Virulence Enzymes Research Articles

Detection Of Some Virulence Enzymes Of Malasseziaspp. Isolated From Patients Of Pityriasis Versicolor And Their Sensitivity To Some Antifungal Agents

This study included isolation and identification of Malassezia spp. from patients of Pityriasis Versicolor (PV) diagnosis by the dermatological consultant at Al-Diwaniyah Teaching Hospital for the period from 1/10/2019 to 5/3/2020, and detection of the virulence enzymes of these species and their sensitivity to some antifungal agents. 87 specimens were collected (38 females 43.7%, and 49 males 56.3%). 73 specimens (84%) were positive while 14 specimens (16%) were negative to direct and indirect examination. The results of isolation and identification of Malassezia spp. based on direct examination of specimens with KOH10%, indirect methods of growing specimens on SDA medium, and biochemical tests showed that the most occurrence isolates is M. furfur with 30 isolates (34.4%) followed by M. globosa with 22 isolates (25.2%), then M. slooffiae with 16 isolates (18.3%), and M. pachydermatis with 5 isolates (5.7%), while 14 specimens (16.0%) that did not give positive growth. The results also showed the ability of some species to produce protease enzyme, the rates of transparent halo diameters around the colonies on the skimmed milk medium ranged from (9.66-11.66) mm, and M. pachydermatis showed the highest ability to produce lipase enzyme with a precipitation diameter (24 mm), followed by M. furfur (20.33 mm), M. globosa (15 mm), then M. slooffiae (13.3 mm), also M. pachydermatis showed the highest ability to produce hemolysin enzyme with a degradation zone diameter. (18.33 mm), followed by M. furfur (17.33 mm), M. slooffiae (13 mm), and M. globosa (11 mm). The antifungal sensitivity of Malassezia spp. was tested using eight tablet, the results showed that the Ketoconazole is the most effective in inhibiting the growth of the fungal species compared to other antifungals agents, as the average diameter of the inhibition zone was (46.66 mm) for M. globosa, which is the highest rate of inhibition compared with other species, followed by M. furfur (45.33 mm), M. slooffiae (43.66 mm), M. pachydermatis (38.66 mm), followed by the antifungal Fluconazole with an inhibition diameter (35.66 mm) for M. furfur, followed by M. slooffiae (33.33 mm), M. pachydermatis (33 mm) and M. globosa (20 mm), while Nystatin showed no effect on the growth of Malassezia spp.

Kan ve Oral Kavite Örneklerinden Soyutlanan Candida albicans Suşlarının Fosfolipaz Aktivitelerinin Araştırılması

Objective: The aim of the present study was to investigate and compare the phospholipase B1, B2, C1 and D1 activities in C.albicans strains isolated from blood cultures and oral cavity specimens. Method: Phospholipase activity of the strains was examined by plate method and reverse transcriptase polymerase chain reaction (RT-PZT). Results: Twenty-six (86.7%) strains isolated from blood and 24 (80.0%) from oral cavities revealed phospholipase activity by plate method. No statistically significant difference (χ2 =0.48; p=0.49) was observed between the groups. However statistical difference was determined between the mean Pz values (0.6138±0.9823; 0.6988±0.9910) (p=0.007). PLB1 expression was detected in all (100%), PLB2 in 29 (96.7%) and 30 (100.0%), PLC1 in 27 (90%) and 22 (73.3%), PLD1 in 27 (90.0%) and 21 (70.0%) of blood and oral strains, respectively. While no significant difference was detected between PLB1 expression values of the groups (t=-0.307; p=0.760), PLB2 and PLC1 expressions were found to be significantly higher in blood (p=0.043) and oral cavity isolates (p<0.001), respectively. No difference was observed between PLD1 expressions (p=0.732). PLB1, PLB2, PLC1 and PLD1 expressions were 100%, 81.7%, 71.6% and 76.7% in agreement with the plate method. The agreement was 83.3% when all the phospholipase genes were considered. No correlation was detected between the Pz values and phospholipase expressions (p=0.602; p=0.555; p=0.241; p=0,096, respectively). Conclusion: The high rates of phospholipase activity of the C.albicans isolates in our study, support the important roles of these enzymes in virulence. Our results may indicate that phospholipase enzymes encoded by PLB2 and PLC1 genes play more important roles for invasive and oral cavity infections, respectively; however large scale studies are needed on this issue.

Candida Pathogenicity and Interplay with the Immune System.

Candida species are opportunistic fungal pathogens that are part of the normal skin and mucosal microflora. Overgrowth of Candida can cause infections such as thrush or life-threatening invasive candidiasis in immunocompromised patients. Though Candida albicans is highly prevalent, several non-albicans species are also isolated from nosocomial infections. Candida sp. are over presented in the gut of people with Crohn's disease and certain types of neurological disorders, with hyphal form and biofilms being the most virulent states. In addition, Candida uses several secreted and cell surface molecules such as pH related antigen 1, High affinity glucose transporter, Phosphoglycerate mutase 1 and lipases to establish pathogenicity. A strong innate immune response is elicited against Candida via dendritic cells, neutrophils and macrophages. All three complement pathways are also activated. Production of proinflammatory cytokines IL-10 and IL-12 signal differentiation of CD4+ cells into Th1 and Th2 cells, whereas IL-6, IL-17 and IL-23 induce Th17 cells. Importance of T-lymphocytes is reflected in depleted T-cell count patients being more prone to Candidiasis. Anti- Candida antibodies also play a role against candidiasis using various mechanisms such as targeting virulent enzymes and exhibiting direct candidacidal activity. However, the significance of antibody response during infection remains controversial. Furthermore, some of the Candida strains have evolved molecular strategies to evade the sophisticated host attack by proteolysis of components of immune system and interfering with immune signalling pathways. Emergence of several non-albicans species that are resistant to current antifungal agents makes treatment more difficult. Therefore, deeper insight into interactions between Candida and the host immune system is required for discovery of novel therapeutic options.

Carbapenem-Resistant Pseudomonas aeruginosa Strains-Distribution of the Essential Enzymatic Virulence Factors Genes.

Pseudomonas aeruginosa is one of the most commonly isolated bacteria from clinical specimens, with increasing isolation frequency in nosocomial infections. Herein, we investigated whether antimicrobial-resistant P. aeruginosa strains, e.g., metallo-beta-lactamase (MBL)-producing isolates, may possess a reduced number of virulence genes, resulting from appropriate genome management to adapt to a changing hospital environment. Hospital conditions, such as selective pressure, may lead to the replacement of virulence genes by antimicrobial resistance genes that are crucial to survive under current conditions. The study aimed to compare, using PCR, the frequency of the chosen enzymatic virulence factor genes (alkaline protease-aprA, elastase B-lasB, neuraminidases-nan1 and nan2, and both variants of phospholipase C-plcH and plcN) to MBL distribution among 107 non-duplicated carbapenem-resistant P. aeruginosa isolates. The gene encoding alkaline protease was noted with the highest frequency (100%), while the neuraminidase-1 gene was observed in 37.4% of the examined strains. The difference in lasB and nan1 prevalence amongst the MBL-positive and MBL-negative strains, was statistically significant. Although P. aeruginosa virulence is generally more likely determined by the complex regulation of the virulence gene expression, herein, we found differences in the prevalence of various virulence genes in MBL-producers.

NOD-like receptor-mediated plant immunity: from structure to cell death.

Animal and plant immune systems use intracellular nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) to detect pathogens, resulting in the activation of immune responses that are often associated with localized host cell death. Whereas vertebrate NLRs detect evolutionarily conserved molecular patterns and have undergone comparatively little copy number expansion, plant NLRs detect virulence factors that have often diversified in plant pathogen populations, and thus plant NLRs have been subject to parallel diversification. Plant NLRs sense the presence of virulence factors with enzymatic virulence activity often indirectly through their modification of host target proteins. By contrast, phytopathogenic virulence factors without enzymatic activity are usually recognized by NLRs directly by their structure. Structural and biochemical analyses have shown that both indirect and direct recognition of plant pathogens trigger the oligomerization of plant NLRs into active complexes. Assembly into three-layered ring-like structures has emerged as a common principle of NLR activation in plants and animals, but with distinct amino-terminal domains initiating different signalling pathways. Collectively, these analyses point to host cell membranes as a convergence point for activated plant NLRs and the disruption of cellular ion homeostasis as a possible major factor in NLR-triggered cell death signalling.

Phenotypic Characterization of Enzymatic and Non-enzymatic Virulence Factors in Etiopathogenesis and Evolution of Drug Resistance among Dermatophytes

The virulence factors of dermatophytes play a vital role in the etio-pathogenesis and emergence of drug resistance. Regular surveillance is essential for constant monitoring of association of virulence factors in the emergence of drug resistant isolates and to guide proper therapeutic strategies to combat infections due to drug resistant isolates of dermatophytes. The aim of this study is to isolate and identify various enzymatic and non-enzymatic virulence factors of dermatophytes isolated from clinically suspected cases of dermatophytosis attending the dermatology outpatient clinic. From 30 clinical samples received from department of Dermatology to the department of Microbiology during the study period July to December 2019, KOH mount and fungal culture was done and only the isolates which grew dermatophytes were used for detecting virulence factors using standard methods. Among 30 affected patients, 13 were male (43.3%) and 17 were female (56.7%) with age range of 13 to 60 years. Skin sample yielded positive growth in 96.6% of the cases. Out of 30 samples, 6 samples were positive for Trichophyton mentagrophytes. Tinea corporis was the common manifestation which yielded positive growth. Female predominance was seen. Male predominance was seen in younger age group while females were more commonly affected in older age group. Adolescents were commonly affected age group (40%). Then people between age 41-50 (middle age group) were mostly affected. The six Trichophyton mentagrophytes were found to have enzymatic and non- enzymatic virulence factors. All the virulence factors included in the study were present in Trichophyton mentagrophytes.

Proteomic analysis deciphers the multi-targeting antivirulence activity of tannic acid in modulating the expression of MrpA, FlhD, UreR, HpmA and Nrp system in Proteus mirabilis

In the present study, the multi-targeting antivirulence activity of tannic acid (TA) was explored against Proteus mirabilis through MS-based proteomic approach. The in vitro biofilm biomass quantification assay and microscopic analysis demonstrated the antibiofilm activity of TA against P. mirabilis in which, minimum biofilm inhibitory concentration (MBIC) of TA was found to be 200 μg/mL concentration. Moreover, the nanoscale liquid chromatography coupled to tandem mass spectrometry (nano LC-MS/MS) analysis revealed that TA (at MBIC) differentially regulated the proteins involved in fimbrial adhesion, flagellar motility, iron acquisition, Fe-S cluster assembly, heat shock response, virulence enzymes, and toxin secretion. Further, the transcriptomic analysis validated the outcomes of proteomic analysis in which, the expression level of virulence genes responsible for MR/P fimbrial adhesion (mrpA), flagellar transcriptional activation (flhD), biosynthesis of urease (ureR), hemolysin (hpmA), non-ribosomal peptide siderophore system (Nrp), oxidative stress responsible enzymes and fitness factors proteins were down-regulated in TA exposed P. mirabilis. These observations were also in correspondence with the in vitro bioassays. Thus, this study reports the feasibility of TA to act as a promising therapeutic agent against multifactorial P. mirabilis infections.

Assessment of different Enzymatic Virulence Traits contributing to pathogenicity of Candida Albicans

Background: Candidiasis is regarded as one of the frequently occurring fungal infections of humans elicited by yeast that belongs to the genus Candida. Among the different species of Candida, Candida albicans is the most pathogenic specie. The pathogenicity of this specie depends on various virulence factors. This study was designed to determine the activity of some important enzymatic virulence factors such as phospholipase, proteinase and hemolysin produced by Candida albicans strains.
 Methodology: A total of 300 clinical isolates of C. albicans were obtained and identified by conventional standard diagnostic criteria. For the evaluation of phospholipase production, the egg yolk agar medium was employed. The minimum agar medium was used for proteinase activity and for hemolysin production, blood plate assay was used. The results were statistically analyzed by SPSS 16.0 version software.
 Results: Of a total of 300 isolates reviewed, the majority of the isolates were recovered from females, i.e. 234 (78%); however, the number of isolates obtained from males were 66 (22%). Amongst all the strains of C. albicans, phospholipase and hemolysin enzymes were produced primarily by 89.3% of the total C. albicans strains. While proteinase enzyme was produced by 86% of the total C. albicans isolates. A p-value < 0.05 was measured as statistically significant.
 Conclusion: From the existing study, it can be determined that these enzymes are produced by most of the pathogenic strains of C. albicans. The detection of these virulence enzymes is tremendously significant in designing of drugs to combat these opportunistic infections in future.

Ethanol extract of propolis decreases the Interleukin-8 (IL-8) expression and blood Malondialdehyde (MDA) level in otitis media rat model induced by Pseudomonas aeruginosa

Background: Pseudomonas aeruginosa is one of the bacteria that cause otitis media, leading to progressive structural damage of the middle ear mucosa due to virulence factor, toxin, and enzyme produced. The excessive inflammatory process can cause oxidative stress, which will aggravate tissue damage, thus difficult to cure. This study aimed to assess the effect of ethanol extract of propolis (EEP) in decreasing IL-8 expression and blood MDA level in the otitis media rat model induced by Pseudomonas aeruginosa.Methods: A randomized controlled trial was conducted among 42 SDF Sprague-Dawley rats. They were divided into 6 groups: normal control, negative control: Otitis Media (OM), positive control: OM + Ciprofloxacin 20 mg/kg body weight/day, OM + EEP 200 mg/kg body weight/day, OM + Ciprofloxacin 20 mg/kg body weight/day + EEP 100 mg/kg body weight, OM + Ciprofloxacin 20 mg/kg body weight/day + EEP 200 mg/kg body weight/day. Induction of Pseudomonas aeruginosa to obtain the OM model was administered on the first day of the study and waited for 28 days before treatments were given. Data were analyzed with SPSS version 23 for Windows.Results: After 14 days of treatment, the expression of IL-8 and MDA level in each treatment group (OM + Ciprofloxacin, OM + EEP 200, OM + Ciprofloxacin + EEP 100, OM + Ciprofloxacin + EEP 200) revealed significant decrease compared to OM (negative control group) (p<0.05). The reduction of IL-8 expression between the Ciprofloxacin group and EEP 200 was not significantly different (p>0.05). There was a significant difference between the Ciprofloxacin group and Ciprofloxacin + EEP 100, and between Ciprofloxacin and Ciprofloxacin + EEP 200 (p<0.05).Conclusion: Ethanol extract of propolis decreased IL-8 expression and MDA level in the OM rat model induced by Pseudomonas aeruginosa.

Identification of subtilisin virulence genes (SUB1-7) in Epidermophyton floccosum isolated from patients with dermatophytosis in Iran

BackgroundDermatophytes are a group of frequent cutaneous infectious agents with different clinical forms, which are highly adapted to invade the enriched keratin tissues, through secretion of different enzymatic and non- enzymatic virulence factors. Since the knowledge of the factors mediating invasion of Epidermophyton floccosum to host tissues is little known, we investigated the harborage of SUB genes family in clinical isolates of E. floccosum. Material and methodsA total, 312 clinically suspected cases of dermatophytosis were investigated in Medical Parasitology and Mycology Laboratory, Tehran University of Medical Sciences. All the obtained specimens underwent direct examination and culture on Sabouraud Dextrose Agar with Chloramphenicol and Cycloheximide. Molecular confirmation of dermatophytes was performed using ITS1 and ITS4 primers. Finally, the presence of SUB gene family in clinical isolates and the reference strain of E. floccosum were evaluated using specific primers. ResultsConsidering our results, the presence of SUB2, SUB3, and SUB7 was demonstrated in 10 isolates of E. floccosum derived from different clinical forms of dermatophytosis, including tinea cruris (n = 7), tinea pedis (n = 2), and tinea corporis (n = 1). Also, SUB1 and SUB6 were only detected in 3 (3/10), and 2 (2/10) isolates of E. floccosum, respectively. Nevertheless, the results of the current study showed that there are no isolates to carry SUB4 and SUB5 genes. ConclusionsIn the current study, the presence of SUB2, SUB3, and SUB7 in all of the investigated isolates probably implies their indispensable role in infection elicitation. Further studies are needed to investigate the key role of subtilisin genes and check the conditions that affect the expression of SUB genes in vitro and in vivo.

Sodium Ascorbate as a Quorum-Sensing Inhibitor Leads to Decreased Virulence in Vibrio campbellii.

Vibrio campbellii is one of the major bacterial pathogens for animals reared in aquaculture, affecting both vertebrates and invertebrates, and causes significant economic losses. It is now evident that the expressions of virulence factors in this pathogen are regulated by the density of the bacterial population. This type of regulation, termed quorum sensing (QS), is mediated by extracellular signal molecules called autoinducers. In this study, the impact of sodium ascorbate (NaAs) on the virulence of V. campbellii was investigated under both in vitro and in vivo conditions, to develop a natural anti-infective strategy to contain V. campbellii infection in aquacultured animals. Results showed that NaAs significantly decreased swimming motility, biofilm production, and the production of virulence enzymes, such as lipase, caseinase, phospholipase, and hemolysin in V. campbellii. Consistent with this, pretreatment of V. campbellii with NaAs before inoculation into the rearing water resulted in significantly increased survival of gnotobiotic brine shrimp larvae, when compared to larvae challenged with untreated V. campbellii. Furthermore, NaAs could interfere with QS-regulated bioluminescence in V. campbellii, suggesting the QS-inhibitory activity largely determines the protective effect of NaAs toward the brine shrimp. In essence, due to the potent anti-virulence effects observed in in vitro studies and the clinical brine shrimp-V. campbellii infection model, NaAs constitute a promising novel strategy for the control of V. campbellii infections in aquaculture.

Virulence Characteristics of mecA-Positive Multidrug-Resistant Clinical Coagulase-Negative Staphylococci.

Coagulase-negative staphylococci (CoNS) are an important group of opportunistic pathogenic microorganisms that cause infections in hospital settings and are generally resistant to many antimicrobial agents. We report on phenotypic and genotypic virulence characteristics of a select group of clinical, mecA-positive (encoding penicillin-binding protein 2a) CoNS isolates. All CoNS were resistant to two or more antimicrobials with S. epidermidis strain 214EP, showing resistance to fifteen of the sixteen antimicrobial agents tested. Aminoglycoside-resistance genes were the ones most commonly detected. The presence of megaplasmids containing both horizontal gene transfer and antimicrobial resistance genetic determinants indicates that CoNS may disseminate antibiotic resistance to other bacteria. Staphylococcus sciuri species produced six virulence enzymes, including a DNase, gelatinase, lipase, phosphatase, and protease that are suspected to degrade tissues into nutrients for bacterial growth and contribute to the pathogenicity of CoNS. The PCR assay for the detection of biofilm-associated genes found the eno (encoding laminin-binding protein) gene in all isolates. Measurement of their biofilm-forming ability and Spearman’s rank correlation coefficient analyses revealed that the results of crystal violet (CV) and extracellular polymeric substances (EPS) assays were significantly correlated (ρ = 0.9153, P = 3.612e-12). The presence of virulence factors, biofilm-formation capability, extracellular enzymes, multidrug resistance, and gene transfer markers in mecA-positive CoNS clinical strains used in this study makes them powerful opportunistic pathogens. The study also warrants a careful evaluation of nosocomial infections caused by CoNS and may be useful in studying the mechanism of virulence and factors associated with their pathogenicity in vivo and developing effective strategies for mitigation.

A STRUCTURE BASED MOLECULAR DOCKING OF Acacia catechu AGAINSTCONTRACTILE PROTEIN Plasmodium falciparum – A MALARIAL DISEASE

The bioactive compounds of Acacia catechu using Gas Chromatography Mass Spectroscopy and the inhibitory activity against contractile protein Plasmodium falciparum against protozoan disease were studied. This research mainly focuses on finding of novel drug screening against malarial enzyme. The compounds of Acacia catechu are screened using Lipinski rule of five with ADMET properties in which the character as well as behaviour of the drug compound is known. The compounds were checked for its dosage level in human and rat as well as distribution properties in blood brain barrier and central neuro system. The compounds 9,12,15- Octadecatrienoic acid has higher affinity with -7.95 Kcal/mol followed by Pthalic acid, butyl 2- pentyl ester -7.35 Kcal/mol and Furo[2,3-d] Pyrimidine-4,6 [5H,7H]-dion -6.24 Kcal/mol were docked using Autodock software. the compound 9,12,15- Octadecatrienoic acid, Pthalic acid butyl 2- pentyl ester, Furo[2,3-d] Pyrimidine-4,6 [5H,7H]-dion has higher affinity such as -7.95 Kcal/mol , -7.35 Kcal/mol and -6.24 Kcal/mol respectively. Thus this research proves that the drug compounds of Acacia catechu have novel therapeutic drug activity against virulent enzymes.

Investigating Drug Properties of Bioactive Compounds of Cymbopogon citratus by Absorption, Distribution, Metabolism, Excretion/Toxicity and Molecular Docking Analysis Against Apolipoprotein N-Acyl Transferase

Delivering a potential drug is a predominant challenge in medicinal chemistry.in this study, bio organic compounds of Cymbopogon citratus was screened by analysing physiochemical properties like solubility, permeability, efficacy, toxicity, and metabolic stability. The optimization of drug potential against virulent protein was calculated by using docking algorithm Autodock 4.2.3. Structure based ligand docking reveals that the compounds having better inhibition potential against virulent enzymes with insoluble and impermeable activities. The organic compounds of Cymbopogon citratus were screened using Lipinski rule of five and ADME/T prediction for drug likeliness. The structure based ligand docking was done between bioactive compounds of plant and virulent protein that cause diseases. The interaction was visualized using Discovery studio and was studies. The molecular docking of bioactive compounds resulted in better inhibition potential with controlled lipophilicity level, without causing toxicity that harms the natural habitat of humans. The compounds, 1,3,4-trimethyl -3cyclohexene-1-carboxaldehyde exhibit binding energy -4.70 Kcal/mol followed by β-myrcene – 4.35 Kcal/mol and Geraniol -4.35 Kcal/mol. Hence, structure based ligand docking and in silico ADME/T studies revealed that the compounds have better inhibition potential against Apolipoprotein by improving the prediction of drug compounds.

Explication of the Potential of 2-Hydroxy-4-Methoxybenzaldehyde in Hampering Uropathogenic Proteus mirabilis Crystalline Biofilm and Virulence.

Proteus mirabilis is an important etiological agent of catheter-associated urinary tract infections (CAUTIs) owing to its efficient crystalline biofilm formation and virulence enzyme production. Hence, the present study explicated the antibiofilm and antivirulence efficacies of 2-hydroxy-4-methoxybenzaldehyde (HMB) against P. mirabilis in a non-bactericidal manner. HMB showed concentration-dependent biofilm inhibition, which was also evinced in light, confocal, and scanning electron microscopic (SEM) analyses. The other virulence factors such as urease, hemolysin, siderophores, and extracellular polymeric substances production as well as swimming and swarming motility were also inhibited by HMB treatment. Further, HMB treatment effectively reduced the struvite/apatite production as well as crystalline biofilm formation by P. mirabilis. Furthermore, the results of gene expression analysis unveiled the ability of HMB to impair the expression level of virulence genes such as flhB, flhD, rsbA, speA, ureR, hpmA, and hpmB, which was found to be in correlation with the results of in vitro bioassays. Additionally, the cytotoxicity analysis divulged the innocuous characteristic of HMB against human embryonic kidney cells. Thus, the present study reports the potency of HMB to act as a promising therapeutic remedy for P. mirabilis-instigated CAUTIs.

Core architecture of a bacterial type II secretion system

Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here we use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae. We show that 7 proteins form a ~2.4 MDa complex that spans the cell envelope. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. The inner membrane assembly platform components PulC, PulE, PulL, PulM and PulN have a relative stoichiometric ratio of 2:1:1:1:1. The PulE ATPase, PulL and PulM combine to form a flexible hexameric hub. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. Our results show that the T2SS has a highly dynamic modular architecture, with implication for pseudo-pilus assembly and substrate loading.

In vivo structure of the Legionella type II secretion system by electron cryotomography.

The type II secretion system (T2SS) is a multiprotein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane (OM) of many Gram-negative bacteria1–3. Here, using electron cryotomography (ECT) and subtomogram averaging methods, we reveal the in vivo structure of an intact T2SS, imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily-related type IV pilus (T4P) system4,5, we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including for instance that the T2SS-ATPase complex is usually present but disengaged from the inner membrane (IM), the T2SS has a much longer periplasmic vestibule, and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our ECT map produced a complete architectural model of the intact T2SS that provides insights into the structure and function of its components, its position within the cell envelope, and the interactions between its different subcomplexes.

A Structure Based Drug Designing of Bioactive Compounds of Gracilaria edulis against Virulent Bacterial Enzyme Aureolysin

The bioactive compounds of Gracilaria edulis were determined by using Gas Chromatography Mass spectroscopy. The drug compounds were screened for analyzing the inhibition potential against the virulent bacterial enzyme. In this research, the protein responsible for bacterial infection was docked against the drug compounds of Gracilaria edulis. The data of the virulent enzymes are studied and retrieved from PDB. The bioactive compounds were screened by Lipinski rule of five and ADMET properties. Using Autodock 4.2.6 the molecular docking analysis were done against virulent enzymes and was visualized by discovery studio 3.1. The bioactive compound eugenol with binding energy -4.42 Kcal/mol followed by 2 Heptene, 2,4,4,6 tetramethyl -3.89 Kcal/mol and 1, 2-Propanediol 2.77 Kcal/mol. The hydrogen and vanderwaals interaction of amino acids were studied. This research work mainly focuses on targeting the virulent enzymes that can reduce clinical costs by designing novel drug.

Typical and Atypical Enteroaggregative Escherichia coli Are Both Virulent in the Galleria mellonella Model.

Enteroaggregative Escherichia coli (EAEC) is an emerging pathotype responsible for acute and persistent diarrhea. It can be classified as typical and atypical strains, respectively, based on the presence or absence of the AggR regulon, suggesting a higher virulence for typical EAEC. This study aims to evaluate in the Galleria mellonella model if there are differences in the virulence profiles among clinical strains of typical and atypical EAEC, prototype strains EAEC C1096, 042 and its aggR mutant. The clinical EAEC strains (n = 20) were analyzed for the presence of 22 putative virulence factors of EAEC or extraintestinal E. coli by PCR, as well as phenotypic characteristics of virulence (enzymes, siderophore, and biofilm). The survival of the larvae was analyzed after inoculation of 104–107 CFU/larva; the monitoring of bacterial growth in vivo and hemocyte quantification was determined after inoculation of the prototype strains (105 CFU/larva) at different periods after infection. The strains of typical and atypical EAEC presented the same virulence profile for the larva, regardless of the amount or type of genes and phenotypic aspects of virulence analyzed. In addition, the EAEC 042 aggR mutant strain showed a significant reduction in the mortality of the inoculated larvae compared to the wild-type strain. In conclusion, the results obtained herein demonstrate that the virulence of EAEC seems to be related to the AggR regulon, but not exclusively, and atypical EAEC strains may be as virulent as typical ones in vivo in the G. mellonella model.

Structural and dynamic studies provide insights into specificity and allosteric regulation of ribonuclease as, a key enzyme in mycobacterial virulence

Ribonuclease AS (RNase AS) is a crucial enzyme for virulence of Mycobacterium tuberculosis. We previously observed that RNase AS structurally resembles RNase T from Escherichia coli, an important enzyme for tRNA maturation and turnover. Here, we combine X-ray crystallography and molecular dynamics (MD) to investigate the specificity and dynamic properties of substrate binding. Both X-ray and MD data provide structural determinants that corroborate the strict substrate specificity of RNase AS to cleave only adenosine residues, due to the structural features of adenine base. Beside suggesting tRNA as most likely substrate of RNase AS, MD and modeling studies identify key enzyme-ligand interactions, both involving the catalytic site and the double helix region of tRNA, which is locked by interactions with a set of arginine residues. The MD data also evidence a ligand-induced conformational change of the enzyme which is transferred from one chain to the adjacent one. These data will explain the dimeric nature of both RNase AS and RNase T, with two catalytic grooves composed of both chains. Also, they account for the dichotomy of tRNA, which contains both the substrate poly(A) chain and an inhibiting double strand RNA. Indeed, they provide a possible mechanism of allosteric regulation, which unlocks one catalytic groove when the second groove is inhibited by the double strand region of tRNA. Finally, a full comprehension of the molecular details of tRNA maturation processes is essential to develop novel strategies to modulate RNA processing, for therapeutic purposes.AbbreviationsMDmolecular dynamicsPDBProtein Data BankRMSDroot mean square deviationRMSFroot mean square fluctuationRNAribonucleotidic acidRNase ASRibonuclease ASCommunicated by Ramasamy H. Sarma