GPCR-MAPK signaling pathways underpin fitness trade-offs in whitefly.

Trade-offs between evolutionary gain and loss are prevalent in nature, yet their genetic basis is not well resolved. The evolution of insect resistance to insecticide is often associated with strong fitness costs; however, how the fitness trade-offs operates remains poorly understood. Here, we show that the mitogen-activated protein kinase (MAPK) pathway and its upstream and downstream actors underlie the fitness trade-offs associated with insecticide resistance in the whitefly Bemisia tabaci. Specifically, we find a key cytochrome P450 gene CYP6CM1, that confers neonicotinoids resistance to in B. tabaci, is regulated by the MAPKs p38 and ERK through their activation of the transcription factor cAMP-response element binding protein. However, phosphorylation of p38 and ERK also leads to the activation of the transcription repressor Cap "n" collar isoform C (CncC) that negatively regulates exuperantia (Ex), vasa (Va), and benign gonial cell neoplasm (Bg), key genes involved in oogenesis, leading to abnormal ovary growth and a reduction in female fecundity. We further demonstrate that the transmembrane G protein-coupled receptor (GPCR) neuropeptide FF receptor 2 (NPFF2) triggers the p38 and ERK pathways via phosphorylation. Additionally, a positive feedback loop between p38 and NPFF2 leads to the continuous activation of the MAPK pathways, thereby constitutively promoting neonicotinoids resistance but with a significant reproductive cost. Collectively, these findings provide fundamental insights into the role of cis-trans regulatory networks incurred by GPCR-MAPK signaling pathways in evolutionary trade-offs and applied knowledge that can inform the development of strategies for the sustainable pest control.

Glutathione S-transferase directly metabolizes imidacloprid in the whitefly, Bemisia tabaci.

The whitefly Bemisia tabaci poses a significant threat to various crops and ornamental plants and causes severe damage to the agricultural industry. Over the past few decades, B. tabaci has developed resistance to several pesticides, including imidacloprid. Therefore, elucidating the mechanism that leads to insecticide detoxification is very important for controlling B. tabaci and managing whitefly resistance to neonicotinoid insecticides. Among insect detoxification enzymes, glutathione S-transferase (GST) is an important phase II detoxification enzyme that helps detoxify exogenous toxic substances. In this study, we cloned the BtGSTz1 gene and observed that its expression level was greater in imidacloprid-resistant populations than sensitive populations of B. tabaci. By silencing BtGSTz1 via RNA interference, we found a significant increase in the mortality of imidacloprid-resistant B. tabaci. Additionally, prokaryotic expression and in vitro metabolism studies revealed that the recombinant BtGSTz1 protein could metabolize 36.36% of the total imidacloprid, providing direct evidence that BtGSTz1 plays a crucial role in the detoxification of imidacloprid. Overall, our study elucidated the role of GSTs in physiological activities related to insecticide resistance, which helps clarify the resistance mechanisms conferred by GSTs and provides useful insights for sustainable integrated pest management.

Cytochrome P450 CYP6EM1 Underpins Dinotefuran Resistance in the Whitefly Bemisia tabaci.

Being a destructive pest worldwide, the whitefly Bemisia tabaci has evolved resistance to neonicotinoid insecticides. The third-generation neonicotinoid dinotefuran has commonly been applied to the control of the whitefly, but its underlying mechanism is currently unknown. On the base of our transcriptome data, here we aim to investigate whether the cytochrome P450 CYP6EM1 underlies dinotefuran resistance in the whitefly. Compared to the susceptible strain, the CYP6EM1 gene was found to be highly expressed in both laboratory and field dinotefuran-resistant populations. Upon exposure to dinotefuran, the mRNA levels of CYP6EM1 were increased. These results demonstrate the involvement of this gene in dinotefuran resistance. Loss and gain of functional studies in vivo were conducted through RNAi and transgenic Drosophila melanogaster assays, confirming the role of CYP6EM1 in conferring such resistance. In a metabolism assay in vitro, the CYP6EM1 protein could metabolize 28.11% of dinotefuran with a possible dinotefuran-dm-NNO metabolite via UPLC-QTOF/MS. Docking of dinotefuran to the CYP6EM1 protein showed a good binding affinity, with an energy of less than -6.0 kcal/mol. Overall, these results provide compelling evidence that CYP6EM1 plays a crucial role in the metabolic resistance of B. tabaci to dinotefuran. Our work provides new insights into the mechanism underlying neonicotinoid resistance and applied knowledge that can contribute to sustainable control of a global pest such as whitefly.

Dual mutations in the whitefly nicotinic acetylcholine receptor ß1 subunit confer target-site resistance to multiple neonicotinoid insecticides.

Neonicotinoid insecticides, which target insect nicotinic acetylcholine receptors (nAChRs), have been widely and intensively used to control the whitefly, Bemisia tabaci, a highly damaging, globally distributed, crop pest. This has inevitably led to the emergence of populations with resistance to neonicotinoids. However, to date, there have been no reports of target-site resistance involving mutation of B. tabaci nAChR genes. Here we characterize the nAChR subunit gene family of B. tabaci and identify dual mutations (A58T&R79E) in one of these genes (BTß1) that confer resistance to multiple neonicotinoids. Transgenic D. melanogaster, where the native nAChR Dß1 was replaced with BTß1A58T&R79E, were significantly more resistant to neonicotinoids than flies where Dß1 were replaced with the wildtype BTß1 sequence, demonstrating the causal role of the mutations in resistance. The two mutations identified in this study replace two amino acids that are highly conserved in >200 insect species. Three-dimensional modelling suggests a molecular mechanism for this resistance, whereby A58T forms a hydrogen bond with the R79E side chain, which positions its negatively-charged carboxylate group to electrostatically repulse a neonicotinoid at the orthosteric site. Together these findings describe the first case of target-site resistance to neonicotinoids in B. tabaci and provide insight into the molecular determinants of neonicotinoid binding and selectivity.

Dynamic monitoring of the insecticide resistance status of Bemisia tabaci across China from 2019-2021.

BACKGROUND: Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a major agricultural insect pest that causes severe economic losses worldwide. Several insecticides have been applied to effectively control this key pest. However, owing to the indiscriminate use of chemical insecticides, B. tabaci has developed resistance against these chemical compounds over the past several years. RESULTS: From 2019 to 2021, 23 field samples of B. tabaci were collected across China. Twenty species were identified as the Mediterranean 'Q' type (MED) and three were identified as MED/ Middle East-Asia Minor 1 mixtures. Subsequently, resistance of the selected populations to different insecticides was evaluated. The results showed that 13 populations developed low levels of resistance to abamectin. An overall upward trend in B. tabaci resistance toward spirotetramat, cyantraniliprole and pyriproxyfen was observed. In addition, resistance to thiamethoxam remained low-to-moderate in the 23 field populations. CONCLUSION: These findings suggest that the overall resistance of the field-collected B. tabaci populations has shown an upward trend over the years in China. We believe our study can provide basic data to support integrated pest management and insecticide resistance management of field B. tabaci in China. © 2023 Society of Chemical Industry.

CYP4CS5-mediated thiamethoxam and clothianidin resistance is accompanied by fitness cost in the whitefly Bemisia tabaci.

BACKGROUND: Understanding the trade-offs between insecticide resistance and the associated fitness is of particular importance to sustainable pest control. One of the most devastating pest worldwide, the whitefly Bemisia tabaci, has developed resistance to various insecticides, especially the neonicotinoid group. Although neonicotinoid resistance often is conferred by P450s-mediated metabolic resistance, the relationship between such resistance and the associated fitness phenotype remains largely elusive. By gene cloning, quantitative reverse transcription (qRT)-PCR, RNA interference (RNAi), transgenic Drosophila melanogaster, metabolism capacity in vitro and 'two sex-age stage' life table study, this study aims to explore the molecular role of a P450 gene CYP4CS5 in neonicotinoid resistance and to investigate whether such resistance mechanism carries fitness costs in the whitefly. RESULTS: Our bioassay tests showed that a total of 13 field-collected populations of B. tabaci MED biotype displayed low-to-moderate resistance to thiamethoxam and clothianidin. Compared to the laboratory susceptible strain, we then found that an important P450 CYP4CS5 was remarkably upregulated in the field resistant populations. Such overexpression of CYP4CS5 had a good match with the resistance level among the whitefly samples. Further exposure to the two neonicotinoids resulted in an increase in CYP4CS5 expression. These results implicate that overexpression of CYP4CS5 is closely correlated with thiamethoxam and clothianidin resistance. RNAi knockdown of CYP4CS5 increased mortality of the resistant and susceptible populations after treatment with thiamethoxam and clothianidin in bioassay, but obtained an opposite result when using a transgenic line of D. melanogaster expressing CYP4CS5. Metabolic assays in vitro revealed that CYP4CS5 exhibited certain capacity of metabolizing thiamethoxam and clothianidin. These in vivo and in vitro assays indicate an essential role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance in whitefly. Additionally, our life-table analysis demonstrate that the field resistant whitefly exhibited a prolonged development time, shortened longevity and reduced fecundity compared to the susceptible, suggesting an existing fitness cost as a result of the resistance. CONCLUSION: Collectively, in addition to the important role of CYP4CS5 in conferring thiamethoxam and clothianidin resistance, this resistance mechanism is associated with fitness costs in the whitefly. These findings not only contribute to the development of neonicotinoids resistance management strategies, but also provide a new target for sustainable whitefly control. © 2023 Society of Chemical Industry.

Over-expression of UDP-glycosyltransferase UGT353G2 confers resistance to neonicotinoids in whitefly (Bemisia tabaci).

The whitefly, Bemisia tabaci, comes up high metabolic resistance to most neonicotinoids in long-term evolution, which is the key problem of pest control. UGT glycosyltransferase, as a secondary detoxification enzyme, plays an indispensable role in detoxification metabolism. In this study, UGT inhibitors, 5-nitrouracil and sulfinpyrazone, dramatically augmented the toxic damage of neonicotinoids to B. tabaci. A UGT named UGT353G2 was identified in whitefly, which was notably up-regulated in resistant strain (3.92 folds), and could be induced by most neonicotinoids. Additionally, the using of RNA interference (RNAi) suppresses UGT353G2 substantially increased sensitivity to neonicotinoids in resistant strain. Our results support that UGT353G2 may be involved in the neonicotinoids resistance of whitefly. These findings will help further verify the functional role of UGTs in neonicotinoid resistance.

Cytochrome P450 CYP6DB3 was involved in thiamethoxam and imidacloprid resistance in Bemisia tabaci Q (Hemiptera: Aleyrodidae).

High level resistance for a variety of insecticides has emerged in Bemisia tabaci, a globally notorious insect. Neonicotinoid insecticides have been applied widely to control B. tabaci. Whether a differentially expressed gene CYP6DB3 discovered from transcriptome data of B. tabaci is involved in the resistance to neonicotinoid insecticides remains unclear. In the study, CYP6DB3 expression was significantly up-regulated in both thiamethoxam- and imidacloprid-resistant strains relative to the susceptive strains. We also found that CYP6DB3 expression was up-regulated after B. tabaci adults were exposed to thiamethoxam and imidacloprid. Moreover, knocking down CYP6DB3 expression via feeding corresponding dsRNA significantly reduced CYP6DB3 mRNA levels by 34.1%. Silencing CYP6DB3 expression increased the sensitivity of B. tabaci Q adults against both thiamethoxam and imidacloprid. Overexpression of CYP6DB3 gene reduced the toxicity of imidacloprid and thiamethoxam to transgenic D. melanogaster. In addition, metabolic studies showed that CYP6DB3 can metabolize 24.41% imidacloprid in vitro. Collectively, these results strongly support that CYP6DB3 plays an important role in the resistance of B. tabaci Q to imidacloprid and thiamethoxam. This work will facilitate a deeper insight into the part of cytochrome P450s in the evolution of insecticide resistance and provide a theoretical basis for the development of new integrated pest resistance management.

Novel_miR-1517 mediates CYP6CM1 to regulate imidacloprid resistance in Bemisia tabaci (Hemiptera: Gennadius).

Bemisia tabaci (Hemiptera: Gennadius) is a notorious pest that is capable of feeding on >600 kinds of agricultural crops. Imidacloprid is critical in managing pest with sucking mouthparts, such as B. tabaci. However, the field population of B. tabaci has evolved resistance because of insecticide overuse. The overexpression of the detoxification enzyme cytochrome P450 monooxygenase is considered the main mechanism of imidacloprid resistance, but the mechanism underlying gene regulation remains unclear. MicroRNAs are a type of endogenous small molecule compounds that is fundamental in regulating gene expression at the post-transcriptional level. Whether miRNAs are related to the imidacloprid resistance of B. tabaci remains unknown. To gain deep insight into imidacloprid resistance, we conducted on miRNAs expression profiling of two B. tabaci Mediterranean (MED) strains with 19-fold resistance through deep sequencing of small RNAs. A total of 8 known and 1591 novel miRNAs were identified. In addition, 16 miRNAs showed significant difference in expression levels between the two strains, as verified by quantitative reverse transcription PCR. Among these, novel_miR-376, 1517, and 1136 significantly expressed at low levels in resistant samples, decreasing by 36.9%, 60.2%, and 15.6%, respectively. Moreover, modulating novel_miR-1517 expression by feeding with 1517 inhibitor and 1517 mimic significantly affected B. tabaci imidacloprid susceptibility by regulating CYP6CM1 expression. In this article, miRNAs related to imidacloprid resistance of B. tabaci were systematically screened and identified, providing important information for the miRNA-based technological innovation for this pest management.

20E biosynthesis gene CYP306A1 confers resistance to imidacloprid in the nymph stage of Bemisia tabaci by detoxification metabolism.

BACKGROUND: Difference in physiology level between the immature and mature stages of insects likely contribute to different mechanisms of insecticide resistance. It is well acknowledged that insect 20-hydroxyecdysone (20E) plays an important role in many biological processes in the immature stage, whether 20E confers insecticide resistance at this specific stage is still poorly understood. By gene cloning, reverse transcription quantitative real-time PCR, RNA interference (RNAi) and in vitro metabolism experiments, this study aimed to investigate the potential role of 20E-related genes in conferring imidacloprid (IMD) resistance in the immature stage of the whitefly Bemisia tabaci Mediterranean. RESULTS: After identification of low to moderate IMD resistance in the whitefly, we found CYP306A1 of the six 20E-related genes was overexpressed in the nymph stage of the three resistant strains compared to a laboratory reference susceptible strain, but not in the adult stage. Further exposure to IMD resulted in an increase in CYP306A1 expression in the nymph stage. These results together imply that CYP306A1 may be implicated in IMD resistance in the nymph stage of the whitefly. RNAi knockdown of CYP306A1 increased the mortality of nymphs after treatment with IMD in bioassay, suggesting a pivotal role of CYP306A1 in conferring IMD resistance in the nymph stage. Additionally, our metabolism experiments in vivo showed that the content of IMD reduced by 20% along with cytochrome P450 reductase and heterologously expressed CYP306A1, which provides additional evidence for the important function of CYP306A1 in metabolizing IMD that leads to the resistance. CONCLUSION: This study uncovers a novel function of the 20E biosynthesis gene CYP306A1 in metabolizing imidacloprid, thus contributing to such resistance in the immature stage of the insect. These findings not only advance our understanding of 20E-mediated insecticide resistance, but also provide a new target for sustainable pest control of global insect pests such as whitefly. © 2023 Society of Chemical Industry.

CYP6CX2 and CYP6CX3 mediate thiamethoxam resistance in field whitefly, Bemisia tabaci (Hemiptera:Aleyrodidae).

Cytochrome P450 monooxygenases (P450s) are well-known for their crucial roles in the detoxification of xenobiotics. However, whether CYP6CX2 and CYP6CX3, 2 genes from our Bemisia tabaci (B. tabaci) MED/Q genome data were associated with detoxification metabolism and confer resistance to thiamethoxam is unclear. In this study, we investigated the role of CYP6CX2 and CYP6CX3 in mediating whitefly thiamethoxam resistance. Our results showed that mRNA levels of CYP6CX2 and CYP6CX3 were up-regulated after exposure to thiamethoxam. Transcriptional levels of 2 genes were overexpressed in laboratory and field thiamethoxam resistant strains by RT-qPCR. These results indicate that the enhanced expression of CYP6CX2 and CYP6CX3 appears to confer thiamethoxam resistance in B. tabaci. Moreover, linear regression analysis showed that the expression levels of CYP6CX2 and CYP6CX3 were positively correlated with thiamethoxam resistance levels among populations. The susceptibility of whitefly adults was markedly increased after silencing 2 genes by RNA interference (RNAi) which further confirming their major role in thiamethoxam resistance. Our findings provide information to better understand the roles of P450s in resistance to neonicotinoids and suggest that these genes may be applied to develop target genes for sustainable management tactic of agricultural pests such as B. tabaci.

Knockdown of the Nicotinic Acetylcholine Receptor ß1 Subunit Decreases the Susceptibility to Five Neonicotinoid Insecticides in Whitefly (Bemisia tabaci).

The sweet potato whitefly, Bemisia tabaci, (Gennadius) (Hemiptera:Aleyrodidae) is a global pest of crops. Neonicotinoids are efficient insecticides used for control of this pest. Insecticidal targets of neonicotinoids are insect nicotinic acetylcholine receptors (nAChRs). Here, we characterized and cloned the full length of the nAChR ß1 subunit (BTß1) in B. tabaci and confirmed the consistency of BTß1 in B. tabaci MEAM1 and MED. Expression levels of BTß1 in different developmental stages and body parts of adults were investigated and compared in B. tabaci MED. dsRNA was prepared to knock down BTß1 in adult B. tabaci and significantly decreases the susceptibility to five neonicotinoid insecticides, including imidacloprid, clothianidin, thiacloprid, nitenpyram, and dinotefuran. This study indicated BTß1 as a notable site influencing the susceptibility of B. tabaci to neonicotinoids.

[Identification of the target site of antimicrobial peptide AMP-17 against Candida albicans].

Candida albicans is one of the major causes of invasive fungal infections and a serious opportunistic pathogen in immunocompromised individuals. The antimicrobial peptide AMP-17 has prominent anti-Candida activity, and proteomic analysis revealed significant differences in the expression of cell wall (XOG1) and oxidative stress (SRR1) genes upon the action of AMP-17 on C. albicans, suggesting that AMP-17 may exert anti-C. albicans effects by affecting the expression of XOG1 and SRR1 genes. To further investigate whether XOG1 and SRR1 genes were the targets of AMP-17, C. albicans xog1Δ/Δ and srr1Δ/Δ mutants were constructed using the clustered regulatory interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system. Phenotypic observations revealed that deletion of two genes had no significant effect on C. albicans growth and biofilm formation, whereas XOG1 gene deletion affected in vitro stress response and mycelium formation of C. albicans. Drug sensitivity assay showed that the MIC80 values of AMP-17 against xog1Δ/Δ and srr1Δ/Δ mutants increased from 8 µg/mL (for the wild type C. albicans SC5314) to 16 µg/mL, while the MIC80 values against srr1Δ/Δ: : srr1 revertants decreased to the level of the wild type SC5314. In addition, the ability of AMP-17 to inhibit biofilm formation of both deletion strains was significantly reduced compared to that of wild type SC5314, indicating that the susceptibility of the deletion mutants to AMP-17 was reduced in both the yeast state and during biofilm formation. These results suggest that XOG1 and SRR1 genes are likely two of the potential targets for AMP-17 to exert anti-C. albicans effects, which may facilitate further exploration of the antibacterial mechanism of novel peptide antifungal drugs.

Over-expression of CP9 and CP83 increases whitefly cell cuticle thickness leading to imidacloprid resistance.

Cuticular proteins (CPs) play an important role in protecting insects from adverse environmental conditions, like neonicotinoid insecticides, which are heavily used for numerous pests and caused environmental problems and public health concerns worldwide. However, the relationship between CPs and insecticides resistance in Bemisia tabaci, a serious and developed high insecticide resistance, is lacking. In this study, 125 CPs genes were identified in B. tabaci. Further phylogenetic tree showed the RR-2-type genes formed large gene groups in B. tabaci. Transcriptional expression levels of CPs genes at different developmental stages revealed that some CPs genes may play a specific role in insect development. The TEM results indicated that the cuticle thickness of susceptible strain was thinner than imidacloprid-resistance strain. Furthermore, 16 CPs genes (5 in RR-1 subfamily, 7 in RR-2 subfamily, 3 in CPAP3 subfamily and 1 in CPCFC subfamily) were activated in response to imidacloprid. And RNAi results indicated that CP9 and CP83 involved in imidacloprid resistance. In conclusion, this study was the first time to establish a basic information framework and evolutionary relationship between CPs and imidacloprid resistance in B. tabaci, which provides a basis for proposing integrated pest management strategies.

CYP6DW3 Metabolizes Imidacloprid to Imidacloprid-urea in Whitefly (Bemisia tabaci).

Bemisia tabaci has developed high resistance to many insecticides and causes substantial agricultural and economic losses annually. The insecticide resistance of whitefly has been widely reported in previous studies; however, the underlying mechanism remains little known. In this study, we cloned two P450 genes: CYP6DW3 and CYP6DW5v1; these genes were markedly overexpressed in imidacloprid-resistant whitefly populations compared with susceptible populations, and knockdown of these genes decreased the imidacloprid resistance of whitefly. Moreover, heterologous expression of whitefly P450 genes in SF9 cells and metabolic studies showed that the CYP6DW3 protein could metabolize 14.11% imidacloprid and produced imidacloprid-urea in vitro. Collectively, the expression levels of CYP6DW3 and CYP6DW5v1 are positively correlated with imidacloprid resistance in B. tabaci. Our study further reveals that cytochrome P450 enzymes affect the physiological activities related to resistance in insects, which helps scholars more deeply understand the resistance mechanism, and contributes to the development of integrated pest management framework.

Antitumor Activity and Mechanism of Action of the Antimicrobial Peptide AMP-17 on Human Leukemia K562 Cells.

Cancer is one of the most common malignant diseases in the world. Hence, there is an urgent need to search for novel drugs with antitumor activity against cancer cells. AMP-17, a natural antimicrobial peptide derived from Musca domestica, has antimicrobial activity against Gram-positive bacteria, Gram-negative bacteria, and fungi. However, its antitumor activity and potential mechanism of action in cancer cells remain unclear. In this study, we focused on evaluating the in vitro antitumor activity and mechanism of AMP-17 on leukemic K562 cells. The results showed that AMP-17 exhibited anti-proliferative activity on K562 cells with an IC50 value of 58.91 ± 3.57 µg/mL. The membrane integrity of K562 was disrupted and membrane permeability was increased after AMP-17 action. Further observation using SEM and TEM images showed that the cell structure of AMP-17-treated cells was disrupted, with depressions and pore-like breaks on the cell surface, and vacuolated vesicles in the cytoplasm. Furthermore, further mechanistic studies indicated that AMP-17 induced excessive production of reactive oxygen species and calcium ions release in K562 cells, which led to disturbance of mitochondrial membrane potential and blocked ATP synthesis, followed by activation of Caspase-3 to induce apoptosis. In conclusion, these results suggest that the antitumor activity of AMP-17 may be achieved by disrupting cell structure and inducing apoptosis. Therefore, AMP-17 is expected to be a novel potential agent candidate for leukemia treatment.

The Antimicrobial Peptide AMP-17 Derived from Musca domestica Inhibits Biofilm Formation and Eradicates Mature Biofilm in Candida albicans.

The biofilm formation of C. albicans represents a major virulence factor during candidiasis. Biofilm-mediated drug resistance has necessitated the search for a new antifungal treatment strategy. In our previous study, a novel antimicrobial peptide named AMP-17 derived from Musca domestica was confirmed to have significant antifungal activity and suppress hyphal growth greatly in C. albicans. In the current work, we aimed to investigate the antibiofilm property of AMP-17 in C. albicans and explore the underlying mechanism. An antifungal susceptibility assay showed that AMP-17 exerted a strong inhibitory efficacy on both biofilm formation and preformed biofilms in C. albicans. Furthermore, AMP-17 was found to block the yeast-to-hypha transition and inhibit the adhesion of biofilm cells with a reduction in cellular surface hydrophobicity. A morphological analysis revealed that AMP-17 indeed suppressed typical biofilm formation and damaged the structures of the preformed biofilm. The RNA-seq showed that the MAPK pathway, biosynthesis of antibiotics, and essential components of the cell were mainly enriched in the biofilm-forming stage, while the citrate cycle (TCA cycle), phenylamine metabolism, and propanoate metabolism were enriched after the biofilm matured. Moreover, the co-expressed DEGs in the two pairwise comparisons highlighted the terms of transmembrane transporter activity, regulation of filamentation, and biofilm formation as important roles in the antibiofilm effect of AMP-17. Additionally, qRT-PCR confirmed that the level of the genes involved in cell adhesion, filamentous growth, MAPK, biofilm matrix, and cell dispersal was correspondingly altered after AMP-17 treatment. Overall, our findings reveal the underlying antibiofilm mechanisms of AMPs in C. albicans, providing an interesting perspective for the development of effective antifungal agents with antibiofilm efficacy in Candida spp.

Identification and functional characterization of ORF19.5274, a novel gene involved in both azoles susceptibility and hypha development in Candida albicans.

Azole resistance is becoming increasingly serious due to the frequent recurrence of fungal infections and the need for long-term clinical prevention. In our previous study, we discovered ORF19.5274 with an unknown function by TMT™ quantitative proteomics technology after fluconazole (FLC) treatment of Candida albicans. In this study, we created the target gene deletion strain using CRISPR-Cas9 editing technology to see if ORF19.5274 regulates azole sensitivity. The data showed that ORF19.5274 was involved in hyphal development and susceptibility to antifungal azoles. Deleting this gene resulted in defective hyphal growth in solid medium, while only a weak lag in the initiation of hyphal development and restoring hyphal growth during the hyphal maintenance phase under liquid conditions. Moreover, intracellular reactive oxygen species (ROS) assay and propidium iodide staining assays showed increased endogenous ROS levels and membrane permeability, but decreased metabolic activity of biofilm in orf19.5274Δ/Δ after treatment with FLC in comparison with either SC5314 or orf19.5274Δ/Δ::ORF19.5274 strains. More importantly, orf19.5274Δ/Δ significantly enhanced the FLC efficacy against C. albicans in infected Galleria mellonella larvae. The above characteristics were fully or partially restored in the complemented strain indicating that the changes caused by ORF19.5274 deletion were specific. In summary, the ORF19.5274 gene is required for hyphal development of C. albicans, and is correlated with the response to antifungal azoles in vitro and in vivo. The identification of ORF19.5275 is promising to expand the potential candidate targets for azoles.

A Cecropin-4 Derived Peptide C18 Inhibits Candida albicans by Disturbing Mitochondrial Function.

Global burden of fungal infections and related health risk has accelerated at an incredible pace, and multidrug resistance emergency aggravates the need for the development of new effective strategies. Candida albicans is clinically the most ubiquitous pathogenic fungus that leads to high incidence and mortality in immunocompromised patients. Antimicrobial peptides (AMPs), in this context, represent promising alternatives having potential to be exploited for improving human health. In our previous studies, a Cecropin-4-derived peptide named C18 was found to possess a broader antibacterial spectrum after modification and exhibit significant antifungal activity against C. albicans. In this study, C18 shows antifungal activity against C. albicans or non-albicans Candida species with a minimum inhibitory concentration (MIC) at 4∼32 µg/ml, and clinical isolates of fluconazole (FLZ)-resistance C. tropicalis were highly susceptible to C18 with MIC value of 8 or 16 µg/ml. Additionally, C18 is superior to FLZ for killing planktonic C. albicans from inhibitory and killing kinetic curves. Moreover, C18 could attenuate the virulence of C. albicans, which includes damaging the cell structure, retarding hyphae transition, and inhibiting biofilm formation. Intriguingly, in the Galleria mellonella model with C. albicans infection, C18 could improve the survival rate of G. mellonella larvae to 70% and reduce C. albicans load from 5.01 × 107 to 5.62 × 104 CFU. For mechanistic action of C18, the level of reactive oxygen species (ROS) generation and cytosolic Ca2 + increased in the presence of C18, which is closely associated with mitochondrial dysfunction. Meanwhile, mitochondrial membrane potential (△Ψm) loss and ATP depletion of C. albicans occurred with the treatment of C18. We hypothesized that C18 might inhibit C. albicans via triggering mitochondrial dysfunction driven by ROS generation and Ca2 + accumulation. Our observation provides a basis for future research to explore the antifungal strategies and presents C18 as an attractive therapeutic candidate to be developed to treat candidiasis.

In vitro Antifungal Activity of a Novel Antimicrobial Peptide AMP-17 Against Planktonic Cells and Biofilms of Cryptococcus neoformans.

BACKGROUND: Cryptococcus neoformans is a common human fungal pathogen in immunocompromised people, as well as a prevalent cause of meningitis in HIV-infected individuals. With the emergence of clinical fungal resistance and the shortage of antifungal drugs, it is urgent to discover novel antifungal agents. AMP-17, a novel antimicrobial peptide from Musca domestica, has antifungal activity against C. neoformans. However, its antifungal and anti-biofilm activities remain unclear. Thus, this study aimed to evaluate the antifungal activity of AMP-17 against planktonic cells and biofilms of C. neoformans. METHODS: The minimum inhibitory concentration (MIC), the biofilm inhibitory and eradicating concentration (BIC and BEC) were determined by the broth microdilution assay or the 2, 3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay, respectively. The inhibitory and killing activities of AMP-17 against C. neoformans were investigated through the time-inhibition/killing kinetic curves. The potential antifungal mechanism of AMP-17 was detected by flow cytometry, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The efficiency of AMP-17 against biofilm formation or preformed biofilm was evaluated by crystal violet staining and XTT reduction assays. The morphology of pre-biofilms was tested by optical microscopy (OM) and CLSM. RESULTS: AMP-17 exhibited in vitro antifungal activity against C. neoformans planktonic cells and biofilms, with MICs of 4~16 µg/ml, BIC80 and BEC80 of 16~32 µg/ml, 64~128 µg/ml, respectively. In addition, the 2× and 4× MIC of AMP-17 exhibited similar inhibition levels compared to the 2× and 4× MIC of the clinical drugs FLC and AMB in C. neoformans growth. Moreover, the time-kill results showed that AMP-17 (8× MIC) did not significantly eliminate colony forming units (CFU) after 6 h of treatment; however, there was 2.9-log reduction in CFU of C. neoformans. Furthermore, increasing of the permeability of the fungal cell membrane was observed with the treatment of AMP-17, since the vast change as fungal leakage and cell membrane disruption. However, the DNA binding assay of AMP-17 indicated that the peptide did not target DNA. Besides, AMP-17 was superior in inhibiting and eradicating biofilms of C. neoformans compared with FLC. CONCLUSION: AMP-17 exhibited potential in vitro antifungal activity against the planktonic cells and biofilms of C. neoformans, and it may disrupt fungal cell membranes through multi-target interactions, which provides a promising therapeutic strategy and experimental basis for Cryptococcus-associated infections.