Comprehensive Review on the Virulence Factors and Therapeutic Strategies with the Aid of Artificial Intelligence against Mucormycosis.

Mucormycosis, a rare but deadly fungal infection, was an epidemic during the COVID-19 pandemic. The rise in cases (COVID-19-associated mucormycosis, CAM) is attributed to excessive steroid and antibiotic use, poor hospital hygiene, and crowded settings. Major contributing factors include diabetes and weakened immune systems. The main manifesting forms of CAM─cutaneous, pulmonary, and the deadliest, rhinocerebral─and disseminated infections elevated mortality rates to 85%. Recent focus lies on small-molecule inhibitors due to their advantages over standard treatments like surgery and liposomal amphotericin B (which carry several long-term adverse effects), offering potential central nervous system penetration, diverse targets, and simpler dosing owing to their small size, rendering the ability to traverse the blood-brain barrier via passive diffusion facilitated by the phospholipid membrane. Adaptation and versatility in mucormycosis are facilitated by a multitude of virulence factors, enabling the pathogen to dynamically respond to various environmental stressors. A comprehensive understanding of these virulence mechanisms is imperative for devising effective therapeutic interventions against this highly opportunistic pathogen that thrives in immunocompromised individuals through its angio-invasive nature. Hence, this Review delineates the principal virulence factors of mucormycosis, the mechanisms it employs to persist in challenging host environments, and the current progress in developing small-molecule inhibitors against them.

LC-MS/MS determination of pyocyanin-N-acetyl cysteine adduct: application for understanding Pseudomonas aeruginosa virulence factor neutralization.

Combating Pseudomonas aeruginosa infection is challenging. It secretes pyocyanin (PCN) pigment that contributes to its virulence. Neutralizing PCN via reaction with thiol-containing compounds may represent a potential therapeutic option. This study investigates the neutralization reaction between PCN and N-acetyl cysteine (NAC) for bacterial inhibition and explores its mechanism of action. The neutralization adduct (PCN-NAC) was synthesized by reacting the purified PCN and NAC. The adduct was analyzed and its structure was elucidated. LC-MS/MS method was developed for the determination of PCN-NAC in P. aeruginosa cultures post-treatment with NAC (0-5 mg/mL). The corresponding anti-bacterial potential was estimated and compared to nanoparticles (NPs) alone and under stress conditions. In silico studies were performed to support explaining the mechanism of action. Results revealed that PCN-NAC was exclusively detected in NAC-treated cultures in a concentration-dependent manner. PCN-NAC concentration (230-915 µg/mL) was directly proportional to the reduction in the bacterial viable count (28.3% ± 7.1-87.5% ± 5.9) and outperformed all tested NPs, where chitosan NPs induced 56.9% ± 7.9 inhibition, followed by zinc NPs (49.4% ± 0.9) and gold NPs (17.8% ± 7.5) even post-exposure to different stress conditions. A concomitant reduction in PCN concentration was detected. In silico studies revealed possible interactions between key bacterial proteins and PCN-NAC rather than the NAC itself. These results pose NAC as a potential choice for the management of P. aeruginosa infection, where it neutralizes PCN via the formation of PCN-NAC adduct.

Ethanolic Extract Propolis-Loaded Niosomes Diminish Phospholipase B1, Biofilm Formation, and Intracellular Replication of Cryptococcus neoformans in Macrophages.

Secretory phospholipase B1 (PLB1) and biofilms act as microbial virulence factors and play an important role in pulmonary cryptococcosis. This study aims to formulate the ethanolic extract of propolis-loaded niosomes (Nio-EEP) and evaluate the biological activities occurring during PLB1 production and biofilm formation of Cryptococcus neoformans. Some physicochemical characterizations of niosomes include a mean diameter of 270 nm in a spherical shape, a zeta-potential of -10.54 ± 1.37 mV, and 88.13 ± 0.01% entrapment efficiency. Nio-EEP can release EEP in a sustained manner and retains consistent physicochemical properties for a month. Nio-EEP has the capability to permeate the cellular membranes of C. neoformans, causing a significant decrease in the mRNA expression level of PLB1. Interestingly, biofilm formation, biofilm thickness, and the expression level of biofilm-related genes (UGD1 and UXS1) were also significantly reduced. Pre-treating with Nio-EEP prior to yeast infection reduced the intracellular replication of C. neoformans in alveolar macrophages by 47%. In conclusion, Nio-EEP mediates as an anti-virulence agent to inhibit PLB1 and biofilm production for preventing fungal colonization on lung epithelial cells and also decreases the intracellular replication of phagocytosed cryptococci. This nano-based EEP delivery might be a potential therapeutic strategy in the prophylaxis and treatment of pulmonary cryptococcosis in the future.

Study on the Interaction of Algal Peptides on Virulence Factors of Helicobacter pylori: In Silico Approach.

In the Asian region, Helicobacter pylori infects about 80% populations, which is most leading cause of peptic ulcers, and it is an asymptomatic infection. Studies reported that the particular bacteria carry specific virulence factors that leads to severe complications. These virulence factors can be used as a drug targets to inhibit their growth and pathogenicity. Chronic infection with H. pylori virulence factors are CagA, VacA and HtrA positive strains the risk factor of gastric cancer. In this study, we aimed to study the antagonistic interaction pattern between the potential eight algal peptides against the virulence factors of H. pylori through in silico analysis intended to treat peptic ulcer and prevent the further complications such as cancer. The proteins of virulent factors are docked using C-Docker algorithm and calculated the bind energy of the complexes. The results showed that the peptide derived from a green alga, Tetradesmus sp. are active against the three virulent factors such as cag-A, vac-A, and Htr-A with multiple hydrogen, vdW, electrostatic interactions, and mild π-hydrophobic bindings with the libdock energy score for CagA, VacA and HtrA are 175.625, 158.603 and 89.397 kcal/mol. These primes and the peptide lead to develop a better and potential inhibitors against H. pylori infection.

Inhibiting Type VI Secretion System Activity with a Biomimetic Peptide Designed To Target the Baseplate Wedge Complex.

Human health is threatened by bacterial infections that are increasingly resistant to multiple drugs. A recently emerged strategy consists of disarming pathogenic bacteria by targeting and blocking their virulence factors. The type VI secretion system (T6SS) is a widespread secretion nanomachine encoded and employed by pathogenic strains to establish their virulence process during host invasion. Given the conservation of T6SS in several human bacterial pathogens, the discovery of an effective broad-spectrum T6SS virulence blocker represents an attractive target for development of antivirulence therapies. Here, we identified and validated a protein-protein interaction interface, TssK-TssG, as a key factor in the assembly of the T6SS baseplate (BP) complex in the pathogen enteroaggregative Escherichia coli (EAEC). In silico and biochemical studies revealed that the determinants of the interface are broadly conserved among pathogenic species, suggesting a role for this interface as a target for T6SS inhibition. Based on the high-resolution structure of the TssKFGE wedge complex, we rationally designed a biomimetic cyclic peptide (BCP) that blocks the assembly of the EAEC BP complex and inhibits the function of T6SS in bacterial cultures. Our BCP is the first compound completely designed from prior structural knowledge with anti-T6SS activity that can be used as a model to target human pathogens. IMPORTANCE New therapeutic options are urgently needed to fight drug-resistant and life-threatening infections. In contrast to antibiotics that inhibit the growth pathways of bacteria, the antivirulence strategy is a promising approach to disarm pathogens by interfering with bacterial virulence factors without exerting evolutionary pressure. The type VI secretion system (T6SS) is used by many pathogens, including members of the antibiotic-resistant ESKAPE bacteria (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.), to establish their virulence during the invasion of the human host. Although the T6SS is undoubtedly involved in pathogenesis, strategies targeting this virulence factor are crucially lacking. Here, we used a combination of genetics, microbiology, biochemical, biophysics, and bioinformatics approaches to rationally design a biomimetic peptide that interferes with T6SS assembly and functioning. This study represents a novel proof of concept for an antivirulence strategy which aims to interfere with the assembly of the T6SS.

New Agents in Development for Sepsis: Any Reason for Hope?

Sepsis is a syndrome which is defined as a dysregulated host response to infection leading to organ failure. Since it remains one of the leading causes of mortality worldwide, numerous drug candidates have already been tested, and continue to be developed, as potential adjunct therapies. Despite convincing mechanisms of action and robust pre-clinical data, almost all drug candidates in the field of sepsis have failed to demonstrate clinical efficacy in the past two decades. Accordingly, the development of new sepsis drugs has markedly decreased in the past few years. Nevertheless, thanks to a better understanding of sepsis pathophysiology and pathways, new promising drug candidates are currently being developed. Instead of a unique sepsis profile as initially suspected, various phenotypes have been characterised. This has  resulted in the identification of multiple targets for new drugs together with relevant biomarkers, and a better understanding of the most appropriate time to intervention. Within the entire sepsis drugs portfolio, those targeting the immune response are probably the most promising. Monoclonal antibodies targeting either cytokines or infectious agents are undoubtedly part of the potential successful therapeutic classes to come.

Targeting Bacterial Sortase A with Covalent Inhibitors: 27 New Starting Points for Structure-Based Hit-to-Lead Optimization.

Because of its essential role as a bacterial virulence factor, enzyme sortase A (SrtA) has become an attractive target for the development of new antivirulence drugs against Gram-positive infections. Here we describe 27 compounds identified as covalent inhibitors of Staphylococcus aureus SrtA by screening a library of approximately 50 000 compounds using a FRET assay followed by NMR-based validation and binding reversibility analysis. Nineteen of these compounds displayed only moderate to weak cytotoxicity, with CC50 against NIH 3T3 mice fibroblast cells ranging from 12 to 740 µM. Analysis using covalent docking suggests that the inhibitors initially associate via hydrophobic interactions, followed by covalent bond formation between the SrtA active site cysteine and an electrophilic center of the inhibitor. The compounds represent good starting points that have the potential to be developed into broad spectrum antivirulence agents as exemplified by hit-to-lead optimization of one of the compounds.

Inhibition of urease activity in the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis by dimethylsulfoxide (DMSO).

AIMS: Urease is a virulence factor for the urinary tract pathogens Staphylococcus saprophyticus and Proteus mirabilis. Dimethylsulfoxide (DMSO) is structurally similar to urea, used as a solvent for urease inhibitors, and an effective treatment for interstitial cystitis/bladder pain syndrome (IC/BPS). The aims of this study were to test DMSO as a urease inhibitor and determine its physiological effects on S. saprophyticus and P. mirabilis. METHODS AND RESULTS: Urease activity in extracts and whole cells was measured by the formation of ammonium ions. Urease was highly sensitive to noncompetitive inhibition by DMSO (Ki about 6 mmol l-1 ). DMSO inhibited urease activity in whole cells, limited bacterial growth in media containing urea, and slowed the increase in pH which occurred in artificial urine medium. CONCLUSIONS: DMSO should be used with caution as a solvent when testing plant extracts or other potential urease inhibitors. Because it can inhibit bacterial growth and delay an increase in pH, it may be an effective treatment for urinary tract infections. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first detailed study of the inhibition of urease by DMSO. Dimethylsulfoxide may be used to treat urinary tract infections that are resistant to antibiotics or herbal remedies.

Fosfomycin Protects Mice From Staphylococcus aureus Pneumonia Caused by α-Hemolysin in Extracellular Vesicles by Inhibiting MAPK-Regulated NLRP3 Inflammasomes.

α-Hemolysin (Hla) is a significant virulence factor in Staphylococcus aureus (S. aureus)-caused infectious diseases such as pneumonia. Thus, to prevent the production of Hla when treating S. aureus infection, it is necessary to choose an antibiotic with good antibacterial activity and effect. In our study, we observed that Fosfomycin (FOM) at a sub-inhibitory concentration inhibited expression of Hla. Molecular dynamics demonstrated that FOM bound to the binding sites LYS 154 and ASP 108 of Hla, potentially inhibiting Hla. Furthermore, we verified that staphylococcal membrane-derived vesicles (SMVs) contain Hla and that FOM treatment significantly reduced the production of SMVs and Hla. Based on our pharmacological inhibition analysis, ERK and p38 activated NLRP3 inflammasomes. Moreover, FOM inhibited expression of MAPKs and NLRP3 inflammasome-related proteins in S. aureus as well as SMV-infected human macrophages (MΦ) and alveolar epithelial cells. In vivo, SMVs isolated from S. aureus DU1090 (an isogenic Hla deletion mutant) or the strain itself caused weaker inflammation than that of its parent strain 8325-4. FOM also significantly reduced the phosphorylation levels of ERK and P38 and expression of NLRP3 inflammasome-related proteins. In addition, FOM decreased MPO activity, pulmonary vascular permeability and edema formation in the lungs of mice with S. aureus-caused pneumonia. Taken together, these data indicate that FOM exerts protective effects against S. aureus infection in vitro and in vivo by inhibiting Hla in SMVs and blocking ERK/P38-mediated NLRP3 inflammasome activation by Hla.

Recombinant N-acyl homoserine lactone-Lactonase AiiAQSI-1 Attenuates Aeromonas hydrophila Virulence Factors, Biofilm Formation and Reduces Mortality in Crucian Carp.

Quorum quenching (QQ) is a promising alternative infection-control strategy to antibiotics that controls quorum-regulated virulence without killing the pathogens. Aeromonas hydrophila is an opportunistic gram-negative pathogen living in freshwater and marine environments. A. hydrophila possesses an N-acyl homoserine lactone (AHL)-based quorum-sensing (QS) system that regulates virulence, so quorum signal-inactivation (i.e., QQ) may represent a new way to combat A. hydrophila infection. In this study, an AHL lactonase gene, aiiA was cloned from Bacillus sp. strain QSI-1 and expressed in Escherichia coli strain BL21(DE3). The A. hydrophila hexanoyl homoserine lactone (C6-HSL) QS signal molecule was degraded by AiiAQSI-1, which resulted in a decrease of bacterial swimming motility, reduction of extracellular protease and hemolysin virulence factors, and inhibited the biofilm formation of A. hydrophila YJ-1 in a microtiter assay. In cell culture studies, AiiAQSI-1 decreased the ability of A. hydrophila adherence to and internalization by Epithelioma papulosum cyprini (EPC) cells. During in vivo studies, oral administration of AiiAQSI-1 via feed supplementation attenuated A. hydrophila infection in Crucian Carp. Results from this work indicate that feed supplementation with AiiAQSI-1 protein has potential to control A. hydrophila aquaculture disease via QQ.

Inhibition of suilysin activity and inflammation by myricetin attenuates Streptococcus suis virulence.

Streptococcus suis (S. suis) is a gram-positive, zoonotic pathogenic bacterium that poses a serious threat to the pig industry and human health. This globally distributed pathogen can cause multiple diseases and fatal infections in both humans and animals. Suilysin (SLY) is an important extracellular secreted toxin regarded as an essential S. suis capsular type 2 (SS2) virulence factor and plays a key role in the infection and cytotoxicity of SS2. In addition, an excessive inflammatory response is also a serious hazard caused by SS2 infection. In this study, we demonstrated that the natural compound myricetin can inhibit the hemolytic activity of SLY and is effective at reducing the production of the inflammatory cytokines TNF-α and IL-1ß and reducing inflammation by downregulating the activation of P38. In addition, myricetin could effectively treat SS2 infections in vitro and in vivo. These findings may aid in the development of promising therapeutic candidates for treating SS2 infections.

Effect of Resveratrol on biofilm formation and virulence factor gene expression of Porphyromonas gingivalis in periodontal disease.

Periodontal disease is an oral inflammatory disease that destroys the tooth supporting periodontal tissues resulting in tooth loss. Porphyromonas gingivalis is a keystone pathogen that plays a significant role in periodontitis. In previous studies, resveratrol has shown significant results by targeting inflammatory and adhesive markers. Virulence factors of P. gingivalis play an important role in the bacterial adhesion and colonization. In this study, we aimed to demonstrate the anti-biofilm and anti-bacterial activity of resveratrol and also study the effect of resveratrol on the expression of virulence factor genes of P. gingivalis using reverse transcriptase polymerase chain reaction (RT-PCR). The anti-microbial and anti-biofilm activity of resveratrol on P. gingivalis was carried out by broth microdilution assay and biofilm adhesion reduction-crystal violet assay, respectively. We carried out the gene expression analysis by RT-PCR with the P. gingivalis treated compound to analyze the change in the expression of virulence factors: fimbriae and gingipain. Minimal inhibitory concentrations (MIC) of resveratrol against P. gingivalis and other clinical strains are in the range of 78.12-156.25 µg/mL. Resveratrol dose-dependently prevented the biofilm formation and also attenuated the virulence of P. gingivalis by reducing the expression of virulence factor genes such as fimbriae (type II and IV) and proteinases (kgp and rgpA). Resveratrol demonstrated superior anti-bacterial and anti-biofilm activity against P. gingivalis. There was significant reduction in the expression of fimbriae and gingipain with the resveratrol-treated compound. The results suggest that resveratrol, due to its multiple actions, may become a simple and inexpensive therapeutic strategy for treating periodontal disease.

Structural determinants of inhibition of Porphyromonas gingivalis gingipain K by KYT-36, a potent, selective, and bioavailable peptidase inhibitor.

Porphyromonas gingivalis is a member of the dysbiotic oral microbiome and a "keystone pathogen" that causes severe periodontal disease, which is among the most prevalent infectious diseases. Part of the virulence factors secreted by P. gingivalis are the essential cysteine peptidases gingipain K (Kgp) and R (RgpA and RgpB), which account for 85% of the extracellular proteolytic activity of the pathogen and are thus prime targets for inhibition. We report the high-resolution (1.20 Å) complex structure of Kgp with KYT-36, a peptide-derived, potent, bioavailable and highly selective inhibitor, which is widely used for studies in vitro, in cells and in vivo. Sub-nanomolar inhibition of Kgp is achieved by tight binding to the active-site cleft, which is covered for its sub-sites S3 through S1' under establishment of nine hydrophobic interactions, 14 hydrogen bonds and one salt bridge. In addition, an inhibitor carbonyl carbon that mimics the scissile carbonyl of substrates is pyramidalized and just 2.02 Å away from the catalytic nucleophile of Kgp, C477Sγ. Thus, the crystal structure emulates a reaction intermediate of the first nucleophilic attack during catalysis of cysteine peptidases. The present study sets the pace for the development of tailored next-generation drugs to tackle P. gingivalis.

Thiazole derivatives act on virulence factors of Cryptococcus spp.

Cryptococcosis is an opportunistic or primary fungal infection considered to be the most prevalent fatal fungal disease worldwide. Owing to the limited number of available drugs, it is necessary to search for novel antifungal compounds. In the present work, we assessed the antifungal efficacy of three thiazole derivatives (1, 2, and 3). We conducted in vitro and in vivo assays to investigate their effects on important virulence factors, such as capsule and biofilm formation. In addition, the phagocytosis index of murine macrophages exposed to compounds 1, 2, and 3 and the in vivo efficacy of 1, 2, and 3 in Galleria mellonella infected with Cryptococcus spp. were evaluated. All compounds exhibited antifungal activity against biofilms and demonstrated a reduction in biofilm metabolic activity by 43-50% for C. gattii and 26-42% for C. neoformans. Thiazole compounds promoted significant changes in the capsule thickness of C. gattii compared to that of C. neoformans. Further examination of these compounds suggests that they can improve the phagocytosis process of peritoneal murine macrophages in vitro, causing an increase in the phagocytosis rate. Survival percentage was examined in the invertebrate model Galleria mellonella larvae, and only compound 3 could increase the survival at doses of 5 mg/kg after infection with C. gattii (P = .0001) and C. neoformans (P = .0007), similar to fluconazole at 10 mg/kg. The results demonstrated that thiazole compounds, mainly compound 3, have potential to be used for future studies in the search for new therapeutics for cryptococcosis.

Identification of FDA-Approved Drugs as Antivirulence Agents Targeting the pqs Quorum-Sensing System of Pseudomonas aeruginosa.

The long-term use of antibiotics has led to the emergence of multidrug-resistant bacteria. A promising strategy to combat bacterial infections aims at hampering their adaptability to the host environment without affecting growth. In this context, the intercellular communication system quorum sensing (QS), which controls virulence factor production and biofilm formation in diverse human pathogens, is considered an ideal target. Here, we describe the identification of new inhibitors of the pqs QS system of the human pathogen Pseudomonas aeruginosa by screening a library of 1,600 U.S. Food and Drug Administration-approved drugs. Phenotypic characterization of ad hoc engineered strains and in silico molecular docking demonstrated that the antifungal drugs clotrimazole and miconazole, as well as an antibacterial compound active against Gram-positive pathogens, clofoctol, inhibit the pqs system, probably by targeting the transcriptional regulator PqsR. The most active inhibitor, clofoctol, specifically inhibited the expression of pqs-controlled virulence traits in P. aeruginosa, such as pyocyanin production, swarming motility, biofilm formation, and expression of genes involved in siderophore production. Moreover, clofoctol protected Galleria mellonella larvae from P. aeruginosa infection and inhibited the pqs QS system in P. aeruginosa isolates from cystic fibrosis patients. Notably, clofoctol is already approved for clinical treatment of pulmonary infections caused by Gram-positive bacterial pathogens; hence, this drug has considerable clinical potential as an antivirulence agent for the treatment of P. aeruginosa lung infections.

The Eukaryotic Host Factor 14-3-3 Inactivates Adenylate Cyclase Toxins of Bordetella bronchiseptica and B. parapertussis, but Not B. pertussis.

Bordetella pertussis, Bordetella bronchiseptica, and Bordetella parapertussis share highly homologous virulence factors and commonly cause respiratory infections in mammals; however, their host specificities and disease severities differ, and the reasons for this remain largely unknown. Adenylate cyclase toxin (CyaA) is a homologous virulence factor that is thought to play crucial roles in Bordetella infections. We herein demonstrate that CyaAs function as virulence factors differently between B. bronchiseptica/B. parapertussis and B. pertussisBbronchiseptica CyaA bound to target cells, and its enzyme domain was translocated into the cytosol similarly to Bpertussis CyaA. The hemolytic activity of Bbronchiseptica CyaA on sheep erythrocytes was also preserved. However, in nucleated target cells, Bbronchiseptica CyaA was phosphorylated at Ser375, which constitutes a motif (RSXpSXP [pS is phosphoserine]) recognized by the host factor 14-3-3, resulting in the abrogation of adenylate cyclase activity. Consequently, the cytotoxic effects of Bbronchiseptica CyaA based on its enzyme activity were markedly attenuated. Bparapertussis CyaA carries the 14-3-3 motif, indicating that its intracellular enzyme activity is abrogated similarly to Bbronchiseptica CyaA; however, Bpertussis CyaA has Phe375 instead of Ser, and thus, was not affected by 14-3-3. In addition, Bpertussis CyaA impaired the barrier function of epithelial cells, whereas Bbronchiseptica CyaA did not. Rat infection experiments suggested that functional differences in CyaA are related to differences in pathogenicity between B. bronchiseptica/Bparapertussis and B. pertussisIMPORTANCEBordetella pertussis, B. bronchiseptica, and B. parapertussis are bacterial respiratory pathogens that are genetically close to each other and produce many homologous virulence factors; however, their host specificities and disease severities differ, and the reasons for this remain unknown. Previous studies attempted to explain these differences by the distinct virulence factors produced by each Bordetella species. In contrast, we indicated functional differences in adenylate cyclase toxin, a homologous virulence factor of Bordetella The toxins of B. bronchiseptica and presumably B. parapertussis were inactivated by the host factor 14-3-3 after phosphorylation in target cells, whereas the B. pertussis toxin was not inactivated because of the lack of the phosphorylation site. This is the first study to show that 14-3-3 inactivates the virulence factors of pathogens. The present results suggest that pathogenic differences in Bordetella are attributed to the different activities of adenylate cyclase toxins.

Inhibition of Biofilm and Virulence Factors of Candida albicans by Partially Purified Secondary Metabolites of Streptomyces chrestomyceticus Strain ADP4.

BACKGROUND: Despite several advancements in antifungal drug discovery, fungal diseases like Invasive Candidiasis (IC) still remain associated with high rates of morbidity and mortality worldwide. Thus there is an enormous need for anti-Candida drugs. OBJECTIVE: The main objectives of the work included: 1. To investigate therapeutically significant classes of secondary metabolites produced by S. chrestomyceticus strain ADP4. 2. To investigate and analyze inhibition of significant virulence attributes of C. albicans, such as, biofilm and secretory hydrolytic enzymes by ADP4 secondary metabolites. 3. Mechanistic analysis of probable compounds for their site of action on Secretary Aspartyl Proteinase 3 (Sap3). METHODS: Metabolite extract-SDB (MESDB) of S. chrestomyceticus strain ADP4 was fractionated on silica gel column chromatography. Fractions were analyzed for anti-Candida activity by disc diffusion assay. Active fractions were further purified by differential solvent treatment. MIC90 values were determined by broth dilution method. MFC was based on counting viable cells. Inhibition of yeast to hyphae transition and that of production of hydrolytic enzymes were estimated by plate assays. GC-MS of MESDB and Partially Purified Metabolite preparations (PPMs) was done. GRIP docking studies with Sap 3 of C. albicans was done using VLife MDS 4.6 software. RESULTS: Chemical profiling showed that ADP4 secondary metabolites contained alkaloids, flavonoids, polyphenols, terpenoids and triterpenes. The MESDB and the PPMs showed low or no cytotoxicity but were able to effectively contain virulence attributes of Candida pathogen. Docking studies revealed that some of the probable compounds have affinity for aspartic acid residue in Sap3 enzyme of C. albicans. CONCLUSION: Secondary metabolite of strain ADP4 included important classes of therapeutically important compounds. Their anti-Candida activity was mediated by inhibition of critical virulence factors of the pathogen.

The structure of urease inactivated by Ag(i): a new paradigm for enzyme inhibition by heavy metals.

The nickel-dependent enzyme urease is a virulence factor for a large number of human pathogens, as well as a negative element for the efficiency of soil nitrogen fertilization for crop production. The use of urease inhibitors to contrast these effects requires the knowledge, at the molecular level, of their mode of action. Among these, silver is an efficient antimicrobial agent and an established inhibitor of this enzyme. The 1.91 Å resolution structure of Sporosarcina pasteurii urease inhibited by silver reveals the presence of two Ag(i) ions bound to the largely conserved triad αCys322/αHis323/αMet367: the first two residues are located on the mobile flap that is essential in modulating the size of the active site cavity and the position of key residues for enzyme catalysis, while αMet367 is on a loop facing the flap at the entrance of the active site cavity. The two Ag(i) ions are bridged by the thiolate Sγ atom of αCys322, and are coordinated, respectively, to the Nδ1 atom of the αHis323 imidazole ring and to the Sδ of αMet367. The binding of the Ag(i) ions at the edge of the active site channel supposedly blocks the movement of the flap, inhibiting the catalytic activity of urease. The structure of the silver-inhibited urease allows us to understand and rationalise all previously acquired kinetic and calorimetric data on this phenomenon, but also provides the details of how silver can exert its antimicrobial action with respect to ureolytic bacteria, a step forward against antibiotic-resistant pathogens.

Receptor-Based Peptides for Inhibition of Leukotoxin Activity.

The Gram-negative bacterium Aggregatibacter actinomycetemcomitans, commonly associated with localized aggressive periodontitis (LAP), secretes an RTX (repeats-in-toxin) protein leukotoxin (LtxA) that targets human white blood cells, an interaction that is driven by its recognition of the lymphocyte function-associated antigen-1 (LFA-1) integrin. In this study, we report on the inhibition of LtxA-LFA-1 binding as an antivirulence strategy to inhibit LtxA-mediated cytotoxicity. Specifically, we designed and synthesized peptides corresponding to the reported LtxA binding domain on LFA-1 and characterized their capability to inhibit LtxA binding to LFA-1 and subsequent cytotoxic activity in human immune cells. We found that several of these peptides, corresponding to sequential ß-strands in the LtxA-binding domain of LFA-1, inhibit LtxA activity, demonstrating the effectiveness of this approach. Further investigations into the mechanism by which these peptides inhibit LtxA binding to LFA-1 reveal a correlation between toxin-peptide affinity and LtxA-mediated cytotoxicity, leading to a diminished association between LtxA and LFA-1 on the cell membrane. Our results demonstrate the possibility of using target-based peptides to inhibit LtxA activity, and we expect that a similar approach could be used to hinder the activity of other RTX toxins.

Thrombospondin-1 protects against pathogen-induced lung injury by limiting extracellular matrix proteolysis.

Acute lung injury is characterized by excessive extracellular matrix proteolysis and neutrophilic inflammation. A major risk factor for lung injury is bacterial pneumonia. However, host factors that protect against pathogen-induced and host-sustained proteolytic injury following infection are poorly understood. Pseudomonas aeruginosa (PA) is a major cause of nosocomial pneumonia and secretes proteases to amplify tissue injury. We show that thrombospondin-1 (TSP-1), a matricellular glycoprotein released during inflammation, dose-dependently inhibits PA metalloendoprotease LasB, a virulence factor. TSP-1-deficient (Thbs1-/-) mice show reduced survival, impaired host defense, and increased lung permeability with exaggerated neutrophil activation following acute intrapulmonary PA infection. Administration of TSP-1 from platelets corrects the impaired host defense and aberrant injury in Thbs1-/- mice. Although TSP-1 is cleaved into 2 fragments by PA, TSP-1 substantially inhibits Pseudomonas elastolytic activity. Administration of LasB inhibitor, genetic disabling of the PA type II secretion system, or functional deletion of LasB improves host defense and neutrophilic inflammation in mice. Moreover, TSP-1 provides an additional line of defense by directly subduing host-derived proteolysis, with dose-dependent inhibition of neutrophil elastase from airway neutrophils of mechanically ventilated critically ill patients. Thus, a host matricellular protein provides dual levels of protection against pathogen-initiated and host-sustained proteolytic injury following microbial trigger.