SAP Genes Research Articles

Molecular identification of SAP9, SAP10 genes with virulence factors and antifungal susceptibility profile among Candida species isolated from women with urinary tract infections

Background. In recent years, there has been a rise in the occurrence of urinary tract infections (UTIs) caused by Candida pathogens. This increase is attributed to the growing resistance of these pathogens to antifungal drugs often used for their treatment. Many virulence factors encoded by virulent genes, which enable opportunistic Candida to invade host tissues and cause infection, for survey the existence of SAP9 andSAP10 genes with determine some virulence factors and antifungal susceptibility profiles among Candida species isolated from women suffering from urinary tract infections. Methods. For this investigation, urine specimens were gathered from female individuals residing in Thi-Qar Province, located in southern Iraq, over the period spanning from December 2022 to March 2023. The identification of all isolates was performed using several laboratory techniques, including assessment of colony characteristics on culture media, Gramme staining, germ tube formation, and analysis of morphological chromatic features on chromogenic agar. The identification was further validated using PCR analysis. All isolates were assessed for their production of extracellular secretory proteinase and hemolysin. Additionally, they were tested for antifungal activity using the disc diffusion method. The DNA was isolated, and the PCR technique was employed to identify the presence of virulence genes (SAP9 and SAP10). Results. A total of 50 Candida species isolates were collected from 150 urine samples. These isolates belonged to four different species within the Candida genus. The most found species was C. albicans, accounting for 54% of the isolates. This was followed by C. krusei at 24%, C. glabrata at 16%, and C. tropicalis at 6%. Proteinase activity was observed in 84% of Candida isolates. All Candida isolates exhibit positive hemolysin production with varying degrees of intensity. Among Candida species showed high resistance to Itraconazole (86%) and Clotrimazole (76%). The prevalence of SAP9 and SAP10 genes among Candida albicans was 62.69% and 100%, respectively. Conclusions. The present study highlights the prevalence of SAP genes and virulence factors with antifungal susceptibility which reflect high pathogenicity of Candida species.

Characterization of Candida species isolated from clinical specimens: insights into virulence traits, antifungal resistance and molecular profiles

BackgroundCandida species have emerged as a significant cause of opportunistic infections. Alongside the expression of various virulence factors, the rise of antifungal resistance among Candida species presents a considerable clinical challenge.AimThis study aimed to identify different Candida species isolated from clinical specimens, evaluate their antifungal sensitivity patterns, identify key genes regulating virulence mechanisms using multiplex PCR and to assess any correlation between their virulence profiles and antifungal resistance patterns.MethodA total of 100 Candida spp. was isolated from 630 different clinical specimens and identified to the species level. Their antifungal susceptibility was phenotypically evaluated in accordance with CLSI guidelines using the Vitek-2 Compact System. Virulence markers, including biofilm formation capacity, protease production, melanin production, coagulase production and hemolysin production, were also phenotypically detected. The genetic determinants for biofilm formation and extracellular hydrolytic enzymes were assessed using a multiplex PCR assay.ResultsThe prevalence of Candida spp. was 15.9%, with C. albicans (48%) and C. glabrata (16%) being the most common. C. albicans showed the highest virulence, with strong biofilm formation, and high proteinase and melanin production. Multiplex PCR revealed Hlp in 22.0%, Hwp in 80.0%, Als in 56.0%, and Sap genes in 56.0% of isolates. Virulence genes were more common in C. albicans than in non-albicans Candida (NAC). Resistance patterns significantly correlated with virulence profiles, with notable associations between flucytosine resistance and the presence of Hlp and Hwp genes.ConclusionThe significant correlation between virulent markers such as germination, coagulase, hemolysin production and resistance patterns among different Candida isolates is crucial for predicting the severity and outcomes of Candida infections. This understanding aids in guiding tailored treatment strategies.

Anti-cancer activity of secreted aspartyl proteinase protein from Candida tropicalis on human cervical cancer HeLa cells

Cervical cancer is the fourth leading cause of cancer-related death in women worldwide. Microbial products are valuable sources of anti-cancer drugs. The aim of this study was to isolate secreted aspartyl proteinase protein from Candida tropicalis, investigate its inhibitory effect on human cervical cancer HeLa cells, and analyze the expression profiling of selected nuclear stem cell-associated transcription factors. The presence of secreted aspartyl proteinase protein was confirmed by the expression of SAP2 and SAP4 genes in C. tropicalis during the yeast-hyphae transition phase. The enzyme was purified and characterized using the aqueous two-phase system purification method, as well as proteolytic activity and the Bradford and micro-Kjeldahl methods, respectively. The in vitro anti-cancer properties of secreted aspartyl proteinase protein were evaluated by MTT assay, microscopic image analysis, nitric oxide (NO) scavenging activity assay, intracellular reactive oxygen species (ROS) production assay, and RT-qPCR. The isolated C. tropicalis secreted aspartyl proteinase protein exhibited proteinase activity with values ranging from 93.72 to 130.70 μg/mL and 89.88–127.72 μg/mL according to the Bradford and micro-Kjeldahl methods, respectively. Secreted aspartyl proteinase showed effective cytotoxicity in HeLa cell line leading to significant morphological changes. Additionally, it exhibited increased free radical scavenging activity compared to the untreated control group, as evidenced by nitrite inhibition. ROS production increased in HeLa cells exposed to secreted aspartyl proteinase. The expression levels of the nuclear stem cell-associated transcription factors octamer-binding transcription factor 4 (OCT4), sex determining region Y-box 2 (SOX2), and Nanog homeobox (NANOG) were significantly downregulated in the HeLa cells treated with secreted aspartyl proteinase. Secreted aspartyl proteinase protein may be a promising anti-cancer agent, as it effectively affects gene expression and may ultimately reduce the development and progression of cervical cancer. Targeting the genes related to nuclear stem cell-associated transcription factors may provide a novel amenable to cancer treatment.

Genotypic and Phenotypic Characterization of Pseudomonas atacamensis EMP42 a PGPR Strain Obtained from the Rhizosphere of Echinocactus platyacanthus (Sweet Barrel).

Plant growth-promoting rhizobacteria (PGPR) are a group of bacteria that associate with the rhizosphere of plants; one of the most abundant bacterial genera in this ecological niche is Pseudomonas, which is constantly expanding due to the emergence of new species such as Pseudomonas atacamensis, whose discovery in 2019 has led to the characterization of several strains from different environments but taxonomically related. The objective of this work was to phenotypically and molecularly characterize P. atacamensis strain EMP42, isolated from the rhizosphere of Echinocactus platyacanthus. The strain EMP42 is able to use different substrates and reduce oxidative stress in plants. It is capable of improving growth parameters such as the number of inflorescences and the height of the aerial body of Arabidopsis thaliana, as well as the germination and seedling survival of the cacti Echinocactus platyacanthus and Astrophytum capricorne. The genetic structure of P. atacamensis EMP42 consists of a closed chromosome of 6.14 Mbp, and 61.1% GC content. It has 5572 genes, including those associated with PGPR activities, such as the trpABCDE, SAP, phoABPRU and acsABC genes, among others, and three ncRNA loci, nine regulatory regions, five complete rRNA operons and three CRISPR-Cas loci, showing phylogenomic similarities with the reference strain P. atacamensis B21-026. Therefore, this study contributes to the understanding of genomic diversity within P. atacamensis and, particularly, highlights the potential application of strain EMP42 as a PGPR.

Molecular Detection of INT, HWP, PLB, SAP and ALS Genes in Candida Species Isolated from Patients with Vulvovaginal Candidiasis

Amaç: Vulvovajinal kandidiyazis, kadınların yaklaşık %75’inde yaşamları boyunca en az bir kez görülen yüzeysel mantar enfeksiyonudur. Bazı genler, Candida türlerinin vajina mukozasına ve epitel hücrelerine yapışmasında ve patogenezinde rol oynamaktadır. Bu çalışmanın amacı, vulvovajinal kandidiyazisli hastalardan izole edilen Candida suşlarındaki HWP1 (hifal duvar proteini), ALS1 (agglutinin benzeri sekans), SAP1 (sekrete aspartil proteaz), PLB1 (fosfolipaz B), INT1 (integrin A) genlerinin sıklığının araştırılmasıdır. 
 Gereç ve Yöntemler: Çalışmaya moleküler teknik kullanılarak tanımlanan 100 vajinal Candida izolatı dahil edildi. İzole edilen tüm Candida suşlarında, enfeksiyon da önemli rol oynayan INT1, HWP1, PLB1, SAP1 ve ALS1 genlerinin varlığı PCR yöntemi kullanılarak araştırıldı.
 Bulgular: SAP1, INT1 ve HWP1 virülans genleri Candida suşları arasında en yaygın, PLB geni en az tespit edilen gen olmuştur. Candida albicans suşlarında SAP1 (%93.0) geni en çok saptanan virülans geni olurken, bunu HWP1 geni (%86) takip etmiştir. Benzer şekilde non-albicans Candida türlerinde saptanan virülans genleri arasında SAP1 geni (%86) en fazla saptanan virülans geni olmuştur. Farklı Candida türlerinin %95’inde VVK’ya neden olabilecek en az iki virülans faktörü tanımlanmıştır.
 Sonuç: Bu çalışmada elde edilen sonuçlar, vulvovajinal kandidiyazis enfeksiyonu sırasında eksprese edilen INT, HWP, PLB, SAP ve ALS genlerinin, Candida suşlarının vajinaya adezyonuna ve biyofilm oluşumuna katkıda bulunmuş olabileceğini düşündürmektedir.

Vitamin D3: A promising antifungal and antibiofilm agent against Candida species.

Candida species are opportunistic fungal pathogens that cause mild to life-threatening infections in both immunocompetent and immunocompromised populations. The increasing prevalence of drug-resistant Candida species has posed a significant challenge to the management of infections in clinical settings. Therefore, this study aimed to investigate the direct antifungal and antibiofilm effect of vitamin D3 against Candida species. The antifungal activity of vitamin D3 was evaluated by broth microdilution method based on the Clinical and Laboratory Standard Institute. Prevention of biofilm formation by Candida albicans was measured using the XTT assay following exposure to different concentrations of vitamin D3. Moreover, expression of Agglutinin-like sequence gene 1 (ALS1), hyphal wall protein gene (HWP1), secreted aspartyl proteinase 6 gene (SAP6), and morphogenesis pathway regulatory gene (EFG1) were analyzed by real-time polymerase chain reaction using the comparative Ct method (ΔΔ Ct) after exposure to vitamin D3. Vitamin D3 showed antifungal activity against Candida species ranging from 1-128 μg/mL. Furthermore, vitamin D3 inhibited biofilm formation in a dose-dependent manner, with IC50 of 7.5 μg/mL. Treatment with vitamin D3 resulted in significant upregulation of the EFG1, ALS1, and SAP6 genes under hypha-inducing conditions to overcome environmental challenges. Results of the current study demonstrated that vitamin D3 has a significant inhibitory effect on Candida growth and biofilm formation. Considering its demonstrated antifungal and antibiofilm properties, vitamin D3 holds promise as a potential agent for medical applications.

RNA-Seq-Pop: Exploiting the sequence in RNA sequencing-A Snakemake workflow reveals patterns of insecticide resistance in the malaria vector Anopheles gambiae.

We provide a reproducible and scalable Snakemake workflow, called RNA-Seq-Pop, which provides end-to-end analysis of RNA sequencing data sets. The workflow allows the user to perform quality control, perform differential expression analyses and call genomic variants. Additional options include the calculation of allele frequencies of variants of interest, summaries of genetic variation and population structure, and genome-wide selection scans, together with clear visualizations. RNA-Seq-Pop is applicable to any organism, and we demonstrate the utility of the workflow by investigating pyrethroid resistance in selected strains of the major malaria mosquito, Anopheles gambiae. The workflow provides additional modules specifically for An.gambiae, including estimating recent ancestry and determining the karyotype of common chromosomal inversions. The Busia laboratory colony used for selections was collected in Busia, Uganda, in November 2018. We performed a comparative analysis of three groups: a parental G24 Busia strain; its deltamethrin-selected G28 offspring; and the susceptible reference strain Kisumu. Measures of genetic diversity reveal patterns consistent with that of laboratory colonization and selection, with the parental Busia strain exhibiting the highest nucleotide diversity, followed by the selected Busia offspring, and finally, Kisumu. Differential expression and variant analyses reveal that the selected Busia colony exhibits a number of distinct mechanisms of pyrethroid resistance, including the Vgsc-995S target-site mutation, upregulation of SAP genes, P450s and a cluster of carboxylesterases. During deltamethrin selections, the 2La chromosomal inversion rose in frequency (from 33% to 86%), supporting a previous link with pyrethroid resistance. RNA-Seq-Pop is hosted at: github.com/sanjaynagi/rna-seq-pop. We anticipate that the workflow will provide a useful tool to facilitate reproducible, transcriptomic studies in An.gambiae and other taxa.

Repurposing HIV Protease Inhibitors Atazanavir and Darunavir as Antifungal Treatments against Candida albicans Infections: An In Vitro and In Vivo Study

Candida albicans is the chief etiological agent of candidiasis, a mycosis prevalent in individuals with acquired immunodeficiency syndrome (AIDS). In recent years, the introduction of human immunodeficiency virus (HIV) protease inhibitors (HIV-PI) has reduced the prevalence of candidiasis in these patients. Seeking new therapeutic strategies based on the perspective of drug repositioning, we evaluated the effects of two second-generation HIV-PIs, atazanavir (ATV) and darunavir (DRV), on virulence factors of C. albicans and experimental candidiasis. For this, clinical strains of C. albicans were subjected to in vitro and in vivo treatments with ATV or DRV. As a result, ATV and DRV exhibited antifungal activity against fungal cells at 512 μg/mL, reduced the viability and biomass of biofilms, and inhibited filamentation of C. albicans. In addition, these HIV-PIs downregulated the expression of SAP2 and BRC1 genes of C. albicans. In an in vivo study, prophylactic use of ATV and DRV prolonged the survival rate of Galleria mellonella larvae infected with C. albicans. Therefore, ATV and DRV showed activity against C. albicans by reducing cell growth, biofilm formation, filamentation, and expression of virulence genes. Furthermore, ATV and DRV decreased experimental candidiasis, suggesting the repurposing of HIV-PIs as antifungal treatments for C. albicans infections.

2,4-Diacetylphloroglucinol Modulates Candida albicans Virulence.

The dimorphic fungus Candida albicans is one of the most important opportunistic pathogens for humankind. The use of fungicides against Candida could be associated with sub-inhibitory effects, which are referred to as fungal stress responses and are undesirable for the host. In this work, we investigated the antifungal action of 2,4-diacetylphloroglucinol (2,4-DAPG) against Candida albicans ATCC 10231 with a focus on their biofilm-forming ability. We found that 2,4-DAPG was able to reduce the ability of Candida cells to form biofilms, but complete inhibition and eradication effects were not achieved. Furthermore, C. albicans cells in the adherent state were characterized by reduced susceptibility to 2,4-DAPG compared to planktonic cells. The investigation of the mechanisms that could explain the antibiofilm action of 2,4-DAPG revealed a reduction in the cell`s surface hydrophobicity and the inhibition of the yeast-to-hyphae transition. The inhibition of the Candida cells filamentation was accompanied by an increase in the expression of the NRG1 gene, which is a negative regulator of hyphal development. In addition, we microscopically visualized the treated biofilms and revealed numerous channels that were decorated with particles and localized on the hyphae. We assumed that these hyphal structures could be associated with the secretion of aspartyl proteases (Sap). The performed assessments revealed an increase in the activity of Sap, which was accompanied by an increase in the expression of the sap2 and sap4 genes. The antifungal action of 2,4-DAPG is known to be associated with affecting the permeability of cellular structures, which leads to H+ATPase malfunction and the disruption of mitochondrial respiration. The subsequent cytosol acidification and generation of ROS trigger the inhibition of Candida filamentation and activation of Sap production. The introduction of antioxidant Trolox simultaneously with 2,4-DAPG leads to a reduction in Sap production. Collectively, the obtained data indicate new aspects of the interaction of fungal cells with 2,4-DAPG, an antimicrobial metabolite of Pseudomonas spp.

P394 Evaluation of diagnostic potential of recombinant secretory aspartyl proteinase 2 (Sap2) protein from Candida parapsilosis for use in systemic candidiasis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesSystemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Along with Candida albicans, 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. Since conventional diagnostics methods for candidiasis show less sensitivity and specificity, novel immunodiagnostic techniques are needed for early diagnosis. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection.MethodsGenomic DNA was isolated from C. parapsilosis, followed by PCR amplification of Sap2 gene using designed gene-specific primers. Sap2 protein expression and purification was performed using Ni-NTA affinity chromatography under denaturing conditions. The denatured protein was subsequently refolded using a multi-step dialysis procedure. CD and FTIR studies were performed to confirm refolding. Mice were immunized with rSap2 protein and serum ELISA assays were performed for testing immunogenicity. Immunoblotting assays and human serum ELISAs were performed using whole-cell Candida and rSap2 protein in proven systemic candidiasis patient serum and controls, recruited at PGIMER, Chandigarh.ResultsSap2 protein from C. parapsilosis was successfully cloned and expressed using an E. coli-based prokaryotic expression system. Protein refolding was performed in vitro using step-wise dialysis. 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 serum from proven systemic candidiasis patients and controls. Our immunoblotting results demonstrate that the recombinant Sap2 protein was recognized as a single band of approximately 41 kDa by systemic candidiasis patient serum samples and no cross reactivity was observed in healthy controls. On evaluating the diagnostic potential of the rSap2 antigen using an ELISA-based approach, our results show that anti-Sap2 Ig, IgG and IgM antibodies could be detected in the sera of proven candidiasis patients. Of note, the differences in Sap2 antibody titers observed amongst patients and controls were similar to the serological response observed when heat-killed whole-cell Candida was used as a coating antigen.ConclusionIn 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. Our results indicate that the rSap2 protein from C. parapsilosis can be used to detect and diagnose systemic candidiasis infection in human patients and can be used as an alternative/replacement of whole-cell Candida-based ELISA procedures, which are currently in use.

P120 Vaccine induced protection by secreted aspartyl proteinase 2 from Candida parapsilosis in Candida tropicalis mediated murine systemic candidiasis: a role of B-cells and antibodies

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesNon-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. These challenges merit the need for developing novel immune therapies against Candida infections. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading Candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. Moreover, while the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. As such, we also investigated the role of humoral immunity in vaccine-mediated protection during murine systemic candidiasis.MethodsThe Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis strains prevalent in India. Groups of wild-type BALb/c mice were vaccinated with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with a lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Histopathological assessment was performed using H&E and PAS staining. Serum cytokine levels and antigen-specific antibody titers were measured by ELISA. Cellular responses were analyzed in detail using flow cytometry. Functional evaluation of antibody role was performed using in vitro (biofilm inhibition and neutrophil-mediated killing) and in vivo (passive transfer) studies. B-cell epitope analysis was carried out using immunoinformatics approaches.ResultsMice vaccinated with rSap2 cloned from C. parapsilosis (Sap2-parapsilosis) showed highest increase in survival time (P = .02) and maximum reduction in organ fungal burden (spleen, kidney, lungs, brain) (P <.05); compared with sham immunized controls. Vaccination with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ∼60% homology across species. Mice vaccinated with rSap2-parapsilosis also exhibited significantly higher levels of IFN gamma, IL-17, and IL-4 cytokine levels just before infection, which correlated with protection. In addition, rSap2-parapsilosis vaccination induced high titers of Sap2-specific antibodies, and a fraction of antibodies could bind whole fungus (which were predominantly of IgM isotype). Notably, sera from rSap2-parapsilosis vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing in vitro. Sap2-specificity was further confirmed by depleting antigen-specific antibodies. Passive transfer of Sap2-parapsilosis immune serum significantly reduced fungal burdens in naive mice, as compared to mice receiving sham-immune serum, upon infection. Higher numbers of total CD19 + B-cells, plasma cells, and Candida-binding B-cells in rSap2-parapsilosis vaccinated mice indicate a role of B-cells during early stages of Sap2-mediated immune response. Epitope analysis performed using identified B-cell epitopes provides insights about including important IgM and IgG epitopes, when designing multivalent or multi-epitope anti-Candida vaccine/s.ConclusionMice immunized with recombinant Sap2 cloned from C. parapsilosis exhibited increased survival during C. tropicalis mediated systemic candidiasis. Both Th1/Th17 mediated cellular immunity and humoral immunity contribute toward protection. Our results establish a role of B-cells and antibodies in generation of protective immune responses against Sap2 vaccine antigen during non-albicans Candida mediated murine systemic candidiasis.

Novel Preclinical Study of Galloylquinic Acid Compounds from Copaifera lucens with Potent Antifungal Activity against Vaginal Candidiasis Induced in a Murine Model via Multitarget Modes of Action.

ABSTRACTVaginal candidiasis is a medical condition characterized by the overgrowth of Candida spp. in the vaginal cavity with complex recurrent pathogenicity as well as tolerance to antifungal therapy and hence is awaiting more safe and effective treatments. This work aimed to assess the potential antifungal activity of galloylquinic acid compounds (GQAs) from Copaifera lucens leaves against vaginal Candida albicans. The antifungal susceptibility test was performed against 20 isolates of multidrug-resistant (MDR) C. albicans using agar diffusion and broth microdilution assays. The results showed that GQAs exhibited strong antagonistic activity against the test isolates, with inhibition zone diameters ranging from 26 to 38 mm and low MICs (1 to 16 μg/mL) as well as minimum fungicidal concentrations (2 to 32 μg/mL). The MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay confirmed the safety of GQAs against the Vero cell line, showing a 50% inhibitory concentration (IC50) of 168.17 mg/mL. A marked difference in the growth pattern of the treated and untreated pathogens was also observed, where a concentration-dependent reduction in the growth rate occurred. Moreover, a pronounced fungicidal effect was demonstrated 6 h after treatment with 1× the minimum fungicidal concentration (MFC), as evidenced by time-kill assays, where the number of survivors was decreased a 6-fold. GQAs effectively inhibited and eradicated about 80% of C. albicans biofilm at 6 μg/mL and 32 μg/mL, respectively. Interestingly, GQAs disturbed the fungal membrane integrity, induced cell lysis, and reduced the virulence factors (proteinase and phospholipase) as well as the catalase activity. Moreover, the ergosterol content in the plasma membrane decreased in a concentration-dependent manner. Additionally, the altered mitochondrial membrane potential was associated with an increased release of cytochrome c from mitochondria to the cytosol, suggesting the initiation of early apoptosis in GQA-treated cells. Transcriptional analysis revealed that all test genes encoding virulence traits, including SAP1, PLB1, LIP1, HWP1, and ALS1, were markedly downregulated in GQA-treated cells compared to the control. The in vivo murine model of vaginal candidiasis further confirmed the therapeutic activity of GQAs (4 mg/kg of body weight) against C. albicans. This work comprehensively evaluated the antifungal, antivirulence, and antibiofilm activities of GQAs against C. albicans isolates using in vitro and in vivo models, providing molecular-level insights into the antifungal mechanism of action and experimental evidence that supports the potential use of GQAs for the treatment of vaginal candidiasis.IMPORTANCE Our work presents a new perspective on the potential use of GQAs as safe and highly effective phytochemicals against MDR C. albicans. This microorganism colonizes the human vaginal epithelium, causing vaginal candidiasis, a condition characterized by recurrent pathogenicity and tolerance to traditional antifungal therapy. Based on the results of in vitro tests, our study reports GQAs antifungal modes of action. These compounds exhibited an anticandidal effect by deactivating the fungal hydrolytic enzymes, reducing ergosterol content in the plasma membrane, altering the potential of the mitochondrial membrane, and inducing apoptosis. Additionally, GQAs showed high activity in eradicating the biofilm formed by the fungus via the downregulation of HWP1, ALS, SAP, PLB, and LIP genes, which are constitutively expressed in the biofilm. In an in vivo murine model of vaginal candidiasis, GQAs further demonstrated strong evidence of their effectiveness as an antifungal therapy. In this regard, our findings provide novel insights into the potential therapeutic use of these phytoactive molecules for vaginal candidiasis treatment.

Molecular Drivers of Multiple and Elevated Resistance to Insecticides in a Population of the Malaria Vector Anopheles gambiae in Agriculture Hotspot of West Cameroon.

(1) Background: Malaria remains a global public health problem. Unfortunately, the resistance of malaria vectors to commonly used insecticides threatens disease control and elimination efforts. Field mosquitoes have been shown to survive upon exposure to high insecticide concentrations. The molecular mechanisms driving this pronounced resistance remain poorly understood. Here, we elucidated the pattern of resistance escalation in the main malaria vector Anopheles gambiae in a pesticide-driven agricultural hotspot in Cameroon and its impact on vector control tools; (2) Methods: Larval stages and indoor blood-fed female mosquitoes (F0) were collected in Mangoum in May and November and forced to lay eggs; the emerged mosquitoes were used for WHO tube, synergist and cone tests. Molecular identification was performed using SINE PCR, whereas TaqMan-based PCR was used for genotyping of L1014F/S and N1575Y kdr and the G119S-ACE1 resistance markers. The transcription profile of candidate resistance genes was performed using qRT-PCR methods. Characterization of the breeding water and soil from Mangoum was achieved using the HPLC technique; (3) Results: An. gambiae s.s. was the only species in Mangoum with 4.10% infection with Plasmodium. These mosquitoes were resistant to all the four classes of insecticides with mortality rates <7% for pyrethroids and DDT and <54% for carbamates and organophophates. This population also exhibited high resistance intensity to pyrethroids (permethrin, alpha-cypermethrin and deltamethrin) after exposure to 5× and 10× discriminating doses. Synergist assays with PBO revealed only a partial recovery of susceptibility to permethrin, alpha-cypermethrin and deltamethrin. Only PBO-based nets (Olyset plus and permaNet 3.0) and Royal Guard showed an optimal efficacy. A high amount of alpha-cypermethrin was detected in breeding sites (5.16-fold LOD) suggesting ongoing selection from agricultural pesticides. The 1014F-kdr allele was fixed (100%) whereas the 1575Y-kdr (37.5%) and the 119S Ace-1R (51.1%) were moderately present. Elevated expression of P450s, respectively, in permethrin and deltamethrin resistant mosquitoes [CYP6M2 (10 and 34-fold), CYP6Z1(17 and 29-fold), CYP6Z2 (13 and 65-fold), CYP9K1 (13 and 87-fold)] supports their role in the observed resistance besides other mechanisms including chemosensory genes as SAP1 (28 and 13-fold), SAP2 (5 and 5-fold), SAP3 (24 and 8-fold) and cuticular genes as CYP4G16 (6 and 8-fold) and CYP4G17 (5 and 27-fold). However, these candidate genes were not associated with resistance escalation as the expression levels did not differ significantly between 1×, 5× and 10× surviving mosquitoes; (4) Conclusions: Intensive and multiple resistance is being selected in malaria vectors from a pesticide-based agricultural hotspot of Cameroon leading to loss in the efficacy of pyrethroid-only nets. Further studies are needed to decipher the molecular basis underlying such resistance escalation to better assess its impact on control interventions.

Effect of Cigarette and E-Cigarette Smoke Condensates on Candida albicans Biofilm Formation and Gene Expression.

Smoking triggers environmental changes in the oral cavity and increases the risk of mucosal infections caused by Candida albicans such as oral candidiasis. While cigarette smoke has a significant impact on C. albicans, how e-cigarettes affect this oral pathogen is less clear. Here, we investigated the effect of cigarette and e-cigarette smoke condensates (CSC and ECSC) on C. albicans growth, biofilm formation, and gene expression. Whereas pure nicotine (N) at the minimum inhibitory concentration (MIC, 4 mg/mL) prevented C. albicans growth, enhanced biofilm formation was observed at 0.1 mg/mL. In contrast, at this nicotine sub-MIC (0.1 mg/mL) concentration, CSC and ECSC had no significant effect on C. albicans biofilm formation. Additionally, N, CSC, and ECSC increased the expression of HWP1 and SAP2 genes. The ECSC group exhibited elevated expression levels of the EAP1 and ALS3 genes, compared to the nicotine-free ECSC (−) control. Moreover, our in vitro study illustrated that the antifungal drugs, fluconazole and amphotericin B, alleviated the effect of nicotine on C. albicans gene expression. Overall, the results of the study indicated nicotine from different sources may affect the pathogenic characteristics of C. albicans, including hyphal growth, biofilm formation, and particularly the expression of virulence-related genes.

Mediation of Candida species growth and virulence by the proinflammatory cytokine IL-6

Abstract 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.

Silver Nanoparticles Biosynthesized with Spruce Bark Extract-A Molecular Aggregate with Antifungal Activity against Candida Species.

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).

XÁC ĐỊNH VÀ PHÂN TÍCH CÁC GENE MÃ HÓA PROTEIN LIÊN KẾT STRESS (SAP) Ở CÂY ĐU ĐỦ (Carica papaya L.) BẰNG PHƯƠNG PHÁP IN SILICO

By using the in silico methods, total of seven stress-associated protein encoding genes have identified in the geneome of papaya (Carica papaya L.). The full-length genomic sequence of papaya SAP genes were ranging from 416 to 813 nucleotides. These genes had single intron or were intronless. The predicted protein sequences included from 114 to 270 amino acids, according to the molecular weight ranged from 13.10 to 29.63 kDa. These proteins were alkaline with a pI value ranging from 8.82 to 9.95. Based on the protein structure and the phylogeneic analysis, the papaya SAP were divided into two groups, I (five members) and II (two members). The papaya SAP had a highly conserved level of structure including two conserved regions, A20-AN21 (group I), or AN21 domains only (group II). RNA-seq analysis showed that all of seven SAP genes expressed in papaya leaves and were induced by freeze thaw awakening treatment (in comparison with control treatment). CpSAP2 showed the highest relative expression level in compared to other SAP genes of papaya. The results of this work have an important significance and are base of the further research on gene cloning, functional analysis of SAP genes and breeding of papaya in response to environmental abiotic stresses.

Insecticide resistance and underlying targets-site and metabolic mechanisms in Aedes aegypti and Aedes albopictus from Lahore, Pakistan

Insecticide resistant Aedes populations have recently been reported in Pakistan, imposing a threat to their control. We aimed to evaluate the susceptibility of Aedes aegypti and Aedes albopictus populations from Lahore to WHO-recommended insecticides and to investigate metabolic and target-site resistance mechanisms. For this purpose, we first carried out bioassays with the larvicides temephos and pyriproxyfen, and the adulticides malathion, permethrin, deltamethrin, alpha-cypermethrin, and etofenprox. We looked for Knockdown resistance mutations (kdr) by qPCR, High-Resolution Melt (HRM), and sequencing. In order to explore the role of detoxifying enzymes in resistance, we carried out synergist bioassay with both species and then checked the expression of CYP9M6, CYP9J10, CYP9J28, CYP6BB2, CCAe3a, and SAP2 genes in Ae. aegypti. Both species were susceptible to organophosphates and the insect growth regulator, however resistant to all pyrethroids. We are reporting the kdr haplotypes 1520Ile + 1534Cys and T1520 + 1534Cys in high frequencies in Ae. aegypti while Ae. albopictus only exhibited the alteration L882M. PBO increased the sensitivity to permethrin in Ae. aegypti, suggesting the participation of P450 genes in conferring resistance, and indeed, CYP928 was highly expressed. We presume that dengue vectors in Lahore city are resistant to pyrethroids, probably due to multiple mechanisms, such as kdr mutations and P450 overexpression.

In vivo and in vitro Pathogenesis and Virulence Factors of Candida albicans Strains Isolated from Cutaneous Candidiasis.

Background:The Candida albicans is one of the most important global opportunistic pathogens, and the incidence of candidiasis has increased over the past few decades. Despite the established role of skin in defense against fungal invasion, little has been documented about the pathogenesis of Candida species when changing from normal flora to pathogens of vaginal and gastrointestinal epithelia. This study was carried out to determine the in vivo and in vitro pathogenesis of clinical C. albicans strains isolated from skin lesions. Methods:In this study, association of in vivo and in vitro pathogenesis of C. albicans isolates with different evolutionary origins was investigated. Oral and systemic experimental candidiasis was established in BALB/C mice. The expression levels of SAP1-3 genes, morphological transformation, and biofilm-forming ability of C. albicans were evaluated. Results:All the strains showed in vitro and in vivo pathogenicity by various extents. The SAP1, SAP2, and SAP3 genes were expressed in 50%, 100%, and 75% of the strains, respectively. The biofilm formation ability was negative in 12% of the strains, while it was considerable in 38% of the strains. Fifty percent of the strains had no phospholipase activity, and no one demonstrated high level of this pathogenesis factor. Relatively all the strains had very low potency to form pseudohyphae. Conclusion:Our findings demonstrated that Candida albicans strains isolated from cutaneous candidiasis were able to cause oral and systemic infections in mice, so they could be considered as the potential agents of life-threatening nosocomial candidiasis in susceptible populations.

Phenotypic and molecular detection of virulence factor genes SAP4 and PLB in Candida albicans isolates from the Western part of India

BackgroundSince the last two decades, substantial increase in fungal infections has been observed in immunocompromised hosts. Virulence factors of Candida albicans play a role in adherence, haemolytic activity, phenotypic switching and production of hydrolytic enzymes. The secreted aspartyl proteinases (SAP family) contribute to adhesion, tissue damage and invasion, while phospholipase (PLB) supports the hydrolysis of phospholipids. Very few studies showed the correlation of phenotypic activity and detection of genes contributing to the similar enzyme activity. Therefore, our study aimed at demonstrating correlation between in vitro phenotypic production of phospholipase and proteinase enzymes with genetic level detection of SAP and PLB genes. MethodsThe present study was carried out on a total of 799 samples over a period of one year. Culturing was carried out on Sabouraud's dextrose agar. Confirmation and speciation of Candida spp. was carried out using cornmeal agar and CHROMagar and by the germ tube test, urease test and automated identification system Vitek-2, and antifungal susceptibility testing was performed on Candida isolates. Phenotypic phospholipase and proteinase activity was analysed, and molecular detection of SAP4 and PLB genes was carried out. ResultsIn our study, we have screened 799 samples for mycological protocol; of which, 269 (33.6%) were Candida species, 44% (119) of which were C. albicans. Proteinase activity was exhibited by 77 (64.7%) and phospholipase activity was exhibited by 73 (61.34%) isolates, while 46.2% exhibited both activities among the C. albicans species. The PLB gene was detected in 97.3% isolates, while SAP4 was detected in 94.7% of C. albicans isolates. ConclusionThe study of in vitro expression of virulence factors and gene detection of C. albicans will help to improve the prognosis despite the nature of the sample.