LasA Protease Research Articles

Regulation of TCA cycle genes by srbA sRNA: Impacts on Pseudomonas aeruginosa virulence and survival

Pseudomonas aeruginosa, an opportunistic bacterial pathogen of public health concern, is known for its metabolic versatility, adaptability in harsh environment, and pathogenic aggressiveness. P. aeruginosa relies on various regulatory networks modulated by small non-coding RNAs, which in turn influence different physiological traits such as metabolism, stress response, and pathogenesis. In this study, srbA sRNA has been shown to play a diverse role in regulating cellular metabolism and the production of different virulence factors in P. aeruginosa. srbA was found to control the TCA cycle, a key regulatory pathway for cellular metabolism and energy production, by regulating three main enzymes: citrate synthase (gltA), isocitrate dehydrogenase (icd), and α-ketoglutarate dehydrogenase E1 subunit (sucA) at both the transcriptional and translational levels. By modulating the TCA cycle, srbA could help the bacteria to adapt nutritional stress by lowering energy consumption. Additionally, srbA has been found to differentially regulate production of various virulence factors such as rhamnolipid, elastase, LasA protease, and pyocyanin under both nutrient-rich and nutrient-limiting conditions. It could also influence motilities in P. aeruginosa, linked to biofilm formation and pathogenicity. Thus, srbA might hold a promise in the research area for identifying virulence pathways and developing novel therapeutic targets to combat the global pathogenic threat of P. aeruginosa.

Attenuation of Quorum Sensing Mediated Virulence Factors and Biofilm Formation in Pseudomonas Aeruginosa PAO1 by Substituted Chalcones and Flavonols.

Flavonoids epitomize structural scaffolds in many biologically active synthetic and natural compounds. They showcase a diverse spectrum of biological activities including anticancer, antidiabetic, antituberculosis, antimalarial, and antibiofilm activities. The antibiofilm activity of a series of new chalcones and flavonols against clinically significant Pseudomonas aeruginosa PAO1 strain was studied. Antivirulence activities were screened by analysing the effect of compounds on the production of virulence factors like pyocyanin, LasA protease, cell surface hydrophobicity, and rhamnolipid. The best ligands towards the quorum sensing proteins LasR, RhlR, and PqsR were recognised using a molecular docking study. The gene expression in P. aeruginosa after treatment with test compounds was evaluated on quorum sensing genes including rhlA, lasB, and pqsE. The antibiofilm potential of chalcones and flavonols was confirmed by the efficient reduction in the production of virulence factors and downregulation of gene expression.

Assessing the antibiofilm activity of flavonol esters against Pseudomonas aeruginosa PAO1 biofilm: an in vitro, molecular docking, and molecular dynamics study

Pseudomonas aeruginosa is one of the opportunistic pathogens that may cause serious health problems and can produce several virulence factors, which are responsible for various infections, particularly in immunocompromised patients. They are responsible for producing infections on indwelling medical devices by attaching on to them and forming a biofilm. Antibiofilm, antivirulence, and gene expression studies of P. aeruginosa biofilm treated with esters of flavonols were evaluated. Pyocyanin, cell surface hydrophobicity, LasA protease estimation, rhamnolipid estimation, and pyoverdine estimation were performed to evaluate the antivirulence activities of the test compounds against P. aeruginosa. Previous studies on the antivirulence activity of flavonoids against P. aeruginosa demonstrate that even if they can inhibit bacterial growth, relatively high concentrations of the compound are generally required for the inhibition of virulence factors. The esters showed more than 40% inhibition in all the tested virulence factors at their sub minimum inhibitory concentration. The gene expression studies of selected esters toward lasB and rhlA genes show downregulation of rhlA which suggests the inhibition in biofilm formation through rhamnolipid inhibition, quorum sensing inhibition, or biofilm formation inhibition. Communicated by Ramaswamy H. Sarma

Biosurfactant derived from probiotic Lactobacillus acidophilus exhibits broad-spectrum antibiofilm activity and inhibits the quorum sensing-regulated virulence.

Antimicrobial resistance by pathogenic bacteria has become a global risk to human health in recent years. The most promising approach to combating antimicrobial resistance is to target virulent traits of bacteria. In the present study, a biosurfactant derived from the probiotic strain Lactobacillus acidophiluswas tested against three Gram-negative bacteria to evaluate its inhibitory potential on their biofilms, and whether it affected the virulence factors controlled by quorum sensing (QS). A reduction in the virulence factors of Chromobacterium violaceum (violacein production), Serratia marcescens (prodigiosin production) and Pseudomonas aeruginosa (pyocyanin, total protease, LasB elastase and LasA protease production) was observed at different sub-MIC concentrations in a dose-dependent manner. Biofilm development was reduced by 65.76%, 70.64% and 58.12% at the highest sub-MIC levels for C. violaceum, P. aeruginosa and S. marcescens, respectively. Biofilm formation on glass surfaces exhibited significant reduction, with less bacterial aggregation and reduced formation of extracellular polymeric materials. Additionally, swimming motility and exopolysaccharides (EPS) production were shown to be reduced in the presence of the L. acidophilus-derived biosurfactant. Furthermore, molecular docking analysis performed on compounds identified through gas chromatography-mass spectrometry (GC-MS) analysis of QS and biofilm proteins yielded further insights into the mechanism underlying the anti-QS activity. Therefore, the present study has clearly demonstrated that a biosurfactant derived from L. acidophilus can significantly inhibit virulence factors of Gram-negative pathogenic bacteria. This could provide an effective method to inhibit the formation of biofilms and QS in Gram-negative bacteria.

Biosynthesized Silver Nanoparticles Derived from Probiotic Lactobacillus rhamnosus (AgNPs-LR) Targeting Biofilm Formation and Quorum Sensing-Mediated Virulence Factors.

In recent years, bacterial pathogens have developed resistance to antimicrobial agents that have created a global threat to human health and environment. As a novel approach to combating antimicrobial resistance (AMR), targeting bacteria's virulent traits that can be explained by quorum sensing (QS) is considered to be one of the most promising approaches. In the present study, biologically synthesized silver nanoparticles derived from Lactobacillus rhamnosus (AgNPs-LR) were tested against three Gram-negative bacteria to determine whether they inhibited the formation of biofilms and triggered the virulence factors controlled by QS. In C. violaceum and S. marcescens, a remarkable inhibition (>70%) of QS-mediated violacein and prodigiosin production was recorded, respectively. A dose-dependent decrease in virulence factors of P. aeruginosa (pyocyanin, pyoverdine, LasA protease, LasB elastase and rhamnolipid production) was also observed with AgNPs-LR. The biofilm development was reduced by 72.56%, 61.70%, and 64.66% at highest sub-MIC for C. violaceum, S. marcescens and P. aeruginosa, respectively. Observations on glass surfaces have shown remarkable reductions in biofilm formation, with less aggregation of bacteria and a reduced amount of extra polymeric materials being formed from the bacteria. Moreover, swimming motility and exopolysaccharides (EPS) was also found to reduce in the presence of AgNPs-LR. Therefore, these results clearly demonstrate that AgNPs-LR is highly effective in inhibiting the development of biofilms and the QS-mediated virulent traits of Gram-negative bacteria. In the future, AgNPs-LR may be used as an alternative to conventional antibiotics for the treatment of bacterial infections after careful evaluation in animal models, especially for the development of topical antimicrobial agents.

Interspecies Interaction between Pseudomonas aeruginosa, Staphylococcus aureus and E. coli in vitro

Microbial interactions are frequently categorized according to how they affect each population in a binary system. We aimed to determine the interaction between P . aeruginosa, S . aureus, and E . coli in-vitro. In this experimental hospitalized patients’ sputum, urine, and blood samples were used to collect a total of 90 clinical isolates for the study in Damanhour Medical National Institute, Behira, Egypt, followed by accurate identification and testing for antibiotic sensitivity. To examine the effect of the supernatant of P. aeruginosa on S. aureus and E. coli determined MIC using broth microdilution method. We also measured the activity of lasA protease by assessing the S. aureus cell lysis potential of P. aeruginosa culture supernatants. Extraction of pyocyanin was made to determine the change in the cell nature of S. aureus upon exposure to pyocyanin by using a scanning electron microscope and the shape of colonies on the culture media was determined. Finally, we detect lasA, operon phz, phzM, phzS and rhlAB genes for P. aeruginosa. P. aeruginosa showed a great impact on S. aureus isolates resistant to different antibiotics as it facilitates their killing and may drive the normal colonies of S. aureus into SCVs. The ability to form biofilm by S. aureus and E. coli decreased in the presence of Pseudomonas supernatant.

Novel Vanillin-based hybrids inhibit quorum sensing and silences phenotypical expressions in Pseudomonas aeruginosa.

In this study, we report the chemical synthesis, computational analysis, and anti-virulent studies of five Vanillin-based hybrids employing phytochemicals. Vanillin (V) is known to have substantial anti-quorum sensing activity against the gram-negative pathogen Pseudomonas aeruginosa. Therefore, with the aim to further enhance the potency of Vanillin, it was chemically conjugated via a triazole (T) linker with five phytochemicals- Zingerone (Z), Eugenol (E), Guaiacol (G), Cinnamaldehyde (C), and Ferulic acid (F) to form the hybrids named as VTZ (1), VTE (2), VTG (3), VTC (4), and VTF (5), respectively. Molecular docking studies revealed the strong binding affinity of the designed hybrids with quorum-sensing receptors (LasR, Rh1R, and PqsR). The synthesized hybrids were also evaluated for anti-quorum sensing activities to examine the efficacy against P. aeruginosa bacterial strains PAO1. The hybrids VTE (2), VTG (3), and VTC (4) displayed improved anti-quorum activity relative to Vanillin. Furthermore, the attenuation of virulence factors of P. aeruginosa (Las-A protease, Las-B elastase, pyocyanin pigmentation, and motility) in the presence of VTE (2), VTG (3), and VTC (4) further authenticated the anti-virulent activity of the hybrids. The new design strategy of the phytochemical-phytochemical scaffolds and their biological evaluation provides a proof of concept for the simultaneous perturbation of well-established anti-virulent targets. This appears to be highly promising and effective strategy to ameliorate the enigma of antimicrobial resistance.

Evaluation of kinetic stability and anti-staphylococcal activity of recombinant LasA protein produced in Escherichia coli.

Objective(s):Staphylococcus aureus has become a major clinical concern due to the growing prevalence of multi-drug resistant (MDR) strains. Enzybioticts are peptidoglycan hydrolases that are recently introduced as an alternative agent to confront the MDR strains with a more effective mechanism than conventional antibiotics. In this regard, our study aimed to evaluate the kinetic stability of LasA protease as a potent enzybiotic in the specific destruction of the S. aureus cell wall. Materials and Methods:The catalytic domain of the Codon-optimized LasA gene was sub-cloned into pET28a vector, and BL21 DE3 cells were used for protein expression. Recombinant LasA protein was affinity purified by Ni-NTA column and staphylolytic activity of the LasA protein against methicillin-resistant strains was evaluated by disk diffusion and MIC test. The kinetic stability was evaluated in different temperatures during 48 hr. Results:Our results revealed that LasA protein can completely prevent the growth of Methicillin-resistant S. aureus (MRSA) strain and inhibit the examined strain at the amount of 4 µg. furthermore, the catalytic domain of LasA protein can tolerate higher temperatures as well. Conclusion:With regard to the failure of conventional antibiotics in treatment of MRSA infections, novel agents to combat multidrug-resistant strains are needed. The present study shows that LasA protein can eradicate MRSA strains, so it can be promising for the treatment of antibiotic-resistant staphylococci infection. The kinetic stability of LasA has also confirmed the possibility of industrial-scale manufacturing for the subsequent use of the enzyme clinically.

Neutrophil extracellular traps in the fight against biofilm-forming microorganisms: hunters or prey?

The review presents up-to-date data on the relationships between neutrophil extracellular traps (NETs) and biofilm-forming microorganisms P aeruginosa, S. aureus, Candida spp. obtained in vitro and in vivo studies. Up to 80% of human microbial infections are associated with biofilm-forming microorganisms. The formation of highly specialized biofilm communities is one of the main strategies for the survival of bacteria and fungi, significantly increasing their tolerance to aggressive and stressful environmental conditions, chemotherapeutic drugs, and immune system factors, contributing to their persistence and chronicity of the infectious process. The formation of NETs in the process of NETosis is one of the biological mechanisms used by neutrophils in protection against pathogens. Chemoattractants of biofilm origin, as well as those secreted by epithelial and immunocompetent cells, attract and activate migrating neutrophils. However, given that bacteria form fairly large cell clusters and aggregates in biofilms, the process of phagocytosis is sometimes difficult or impossible. Under these conditions, it is logical to assume that the importance of NETs in anti-biofilm immunity increases. However, due to the components of the extracellular biofilm matrix (e.g., Psl exopolysaccharide P aeruginosa), quorum sensing (QS) molecules (e.g., LasR QS system P aeruginosa), enzymes (e.g., LasA protease and LasB elastase P. aeruginosa), toxins (e.g., Panton-Valentine leukocidin and AB Y-hemolysin S. aureus) and probably other factors yet to be studied, the microorganisms in biofilms are able to influence the signaling systems involved in NETosis, the intensity of the formation of NETs, the sequestration and killing mechanisms in them, sometimes subordinating and using NETs components for their own purposes.

Iron facilitates the RetS-Gac-Rsm cascade to inversely regulate protease IV (piv) expression via the sigma factor PvdS in Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces several proteases, such as an elastase (LasB protease), a LasA protease, and protease IV (PIV), which are thought as significant virulence factors during infection. Regulators of LasA and LasB expression have been identified and well characterized; however, the molecular details of this regulation of protease IV (PIV) remained largely unknown. Here, we describe the interaction between protease IV and the RetS/Rsm signalling pathway, which plays a central role in controlling the production of multiple virulence factors and the switch from planktonic to biofilm lifestyle. We show that the expression of piv was reduced in ΔretS or ΔrsmA strain grown under restrictive conditions but was induced in ΔretS or ΔrsmA mutant grown under rich conditions as compared with wild-type parent. We compare the expression of piv under various conditions and found that iron facilitates RetS/Rsm system to lead this inverse regulation. Moreover, we reveal that the RetS/Rsm pathway regulates PIV production dependent on the alternative sigma factor PvdS. Collectively, this study extends the understanding of the RetS/Rsm regulatory cascade in response to environmental signals and provides insights into how P. aeruginosa adapts to the complex conditions.

Characterisation of the antimicrobial mode of action of gallium maltolate

To human health worldwide. Existing treatments are becoming inefficacious and therefore there is an urgent need for the development of treatments with alternative modes of action. The use of gallium as an antimicrobial agent has been of interest due to its unconventional mode of action involving the inhibition of iron acquisition and metabolism. The structural similarity and inability to reduce from a trivalent to divalent form under normal physiological conditions allows gallium to act as an iron mimetic and inhibit many iron-dependent biological pathways, respectively. The antimicrobial potential of gallium maltolate (GaM), Ga(III) coordination complex of maltol, was investigated on the opportunistic pathogen Pseudomonas aeruginosa. In vitro and in vivo analyses using Galleria mellonella (greater wax moth) larvae demonstrated the potent bacteriostatic and non-toxic effect of the complex. Subsequent analysis of GaM treated P. aeruginosa via label-free quantitative proteomics provided an insight into the intrinsic mechanisms of action of GaM. Increased expression of iron-storage protein Bacterioferritin B, the HemO component of iron-sulfur clusters and several stress response proteins (Chaperone Proteins ClpB, HtpG and DnaJ) indicate cell stress in response to inhibited iron uptake. Decreased expression of LasA Protease and LasB Elastase quorum-sensing proteins and flagellar motility proteins FlgM and FlgG further demonstrate the growth inhibitory effect of GaM. These findings provide a basis for a better understanding of the mode of action of GaM, a requirement for the improvement of synthesis and efficacy of the treatment.

Coordination of las regulated virulence factors with Multidrug-Resistant and extensively drug-resistant in superbug strains of P. aeruginosa.

Successful pathogenicity often resulted from a complicated association between virulence and antibiotic resistance in Pseudomonas aeruginosa infections. Therefore, the current study aimed to investigate the relationship between the las system and antibiotic resistance. Seventy-three (73) P. aeruginosa isolates were collected from burn wounds (26.02%), blood cultures (30.13%), catheters (12.32%), and urine culture (31.50%). Among the 73 collected isolates, 22 isolates were considered as multi-drug resistant (MDR) and 11 isolates as extensively-drug resistant (XDR). Furthermore, phenazines and LasA protease were detected among 21.91% and 32.87% of isolates, respectively. Quantitative real-time PCR assessment of KPC, MBL, and lasI/R indicated that resistance and virulence factors are more expressed in XDR strains than MDR strains. Also, the expression level of KPC and MBL reduced in non-biofilm forming strains. However, increased expression levels of lasI, lasR, and the KPC genes were observed in LasA and LasB protease producing strains. Interestingly, 16 known sequence types (including ST108, ST260, ST217) and three novel STs (ST2452, ST2427, and ST2542) were characterized among the collected isolates, which are related to the virulence and resistance. In MDR-XDR strains, a strong correlation between lasI/R and the variants of antibiotic resistance genes was found. In conclusion, the pathogenicity of P. aeruginosa may increase the prevalence of antibiotic-resistant strains.

Musa acuminata and its bioactive metabolite 5-Hydroxymethylfurfural mitigates quorum sensing (las and rhl) mediated biofilm and virulence production of nosocomial pathogen Pseudomonas aeruginosa in vitro.

Musa acuminata, a tropical plant belongs to the family Musaceae. The fruit peels of this plant have been well documented for their therapeutic value in Asia and Africa. It has also been previously reported for numerous biological applications such as antimicrobial, antioxidant, itching, psoriasis and anti-diarrheal activities. Moreover, M. acuminata peels have been well known for its anti-healing and antiseptic properties and most commonly used for healing wounds and heat burns in South Asian and African traditional medicines. To evaluate the QS-mediated antibiofilm and antivirulence potential of M. acuminata, and its bioactive metabolites 5-Hydroxymethylfurfural (5HMF) against Pseudomonas aeruginosa. The M. acuminata peel methanol extract (MAM) was evaluated for its antibiofilm potential against P. aeruginosa with increasing concentration. Besides, biofilm related phenomenon's such as total biofilm proteins, microcolony formation exopolysaccharides (EPS) and cell surface hydrophobicity (CSH) productions were also examined to support the antibiofilm potential of MAM. Further, MAM was evaluated for its antivirulence efficacy against P. aeruginosa by assessing the protease, LasA protease, LasB elastase, pyocyanin, alginate and rhamnolipid productions at 400 μg ml-1 concentration. Transcriptional analysis of QS regulated virulence genes expression level was also done by real-time PCR analysis. Then, the MAM was subjected to column chromatography for further fractions and the bioactive compounds present in MAM were identified by gas chromatograph-mass spectrometry analysis. Further, the major compounds such as 5-hydroxymethylfurfural, vaccenic acid and pentanoic acid identified from active fraction of MAM were evaluated for their antibiofilm and antivirulence potential against P. aeruginosa. MAM significantly inhibited the biofilm formation in P. aeruginosa at 400 μg ml-1 concentration which also inhibited the production of biofilm proteins, biofilm adherence, EPS and CSH productions to the level of 79%, 82% and 77% respectively. Further, the antivirulence potential was confirmed through numerous virulence inhibition assays. The MAM at 400 μg ml-1 concentration inhibited the QS-mediated virulence production such as protease, LasA protease, LasB elastase, pyocyanin, alginate and rhamnolipid productions to the level of 77%, 75%, 68%, 80%, 78% and 69% respectively. Moreover, the results of qPCR analysis confirmed the downregulation of QS regulated virulence genes expression upon treatment with MAM. The chromatographic analysis revealed the presence of 5-Hydroxymethylfurfural (5HMF), vaccenic acid and pentanoic acid in MAM and the potential bioactive compounds with antibiofilm and antivirulence was identified as 5-hydroxymethylfurfural, without exerting any growth inhibition in P. aeruginosa. This study investigated the ideal antibiofilm and antivirulence potential of MAM and its bioactive compound 5HMF, and confirms the ethnopharmacological value of these peels against P. aeruginosa infections.

Regulation of Pseudomonas aeruginosa-Mediated Neutrophil Extracellular Traps.

Pseudomonas aeruginosa is the most prevalent opportunistic pathogen in the airways of cystic fibrosis (CF) patients. The pulmonary disorder is characterized by recurrent microbial infections and an exaggerated host inflammatory immune response led primarily by influx of neutrophils. Under these conditions, chronic colonization with P. aeruginosa is associated with diminished pulmonary function and increased morbidity and mortality. P. aeruginosa has a wide array of genetic mechanisms that facilitate its persistent colonization of the airway despite extensive innate host immune responses. Loss of function mutations in the quorum sensing regulatory gene lasR have been shown to confer survival advantage and a more pathogenic character to P. aeruginosa in CF patients. However, the strategies used by LasR-deficient P. aeruginosa to modulate neutrophil-mediated bactericidal functions are unknown. We sought to understand the role of LasR in P. aeruginosa-mediated neutrophil extracellular trap (NET) formation, an important anti-microbial mechanism deployed by neutrophils, the first-line responder in the infected airway. We observe mechanistic and phenotypic differences between NETs triggered by LasR-sufficient and LasR-deficient P. aeruginosa strains. We uncover that LasR-deficient P. aeruginosa strains fail to induce robust NET formation in both human and murine neutrophils, independently of bacterial motility or LPS expression. LasR does not mediate NET release via downstream quorum sensing signaling pathways but rather via transcriptional regulation of virulence factors, including, but not restricted to, LasB elastase and LasA protease. Finally, our studies uncover the differential requirements for NADPH oxidase in NET formation triggered by different P. aeruginosa strains.

Potassium 2-methoxy-4-vinylphenolate: a novel hit exhibiting quorum-sensing inhibition in Pseudomonas aeruginosa via LasIR/RhlIR circuitry.

The emergence of multidrug-resistant (MDR) bacterial strains in the last decade is astonishingly alarming. Many of the widely used antibiotics have failed to exhibit clinical efficacy against such strains. Eventually we will exhaust all the resources in our antibiotic armamentarium. As a need of the hour, novel strategies are desperately required not only to curb, but also to reverse, the development of resistance in these pathogens, thereby maintaining their sensitivity towards current antibiotics. Intervention of bacterial virulence, rather than killing them, by inhibiting specific pathways/targets has emerged as a novel approach to tackle the drug resistance problem. The bacterial virulence is regulated via quorum-sensing, a cell–cell communication process precisely controlled by autoinducer molecules such as acyl homoserine lactone (AHL). The present study aimed at identifying promising quorum-sensing inhibitors in Pseudomonas aeruginosa, an opportunistic human pathogen especially associated with nosocomial infections, yielding four potential hits. Out of these, potassium 2-methoxy-4-vinylphenolate was the most potent quorum-sensing inhibitor targeting P. aeruginosa LasIR/RhlIR circuitry. It also inhibited biofilm formation, various virulence factors like LasA protease, LasB elastase and pyocyanin, and motility of bacteria like swarming and twitching.

Anti-quorum sensing and antibiofilm potential of Alternaria alternata, a foliar endophyte of Carica papaya, evidenced by QS assays and in-silico analysis

In the present study, secondary metabolites from an endophytic fungus, Alternaria alternata, colonizing Carica papaya, demonstrated antiquorum sensing properties against Pseudomonas aeruginosa. This study reports the antagonistic effects of fungal crude extract of A. alternata against the various quorum sensing (QS) associated virulent factors such as percentage decrease in production of pyocyanin, alginate, chitinase and rhamnolipid; significant decrease in proteases activity such as LasA protease activity, staphylolytic activity, Las B elastase; and a marked decrease in biofilm formation and associated factors such as exopolysaccharide (EPS) production and cell surface hydrophobicity (CSH). Further, motility pattern i.e., swimming and swarming was also found to be inhibited. This down regulation of QS and associated factors are further supported by in-silico analysis of interaction between QS receptor LasR and bioactive molecules viz., sulfurous acid, 2-propyl tridecyl ester and 1,2-benzenedicarboxylic acid, bis(2-methylpropyl) ester present in fungal crude extract, found based on GCMS analysis, sketches the modulating ability of QS expression. This is the first report on an endophytic fungus of C. papaya having a role in QS inhibition against P. aeruginosa and lays a platform to explore further the endophytes for potent therapeutic agents in QS.

Aspergillus ochraceopetaliformis SSP13 modulates quorum sensing regulated virulence and biofilm formation in Pseudomonas aeruginosa PAO1

Pseudomonas aeruginosa is an opportunistic nosocomial pathogen causing the majority of acute and persistent infections in human beings. The ability to form biofilm adds a new dimension to its resistance to conventional therapeutic agents. In the present study, down-regulation of quorum sensing regulated virulence and biofilm development resulting from exposure to Aspergillus ochraceopetaliformis SSP13 extract was investigated. The in vitro results inferred impairment in the production of LasA protease, LasB elastase, chitinase, pyocyanin, exopolysaccharides and rhamnolipids. In addition, motility and biofilm formation by P. aeruginosa PAO1 was significantly altered. The in vitro results were further supported by molecular docking studies of the metabolites obtained from GC-MS analysis depicting the quorum sensing attenuation by targeting the receptor proteins LasR and RhlR. The in vitro and in silico studies suggested new avenues for the development of bioactive metabolites from A. ochraceopetaliformis SSP13 extract as potential anti-infective agents.

Attenuation of quorum sensing regulated virulence and biofilm development in Pseudomonas aeruginosa PAO1 by Diaporthe phaseolorum SSP12

In recent years, Pseudomonas aeruginosa PAO1 emerged as the significant pathogenic microorganism in majority of the hospital-acquired infections due to its resistance to the conventional antibiotics by virtue of its highly organized quorum sensing and associated biofilm formation. In the present study, quorum sensing attenuation potential of Diaporthe phaseolorum SSP12 extract was investigated against P. aeruginosa PAO1 amply supported by molecular docking studies. D. phaseolorum SSP12 extract significantly inhibited the production of LasI/R mediated LasA protease, LasB elastase and chitinase with 66.52 ± 5.41, 71.26 ± 4.58 and 61.16 ± 4.28% of inhibition respectively at a concentration of 750 μg mL−1. In addition, RhlI/R mediated production of pyocyanin, exopolysaccharides and rhamnolipids were also down-regulated by 74.71 ± 3.97, 66.41 ± 3.62 and 63.75 ± 3.76% respectively on treatment with sub-MIC concentration of D. phaseolorum SSP12. The light, fluorescence and confocal laser scanning microscopic (CLSM) analysis confirmed the significant disruption in biofilm formation. The presence of bioactive constituents such as phenyl ethylalcohol, 2, 4-di-tert-butylphenol, fenaclon, 1, 4-phenylenediacetic acid, and benzyl hydrazine in D. phaseolorum SSP12 extract was evident from Gas chromatography-mass spectrophotometric (GC-MS) analysis. From the in silico molecular docking studies, fenaclon and 2, 4-di-tert-butylphenol competitively binds to QS receptors LasR and RhlR and alters the binding of its cognate ligands and modulates the expression of virulence phenotypes. The promising anti quorum sensing efficacy of D. phaseolorum SSP12 extract suggested new avenues for development of anti-infective drugs from fungal derived metabolites to counteract the problems associated with conventional antibiotic therapies.

Effects of null mutation of the heat-shock gene htpG on the production of virulence factors by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is one of the most clinically important opportunistic pathogen in humans. The aim of the project was to study effects of HtpG on the selected virulence factors responsible for pathogenesis and biofilm formation of P. aeruginosa. By characterizing a htpG null mutant of P. aeruginosa, we have identified the role of HtpG in the production of selected factors. We showed that ΔhtpG mutant affects many physiological processes containing: decreased activity of the LasA protease, reduction of biofilm formation, decreased motility, and diminished amount of rhamnolipids and pyoverdine/pyocyanin. These defects were most evident when the ΔhtpG strain was cultured at 42°C. Our findings demonstrate the unexplored role of HtpG in the pathogenicity of P. aeruginosa, and indicate potential targets for antibacterial therapeutics. [Formula: see text].

Hospitalization length survey and relation with distribution of LasA protease and type III secretion system encoding-genes in multi-drug resistant Pseudomonas aeruginosa isolates from burn wounds in southwest of Iran

Virulence factors are the main reason of bacterial pathogenicity. Pseudomonas aeruginosa is an opportunistic bacterium which has been armed with many virulence factors involved in its acute infection ability. Some of these importance factors have been involved in tissue destruction are proteases like elastase and exoenzymes. According to the importance of elastase activity in preventing wound healing and the activity of type III secretion system (T3SS) in injection secreted proteins to the host cells, we aimed that virulence factors pattern in Pseudomonas aeruginosa isolates from burn wounds may change the length of burn patients hospitalization time. Therefore, Pseudomonas aeruginosa isolates from Burn wounds were evaluated for presence of elastase activity and T3SS and their relation to the hospitalization length of patients In this cross sectional study (January 2015–January 2016) total of 38 Pseudomonas aeruginosa isolates were collected from burn wounds (one isolate from one patient) hospitalized in Amir-Al-Momenin burn hospital affiliated with Shiraz University of Medical Sciences, Shiraz, Iran. Isolates were detected and confirmed with specific bacteriology standard methods. All of the isolates were evaluated for antimicrobial sensitivity pattern, and presence of LasA and PcrH genes related to Elastase activity and T3SS respectively. The presence of these genes and hospitalization time of patients were compared with chi-square analytical analysis (SPSS software, V.18). The prevalence of Pseudomonas aeruginosa was 15.2% at the time of study, where >85% of them were detected multidrug resistant. Separate results for LasA and PcrH presentation was 73.7% and 76.3% respectively, while about 63% of isolates harbor both genes simultaneously and this group of patients have more length of stay in hospital (P value≤0.003). According to the results we concluded that presenting of several factors by a circulating infectious agents simultaneously make the situations of infected patients more critical. Either we highly recommended that the clinical centers such as burn clinics prioritize the determining of the sensitivity pattern of their prevalent infections. Knowing these algorithms will help patients to be treated faster and decrease all of the related costs.