Hyphal Genes Research Articles

Mechanism and bioinformatics analysis of the effect of berberine-enhanced fluconazole against drug-resistant Candida albicans

Biofilms produced by Candida albicans present a challenge in treatment with antifungal drug. Enhancing the sensitivity to fluconazole (FLC) is a reasonable method for treating FLC-resistant species. Moreover, several lines of evidence have demonstrated that berberine (BBR) can have antimicrobial effects. The aim of this study was to clarify the underlying mechanism of these effects. We conducted a comparative study of the inhibition of FLC-resistant strain growth by FLC treatment alone, BBR treatment alone, and the synergistic effect of combined FLC and BBR treatment. Twenty-four isolated strains showed distinct biofilm formation capabilities. The antifungal effect of combined FLC and BBR treatment in terms of the growth and biofilm formation of Candida albicans species was determined via checkerboard, time-kill, and fluorescence microscopy assays. The synergistic effect of BBR and FLC downregulated the expression of the efflux pump genes CDR1 and MDR, the hyphal gene HWP1, and the adhesion gene ALS3; however, the gene expression of the transcriptional repressor TUP1 was upregulated following treatment with this drug combination. Furthermore, the addition of BBR led to a marked reduction in cell surface hydrophobicity. To identify resistance-related genes and virulence factors through genome-wide sequencing analysis, we investigated the inhibition of related resistance gene expression by the combination of BBR and FLC, as well as the associated signaling pathways and metabolic pathways. The KEGG metabolic map showed that the metabolic genes in this strain are mainly involved in amino acid and carbon metabolism. The metabolic pathway map showed that several ergosterol (ERG) genes were involved in the synthesis of cell membrane sterols, which may be related to drug resistance. In this study, BBR + FLC combination treatment upregulated the expression of the ERG1, ERG3, ERG4, ERG5, ERG24, and ERG25 genes and downregulated the expression of the ERG6 and ERG9 genes compared with fluconazole treatment alone (p < 0.05).

Temporal dynamics of Candida albicans morphogenesis and gene expression reveals distinctions between in vitro and in vivo filamentation.

Candida albicans is a common human fungal pathogen that is also a commensal of the oral cavity and gastrointestinal tract. C. albicans pathogenesis is linked to its transition from budding yeast to filamentous morphologies including hyphae and pseudohyphae. The centrality of this virulence trait to C. albicans pathobiology has resulted in extensive characterization of a wide range of factors associated with filamentation with a strong focus on transcriptional regulation. The vast majority of these experiments have used in vitro conditions to induce the yeast-to-filament transition. Taking advantage of in vivo approaches to quantitatively characterize both morphology and gene expression during filamentation during mammalian infection, we have investigated the dynamics of these two aspects of filamentation in vivo and compared them to in vitro filament induction with "host-like" tissue culture media supplemented with serum at mammalian body temperature. Although filamentation shares many common features in the two conditions, we have found two significant differences. First, alternative carbon metabolism genes are expressed early during in vitro filamentation and late in vivo, suggesting significant differences in glucose availability. Second, C. albicans begins a hyphae-to-yeast transition after 4-h incubation while we find little evidence of hyphae-to-yeast transition in vivo up to 24 h post-infection. We show that the low rate of in vivo hyphae-to-yeast transition is likely due to the very low expression of PES1, a key driver of lateral yeast in vitro and that heterologous expression of PES1 is sufficient to trigger lateral yeast formation in vivo.IMPORTANCECandida albicans filamentation is correlated with virulence and is an intensively studied aspect of C. albicans biology. The vast majority of studies on C. albicans filamentation are based on in vitro induction of hyphae and pseudohyphae. Here we used an in vivo filamentation assay and in vivo expression profiling to compare the tempo of morphogenesis and gene expression between in vitro and in vivo filamentation. Although the hyphal gene expression profile is induced rapidly in both conditions, it remains stably expressed over a 12-h time course in vivo while it peaks after 4 h in vitro and is reduced. This reduced hyphal gene expression in vitro correlates with reduced hyphae and increased hyphae-to-yeast transition. By contrast, there is little evidence of hyphae-to-yeast transition in vivo.

Inhibiting pathogenicity of vaginal Candida albicans by lactic acid bacteria and MS analysis of their extracellular compounds.

Maintaining healthy vaginal microflora post-puberty is critical. In this study we explore the potential of vaginal lactic acid bacteria (LAB) and their extracellular metabolites against the pathogenicity of Candida albicans. The probiotic culture free supernatant (PCFS) from Lactobacillus crispatus, L. gasseri, and L. vaginalis exhibit an inhibitory effect on budding, hyphae, and biofilm formation of C. albicans. LGPCFS manifested the best potential among the LAB PCFS, inhibiting budding for 24 h and restricting hyphae formation post-stimulation. LGPCFS also pre-eminently inhibited biofilm formation. Furthermore, L. gasseri itself grew under RPMI 1640 stimulation suppressing the biofilm formation of C. albicans. The PCFS from the LAB downregulated the hyphal genes of C. albicans, inhibiting the yeast transformation to fungi. Hyphal cell wall proteins HWP1, ALS3, ECE1, and HYR1 and transcription factors BCR1 and CPH1 were downregulated by the metabolites from LAB. Finally, the extracellular metabolome of the LAB was studied by LC-MS/MS analysis. L.gasseri produced the highest antifungal compounds and antibiotics, supporting its best activity against C. albicans. Vaginal LAB and their extracellular metabolites perpetuate C. albicans at an avirulent state. The metabolites produced by these LAB in vitro have been identified, and can be further exploited as a preventive measure against vaginal candidiasis.

The short-chain fatty acid crotonate reduces invasive growth and immune escape of Candida albicans by regulating hyphal gene expression.

Macrophages curtail the proliferation of the pathogen Candida albicans within human body niches. Within macrophages, C. albicans adapts its metabolism and switches to invasive hyphal morphology. These adaptations enable fungal growth and immune escape by triggering macrophage lysis. Transcriptional programs regulate these metabolic and morphogenetic adaptations. Here we studied the roles of chromatin in these processes and implicate lysine crotonylation, a histone mark regulated by metabolism, in hyphal morphogenesis and macrophage interactions by C. albicans. We show that the short-chain fatty acid crotonate increases histone crotonylation, reduces hyphal formation within macrophages, and slows macrophage lysis and immune escape of C. albicans. Crotonate represses hyphal gene expression, and we propose that C. albicans uses diverse acylation marks to regulate its cell morphology in host environments. Hyphal formation is a virulence property of C. albicans. Therefore, a further importance of our study stems from identifying crotonate as a hyphal inhibitor.

Collaboration between Antagonistic Cell Type Regulators Governs Natural Variation in the Candida albicans Biofilm and Hyphal Gene Expression Network.

ABSTRACTCandida albicans is among the most significant human fungal pathogens. However, the vast majority of C. albicans studies have focused on a single clinical isolate and its marked derivatives. We investigated natural variation among clinical C. albicans isolates in gene regulatory control of biofilm formation, a process crucial to virulence. The transcription factor Efg1 is required for biofilm-associated gene expression and biofilm formation. Previously, we found extensive variation in Efg1-responsive gene expression among 5 diverse clinical isolates. However, chromatin immunoprecipitation sequencing analysis showed that Efg1 binding to genomic loci was uniform among the isolates. Functional dissection of strain differences identified three transcription factors, Brg1, Tec1, and Wor1, for which small changes in expression levels reshaped the Efg1 regulatory network. Brg1 and Tec1 are known biofilm activators, and their role in Efg1 network variation may be expected. However, Wor1 is a known repressor of EFG1 expression and an inhibitor of biofilm formation. In contrast, we found that a modest increase in WOR1 RNA levels, reflecting the expression differences between C. albicans strains, could augment biofilm formation and expression of biofilm-related genes. The analysis of natural variation here reveals a novel function for a well-characterized gene and illustrates that strain diversity offers a unique resource for elucidation of network interactions.

Elucidation of Bacterial Peptidoglycan Signaling and Recognition in the Human Commensal Candida. albicans through the LRR domain of the peripheral Membrane Protein Cyr1p

The human commensal Candida. albicans (C. albicans) is the most common fungal resident of the human microbiome. In healthy individuals this fungus occupies a budding morphology, but during immune incompetence morphologically transitions to a pathogenic hyphal form. The long filamentous form of hyphae readily invades human epithelial cells and if not successfully treated, cause lethal systemic infections. One of the key molecular integrators in this morphological transition is the binding of bacterial peptidoglycan (PG) fragments to the Leucine Rich Repeat (LRR) domain of the adenyl cyclase Cyr1p. PG is a highly complex polymer consisting of repeating units of the disaccharide N‐acetyl glucosamine‐N acetyl muramic acid, which are crosslinked by pentapeptide side chains. Considerable diversity exists in this polymer within and among Gram (‐) and Gram (+) species of bacteria, rendering a plethora of potential ligands for Cyr1p. Identification of specific PG fragments that are able to induce hyphal formation in C. albicans, and stabilize Cyr1p through the LRR domain can elude to small molecule inhibitors to combat these lethal fungal infections that are continuously becoming resistant to current antifungal therapies. To approach a congruency of PG fragments from both Gram (‐) and Gram (+) bacterium that can serve as ligands for the LRR domain, we began by characterizing recombinant LRR and in doing so have determined this protein is a peripheral membrane protein that adopts an alpha helical conformation. Furthermore, to assess Cyr1p's ability to interact with a repertoire of PG fragments, we conducted Cellular Thermal Shift Assays (CETSA) with various PG fragments and have observed a temperature induced stabilization in comparison to apo‐protein, suggesting that there is a positive interaction with the LRR domain and the fragments. We further investigated the ability of these PG fragments to induce hyphal formation in WT C. albicans cells via microscopy assays and evaluated upregulation of hyphae specific genes at various timepoints via RT‐PCR. We observed differential hyphal gene regulation at various timepoints, suggesting that some fragments although able to initiate hyphal growth, failed to maintain growth and subsequent elongation. In addition, we observed that despite a positive association of the LRR with some fragments through CETSA assays, these fragments failed to illicit hyphal formation in WT cells. We infer that stabilization of the LRR is not linearly attributed to hyphal elongation, although hyphal initiation may be present. Collectively, these data help to identify new mechanisms to treat pathogenic fungal infections through the elucidation of promiscuity of the Cyr1p LRR domain, and steers us towards the identification of PG decoys and/or small molecule inhibitors for the treatment of these infections.

Regulation of Candida albicans Hyphal Morphogenesis by Endogenous Signals.

Candida albicans is a human commensal fungus that is able to assume several morphologies, including yeast, hyphal, and pseudohyphal. Under a range of conditions, C. albicans performs a regulated switch to the filamentous morphology, characterized by the emergence of a germ tube from the yeast cell, followed by a mold-like growth of branching hyphae. This transition from yeast to hyphal growth has attracted particular attention, as it has been linked to the virulence of C. albicans as an opportunistic human pathogen. Signal transduction pathways that mediate the induction of the hyphal transcription program upon the imposition of external stimuli have been extensively investigated. However, the hyphal morphogenesis transcription program can also be induced by internal cellular signals, such as inhibition of cell cycle progression, and conversely, the inhibition of hyphal extension can repress hyphal-specific gene expression, suggesting that endogenous cellular signals are able to modulate hyphal gene expression as well. Here we review recent developments in the regulation of the hyphal morphogenesis of C. albicans, with emphasis on endogenous morphogenetic signals.

A Review of Esophagitis Due to Candida Species in Human Immunodeficiency Virus (HIV) Infected Patients

The present study aimed to provide an overview of epidemiology, pathogenicity, clinical diagnosis, and treatment of Candida esophagitis in human immunodeficiency virus (HIV)-infected patients. The review process involved studying all the existing literature published on this Candida infection. Esophageal candidiasis (EC) is the most common manifestation of mucosal candidiasis and patients with HIV are predominantly at the risk of this opportunistic infection. The prevalence of EC indicated diverse ranges among HIV patients in different geographic areas due to antiretroviral therapy (ART). The main factors for EC were gastric ulcers, CD4+cell count 400 cells/mL in the ART era. However, a low CD4+ cell count (<200 cells/mL) was significantly associated with EC in the pre-ART era. The interactions between the Candida virulence factor and host immune defense lead to the host responses against this fungal pathogen. During the Candida albicans invasion, secretion of candidalysin which is encoded by the hyphal gene ECE1 has a potential role in epithelial cell damage and secretion of stimulated cytokine. Early trials of the empirical antifungal therapy are recommended before an endoscopic examination. Esophageal biopsy should be considered in patients with a failure of empiric antifungal treatment as it may allow the possibility of drug-resistant Candida and other opportunistic pathogens. The first-line induction treatment of Candida esophagitis is based on oral fluconazole. The shift from C. albicans to non-albicans Candida (NAC) may be correlated with the development of fluconazole resistance and relapse or therapeutic failure in this infection. An increase in the intrinsic and acquired resistance has raised the significance of the optimal antifungal therapy for the critically ill patient. Candida esophagitis requires a systematic suspicion for early diagnosis and appropriate management of HIV infected patients in order to prevent delayed treatment related to undesirable morbidity or even mortality scores.

Boric acid destabilizes the hyphal cytoskeleton and inhibits invasive growth of Candida albicans.

Exposure of Candida albicans to sub-lethal concentrations of boric acid (BA) restricts the dimorphic fungus to its yeast morphology and prevents the formation of invasive hyphae on solid substrates. Exposure to BA causes a rapid and reversible disappearance of polarisome and Spitzenkörper in growing hyphae. In BA-treated hyphae of C. albicans, actin quickly reorganizes from cytoplasmic cables to cortical patches and cell wall growth switches from an apical to an isotropic pattern. As a result of the cytoskeletal changes, the hyphal tips broaden and directional growth of hyphae ceases in the presence of BA. An analysis of homozygous deletion strains showed that mutants with constitutive or enhanced hyphal growth (tup1, nrg1, ssn6, rbf1) are BA-sensitive, demonstrating that cellular morphology is a major determinant of BA tolerance. The screening of deletion mutants also showed that deficiencies of the main activator of hyphal gene expression, Efg1, and the Rim101-signalling cascade, leading to Efg1 activation, cause BA resistance. Taken together, the data presented show that the selective inhibitory effect on BA on C. albicans hyphae is rooted in a disruption of apical cytoskeletal elements of growing hyphae.

Hyphal growth in Candida albicans does not require induction of hyphal-specific gene expression.

Various stimuli, including N-acetylglucosamine (GlcNAc), induce the fungal pathogen Candida albicans to switch from budding to hyphal growth. Previous studies suggested that hyphal morphogenesis is stimulated by transcriptional induction of a set of genes that includes known virulence factors. To better understand hyphal development, we examined the role of GlcNAc metabolism using a triple mutant lacking the genes required to metabolize exogenous GlcNAc (hxk1Δ nag1Δ dac1Δ). Surprisingly, at low ambient pH (∼pH 4), GlcNAc stimulated this mutant to form hyphae without obvious induction of hyphal genes. This indicates that GlcNAc can stimulate a separate signal to induce hyphae that is independent of transcriptional responses. Of interest, GlcNAc could induce the triple mutant to express hyphal genes when the medium was buffered to a higher pH (>pH 5), which normally occurs after GlcNAc catabolism. Catabolism of GlcNAc raises the ambient pH rather than acidifying it, as occurs after dextrose catabolism. This synergy between alkalinization and GlcNAc to induce hyphal genes involves the Rim101 pH-sensing pathway; GlcNAc induced rim101Δ and dfg16Δ mutants to form hyphae, but hyphal gene expression was partially defective. These results demonstrate that hyphal morphogenesis and gene expression can be regulated independently, which likely contributes to pathogenesis at different host sites.

Signaling Through Protein Kinases and Transcriptional Regulators in Candida albicans

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. Several signaling pathways that regulate morphogenesis have been identified, including various transcription factors that either activate or repress hypha-specific genes. Two well-characterized pathways include the MAP kinase cascade and cAMP-dependent protein kinase pathway that regulate the transcription factors Cph1p and Efg1p, respectively. cAMP also appears to interplay with other second messengers: Ca2+, inositol tri-phosphates in regulating yeast-hyphal transition. Other, lesscharacterized pathways include two component histidine kinases, cyclin-dependent kinase pathway, and condition specific pathways such as pH and embedded growth conditions. Nrg1 and Rfg1 function as transcriptional repressors of hyphal genes via recruitment of Tup1 co-repressor complex. Different upstream signals converge into a common downstream output during hyphal switch. The levels of expression of several genes have been shown to be associated with hyphal morphogenesis rather than with a specific hypha-inducing condition. Hyphal development is also linked to the expression of a range of other virulence factors. This review explains the relative contribution of multiple pathways that could be used by Candida albican cells to sense subtle differences in the growth conditions of its native host environment.

Repression of hyphal proteinase expression by the mitogen-activated protein (MAP) kinase phosphatase Cpp1p of Candida albicans is independent of the MAP kinase Cek1p.

Cpp1p is a putative mitogen-activated protein (MAP) kinase phosphatase that suppresses Candida albicans hyphal formation at 25 degrees C through its probable substrate, the Cek1p filamentation MAP kinase. Here we report that expression of the serum-induced genes SAP4-6 and HYR1 increased several fold in hyphal forms of a cpp1/cpp1 null mutant, while the rate and extent of hyphal development up to 5 h were normal. Therefore, we provide evidence that Cpp1p represses hyphal gene expression by acting through a Cek1p-independent mechanism. SAP4-6 and HYR1 transcripts were undetectable in a null mutant of another key regulator of filamentation, Efg1p; thus, Efg1p and Cpp1p oppose each other during the expression of these genes in hyphal forms.