Aspartate Semialdehyde Dehydrogenase Research Articles

Campylobacter fetus subspecies specific PCR assays inferred from comparative genomic analysis for accurate subspecies identification

Bovine Genital Campylobacteriosis (BGC) is caused by Campylobacter fetus subsp. venerealis and is a notifiable disease to the WOAH (World Organisation for Animal Health). For an effective BGC control program, the reliable differentiation of Campylobacter fetus subsp. venerealis (Cfv) from the closely related Campylobacter fetus subsp. fetus (Cff) is required. However, the available molecular C. fetus subspecies identification assays lack sensitivity and specificity to differentiate C. fetus isolates based on their phenotypic or genotypic differences. Furthermore, the current biochemical subspecies identification is not fully congruent with the genomic differentiation of C. fetus strains.In this study, the genome sequences of 41C. fetus strains with well identified subspecies, were analyzed with the large-scale BLAST score ratio (LS-BSR) pipeline to identify Cff and Cfv specific sequences. With this analysis, the asd gene encoding an aspartate-semialdehyde dehydrogenase was identified, which contained a 6-bp Cff-specific sequence, and this 6-bp sequence was absent in the asd gene of Cfv strains. This sequence was used for the development of PCR assays to differentiate Cff and Cfv strains. The C. fetus subspecies identification of the developed asd PCR assays was in full congruence with the genomic classification of strains and are recommended for molecular identification of C. fetus subspecies in BGC control programs.The asd PCR can be assessed on sequenced genomes using a web interface containing the Cfvcatch tool, which includes placement of the tested genome in a phylogenetic tree with reference C. fetus genomes to distinguish the two subspecies and to detect antimicrobial resistance genes.

An In Vitro System Mimics the Intestinal Microbiota of Striped Beakfish (Oplegnathus fasciatus) and Inhibits Vibrio alginolyticus by Limosilactobacillus reuteri-Derived Extracellular Vesicles.

Extracellular vesicles (EVs) are functional substances secreted by microbes and host cells, and it has been discovered that they participate in the interactions between different microorganisms. Our recent findings indicate that Limosilactobacillus reuteri-derived EVs have the potential to improve the intestinal microbiota of Oplegnathus fasciatus fish and inhibit pathogenic bacteria. Previous research has reported that the host intestinal cells play a regulatory role in the intestinal microbiota. This suggested that to investigate the mechanisms through which L. reuteri-derived EVs regulate the intestinal microbiota, a system that excludes interference from host intestinal cells should be established. In this study, an in vitro cultured intestinal bacteria system, without host factors, was used to simulate the intestinal microbiota of O. fasciatus fish. After adding L. reuteri-derived EVs to the system, the changes in the microbiota were analyzed. The results showed that L. reuteri-derived EVs effectively reduced the abundance of Vibrio spp. In the results of the in vitro experiments, it was also observed that L. reuteri-derived EVs have the ability to inhibit Vibrio alginolyticus. We further sequenced the small RNA contained in L. reuteri-derived EVs and found that these small RNAs can interfere with genes (LysR, pirin, MIpA/OmpV, CatB, and aspartate-semialdehyde dehydrogenase) related to the growth of V. alginolyticus. Taken together, the results indicate that in the absence of host involvement, the small RNAs present in L. reuteri-derived EVs have the function of inhibiting pathogenic bacteria and exhibit the potential to regulate the intestinal microbiota.

Osmotic response in Leptospirillum ferriphilum isolated from an industrial copper bioleaching environment to sulfate

Iron and sulfur-oxidizing microorganisms play important roles in several natural and industrial processes. Leptospirillum (L.) ferriphilum, is an iron-oxidizing microorganism with a remarkable adaptability to thrive in extreme acidic environments, including heap bioleaching processes, acid mine drainage (AMD) and natural acidic water. A strain of L. ferriphilum (IESL25) was isolated from an industrial bioleaching process in northern Chile. This strain was challenged to grow at increasing concentrations of sulfate in order to assess changes in protein expression profiles, cells shape and to determine potential compatible solute molecules. The results unveiled changes in three proteins: succinyl CoA (SCoA) synthetase, isocitrate dehydrogenase (IDH) and aspartate semialdehyde dehydrogenase (ASD); which were notably overexpressed when the strain grew at elevated concentrations of sulfate. ASD plays a pivotal role in the synthesis of the compatible solute ectoine, which was identified along with hydroxyectoine by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The relationship between IDH, SCoA, and ectoine production could be due to the TCA cycle, in which both enzymes produce metabolites that can be utilized as precursors or intermediates in the biosynthesis of ectoine. In addition, distinct filamentous cellular morphology in L. ferriphilum IESL25 was observed when growing under sulfate stress conditions. This study highlights a new insight into the possible cellular responses of L. ferriphilum under the presence of high sulfate levels, commonly found in bioleaching of sulfide minerals or AMD environments.

Bridging drug discovery through hierarchical subtractive genomics against asd, trpG, and secY of pneumonia causing MDR Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is an opportunistic gram-positive, non-motile, and non-sporulating bacteria that induces pneumonia, a provocative lung infection affecting mainly the terminal bronchioles and the small air sacs known as alveoli. Recently, it has developed antibiotic resistance to the available consortium as per the WHO reports; thereby, novel remedial targets and resilient medications to forestall and cure this illness are desperately needed. Here, using pan-genomics, a total of 1,387 core proteins were identified. Subtractive proteome analyses further identified 12 proteins that are vital for bacteria. One membrane protein (secY) and two cytoplasmic proteins (asd and trpG) were chosen as possible therapeutic targets concerning minimum % host identity, essentiality, and other cutoff values, such as high resistance in the MDR S. aureus. The UniProt AA sequences of the selected targets were modelled and docked against 3 drug-like chemical libraries. The top-ranked compounds i.e., ZINC82049692, ZINC85492658 and 3a of Isosteviol derivative for Aspartate-semialdehyde dehydrogenase (asd); ZINC38222743, ZINC70455378, and 5m Isosteviol derivative for Anthranilate synthase component II (trpG); and finally, ZINC72292296, ZINC85632684, and 7m Isosteviol derivative for Protein translocase subunit secY (secY), were further subjected to molecular dynamics studies for thermodynamic stability and energy calculation. Our study proposes new therapeutic targets in S. aureus, some of which have previously been reported in other pathogenic microorganisms. Owing to further experimental validation, we anticipate that the adapted methodology and the predicted results in this work could make major contributions towards novel drug discovery and their targets in S. aureus caused pneumonia.

AraC transcriptional regulator, aspartate semialdehyde dehydrogenase and acyltransferase: Three putative genes in phenol catabolic pathway of Acinetobacter sp. Strain DF4

The objective of this study was to identify genes associated with the biodegradation of phenol by Acinetobacter sp. strain DF4 through the use of differential display (DD) methodology. The bacteria were grown in YEPG medium, and total RNA was extracted and analyzed using labeled primers to detect gene expression differences. Three distinctively expressed cDNA bands (ph1, ph2, and ph3) were identified, cloned, and sequenced. DNA analysis involved searching for open reading frames (ORFs), verifying results with the NCBI database, predicting promoter regions, and constructing phylogenetic trees using bioinformatics tools. The ph1 gene displayed a 97% identity with the AraC transcriptional regulator, suggesting its potential role in regulating the ortho-catabolic pathway of phenol. The ph2 gene showed a 98% identity with aspartate semialdehyde dehydrogenase, which is involved in phenol degradation. The ph3 gene had a 93% identity with acetyltransferase. Essential transcription factors, such as TATA, GTGTGT, CACA, and CTTTT, were detected, and the three genes promoter regions were predicted. This study successfully identified functional genes involved in the metabolism of cyclic chemicals, particularly phenol, using the DD technique. These findings provide insights into the biodegradation pathways of phenol by Acinetobacter sp. Strain DF4 and may contribute to the development of more efficient bioremediation strategies for phenol-contaminated environments.

Metabolic engineering combined with enzyme engineering for overproduction of ectoine in Escherichia coli

Ectoine, a natural protective agent, is naturally synthesized at low titers by some extreme environment microorganisms that are usually difficult to culture. There is a need for an efficient and eco-friendly ectoine production process. In this study, Escherichia coli BL21(DE3) with the ectABC gene cluster from Halomonas venusta achieved 1.7 g/L ectoine. After optimizing the expression plasmid, 2.1 g/L ectoine was achieved. Besides, the aspartate kinase mutant LysCT311I from Corynebacterium glutamicum and aspartate semialdehyde dehydrogenase from Halomonas elongata were overexpressed to increase precursors supply. Furthermore, the rate-limiting enzyme EctB was semirationally engineered, and the E407D mutation enhanced ectoine production by 13.8 %. To improve acetyl-CoA supply, the non-oxidative glycolysis pathway was introduced. Overall, the optimized strain ECT9-5 produced 67.1 g/L ectoine by fed-batch fermentation with a 0.3 g/g of glucose and the kinetic model resulted in a good fit.

Crystal Structure of Aspartate Semialdehyde Dehydrogenase from Porphyromonas gingivalis

Aspartate semialdehyde dehydrogenase (ASADH) catalyzes the biosynthesis of several essential amino acids, including lysine, methionine, and threonine, and bacterial cell components. Thus, ASADH is a crucial target for developing new antimicrobial agents that can potentially disrupt the biosynthesis of essential amino acids, thereby inhibiting the growth of pathogens. Herein, the crystal structures of ASADH obtained from Porphyromonas gingivalis (PgASADH, UniProtKB code A0A1R4DY25) were determined in apo- and adenosine-2′-5′-diphosphate (2′,5′-ADP)-bound complex forms at a resolution of 1.73 Å. The apo- and 2′,5′-ADP-complexed crystals of PgASADH belonged to the space groups of I212121 and C2221, respectively. Analytical size-exclusion chromatography showed a stable PgASADH dimer in a solution. Clustering analysis and structural comparison studies performed on PgASADH and previously known ASADHs revealed that ASADHs, including PgASADH, can be classified into three types depending on sequential and structural differences at the α-helical subdomain region. These findings provide valuable insights into developing structure-based species-specific new antibacterial drugs against the oral pathogen P. gingivalis.

Whole-genome analysis of Haemophilus influenzae strains isolated from persons with cystic fibrosis.

Introduction. Haemophilus influenzae is a commensal of the respiratory tract that is frequently present in cystic fibrosis (CF) patients and may cause infection. Antibiotic resistance is well described for CF strains, and virulence factors have been proposed.Hypothesis/Gap. The genetic diversity of H. influenzae strains present in the lungs of persons with CF is largely unknown despite the fact that this organism is considered to be a pathogen in this condition. The aim was to establish the genetic diversity and susceptibility of H. influenzae strains from persons with CF, and to screen the whole genomes of these strains for the presence of antibiotic resistance determinants and proposed virulence factors.Methods. A total of 67 strains, recovered from respiratory samples from persons with CF from the UK (n=1), Poland (n=2), Spain (n=24) and the Netherlands (n=40), were subjected to whole-genome sequencing using Illumina technology and tested for antibiotic susceptibility. Forty-nine of these strains (one per different sequence type) were analysed for encoded virulence factors and resistance determinants.Results. The 67 strains represented 49 different sequence types. Susceptibility testing showed that all strains were susceptible to aztreonam, ciprofloxacin, imipenem and tetracycline. Susceptibility to ampicillin, ampicillin/sulbactam, amoxicillin/clavulanic acid, cefuroxime, cefixime, ceftriaxone, cefepime, meropenem, clarithromycin, co-trimoxazole and levofloxacin ranged from 70.2-98.5%. Only 6/49 strains (12.2%) harboured acquired resistance genes. Mutations associated with a ß-lactamase-negative ampicillin-resistant phenotype were present in four strains (8.2 %). The potential virulence factors, urease, haemoglobin- and haptoglobin-binding protein/carbamate kinase, and OmpP5 (OmpA), were encoded in more than half of the strains. The genes for HMW1, HMW2, H. influenzae adhesin, a IgA-specific serine endopeptidase autotransporter precursor, a TonB-dependent siderophore, an ABC-transporter ATP-binding protein, a methyltransferase, a BolA-family transcriptional regulator, glycosyltransferase Lic2B, a helix-turn-helix protein, an aspartate semialdehyde dehydrogenase and another glycosyltransferase were present in less than half of the strains.Conclusion. The H. influenzae strains showed limited levels of resistance, with the highest being against co-trimoxazole. Sequences encoding a carbamate kinase and a haemoglobin- and haemoglobin-haptoglobin-binding-like protein, a glycosyl transferase and an urease may aid the colonization of the CF lung. The adhesins and other identified putative virulence factors did not seem to be necessary for colonization.

Structural characterization of aspartate-semialdehyde dehydrogenase from Pseudomonas aeruginosa and Neisseria gonorrhoeae

Gonorrhoea infection rates and the risk of infection from opportunistic pathogens including P. aeruginosa have both risen globally, in part due to increasing broad-spectrum antibiotic resistance. Development of new antimicrobial drugs is necessary and urgent to counter infections from drug resistant bacteria. Aspartate-semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate biosynthetic pathway, which is critical for amino acid and metabolite biosynthesis in most microorganisms including important human pathogens. Here we present the first structures of two ASADH proteins from N. gonorrhoeae and P. aeruginosa solved by X-ray crystallography. These high-resolution structures present an ideal platform for in silico drug design, offering potential targets for antimicrobial drug development as emerging multidrug resistant strains of bacteria become more prevalent.

Evaluation of a novel inhibitor of aspartate semialdehyde dehydrogenase as a potent antitubercular agent against Mycobacterium tuberculosis.

The in vitro activity of IMB-XMA0038, a novel inhibitor targeting Mycobacterial tuberculosis (Mtb) aspartate semialdehyde dehydrogenase, was evaluated. Minimum inhibitory concentrations (MICs) of IMB-XMA0038 were against 20 Mtb isolates, including H37Rv (ATCC 27294), ten clinical pan-sensitive isolates, and nine clinical multidrug-resistant (MDR) isolates. In addition, minimum bactericidal concentrations (MBCs) were also determined against the H37Rv and 6 MDR isolates (the background information is same as above in order). A model was generated to evaluate IMB-XMA0038 activity against dormant Mtb. The post-antibiotic effect (PAE), an important indicator of antimicrobial drug dosing schedules to obtain efficacy, was determined based on time required for regrowth of Mtb to 50% of the OD600max value after treatment with various concentrations of IMB-XMA0038 and INH. In addition, interactions between IMB-XMA0038 and other anti-tuberculosis drugs, measured using a checkerboard assay, revealed that IMB-XMA0038 MICs of 0.5-1 μg/mL could be achieved in combinations. Synergistic effects were observed for IMB-XMA0038 when used together with almost all other anti-tuberculosis drugs against most Mtb isolates. IMB-XMA0038 exhibited greater activity than rifampin against Mtb under hypoxic conditions, as reflected by CFU decreases of 1.1-log-unit versus 0.8-log-unit, respectively, for IMB-XMA0038 and rifampin concentrations of 4 × MIC. IMB-XMA0038-induced PAEs (9, 10, 11 days) were comparable to INH PAEs (10, 11, 12 days). These findings suggest that addition of IMB-XMA0038 to current therapeutic regimens could be useful to improve the efficacy of treatments for drug-resistant and drug-susceptible TB.

Qipengyuania pacifica sp. nov., a Novel Carotenoid-Producing Marine Bacterium of the Family Erythrobacteraceae, Isolated from Sponge (Demospongiae), and Antimicrobial Potential of Its Crude Extract

A marine Alphaproteobacterium designated as strain NZ-96T was isolated in February 2021, from a sponge species (Demospongiae) collected in muddy sediments with boulders and old chimneys in Otago/Canterbury Slope, Pacific Ocean, New Zealand. The isolate was found to be Gram-negative, rod-shaped, aerobic, motile, and produced yellow-colored colonies. The isolate was positive for alkaline phosphatase, leucine arylamidase, trypsin, catalase, and oxidase and negative for α-galactosidase and urease. It was resistant to many antibiotics including hygromycin, trimethoprim, spectinomycin, ampicillin, oxytetracycline, cephalosporin, bacitracin, and polymyxin. The 16S rRNA gene-based phylogenetic analyses exhibited that strain NZ-96T belonged to the genus Qipengyuania and showed 98.3–98.8% 16S rRNA gene sequence similarity to its closest relatives. The major respiratory quinone was ubiquinone-10 (Q-10). The polar lipid profile consisted of phosphatidylcholine, sphingoglycolipid, phosphatidylglycerol, one unknown polar lipid, and three unknown glycolipids. The major fatty acids were C18:1ω12t, C16:0, C16:1ω7c, C17:1ω6c, C16:02-OH, and C14:0 2-OH. Carotenoid were produced. The crude extract showed pronounced activity against Staphylococcus aureus Newman and Bacillus subtilis DSM 10. Pairwise ANI and dDDH values of strain NZ-96T and closely related phylogenetic hits were below the threshold values of 95% and 70%, respectively. Genes for trehalose biosynthesis, aspartate-semialdehyde dehydrogenase, flagellar biosynthesis, fatty acid biosynthesis, and antibiotics resistance were present, which aids in isolate survival in a sea or ocean environment. The DNA G+C content was 60.8% (by genome). Based on data obtained by the polyphasic approach, strain NZ-96T (= DSM 112811T = NCCB 100842T) represents a novel species of the genus Qipengyuania, for which the name Qipengyuania pacifica sp. nov. is proposed.

Engineering Escherichia coli for high-yield production of ectoine

Ectoine is a natural macromolecule protector and synthesized by some extremophiles. It provides protections against radiation-mediated oxidative damages and is widely used as a bioactive ingredient in pharmaceutics and cosmetics. To meet its growing commercial demands, we engineered Escherichia coli strains for the high-yield production of ectoine. The ectABC gene cluster from the native ectoine producer Halomonas elongata was introduced into different Escherichia coli (E. Coil) strains via plasmids and 0.8 g L-1 of ectoine was produced in flask cultures by engineered E. coli BL21 (DE3). Subsequently, we designed the ribosome-binding sites of the gene cluster to fine-tune the expressions of genes ectA, ectB, and ectC, which increased the ectoine yield to 1.6 g L-1. After further combinatorial overexpression of Corynebacterium glutamicum aspartate kinase mutant (G1A, C932T) and the H. elongate aspartate-semialdehyde dehydrogenase to increase the supply of the precursor, the titer of ectoine reached to 5.5 g L-1 in flask cultures. Finally, the engineered strain produced 60.7 g L-1 ectoine in fed-batch cultures with a conversion rate of 0.25 g/g glucose.

Design and testing of selective inactivators against an antifungal enzyme target.

Systemic infections from fungal organisms are becoming increasingly difficult to treat as drug resistance continues to emerge. To substantially expand the antifungal drug landscape new compounds must be identified and developed with novel modes of action against previously untested drug targets. Most drugs block the activity of their targets through reversible, noncovalent interactions. However, a significant number of drugs form irreversible, covalent bonds with their selected targets. While more challenging to develop, these irreversible inactivators offer some significant advantages as novel antifungal agents. Vinyl sulfones contain a potentially reactive functional group that could function as a selective enzyme inactivator, and members of this class of compounds are now being developed as inactivators against an antifungal drug target. The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a key step in an essential microbial pathway and is essential for the survival of every microorganism examined. A series of vinyl sulfones have been designed, guided by molecular modeling and docking studies to enhance their affinity for fungal ASADHs. These newly synthesized compounds have been examined against this target enzyme from the pathogenic fungal organism Candida albicans. Vinyl sulfones containing complementary structural elements inhibit this enzyme with inhibition constants in the low-micromolar range. These inhibitors have also led to the rapid and irreversible inactivation of this enzyme, and show some initial selectivity when compared to the inactivation of a bacterial ASADH. The best inactivators will serve as lead compounds for the development of potent and selective antifungal agents.

Quest for Novel Preventive and Therapeutic Options Against Multidrug-Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa (P. aeruginosa) is a critical healthcare challenge due to its ability to cause persistent infections and the acquisition of antibiotic resistance mechanisms. Lack of preventive vaccines and rampant drug resistance phenomenon has rendered patients vulnerable. As new antimicrobials are in the preclinical stages of development, mining for the unexploited drug targets is also crucial. In the present study, we designed a B- and T-cell multi-epitope vaccine against P. aeruginosa using a subtractive proteomics and immunoinformatics approach. A total of five proteins were shortlisted based on essentiality, extracellular localization, virulence, antigenicity, pathway association, hydrophilicity, and low molecular weight. These include two outer membrane porins; OprF (P13794) and OprD (P32722), a protein activator precursor pra (G3XDA9), a probable outer membrane protein precursor PA1288 (Q9I456), and a conserved hypothetical protein PA4874 (Q9HUT9). These shortlisted proteins were further analyzed to identify immunogenic and antigenic B- and T-cell epitopes. The best scoring epitopes were then further subjected to the construction of a polypeptide multi-epitope vaccine and joined with cholera toxin B subunit adjuvant. The final chimeric construct was docked with TLR4 and confirmed by normal mode simulation studies. The designed B- and T-cell multi-epitope vaccine candidate is predicted immunogenic in nature and has shown strong interactions with TLR-4. Immune simulation predicted high-level production of B- and T-cell population and maximal expression was ensured in E. coli strain K12. The identified drug targets qualifying the screening criteria were: UDP-2-acetamido-2-deoxy-d-glucuronic acid 3-dehydrogenase WbpB (G3XD23), aspartate semialdehyde dehydrogenase (Q51344), 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (Q9HV71), 3-deoxy-D-manno-octulosonic-acid transferase (Q9HUH7), glycyl-tRNA synthetase alpha chain (Q9I7B7), riboflavin kinase/FAD synthase (Q9HVM3), aconitate hydratase 2 (Q9I2V5), probable glycosyltransferase WbpH (G3XD85) and UDP-3-O-[3-hydroxylauroyl] glucosamine N-acyltransferase (Q9HXY6). For druggability and pocketome analysis crystal and homology structures of these proteins were retrieved and developed. A sequence-based search was performed in different databases (ChEMBL, Drug Bank, PubChem and Pseudomonas database) for the availability of reported ligands and tested drugs for the screened targets. These predicted targets may provide a basis for the development of reliable antibacterial preventive and therapeutic options against P. aeruginosa.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10989-021-10255-3.

Combinatorial Design to Decipher Novel Lead Molecule against Mycobacterium tuberculosis

Aspartate-semialdehyde dehydrogenase (ASADH) of DAP/lysine pathway plays a crucial role in sustainable growth and pathogenicity of Mycobacterium tuberculosis (Mtb) via reductive dephosphorylation of the β-aspartyl phosphate (AP). Inhibition of ASADH through different lead molecules has been gaining high impetus due to its indispensable role in the pathogen’s survival. In the present study, we aimed to decipher the novel lead molecule against Mtb. The AP, a substrate of the DAP/lysine pathway, was used as a template to design new lead molecules to advance the understanding of the molecular inhibition mechanism of Mtb-ASADH. Monodentate and bidentate groups at three different substitution sites of AP were considered to generate a virtual library of new molecules using the combinatorial approach of the LeadGrow module of the VLifeMDS package. These substrate analogs were sifted through ADRXWS drug-likeness descriptors of the module above. Multi-scoring docking was achieved using Biopredicta, Molecular Virtual Docker, and AutoDock Tools. The adopted combinatorial approach yielded 6000 new molecules that reduced to 4979 plausible hits after lead-like filtration. The post-analysis of ADMET and molecular docking exhibited two pro-lead molecules, namely AP0600 and AP0639. The study delineates the substantial understanding of the Mtb-ASADH inhibition mechanism that would undoubtedly accelerate the pace of antitubercular design, thereby gaining more in-depth knowledge to eradicate tuberculosis across the globe.

IMB-XMA0038, a new inhibitor targeting aspartate-semialdehyde dehydrogenase of Mycobacterium tuberculosis

The emergence of drug-resistant tuberculosis (TB) constitutes a major challenge to TB control programs. There is an urgent need to develop effective anti-TB drugs with novel mechanisms of action. Aspartate-semialdehyde dehydrogenase (ASADH) is the second enzyme in the aspartate metabolic pathway. The absence of the pathway in humans and the absolute requirement of aspartate in bacteria make ASADH a highly attractive drug target. In this study, we used ASADH coupled with Escherichia coli type III aspartate kinase (LysC) to establish a high-throughput screening method to find new anti-TB inhibitors. IMB-XMA0038 was identified as an inhibitor of MtASADH with an IC50 value of 0.59 μg/mL through screening. The interaction between IMB-XMA0038 and MtASADH was confirmed by surface plasmon resonance (SPR) assay and molecular docking analysis. Furthermore, IMB-XMA0038 was found to inhibit various drug-resistant MTB strains potently with minimal inhibitory concentrations (MICs) of 0.25-0.5 μg/mL. The conditional mutant strain MTB::asadh cultured with different concentrations of inducer (10−5 or 10−1 μg/mL pristinamycin) resulted in a maximal 16 times difference in MICs. At the same time, IMB-XMA0038 showed low cytotoxicity in vitro and vivo. In mouse model, it encouragingly declined the MTB colony forming units (CFU) in lung by 1.67 log10 dosed at 25 mg/kg for 15 days. In conclusion, our data demonstrate that IMB-XMA0038 is a promising lead compound against drug-resistant tuberculosis.

Molecular evolution, binding site interpretation and functional divergence of aspartate semialdehyde dehydrogenase

Aspartate Semialdehyde Dehydrogenase (ASDH) is an important enzyme essential for the viability of pathogenic microorganisms. ASDH is mainly involved in amino acid and cell wall biosynthesis of microorganisms, hence it is considered to be a promising target for drug design. This enzyme depicts similar mechanistic function in all microorganisms; although, the kinetic efficiency of an enzyme differs according to their active site residual composition. Therefore, understanding the residual variation and kinetic efficiency of the enzyme would pave new insights in structure-based drug discovery and a novel drug molecule against ASDH. Here, ASDH from Wolbachia endosymbiont of Brugia malayi is used as a prime enzyme to execute evolutionary studies. The phylogenetic analysis was opted to classify 400 sequences of ASDH enzymes based on their structure and electrostatic surfaces. Analysis resulted in 37 monophyletic clades of diverse pathogenic and non-pathogenic organisms. The representative structures of 37 ASDHs from different clades were further deciphered to structural homologues. These enzymes exhibited presence of more positively charged surfaces than negatively charged surfaces in the active site pocket which restrains evolutionary significance. Docking studies of NADP+ with 37 enzymes reveals that site-specific residual variation in the active site pocket modulates the binding affinity (ranges of −13 to −9 kcal/mol). Type-I and Type-II divergence studies show, no significant functional divergence among ASDH, but residual changes were found among the enzyme that modulates the biochemical characteristics and catalytic efficiency. The present study not only explores residual alteration and catalytic variability, it also aids in the design of species-specific inhibitors. Communicated by Ramaswamy H. Sarma

Cinnamomum zeylanicum (Cinnamon) Derived Phytochemicals against Aspartate Semialdehyde Dehydrogenase of Aspergillus fumigatus Causing Aspergillosis

Aspergillosis is an infection caused by a type of fungus. The illnesses is caused by aspergillosis infection and usually attack the respiratory system, but their signs and severity vary greatly. It has already been established that plant Cinnamomum zeylanicum belonging to the Lauraceae family has the potential to help controlling aspergillosis. This work is focused on the identification of the particular phytochemical responsible for inhibiting and decreasing the effect of aspergillosis. Molecular docking method applied using “Biovia Discovery Studio”. “High positive values of -CDOCKER energy and -CDOCKER interaction energy” suggested that cinnamic acid can effectively deactivate aspergillosis.

Aspartate semialdehyde dehydrogenase inhibition suppresses the growth of the pathogenic fungus Candida albicans.

Potent inhibitors of an essential microbial enzyme have been shown to be effective growth inhibitors of Candida albicans, a pathogenic fungus. C. albicans is the main cause of oropharyngeal candidiasis, and also causes invasive fungal infections, including systemic sepsis, leading to serious complications in immunocompromised patients. As the rates of drug-resistant fungal infections continue to rise novel antifungal treatments are desperately needed. The enzyme aspartate semialdehyde dehydrogenase (ASADH) is critical for the functioning of the aspartate biosynthetic pathway in microbes and plants. Because the aspartate pathway is absent in humans, ASADH has the potential to be a promising new target for antifungal research. Deleting the asd gene encoding for ASADH significantly decreases the survival of C. albicans, establishing this enzyme as essential for this organism. Previously developed ASADH inhibitors were tested against several strains of C. albicans to measure their possible therapeutic impact. The more potent inhibitors show a good correlation between enzyme inhibitor potency and fungal growth inhibition. Growth curves generated by incubating different C. albicans strains with varying enzyme inhibitor levels show significant slowing of fungal growth by these inhibitors against each of these strains, similar to the effect observed with a clinical antifungal drug. The most effective inhibitors also demonstrated relatively low cytotoxicity against a human epithelial cell line. Taken together, these results establish that the ASADH enzyme is a promising new target for further development as a novel antifungal treatment against C. albicans and related fungal species.

Ectoine degradation pathway in halotolerant methylotrophs.

BackgroundMicroorganisms living in saline environments are forced to regulate turgor via the synthesis of organic osmoprotective compounds. Microbial adaptation to fluctuations in external salinity includes degradation of compatible solutes. Here we have examined the biochemical pathway of degradation of the cyclic imino acid ectoine, the major osmoprotector in halotolerant methane-utilizing bacteria.MethodsThe BLAST search of the genes involved in ectoine degradation in the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z was performed with the reference sequences of Halomonas elongata. The genes for the key enzymes of the pathway were disrupted by insertion mutagenesis and the cellular metabolites in the methanol extracts of mutant cells were analyzed by HPLC. The doeA gene from Mm. alcaliphilum 20Z was heterologously expressed in Escherichia coli to identify the product of ectoine hydrolysis catalyzed by ectoine hydrolase DoeA.ResultsWe have shown that the halotolerant methanotroph Mm. alcaliphilum 20Z possesses the doeBDAC gene cluster coding for putative ectoine hydrolase (DoeA), Nα-acetyl-L-2,4-diaminobutyrate deacetylase (DoeB), diaminobutyrate transaminase (DoeD) and aspartate-semialdehyde dehydrogenase (DoeC). The deletion of the doeA gene resulted in accumulation of the higher level of ectoine compared to the wild type strain. Nγ-acetyl-L-2,4-diaminobutyrate (Nγ-acetyl-DAB), a substrate for ectoine synthase, was found in the cytoplasm of the wild type strain. Nα-acetyl-L-2,4-diaminobutyrate (Nα-acetyl-DAB), a substrate for the DoeB enzyme, appeared in the cells as a result of exposure of the doeB mutant to low osmotic pressure. The genes for the enzymes involved in ectoine degradation were found in all aerobic methylotrophs capable of ectoine biosynthesis. These results provide the first evidence for the in vivo operation of the ectoine degradation pathway in methanotrophs and thus expand our understanding of the regulation mechanisms of bacterial osmoadaptation.ConclusionsDuring adaptation to the changes in external osmolarity, halophilic and halotolerant methylotrophs cleave ectoine, thereby entering the carbon and nitrogen of the compatible solute to the central metabolic pathways. The biochemical route of ectoine degradation in the halotolerant methanotroph Mm. alcaliphilum 20Z is similar to that in heterotrophic halophiles. We have shown that ectoine hydrolase DoeA in this methanotroph hydrolyzes ectoine with the formation of the only isomer: Nα-acetyl-DAB. All aerobic methylotrophs capable of ectoine biosynthesis harbor the genetic determinants for ectoine degradation.