vp AHPND as the ΔqseC mutant exhibited a decreased expression of genes related to type IV pilin, flagellar motility, and biofilm formation, while an overexpression of type VI secretion system and cell wall biosynthesis. Subsequently, the bacterial catecholamine receptor antagonist LED209 not only neutralized the stimulatory effects of host catecholamines on the growth and motility of Vp AHPND in vitro, but also attenuated the virulence of Vp AHPND towards brine shrimp larvae and white shrimp in vivo. Additionally, LED209 presented no interference with pathogen growth, nor the toxicity to the experimental animals. These results suggest that QseC can be an attractive antivirulence therapy target, and LED209 is a promising candidate for development of broad-spectrum antivirulence agents. This is the first study that demonstrated the role of QseC in the global regulation of Vp AHPND infection and demonstrated the antivirulence potential of LED209, which provides insight into the use of an antivirulence approach for targeting not only Vp AHPND, but also a much larger collection of pathogenic bacteria.

Anti-Virulence Therapeutic Approaches for Neisseria gonorrhoeae.

While antimicrobial resistance (AMR) is seen in both Neisseria gonorrhoeae and Neisseria meningitidis, the former has become resistant to commonly available over-the-counter antibiotic treatments. It is imperative then to develop new therapies that combat current AMR isolates whilst also circumventing the pathways leading to the development of AMR. This review highlights the growing research interest in developing anti-virulence therapies (AVTs) which are directed towards inhibiting virulence factors to prevent infection. By targeting virulence factors that are not essential for gonococcal survival, it is hypothesized that this will impart a smaller selective pressure for the emergence of resistance in the pathogen and in the microbiome, thus avoiding AMR development to the anti-infective. This review summates the current basis of numerous anti-virulence strategies being explored for N. gonorrhoeae.

Antivirulence Strategies for the Treatment of Staphylococcus aureus Infections: A Mini Review.

Staphylococcus aureus is a Gram-positive bacterium capable of infecting nearly all host tissues, causing severe morbidity and mortality. Widespread antimicrobial resistance has emerged among S. aureus clinical isolates, which are now the most frequent causes of nosocomial infection among drug-resistant pathogens. S. aureus produces an array of virulence factors that enhance in vivo fitness by liberating nutrients from the host or evading host immune responses. Staphylococcal virulence factors have been identified as viable therapeutic targets for treatment, as they contribute to disease pathogenesis, tissue injury, and treatment failure. Antivirulence strategies, or treatments targeting virulence without direct toxicity to the inciting pathogen, show promise as an adjunctive therapy to traditional antimicrobials. This Mini Review examines recent research on S. aureus antivirulence strategies, with an emphasis on translational studies. While many different virulence factors have been investigated as therapeutic targets, this review focuses on strategies targeting three virulence categories: pore-forming toxins, immune evasion mechanisms, and the S. aureus quorum sensing system. These major areas of S. aureus antivirulence research demonstrate broad principles that may apply to other human pathogens. Finally, challenges of antivirulence research are outlined including the potential for resistance, the need to investigate multiple infection models, and the importance of studying antivirulence in conjunction with traditional antimicrobial treatments.