A few Candida species are among the most pathogenic human fungi. Depending on the underlying host defect, Candida species cause a variety of infections, ranging from superficial mucocutaneous candidiasis to blood-stream infections [1-3]. The incidence of candidiasis has been notably increased over the recent decades due to the increasing in the number of immunocompromised patients. Among over 200 identiﬁed Candida species, Candida albicans remains the major fungal pathogen of human, followed by non-Candida albicans species, which has a remarkably growing prevalence [4-7].
This genus has been characterized with a set of virulence factors, such as reversible morphological transition between yeast, pseudohyphal, and hyphal, adhesion to biological substrates, transmigrationby enzymatic and/or physical processes, and modulation of host immune defense [8-10]. During the last decades, the molecular methods applied different genetic markers, such as ITS and D1/D2 regions in rDNA, for the identiﬁcation of Candida species .
The hyphal wall protein (HWP1) is a main adhesin protein, commonly expressed on the germ tube and hyphal surface of Candida species as a substrate attach covalently to host cells transglutaminases and cross-links this genus to epithelial cells of mucosa [10, 12]. Moreover, in the in vivo model for bioﬁlm formation, it is proposed that HWP1 adhesin retains Candida in the bioﬁlm .
The nucleotide sequences of HWP1 gene were previously amplified only for C. albicans, C. dubliniensis and C. africana by Romeo and Criseo . Nevertheless, there are no available data regarding the nucleotide or amino acid sequences of this gene for other species. Furthermore, the efficacy of HWP1 gene in the identification of non-albicans Candida species has not been reported. With this background in mind, we providedthe sequence data, phylogenetic analysis, and polymerase chain reaction (PCR) discriminatory pattern for Candida isolates of four different species, including C. parapsilosis, C. orthopsilosis, C. tropicalis, and C. glabrata.
Materials and Methods
For the purpose of the study, 70 clinical and 7 reference strains of Candida species isolates were used for the optimization of the PCR reaction. The reference strains, including C. albicans (CBS 2747), C. dubliniensis (CBS 8501), C. tropicalis (ATCC 750), C. parapsilosis (ATCC 90018), C. orthopsilosis (ATCC 96139), C. africana (IFRC 707), and C. glabrata (CBS 138) were obtained from the American Type Culture Collection, CBS-KNAW Fungal Biodiversity Centre, and Invasive Fungi Research Center.
We investigated 70 Candida isolates from a variety of specimens, including C. albicans (n=4), C. africana (n=4), C. glabrata (n=15), C. parapsilosis (n=7), C. tropicalis (n=15), C. dubliniensis (n=18), and C. orthopsilosis (n=7). These specimens were obtained from the medical mycology laboratories in Tehran, Iran and Mazandaran University of Medical Sciences.
All samples were cultured on Sabouraud dextrose agar (S) and incubated at 35°C. Species identification of the clinical isolates was initially performed based on the conventional methods, including morphology on cornmeal agar and colony color on CHROMagar. The results were confirmed by performing the PCR-restriction fragment length polymorphism (RFLP) on the secondary alcohol dehydrogenase-encoding gene (SADH) and ITS1-5.8S-ITS2 rDNA region [13, 14].
Four primer pairs (Table 1) were specifically designed based on HWP1 gene sequences of various Candida species. These Candida species including C. parapsilosis, C. orthopsilosis, C. tropicalis,and C. dubliniensis retrieved from the NCBI (http://www.ncbi.nlm.nih.gov/pubmed/). Primer des-ignning was carried out using the AlleleID software, version 7.0 (Premier Biosoft International, Palo Alto, CA, USA).
The name, sequences, and length of these primers are displayed in Table 1. The PCR amplification of HWP1 gene of other species, including C. glabrata, C. africana, and C. albicans, was performed using the primers designed by Romeo and Criseo  as follows: forward,5′ -GCTACCACTTCAGAATCATCATC-3′ and reverse, 5′- GCACCTTCAGTCGTAGAGACG-3′).
|Species and primers used||Sequence||Fragment length||PCR program||Accession numbers|
|C. glabrata ForwardReverse||GCT ACC ACT TCA GAA TCA TCA TC GCA CCT TCA GTC GTA GAG ACG||250 bp||95◦C||5'||KX758626-28|
|C. parapsilosis ForwardReverse||CGA GGT GAA TAT GAT GCT TGT A CCA ACA GAA TTG CTT AAT ACC ATA||840 bp||95◦C||5'||KX758617/KX758618KX758619/KX758620KX758621/KX758622KX758629|
|C. orthopsilosis ForwardReverse||ACC ACCACC TAG TTC TGA G TCA CTT GGA AGA TTG AGA ATA ACA||900 bp||95◦C||5'||KX758615-16|
|C. tropicalis ForwardReverse||TAC TGT TAC TTC TTG CTC CAG GCT TGC CAT TGC TTA GTG||1236 bp||95◦C||5'||KX898983-85|
|C. dubliniensis ForwardReverse||ACA GGA ATC TCC AAT AGT CACAGA ACA GAC ACG GAT TCA G||1300 bp||95◦C||5'||KX758623-25|
DNA extraction and polymerase chain reaction amplification
DNA of the Candida strains was extracted from the fresh colonies using the previously described methods [15, 16]. The PCR reactions consisted of 12.5 μl Master Mix Red (Ampliqon, Copenhagen, Denmark), 1 μl template DNA, 1 μl of each forward and reverse primers, and enough water up to a final volume of 25 μl. The PCR programs are illustrated in Table 1.
Sequencing and Sequence Analysis
The PCR products of HWP1 gene were puriﬁed using QIAquick Puriﬁcation Kit (Qi-agen, Valencia, CA, USA) and subjected to ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). For all isolates, the forward and reverse primers were used for the sequencing of purified gene fragments by an automated DNA sequencer (ABI PrismTM 3730 Genetic Analyzer, Applied Biosystems). The sequences were assembled and edited with CLC Genome Workbench (version 7), Bioedit (version 7.2), and GENEIOUS softwares (http://www.geneious.com). The consensus sequences were annotated and deposited in the GenBank, and the accession numbers of the sequences were received.
The pairwise and multiple comparisons of sequences were performed to evaluate the levelsof similarity and difference between nucleotide and amino acid sequences using GENEIOUSand MEGA softwares version 6. Phylogenetic evaluation used the neighbor joining method with 1,000 bootstrap simulations, conducted with the CLC Genome Workbench, GENEIOUS, and MEGA softwares. Bootstrap values greater than 70% were regarded significant. Scheffersomyces stipitis was used as the outgroup.
This research was approved by the Ethics Committee of the university with the ethical code of IR.MAZUMS.REC.94-1816.
In this study, a newly simple PCR on HWP1 gene was optimized and tested on 70 clinical and 7 reference strains of Candida species isolates. The HWP1 gene was successfully ampliﬁed for all strains using new specific primers and generated PCR products ranging in size from 840 bp forC. parapsilosis, 900 bp for C. orthopsilosis, 1236 bp for C. tropicalis to 1300 bp for C. dubliniensis, providing specific patterns for the differentiation of these species (Figure 1, A-D).
Furthermore, HWP1 gene  was partially ampliﬁed for C. glabrata, C. albicans, and C. africana using previously designed primers, yielding a single band with sizes of 250, 941, and 700 bp, respectively (Figure 1, E). Table 1 presents the exact size of partial ampliﬁed products of the HWP1 gene for all species. The GenBank accession numbers of the 18 reference and clinical strains are displayed in Table 1.
Based on the sequence analysis performed through the Geneious software, the pairwise and total sequence homology of partial HWP1 gene among species were 43% and 35.2%, respectively. The nucleotide and amino acid sequence alignment of the strains revealed that the similarity rate of C. parapsilosis with C. orthopsilosis was 60% and 23.07%, respectively. Among all isolates, C. albicans had the most similarity rate withC. africana and C. dubliniensis with 88.9% and 60.34% for nucleotide sequences and 9.52% and 38.36% for amino acid sequences, respectively.
Notably, the comparison of nucleotide and amino acid sequences displayed that the most pairwise homology belonged to C. albicans and C. africana, rendering rates of 90.1% and 89.6%, respectively. Furthermore, the least similarity of nucleotide and amino acid between species was observed in C. orthopsilosis and C. dubliniensis (50.8%) as well as C. tropicalis and C. dubliniensis (0%), respectively. Intra-species variation was observed only in C. tropicalis strains with two DNA types.
Phylogenetic analysis on hyphal wall protein 1 gene
The maximum likelihood phylogenetic tree constructed with HWP1 gene sequences for the representative strains of each species (Figure 2) showed that the members of the genus Candida were divided into three clades. The C. africana,C. dubliniensis, and C. albicans were placed in clade I. The C. glabrata and C. tropicalis were located in clade II, and C. parapsilosis and C. orthopsilosis were in clade III. The phylogeny of Candida strains inferred from each HWP1 gene was compared with the phylogenetic tree obtained from the Maximum Likelihood analysis of the ITS1-5.8S-ITS2 rRNA gene (Figure 3).
In spite of advances in the diagnosis and treatment of invasive candidiasis, this disease is still the principal cause of death in the critically ill patients. Given the remarkable expansion of Candida species infections as well as the differences in pathogenicity and antifungal susceptibility pattern, the rapid and accurate identification at the species level seems to be crucial for clinical management [ 17 ].
During the last decades, the utility of the PCR-based techniques focusing on various genetic markers have gained popularity to identify the pathogenic fungi. This increased application is due to the fact that the PCR-based techniques have higher speed, sensitivity, speciﬁcity, and reproducibility, compared to the conventional methods . Several authors have previously described the PCR-based procedures, which used specific primers to amplify the DNA fragment of different genes, such as pH-regulated PHR1 and PHR2 , mitochondrial cytochrome b , pH-regulated KER1 , and HWP1  for the discrimination of C. albicans, C. dubliniensis, and C. africana.
In the current study, we focused on HWP1 gene as a marker for the identiﬁcation of the common species of non- albicans Candida, including C. glabrata, C. tropicalis, C. parapsilosis, and C. orthopsilosis. However, the previous reports have demonstrated that this gene is highly expressed after transition from blastoconidia to germ tube and hyphae in C. albicans, C. dubliniensis, and C. africana species .
Currently, only few genetic markers have been reported for the specific identification of C. parapsilosis complex. Some of the markers introduced in different studies include the SADH [ 14 ], RPS0 [ 23 ], mitochondrial antioxidant manganese superoxide dismutase [ 24 ], vacuolar membrane ATPase, and intein genes [ 25 ]. Mirhendi et al. [ 14 ] developed a PCR-RFLP method forthe species identiﬁcation of C. orthopsilosis, C. metapsilosis, and C. parapsilosis. In their study, the PCR amplification of the secondary alcohol dehydrogenase-encoding gene (SADH) followed by digestion with a single restriction enzyme, NlaIII. They found that this genetic marker is suitable for separation of the species within the C. parapsilosis complex. In the present study, a simple and cost-effective PCR test was described to differentiate the two closely related species of C. parapsilosis complex (i.e., C. parapsilosis and C. orthopsilosis) using two specific primers. Our findings revealed that C. orthopsilosis, which is phenotypically indistinguishable from C. parapsilosis, can be easily identified with our strategy. The C. parapsilosis strains have less susceptibility to triazole compounds in comparison to C. orthopsilosis; furthermore, they have a key role as the first-line therapy for candidiasis. Regarding this, the accurate and rapid differentiation between these species may have important therapeutic effects .
The majority of molecular procedures have focused on ITS1-5.8S-ITS2 of rRNA gene for the identification of C. glabrata species, using such methods as PCR-RFLP [ 27 ], multiplex nested PCR [ 28 ], and Real-Time PCR [ 29 ]. Therefore, this study was the first attempt for finding a unique discriminatory pattern for C. glabrata isolates based on HWP1 gene. However, this species is a non-dimorphic unicellular budding yeast, which does not form germ tube and pseudohyphae; therefore, HWP1 gene is not expressed in C. glabrata isolates .
In contrast to C. glabrata isolates, C. tropicalis undergoes the yeast-to-germ tube and hyphal switching and are shown to have HWP1 expression [30-32]. There are a limited studies on the role of adhesion proteins, such as HWP1, in C. tropicalis [33, 34]. Our study provided the nucleotide and amino acid sequences of HWP1 deposited in the Genbank as well as a specific PCR pattern for the rapid identification of this species.
Previously, Cornet et al.  and mirhendi et al.  introduced the PCR-RFLP, targeting the inter-genic spacer and ITS1-5.8S-ITS2 of ribosomal DNA, respectively, as a reliable method to differentiate C. tropicalis from other common pathogenic species of Candida. The phylogenetic analysis provided statistically signiﬁcant support for the clustering of Candida species based on HWP1 gene. The topology was largely consistent with the phylogenetic tree obtained from the previously analyzed genes, such as COX3, SADH, SYA1, ALS, and ribosomal RNA [35-38].
Consistent with the last phylogenetic evidence [17, 39], we found that C. dubliniensis and C. africana fell into C. albicans complex as shown for ITS1-5.8S-ITS2 gene in Figure 3. The phylogenetic tree of HWP1 gene indicated that the C. dubliniensis isolates formed a monophyletic group nested within the clade of C. albicans.
In addition, the phylogeny of C. parapsilosis complex based on the HWP1 gene showed a completely similar topology with the phylogenetic tree based on the D1/D2 region of rDNA reported by Herkert et al.  and ITS (Figure 3), SADH, and 26S rRNA gene sequences presented by Mirhendi et al. . However, in our study, HWP1 gene was not amplified for C. metapsilosis. Our ﬁnding confirmed the previous analysis in which C. parapsilosis and C. orthopsilosis were placed in "psilosis" clade [38, 40, 41].
In agreement with the phylogenetic tree based on D1/D2 region of rDNA , ALS , and cytochrome b genes , the current phylogeny inferred from HWP1 displayed that C. tropicalis was phylogenetically distinct from other species of Candida and a sister taxon (75% bootstrap support) of the C. glabrata species. While in the phylogenetic tree of the DNA topoisomerase II and ITS1-5.8S-ITS2 genes (Figure 3) reconstructed by the neighbor-joining and Maximum Likelihood methods, respectively , C. tropicalis placed in a separate cluster next to C. albicans, C. dubliniensis, and C. parapsilosis isolates. Substantially, the differences between phylogenetic results seem to be related to the methods of analysis and genetic markers .
The analysis of HWP1 gene revealed that except for C. africana and C. albicans, no other species showed more than 90% homology based on nucleotide sequences. Therefore, this gene presents an excellent genetic marker for the identification of different species of Candida. In the previous studies, the sequence homology rate for the different species of Candida was reported as 89.9%, 97.52%, and 97.9% for cytochrome b, V3 variable region of the LSU of rRNA gene, and ACT1, respectively .
The genetic diversity of HWP1 gene has already been reported for C. albicans isolates originated from Yaound´e HIV-infected patients . In a study, Kammalac Ngouana et al. described five genotypes (i.e., H1-H5) using the amplification of HWP1 gene. In the current study, we obtained two single-nucleotide polymorphisms in one isolate of C. tropicalis.
The present study was the ﬁrst report of HWP1 gene analysis for the identiﬁcation and phylogenetic evaluation in some Candida species, including C. parapsilosis, C. orthopsilosis,C. tropicalis, and C. glabrata. In this study, 77 Candida isolates causing different infections were identiﬁed at the species level based on the developed PCR pattern on HWP1 gene. We concluded that the proposed simple PCR test was a rapid and cost-effective method. Therefore, this method could be used routinely in the research and clinical laboratories for the reliable identification of the most common species of Candida.
Finally, we found that HWP1 gene was an excellent marker for the identification of non-albicans Candida species.
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