ResearchPad - candida-albicans https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Abrogation of pathogenic attributes in drug resistant <i>Candida auris</i> strains by farnesol]]> https://www.researchpad.co/article/elastic_article_7651 Candida auris, a decade old Candida species, has been identified globally as a significant nosocomial multidrug resistant (MDR) pathogen responsible for causing invasive outbreaks. Biofilms and overexpression of efflux pumps such as Major Facilitator Superfamily and ATP Binding Cassette are known to cause multidrug resistance in Candida species, including C. auris. Therefore, targeting these factors may prove an effective approach to combat MDR in C. auris. In this study, 25 clinical isolates of C. auris from different hospitals of South Africa were used. All the isolates were found capable enough to form biofilms on 96-well flat bottom microtiter plate that was further confirmed by MTT reduction assay. In addition, these strains have active drug efflux mechanism which was supported by rhodamine-6-G extracellular efflux and intracellular accumulation assays. Antifungal susceptibility profile of all the isolates against commonly used drugs was determined following CLSI recommended guidelines. We further studied the role of farnesol, an endogenous quorum sensing molecule, in modulating development of biofilms and drug efflux in C. auris. The MIC for planktonic cells ranged from 62.5–125 mM, and for sessile cells was 125 mM (4h biofilm) and 500 mM (12h and 24h biofilm). Furthermore, farnesol (125 mM) also suppresses adherence and biofilm formation by C. auris. Farnesol inhibited biofilm formation, blocked efflux pumps and downregulated biofilm- and efflux pump- associated genes. Modulation of C. auris biofilm formation and efflux pump activity by farnesol represent a promising approach for controlling life threatening infections caused by this pathogen.

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<![CDATA[Candida lusitaniae in Kuwait: Prevalence, antifungal susceptibility and role in neonatal fungemia]]> https://www.researchpad.co/article/5c8accced5eed0c484990063

Objectives

Candida lusitaniae is an opportunistic yeast pathogen in certain high-risk patient populations/cohorts. The species exhibits an unusual antifungal susceptibility profile with tendency to acquire rapid resistance. Here, we describe prevalence of C. lusitaniae in clinical specimens in Kuwait, its antifungal susceptibility profile and role in neonatal fungemia.

Methods

Clinical C. lusitaniae isolates recovered from diverse specimens during 2011 to 2017 were retrospectively analyzed. All isolates were identified by germ tube test, growth on CHROMagar Candida and by Vitek 2 yeast identification system. A simple species-specific PCR assay was developed and results were confirmed by PCR-sequencing of ITS region of rDNA. Antifungal susceptibility was determined by Etest. Minimum inhibitory concentrations (MICs) were recorded after 24 h incubation at 35°C.

Results

Of 7068 yeast isolates, 134 (1.89%) were identified as C. lusitaniae including 25 (2.52%) among 990 bloodstream isolates. Species-specific PCR and PCR-sequencing of rDNA confirmed identification. Of 11 cases of neonatal candidemia, 9 occurred in NICU of Hospital A and are described here. Eight of 9 neonates received liposomal amphotericin B, which was followed by fluconazole in 7 and additionally by caspofungin in 2 cases as salvage therapy. Three of 8 (37.5%) patients died. No isolate exhibited reduced susceptibility to amphotericin B, fluconazole, voriconazole, caspopfungin, micafungin and anidulafungin. The MIC ± geometric mean values for amphotericin B, fluconazole, voriconazole, and caspofungin were as follows: 0.072 ± 0.037 μg/ml, 2.32 ± 0.49 μg/ml, 0.09 ± 0.01 μg/ml and 0.16 ± 0.08 μg/ml, respectively. Only two isolates exhibited reduced susceptibility to fluconazole.

Conclusions

This study describes the prevalence and antifungal susceptibility profile of clinical C. lusitaniae isolates in Kuwait. No isolate showed reduced susceptibility to amphotericin B. The study highlights the emerging role of C. lusitaniae as a healthcare-associated pathogen capable of causing fungemia in preterm neonates and causing significant mortality.

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<![CDATA[A prospective, multi-center study of Candida bloodstream infections in Chile]]> https://www.researchpad.co/article/5c8c1960d5eed0c484b4d4f3

Background

Active surveillance is necessary for improving the management and outcome of patients with candidemia. The aim of this study was to describe epidemiologic and clinical features of candidemia in children and adults in tertiary level hospitals in Chile.

Methods

We conducted a prospective, multicenter, laboratory-based survey study of candidemia in 26 tertiary care hospitals in Chile, from January 2013 to October 2017.

Results

A total of 780 episodes of candidemia were included, with a median incidence of 0.47/1,000 admissions. Demographic, clinical and microbiological information of 384 cases of candidemia, from 18 hospitals (7,416 beds), was included in this report. One hundred and thirty-four episodes (35%) occurred in pediatric patients and 250 (65%) in adult population. Candida albicans (39%), Candida parapsilosis (30%) and Candida glabrata (10%) were the leading species, with a significant difference in the distribution of species between ages. The use of central venous catheter and antibiotics were the most frequent risk factors in all age groups (> 70%). Three hundred and fifteen strains were studied for antifungal susceptibility; 21 strains (6.6%) were resistant to fluconazole, itraconazole, voriconazole, anidulafungin or micafungin. The most commonly used antifungal therapies were fluconazole (39%) and echinocandins (36%). The overall 30-day survival was 74.2%, significantly higher in infants (82%) and children (86%) compared with neonates (72%), adults (71%) and elderly (70%).

Conclusions

Our prospective, multicenter surveillance study showed a low incidence of candidemia in Chile, with high 30-day survival, a large proportion of elderly patients, C. glabrata as the third most commonly identified strain, a 6.6% resistance to antifungal agents and a frequent use of echinocandins.

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<![CDATA[Efficacy of liposomal amphotericin B and anidulafungin using an antifungal lock technique (ALT) for catheter-related Candida albicans and Candida glabrata infections in an experimental model]]> https://www.researchpad.co/article/5c75ac7dd5eed0c484d088b2

Objective

The aims of this study were as follows. First, we sought to compare the in vitro susceptibility of liposomal amphotericin B (LAmB) and anidulafungin on Candida albicans and Candida glabrata biofilms growing on silicone discs. Second, we sought to compare the activity of LAmB versus anidulafungin for the treatment of experimental catheter-related C. albicans and C. glabrata infections with the antifungal lock technique in a rabbit model.

Methods

Two C. albicans and two C. glabrata clinical strains were used. The minimum biofilm eradication concentration for 90% eradication (MBEC90) values were determined after 48h of treatment with LAmB and anidulafungin. Confocal microscopy was used to visualize the morphology and viability of yeasts growing in biofilms. Central venous catheters were inserted into New Zealand rabbits, which were inoculated of each strain of C. albicans and C. glabrata. Then, catheters were treated for 48h with saline or with antifungal lock technique using either LAmB (5mg/mL) or anidulafungin (3.33mg/mL).

Results

In vitro: anidulafungin showed greater activity than LAmB against C. albicans and C. glabrata strains. For C. albicans: MBEC90 of anidulafungin versus LAmB: CA176, 0.03 vs. 128 mg/L; CA180, 0.5 vs. 64 mg/L. For C. glabrata: MBEC90 of anidulafungin versus LAmB: CG171, 0.5 vs. 64 mg/L; CG334, 2 vs. 32 mg/L. In vivo: for C. albicans species, LAmB and anidulafungin achieved significant reductions relative to growth control of log10 cfu recovered from the catheter tips (CA176: 3.6±0.3 log10 CFU, p≤0.0001; CA180: 3.8±0.1 log10 CFU, p≤0.01). For C. glabrata, anidulafungin lock therapy achieved significant reductions relative to the other treatments (CG171: 4.8 log10 CFU, p≤0.0001; CG334: 5.1 log10 CFU, p≤0.0001)

Conclusions

For the C. albicans strains, both LAmB and anidulafungin may be promising antifungal lock technique for long-term catheter-related infections; however, anidulafungin showed significantly higher activity than LAmB against the C. glabrata strains.

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<![CDATA[Exposure of Candida albicans β (1,3)-glucan is promoted by activation of the Cek1 pathway]]> https://www.researchpad.co/article/5c5ca280d5eed0c48441e4da

Candida albicans is among the most common causes of human fungal infections and is an important source of mortality. C. albicans is able to diminish its detection by innate immune cells through masking of β (1,3)-glucan in the inner cell wall with an outer layer of heavily glycosylated mannoproteins (mannan). However, mutations or drugs that disrupt the cell wall can lead to exposure of β (1,3)-glucan (unmasking) and enhanced detection by innate immune cells through receptors like Dectin-1, the C-type signaling lectin. Previously, our lab showed that the pathway for synthesizing the phospholipid phosphatidylserine (PS) plays a role in β (1,3)-glucan masking. The homozygous PS synthase knockout mutant, cho1Δ/Δ, exhibits increased exposure of β (1,3)-glucan. Several Mitogen Activated Protein Kinase (MAPK) pathways and their upstream Rho-type small GTPases are important for regulating cell wall biogenesis and remodeling. In the cho1Δ/Δ mutant, both the Cek1 and Mkc1 MAPKs are constitutively activated, and they act downstream of the small GTPases Cdc42 and Rho1, respectively. In addition, Cdc42 activity is up-regulated in cho1Δ/Δ. Thus, it was hypothesized that activation of Cdc42 or Rho1 and their downstream kinases cause unmasking. Disruption of MKC1 does not decrease unmasking in cho1Δ/Δ, and hyperactivation of Rho1 in wild-type cells increases unmasking and activation of both Cek1 and Mkc1. Moreover, independent hyperactivation of the MAP kinase kinase kinase Ste11 in wild-type cells leads to Cek1 activation and increased β (1,3)-glucan exposure. Thus, upregulation of the Cek1 MAPK pathway causes unmasking, and may be responsible for unmasking in cho1Δ/Δ.

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<![CDATA[Toxicity and oviposition deterrence of essential oils of Clinopodium nubigenum and Lavandula angustifolia against the myiasis-inducing blowfly Lucilia sericata]]> https://www.researchpad.co/article/5c76fe5ad5eed0c484e5b930

Cutaneous myiasis is a severe worldwide medical and veterinary issue. In this trial the essential oil (EO) of the Andean medicinal plant species Clinopodium nubigenum (Kunth) Kuntze was evaluated for its bioactivity against the myiasis-inducing blowfly Lucilia sericata (Meigen) (Diptera Calliphoridae) and compared with that of the well-known medicinal plant species Lavandula angustifolia Mill. The EOs were analysed and tested in laboratory for their oviposition deterrence and toxicity against L. sericata adults. The physiology of EO toxicity was evaluated by enzymatic inhibition tests. The antibacterial and antifungal properties of the EOs were tested as well. At 0.8 μL cm-2, both EOs completely deterred L. sericata oviposition up to 3 hours. After 24 h, the oviposition deterrence was still 82.7% for L. angustifolia and the 89.5% for C. nubigenum. The two EOs were also toxic to eggs and adults of L. sericata. By contact/fumigation, the EOs, the LC50 values against the eggs were 0.07 and 0.48 μL cm-2 while, by topical application on the adults, LD50 values were 0.278 and 0.393 μL per individual for C. nubigenum and L. angustifolia EOs, respectively. Inhibition of acetylcholine esterase of L. sericata by EOs (IC50 = 67.450 and 79.495 mg L-1 for C. nubigenum and L. angustifolia, respectively) suggested that the neural sites are targets of the EO toxicity. Finally, the observed antibacterial and antifungal properties of C. nubigenum and L. angustifolia EOs suggest that they could also help prevent secondary infections.

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<![CDATA[Dissection of the regulatory role for the N-terminal domain in Candida albicans protein phosphatase Z1]]> https://www.researchpad.co/article/5c5df31ad5eed0c484580d1d

The novel type, fungus specific protein phosphatase Z1 of the opportunistic pathogen, Candida albicans (CaPpz1) has several important physiological roles. It consists of a conserved C-terminal catalytic domain and a variable, intrinsically disordered, N-terminal regulatory domain. To test the function of these domains we modified the structure of CaPpz1 by in vitro mutagenesis. The two main domains were separated, four potential protein binding regions were deleted, and the myristoylation site as well as the active site of the enzyme was crippled by point mutations G2A and R262L, respectively. The in vitro phosphatase activity assay of the bacterially expressed recombinant proteins indicated that the N-terminal domain was inactive, while the C-terminal domain became highly active against myosin light chain substrate. The deletion of the N-terminal 1–16 amino acids and the G2A mutation significantly decreased the specific activity of the enzyme. Complementation of the ppz1 Saccharomyces cerevisiae deletion mutant strain with the different CaPpz1 forms demonstrated that the scission of the main domains, the two point mutations and the N-terminal 1–16 deletion rendered the phosphatase incompetent in the in vivo assays of LiCl tolerance and caffeine sensitivity. Thus our results confirmed the functional role of the N-terminal domain and highlighted the significance of the very N-terminal part of the protein in the regulation of CaPpz1.

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<![CDATA[Metagenomic quorum quenching enzymes affect biofilm formation of Candida albicans and Staphylococcus epidermidis]]> https://www.researchpad.co/article/5c58d622d5eed0c4840316f5

Biofilm formation in the clinical environment is of increasing concern since a significant part of human infections is associated, and caused by biofilm establishment of (opportunistic) pathogens, for instance Candida albicans and Staphylococcus epidermidis. The rapidly increasing number of antibiotic-resistant biofilms urgently requires the development of novel and effective strategies to prevent biofilm formation ideally targeting a wide range of infectious microorganisms. Both, synthesis of extracellular polymeric substances and quorum sensing are crucial for biofilm formation, and thus potential attractive targets to combat undesirable biofilms.We evaluated the ability of numerous recently identified metagenome-derived bacterial quorum quenching (QQ) proteins to inhibit biofilm formation of C. albicans and S. epidermidis. Here, proteins QQ-5 and QQ-7 interfered with the morphogenesis of C. albicans by inhibiting the yeast-to-hyphae transition, ultimately leading to impaired biofilm formation. Moreover, QQ5 and QQ-7 inhibited biofilm formation of S. epidermidis; in case of QQ7 most likely due to induced expression of the icaR gene encoding the repressor for polysaccharide intercellular adhesin (PIA) synthesis, the main determinant for staphylococcal biofilm formation. Our results indicate that QQ-5 and QQ-7 are attractive potential anti-biofilm agents in the prevention and treatment of C. albicans and S. epidermidis mono-species biofilms, and potentially promising anti-biofilm drugs in also combating multi-species infections.

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<![CDATA[Vacuolar proton-translocating ATPase is required for antifungal resistance and virulence of Candida glabrata]]> https://www.researchpad.co/article/5c52186dd5eed0c4847982ae

Vacuolar proton-translocating ATPase (V-ATPase) is located in fungal vacuolar membranes. It is involved in multiple cellular processes, including the maintenance of intracellular ion homeostasis by maintaining acidic pH within the cell. The importance of V-ATPase in virulence has been demonstrated in several pathogenic fungi, including Candida albicans. However, it remains to be determined in the clinically important fungal pathogen Candida glabrata. Increasing multidrug resistance of C. glabrata is becoming a critical issue in the clinical setting. In the current study, we demonstrated that the plecomacrolide V-ATPase inhibitor bafilomycin B1 exerts a synergistic effect with azole antifungal agents, including fluconazole and voriconazole, against a C. glabrata wild-type strain. Furthermore, the deletion of the VPH2 gene encoding an assembly factor of V-ATPase was sufficient to interfere with V-ATPase function in C. glabrata, resulting in impaired pH homeostasis in the vacuole and increased sensitivity to a variety of environmental stresses, such as alkaline conditions (pH 7.4), ion stress (Na+, Ca2+, Mn2+, and Zn2+ stress), exposure to the calcineurin inhibitor FK506 and antifungal agents (azoles and amphotericin B), and iron limitation. In addition, virulence of C. glabrata Δvph2 mutant in a mouse model of disseminated candidiasis was reduced in comparison with that of the wild-type and VPH2-reconstituted strains. These findings support the notion that V-ATPase is a potential attractive target for the development of effective antifungal strategies.

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<![CDATA[Analysis of antifungal resistance genes in Candida albicans and Candida glabrata using next generation sequencing]]> https://www.researchpad.co/article/5c40f7d3d5eed0c484386a4b

Introduction/Objectives

An increase in antifungal resistant Candida strains has been reported in recent years. The aim of this study was to detect mutations in resistance genes of azole-resistant, echinocandin-resistant or multi-resistant strains using next generation sequencing technology, which allows the analysis of multiple resistance mechanisms in a high throughput setting.

Methods

Forty clinical Candida isolates (16 C. albicans and 24 C. glabrata strains) with MICs for azoles and echinocandins above the clinical EUCAST breakpoint were examined. The genes ERG11, ERG3, TAC1 and GSC1 (FKS1) in C. albicans, as well as ERG11, CgPDR1, FKS1 and FKS2 in C. glabrata were sequenced.

Results

Fifty-four different missense mutations were identified, 13 of which have not been reported before. All nine echinocandin-resistant Candida isolates showed mutations in the hot spot (HS) regions of FKS1, FKS2 or GSC1. In ERG3 two homozygous premature stop codons were identified in two highly azole-resistant and moderately echinocandin-resistant C. albicans strains. Seven point mutations in ERG11 were determined in azole-resistant C. albicans whereas in azole-resistant C. glabrata, no ERG11 mutations were detected. In 10 out of 13 azole-resistant C. glabrata, 12 different potential gain-of-function mutations in the transcription factor CgPDR1 were verified, which are associated with an overexpression of the efflux pumps CDR1/2.

Conclusion

This study showed that next generation sequencing allows the thorough investigation of a large number of isolates more cost efficient and faster than conventional Sanger sequencing. Targeting different resistance genes and a large sample size of highly resistant strains allows a better determination of the relevance of the different mutations, and to differentiate between causal mutations and polymorphisms.

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<![CDATA[Plasma membrane architecture protects Candida albicans from killing by copper]]> https://www.researchpad.co/article/5c424382d5eed0c4845e0414

The ability to resist copper toxicity is important for microbial pathogens to survive attack by innate immune cells. A sur7Δ mutant of the fungal pathogen Candida albicans exhibits decreased virulence that correlates with increased sensitivity to copper, as well as defects in other stress responses and morphogenesis. Previous studies indicated that copper kills sur7Δ cells by a mechanism distinct from the known resistance pathways involving the Crp1 copper exporter or the Cup1 metallothionein. Since Sur7 resides in punctate plasma membrane domains known as MCC/eisosomes, we examined overexpression of SUR7 and found that it rescued the copper sensitivity of a mutant that fails to form MCC/eisosomes (pil1Δ lsp1Δ), indicating that these domains act to facilitate Sur7 function. Genetic screening identified new copper-sensitive mutants, the strongest of which were similar to sur7Δ in having altered plasma membranes due to defects in membrane trafficking, cortical actin, and morphogenesis (rvs161Δ, rvs167Δ, and arp2Δ arp3Δ). Consistent with the mutants having altered plasma membrane organization, they were all more readily permeabilized by copper, which is known to bind phosphatidylserine and phosphatidylethanolamine and cause membrane damage. Although these phospholipids are normally localized to the intracellular leaflet of the plasma membrane, their exposure on the surface of the copper-sensitive mutants was indicated by increased susceptibility to membrane damaging agents that bind to these phospholipids. Increased copper sensitivity was also detected for a drs2Δ mutant, which lacks a phospholipid flippase that is involved in maintaining phospholipid asymmetry. Copper binds phosphatidylserine with very high affinity, and deleting CHO1 to prevent phosphatidylserine synthesis rescued the copper sensitivity of sur7Δ cells, confirming a major role for phosphatidylserine in copper sensitivity. These results highlight how proper plasma membrane architecture protects fungal pathogens from copper and attack by the immune system, thereby opening up new avenues for therapeutic intervention.

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<![CDATA[Subgingival areas as potential reservoirs of different Candida spp in type 2 diabetes patients and healthy subjects]]> https://www.researchpad.co/article/5c40f77fd5eed0c48438628b

Objectives

The aim of this cross-sectional observational study was to compare the prevalence of different oral Candida spp. in patients with Type 2 Diabetes and chronic periodontitis in two oral sites: dorsal surface of the tongue and subgingival area. In order to determine subgingival areas as potential reservoirs of yeasts, this study aimed to find differences in the yeasts’ detection between the dorsum of the tongue, as the oral site most commonly inhabited with microorganisms, and subgingival samples. Additionally, potential predictors for the yeasts prevalence were determined.

Material and methods

Subjects (N = 146) were divided into four groups: group A- healthy individuals without periodontitis, group B- healthy individuals with chronic periodontitis, group C- Type 2 Diabetes patients with good glycoregulation and Chronic periodontitis and group D- Type 2 Diabetes patients with poor glycoregulation and Chronic periodontitis. Samples were obtained from the tongue by swabbing. Subgingival plaque samples were taken by paper points and periodontal curette. Isolation and identification of different Candida spp. was done using ChromAgar medium. In addition, germ-tube production and carbohydrate assimilation tests were performed.

Results

The prevalence of Candida spp. was higher in diabetics with poor glycoregulation. The most frequently isolated species was Candida albicans followed by Candida glabrata and Candida tropicalis. In 15.6% of cases, Candida spp. was present in the subgingival area while absent on the tongue. Multivariate regression model showed that HbA1c was Candida spp. predictor for both locations.

Conclusions

Our results confirmed that there are Candida spp. carriers among subjects with clinically healthy oral mucosa. Also, this study identified subgingival areas as potential reservoirs of these pathogenic species. Glycoregulation has been recognized as a positive predictor factor of Candida spp.

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<![CDATA[Oral Candida administration in a Clostridium difficile mouse model worsens disease severity but is attenuated by Bifidobacterium]]> https://www.researchpad.co/article/5c478c89d5eed0c484bd2ed3

Gut fungi may influence the course of Clostridium difficile infection either positively or negatively for the host. Fungi are not prominent in the mouse gut, and C. albicans, the major human gastrointestinal commensal yeast, is in low abundance or absent in mice. Bifidobacterium is one of the probiotics that may attenuate the severity of C. difficile infection. Inflammatory synergy between C. albicans and C. difficile, in gut, may provide a state that more closely resembles human infection and be more suitable for testing probiotic effects. We performed fecal mycobiota analysis and administered C. albicans at 1 day prior to C. difficile dosing. Fecal eukaryotic 18S rDNA analysis demonstrated the presence of Ascomycota, specifically, Candida spp., after oral antibiotics, despite negative fecal fungal culture. C. albicans administration enhanced the severity of the C. difficile infection model as determined by mortality rate, weight loss, gut leakage (FITC-dextran assay), and serum and intestinal tissue cytokines. This occurred without increased fecal C. difficile or bacteremia, in comparison with C. difficile gavage alone. Candida lysate with C. difficile increased IL-8 production from HT-29 and Caco-2 human intestinal epithelial cell-lines. Bifidobacterium attenuated the disease severity of the C. difficile plus Candida model. The reduced severity was associated with decreased Candida burdens in feces. In conclusion, gut C. albicans worsened C. difficile infection, possibly through exacerbation of inflammation. Hence, a mouse model of Clostridium difficile infection with C. albicans present in the gut may better model the human patient condition. Gut fungal mycobiome investigation in patients with C. difficile is warranted and may suggest therapeutic targets.

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<![CDATA[Diversification of DNA binding specificities enabled SREBP transcription regulators to expand the repertoire of cellular functions that they govern in fungi]]> https://www.researchpad.co/article/5c33c3afd5eed0c48459e8a0

The Sterol Regulatory Element Binding Proteins (SREBPs) are basic-helix-loop-helix transcription regulators that control the expression of sterol biosynthesis genes in higher eukaryotes and some fungi. Surprisingly, SREBPs do not regulate sterol biosynthesis in the ascomycete yeasts (Saccharomycotina) as this role was handed off to an unrelated transcription regulator in this clade. The SREBPs, nonetheless, expanded in fungi such as the ascomycete yeasts Candida spp., raising questions about their role and evolution in these organisms. Here we report that the fungal SREBPs diversified their DNA binding preferences concomitantly with an expansion in function. We establish that several branches of fungal SREBPs preferentially bind non-palindromic DNA sequences, in contrast to the palindromic DNA motifs recognized by most basic-helix-loop-helix proteins (including SREBPs) in higher eukaryotes. Reconstruction and biochemical characterization of the likely ancestor protein suggest that an intrinsic DNA binding promiscuity in the family was resolved by alternative mechanisms in different branches of fungal SREBPs. Furthermore, we show that two SREBPs in the human commensal yeast Candida albicans drive a transcriptional cascade that inhibits a morphological switch under anaerobic conditions. Preventing this morphological transition enhances C. albicans colonization of the mammalian intestine, the fungus’ natural niche. Thus, our results illustrate how diversification in DNA binding preferences enabled the functional expansion of a family of eukaryotic transcription regulators.

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<![CDATA[Functional divergence of a global regulatory complex governing fungal filamentation]]> https://www.researchpad.co/article/5c3d00ebd5eed0c484036b17

Morphogenetic transitions are prevalent in the fungal kingdom. For a leading human fungal pathogen, Candida albicans, the capacity to transition between yeast and filaments is key for virulence. For the model yeast Saccharomyces cerevisiae, filamentation enables nutrient acquisition. A recent functional genomic screen in S. cerevisiae identified Mfg1 as a regulator of morphogenesis that acts in complex with Flo8 and Mss11 to mediate transcriptional responses crucial for filamentation. In C. albicans, Mfg1 also interacts physically with Flo8 and Mss11 and is critical for filamentation in response to diverse cues, but the mechanisms through which it regulates morphogenesis remained elusive. Here, we explored the consequences of perturbation of Mfg1, Flo8, and Mss11 on C. albicans morphogenesis, and identified functional divergence of complex members. We observed that C. albicans Mss11 was dispensable for filamentation, and that overexpression of FLO8 caused constitutive filamentation even in the absence of Mfg1. Harnessing transcriptional profiling and chromatin immunoprecipitation coupled to microarray analysis, we identified divergence between transcriptional targets of Flo8 and Mfg1 in C. albicans. We also established that Flo8 and Mfg1 cooperatively bind to promoters of key regulators of filamentation, including TEC1, for which overexpression was sufficient to restore filamentation in the absence of Flo8 or Mfg1. To further explore the circuitry through which Mfg1 regulates morphogenesis, we employed a novel strategy to select for mutations that restore filamentation in the absence of Mfg1. Whole genome sequencing of filamentation-competent mutants revealed chromosome 6 amplification as a conserved adaptive mechanism. A key determinant of the chromosome 6 amplification is FLO8, as deletion of one allele blocked morphogenesis, and chromosome 6 was not amplified in evolved lineages for which FLO8 was re-located to a different chromosome. Thus, this work highlights rewiring of key morphogenetic regulators over evolutionary time and aneuploidy as an adaptive mechanism driving fungal morphogenesis.

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<![CDATA[Antifungal activity of well-defined chito-oligosaccharide preparations against medically relevant yeasts]]> https://www.researchpad.co/article/5c3e4fd7d5eed0c484d79991

Due to their antifungal activity, chitosan and its derivatives have potential to be used for treating yeast infections in humans. However, to be considered for use in human medicine, it is necessary to control and know the chemical composition of the compound, which is not always the case for polymeric chitosans. Here, we analyze the antifungal activity of a soluble and well-defined chito-oligosaccharide (CHOS) with an average polymerization degree (DPn) of 32 and fraction of acetylation (FA) of 0.15 (C32) on 52 medically relevant yeast strains. Minimal inhibitory concentrations (MIC) varied widely among yeast species, strains and isolates (from > 5000 to < 9.77 μg mL-1) and inhibition patterns showed a time- and dose-dependencies. The antifungal activity was predominantly fungicidal and was inversely proportional to the pH, being maximal at pH 4.5, the lowest tested pH. Furthermore, antifungal effects of CHOS fractions with varying average molecular weight indicated that those fractions with an intermediate degree of polymerization, i.e. DP 31 and 54, had the strongest inhibitory effects. Confocal imaging showed that C32 adsorbs to the cell surface, with subsequent cell disruption and accumulation of C32 in the cytoplasm. Thus, C32 has potential to be used as a therapy for fungal infections.

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<![CDATA[Therapeutic potential of thymoquinone liposomes against the systemic infection of Candida albicans in diabetic mice]]> https://www.researchpad.co/article/5c2e7fa1d5eed0c48451a3f0

The present study was aimed to develop a liposomal formulation of thymoquinone (Lip-TQ) to treat Candida albicans infection in diabetic mice. Streptozotocin (STZ) was injected to induce hyperglycemia and on day 3 post STZ administration, mice were intravenously infected with C. albicans. Various doses (2, 5 and 10 mg/kg) of Free or Lip-TQ were administered in C. albicans infected diabetic mice. The effect of Lip-TQ was also determined on the organ indices, liver and kidney function parameters. Lip-TQ at a dose of 10 mg/kg significantly reduced the level of the blood glucose and alleviated the systemic C. albicans infection in diabetic mice. C. albicans infected diabetic mice treated with Lip-TQ at a dose of 10 mg/kg showed the survival rate of 70% as compared to that of 20% in the group treated with free TQ. The treatment with Lip-TQ resulted in the recovery of the organ indices, liver inflammation, kidney functioning and pancreas regeneration in diabetic mice. Moreover, TQ formulations also showed the direct therapeutic effect against candidiasis in the untreated or metformin-treated diabetic mice. Therefore, the findings of the present study support the use of Lip-TQ in the treatment of candidiasis in the diabetic patients.

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<![CDATA[TAMMiCol: Tool for analysis of the morphology of microbial colonies]]> https://www.researchpad.co/article/5c0ed741d5eed0c484f13db7

Many microbes are studied by examining colony morphology via two-dimensional top-down images. The quantification of such images typically requires each pixel to be labelled as belonging to either the colony or background, producing a binary image. While this may be achieved manually for a single colony, this process is infeasible for large datasets containing thousands of images. The software Tool for Analysis of the Morphology of Microbial Colonies (TAMMiCol) has been developed to efficiently and automatically convert colony images to binary. TAMMiCol exploits the structure of the images to choose a thresholding tolerance and produce a binary image of the colony. The images produced are shown to compare favourably with images processed manually, while TAMMiCol is shown to outperform standard segmentation methods. Multiple images may be imported together for batch processing, while the binary data may be exported as a CSV or MATLAB MAT file for quantification, or analysed using statistics built into the software. Using the in-built statistics, it is found that images produced by TAMMiCol yield values close to those computed from binary images processed manually. Analysis of a new large dataset using TAMMiCol shows that colonies of Saccharomyces cerevisiae reach a maximum level of filamentous growth once the concentration of ammonium sulfate is reduced to 200 μM. TAMMiCol is accessed through a graphical user interface, making it easy to use for those without specialist knowledge of image processing, statistical methods or coding.

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<![CDATA[Candida–streptococcal interactions in biofilm-associated oral diseases]]> https://www.researchpad.co/article/5c1c0b11d5eed0c484427391 ]]> <![CDATA[TPP riboswitch-dependent regulation of an ancient thiamin transporter in Candida]]> https://www.researchpad.co/article/5b28b39a463d7e126303d2ab

Riboswitches are non-coding RNA molecules that regulate gene expression by binding to specific ligands. They are primarily found in bacteria. However, one riboswitch type, the thiamin pyrophosphate (TPP) riboswitch, has also been described in some plants, marine protists and fungi. We find that riboswitches are widespread in the budding yeasts (Saccharomycotina), and they are most common in homologs of DUR31, originally described as a spermidine transporter. We show that DUR31 (an ortholog of N. crassa gene NCU01977) encodes a thiamin transporter in Candida species. Using an RFP/riboswitch expression system, we show that the functional elements of the riboswitch are contained within the native intron of DUR31 from Candida parapsilosis, and that the riboswitch regulates splicing in a thiamin-dependent manner when RFP is constitutively expressed. The DUR31 gene has been lost from Saccharomyces, and may have been displaced by an alternative thiamin transporter. TPP riboswitches are also present in other putative transporters in yeasts and filamentous fungi. However, they are rare in thiamin biosynthesis genes THI4 and THI5 in the Saccharomycotina, and have been lost from all genes in the sequenced species in the family Saccharomycetaceae, including S. cerevisiae.

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