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Multi-Locus Next-Generation Sequence Typing of DNA Extracted From Pooled Colonies Detects Multiple Unrelated Candida albicans Strains in a Significant Proportion of Patient Samples.
(Frontiers Media S.A., 2018-06-05) Zhang N; Wheeler D; Truglio M; Lazzarini C; Upritchard J; McKinney W; Rogers K; Prigitano A; Tortorano AM; Cannon RD; Broadbent RS; Roberts S; Schmid J; Sanglard D
The yeast Candida albicans is an important opportunistic human pathogen. For C. albicans strain typing or drug susceptibility testing, a single colony recovered from a patient sample is normally used. This is insufficient when multiple strains are present at the site sampled. How often this is the case is unclear. Previous studies, confined to oral, vaginal and vulvar samples, have yielded conflicting results and have assessed too small a number of colonies per sample to reliably detect the presence of multiple strains. We developed a next-generation sequencing (NGS) modification of the highly discriminatory C. albicans MLST (multilocus sequence typing) method, 100+1 NGS-MLST, for detection and typing of multiple strains in clinical samples. In 100+1 NGS-MLST, DNA is extracted from a pool of colonies from a patient sample and also from one of the colonies. MLST amplicons from both DNA preparations are analyzed by high-throughput sequencing. Using base call frequencies, our bespoke DALMATIONS software determines the MLST type of the single colony. If base call frequency differences between pool and single colony indicate the presence of an additional strain, the differences are used to computationally infer the second MLST type without the need for MLST of additional individual colonies. In mixes of previously typed pairs of strains, 100+1 NGS-MLST reliably detected a second strain. Inferred MLST types of second strains were always more similar to their real MLST types than to those of any of 59 other isolates (22 of 31 inferred types were identical to the real type). Using 100+1 NGS-MLST we found that 7/60 human samples, including three superficial candidiasis samples, contained two unrelated strains. In addition, at least one sample contained two highly similar variants of the same strain. The probability of samples containing unrelated strains appears to differ considerably between body sites. Our findings indicate the need for wider surveys to determine if, for some types of samples, routine testing for the presence of multiple strains is warranted. 100+1 NGS-MLST is effective for this purpose.
Glucose induced germ tube formation in Candida albicans : a thesis presented to Massey University in fulfilment of the requirements for a Masters of Science degree in Microbiology
(Massey University, 2004) Sciascia, Miriama
Candida albicans is an opportunistic fungal pathogen that can cause a wide range of superficial and systemic infections. One of the many factors that have been implicated in C. albicans success as a pathogen is its ability to reversibly switch between a yeast form and a hyphal form (dimorphism). The dimorphic switch is triggered by a wide variety of stimuli which include temperature alone, pH alone, and serum. Serum is a potent inducer of germ tube formation and remains the medium of choice for rapid identification of C. albicans from other non-albicans Candida species. Recently it was shown that, in serum, glucose is the primary inducer of germ tubes in C. albicans strain A72 (Hudson and Farley, unpublished). In this study the ability of glucose, dialysed serum and serum filtrate to induce germ tube formation in a randomly chosen panel of clinical isolates of C. albicans was studied, and the role ot two putative glucose receptors and a putative glucose transporter in the transduction of the glucose signal was investigated. Dialysed serum (molecular weight, > 10 kDa) was less effective (P > 0.05,Students t- test) at inducing germ tube formation than serum. The addition of exogenous glucose alone to dialysed serum restored its ability to induce germ tube formation levels to those seen in serum in seven of the nine clinical isolates tested. Serum filtrate (molecular weight, > 10 kDa) induced germ tubes to levels indistinguishable trom those seen in serum (P > 0.05, Students t-test) in all but one of the clinical isolates tested. Buffered glucose was also able to induce germ tubes in all the clinical isolates tested and the percentage germ tube formation was not statistically significantly different from that obtained with serum in ten out of sixteen clinical isolates tested. The addition of urea to these assays had no statistically significant effect on the induction of germ tube formation. It was proposed that the induction of germ tube formation by glucose was mediated by a surface receptor and therefore the C. albicans genome was examined for genes encoding putative glucose receptors. Identified as possible receptors were orf19.1944 and orf19.5962. Orf19.3668, a putative glucose transporter, was also examined because its expression had been reported to increase during serum induced germ tube formation. Strains carrying homozygous deletions of each ORF were made and the phenotypes of the mutants investigated. None of the ORFs were found to be involved in glucose or serum mediated germ tube formation. However, orf19.1944 was shown to play a role in germ tube formation under embedded conditions.
A search for contingency genes in Candida albicans : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
(Massey University, 2014) Wattimena, Synodalia Chrisma
Many microbial pathogens have been known to use repeats in their cell wall pro- teins to generate diversity, and this has been found to contribute to their virulence. In bacteria, these genes are called contingency genes, and function to facilitate adap- tation of bacteria to the host environments as they invade di erent host parts and to evade the host's constantly evolving immune system. In the diploid Candida albicans, few genes have been classi ed as contingency genes due to the variation in the length of their repeat regions in di erent clinical isolates. This study attempts to answer a question of whether YWP1, HWP1, and EAP1 of C. albicans are contingency genes. These three genes encode cell wall proteins and contain repeats. For this purposes, allelic distributions of the genes in the general purpose genotype (GPG) and non-GPG strains (two groups with di erent genetic backgrounds), in commensal and infection strains, and in strains isolated from di erent sites of the humans body were examined. Based on the allelic distributions of the genes in GPG and non-GPG strains, it can be inferred that YWP1 and HWP1 can be categorized as contingency genes, while EAP1 cannot be categorized as a contingency gene. The allelic distributions of the genes in commensal and infection strains indicate that YWP1, HWP1, and EAP1 do not act as contingency genes when C. albicans state changes from commensal to pathogenic. Although the allelic distributions of the genes cannot distinguish com- mensal from infection strains, the non-random association between alleles of YWP1, HWP1, and EAP1 does distinguish these two groups, i.e. the YWP1 -HWP1 -EAP1 association is stronger in commensal strains that it is in infection strains. Based on the allelic distribution of the genes in strains isolated from di erent sites of the human body, it can be inferred that YWP1 and EAP1 do not act as contingency genes, but HWP1 may act as a contingency gene, when C. albicans moves to particular sites of the human body.
Molecular typing and phylogenetic analysis of Candida albicans isolates from different patient populations : a thesis presented in partial fulfilment of the requirements for the degree of PhD in Molecular Biology and Genetics at Massey University, Palmerston North, New Zealand
(Massey University, 2000) Maltzahn, Nicole Beate
An important question in understanding the epidemiology of Candida albicans is to determine whether certain strains, e.g. HSP strains (highly successful strains), more prevalent in Candidosis patients than in healthy individuals, replace commensal strains under certain conditions and whether the replacing strains may cause Candidosis. This question was investigated in experiments which monitored the genetic diversity of commensal C. albicans isolates obtained from individuals before and after they were exposed to conditions that may predispose them to the development of Candidosis. The distinctiveness of C.albicans strains isolated from individuals was analysed using the phylogenetic method of split decomposition. The method was found to provide a good representation of the phylogenetic information in strain replacement Ca3 data. Our study highlighted difficulties in monitoring of strain replacement with Ca3 methodology. An indication for strain replacement was observed in one patient at low risk to acquire Candidosis. However, the observations that cancer patients, who were at a high risk of developing Candidosis, were colonised with diverse strains and that healthy individuals could be colonised with different commensal C.albicans strains within one body location, cautioned against overinterpretation of this finding. These results demonstrated the need for extensive sampling of larger numbers of isolates from different body locations when evaluating replacement hypotheses. In investigating potential sources of C.albicans infections we successfully isolated this fungus from the hospital environment of high risk patients, demonstrating the potential of the hospital environments as a source for infection causing strains. In characterising strains, a nonradioactive fingerprinting protocol was developed for a more convenient use of Ca3 fingerprinting. Until now the existence of the HSP group has been based entirely on Ca3 fingerprinting data. To test for the existence of this group we have analysed amplified fragment length polymorphisms (AFLP) of 36 C.albicans isolates from different geographical regions. Phylogenetic reconstruction from both data forms (Ca3 and AFLP data) were highly congruent and suggest a worldwide distribution of HSP strains. Study of the tree building properties of AFLP and Ca3 data using Quartet Puzzling and a tree comparison metric showed that AFLP data were more treelike than the Ca3 data. However, whilst both AFLP and Ca3 methods provided high resolution data to identify strains and substrains of C.albicans, the need for population based studies to test for strain replacement makes the use of either method limited. For this reason, both nuclear ITS and AFLP derived PCR markers were investigated for their potential use in such studies. In particular, one AFLP derived PCR marker that was partially characterised appears very promising for future strain replacement studies. It is likely to provide a simple diagnostic test for rapid identification of HSP strains.
Sex has no detectable net benefits for Candida albicans : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Molecular Microbiology at Massey University, Palmerston North, New Zealand
(Massey University, 2012) Zhang, Ningxin
Like many other important opportunistic human fungal pathogens, for more than a century Candida albicans was thought to be strictly asexual until a parasexual cycle was recently discovered in the laboratory. It is uncertain, however, whether sex is still a viable reproductive strategy for C. albicans. In this study I tested whether or not mating enhanced survival of parental genes in this yeast, by mating 10 clinical isolates and testing recombinants’ fitness. Clinical isolates of C.albicans usually are diploid, carrying both the MTLa and MTLα mating type alleles, each on a different copy of chromosome 5. These strains are apparently incapable of meiosis and cannot mate with each other, because the a1-α2 heterodimer suppresses mating. Through selection on sorbose-containing agar I induced loss of MTL-heterozygosity and generated 5 MTLa and 5 MTLα derivatives of clinical isolates. Existing mating techniques involve the use of auxotrophic markers, requiring time-consuming sequential disruption of two copies of biosynthetic genes if wild-type clinical isolates are to be mated. Furthermore, auxotrophy affects the virulence of a strain, and this can potentially interfere with comparing the fitness of recombinants with that of their parents. I therefore developed a method for mating clinical isolates marked with two drug resistance markers, the mycophenolic acid (MPA) resistance-conferring allele of IMH3 and the nourseothricin (NAT) resistance gene CaNAT1, allowing selection of recombinants on the basis of resistance to both agents. I marked all MTLa strains with the MPA resistance gene and all MTLα strains with the NAT resistance gene. This allowed 25 combinations for mating. Recombinants were obtained from 15 combinations of 9 strains. It was found that not all C. albicans clinical isolates could mate. Using growth rate as the criterion, I tested the fitness of clinical isolates, MTLhomozygous derivatives with and without resistance markers and recombinants during adaptation to a novel environment (YPD medium), maximizing the potential benefits of sex. After computationally correcting for the impact of experimental manipulations, I calculated the net benefit of sex as the difference in the number of offspring from two cells that become mating competent and engage in sex compared to the offspring they could have produced by continued clonal reproduction. My results indicated that, as a rule, engaging in sex reduces the chances of survival of C. albicans’ genes, in part because MTL homozygosis significantly reduced growth rates. Through fitness increase after recombination, sex may eventually confer a net benefit for some strain combinations in the laboratory, but this probably occurs too late to prevent elimination of recombinants by competition and genetic drift in nature. Sex in C. albicans therefore diminished parents’ chances to pass on their genes to future generations. These findings have a significant impact on the assessment of the role of sex in C. albicans and other “asexual” human fungal pathogens. Recent loss of the function of sex and incomplete decay of the sex machinery are the most likely explanation of C. albicans’s residual ability to mate, and one that also needs to be considered in other fungal pathogens.
The secreted aspartic proteinases of Candida albicans : a thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular BioSciences at Massey University, New Zealand
(Massey University, 1999) Sullivan, Michelle E.
The common human fungal pathogen, Candida albicans, possesses at least nine genes encoding secreted aspartic proteinases (SAPs). Saps are widely regarded as virulence factors, despite historical controversy surrounding their actual roles in the onset and development of candidosis. While Sap1, Sap2 and Sap3 had been previously studied at the biochemical level, Sap4 and Sap5 had not been detected, purified or characterised. To facilitate analysis of the proteins, SAP4 and SAP5 were amplified by PCR and cloned. The nucleotide changes in SAP4 were silent, and SAP5 contained one conserved amino acid substitution, compared with the published sequences. The methylotrophic yeast Pichia pastoris was used as a host for heterologous expression of SAP4 and SAP5, and the respective proteins were purified to homogeneity. Purification of Sap4 involved Mono Q anion exchange chromatography at pH 7.0, while Sap5 was purified by cation exchange chromatography on Resource S at pH 7.0. C. albicans SAP1 was also over-expressed in P. pastoris as a control. Biochemical analysis of the recombinant proteins revealed that Sap4 and Sap5 were optimally active at pH 4.5, 1-2 pH units higher than the optima of Saps1, 2 and 3. At optimum pH, the specific activities of Sap4 and Sap5 were 239 and 33 µg tyrosine equivalents/min, respectively. These isoenzymes also retained significant activity at pH 7.0, which suggested roles in the disease process at host sites of neutral pH. Sap4 and Sap5 showed decreased specific activity towards denatured globin, and increased specific activity towards a fluorocasein substrate, when compared with Saps1-3. Substrate specificity analyses (performed using a peptide substrate, glucagon, and MALDI-TOF of the purified peptide fragments), showed that Sap4 and Sap5 hydrolysed the glucagon at the same sites, but the analysis did not reveal a consensus cleavage sequence. The deduced masses of Sap1, Sap4 and Sap5 were 36,179, 36,995 and 37,256, respectively. However, ES-MS indicated the masses of the recombinant Sap1 and Sap4 were larger than expected, by 2232 and 2041 respectively. Glycopeptide fragment ion analysis suggested the additional mass was due to attached sugar residues. Carbohydrate chromatography confirmed the presence of mannose and N-acetyl glucosamine. The presence of N-acetyl glucosamine species, and the lack of consensus N-linked glycosylation sites in the Sap1, Sap4 and Sap5 proteins suggests a novel pattern of O-linked glycosylation in P. pastoris. The purified enzymes were subjected to crystallisation trials and some promising crystals were produced. Previous studies showed that SAP4, 5 and 6 are expressed during serum-induced germ-tube formation, but this was not confirmed in this study.
Evidence that SSR1 can act as a hypermutable contingency gene in Candida albicans : a thesis presented in partial fulfillment of the requirement for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
(Massey University, 2010) Zhou, Zhuo
During adaptation to the host environment, many microorganisms undergo rapid variation in cell surface phenotype through genetic alteration in hypermutable contingency genes. One of the main mechanisms underlying these changes is alteration in the number of DNA repeat units that results in a large and flexible repertoire of similar but non-identical surface proteins. SSR1, a gene in the opportunistic pathogen Candida albicans, encodes a repeat-containing cell wall protein which may play a role in maintaining cell wall strength. This gene contains 2 regions with multiple 6 bp tandem repeat units, encoding the amino acids serine and alanine, separated by a 200 bp non-repetitive DNA region. This study investigated whether SSR1 was a hypermutable contingency gene. Among a worldwide collection of 96 infection-causing C. albicans strains, 24 alleles and 40 allele combinations were identified by fluorescent-based genotyping of SSR1 PCR products. Sequencing results confirmed that the differences in allele size were caused by variation in number of tandem repeats. Two very similar allele combinations were overrepresented (30% and 28%) among a cluster of generalpurpose genotype (GPG) strains (which is the most widespread cluster) compared with non-GPG strains (Fisher’s exact test, P=0.0001 and P<0.0001). Among a worldwide collection of 36 commensal GPG C. albicans strains, 8 allele combinations were identified by genotyping. One of the two allele combinations that were overrepresented in GPG infection-causing strains was found significantly less in GPG commensal strains (Fisher’s exact test, P=0.0004). After culture of C. albicans cells in vitro for 300 generations, mutation of repeats in SSR1 occurred, giving a high mutation rate of 1.11×10-4 per cell division. The results indicate that SSR1 is a hypermutable gene and that it shows clade-specificity with the GPG cluster. Growth in a rat model did not seem to cause variation in SSR1 and human host body sites did not seem to be associated with specific SSR1 alleles, suggesting that SSR1 is not used for short-term adaptation in these environments. However, the different allele distribution in commensal and infection-causing GPG strains suggest that SSR1 may have a role in short-term adaptation in GPG strains, contributing to the change between commensalism and infection. In this case, SSR1 may act as a hypermutable contingency gene.
Impact of genetic background on allele selection in a highly mutable Candida albicans gene, PNG2
(PLoS ONE, 2010) Zhang N; Cannon RD; Holland BR; Patchett ML; Schmid J
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 2.8x10(-5) (confidence interval 3.3x10(-6)-9. 9x10(-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.

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