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Introduction Intercellular communication helps regulate the cell density and the virulence traits in yeasts and bacteria. The study aims to identify the effects of quorum sensing molecules (QSMs) like farnesol and tyrosol on Candida spp. virulence traits. Methods The effects of farnesol and tyrosol were studied on the growth rate of Candida albicans, Candida parapsilosis, Candida krusei, Candida auris, and Candida guilliermondii at different time points, on a 48 hours incubation period. The growth rate was assessed spectrophotometrically. The biofilm formation abilities of Candida spp. were assessed by crystal violet staining technique. Moreover, the expression of C. albicans virulence genes (ALS3, HSP70, SAP2) was analyzed as a response to 100 µM farnesol and tyrosol, by RT-PCR. Results Generally, farnesol was found to inhibit the growth rate and biofilm formation mostly in non-albicans species, while tyrosol exerted a non-consistent response on the different Candida species. The expression of ALS3 and HSP70 in C. albicans was upregulated by the QSMs. Conclusions Both farnesol and tyrosol are involved in the regulation of Candida spp. virulence mechanisms, dependent on the used concentration and exposure time and in a species-dependent manner. [ABSTRACT FROM AUTHOR]

Objective To analyse the effects of Candida albicans virulence factor SAP2 and transcription factor MRR2 on Itraconazole (ITR) resistance in the planktonic and biofilm states. Methods Sequencing of SAP! and MRR2 genes on 10 ITR monoresistant strains and 10 FCA/ITR /VRC fully susceptible strains to analyse the effect of mutations on drug resistance. The formation of Candida albicans biofilms under different time periods was observed using an inverted microscope and laser confocal microscope. The relationship between biofilm formation ability and drug resistance was assessed by measuring the optical density (OD) value of the biofilm using an enzyme marker. Detection and analysis of SAP2 and MRR2 expression in clinical isolates of Candida albicans in planktonic and biofilm states respectively, by real-time fluorescence quantitative polymerase chain reaction ( RT-PCR) technology. We also examined the expression of SAP2 in MRR2A/A strains in planktonic and biofilm states to further clarify the relationship between MRR2 and SAP2. Results Gene sequencing revealed no missense mutations in the SAP2 gene, and sequencing of the MRR2 gene revealed 12 missense mutation sites. It was found that the expression of SAP2 [ planktonic/biofilm: (1.30 ± 0.39)/(4.29 ± 1.76)] and MRR2 [planktonic/biofilm: (0.95 ± 0.14)/(1.77 ±0.80)] were higher in resistant strain group than sensitive strain group in different states [SAP2 planktonic/ biofilm: (0.80 ± 0.48)/(2.59 ± 0.93); MRR2 planktonic/biofilm: (0.49 ± 32)/(0.98 ± 0.64), P < 0.05 ]. The expression levels of both SAP2 (3.44 ± 0.63) and MRR2 (1.19 ±0. 99) were up-regulated in the biofilm state compared to the planktonic state [SAP2/MRR2, (1.05 ± 0.50)/(0.81 ± 0.62)] (P < 0.05), and there was a positive correlation between the two genes in the planktonic state (r = 0.66, P <0.05), the expression of MRR2A/A strain SAP2 was significandy down-regulated in both planktonic and biofilm states [planktonic/biofilm; (0.80 ± 0.06)/(2.15 ±0.23)] relative to the standard strain ATCC11006 [planktonic/ biofilm: (1.14 ± 0.07)/(2.90 ± 0.10)] (P < 0.05). All strains formed biofilms with interlocking hyphae and spores, and it was found that the biofilm formation ability of the drug-resistant strain group (0.24 ±0.02) was significantly higher than the sensitive group (0.15 ± 0.02, P < 0.001), and that the MRR2A/A strain's biofilm formation ability (0.12 ± 0.12) was significantly lower than the standard strain group (0.16 ±0.00, P < 0.001). Conclusion ITR resistance may be associated with Candida albicans MRR2 gene mutations. The high expression of SAP2 and MRR2 in both planktonic and biofilm states may affect ITR resistance in Candida albicans. Furthermore, there is evidence to suggest that the high expression of MRR2 may positively regulate SAP2, which in turn affects its drug resistance. [ABSTRACT FROM AUTHOR]

Abstract Background Globally, millions of people suffer from repeated fungal infections affecting the skin, keratinized tissues, and mucosal membranes. Approximately 1.7 million cases result in death with an elevated incidence rate among immunocompromised people that may later result in severe infections. Among the causative agents, Candida albicans are the most prevalent fungi inducing subcutaneous to invasive candidiasis. Although they are commensals in human body alteration in gut or prolonged treatments results in candidiasis. Several virulence proteins of C. albicans are involved in infections and secreted aspartic proteases2 (SAP2) plays an important role among them by causing damage to the reconstituted human epithelium. In the present study, phytochemicals from Heliotropium indicum, Grona triflora, Ziziphus mauritiana, Atalantia racemosa, Coccinia grandis, Caryota urens, Aristolochia bracteolata, Evolvulus alsinoides, Pyrus communis and Commelina benghalensis were studied against SAP2 with the help of bioinformatic tools to understand their binding efficiency. Results The phytochemical structures were retrieved from PubChem database and the target protein structure was retrieved from PDB database with ID:3PVK. ADME profiling for phytochemicals was performed with Qikprop module, followed by docking with protein using Schrodinger software. Docking studies showed that Indicine-N-Oxide from H. indicum scored the significant glide score of − 5.54 kcal/mol. Finally, antifungal studies against C. albicans were conducted using several extracts of plants containing phytochemicals with considerable glide scores on docking studies. The Ethyl acetate leaf extract of H. indicum prominently inhibited the fungal growth when compared with the control. Conclusion Identification of effective therapeutic candidates for the treatment of fungal infection is facilitated by the potential of H. indicum to hinder fungal growth and the interaction of their phytocompounds with fungal targets paves a way for developing a novel drug.

Background: SAP2 is closely associated with the pathogenicity and drug resistance of Candida albicans (C. albicans). Our study aimed to construct C. albicans with SAP2 overexpression (pRB895-SAP2-SC5314) to explore the influence of SAP2 on the adhesion of C. albicans and predict the interaction between magnolol and Sap2 by molecular docking., Methods: The recombinant plasmid pRB895-SAP2 with high SAP2 expression was prepared using a plasmid extraction kit and transformed into C. albicans strain SC5314 using an improved lithium acetate conversion method to construct PRB895-SAP2-SC5314. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to detect the expression of adhesion-related genes in the different strains. Molecular docking and visual analysis of magnolol and Sap2 were performed using the CB-DOCK2 platform., Results: Compared with the control SC5341 and SC5341 transfected with pRB895, SAP2 expression was significantly higher in the pRB895-SAP2-SC5314 strain (p < 0.05). Based on the sequencing and mapping results, the pRB895-SAP2-SC5314 strain was successfully prepared. SAP2 overexpression significantly downregulated ALS1 expression (p < 0.05), whereas ALS3, TEC1, HOG1, PHR1, and TUP1 expression was downregulated in C. albicans (p < 0.05). The optimal docking result for magnolol and Sap2 protein was -8.1 kcal/mol of vina score, which was considered good docking., Conclusions: SAP2 overexpression may strengthen the adhesion and pathogenicity of C. albicans, and magnolol may act as an Sap2 inhibitor that affects the adhesion of C. albicans., (© 2024 The Author(s). Journal of Clinical Laboratory Analysis published by Wiley Periodicals LLC.)

Abstract Background Candida albicans, the main human fungal pathogen, can cause fungal infection and seriously affect people's health and life. This study aimed to investigate the effects of ritonavir (RIT) on C. albicans and the correlation between SAP2 as well as ERG11 and drug resistance. Results Secreted aspartyl proteinases (Saps) activities and pathogenicity of C. albicans with different drug resistance were measured. M27‐A4 broth microdilution method was used to analyze the drug sensitivity of RIT combined with fluconazole (FCA) on C. albicans. After that, SAP2 and ERG11 mutations were examined by polymerase chain reaction (PCR) and sequencing, and quantitative real‐time PCR was utilized to determine the expression of the two genes. By analyzing pz values, the Saps activity of cross‐resistant strains was the highest, followed by voriconazole (VRC)‐resistant strains, FCA‐resistant strains, itraconazole (ITR)‐resistant strains, and sensitive strains. The pathogenicity of C. albicans in descending order was as follows: cross‐resistant strains, VRC‐resistant strains, ITR‐resistant strains, FCA‐resistant strains, and sensitive strains. With the increase of RIT concentrations, the Saps activity was gradually inhibited. Drug sensitivity results showed that there was no synergistic effect between RIT and FCA. Additionally, no gene mutation sites were found in SAP2 sequencing, and 17 synonymous mutations and 6 missense mutations occurred in ERG11 sequencing. Finally, the expression of SAP2 and ERG11 was significantly higher in the resistant strains compared with the sensitive strains, and there was a positive liner correlation between SAP2 and ERG11 messenger RNA expression (r = .6655, p

Interleukin-6 (IL-6) is a cytokine with pleiotropic effects that might also influence the virulence traits of some microorganisms, but its direct influence over Candida spp. is currently unknown. The objective of the study is to determine the influence of IL-6 (250 pg/ml) on the growth rate and biofilm formation of C. albicans, C. parapsilosis, C. krusei, C. auris, and C. guilliermondii, as well as to analyze the influence of this citokine on the expression of three virulence genes (ALS3, HSP70, SAP2), respectively on the germ tube formation ability of C. albicans. The influence of IL-6 on growth rate was assessed by incubating the fungal cells in presence of IL-6 for 48 hours and assessing the optical density of the samples at five timepoints. The biofilm production in presence of IL-6 was studied in microtiter plates, using crystal-violet assay, the intensity of biofilms being evaluated by spectrophotometry. The expression of ALS3, HSP70, and SAP2 in C. albicans was studied by RT-PCR, reported to ACT1 housekeeping gene. The germ-tube test was performed to assess the influence of IL-6 on the filamentation rate of C. albicans. All test results were normalized against control, without added IL-6. The results showed that IL-6 influences the growth rate of C. albicans, C. parapsilosis, C. krusei, C. auris, and C. guilliermondii in a time-dependent way. Also, IL-6 inhibited the formation of biofilms for C. albicans and C. guilliermondii. In C. albicans cells, IL-6 induced upregulation of ALS3 and HSP70, while it down-regulated the SAP2 gene. IL-6 did not influence the germ-tube formation in C. albicans. In conclusion, IL-6 might exert, in vitro, direct effects on the virulence traits of Candida spp., and its influence is dependent on the exposure time. Non-albicans Candida species presented particular responses to IL-6. [ABSTRACT FROM AUTHOR]

Background: Candida albicans, the main human fungal pathogen, can cause fungal infection and seriously affect people's health and life. This study aimed to investigate the effects of ritonavir (RIT) on C. albicans and the correlation between SAP2 as well as ERG11 and drug resistance. Results: Secreted aspartyl proteinases (Saps) activities and pathogenicity of C. albicans with different drug resistance were measured. M27‐A4 broth microdilution method was used to analyze the drug sensitivity of RIT combined with fluconazole (FCA) on C. albicans. After that, SAP2 and ERG11 mutations were examined by polymerase chain reaction (PCR) and sequencing, and quantitative real‐time PCR was utilized to determine the expression of the two genes. By analyzing pz values, the Saps activity of cross‐resistant strains was the highest, followed by voriconazole (VRC)‐resistant strains, FCA‐resistant strains, itraconazole (ITR)‐resistant strains, and sensitive strains. The pathogenicity of C. albicans in descending order was as follows: cross‐resistant strains, VRC‐resistant strains, ITR‐resistant strains, FCA‐resistant strains, and sensitive strains. With the increase of RIT concentrations, the Saps activity was gradually inhibited. Drug sensitivity results showed that there was no synergistic effect between RIT and FCA. Additionally, no gene mutation sites were found in SAP2 sequencing, and 17 synonymous mutations and 6 missense mutations occurred in ERG11 sequencing. Finally, the expression of SAP2 and ERG11 was significantly higher in the resistant strains compared with the sensitive strains, and there was a positive liner correlation between SAP2 and ERG11 messenger RNA expression (r =.6655, p <.001). Conclusion: These findings may help to improve our understanding of azole‐resistant mechanisms of C. albicans and provide a novel direction for clinical therapeutics of C. albicans infection. [ABSTRACT FROM AUTHOR]

Background: Candidiasis has gained much attention in recent decades due to its increasing prevalence in immunocompromised patients. Usually, antifungals such as fluconazole and amphotricin B are used for treatment of candidiasis, but one of the major clinical problems is the emergence of antifungal resistance. Combination antifungal therapy is one of the most commonly used methods to alleviate the problem of antifungal resistance. Methods: The effect of fluconazole alone and in combination with amphotericin B on C. tropicalis isolates were performed using the Clinical and Laboratory Standards Institute (CLSI) reference method. Eventually hypha formation, time kill study, proteinase and phospholipase activity and expression of PLB and SAP2 genes were carried out to investigate the enzymes inhibitory properties of antifungal tested against C. tropicalis. Results: Results showed the significant synergic effect of fluconazole in combination with amphotericin B in inhibiting the growth of C. tropicalis isolates, with fractional inhibitory concentration indices ranging from 0.06 to 0.5. The combination of fluconazole with amphotericin B reduced the number of yeast form and inhibited the yeast to hyphae transition in C. tropicalis. The antifungals tested were able to show the effect of down regulating expression of the selected genes significantly in fluconazole/amphotericin B ranging from 1.42- to 2.27-fold. Conclusion: Our results demonstrated that the synergistic interaction of fluconazole/amphotericin B would be worth exploring for the management of candidiasis. In addition, PLB and SAP2 genes could be probable molecular targets in the synergistic interaction of fluconazole/amphotericin B in C. tropicalis.

Candida albicans is colonizes in oral, gastrointestinal tract, skin, and vagina at many of anatomically distinct sites, a commensal C. albicans how harmless, It's not certain whether become a pathogen of human as opportunities come, This review will focus on the dissection of confirmatory diagnosis of C. albicans isolates by 18srRNA and study some Virulence factor genes that is importance and its effect on the female reproductive system, It also affects fetuses in pregnant women include With its impression share (INT1 (31.42%), PlB1(100%), Hwp1(100%), SAP1(48.57%), SAP2(100%), ALS1(45,71%), CPH1(40%), CDR1(92.85%), MDR1(78.57%), from (300) female genital tract secretions specimens isolate, The number of bacterial isolates was 165 (55%) and the number of yeast isolates was 1 10 (36,6%) and no growth 25(8,33%).The current study was isolated some types of yeast from women with vaginitis, belong to a genus Candida, C. albicans, C. krusei, C. tropicalis, C. glabrata, C. parapsilosisas, where the largest number and percentage was for C. albicans 70(63,6%),18 (16.3%),10(9.09%), 7 (6.36%), 5 (4.54%) Respectively, The results also showed that the resistance and sensitivity of the different types of antifungals (Nystatin, ketoconazole, Amphotericin B, Miconazole) used in the C. albicans fungus, The ratio of resistance (50 (71.4%), 42(60%), 37(52.8%), 30(42.8%) Respectively [ABSTRACT FROM AUTHOR]

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy.

Due to their high content of biomolecules, combined with silver’s well known antimicrobial potential, silver nanoparticles biosynthesized using spruce bark (AgNP SBEs) demonstrate antibacterial and antioxidant activity, making them a versatile option for developing new antimicrobial agents that might be used for medical treatment or as adjuvants for the classical agents. This study aims to analyze if silver nanoparticles (AgNPs) mediated by spruce bark extract (SBE) and silver salts (AgNP SBE Acetate, AgNP SBE Nitrate) presents antifungal activity against five different Candida spp., synergistic activity with fluconazole, and if they interact with some virulence factors of C. albicans. AgNP SBEs presented MICs (minimum inhibitory concentrations) for all the five tested Candida spp. AgNP SBEs inhibited the growth of C. parapsilosis, C. krusei, and C. guilliermondii, exerted synergistic activity with fluconazole for C. parapsilosis and C. guilliermondii, and inhibited biofilm production for C. albicans, C. auris, and C. guilliermondii. MICs of AgNP SBE Acetate significantly inhibited the production of germ tubes of C. albicans. The expression of C. albicans SAP2 gene was down-regulated by the short-time treatment with MICs of AgNP SBE Acetate, while ALS3 and HSP70 genes were up-regulated by the AgNPs MICs. These results emphasize the potential of using the AgNP SBEs as treatments/adjuvants options, not only against the redundant C. albicans but also for the non-albicans Candida species (which are not as frequently involved in human pathologies, but, sometimes, can be more aggressive).

In acute vulvovaginal candidiasis (VVC), the fungus Candida albicans activates inflammasome receptors of vaginal epithelial cells through the production of virulence and immuno-inflammatory factors. Here, we show that in VVC patients, genes encoding some of the above factors (SAP2, SAP5, SAP6, ECE1, and HWP1) are expressed in a correlated fashion. Cytological observations pointed out that pseudohyphal filaments with yeast cells are dominant at the acidic vaginal pH, and this is coupled with co-expression, at roughly similar level, of SAP2, a typical yeast and ECE1, a typical hyphae-associated genes. In contrast, vigorous hyphal growth dominated at the neutral vaginal pH of mice experimentally infected with C. albicans isolates from VVC subjects, and this is coupled with a high ratio of ECE1 to SAP2 expression. We suggest that the pseudohyphal rather than true hyphal cells of C. albicans play a critical role in VVC, possibly through the activity of multiple inflammasome inducers.

Multilocus sequence typing is a powerful method for genotyping of clinical isolates of Candida albicans. Cross-contamination between the patients is an important reason for nosocomial infections. Oropharyngeal candidiasis (OPC) caused by C. albicans is an important problem in patients with head and neck cancer, in Cancer Institute of Tehran. Here we studied the endemic genotypes of C. albicans isolates and the relationship between geographic distributions, potential cross-contaminations and the expression of SAP2 gene, an important gene in oral candidiasis, with MLST groups. A total of 32 clinical strains of C. albicans isolated from head and neck cancer patients with oropharyngeal candidiasis were subjected to MLST analysis and SAP2 gene expression was analyzed by Real-time PCR. We identified 75 polymorphic sites in 7 loci of C. albicans isolates and 30 diploid sequence types which 27 of them were found as new. After eBURST analysis, our results determined that CC 124 was the most prevalent group among all CCs. SAP2 gene showed high expression in almost all OPC patients' isolates, compared to the control. We found few genetically-related as well as identical isolates among the 32 Candida strains which indicated low cross-contaminations among the patients. There was no relationship between C. albicans MLST profiles and their geographic distribution, cancer type and SAP2 gene expression. This lack of correlation was possibly due to the small understudy population; hence, finding more relevance requires studies with a higher number of samples. [ABSTRACT FROM AUTHOR]

In acute vulvovaginal candidiasis (VVC), the fungus Candida albicans activates inflammasome receptors of vaginal epithelial cells through the production of virulence and immuno-inflammatory factors. Here, we show that in VVC patients, genes encoding some of the above factors (SAP2 , SAP5 , SAP6 , ECE1 , and HWP1) are expressed in a correlated fashion. Cytological observations pointed out that pseudohyphal filaments with yeast cells are dominant at the acidic vaginal pH, and this is coupled with co-expression, at roughly similar level, of SAP2 , a typical yeast and ECE1 , a typical hyphae-associated genes. In contrast, vigorous hyphal growth dominated at the neutral vaginal pH of mice experimentally infected with C. albicans isolates from VVC subjects, and this is coupled with a high ratio of ECE1 to SAP2 expression. We suggest that the pseudohyphal rather than true hyphal cells of C. albicans play a critical role in VVC, possibly through the activity of multiple inflammasome inducers. [ABSTRACT FROM AUTHOR]

Background: The secreted aspartyl proteinases 2 (Sap2) of Candida albicans (C. albicans) is a potential marker of candididasis. It is a virulence factor associated with adherence and tissue invasion. Aim: In order to detect Sap2 in clinical sera, we developed an indirect competitive enzyme-linked immunosorbent assay (ELISA). Materials and Methods: Polyclonal antibodies were produced for Sap2 by injecting Sap2 into a New Zealand White inbred rabbit. They could be used at a dilution exceeding 1:1200 in an indirect ELISA, and detected Sap2 concentration up to 1 ng/mL. Results: Of the 286 cancer serum samples tested, 16.8% were found as candidiasis. The test was simple and economical to perform and had a level of sensitivity for detection of low-titer positive sera; thus, it may be proven to be of value in epidemiological studies on candidiasis.

Candida albicans is an opportunistic fungal pathogen that causes severe systemic infections in immunosuppressed individuals. C. albicans resistance to antifungal drugs is a severe problem in patients receiving prolonged therapy. Moreover, trailing yeast growth, which is defined as a resistant MIC after 48 h of incubation with triazole antifungal agents but a susceptible MIC after 24 h, has been noted in tests of antifungal susceptibility against some C. albicans isolates. In this context, we recently noticed this phenomenon in our routine susceptibility tests with fluconazole/itraconazole and C. albicans clinical isolates. In the present study, we investigated the production of cell-associated and secreted aspartyl peptidases (Saps) in six trailing clinical isolates of C. albicans, since this class of hydrolytic enzymes is a well-known virulence factor expressed by this fungal pathogen. Sap2, which is the best-studied member of the Sap family, was detected by flow cytometry on the cell surface of yeasts and as a 43-kDa polypeptide in the culture supernatant, as demonstrated by Western blotting assay using an anti-Sap1-3 polyclonal antibody. Released aspartyl peptidase activity was measured with BSA hydrolysis and inhibited by pepstatin A, showing distinct amounts of proteolytic activity ranging from 5.7 (strain 44B) to 133.2 (strain 11) arbitrary units. Taken together, our results showed that trailing clinical isolates of C. albicans produced different amounts of both cellular and secreted aspartyl-type peptidases, suggesting that this phenotypic feature did not generate a regular pattern regarding the expression of Sap.

The opportunistic pathogenic yeast Candida albicans employs several mechanisms to interfere with the human complement system. This includes the acquisition of host complement regulators, the release of molecules that scavenge complement proteins or block cellular receptors, and the secretion of proteases that inactivate complement components. Secreted aspartic protease 2 (Sap2) was previously shown to cleave C3b, C4b and C5. C. albicans also recruits the complement inhibitor factor H (FH), but yeast-bound FH can enhance the antifungal activity of human neutrophils via binding to complement receptor type 3 (CR3). In this study, we characterized FH binding to human monocyte-derived macrophages. Inhibition studies with antibodies and siRNA targeting CR3 (CD11b/CD18) and CR4 (CD11c/CD18), as well as analysis of colocalization of FH with these integrins indicated that both function as FH receptors on macrophages. Preincubation of C. albicans yeast cells with FH induced increased production of IL-1β and IL-6 in macrophages. Furthermore, FH enhanced zymosan-induced production of these cytokines. C. albicans Sap2 cleaved FH, diminishing its complement regulatory activity, and Sap2-treatment resulted in less detectable CR3 and CR4 on macrophages. These data show that FH enhances the activation of human macrophages when bound on C. albicans. However, the fungus can inactivate both FH and its receptors on macrophages by secreting Sap2, which may represent an additional means for C. albicans to evade the host innate immune system. [ABSTRACT FROM AUTHOR]

Fungal virulence factors represent a strategy for the design of new compounds with effective activities against Candida spp. Dichloromethyl-4-chloro-3-nitrophenylsulfone (named Compound 1) and chlorodibromomethyl-4-hydrazino-3-nitrophenylsulfone (Compound 2) versus Candida albicans virulence factors ( SAP2 expression and adhesion to Caco-2 cell line) were investigated. Candida albicans SC5314 and its mutants: Δsap9, Δsap10, Δsap9/10 were used. MICs of the Compounds (concentrated at 0.0313-16 µg/ml) were determined using M27-A3. Percentage of cell inhibition was assessed spectrophotometrically (OD) after 48 h at 35 °C. The SAP2 expression was analyzed with the use of RT-PCR; relative quantification was normalized against ACT1 in cells grown in YEPD and on Caco-2. Adherence assay of C. albicans to Caco-2 was performed in a 24-well-plate. Compound 1 showed higher activity (% = 100 at 4 µg/ml) than Compound 2 (MIC = 16 µg/ml). Dichloromethyl at the para position of the phenyl ring exerted anti- Candidal potential. Under Compound 1, SAP2 was down-regulated in all the strains ( P ≤ 0.05). Conversely, SAP2 was over-expressed in Δsap9- 10 (untreated cells) compared with the wild-type. The Compounds significantly affected adherence to epithelium ( P ≤ 0.05). The tested sulfones interfered with the adhesion of C. albicans cells to the epithelial tissues without affecting their viability after 90-min of incubation. The Compounds' mode of action was attributed to the reduced adhesiveness and the lower SAP2 expression. Saps9-10 play a role in C. albicans adhesion and they can be involved in the sulfone resistance mechanisms. [ABSTRACT FROM AUTHOR]

Background: Smokers are more prone to oral infections than are non-smokers. Cigarette smoke reaches the host cells but also microorganisms present in the oral cavity. The contact between cigarette smoke and oral bacteria promotes such oral diseases as periodontitis. Cigarette smoke can also modulate C. albicans activities that promote oral candidiasis. The goal of this study was to investigate the effect of cigarette smoke condensate on C. albicans adhesion, growth, and biofilm formation as well as the activation of EAP1, HWP1 and secreted aspartic protease 2. Results: Cigarette smoke condensate (CSC) increased C. albicans adhesion and growth, as well as biofilm formation. These features may be supported by the activation of certain important genes. Using quantitative RT-PCR, we demonstrated that CSC-exposed C. albicans expressed high levels of EAP1, HWP1 and SAP2 mRNA and that this gene expression increased with increasing concentrations of CSC. Conclusion: CSC induction of C. albicans adhesion, growth, and biofilm formation may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced oral diseases. [ABSTRACT FROM AUTHOR]

This study aimed to explore the influences of SAP2 and CAP1 on itraconazole (ITR) resistance of Candida albicans at different states. A total of 10 ITR-resistant strains and 10 ITR-sensitive strains were used for SAP2 sequencing and CAP1 sequencing. SAP2 sequencing showed no missense mutation, and three synonymous mutations. CAP1 gene sequencing identified two missense mutations M140I (8) and K191Q (4), and 14 synonymous mutations G201A (1), A246C (5), C282T (6), G288A (6), C321T (7), A399C (16), C432T (16), C465T (11), G552A (16), G669T (1), G672A (1), G681T (2), T783C (1), and T819A (2). The biofilm formation capacity of resistant C. albicans strains, including the CAP1 ∆/∆ strain, was stronger. Afterward, real-time quantitative PCR was used to analyze the expression of SAP2 and CAP1 . Compared with the sensitive strains, SAP2 and CAP1 expressions were both significantly upregulated in resistant strains at planktonic and biofilm states ( P  < 0.05). Compared with the strains at planktonic state, SAP2 was significantly upregulated, while CAP1 was significantly downregulated at biofilm states ( P  < 0.05). Additionally, SAP2 expression in the CAP1 knocked down strain of C. albicans was significantly upregulated, and SAP2 expression was evidently downregulated in the CAP1 ∆/∆ strain at biofilm states compared with that at planktonic states ( P  < 0.05). Loss of CAP1 can increase SAP2 level and may influence the biofilm formation of C. albicans , thus increasing ITR resistance of C. albicans .

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy. [ABSTRACT FROM AUTHOR]

Due to their high content of biomolecules, combined with silver's well known antimicrobial potential, silver nanoparticles biosynthesized using spruce bark (AgNP SBEs) demonstrate antibacterial and antioxidant activity, making them a versatile option for developing new antimicrobial agents that might be used for medical treatment or as adjuvants for the classical agents. This study aims to analyze if silver nanoparticles (AgNPs) mediated by spruce bark extract (SBE) and silver salts (AgNP SBE Acetate, AgNP SBE Nitrate) presents antifungal activity against five different Candida spp., synergistic activity with fluconazole, and if they interact with some virulence factors of C. albicans. AgNP SBEs presented MICs (minimum inhibitory concentrations) for all the five tested Candida spp. AgNP SBEs inhibited the growth of C. parapsilosis, C. krusei, and C. guilliermondii, exerted synergistic activity with fluconazole for C. parapsilosis and C. guilliermondii, and inhibited biofilm production for C. albicans, C. auris, and C. guilliermondii. MICs of AgNP SBE Acetate significantly inhibited the production of germ tubes of C. albicans. The expression of C. albicans SAP2 gene was down-regulated by the short-time treatment with MICs of AgNP SBE Acetate, while ALS3 and HSP70 genes were up-regulated by the AgNPs MICs. These results emphasize the potential of using the AgNP SBEs as treatments/adjuvants options, not only against the redundant C. albicans but also for the non-albicans Candida species (which are not as frequently involved in human pathologies, but, sometimes, can be more aggressive). [ABSTRACT FROM AUTHOR]

Biosynthesis is a green method for the synthesis of silver nanoparticles (AgNPs). This study aimed to assess the antifungal activity of two silver nanoparticle solutions, synthesized using beech bark extract (BBE) and acetate and nitrate silver salts (AgNP Acetate BBE and AgNP Nitrate BBE), their influence on biofilm production, their potential synergistic effects with fluconazole, on different Candida spp., and their influence on virulence factors of C. albicans (germ tube production, gene expression for ALS3, SAP2, HSP70). Both the AgNP BBEs presented different minimum inhibitory concentrations for all the studied Candida spp., but biofilm production was inhibited only for C. albicans and C. guilliermondii. The growth rates of all the studied Candida spp. were inhibited in the presence of both AgNP BBEs, except for C. auris. Synergistic activity was observed for C. parapsilosis and C. guilliermondii, for different combinations of fluconazole with both the AgNP BBEs. The germ tube production of C. albicans was slightly inhibited by the AgNP BBEs. Only AgNP Acetate BBE was able to down-regulate the expression of SAP2. Overall, we can conclude that, even if more studies are necessary, AgNPs synthesized with beech bark extract might be an interesting alternative to classic antifungal treatments. [ABSTRACT FROM AUTHOR]

Objective: Multilocus sequence typing is a powerful method for genotyping of clinical isolates of Candidaalbicans. Cross-contamination between the patients is an important reason for nosocomial infections. Oropharyngeal candidiasis (OPC) caused by C. albicans is an important problem in patients with head and neck cancer, in Cancer Institute of Tehran. Here we studied the endemic genotypes of C. albicans isolates and the relationship between geographic distributions, potential cross-contaminations and the expression of SAP2 gene, an important gene in oral candidiasis, with MLST groups., Material and Methods: A total of 32 clinical strains of C. albicans isolated from head and neck cancer patients with oropharyngeal candidiasis were subjected to MLST analysis and SAP2 gene expression was analyzed by Real-time PCR., Results: We identified 75 polymorphic sites in 7 loci of C. albicans isolates and 30 diploid sequence types which 27 of them were found as new. After eBURST analysis, our results determined that CC 124 was the most prevalent group among all CCs. SAP2 gene showed high expression in almost all OPC patients' isolates, compared to the control., Conclusion: We found few genetically-related as well as identical isolates among the 32 Candida strains which indicated low cross-contaminations among the patients. There was no relationship between C. albicans MLST profiles and their geographic distribution, cancer type and SAP2 gene expression. This lack of correlation was possibly due to the small understudy population; hence, finding more relevance requires studies with a higher number of samples., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The polymorphic fungus Candida albicans (C. albicans) can live as an aggressive pathogen and cause many diseases in hosts, for which no effective vaccine exists. The secreted aspartyl proteinase 2 (Sap2) plays a protective role in systemically infected BALB/c mice. Protective cellular immune responses can be preferentially induced when antigens are displayed on small particles. Therefore, the emphasis is placed on developing new phage vaccine to inhibit C. albicans infection. In this study, the ability of the hybrid phage displaying the epitope SLAQVKYTSASSI and recombinant protein of Sap2 (rSap2) for inducing immune protective responses against C. albicans infection was evaluated by lymphoproliferative assay, to gather cytokine and antibody measurements in BALB/c mice. Our results showed that, strong cellular and humoral immune responses were induced in a mouse model immunized with hybrid phage or rSap2. Furthermore, the protection against lethal challenge with C. albicans was observed in mice vaccinated hybrid phage without adjuvant. These findings demonstrate that the hybrid phage displaying the epitope SLAQVKYTSASSI might be a potential vaccine against C. albicans infections.