Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen

The survival of all microbes depends upon their ability to respond to environmental challenges. To establish infection, pathogens such as Candida albicans must mount effective stress responses to counter host defences while adapting to dynamic changes in nutrient status within host niches. Studies of C. albicans stress adaptation have generally been performed on glucose‐grown cells, leaving the effects of alternative carbon sources upon stress resistance largely unexplored. We have shown that growth on alternative carbon sources, such as lactate, strongly influence the resistance of C. albicans to antifungal drugs, osmotic and cell wall stresses. Similar trends were observed in clinical isolates and other pathogenic Candida species. The increased stress resistance of C. albicans was not dependent on key stress (Hog1) and cell integrity (Mkc1) signalling pathways. Instead, increased stress resistance was promoted by major changes in the architecture and biophysical properties of the cell wall. Glucose‐ and lactate‐grown cells displayed significant differences in cell wall mass, ultrastructure, elasticity and adhesion. Changes in carbon source also altered the virulence of C. albicans in models of systemic candidiasis and vaginitis, confirming the importance of alternative carbon sources within host niches during C. albicans infections.     

Impact of Genetic Background on Allele Selection in a Highly Mutable Candida albicans Gene,PNG2

In many microbes rapid mutation of highly mutable contingency genes continually replenishes a pool of variant alleles from which the most suitable are selected, assisting in rapid adaptation and evasion of the immune response. In some contingency genes mutability is achieved through DNA repeats within the coding region. The fungal human pathogen Candida albicans has 2600 repeat-containing ORFs. For those investigated (ALS genes, HYR1, HYR2, CEK1, RLM1) many protein variants with differing amino acid repeat regions exist, as expected for contingency genes. However, specific alleles dominate in different clades, which is unexpected if allele variation is used for short-term adaptation. Generation of new alleles of repeat-containing C. albicans ORFs has never been observed directly. Here we present evidence for restrictions on the emergence of new alleles in a highly mutable C. albicans repeat-containing ORF, PNG2, encoding a putative secreted or cell surface glycoamidase. In laboratory cultures new PNG2 alleles arose at a rate of 2.8×10⁻⁵ (confidence interval 3.3×10⁻⁶−9. 9×10⁻⁵) per cell per division, comparable to rates measured for contingency genes. Among 80 clinical isolates 17 alleles of different length and 23 allele combinations were distinguishable; sequence differences between repeat regions of identical size suggest the existence of 36 protein variants. Specific allele combinations predominated in different genetic backgrounds, as defined by DNA fingerprinting and multilocus sequence typing. Given the PNG2 mutation rate, this is unexpected, unless in different genetic backgrounds selection favors different alleles. Specific alleles or allele combinations were not preferentially associated with C. albicans isolates from particular body sites or geographical regions. Our results suggest that the mutability of PNG2 is not used for short-term adaptation or evasion of the immune system. Nevertheless the large number of alleles observed indicates that mutability of PNG2 may assist C. albicans strains from different genetic backgrounds optimize their interaction with the host in the long term.     

An Extensive Circuitry for Cell Wall Regulation in Candida albicans

Protein kinases play key roles in signaling and response to changes in the external environment. The ability of Candida albicans to quickly sense and respond to changes in its environment is key to its survival in the human host. Our guiding hypothesis was that creating and screening a set of protein kinase mutant strains would reveal signaling pathways that mediate stress response in C. albicans. A library of protein kinase mutant strains was created and screened for sensitivity to a variety of stresses. For the majority of stresses tested, stress response was largely conserved between C. albicans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. However, we identified eight protein kinases whose roles in cell wall regulation (CWR) were not expected from functions of their orthologs in the model fungi Saccharomyces cerevisiae and Schizosaccharomyces pombe. Analysis of the conserved roles of these protein kinases indicates that establishment of cell polarity is critical for CWR. In addition, we found that septins, crucial to budding, are both important for surviving and are mislocalized by cell wall stress. Our study shows an expanded role for protein kinase signaling in C. albicans cell wall integrity. Our studies suggest that in some cases, this expansion represents a greater importance for certain pathways in cell wall biogenesis. In other cases, it appears that signaling pathways have been rewired for a cell wall integrity response.     

A comparative study on cell wall antigens and cell surface hydrophobicity in clinical isolates ofCandida albicans

Characterization of common cell surface-bound antigens inCandida albicans strains, particularly those expressed in the walls of mycelial cells might be useful in the diagnosis of systemic candidiasis. Hence, antigenic similarities among wall proteins and mannoproteins fromC. albicans clinical serotype A and B isolates, were studied using polyclonal (mPAbs) and monoclonal (MAb 4C12) antibodies raised against wall antigens from the mycelial form of a commonC. albicans serotype A laboratory strain (ATCC 26555). Zymolyase digestion of walls isolated from cells of the different strains studied grown at 37°C (germination conditions), released, in all cases, numerous protein and mannoprotein components larger than 100 kDa, along with a 33–34 kDa species. The pattern of major antigens exhibiting reactivity towards the mPAbs preparation was basically similar for all the serotype A and B isolates, though minor strain-specific bands were also observed. The immunodeterminant recognized by MAb 4C12 was found to be absent or present in very low amounts inC. albicans isolates other than the ATCC 26555 strain, yet high molecular weight species similar in size (e.g., 260 kDa) to the wall antigen against which MAb 4C12 was raised, were observed, particularly in wall digests from serotype A strains. Cell surface hydrophobicity, an apparently important virulence factor inC. albicans, of the cell population of each serotype B strain was lower than that of the corresponding serotype A counterparts, which is possibly due to the fact that the former strains exhibited a reduced ability to form mycelial filaments under the experimental conditions used.Abbreviations CSH cell surface hydrophobicity - IIF indirect immunofluorescence     

The plant defensin RsAFP2 induces cell wall stress, septin mislocalization and accumulation of ceramides in Candida albicans

The antifungal plant defensin RsAFP2 isolated from radish interacts with fungal glucosylceramides and induces apoptosis in Candida albicans. To further unravel the mechanism of RsAFP2 antifungal action and tolerance mechanisms, we screened a library of 2868 heterozygous C. albicans deletion mutants and identified 30 RsAFP2‐hypersensitive mutants. The most prominent group of RsAFP2 tolerance genes was involved in cell wall integrity and hyphal growth/septin ring formation. Consistent with these genetic data, we demonstrated that RsAFP2 interacts with the cell wall of C. albicans, which also contains glucosylceramides, and activates the cell wall integrity pathway. Moreover, we found that RsAFP2 induces mislocalization of septins and blocks the yeast‐to‐hypha transition in C. albicans. Increased ceramide levels have previously been shown to result in apoptosis and septin mislocalization. Therefore, ceramide levels in C. albicans membranes were analysed following RsAFP2 treatment and, as expected, increased accumulation of phytoC24‐ceramides in membranes of RsAFP2‐treated C. albicans cells was detected. This is the first report on the interaction of a plant defensin with glucosylceramides in the fungal cell wall, causing cell wall stress, and on the effects of a defensin on septin localization and ceramide accumulation.     

Molecular Analysis of <Emphasis Type="Italic">Candida albicans</Emphasis> Isolates from Clinical Specimens

The aim of this study was to genotype Candida albicans strains isolated from various clinical specimens by using CA-INT-R and CA-INT-L primer pairs designed to span the region that includes the site of the transposable group-1 intron in the 25S rRNA gene. A total of 194 C. albicans isolates (28 invasive and 166 noninvasive) were genotyped. The frequencies of genotypes A, B, C and D were found as 51.0, 29.4, 19.1 and 0.5%, respectively. Statistically significant difference was determined between frequency of genotype distribution between invasive and noninvasive isolates (P < 0.001). Genotype C was more prevalent among invasive isolates while genotype A was in noninvasive ones. Furthermore, six different subtypes were determined among genotype A C. albicans isolates by restriction endonuclease analysis using a previously constructed differentiation scheme consisting of HaeIII and MspI digestions. This study demonstrated the genetic diversity of clinical isolates of C. albicans in our hospital.     

Microsatellite DNA Identification and Genotyping of <Emphasis Type="Italic">Candida albicans</Emphasis> from Lebanese Clinical Isolates

The present study involves collecting 125 isolates labeled as C. albicans from five different Lebanese hospitals and utilizing the microsatellite genotyping test to determine the following: first, the accuracy of hospital identification by comparing microsatellite results to hospital results. Second, the frequency and genotypes of infectious strains present relative to tissue and hospital location- a possible indicator of nosocomial infection, and third, a possible relationship between lack of microsatellite heterozygosity to azole resistance. Our results showed that the error in hospital identification varied from 2 to 33%, averaging at 7%, with the highest identification error in stool. Misidentified isolates were mainly Candida tropicalis followed by C. glabrata and C. parapsilosis. Strains with similar genotypes were also found to occur within certain hospitals suggesting the possibility of nosocomial infection. Finally, a relationship between lack of heterozygosity and azole resistance was observed since nine out of 10 homozygous isolates sharing a common allele with a heterozygote strain were sensitive to all drugs tested, whereas the homozygous genotype was resistant to at least one drug.     

Acid proteinase,phospholipase, and biofilm production of <Emphasis Type="Italic">Candida</Emphasis> species isolated from blood cultures

Three virulence factors comprising proteinase, phospholipase, and biofilm among 68 Candida albicans and 31 non-albicans Candida strains (11 C. tropicalis, 8 C. parapsilosis, 6 C. glabrata, 4 C. guillermondii, 2 C. krusei) isolated from blood cultures were analyzed. In total, 61 (89.7%) C. albicans strains were detected as proteinase positive whereas eight (25.8%) non-albicans Candida strains were proteinase positive (P < 0.05). Phospholipase production was detected in 41 (60.3%) C. albicans strains. All non-albicans Candida strains were phospholipase negative. Biofilm production was determined by both visual and spectrophotometric methods. Eight (11.8%) of C. albicans strains and 13 (41.93%) of 31 non-albicans Candida strains were biofilm positive with two of the methods (P < 0.05). According to our results, we may suggest that detection of hydrolytic enzyme and biofilm production abilities of the Candida isolates in clinical mycology laboratories may warn the clinican for a possible hematogenous infection.     

Factors influencing the expression in vitro of Candida albicans stress mannoproteins reactive with salivary secretory IgA

We have examined the influence of subinhibitory concentrations of several antifungals, the different glucose and ammonium sulphate concentrations in the culture medium as well as the strain variability on the expression in vitro of stress mannoproteins reactive with salivary sIgA in C. albicans and other Candida spp isolates. Irrespective of the conditions used, no reactivity with salivary sIgA was observed in yeast cells grown at 25 °C. However, when grown at 37 °C, all of the 10 C. albicans strains, but only 9 out 28 non-C.albicans isolates studied showed reactivity with salivary sIgA. Cells grown at 37 °C in medium containing maximum concentrations of glucose and ammonium sulphate expressed the antigens reactive with sIgA during longer periods of time than the cells grown in medium with minimal concentrations of the same compounds. The regulatory role showed by the concentration of glucose and ammonium sulphate on the antigenic expression was subordinated, nevertheless, to the most important factor, the temperature of incubation. Only isolates showing low susceptibility expressed the antigens reactive with sIgA under the influence of subinhibitory concentration of antifungals. However, induced resistance to one of the antifungals tested (5 fluorocytosine) allowed the antigenic expression at elevated subinhibitory concentrations even in previous susceptible strains. In conclusion, in addition to the temperature, factors such as characteristics of the strain, the concentration of glucose and ammonium sulphate in the culture medium and the resistance to antifungals played a role on the expression of C. albicans antigens reactive with sIgA, which could be of clinical relevance in the course of infection.This revised version was published online in October 2005 with corrections to the Cover Date.     

Use of CHROMagar in the Differentiation of Common Species of <Emphasis Type="Italic">Candida</Emphasis>

CHROMagar has been reported to be useful for the rapid and accurate identification of Candida species. We tested 135 isolates of Candida species isolated from oropharyngeal candidiasis in HIV patients and found that it was useful in the presumptive identification of Candida albicans and Candida krusei. Occasional strains of C. tropicalis produced colonies with a greenish tinge making it difficult to differentiate from C. albicans.     

The synergy of honokiol and fluconazole against clinical isolates of azole‐resistant Candida albicans

Aims: To evaluate the interaction of fluconazole (FLC) and honokiol (HNK) in vitro and vivo against azole‐resistant (azole‐R) clinical isolates of Candida albicans. Methods and Results: A checkerboard microdilution method was used to study the in vitro interaction of FLC and HNK in 24 azole‐R clinical isolates of C. albicans. In vivo antifungal activity was performed to further analyse the interaction between FLC and HNK. In the in vitro study, synergism was observed in all 24 FLC‐resistant strains tested as determined by fractional inhibitory concentration index (FICI), and in 22 strains by ΔE models. No antagonistic activity was observed in any of the strains tested. These positive interactions were also confirmed by using the time‐killing test for the selected strain C. albicans YL371, which shows strong susceptible to the combination of HNK and FLC. In the in vivo study, the mice with candidiasis were treated successfully by a combination therapy of HNK with FLC, the results showed a decrease of the colony forming unit in infected and treated animals compared to the controls, at the conditions of the treatment used in this study. Conclusions: Synergistic activity of HNK and FLC against clinical isolates of FLC‐resistant C. albicans was observed in vitro and in vivo. Significance and Impact of the Study: This report might provide a potential therapeutic method to overcome the problem of drug‐resistance in C. albicans.     

Genetic diversity analysis of a world-wide collection of <Emphasis Type="Italic">Ascochyta rabiei</Emphasis> isolates using sequence tagged microsatellite markers

A previously characterized compound microsatellite locus ARMS1, containing penta- and decameric repeat units, has been reported to reveal genetic diversity in Ascochyta rabiei (Pass.) Labr. isolates. Therefore, 37 isolates of Ascochyta rabiei collected from different states of India and 38 isolates from fifteen other countries in the world were examined for their diversity at this locus. Twenty-six alleles on the basis of size (228--451 base pairs) were detected in the world isolates examined, while 15 alleles (287--418 base pairs) were observed in isolates from the Indian subcontinent. To the best of our knowledge, this study is the first to demonstrate diversity in representative Ascochyta rabiei isolates from different parts of the world at the ARMS1 locus.     

Identification of <Emphasis Type="Italic">Candida</Emphasis> Species Isolated from Bovine Mastitic Milk and Their In Vitro Hemolytic Activity in Western Turkey

In this study, identification of 207 Candida isolates, previously isolated from mastitic bovine quarter milk samples at the level of genus, was made using API 20 C AUX system. The most frequently isolated species were Candida krusei (34.8%), followed by Candida rugosa (16.4%), Candida kefyr (12.6%), Candida albicans (10.1%), and Candida tropicalis (9.2%). Less common isolates were Candida zeylanoides (5.8%), Candida parapsilosis (4.3%), Candida guilliermondii (3.4%), Candida famata (1.9%), and Candida glabrata (1.5%). Additionally, in vitro hemolytic activity of all Candida strains were also examined in the present study. C. krusei (72 isolates), C. kefyr (26), C. albicans (21), C. tropicalis (19), C. zeylanoides (12), and C. glabrata (3) demonstrated both alpha and beta hemolysis at 48-h postinoculation. Only alpha hemolysis was detected in C. rugosa (34), C. guilliermondii (7), and C. famata (4), while C. parapsilosis (9) did not show any hemolytic activity after incubation for 72 h. Statistically significant difference (P < 0.001) was determined between the beta-hemolytic activities of Candida strains. The hemolytic activities of C. zeylanoides, C. albicans and C. kefyr were higher than other strains. This is the first study to describe variable hemolysis types exhibited by different Candida strains isolated from bovine mastitic milk in Turkey.     

Evaluation of the V404I and V509M amino acid substitutions of <Emphasis Type="Italic">ERG11</Emphasis> gene in <Emphasis Type="Italic">Candida albicans</Emphasis> isolates by pyrosequencing

The molecular mechanisms underlying fluconazole resistance in C. albicans involve mutations and the overexpression of the ERG11 gene and membrane transport proteins. We examined the relationship between the reduced fluconazole susceptibility of C. albicans and mutations of V404I and V509M in the ERG11 gene in 182 C. albicans clinical isolates using the Pyrosequencing™ method. DNAs from these clinical isolates with different levels of in-vitro fluconazole susceptibility — one resistant, five susceptible dose-dependent (SDD), four trailer, and 172 susceptible — were analyzed. None of the fluconazole-susceptible, SDD, trailer or resistant isolates had mutations of V404I or V509M. Our results showed that no correlation can be found between the V404I or V509M mutation and fluconazole susceptibility in C. albicans.     

A proteomic approach to understanding the development of multidrug-resistant<Emphasis Type="Italic"> Candida albicans</Emphasis> strains

Resistance of the pathogenic yeast Candida albicans to the antifungal agent fluconazole is often caused by the overexpression of genes that encode multidrug efflux pumps (CDR1, CDR2, or MDR1). We have undertaken a proteomic approach to gain further insight into the regulatory network controlling efflux pump expression and drug resistance in C. albicans. Three pairs of matched fluconazole-susceptible and resistant clinical C. albicans isolates, in which drug resistance correlated with stable activation of MDR1 or CDR1/2, were analyzed for differences in their protein expression profiles. In two independent, MDR1-overexpressing, strains, additional up-regulated proteins were identified, which are encoded by the YPR127 gene and several members of the IFD (YPL088) gene family. All are putative aldo-keto reductases of unknown function. These proteins were not up-regulated in a fluconazole-resistant strain that overexpressed CDR1 and CDR2 but not MDR1, indicating that expression of the various efflux pumps of C. albicans is controlled by different regulatory networks. To investigate the possible role of YPR127 in the resistance phenotype of the clinical isolates, we constitutively overexpressed the gene in a C. albicans laboratory strain. In addition, the gene was deleted in a C. albicans laboratory strain and in one of the drug-resistant clinical isolates in which it was overexpressed. Neither forced overexpression nor deletion of YPR127 affected the susceptibility of the strains to drugs and other toxic substances, suggesting that the regulatory networks which control the expression of efflux pumps in C. albicans also control genes involved in cellular functions not related to drug resistance.Communicated by D. Y. Thomas     

Aurora kinase Ipl1 facilitates bilobed distribution of clustered kinetochores to ensure error‐free chromosome segregation in Candida albicans

Candida albicans, an ascomycete, has an ability to switch to diverse morphological forms. While C. albicans is predominatly diploid, it can tolerate aneuploidy as a survival strategy under stress. Aurora kinase B homolog Ipl1 is a critical ploidy regulator that controls microtubule dynamics and chromosome segregation in Saccharomyces cerevisiae. In this study, we show that Ipl1 in C. albicans has a longer activation loop than that of the well‐studied ascomycete S. cerevisiae. Ipl1 localizes to the kinetochores during the G1/S phase and associates with the spindle during mitosis. Ipl1 regulates cell morphogenesis and is required for cell viability. Ipl1 monitors microtubule dynamics which is mediated by separation of spindle pole bodies. While Ipl1 is dispensable for maintaining structural integrity and clustering of kinetochores in C. albicans, it is required for the maintenance of bilobed distribution of clustered kinetochores along the mitotic spindle. Depletion of Ipl1 results in erroneous kinetochore‐microtubule attachments leading to aneuploidy due to which the organism can survive better in the presence of fluconazole. Taking together, we suggest that Ipl1 spatiotemporally ensures bilobed kinetochore distribution to facilitate bipolar spindle assembly crucial for ploidy maintenance in C. albicans.     

Sodium Choleate (NaCho) Effects on <Emphasis Type="Italic">Candida albicans</Emphasis>: Implications for Its Role as a Gastrointestinal Tract Inhabitant

Candida albicans at times resides in the intestinal tract, where it experiences exposure to bile salts suggesting a study of the effects of crude bile salts in the form of sodium choleate (NaCho) on C. albicans growth, expression of virulent phenotypes, and adaptation to physiological challenges in vitro. Growth and phenotype alteration was examined by challenging clinical isolates of C. albicans with a wide range of NaCho concentrations by using conventional microbial physiology methods. Our results showed that (1) NaCho did not inhibit growth of yeast cells, up to 40 mg/ml; (2) NaCho powerfully stimulated the hypha formation; (3) NaCho at 2.5 and 5 mg/ml significantly induced CDR1p and biofilm formation, but these effects decreased at higher NaCho concentrations; (4) loss of cell integrity with exposure to 56°C for 15 min, was exacerbated by increasing levels of NaCho; (5) NaCho protected yeast from hydrogen peroxide damage in a dose-dependent manner; and (6) catalase activity was increased by NaCho exposure.