MFS Transporter Research Articles

Molecular insights into biological transformation mechanism of sulfathiazole by Chlorella sorokiniana: Deciphering the uptake, translocation, and biotransformation

As a sustainable approach to wastewater treatment, microalgae have been extensively used to degrade antibiotics. However, the underlying mechanisms involved in the degradation process remain unclear. Therefore, this study investigated the biotransformation mechanism of sulfathiazole (STZ) by Chlorella sorokiniana (C. sorokiniana) at the molecular level. The results show that C. sorokiniana could efficiently degrade STZ, achieving a maximum degradation rate of 94.74 %, mainly through biodegradation routes. Transcriptome analysis has elucidated the potential biological transformation mechanisms driving the degradation of STZ by microalgae, focusing on the uptake, translocation, and biotransformation as key metabolic processes. In particular, STZ induced the up-regulation of genes associated with cell adhesion, membrane protein, and lipopolysaccharide, suggesting their involvement in the uptake of STZ by microalgae. Furthermore, ABC, MATE, and MFS transporters were identified as crucial for the transmembrane transport of STZ by microalgae. A plausible biotransformation pathway for STZ degradation was proposed, identifying hydroxylation, oxidation, ring cleavage, and formylation as the primary transformation processes. The up-regulation of key enzymes such as monooxygenases, dioxygenases, hydrolases, and transferases suggested their pivotal role in the biodegradation of STZ. This research provides valuable insights into the biotransformation mechanisms of STZ by microalgae, thereby laying a theoretical framework to advance the implementation of microalgae in the treatment of antibiotic-contaminated wastewater.

MFS Transporter as the Molecular Switch Unlocking the Production of Cage-Like Acresorbicillinol C.

Sorbicillinoids are a class of fungal polyketides with diverse structures and distinguished bioactivities. Although remarkable progress has been achieved in their chemistry and biosynthesis, the efflux of sorbicillinoids is poorly understood. Here, we found MFS transporter AcsorT was responsible for the biosynthesis of sorbicillinoids in Acremonium chrysogenum. Combinatorial knockout and subcellular location demonstrated that the plasma membrane-associated AcsorT was responsible for the transportation of sorbicillinol and subsequent formation of oxosorbicillinol and acresorbicillinol C via the berberine bridge enzyme-like oxidase AcsorD in the periplasm. Homology modeling and site-directed mutation revealed that Tyr303 and Arg436 were the key residues of AcsorT, which was further explained by molecular dynamics simulation. Based on our study, it was suggested that AcsorT modulates sorbicillinoid production by coordinating its biosynthesis and export, and a transport model of sorbicillinoids was proposed in A. chrysogenum.

Corynebacterium pseudotuberculosis: Whole genome sequencing reveals unforeseen and relevant genetic diversity in this pathogen.

Corynebacterium pseudotuberculosis (CPS) is an important bacterial animal pathogen. CPS causes chronic, debilitating and currently incurable infectious diseases affecting a wide range of livestock and wild herbivores including camelids worldwide. Belonging to the Corynebacterium diphtheriae complex, this pathogen can also infect humans. The classical characterization of CPS is typically based on the testing of nitrate reductase activity, separating the two biovars Equi and Ovis. However, more refined resolutions are required to unravel routes of infection. This was realized in our study by generating and analyzing whole genome sequencing (WGS) data. Using newly created core genome multilocus sequence typing (cgMLST) profiles we were the first to discover isolates grouping in a cluster adjacent to clusters formed by CPS biovar Equi isolates. This novel cluster includes CPS isolates from alpacas, llamas, camels and dromedaries, which are characterized by a lack of nitrate reductase activity as encountered in biovar Ovis. This is of special interest for molecular epidemiology. Nevertheless, these isolates bear the genes of the nitrate locus, which are characteristic of biovar Equi isolates. However, sequence analysis of the genes narG and narH of the nitrate locus revealed indels leading to frameshifts and inactivity of the enzymes involved in nitrate reduction. Interestingly, one CPS isolate originating from another lama with an insertion in the MFS transporter (narT) is adjacent to a cluster formed by ovine CPS isolates biovar Equi. Based on this knowledge, the combination of biochemical and PCR based molecular biological nitrate reductase detection can be used for a fast and uncomplicated classification of isolates in routine diagnostics in order to check the origin of camelid CPS isolates. Further analysis revealed that partial sequencing of the ABC transporter substrate binding protein (CP258_RS07935) is a powerful tool to assign the biovars and the novel genomovar.

ARTP mutagenesis of Aureobasidium pullulans RM1603 for high pullulan production and transcriptome analysis of mutants.

Pullulan is a microbial exopolysaccharide produced by Aureobasidium spp. with excellent physical and chemical properties, resulting in great application value. In this study, a novel strain RM1603 of Aureobasidium pullulans with high pullulan production of 51.0 ± 1.0g·L- 1 isolated from rhizosphere soil was subjected to atmospheric and room temperature plasma (ARTP) mutagenesis, followed by selection of mutants to obtain pullulan high-producing strains. Finally, two mutants Mu0816 and Mu1519 were obtained, with polysaccharide productions of 58.7 ± 0.8 and 60.0 ± 0.8g∙L- 1 after 72-h fermentation, representing 15.1 and 17.6% increases compared with the original strain, respectively. Transcriptome analysis of the two mutants and the original strain revealed that the high expression of α/β-hydrolase (ABHD), α-amylase (AMY1), and sugar porter family MFS transporters (SPF-MFS) in the mutants may be related to the synthesis and secretion of pullulan. These results demonstrated the effectiveness of ARTP mutagenesis in A. pullulans, providing a basis for the investigation of genes related to pullulan synthesis and secretion.

A new understanding of the regulatory mechanism by which Fe/Mn nanoparticles boost Bisphenol A removal using Comamonas testosteroni

Green synthesized iron/manganese nanoparticles (Fe/Mn NPs), acted as an exogenous promoter to enhance the lignin-degrading bacteria Comamonas testosteroni FJ17 resulting in more efficient removal of bisphenol A (BPA). Batch experiments demonstrated that removal efficiency of BPA via cells at a BPA concentration of 10 mg·L−1 increased by 20.9 % when exposed to 100 mg·L−1 Fe/Mn NPs after 48 h (93.63 %) relative to an unexposed control group (72.70 %). TEM and 3D-EEM analysis confirmed that the cell membrane thickness increased from 47 to 80 nm under Fe/Mn NPs exposure, and the TB-EPS secretion was promoted. Meanwhile, Fe/Mn NPs facilitated greater electron transfer capacity of c-cytochrome (0.55 V reduction peak) and an unknown cytochrome substance (0.7 V oxidation peak) on the surface of cells. Studies of the effect of Fe/Mn NPs on both the growth and activity of laccase cells showed that both biomass and laccase secretion increased significantly during the logarithmic growth period (6–36 h). LC-MS analysis and toxicity assessment indicated that Fe/Mn NPs decreased the degradation time of BPA and efficiently reduced the toxicity of its by-products. Transcriptomic analysis revealed 315 up-regulation of the key genes associated with energy supply, membrane translocation, and metabolic pathways upon exposure to Fe/Mn NPs. Such as MFS transporter (2.27-fold), diguanylate cyclase (1.76-fold) and protocatechuate-3,4-dioxygenase (1.62-fold). Overall, Fe/Mn NPs accelerated proliferation by enhancing metabolic capacity and nutrient transport processes, which serves to improve the efficiency of BPA removal.

Transcriptomic Analysis of Fludioxonil Resistance Mechanisms in Botrytis cinerea

Botrytis cinerea is a destructive plant pathogen that infects a wide range of economically important crops. Limiting pathogen infection during production and after harvest is largely dependent on fungicide applications; fungicide-resistant isolates of B. cinerea have been recovered from various hosts. Resistance of B. cinerea to fludioxonil has been associated with overexpression of transporter genes ( BcatrB and mfsM2) and mutations on histidine kinase proteins ( Bos1, Bchhk2, Bchhk17). To identify possible mechanisms associated with fludioxonil resistance, the genomic expression of three sensitive and three low-resistant isolates was studied. Overexpression of BcatrB was observed when comparing low-resistant and sensitive isolates but was not specific to the fludioxonil treatment. Seven amino acid substitutions and one deletion were identified in the transcription factor Bcmrr1 in low-resistant isolates, associated with overexpression of BcatrB. The L497 deletion, previously associated with highly resistant isolates (MDR1h), was observed in two low-resistant isolates. Other differentially expressed genes associated with transmembrane transport, oxidoreductase activity, and lipid metabolic processes could be key in understanding the fungicidal mechanism(s) of fludioxonil. Expression profiles were isolate-specific. Following fludioxonil exposure, two sensitive isolates of B. cinerea sensu stricto showed a change in gene expression levels associated with cell membrane and peroxidase activity. In one low-resistant isolate of B. cinerea group S, fludioxonil exposure resulted in the overexpression of stress response genes and MFS transporter Bcstl1; one sensitive and two low-resistant isolates showed no significant changes in gene expression profiles. This work provides insight into the effect of fludioxonil on B. cinerea and potential fungicide resistance mechanisms. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Elucidation of novel drought-responsive genes from tuber transcriptome of cassava under water deficit stress

Cassava (Manihot esculenta Carntz) is the major starchy root crop grown in the tropical parts of the world. Two cassava clones contrasting for their drought tolerance capacity (8S501- drought tolerant and H97-drought sensitive) were selected and grown in the field for studying the agronomical and molecular changes in both the clones under drought stress conditions. A reduction in the number of tuberous roots per plant, tuberous root length, average single tuber weight and tuber yield per plant was observed under drought stress when compared with the control conditions and also significantly lower mean values were observed for the sensitive clone H97 than that of the tolerant clone 8S501 when measured at 60 days of stress. Transcriptome sequencing of the tuber tissues of the two clones was done at 60 days of imposing drought stress. Different genes belonging to various groups like aquaporins, transporter genes, growth regulator related genes, calcium binding and mineral transfer-related genes, transferase genes and transcription factors were differentially expressed in the tubers under drought stress. Comparative transcriptomics revealed that acyl-CoA oxidase, primary-amine oxidase, glutathione peroxidase, vesicle transport, late embryogenesis abundant protein related, histone demethylase, BR-signaling kinase, palmitoyltransferase, DNA mismatch repair protein, heat shock protein related, starch/sucrose metabolic pathway related and MFS transporter genes were differentially expressed in the tubers of the tolerant clone 8S501 relative to the susceptible clone H97.

The Comparative Genomics of Botryosphaeriaceae Suggests Gene Families of Botryosphaeria dothidea Related to Pathogenicity on Chinese Hickory Tree.

Trunk canker poses a major threat to the production of Chinese hickory tree (Carya cathayensis Sarg.), which is primarily determined by Botryosphaeriaceae. In our previous work, we identified Botryosphaeria dothidea as the predominant pathogen of this disease. However, it is still unclear about corresponding gene families and mechanisms associated with B. dothidea's pathogenicity on Chinese hickory tree. Here, we present a comparative analysis of high-quality genome assemblies of Botryosphaeria dothidea and other isolated pathogens, showing highly syntenic relationships between B. dothidea and its closely related species and the conservative evolution of the Botryosphaeriaceae family. Higher GC contents were found in the genomes of B. dothidea and three other isolated pathogens (Botryshaeria cortices, Botryshaeria fabicerciana, and Botryshaeria qingyuanensis) compared to Macrophomina phaseolina, Neofusicoccum parvum, Diplodia corticola, and Lasiodiplodia theobromae. An investigation of genes specific to or expanded in B. dothidea revealed that one secreted glucanase, one orsellinic acid biosynthesis enzyme, and two MFS transporters positively regulated B. dothidea's pathogenicity. We also observed an overrepresentation of viral integrase like gene and heterokaryon incompatibility proteins in the B. dothidea's genome. In addition, we observed one LRR-domain-containing protein and two Sec-domain-containing proteins (Sec_1 and Sec_7) that underwent positive selection. This study will help to understand B. dothidea's pathogenicity and potential influence on the infection of Chinese hickory, which will help in the development of disease control and ensure the security of Chinese hickory production.

Integrated transcriptomics and metabolomics analyses reveal the aerobic biodegradation and molecular mechanisms of 2,3′,4,4′,5-pentachlorodiphenyl (PCB 118) in Methylorubrum sp. ZY-1

2,3′,4,4′,5-pentachlorodiphenyl (PCB 118), a highly representative PCB congener, has been frequently detected in various environments, garnering much attention across the scientific community. The degradation of highly chlorinated PCBs by aerobic microorganisms is challenging due to their hydrophobicity and persistence. Herein, the biodegradation and adaptation mechanisms of Methylorubrum sp. ZY-1 to PCB 118 were comprehensively investigated using an integrative approach that combined degradation performance, product identification, metabolomic and transcriptomic analyses. The results indicated that the highest degradation efficiency of 0.5 mg L−1 PCB 118 reached 75.66% after seven days of inoculation when the bacteria dosage was 1.0 g L−1 at pH 7.0. A total of eleven products were identified during the degradation process, including low chlorinated PCBs, hydroxylated PCBs, and ring-opening products, suggesting that strain ZY-1 degraded PCB 118 through dechlorination, hydroxylation, and ring-opening pathways. Metabolomic analysis demonstrated that the energy supply and redox metabolism of strain ZY-1 was disturbed with exposure to PCB 118. To counteract this environmental stress, strain ZY-1 adjusted both the fatty acid synthesis and purine metabolism. The analysis of transcriptomics disclosed that multiple intracellular and extracellular oxidoreductases (e.g., monooxygenase, alpha/beta hydrolase and cytochrome P450) participated in the degradation of PCB 118. Besides, active efflux of PCB 118 and its degradation intermediates mediated by multiple transporters (e.g., MFS transporter and ABC transporter ATP-binding protein) might enhance bacterial resistance against these substances. These discoveries provided the inaugural insights into the biotransformation of strain ZY-1 to PCB 118 stress, illustrating its potential in the remediation of contaminated environments.

Molecular Mechanisms and Biological Characteristics of Botrytis cinerea Field Isolate Resistance to Pyrisoxazole in Liaoning Province.

Botrytis cinerea is a broad-host-range necrotrophic phytopathogen responsible for serious diseases in leading crops worldwide. The novel sterol 14α-demethylase inhibitor (DMI) pyrisoxazole was recently registered for the control of tomato gray mold caused by B. cinerea in China. One hundred fifty-seven isolates of B. cinerea were collected from tomato greenhouses in 14 cities of Liaoning Province from 2016 to 2021 and examined for sensitivity to pyrisoxazole, with a mean EC50 value of 0.151 μg/ml. Three highly resistant isolates, XD-5, DG-4, and GQ-3, were screened, and the EC50 values were 0.734, 0.606, and 0.639 μg/ml with corresponding resistance factors of 12.88, 10.63, and 11.21, respectively. Compared with field-sensitive strains, the highly resistant isolate XD-5 exhibited fitness defects in traits, including mycelial growth, conidial production, and pathogenicity, but DG-4 and GQ-3 did not experience fitness costs. Positive cross-resistance was observed only between pyrisoxazole and the DMIs tebuconazole and prochloraz but not between pyrisoxazole and the non-DMIs iprodione, procymidone, pyrimethanil, fludioxonil, fluazinam, and fluopyram. Sequence alignment of the CYP51 gene indicated that three point mutations were observed in the highly resistant mutant, namely, V24I in XD-5, G461S in GQ-3, and R464K in DG-4. When exposed to pyrisoxazole, the induced expression levels of the ABC transporter AtrD and MFS transporter Mfs1 increased in the resistant isolates compared with those in the sensitive isolates, whereas the expression level of the CYP51 gene did not change significantly. Molecular docking suggested that the G461S and R464K mutations both led to a decrease in the binding energy between CYP51 and pyrisoxazole, whereas no change was found with the V24I mutation. Thus, two point mutations in the CYP51 protein combined with induced expression of the Mfs1 and AtrD genes appeared to mediate the pyrisoxazole resistance of the highly resistant mutants DG-4 and GQ-3, while the overexpression of the Mfs1 and AtrD genes was responsible for the highly resistant mutant XD-5.

Identification of the Ilex macrocarpa anthracnose pathogen and the antifungal potential of the cell-free supernatant of Bacillus velezensis against Colletotrichum fioriniae.

Anthracnose is a significant fungal disease that affects tree growth and development, with Colletotrichum spp. exhibiting host non-specificity and targeting various organs, making disease control challenging. This study aimed to identify the pathogenic species causing anthracnose in Ilex macrocarpa in Nanchong, Sichuan Province, and screen effective fungicides, particularly biological ones. The pathogen was identified as Colletotrichum fioriniae through morphological observation, pathogenicity assays, and molecular biological methods. Three biological and five chemical fungicides were evaluated for their effects on the mycelial growth and spore germination rate of the pathogen. The results indicated that prochloraz was the most effective chemical fungicide, while the cell-free supernatant (CFS) of Bacillus velezensis had the most significant inhibitory effect among the biological fungicides. Transcriptome analysis revealed that the CFS of B. velezensis significantly reduced the expression of genes associated with ribosomes, genetic information processing, membrane lipid metabolism, and sphingolipid biosynthesis in C. fioriniae. Additionally, the glutathione pathway's expression of various genes, including key genes such as GST, GFA, Grx, TRR, and POD, was induced. Furthermore, the expression of 17 MFS transporters and 9 ABC transporters was increased. Autophagy-related ATGs were also affected by the B. velezensis CFS. These findings suggest that the B. velezensis CFS may inhibit C. fioriniae through interference with ribosomes, genetic information processing, cell membrane metabolism, and energy metabolism. These results provide potential target genes for the B. velezensis CFS and insights into the antifungal mechanism by which B. velezensis inhibits C. fioriniae.

Effect of Atractylodes essential oil on different Colletotrichum species causing tea brown blight disease

As a traditional Chinese medicine, atractylodes has the potential to be an antifungal agent for controlling fungal diseases. Essential oils with the advantage of vapor phase bioactivity makes them have wider potential applications. In this study, 5 different species in genus Colletotrichum causing tea brown blight disease were firstly employed to investigate the antifungal activities of atractylodes essential oil (AO) by direct contact and fumigation methods. Our results indicated that the tested AO had significantly higher antifungal effect in contact phase than that in vapor phase (P < 0.05). It is important that AO showed different antifungal activities against C. karstii, C. gloeosporioides, C. camelliae, C. fioriniae and C. chongqingense with EC50 of 0.089, 0.165, 0.108, 0.205 and 0.092 mg·mL−1, respectively. The growth of Colletotrichum species were all inhibited by influencing the morphology of conidia and hyphae, damaging the cell walls and then increasing the membrane permeability. After AO treatment, total 949 up-regulated and 1360 down-regulated genes were detected in treated C. gloeosporioides, and the significantly differential genes were mainly related to ABC transporters, MFS transporters and cytochrome P450s. KEGG pathways analysis revealed the significantly differential genes were mainly enriched in carbohydrate metabolism especially fructose and mannose metabolism, which may act as the main energy source for the activities of fungi. Five differential genes related to ABC, MFS and CYP families showed significantly different expression levels among different Colletotrichum species by qRT-PCR analysis. This study fully reveals the differential antifungal activities and mechanism of different Colletotrichum species against AO.

The Escherichia coli MFS-type transporter genes yhjE, ydiM, and yfcJ are required to produce an active bo3 quinol oxidase.

Heme-copper oxygen reductases are membrane-bound oligomeric complexes that are integral to prokaryotic and eukaryotic aerobic respiratory chains. Biogenesis of these enzymes is complex and requires coordinated assembly of the subunits and their cofactors. Some of the components are involved in the acquisition and integration of different heme and copper (Cu) cofactors into these terminal oxygen reductases. As such, MFS-type transporters of the CalT family (e.g., CcoA) are required for Cu import and heme-CuB center biogenesis of the cbb3-type cytochrome c oxidases (cbb3-Cox). However, functionally homologous Cu transporters for similar heme-Cu containing bo3-type quinol oxidases (bo3-Qox) are unknown. Despite the occurrence of multiple MFS-type transporters, orthologs of CcoA are absent in bacteria like Escherichia coli that contain bo3-Qox. In this work, we identified a subset of uncharacterized MFS transporters, based on the presence of putative metal-binding residues, as likely candidates for the missing Cu transporter. Using a genetic approach, we tested whether these transporters are involved in the biogenesis of E. coli bo3-Qox. When respiratory growth is dependent on bo3-Qox, because of deletion of the bd-type Qox enzymes, three candidate genes, yhjE, ydiM, and yfcJ, were found to be critical for E. coli growth. Radioactive metal uptake assays showed that ΔydiM has a slower 64Cu uptake, whereas ΔyhjE accumulates reduced 55Fe in the cell, while no similar uptake defect is associated with ΔycfJ. Phylogenomic analyses suggest plausible roles for the YhjE, YdiM, and YfcJ transporters, and overall findings illustrate the diverse roles that the MFS-type transporters play in cellular metal homeostasis and production of active heme-Cu oxygen reductases.

Mutation in the two-component regulator BaeSR mediates cefiderocol resistance and enhances virulence in Acinetobacter baumannii.

Acinetobacter baumannii has become one of the most challenging pathogens in many countries with limited treatment options available. Cefiderocol, a novel siderophore-conjugated cephalosporin, shows potent in vitro activity against A. baumannii, including isolates resistant to carbapenems. To date, few reports on the mechanisms of cefiderocol resistance are available. In order to investigate potential mechanisms of cefiderocol resistance in A. baumannii, we performed in vitro evolution experiments at sub-lethal concentrations of the antibiotic. All four cefiderocol-resistant strains obtained harbored mutations in two-component system BaeS-BaeR. When we engineered the mutations of BaeS (D89V) and BaeR (S104N) into the genome of ATCC 17978, these mutations increased cefiderocol minimum inhibitory concentrations (MICs) by 8-fold to 16-fold. Transcriptome analyses showed that the expression of MacAB-TolC and MFS transporters was up-regulated in BaeSR mutants. Strains over-expressing MFS transporter and MacAB-TolC displayed higher MICs and higher median inhibition concentration (IC50) values, while MICs and IC50 decreased when efflux pump genes were knocked out. In a BaeR mutant with up-regulated csu operon, we observed a higher number of pili, enhanced surface motility, and increased biofilm formation compared to wild-type ATCC 17978. Using the Galleria mellonella infection model, we found that the BaeS mutant in which paa operon was up-regulated exhibited increased virulence. In conclusion, the mutations in BaeSR decreased cefiderocol susceptibility of A. baumannii through up-regulating efflux pumps gene expression. BaeS or BaeR also controls the expression of csu and paa, influencing biofilm formation, surface motility, and virulence in A. baumannii. IMPORTANCE The widespread prevalence of multi-drug-resistant A. baumannii (MDRAB) poses a significant therapeutic challenge. Cefiderocol is considered a promising antibiotic for the treatment of MDRAB infections. Therefore, it is necessary to study the potential resistance mechanisms of cefiderocol to delay the development of bacterial resistance. Here, we demonstrated that mutations in baeS and baeR reduced the susceptibility of A. baumannii to cefiderocol by up-regulating the expression of the MFS family efflux pump and MacAB-TolC efflux pump. We propose that BaeS mutants increase bacterial virulence by up-regulating the expression of the paa operon. This also reports the regulatory effect of BaeSR on csu operon for the first time. This study provides further insights into the role of BaeSR in developing cefiderocol resistance and virulence in A. baumannii.

An oxidoreductase gene CtnD involved in citrinin biosynthesis in Monascus purpureus verified by CRISPR/Cas9 gene editing and overexpression.

Monascus produces a kind of mycotoxin, citrinin, whose synthetic pathway is still not entirely clear. The function of CtnD, a putative oxidoreductase located upstream of pksCT in the citrinin gene cluster, has not been reported. In this study, the CtnD overexpressed strain and the Cas9 constitutively expressed chassis strain were obtained by genetic transformation mediated by Agrobacterium tumefaciens. The pyrG and CtnD double gene-edited strains were then obtained by transforming the protoplasts of the Cas9 chassis strain with in vitro sgRNAs. The results showed that overexpression of CtnD resulted in significant increases in citrinin content of more than 31.7% and 67.7% in the mycelium and fermented broth, respectively. The edited CtnD caused citrinin levels to be reduced by more than 91% in the mycelium and 98% in the fermented broth, respectively. It was shown that CtnD is a key enzyme involved in citrinin biosynthesis. RNA-Seq and RT-qPCR showed that the overexpression of CtnD had no significant effect on the expression of CtnA, CtnB, CtnE, and CtnF but led to distinct changes in the expression of acyl-CoA thioesterase and two MFS transporters, which may play an unknown role in citrinin metabolism. This study is the first to report the important function of CtnD in M. purpureus through a combination of CRISPR/Cas9 editing and overexpression.

Novel antifungal triazoles with alkynyl-methoxyl side chains: Design, synthesis, and biological activity evaluation

Previous work led to the rational design, synthesis and testing of novel antifungal triazole analogues bearing alkynyl-methoxyl side chains. Tests of in vitro antifungal activity showed Candida albicans SC5314 and Candida glabrata 537 gave MIC values of ≤0.125 μg/mL for most of the compounds. Among these, compounds 16, 18, and 29 displayed broad-spectrum antifungal activity against seven human pathogenic fungal species, two fluconazole-resistant C. albicans isolates and two multi-drug resistant Candida auris isolates. Moreover, 0.5 μg/mL of 16, 18, and 29 was more effective than 2 μg/mL of fluconazole at inhibiting fungal growth of the strains tested. The most active compound (16) completely inhibited the growth of C. albicans SC5314 at 16 μg/mL for 24 h, affected biofilm formation and destroyed the mature biofilm at 64 μg/mL. Several Saccharomyces cerevisiae strains, overexpressing recombinant Cyp51s or drug efflux pumps, indicated 16, 18, and 29 targeted Cyp51 without being significantly affected by a common active site mutation, but were susceptible to target overexpression and efflux by both MFS and ABC transporters. GC-MS analysis demonstrated that 16, 18, and 29 interfered with the C. albicans ergosterol biosynthesis pathway by inhibition at Cyp51. Molecular docking studies elucidated the binding modes of 18 with Cyp51. The compounds showed low cytotoxicity, low hemolytic activity and favorable ADMT properties. Importantly, compound 16 showed potent in vivo antifungal efficacy in the G. mellonella infection model. Taken together, this study presents more effective, broad-spectrum, low toxicity triazole analogues that can contribute to the development of novel antifungal agents and help overcome antifungal resistance.

A novel transporter ThuT, couples the biosynthesis and secretion of thuringiensin

Thuringiensin is a thermostable nucleoside metabolite produced by Bacillus thuringiensis and has insecticidal activity against various pests. The synthesis cluster of thuringiensin was cloned and the biosynthesis pathway deduced and clarified in a previous study. ThuE catalyzes phosphorylation from precursor C to thuringiensin in the final step of thuringiensin maturation. However, the transport mechanism of thuringiensin remains unclear. Here, we identify a novel predicted MFS transporter named ThuT (thuringiensin transporter) which is necessary for the biosynthesis and secretion of thuringiensin. A thuT gene deletion resulted in a massive loss of thuringiensin in the extracellular space and a small accumulation in the intracellular space, implying its role in the transport of thuringiensin. The overall abundance of thuringiensin was significantly reduced, while the accumulation of precursor C in the thuT mutant suggests its role in thuringiensin biosynthesis. Further proteinprotein interaction analysis demonstrated that ThuT forms a complex with ThuE, which suggests the mechanism by which ThuT participates in thuringiensin maturation. We have identified the first transporter of thuringiensin and propose that the ThuT-ThuE complex could couple the maturation and transport of thuringiensin to avoid thuringiensin accumulation. A novel high-efficiency model coupling the synthesis and secretion of antibiotics has been revealed in B.thuringiensis.

The Major Facilitator Superfamily and Antimicrobial Resistance Efflux Pumps of the ESKAPEE Pathogen Staphylococcus aureus

The ESKAPEE bacterial pathogen Staphylococcus aureus has posed a serious public health concern for centuries. Throughout its evolutionary course, S. aureus has developed strains with resistance to antimicrobial agents. The bacterial pathogen has acquired multidrug resistance, causing, in many cases, untreatable infectious diseases and raising serious public safety and healthcare concerns. Amongst the various mechanisms for antimicrobial resistance, integral membrane proteins that serve as secondary active transporters from the major facilitator superfamily constitute a chief system of multidrug resistance. These MFS transporters actively export structurally different antimicrobial agents from the cells of S. aureus. This review article discusses the S. aureus-specific MFS multidrug efflux pump systems from a molecular mechanistic perspective, paying particular attention to structure-function relationships, modulation of antimicrobial resistance mediated by MFS drug efflux pumps, and direction for future investigation.

Insights into β-manno-oligosaccharide uptake and metabolism in Bifidobacterium adolescentis DSMZ 20083 from whole-genome microarray analysis

Metabolism of non-digestible dietary glycans directly influences the structure and composition of human gut microbiota and, in turn, the host health. β-Mannans form an integral component of the modern diet as naturally occurring dietary fibre or additives in processed foods. In the present study, in vitro fermentation and TLC studies were used to determine the ability of adult-associated Bifidobacterium adolescentis DSMZ 20083 to utilise β-manno-oligosaccharides from guar gum, locust bean gum, konjac root, and copra meal generated using GH26 endo-β-mannanase (ManB-1601). Further, to gain insights into the underlying molecular mechanism, a whole-genome microarray analysis, RT-qPCR, and molecular docking studies were employed to reconstruct the copra meal β-manno-oligosaccharides (CM-β-MOS) utilisation pathway in B. adolescentis DSMZ 20083. B. adolescentis DSMZ 20083 grew appreciably (O.D600 nm up to 0.8) on all tested β-manno-oligosaccharides but maximally on CM-β-MOS. CM-β-MOS having DP2–3 were found to deplete from the fermentation media. Whole-genome transcriptome analysis, RT-qPCR, and molecular docking studies suggested that in B. adolescentis DSMZ 20083, ABC & MFS transporters are possibly involved in the uptake of DP ≥ 2 and DP ≥ 3 linear CM-β-MOS, respectively, while GH1 β-glucosidase, and GH32 β-fructofuranosidase possibly cleave linear CM-β-MOS into monosaccharides. Sugar absorption and utilisation pathways; Bifid shunt, ABC transport system, pyruvate metabolism, glycolysis/gluconeogenesis, pentose, and glucouronate inter-conversions were also found up-regulated following the growth on CM-β-MOS. This is the first study reporting on possible molecular determinants used by B. adolescentis DSMZ 20083 to utilise β-manno-oligosaccharides. Our studies can prove resourceful to food and nutraceutical industries, aiming at precision microbiome modulation using β-manno-oligosaccharides.

P113 Expression profile of ABC and MFS multi-drug transporter genes in terbinafine- and fluconazole-resistant Trichophyton spp.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesOver the past few decades, an unprecedented increase in recalcitrant and chronic dermatophytosis with atypical clinical presentations have been noted in the Indian subcontinent. Recent studies have reported Trichophyton mentagrophytes/interdigitale complex as predominant etiological agent of chronic/recurrent dermatophytosis followed by T. rubrum. Multiple factors, including unregulated use of antifungal drugs, poor compliance to treatment, and irrational fixed-drug combinations are associated with emerging resistance. However, molecular studies investigating the underlying mechanisms related to terbinafine and fluconazole resistance in dermatophytes are sparse.The present study was designed to explore the role of multidrug efflux transporters in terbinafine- and fluconazole-resistant T. mentagrophytes/interdigitale complex and T. rubrum.MethodsThe expression of MDR transporter genes was evaluated in 36 isolates with terbinafine resistance (with or without F397L or L393F mutation in squalene epoxidase gene, SE) of T. mentagrophytes/interdigitale complex (n = 26), and T. rubrum (n = 10) isolates along with 16 susceptible wild-type isolates of T. mentagrophytes/interdigitale complex (n = 10), and T. rubrum (n = 6) isolates. In addition, 19 fluconazole-resistant T. mentagrophytes/interdigitale complex (n = 11), and T. rubrum (n = 8) isolates along with 13 susceptible T. mentagrophytes/interdigitale complex (n = 9), and T. rubrum (n = 4) were also included. Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of ABC transporter (MDR1, MDR2, MDR3, MDR4, and MDR5) and MFS transporter genes (MFS1 and MFS2).ResultsIn terbinafine-resistant T. mentagrophytes/interdigitale complex, the mean expression of MDR1 and MDR2 was 4.98 and 5.27-fold in isolates with wild-type SE, compared with 0.64 and 1.04-fold in isolates with non-wildtype SE gene (P <.0001) and susceptible isolates at 0.4, and 0.88-fold (P <.0001), respectively. For MFS1 gene, 3.4-fold-change expression was noted in isolates with wild-type SE compared with 1.51-fold in isolates with non-wild type SE (P <.001). In resistant isolates with non-WT SE, the upregulation of the SE gene was noted at 6.3-fold compared with 1.8-fold in isolates with wild-type SE (P <.001), and 0.63-fold in susceptible isolates (P <.0001). However, there was no significant upregulation of MDR3, MDR4, MDR5, and MFS2 genes among the three groups. In terbinafine-resistant T. rubrum, the mean inducible expression of MDR1 was significantly higher in isolates with wild-type SE at 3.88-fold compared to isolates with non-wild type SE at 0.5-fold (P <.05) and susceptible isolates at 0.33-fold (P <.01). Only resistant isolates with wild-type SE showed significant up-regulation of MDR2 (P <.01) compared to susceptible isolates. Other genes, MDR3, MDR4, MDR5, MFS1, MFS2, and SE, did not show substantial overexpression among the groups (Resistant WT vs. Resistant NWT vs. susceptible WT). In fluconazole-resistant and -susceptible isolates of T. mentagrophytes/interdigitale complex, and T. rubrum, similar findings were observed. In both the species, the mean inducible expression of MDR1, MDR2, and MDR3 was significantly higher (P <.05) in resistant isolates compared to susceptible isolates.ConclusionIn conclusion, this study demonstrates the overexpression of MDR1 and MDR2 in terbinafine-resistant Trichophyton spp. isolates lacking drug-target gene mutation, suggesting the role of multidrug transporters in resistance. This study also suggests the importance of MDR2, and MDR3 transporter genes in imparting fluconazole resistance in Trichophyton spp.