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Background: Invasive fungal infections (IFIs) are primarily caused by Candida spp., Cryptococcus neoformans, Aspergillus spp., Mucor spp., Sporothrix spp., and Pneumocystis spp., which attack human organs with a strong pathogenicity and exhibit drug resistance against commonly used chemical drugs. Therefore, the search for alternative drugs with high efficacy, low resistance rates, few side effects, and synergistic antifungal effects remains a major challenge. The characteristics of natural products with structural and bioactive diversity, lower drug resistance, and rich resources make them a major focus of the development of antifungal drugs. Objective: This review attempts to summarize the origin, structure, and antifungal activity of natural products and their derivatives with MIC ≤ 20 µg/mL or 100 µM, focusing on their MoA and SAR. Methods: All pertinent literature databases were searched. The search keywords were antifungal or antifungals, terpenoids, steroidal saponins, alkaloid, phenols, lignans, flavonoids, quinones, macrolide, peptide, tetramic acid glycoside, polyene, polyketide, bithiazole, natural product, and derivatives. All the related literature (covering the past 20 years, 2001-2022) was evaluated. Results: In total, 340 natural products and 34 synthesized derivatives with antifungal activity from 301 studies were included in this review. These compounds were derived from terrestrial plants, ocean life, and microorganisms and exhibited in vitro and in vivo potent antifungal activity alone or in combination. The MoA and SARs of reported compounds were summarized whenever applicable. Conclusion: In this review, we attempted to review the available literature on natural antifungal products and their derivatives. Most of the studied compounds showed potent activity against Candida species, Aspergillus species, or Cryptococcus species. Some of the studied compounds also demonstrated the ability to impair the cell membrane and cell wall, inhibit hypha and biofilms, and cause mitochondrial dysfunction. Alth

Genome instability is a hallmark of C. albicans . Aneuploidy usually causes fitness loss in the absence of stress but confers better fitness under particular stress conditions.

Antifungal drug tolerance is a response distinct from resistance, in which cells grow slowly above the minimum inhibitory drug concentration (MIC). Here we found that the majority (69.2%) of 133Candida albicansclinical isolates, including standard lab strain SC5314, exhibitedtemperature-enhancedtolerance at 37°C and 39°C, and were not tolerant at 30°C. Other isolates were eitheralwaystolerant (23.3%) ornevertolerant (7.5%) at these three temperatures, suggesting that tolerance requires different physiological processes in different isolates. At supra-MIC fluconazole concentrations (8-128 μg/ml), tolerant colonies emerged rapidly at a frequency of ~10−3. In liquid passages over a broader range of fluconazole concentrations (0.25-128 μg/ml), tolerance emerged rapidly (within one passage) at supra-MIC concentrations. By contrast, resistance appeared at sub-MIC concentrations after 5 or more passages. Of 155 adaptors that evolved higher tolerance, all carried one of several recurrent aneuploid chromosomes, often including chromosome R, alone or in combination with other chromosomes. Furthermore, loss of these recurrent aneuploidies was associated with a loss of acquired tolerance, indicating that specific aneuploidies confer fluconazole tolerance. Thus, genetic background and physiology, and the degree of drug stress (above or below the MIC) influence the evolutionary trajectories and dynamics with which antifungal drug resistance or tolerance emerges.ImportanceAntifungal drug tolerance differs from drug resistance: tolerant cells grow slowly in drug, while resistant cells usually grow well, due to mutations in a few known genes. More than half ofCandida albicansclinical isolates have higher tolerance at body temperature than they do at the lower temperatures used for most lab experiments. This implies that different isolates achieve drug tolerance via several cellular processes. When we evolved different strains at a range of high drug concentrations above inhibitory levels, tolerance emerged rapidly and at high frequency (one in 1000 cells) while resistance only appeared later at very low drug concentrations. An extra copy of all or part of chromosome R was associated with tolerance, while point mutations or different aneuploidies were seen with resistance. Thus, genetic background and physiology, temperature, and drug concentration all influence how drug tolerance or resistance evolves.

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Objectives Cryptococcus neoformans is a worldwide threat causing global pulmonary and systemic infections in humans. However, only three drugs are available for the treatment of cryptococcosis: fluconazole, amphotericin B, and flucytosine. Drug repurposing, the process of using a drug for an indication different from the initial indication, is an emerging approach in the discovery of new antifungal drugs. We've set out to identify repurposable drugs for cryptococossis. Methods We screened the United States FDA-approved drugs for antifungal activity against C. albicans lab strain SC5314. We tested the antifungal profile against major human fungal pathogens including C. neoformans. In order to uncover the mechanism of action against C. neoformans, lab strain H99 was used to evolve drug resistant adaptors. Next-generation sequencing technology was used to investigate the genome change of drug resistant adaptors. SNP calling was also performed to identify possible mutations causing drug resistance. Results Here we found that otilonium bromide (OTB), which is extensively used to treat patients affected by the irritable bowel syndrome, had broad-spectrum antifungal activity. OTB was active against fluconaozle-resistant and flucotysine-resistant C. neoformans strains. Furthermore, we found resistance to OTB was mostly due to duplication of chromosome 6. Further work will be on the identification of the Candidate gene on chromosome 6 which is required OTB resistance. Conclusion This study highlights the potential application of OTB as a new antifungal drug against C. neoformans strains susceptible or resistant to commonly used antifungal drugs.

S5.4 Free oral paper session, September 22, 2022, 3:00 PM - 4:30 PM Objectives Cryptococcus neoformans is an environmental fungal pathogen causing fatal pulmonary and disseminated infections in humans, especially in AIDS patients. However, only three antifungal drugs are available for the treatment of cryptococcosis: fluconazole, amphotericin B, and flucytosine. C. neoformans is worldwide distributed in nature, but environmental factors influencing antifungal drug susceptibility are largely unknown. Methods We tested the antifungal effect of agrochemicals against C. neoformans lab strain H99. We obtained drug-resistant adaptors. We sequenced the adaptors and got a list of adaptors bearing unique aneuploidy. We asked if aneuploidy caused multidrug resistance to antifungal drugs and if aneuploidy altered virulence in vitro and in vivo. We also investigated the molecular mechanism of aneuploidy-mediated multidrug resistance. Results Here we found tebuconazole and benomyl, belonging to two distinct classes of agrochemicals, could inhibit the growth of H99. However, H99 could gain resistance to both drugs mainly via aneuploidy. Importantly, particular aneuploidy also caused multidrug resistance to fluconazole, amphotericin B, and flucytosine. Furthermore, aneuploidy also altered virulence in vitro and in vivo. We found copy number variation of genes on the aneuploid chromosome caused a proportional increase in transcript and protein levels. Conclusion Therefore, we posit agrochemicals could enhance multidrug resistance and alter the virulence of C. neoformans via the hitch-hiking effect of aneuploidy.

Baicalein could inhibit the growth and biofilm formation of Candida albicans, the most common clinical fungal pathogen. However, the antifungal mechanism of baicalein has not been elucidated. In this study, isobaric tags for relative and absolute quantification (iTRAQ) was used to verify the mechanism of antifungal fluconazole and baicalein. A total of 58 common proteins were detected in cells treated with fluconazole. These proteins encompassed fluconazole-targeted sterol synthesis pathway, including Erg11p, Erg6p, Erg3p, Erg25p, Erg5p, Erg10p, and Ncp1p. Next, iTRAQ was applied to the comparison of baicalein-treated C. albicans proteins, which detected 16 common proteins. The putative NADH dehydrogenase Cpd2p and the ATP-binding cassette transporter Snq2p were the most upregulated proteins with the treatment of baicalein. Our results showed that CPD2 disruption elevated C. albicans resistance to baicalein significantly both in vitro and in vivo. Further in-depth studies revealed that CPD2 disruption reduced the activation of C. albicans metacaspase and partially restored the mitochondrial membrane potential reduction caused by the treatment of baicalein, which indicated that CPD2 was involved in the apoptosis induced by baicalein. Consistently, under the treatment of baicalein, CPD2Δ/Δ mutant produced lower reactive oxygen species that was critical in causing oxidative damage and apoptosis in C. albicans. These results indicated that baicalein could increase intracellular oxidative damage by upregulating the expression of Cpd2p so as to inhibit the growth of C. albicans, which provides new insights for investigating the antifungal target of baicalein.In our study, isobaric tags for relative and absolute quantification (iTRAQ) was used to study the antifungal mechanisms of fluconazole and baicalein. Baicalein could enhance the oxidative stress of Candida albicans by upregulating CPD2 expression.

The high morbidity and mortality of cryptococcal meningitis is due to the limited range of therapeutic options: only three classes of antifungal drugs are available (polyenes [amphotericin B], azoles [fluconazole], and pyrimidine analogues [flucytosine]). Fluconazole is the most widely used antifungal drug in sub-Saharan Africa, where cryptococcal meningitis is a major cause of death in patients infected with HIV. In this study, we found that exposure to fluconazole, even for short times (48 h) at subinhibitory concentrations, drove rapid adaptation of Cryptococcus neoformans serotype A strain H99 via the acquisition of different aneuploid chromosomes. These aneuploidies conferred heteroresistance to fluconazole. Importantly, most of the adaptors were cross-tolerant to flucytosine. Some of the aneuploid adaptors were not heteroresistant to fluconazole but were tolerant to amphotericin B. Thus, exposure to one antifungal drug class can promote adaptation to two antifungal drug classes, highlighting the plasticity of the C. neoformans genome and raising concerns about the rapid reduction in the range of treatment options for cryptococcal infections. IMPORTANCE Cryptococcosis is a globally distributed invasive fungal infection caused by infections with Cryptococcus neoformans or Cryptococcus gattii. Only three classes of therapeutic drugs are clinically available for treating cryptococcosis: polyenes (amphotericin B), azoles (fluconazole), and pyrimidine analogues (flucytosine). Fluconazole is the primary drug available in resource-limited countries. Aneuploidy is a genomic state due to the gain or loss of chromosomes. We found that C. neoformans rapidly adapted to fluconazole by acquiring diverse aneuploidies and that specific aneuploidies enabled improved growth of isolates susceptible (tolerance) to amphotericin B and/or cross-tolerance to both fluconazole and flucytosine. Therefore, aneuploidy is an underlying mechanism of drug tolerance that not only arises rapidly during growth in fluconazole but can also confer tolerance to other antifungal drugs without prior exposure to those drugs. Resistant isolates have high MICs, and all cells grow similarly in medium with the drug, while tolerant isolates test as susceptible and grow slowly at drug concentrations above the MIC.

Candida species are the most common human fungal pathogens worldwide. Although C. albicans remains the predominant cause of candidiasis, infections caused by non-albicans Candida species, including C. parapsilosis, are increasing. In C. albicans, genome plasticity has been shown to be a prevalent strategy of adaptation to stresses. However, the role of aneuploidy in C. parapsilosis is largely unknown. In this study, we found that six different aneuploid karyotypes conferred adaptation to the endoplasmic reticulum stress inducer tunicamycin (TUN) in C. parapsilosis. Interestingly, a specific aneuploidy including trisomy of chromosome 6 (Chr6x3) also enabled cross-tolerance to aureobasidin A (AbA), a sphingolipid biosynthesis inhibitor. Consistent with this, selection on AbA identified adaptors with three different aneuploid karyotypes, including Chr6x3, which also enabled cross-tolerance to both AbA and TUN. Therefore, as in other Candida species, recurrent aneuploid karyotypes enable the adaptation of C. parapsilosis to specific stresses, and specific aneuploidies enable cross-adaptation to different stresses. IMPORTANCE Candida parapsilosis is an emerging human fungal pathogen, especially prevalent in neonates. Aneuploidy, having uneven numbers of chromosomes, is a well-known mechanism for adapting to stress in Candida albicans, the most common human fungal pathogen. In this study, we exposed C. parapsilosis to two very different drugs and selected for rare cells that grew in one of the drugs. We found that the majority of isolates that grew in the drugs had acquired an extra copy of one of several aneuploid chromosomes and that specific aneuploid chromosomes appeared in several independent cell clones. Importantly, an extra copy of chromosome 6 was detected following selection in either one of the drugs, and this extra chromosome conferred the ability to grow in both drugs, a property called cross-adaptation, or cross-tolerance. Thus, this study highlights the genome plasticity of C. parapsilosis and the ability of an extra copy of a single chromosome to promote cell growth in the presence of more than one drug.

Candida albicans is a prevalent opportunistic human fungal pathogen for which treatment is limited to only four main classes of antifungal drugs, with the azole and echinocandin classes being used most frequently. Drug tolerance, the ability of some cells to grow slowly in supra-MIC drug concentrations, decreases the number of available treatment options. Here, we investigated factors affecting tolerance and resistance to ketoconazole in C. albicans. We found both temperature and the composition of growth medium significantly affected tolerance with little effect on resistance. In deletion analysis of known efflux pump genes, CDR1 was partially required for azole tolerance, while CDR2 and MDR1 were dispensable. Tolerance also required Hsp90 and calcineurin components; CRZ1, which encodes a transcription factor downstream of calcineurin, was required only partially. Deletion of VMA11, which encodes a vacuolar ATPase subunit, and concanamycin A, a V-ATPase inhibitor, abolished tolerance, indicating the importance of vacuolar energy transactions in tolerance. Thus, tolerance to ketoconazole is regulated by multiple factors, including physiological and genetic mechanisms. IMPORTANCE Due to the ever-expanding range of invasive medical procedures and treatments, invasive fungal infections now pose a serious global threat to many people living in an immunocompromised status. Like humans, fungi are eukaryotic, which significantly limits the number of unique antifungal targets; the current arsenal of antifungal agents is limited to just three frontline drug classes. Additional treatment complexities result from the development of drug tolerance and resistance, which further narrows therapeutic options; however, the difference between tolerance and resistance remains largely unknown. This study demonstrates that tolerance and resistance are regulated by multiple genetic and physiological factors. It is prudent to note that some factors affect tolerance only, while other factors affect both tolerance and resistance. The complex underlying mechanisms of these drug responses are highlighted by the fact that there are both shared and distinct mechanisms that regulate tolerance and resistance.

Fungi belong to a unique kingdom of organisms closely related to human beings. Monitoring the vital species in fungal cells, H+ as an example, reveals tremendous physiological and pathological processes that are crucial for everyone. Herein, we introduced fluorescence imaging with designed fluorophores into the pH imaging in fungal cells. A rhodamine-coumarin based fluorescence resonance energy transfer (FRET) ratiometric probe was successfully applied in visualizing pH and acidification in pathogenic Candida albicans. The probe was designed for pH through the H+-triggered reversible closed-open process of spiroboronate with high sensitivity and selectivity. It turns out that the coumarin group is more than a FRET donor, it significantly pushed the closed-open equilibrium constant of the probe from >8.0 into 6.0, the region that suits pH imaging of fungal cells. With excellent cellular permeability and ignorable biotoxicity in fungi, the probe manifested precise pH change from 7.5 to 6.5 in amiodarone treated fungal cells. We hope this work can shed some light on the fluorescence imaging of fungal cells with versatile molecular probes and invoke more interests in fungal cells.

Candida albicans is a prevalent human fungal pathogen. Rapid genomic change, due to aneuploidy, is a common mechanism that facilitates survival from multiple types of stresses including the few classes of available antifungal drugs. The stress survival of aneuploids occurs despite the fitness costs attributed to most aneuploids growing under idealized lab conditions. Systematic study of the aneuploid state in C. albicans has been hindered by the lack of a comprehensive collection of aneuploid strains. Here, we describe a collection of diploid C. albicans aneuploid strains, each carrying one extra copy of each chromosome, all from the same genetic background. We tested the fitness of this collection under several physiological conditions including shifts in pH, low glucose, oxidative stress, temperature, high osmolarity, membrane stress, and cell wall stress. We found that most aneuploids, under most conditions, were less fit than their euploid parent, yet there were specific conditions under which specific aneuploid isolates provided a fitness benefit relative to the euploid parent strain. Importantly, this fitness benefit was attributable to the change in the copy number of specific chromosomes. Thus, C. albicans can tolerate aneuploidy of each chromosome and some aneuploids confer improved growth under conditions that the yeast encounters in its host niches.

Candida albicans is a prevalent human fungal pathogen. Rapid genomic change, due to aneuploidy, is a common mechanism that facilitates survival from multiple types of stresses including the few classes of available antifungal drugs. The stress survival of aneuploids occurs despite the fitness costs attributed to most aneuploids growing under idealized lab conditions. Systematic study of the aneuploid state in C. albicans has been hindered by the lack of a comprehensive collection of aneuploid strains. Here, we describe a collection of diploid C. albicans aneuploid strains, each carrying one extra copy of each chromosome, all from the same genetic background. We tested the fitness of this collection under several physiological conditions including shifts in pH, low glucose, oxidative stress, temperature, high osmolarity, membrane stress and cell wall stress. We found that, most aneuploids, under most conditions, were less fit than their euploid parent, yet there were specific conditions under which specific aneuploid isolates provided a fitness benefit relative to the euploid parent strain. Importantly, this fitness benefit was attributable to the change in the copy number of specific chromosomes. Thus, C. albicans can tolerate aneuploidy of each chromosome and some aneuploids confer improved growth under conditions that the yeast encounters in its host niches.

The sudden epidemic has a huge impact on the global economy. This paper takes the International crude oil and the SSE Industry Index as the research objects to explore the linkage between the two markets under COVID-19. We use DCC-GARCH to study the dynamic correlation between the two markets before and after the outbreak. The PCA-GARCH model is further used to verify whether there is a spillover effect between the two markets, and finally the time-varying spillover index is used to quantify the spillover effect. The results show that the epidemic has strengthened the overall connection between the two markets. In particular, the correlation between SSE Public and International crude oil has the greatest impact. During the epidemic, crude oil has the most volatility, and most of the volatility series can reach the peak state. There are positive spillover effects among SSE Material, SSE Energy, and SSE Industry. In the total spillover index table, the conclusion of the PCA-GARCH model is verified, that is, the spillover index value is larger when there is a spillover effect. After the outbreak, the total spillover index rose by 10%. Before and after the outbreak, crude oil changed from a volatility sender to a receiver.

Candida albicans is a common fungal pathogen in humans that colonizes the skin and mucosal surfaces of the majority healthy individuals. How C. albicans disseminates into the bloodstream and causes life-threatening systemic infections in immunocompromised patients remains unclear. Plasminogen system activation can degrade a variety of structural proteins in vivo and is involved in several homeostatic processes. Here, for the first time, we characterized that C. albicans could capture and “subvert” host plasminogen to invade host epithelial cell surface barriers through cell-wall localized Eno1 protein. We found that the “subverted” plasminogen system plays an important role in development of invasive infection caused by C. albicans in mice. Base on this finding, we discovered a mouse monoclonal antibody (mAb) 12D9 targeting C. albicans Eno1, with high affinity to the 254FYKDGKYDL262 motif in α-helices 6, β-sheet 6 (H6S6) loop and direct blocking activity for C. albicans capture host plasminogen. mAb 12D9 could prevent C. albicans from invading human epithelial and endothelial cells, and displayed antifungal activity and synergistic effect with anidulafungin or fluconazole in proof-of-concept in vivo studies, suggesting that blocking the function of cell surface Eno1 was effective for controlling invasive infection caused by Candida spp. In summary, our study provides the evidence of C. albicans invading host by “subverting” plasminogen system, suggesting a potential novel treatment strategy for invasive fungal infections.

Candida albicans is the most common fungal microorganism in healthy individuals, as well as the cause of high mortality infections in high-risk hosts such as immunocompromised patients. Antifungal vaccines and monoclonal antibodies useful for active or passive immunizations have recently generated considerable excitement for the treatment of fungal infections. The cell wall proteins of C. albicans, which are crucial for virulence and pathogenicity, are attractive target antigens. Als3p, a member of the C. albicans agglutinin-like sequence (ALS) family, is a hyphal-specific glycophosphatidylinositol cell wall protein that plays a key role in the interaction with host cells. The abundance of Als3p on the hyphal surface makes it an attractive target. For example, the NDV-3 vaccine, targeted at the N-terminus of Als3p, has entered a preparation of Phase 2 clinical trial. The Als3p-specific antibodies include monoclonal antibodies (MAbs) 3-A5, MAb 113, and scFv3. In addition, MAb C7, MAb 3D9.3 and MAb 2G8, which were supposed to be identifying other targets, have also provided good protection by recognizing Als3p. In this review, we summarize the functions of Als3p and highlight the development of the vaccines and the antibodies that are associated, directly or indirectly, with this protein.

Candida albicans is the most common opportunistic fungal pathogen which can cause life-threatening bloodstream infections known as candidaemia. It is very important to discover new drugs and targets for the treatment of candidaemia. In this study, we first investigated the combination antifungal effects of the small molecule ENOblock and fluconazole (FLC) against FLC-resistant C. albicans. A checkerboard microdilution assay showed that ENOblock has a significant synergistic effect in combination with FLC against FLC-resistant C. albicans. The time-kill curve further confirmed the synergism of this compound with FLC against FLC-resistant C. albicans. Moreover, we demonstrated the significant inhibitory effects of ENOblock alone and in combination with FLC against C. albicans hypha and biofilm formation. Furthermore, the XTT assay showed that ENOblock has relatively low toxicity to human umbilical vein endothelial cells. The in vivo antifungal efficacy of ENOblock was further assessed in a murine model of systemic C. albicans infection. Although ENOblock alone was not sufficient to treat C. albicans infection, the combination of FLC and ENOblock showed significant in vivo activity against FLC-resistant C. albicans. Finally, using surface plasmon resonance analysis as well as an inhibition assay, we determined that ENOblock directly interacted with CaEno1 and significantly inhibited the transglutaminase activity of this enzyme, which is involved in the growth and morphogenesis of C. albicans. In summary, these results demonstrate the synergistic effects of FLC and ENOblock against FLC-resistant C. albicans, and indicate that inhibition of the transglutaminase activity of CaEno1 by ENOblock might confer an advantage for the synergism of FLC and ENOblock, suggesting the potential of ENOblock as a new antifungal candidate.

In the past decades, the incidence of cryptococcosis has increased dramatically, which poses a new threat to human health. However, only a few drugs are available for the treatment of cryptococcosis. Here, we described a leading compound, NT-a9, an analogue of isavuconazole, that showed strong antifungal activities in vitro and in vivo. NT-a9 showed a wide range of activities against several pathogenic fungi in vitro, including Cryptococcus neoformans, Cryptococcus gattii, Candida albicans, Candida krusei, Candida tropicalis, Candida glabrata, and Candida parapsilosis, with MICs ranging from 0.002 to 1 μg/ml. In particular, NT-a9 exhibited excellent efficacy against C. neoformans, with a MIC as low as 0.002 μg/ml. NT-a9 treatment resulted in changes in the sterol contents in C. neoformans, similarly to fluconazole. In addition, NT-a9 possessed relatively low cytotoxicity and a high selectivity index. The in vivo efficacy of NT-a9 was assessed using a murine disseminated-cryptococcosis model. Mice were infected intravenously with 1.8 × 10(6) CFU of C. neoformans strain H99. In the survival study, NT-a9 significantly prolonged the survival times of mice compared with the survival times of the control group or the isavuconazole-, fluconazole-, or amphotericin B-treated groups. Of note, 4 and 8 mg/kg of body weight of NT-a9 rescued all the mice, with a survival rate of 100%. In the fungal-burden study, NT-a9 also significantly reduced the fungal burdens in brains and lungs, while fluconazole and amphotericin B only reduced the fungal burden in lungs. Taken together, these data suggested that NT-a9 is a promising antifungal candidate for the treatment of cryptococcosis infection.

Loureirin A is a major active component of Draconis sanguis, a traditional Chinese medicine. This work aimed to investigate the activity of loureirin A against Candida albicans biofilms. 2, 3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT)reduction assay and scanning electron microscopy were used to investigate the anti-biofilm effect. Minimal inhibitory concentration testing and time-kill curve assay were used to evaluate fungicidal activity. Cell surface hydrophobicity (CSH) assay and hyphal formation experiment were respectively carried out to investigate adhesion and morphological transition, two virulence traits of C. albicans. Real-time RT-PCR was used to investigate gene expression. Galleria mellonella-C. albicans and Caenorhabditis elegans-C. albicans infection models were used to evaluate the in-vivo antifungal effect. Human umbilical vein endothelial cells and C. elegans nematodes were used to evaluate the toxicity ofloureirin A. Our data indicated that loureirin A had a significant effect on inhibiting C. albicans biofilms, decreasing CSH, and suppressing hyphal formation. Consistently, loureirin A down-regulated the expression of some adhesion-related genes and hypha/biofilm-related genes. Moreover, loureirin A prolonged the survival of Galleria mellonella and Caenorhabditis elegans in C. albicans infection models and exhibited low toxicity. Collectively, loureirin A inhibits fungal biofilms, and this effect may be associated with the suppression of pathogenic traits, adhesion and hyphal formation.

There are increasing invasive fungal infections associated with non-albicans, which causes mortal infections in immune deficiency population. Candida krusei is a major non-albicans that exhibits intrinsic resistance to fluconazole and makes clinical treatment difficult. Previous studies revealed that C-type lectin receptors (CLRs) Dectin-1 plays critical roles in host defense against C. albicans infections. C. krusei and C. albicans are phylogenetically different although in the same genus. Whether Dectin-1 contributes to host immune response against C. krusei infection is still unknown. In the present study, we explored the potential roles of the Dectin-1 in host defense against C. krusei. We found that Dectin-1 ligand β-(1,3)-glucan markedly exposed on the cell surface of C. krusei, while β-(1,3)-glucan of C. albicans is masked. Dectin-1 is required for host myeloid cells recognition, killing of C. krusei, and development of subsequent Th1 and Th17 cell-mediated adaptive immune response. Furthermore, Dectin-1-deficient mice (Dectin-1(-/-)) are more susceptible to C. krusei infection. Together, we confirmed the important roles of Dectin-1 in host defense against C. krusei infection, demonstrating a previously unknown mechanism for C. krusei infection. Our study, therefore, provides a further understanding of host immune response against C. krusei.

Sterols are the basal components of the membranes of the fungal pathogen Candida albicans, and these membranes determine the susceptibility of C. albicans cells to a variety of stresses, such as ionic, osmotic and oxidative pressures, and treatment with antifungal drugs. The common antifungal azoles in clinical use are targeted to the biosynthesis of ergosterol. In the past years, the synthesis, storage and metabolism of ergosterol in Saccharomyces cerevisiae has been characterized in some detail; however, these processes has not been as well investigated in the human opportunistic pathogen C. albicans. In this review, we summarize the genes involved in ergosterol synthesis and regulation in C. albicans. As well, genes in S. cerevisiae implicated in ergosterol storage and conversions with other lipids are noted, as these provide us clues and directions for the study of the homologous genes in C. albicans. In this report we have particularly focused on the essential roles of ergosterol in the dynamic process of cell biology and its fundamental status in the biological membrane system that includes lipid rafts, lipid droplets, vacuoles and mitochondria. We believe that a thorough understanding of this classic and essential pathway will give us new ideas about drug resistance and morphological switching in C. albicans.

Candida albicans is a polymorphic fungus which is the predominant cause of superficial and deep tissue fungal infections. This microorganism has developed efficient strategies to invade the host and evade host defense systems. However, the host immune system will be prepared for defense against the microbe by recognition of receptors, activation of signal transduction pathways and cooperation of immune cells. As a consequence, C. albicans could either be eliminated by immune cells rapidly or disseminate hematogenously, leading to life-threatening systemic infections. The interplay between Candida albicans and the host is complex, requiring recognition of the invaded pathogens, activation of intricate pathways and collaboration of various immune cells. In this review, we will focus on the effects of innate immunity that emphasize the first line protection of host defense against invaded C. albicans including the basis of receptor-mediated recognition and the mechanisms of cell-mediated immunity.

This study aimed to investigate the antifungal activity of Rubus chingii extract in combination with fluconazole (FLC) against FLC-resistant Candida albicans 100 in vitro. A R. chingii extract and FLC-resistant C. albicans fungus suspension were prepared. The minimum inhibitory concentration and fractional inhibitory concentration index of R. chingii extract combined with FLC against C. albicans were determined, after which growth curves for C. albicans treated with R. chingii extract, FLC alone and a combination of these preparations were constructed. Additionally, the mechanisms of drug combination against C. albicans were explored by flow cytometry, gas chromatographic mass spectrometry and drug efflux pump function detection. R. chingii extract combined with FLC showed significant synergy. Flow cytometry suggested that C. albicans cells mainly arrest in G1 and S phases when they have been treated with the drug combination. The drug combination resulted in a marked decrease in the ergosterol content of the cell membrane. Additionally, efflux of Rhodamine 6G decreased with increasing concentrations of R. chingii extract. R. chingii extract combined with FLC has antifungal activity against FLC-resistant C. albicans.

The aim of the present study was to investigate the role of nitric oxide (NO) in the antifungal activity of Shikonin (SK) against Candida albicans (C. albicans) and to clarify the underlying mechanism. The results showed that the NO donors S-nitrosoglutathione (GSNO) and L-arginine could enhance the antifungal activity of SK, whereas the NO production inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) attenuated antifungal action. Using the fluorescent dye 3-amino,4-aminomethyl-2′, 7-difluorescein, diacetate (DAF-FM DA), we found that the accumulation of NO in C. albicans was increased markedly by SK in a time- and dose-dependent manner. In addition, the results of real-time reverse transcription-PCR (RT-PCR) demonstrated that the transcription level of YHB1 in C. albicans was greatly increased upon incubation of SK. Consistently, the YHB1-null mutant (yhb1Δ/Δ) exhibited a higher susceptibility to SK than wild-type cells. In addition, although the transcription level of CTA4 in C. albicans was not significantly changed when exposed to SK, the CTA4-null mutant (cta4Δ/Δ) was more susceptible to SK. Collectively, SK is the agent found to execute its antifungal activity directly via endogenous NO accumulation, and NO-mediated damage is related to the suppression of YHB1 and the function of CTA4.

Antifungal azole drugs inhibit the synthesis of ergosterol and cause the accumulation of sterols containing a 14α-methyl group, which is related to the properties of cell membrane. Due to the frequent recurrence of fungal infections and clinical long-term prophylaxis, azole resistance is increasing rapidly. In our research, Nsg2p, encoded by the ORF19.273 in Candida albicans, is found to be involved in the inhibition of 14α-methylated sterols and resistance to azoles. Under the action of fluconazole, nsg2Δ/Δ mutants are seriously damaged in the integrity and functions of cell membranes with a decrease of ergosterol ratio and an increase of both obtusifoliol and 14α-methylfecosterol ratio. The balance between ergosterol and 14α-methyl sterols mediated by NSG2 plays an important role in C. albicans responding to azoles in vitro as well as in vivo. These phenotypes are completely different from those of Nsg2p in Saccharomyces cerevisiae, which is proved to increase the stability of HMG-CoA and resistance to lovastatin. Based on the evidence above, it is indicated that the decrease of 14α-methylated sterols is an azole-resistant mechanism in C. albicans, which may provide new strategies for overcoming the problems of azole resistance.

Widespread and repeated use of azoles has led to the rapid development of drug resistance in Candida albicans. Our previous study found Rta2p, a membrane protein with 7 transmembrane domains, was involved in calcineurin-mediated azole resistance and sphingoid long-chain base release in C. albicans. Conserved amino acids in the transmembrane domain of Rta2p were subjected to site-directed mutagenesis. The sensitivity of C. albicans to fluconazole in vitro was examined by minimum inhibitory concentration and killing assay, and the therapeutic efficacy of fluconazole in vivo was performed by systemic mice candidiasis model. Furthermore, dihydrosphingosine transport activity was detected by NBD labeled D-erythro-dihydrosphingosine uptake and release assay, and the sensitivity to sphingolipid biosynthesis inhibitors. We successfully constructed 14 mutant strains of Rta2p, screened them by minimum inhibitory concentration and found Ca(2+) did not completely induce fluconazole resistance with G158E and G234S mutations. Furthermore, we confirmed that G234S mutant enhanced the therapeutic efficacy of fluconazole against systemic candidiasis and significantly increased the accumulation of dihydrosphingosine by decreasing its release. However, G158E mutant didn't affect drug therapeutic efficacy in vivo and dihydrosphingosine transport in C. albicans. G234 of Rta2p in C. albicans is crucial in calcineurin-mediated fluconazole resistance and dihydrosphingosine transport.

Fungi can shield surface pathogen-associated molecular patterns (PAMPs) for evading host immune attack. The most common and opportunistic human pathogen, Candida albicans , can shield β-(1 3)-glucan on the cell wall, one of the major PAMPs, to avoid host phagocyte Dectin-1 recognition. The way to interfere in the shielding process for more effective antifungal defense is not well established. In this study, we found that deletion of the C. albicans GPI7 gene, which was responsible for adding ethanolaminephosphate to the second mannose in glycosylphosphatidylinositol (GPI) biosynthesis, could block the attachment of most GPI-anchored cell wall proteins (GPI-CWPs) to the cell wall and subsequently unmask the concealed β-(1,3)-glucan. Neutrophils could kill the uncloaked gpi7 mutant more efficiently with an augmented respiratory burst. The gpi7 mutant also stimulated Dectin-1-dependent immune responses of macrophages, including activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and secretion of specific cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-12p40. Furthermore, the gpi7 null mutant could induce an enhanced inflammatory response through promoting significant recruitment of neutrophils and monocytes and could stimulate stronger Th1 and Th17 cell responses to fungal infections in vivo . These in vivo phenotypes also were Dectin-1 dependent. Thus, we assume that GPI-CWPs are involved in the immune mechanism of C. albicans escaping from host recognition by Dectin-1. Our studies also indicate that the blockage of GPI anchor synthesis is a strategy to inhibit C. albicans evading host recognition.

Kehan Xu,1,* Lei Huang,1,* Zheng Xu,2 Yanwei Wang,1,3 Guojing Bai,1 Qiuye Wu,1 Xiaoyan Wang,1 Shichong Yu,1 Yuanying Jiang1 1School of Pharmacy, Second Military Medical University, Shanghai, 2Shanghai Changzheng Hospital, Second Military Medical University, Shanghai, 3Number 422 Hospital of PLA, Zhanjiang, People’s Republic of China *These authors contributed equally to this work Abstract: In previous studies undertaken by our group, a series of 1-(1H-1,2,4-triazole-1-yl)-2-(2,4-difluorophenyl)-3-substituted-2-propanols (1a–r), which were analogs of fluconazole, was designed and synthesized by click chemistry. In the study reported here, the in vitro antifungal activities of all the target compounds were evaluated against eight human pathogenic fungi. Compounds 1a, 1q, and 1r showed the more antifungal activity than the others. Keywords: triazole, synthesis, antifungal activity, CYP51

Candida albicans is both a commensal microorganism in healthy individuals and a major fungal pathogen causing high mortality in immunocompromised patients. Yeast-hypha morphological transition is a well known virulence trait of C. albicans. Host innate immunity to C. albicans critically requires pattern recognition receptors (PRRs). In this review, we summarize the PRRs involved in the recognition of C. albicans in epithelial cells, endothelial cells, and phagocytic cells separately. We figure out the differential recognition of yeasts and hyphae, the findings on PRR-deficient mice, and the discoveries on human PRR-related single nucleotide polymorphisms (SNPs).

Candida albicans is the most common cause of hematogenously disseminated candidiasis, and this disease is particularly prevalent in immunocompromised patients. The mortality of invasive candidiasis remains 40% to 50% even with the proper treatment with current antifungal drugs. Recently, with the better understanding of host-fungus interactions, notable progress has been made in antifungal vaccine research. Most antifungal vaccines exert protection by inducing either (or both) B-cell and T-cell responses. Here we summarize the current available information on C. albicans vaccines, highlight the obstacles that researchers identified, and offer several suggestions.