Andida Albicans Research Articles

Characterization of a Candida albicans isolate from a recurrent cervical lymphadenitis patient

ABSTRACTCandida albicans is the most frequently isolated opportunistic fungal pathogen in humans. However, patients with cervical lymphadenitis caused by Candida infection are rarely reported, and few studies have focused on the mechanisms underlying chronic Candida infection. In this study, we isolated a C. albicans strain (JL01) from a recurrent cervical lymphadenitis patient. The clinical isolate was identified by morphological observation and confirmed by DNA sequencing of the internal transcribed spacer (ITS) regions. Strain JL01 is resistant to azole antifungal drugs, but sensitive to amphotericin B. The strain is able to adapt to oxidative and osmotic stresses but is defective in filamentous and invasive growth. The strain displays attenuated virulence in a murine systemic infection model. RNA-sequencing analysis revealed that JL01 has a distinct gene expression profile compared with C. albicans reference strain SC5314; hundreds of transcripts were significantly dysregulated, including those related to morphogenesis and pathogenesis. Taken together, our clinical, virulence, morphological, and biological analyses suggest that the azole resistance, oxidative and osmotic stress tolerance, invasive defect, hypovirulence, and impaired interaction with the host immune system of strain JL01 may correlate with its ability to cause cervical lymphadenitis in the patient. Our research may contribute to elucidating the mechanism(s) underlying the drug resistance and immune escape of C. albicans in chronic fungal infection.

Molecular and genetic basis of azole antifungal resistance in the opportunistic pathogenic fungus Candida albicans.

Candida albicans is an opportunistic yeast and the major human fungal pathogen in the USA, as well as in many other regions of the world. Infections with C. albicans can range from superficial mucosal and dermatological infections to life-threatening infections of the bloodstream and vital organs. The azole antifungals remain an important mainstay treatment of candidiasis and therefore the investigation and understanding of the evolution, frequency and mechanisms of azole resistance are vital to improving treatment strategies against this organism. Here the organism C. albicans and the genetic changes and molecular bases underlying the currently known resistance mechanisms to the azole antifungal class are reviewed, including up-regulated expression of efflux pumps, changes in the expression and amino acid composition of the azole target Erg11 and alterations to the organism's typical sterol biosynthesis pathways. Additionally, we update what is known about activating mutations in the zinc cluster transcription factor (ZCF) genes regulating many of these resistance mechanisms and review azole import as a potential contributor to azole resistance. Lastly, investigations of azole tolerance in C. albicans and its implicated clinical significance are reviewed.

FORMULASI SABUN PEMBERSIH KEWANITAAN (Feminime Hygiene) DARI MINYAK ATSIRI RIMPANG LENGKUAS PUTIH (Alpinia galanga L) DAN UJI AKTIFITAS ANTISEPTIK TERHADAP Candida albicans

ABSTRACTCandida albicans is a normal flora in the female genitalia which can cause vaginal discharge. White galangal rhizome contains essential oils of eugenol which have activities that inhibit the growth of Candida albicans. This study formulated the white galangal rhizome essential oil in the form of feminime hygiene to make it easier and more comfortable to use and to determine the antiseptic feminime hygiene activity of the white galangal rhizome oil of Candida albicans. The essential oil of white galangal rhizome is formulated in three variations of concentration namely 10%, 15% and 20%. Feminime hygiene of essential white galangal rhizome oil was evaluated such as organoleptic, homogeneity, specific gravity, foam power test, viscosity, pH and stability. Antiseptic activity testing was tested with SDA (Sabouraud Dextrose Agar) media and using agar diffusion method. The results of the feminime hygiene evaluation formulated showed results similar to the results of comparative evaluations (BN). The parameters of antiseptic activity are indicated by the formation of inhibitory zone diameters where F0 (negative control), F1 (10%), F2 (15%), and F3 (20%) respectively have an average diameter value of 11 mm, 14.65 mm 17.58 mm and 25.41 mm. The classification category of the response to microbial growth according to CLSI, F0 and F1 includes the weak group, the F2 is the moderate group and the F3 is the strong group. Based on the results of one-way ANOVA statistical analysis there were significant differences in liquid soap preparations F0, F1, F2 with F3 at (P <0.05). White galangal rhizome essential oil can be formulated into feminine hygiene and has antiseptic activity against the fungus Candida albicans.Keywords: white galangal, antiseptic, candida albicans

Processing of Candida albicans Ece1p Is Critical for Candidalysin Maturation and Fungal Virulence.

ABSTRACTCandida albicans is an opportunistic fungal pathogen responsible for superficial and life-threatening infections in humans. During mucosal infection, C. albicans undergoes a morphological transition from yeast to invasive filamentous hyphae that secrete candidalysin, a 31-amino-acid peptide toxin required for virulence. Candidalysin damages epithelial cell plasma membranes and stimulates the activating protein 1 (AP-1) transcription factor c-Fos (via p38–mitogen-activated protein kinase [MAPK]), and the MAPK phosphatase MKP1 (via extracellular signal-regulated kinases 1 and 2 [ERK1/2]–MAPK), which trigger and regulate proinflammatory cytokine responses, respectively. The candidalysin toxin resides as a discrete cryptic sequence within a larger 271-amino-acid parental preproprotein, Ece1p. Here, we demonstrate that kexin-like proteinases, but not secreted aspartyl proteinases, initiate a two-step posttranslational processing of Ece1p to produce candidalysin. Kex2p-mediated proteolysis of Ece1p after Arg61 and Arg93, but not after other processing sites within Ece1p, is required to generate immature candidalysin from Ece1p, followed by Kex1p-mediated removal of a carboxyl arginine residue to generate mature candidalysin. C. albicans strains harboring mutations of Arg61 and/or Arg93 did not secrete candidalysin, were unable to induce epithelial damage and inflammatory responses in vitro, and showed attenuated virulence in vivo in a murine model of oropharyngeal candidiasis. These observations identify enzymatic processing of C. albicans Ece1p by kexin-like proteinases as crucial steps required for candidalysin production and fungal pathogenicity.

Development of Anti-Virulence Approaches for Candidiasis via a Novel Series of Small-Molecule Inhibitors of Candida albicans Filamentation.

ABSTRACTCandida albicans remains the main etiologic agent of candidiasis, the most common fungal infection and now the third most frequent infection in U.S. hospitals. The scarcity of antifungal agents and their limited efficacy contribute to the unacceptably high morbidity and mortality rates associated with these infections. The yeast-to-hypha transition represents the main virulence factor associated with the pathogenesis of C. albicans infections. In addition, filamentation is pivotal for robust biofilm development, which represents another major virulence factor for candidiasis and further complicates treatment. Targeting pathogenic mechanisms rather than growth represents an attractive yet clinically unexploited approach in the development of novel antifungal agents. Here, we performed large-scale phenotypic screening assays with 30,000 drug-like small-molecule compounds within ChemBridge’s DIVERSet chemical library in order to identify small-molecule inhibitors of C. albicans filamentation, and our efforts led to the identification of a novel series of bioactive compounds with a common biaryl amide core structure. The leading compound of this series, N-[3-(allyloxy)-phenyl]-4-methoxybenzamide, was able to prevent filamentation under all liquid and solid medium conditions tested, suggesting that it impacts a common core component of the cellular machinery that mediates hypha formation under different environmental conditions. In addition to filamentation, this compound also inhibited C. albicans biofilm formation. This leading compound also demonstrated in vivo activity in clinically relevant murine models of invasive and oral candidiasis. Overall, our results indicate that compounds within this series represent promising candidates for the development of novel anti-virulence approaches to combat C. albicans infections.

Identification and Mode of Action of a Plant Natural Product Targeting Human Fungal Pathogens.

ABSTRACTCandida albicans is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). In vitro susceptibility profiling experiments identified 33 NPs with activity against C. albicans (MIC50s ≤ 32 μg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato (Solanum lycopersicum) and exhibited high levels of fungistatic activity against Candida species (MIC50s ≤ 1 μg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated C. albicans cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in Saccharomyces cerevisiae coupled with whole-genome sequencing identified 2 nonsynonymous mutations in ERG6 (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the in vivo efficacy of tomatidine in a mouse model of C. albicans systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent.

Global regulatory roles of the cAMP/PKA pathway revealed by phenotypic, transcriptomic and phosphoproteomic analyses in a null mutant of the PKA catalytic subunit in Candida albicans.

The conserved cAMP-dependent protein kinase (PKA) plays critical roles in the regulation of morphological transitions and virulence in the human fungal pathogen Candida albicans. It has long been thought that the PKA catalytic subunit is essential for cell viability in this fungus. Paradoxically, the single adenylyl cyclase-encoding gene, CYR1, which is required for the production of cAMP in C. albicans, is not essential for cell growth. Here, a double mutant of TPK1 and TPK2 (tpk2/tpk2 tpk1/tpk1, t2t1), which encode two isoforms of the PKA catalytic subunit was successfully generated, suggesting that this subunit is not essential for cell viability. Inactivation of the PKA catalytic subunit blocked filamentation and dramatically attenuated white-to-opaque switching, but promoted sexual mating. Comparative transcriptomic analyses demonstrated that the t2t1 and cyr1/cyr1 mutants exhibited similar global gene expression profiles. Compared with the WT strain, the general transcriptional activity and metabolism were significantly decreased in both the t2t1 and cyr1/cyr1 mutants. Using combined phosphoproteomic and bioinformatic analyses, we identified 181 potential PKA phosphorylation targets, which represent 148 unique proteins involved in a wide spectrum of biological processes. The study sheds new insights into the global regulatory features of the cAMP/PKA pathway in C. albicans.

Post-translational modification directs nuclear and hyphal tip localization of Candida albicans mRNA-binding protein Slr1.

SummaryThe morphological transition of the opportunistic fungal pathogen Candida albicans from budding to hyphal growth has been implicated in its ability to cause disease in animal models. Absence of SR‐like RNA‐binding protein Slr1 slows hyphal formation and decreases virulence in a systemic candidiasis model, suggesting a role for post‐transcriptional regulation in these processes. SR (serine–arginine)‐rich proteins influence multiple steps in mRNA metabolism and their localization and function are frequently controlled by modification. We now demonstrate that Slr1 binds to polyadenylated RNA and that its intracellular localization is modulated by phosphorylation and methylation. Wildtype Slr1‐GFP is predominantly nuclear, but also co‐fractionates with translating ribosomes. The non‐phosphorylatable slr1‐6SA‐GFP protein, in which six serines in SR/RS clusters are substituted with alanines, primarily localizes to the cytoplasm in budding cells. Intriguingly, hyphal cells display a slr1‐6SA‐GFP focus at the tip near the Spitzenkörper, a vesicular structure involved in molecular trafficking to the tip. The presence of slr1‐6SA‐GFP hyphal tip foci is reduced in the absence of the mRNA‐transport protein She3, suggesting that unphosphorylated Slr1 associates with mRNA–protein complexes transported to the tip. The impact of SLR1 deletion on hyphal formation and function thus may be partially due to a role in hyphal mRNA transport.

Extensive functional redundancy in the regulation of Candida albicans drug resistance and morphogenesis by lysine deacetylases Hos2, Hda1, Rpd3 and Rpd31.

Current treatment efforts for fungal infections are hampered by the limited availability of antifungal drugs and by the emergence of drug resistance. A powerful strategy to enhance the efficacy of antifungal drugs is to inhibit the molecular chaperone Hsp90. Hsp90 governs drug resistance, morphogenesis and virulence in a leading fungal pathogen of humans, Candida albicans. Our previous work with Saccharomyces cerevisiae established acetylation as a novel mechanism of posttranslational control of Hsp90 function in fungi. We implicated lysine deacetylases (KDACs) as key regulators of resistance to the most widely deployed class of antifungals, the azoles, in both S. cerevisiae and C. albicans. Here, we demonstrate high levels of functional redundancy among the KDACs that are important for regulating Hsp90 function. We identify Hos2, Hda1, Rpd3 and Rpd31 as the KDACs mediating azole resistance and morphogenesis in C. albicans. Furthermore, we identify lysine 30 and 271 as critical acetylation sites onC. albicans Hsp90, and substitutions at these residues compromise Hsp90 function. Finally, we show that pharmacological inhibition of KDACs phenocopies pharmacological inhibition of Hsp90 and abrogates Hsp90-dependent azole resistance in numerous Candida species. This work illuminates new facets to the impact of KDACs on fungal drug resistance and morphogenesis, provides important insights into the divergence of the C. albicans Hsp90 regulatory network and reveals new targets for development of antifungal drugs.

Dual-species transcriptional profiling during systemic candidiasis reveals organ-specific host-pathogen interactions.

Candida albicans is a common cause of life-threatening fungal bloodstream infections. In the murine model of systemic candidiasis, the kidney is the primary target organ while the fungal load declines over time in liver and spleen. To better understand these organ-specific differences in host-pathogen interaction, we performed gene expression profiling of murine kidney, liver and spleen and determined the fungal transcriptome in liver and kidney. We observed a delayed transcriptional immune response accompanied by late induction of fungal stress response genes in the kidneys. In contrast, early upregulation of the proinflammatory response in the liver was associated with a fungal transcriptome resembling response to phagocytosis, suggesting that phagocytes contribute significantly to fungal control in the liver. Notably, C. albicans hypha-associated genes were upregulated in the absence of visible filamentation in the liver, indicating an uncoupling of gene expression and morphology and a morphology-independent effect by hypha-associated genes in this organ. Consistently, integration of host and pathogen transcriptional data in an inter-species gene regulatory network indicated connections of C. albicans cell wall remodelling and metabolism to the organ-specific immune responses.

Integrating Candida albicans metabolism with biofilm heterogeneity by transcriptome mapping.

Candida albicans biofilm formation is an important virulence factor in the pathogenesis of disease, a characteristic which has been shown to be heterogeneous in clinical isolates. Using an unbiased computational approach we investigated the central metabolic pathways driving biofilm heterogeneity. Transcripts from high (HBF) and low (LBF) biofilm forming isolates were analysed by RNA sequencing, with 6312 genes identified to be expressed in these two phenotypes. With a dedicated computational approach we identified and validated a significantly differentially expressed subnetwork of genes associated with these biofilm phenotypes. Our analysis revealed amino acid metabolism, such as arginine, proline, aspartate and glutamate metabolism, were predominantly upregulated in the HBF phenotype. On the contrary, purine, starch and sucrose metabolism was generally upregulated in the LBF phenotype. The aspartate aminotransferase gene AAT1 was found to be a common member of these amino acid pathways and significantly upregulated in the HBF phenotype. Pharmacological inhibition of AAT1 enzyme activity significantly reduced biofilm formation in a dose-dependent manner. Collectively, these findings provide evidence that biofilm phenotype is associated with differential regulation of metabolic pathways. Understanding and targeting such pathways, such as amino acid metabolism, is potentially useful for developing diagnostics and new antifungals to treat biofilm-based infections.

Global Identification of Biofilm-Specific Proteolysis in Candida albicans.

ABSTRACTCandida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans. Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation.

Novel nanoscale bacteriophage-based single-domain antibodies for the therapy of systemic infection caused by Candida albicans.

Candida albicans (C. albicans) is an important human commensal and opportunistic fungal pathogen. Secreted aspartyl proteinases (Saps) are a major virulence trait of C. albicans, and among these proteases Sap2 has the highest expression levels. It is possible that antibodies against Sap2 could provide an antifungal effect. In this study, two phages displaying anti-rSap2 single chain variable fragments (scFvs) were screened from human single fold scFv libraries, and their potential therapeutic roles were evaluated using a murine model infected by C. albicans. The in vivo efficacies were assessed by mortality rates, fungal burden and histological examination. Overall survival rates were significantly increased while the colony counts and infectious foci were significantly decreased after treatment with the scFv-phages relative to the control groups. In order to investigate the immune response provoked by scFv-phages, three kinds of cytokines (Th1, Th2 and Th17 types) were measured and a clear immune response was observed. These findings suggest that anti-rSap2 scFv-phages have potential in the therapy of systemic infection caused by C. albicans.

Morphological and physiological changes induced by contact-dependent interaction between Candida albicans and Fusobacterium nucleatum.

Candida albicans and Fusobacterium nucleatum are well-studied oral commensal microbes with pathogenic potential that are involved in various oral polymicrobial infectious diseases. Recently, we demonstrated that F. nucleatum ATCC 23726 coaggregates with C. albicans SN152, a process mainly mediated by fusobacterial membrane protein RadD and Candida cell wall protein Flo9. The aim of this study was to investigate the potential biological impact of this inter-kingdom interaction. We found that F. nucleatum ATCC 23726 inhibits growth and hyphal morphogenesis of C. albicans SN152 in a contact-dependent manner. Further analysis revealed that the inhibition of Candida hyphal morphogenesis is mediated via RadD and Flo9 protein pair. Using a murine macrophage cell line, we showed that the F. nucleatum-induced inhibition of Candida hyphal morphogenesis promotes C. albicans survival and negatively impacts the macrophage-killing capability of C. albicans. Furthermore, the yeast form of C. albicans repressed F. nucleatum-induced MCP-1 and TNFα production in macrophages. Our study suggests that the interaction between C. albicans and F. nucleatum leads to a mutual attenuation of virulence, which may function to promote a long-term commensal lifestyle within the oral cavity. This finding has significant implications for our understanding of inter-kingdom interaction and may impact clinical treatment strategies.

Inhibitory Effect of Sophorolipid on Candida albicans Biofilm Formation and Hyphal Growth.

Candida albicans causes superficial and life-threatening systemic infections. These are difficult to treat often due to drug resistance, particularly because C. albicans biofilms are inherently resistant to most antifungals. Sophorolipid (SL), a glycolipid biosurfactant, has been shown to have antimicrobial and anticancer properties. In this study, we investigated the effect of SL on C. albicans biofilm formation and preformed biofilms. SL was found to inhibit C. albicans biofilm formation as well as reduce the viability of preformed biofilms. Moreover, SL, when used along with amphotericin B (AmB) or fluconazole (FLZ), was found to act synergistically against biofilm formation and preformed biofilms. Effect of SL on C. albicans biofilm formation was further visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), which revealed absence of hyphae, typical biofilm architecture and alteration in the morphology of biofilm cells. We also found that SL downregulates the expression of hypha specific genes HWP1, ALS1, ALS3, ECE1 and SAP4, which possibly explains the inhibitory effect of SL on hyphae and biofilm formation.

SUMOylation of Wor1 by a novel SUMO E3 ligase controls cell fate in Candida albicans.

Candida albicans is the most common human fungal pathogen, yet is a normal commensal resident of the human gut. CO(2) levels in the gut are much higher than in air, and it is known that elevated CO(2) concentration promotes C. albicans cells to undergo a phenotypic switch from white to opaque phase. Wor1, the master regulator of opaque cell formation, is required for both the white to opaque transition and opaque maintenance. To elucidate the regulatory mechanism of Wor1, we set out to identify Wor1-interacting proteins using a yeast two-hybrid screen. A SUMO E3 ligase named Wos1 (Wor1 SUMO-ligase 1) was identified to interact with Wor1 and regulate Wor1 SUMOylation. WOS1 expression is upregulated in response to high CO(2), and the induction by CO(2) is dependent on the transcription factor Flo8. Under high CO(2) conditions, Wos1 is required for the white to opaque switch and acts downstream of Flo8. At atmospheric CO(2) levels, overexpression of Wos1 enhances Wor1 SUMOylation and promotes the white to opaque switch. Wor1 is found to be SUMOylated at lysine 385, and loss of this mark by point mutation leads to a defect in CO(2) -mediated opaque cell induction. Together, our genetic and biological data show that Wos1-mediated Wor1 SUMOylation contributes to the regulation of CO(2) -induced white to opaque switching as well as heritable maintenance of the opaque cell type.

Dressed to impress: impact of environmental adaptation on the Candida albicans cell wall.

Summary C andida albicans is an opportunistic fungal pathogen of humans causing superficial mucosal infections and life‐threatening systemic disease. The fungal cell wall is the first point of contact between the invading pathogen and the host innate immune system. As a result, the polysaccharides that comprise the cell wall act as pathogen associated molecular patterns, which govern the host–pathogen interaction. The cell wall is dynamic and responsive to changes in the external environment. Therefore, the host environment plays a critical role in regulating the host–pathogen interaction through modulation of the fungal cell wall. This review focuses on how environmental adaptation modulates the cell wall structure and composition, and the subsequent impact this has on the innate immune recognition of C . albicans.

The WOR1 5' untranslated region regulates white-opaque switching in Candida albicans by reducing translational efficiency.

SummaryThe human fungal pathogen C andida albicans undergoes white‐opaque phenotypic switching, which enhances its adaptation to host niches. Switching is controlled by a transcriptional regulatory network of interlocking feedback loops acting on the transcription of WOR 1, the master regulator of white‐opaque switching, but regulation of the network on the translational level is not yet explored. Here, we show that the long 5′ untranslated region of WOR 1 regulates the white‐opaque phenotype. Deletion of the WOR 1 5′ UTR promotes white‐to‐opaque switching and stabilizes the opaque state. The WOR 1 5′ UTR reduces translational efficiency and the association of the transcript with polysomes. Reduced polysome association was observed for additional key regulators of cell fate and morphology with long 5′ UTR as well. Overall, we find a novel regulatory step of white‐opaque switching at the translational level. This translational regulation is implicated for many key regulators of cell fate and morphology in C . albicans.

Fungal-specific subunits of the Candida albicans mitochondrial complex I drive diverse cell functions including cell wall synthesis.

Our published research has focused on the role of Goa1p, an apparent regulator of the Candida albicans mitochondrial complex I (CI). Lack of Goa1p affects optimum cell growth, CI activity and virulence. Eukaryotic CI is composed of a core of 14 alpha-proteobacterial subunit proteins and a variable number of supernumerary subunit proteins. Of the latter group of proteins, one (NUZM) is fungal specific and the other (NUXM) is found in fungi, algae and plants, but is not a mammalian CI subunit protein. We have established that NUXM is orf19.6607 and NUZM is orf19.287 in C. albicans. Herein, we validate both subunit proteins as NADH:ubiquinone oxidoreductases (NUO) and annotate their gene functions. To accomplish these objectives, we compared null mutants of each with wild type (WT) and gene-reconstituted strains. Genetic mutants of genes NUO1 (orf19.6607) and NUO2 (orf19.287), not surprisingly, each had reduced oxygen consumption, decreased mitochondrial redox potential, decreased CI activity, increased reactive oxidant species (ROS) and decreased chronological ageing in vitro. Loss of either gene results in disassembly of CI. Transcriptional profiling of both mutants indicated significant down-regulation of genes of carbon metabolism, as well as up-regulation of mitochondrial-associated gene families that may occur to compensate for the loss of CI activity. Profiling of both mutants also demonstrated a loss of cell wall β-mannosylation but not in a conserved CI subunit (ndh51Δ). The profiling data may indicate specific functions driven by the enzymatic activity of Nuo1p and Nuo2p. Of importance, each mutant is also avirulent in a murine blood-borne, invasive model of candidiasis associated with their reduced colonization of tissues. Based on their fungal specificity and roles in virulence, we suggest both as drug targets for antifungal drug discovery.

SCREENING ANTIBACTERIAL AND ANTIFUNGAL ACTIVITIES AND EVALUATION OF THE EXHAUSTIVE EXTRACTIONS YIELDS FOR VERBASCUM SINUATUM L.

T his study aimed to evaluate antibacterial and antifungal activities and exhaustive extraction yields of the aqueous and organic extract s of V erbascum sinuatum L . , against possible human pathogens, which are the fungus Candida albicans , gram positive bacteria Bacillus subtilis, Staphylococcus aureus , Staphylococcus epidermidis and the gram negative bacteria Eschrichia coli and Pseudomonas aeruginosa . Well diffusion method was used in screening of antimicrobial activity for the plant extracts, in which the diameters of inhibition zones were measured and compared to a positive control. S erial dilution method wa s used for measuring the minimum inhibition concentrations for each microorganism. In well diffusion method, the plant’s aqueous extract has antimicrobial activity to all the tested organisms except Pseudomonas aeruginosa and C andida albicans, with variabl e diameters of inhibition zone. The percent inhibition c o mpared to the positive control imipene m was 39.13 % for Staphylococcus aureus , 37.5 % for Staphylococcus epidermidis, 30.55 % for Eschrichia coli and the least 30.43 % for Bacillus subtilis . The orga nic extract exhibited inhibition activity 26.08 % against Bacillus subtilis and 50 % against Eschrichia coli . In the serial dilution method, the aqueous extract exhibited inhibition for all the test microorganisms. At initial concentration of 20 mg/ml, the lowest MIC value was for Staphylococcus aureus 1.28 µg/ml, and highest for S taphylococcus epidermidis 4000 µg/ml. The MIC values for P seudomonas aeruginosa 160 µg/ml, 800 µg/ml for B acillus subtilis , 800 µg/ml for Eschrichia coli and 32 µg/ml for C andida albicans respectively . This study showed that V . sinuatum extract has a broad spectrum activity against gram positive and gram negative bacteria, as well as anti candidal activity.