Development of quick and accurate methods for identifying human pathogenic yeast by targeting the phospholipase B gene, topoisomerase II gene, Candida drug resistance gene, and species-specific ITS 2

1. Introduction

In recent years, fungal infections, particularly Candida, Aspergillus, Mucor, and Cryptococcus species, have significantly increased in the majority of immunocompromised patients, mostly cancer patients (Tufa et al., 2023; Gnat et al., 2021; Shariati et al., 2020). Compared to others, Candida species mostly cause yeast infections. Nevertheless, their clinical manifestations and prognoses may vary. Candidiasis, a fungal infection caused by Candida species, is a significant threat to human health. Notably, Candida albicans is the predominant etiological agent responsible for invasive yeast infections (Pappas et al., 2018; Lass-Flörl et al., 2024). However, recent epidemiological studies have revealed an alarming surge in candidiasis cases attributed to non-albicans Candida species, including C. guilliermondii, C. parapsilosis, C. krusei, C. tropicalis, C. kefyr, C. glabrata, and C. dubliniensis, particularly in immunocompromised individuals, like those with cancer (Arafa et al., 2023; Aydemir et al., 2017; Taei et al., 2019). Another form of yeast infection caused by Rhodotorula species has emerged in patients with cancer. Although Rhodotorula species, including R. minuta, R. glutinis, and R. mucilaginosa, have been identified as human pathogens, members of the Rhodotorula genus were previously believed to be non-pathogenic. The majority of the infections were caused by fungemia, that is, the presence of yeast infection in the blood (Miglietta et al., 2015; FaqeAbdulla, 2024). Fungal diseases are responsible for an estimated 1.6 million deaths per year, and more than a billion individuals suffer from serious fungal diseases (Almeida et al., 2019; Rokas, 2022).

Several approaches for the rapid and accurate identification of yeasts have been developed. Numerous yeast identification tests are available, ranging from traditional methods to molecular methods. Clinical microbiology laboratories face significant challenges in selecting an accurate, cost-effective, easy-to-interpret, and reasonably fast yeast identification system (Bharathi, 2018). Fungal identification primarily relies on morphological and physiological characteristics. However, the distinctive properties of fungi make morphological identification and classification difficult (Fajarningsih, 2016; Arifah et al., 2023). Furthermore, identification processes require specialized knowledge, can yield misleading results, and are typically time-consuming, delaying an accurate diagnosis. Alternative diagnostic methods that directly detect diagnostic molecules are becoming increasingly popular (Gabaldón, 2019).

Specific primers derived from genome sequences were created, and their specificities were verified by PCR-based identification, enabling quick species-level detection of Candida (Harmal et al., 2013), and a single, targeted DNA fragment from the pertinent genomic DNA of the Candida species was amplified by each primer (Kanbe et al., 2002).

Phospholipases (PLB) are enzymes found in most living organisms, particularly eukaryotes (Cerminati et al., 2019). They help balance membrane contents, obtain nutrients, and produce bioactive molecules (Köhler et al., 2006; Giriraju et al., 2023). The PLB gene of Candida species was selected as the place to make primers for Candida species because it is a new target, and the sequences of Candida species vary greatly. This resulted in a few unique sequences for each species of Candida, making it possible to create primers for each species that target the unique sequences of the PLB gene (Harmal et al., 2013).

DNA topoisomerases are enzymes that catalyze topological changes in DNA by reuniting double-helical strands. Type II enzymes simultaneously cut and seal both strands, eliminating twists and knots ( Matias-Barrios, & Dong, 2023 ;Uusküla-Reimand & Wilson, 2022; Wang, 1996). They play important roles in gene expression, recombination, repair, and DNA replication. DNA topoisomerases are potential therapeutic targets. However, research on fungal DNA topoisomerase II is limited (Keller et al., 1997; Madabhushi, 2018). The nucleotide sequences of the gene in Aspergillus nidulans and Candida species have been determined (Kato et al., 2001). This gene is a useful target to research phylogenetic links between fungal species, evolution of fungal genes, and the molecular identification of several fungal species (Kanbe et al., 2002).

Candida species are resistant to clinical fungicides because of their multidrug efflux pumps, which help move drugs out of cells (Krishnasamy et al., 2018). This resistance is linked to the growth of gene families and increased gene expression. Specifically, ATP-Binding Cassette (ABC) and Major Facilitator Superfamily (MFS) transporters are involved; notably, CDR1 had the highest expression. CDR1 is an ABC transporter with the highest basal expression level among the ABC transporters. The Candida drug resistance (CDR) genes, including CDR1, facilitate the transport of azoles and other compounds. (de Oliveira Santos et al., 2018; Rybak et al., 2019). The foundation for the use of ribosomal DNA (rDNA) genes for the identification of fungal species was established by identifying conserved areas in 5.8S and 28S rDNA that permit the amplification of the Internal transcribed spacer (ITS2)c region between these two genes (Keller et al., 2009). Recent research suggests that sequence variations and sizes of fungal ITS2 areas are relevant for quick identification of therapeutically important fungi (Turenne et al., 1999). PCR using fungus-specific primers that target conserved regions of 5.8S and 28S ribosomal DNA (rDNA) caused the species-specific ITS2 regions, whose amplicon lengths differed, to be amplified (Turenne et al., 1999).

This study involved the development of a PCR technique tailored to specific species. The technique targets specific genes, such as phospholipases, DNA topoisomerases, Candida drug resistance, and ITS2 found in yeast species. This enabled the identification and differentiation of the most prevalent, clinically, and medically important Candida and pink yeast strains.

2. Material and Methods

3. Results

Amplicons of different sizes were produced during the PCR process because of the variation in length between the ITS 1 and ITS regions of distinct Candida and pink species. The PCR products of, C. tropicalis, C. lusitaniae, C. albicans C. glabrata, C. dubliniensis, C. parapsilosis, C. gattii, and R.mucilaginosa generated using ITS 1and ITS 4 universal fungal primers, were roughly 524bp ,377bp, 535bp, 871bp, 540bp, 520bp, 600bp, and 630bp, respectively.

3.1 Phospholipase B gene PCR amplification

Species-specific primers of six Candida species viz., C. dubliniensis, C. lusitaniae, C. parapsilosis, C. glabrata, C. albicans, C. tropicalis, and C. gattii were designed from a region of the PLB gene using Primer 3 online software tools. Distinct band-size PCR products were produced on a 2% agarose gel by all six Candida species. The amplified PCR bands were observed at, 230bp, 225bp, 234bp, 164bp, 160bp, 177bp, and 205bp and respectively identified as C. tropicalis, C. dubliniensis, C. parapsilosis, C. albicans, C. glabrata, C. lusitaniae, and C. gattii (Figs. 1 and 2).

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Fig. 1.

DNA ladder 100bp (Transgen Biotech), C. albicans, (1,2): C. tropicalis (3,4) : C. lusitaniae (5,6) : C. dubliniensis (7,8): C. glabrata (9,10), 11: Cryptococcus gattii (11): C: negative control (12) 1 M: DNA molecular marker

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Fig. 2.

DNA ladder 100bp (Transgen Biotech), C. parapsilosis (1,2,3,4).

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3.2 PCR amplification of topoisomerase II gene

The topoisomerase II gene of Candida spp. has been selected as a novel target for species identification. In this study, primers specific to each species were created for three different Candida species, including Lusitaniae, C. parapsilosis, and C. glabrata, because of variation in their topoisomerase II genes. Visualization on 2% gel showed that C. glabrata produced 154bp pair PCR amplified products, whereas C. parapsilosis gave 220bp and C. lusitaniae gave 219bp PCR products (Fig. 3).

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Fig. 3.

PCR products of three (3) different Candida species viz., C. glabrata (1,2), C. lusitaniae (3,4), C. parapsilosis (5,6) 100bp DNA Ladder (M).

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3.3 PCR Amplification of Candida Drug Resistance (CDR) Gene

Species-specific primers for the CDR1 gene were also used to specifically identify the four Candida species. When PCR-amplified products of these Candida species were visualized on a 2% gel, C. parapsilosis, C. Tropicalis, C. glabrata, and C. dubliniensis showed characteristic PCR bands of 241bp, 163bp,188bp, and 221bp, identified correctly (Fig. 4).

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Fig. 4.

DNA marker (M), Candida tropicalis (1,2), C. dubliniensis (3,4), C. glabrata (5,6), C. parapsilosis (7,8), negative control (9) and DNA marker (100bp).

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3.4 PCR amplification of ITS2 gene

Species-specific ITS2 primers were designed using Primer 3 online tool for the pink yeast R. mucilaginosa. PCR was performed using template DNA from all 29 pink yeast cultures of R. mucilaginosa and the ITS2 primer set (ITS2F&R). The targeted region of the ITS2 gene of all cultures of R. mucilaginosa was successfully amplified using these species-specific primers and examined using 2% gel electrophoresis. The PCR findings revealed a distinct band of the same size of around 187 base pairs (Fig. 5).

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Fig. 5.

PCR amplified products of ITS2 gene of Rhodotorula mucilaginosa (lane 1-7) 100bp DNA marker (M) (Transgen Biotech) and negative control (C).

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4. Discussion

Fungal infections can cause a range of symptoms from mild superficial to life-threatening invasive ones. They cause diseases in immunocompromised individuals such as those with human immunodeficiency virus (HIV), cancer, diabetes, transplant, or immunosuppression, such as those receiving chemotherapy, radiotherapy, or immunosuppressive drugs. Effective detection and treatment of high-risk populations are crucial to prevent most deaths (Bongomin et al., 2017; Pathakumari et al., 2020). We created a PCR test to detect and identify the most clinically significant yeast species through species-specific amplification of target genes.

Phospholipases are enzymes found in most living organisms, including eukaryotes, that help maintain the membrane balance, obtain nutrients, and produce bioactive compounds (Aloulou et al., 2018; Köhler et al., 2006). Unlike rRNA genes, PLB genes contain limited variable areas (Harmal et al., 2013) and can therefore be used for specific identification. Because of the significant sequence variability between Candida species, PLB was chosen as the locus for designing Candida species-specific primers. Consequently, this yielded distinct and exclusive sequences for each Candida species, facilitating the development of primers that specifically target individual sequences of the PLB gene (Harmal et al., 2013). Species-specific primers were designed to amplify specific regions of PLB genes for the identification of six Candida spp. and C. gattii. All designed primers successfully amplified the target and specific regions. The product sizes were exactly the same as the predicted sizes, and Candida species, that is, C. albicans (164bp), C. glabrata (160bp), C. lusitaniae (177bp), C. dubliniensis (225bp), C. parapsilosis (234bp), C. tropicalis (230bp), and Cryptococcus gattii (205bp) were correctly identified, corroborating the observations of Harmal et al. (2013) and Pfaller et al. (2010).

DNA topoisomerases catalyze topological changes in DNA by breaking and reuniting double-helical strands (Wang, 1996). These drugs are crucial therapeutic targets. However, few studies have focused on fungal DNA topoisomerase II (Keller et al., 1997). Phylogenetic relationships and characteristics of Candida species have been revealed through isolation and sequencing of their DNA topoisomerase II genes. This gene is crucial for understanding fungal gene evolution and the molecular identification of multiple species (Kato et al., 2001).

To analyze fungal DNA topoisomerase II genes for phylogenetic studies and patient-infecting fungal species, researchers have identified Aspergillus nidulans and numerous Candida species (Kanbe et al., 2002; Kato et al., 2001). The DNA topoisomerase II-encoding gene was selected as the target because it is present in all organisms and has a sequence that is highly similar and unique to each species (Al-Tekreeti et al., 2018; Kanbe et al., 2002). In this experiment we designed primers for three Candida species, C. glabrata, C. lusitaniae, and C. parapsilosis, based on variation in their topoisomerase II genes. Results showed C. glabrata as producing 154bp PCR products, while C. parapsilosis produced 220bp and C. lusitaniae produced 219bp.

Candida species develop resistance to clinical fungicides owing to multidrug efflux pumps, which help them move drugs out of their cells. This resistance can occur because of gene family growth or increased expression (Morschhäuser et al., 2007). Five CDR genes, including CDR1, have been identified in Candida spp. to transport azoles and other drugs, such as cycloheximide and chloramphenicol (Maras et al., 2021). CDR genes have been used in drug expression testing; however, their use for yeast species identification through PCR amplification remains limited. We also used species-specific primers for the CDR1 gene to identify four Candida species, including C. tropicalis, C. Dubliniensis, C. parapsilosis, and C. glabrata. The PCR bands for these Candida spp. were recorded at 163bp,221bp, 241bp, and 188bp, respectively, as expected.

rDNA genes were used to specifically identify fungal species by amplification of the ITS2 region between 5.8S and 28S rDNA (James et al. 1996). ITS2 regions specific to a species are amplified by PCR using primers specific to fungi, and amplicon length varies (Turenne et al., 1999). In the present study, species-specific ITS2 primers were created for the pink yeast R. mucilaginosa (rubra). The targeted region of the ITS2 gene was successfully amplified in all R. mucilaginosa cultures and 187bp PCR products were resolved using 2% agarose gel electrophoresis and observed under UV light.

Fungal infections can range from mild superficial infections to life-threatening invasive ones, causing disease in immunocompromised individuals like those with HIV, cancer, diabetes, transplant, or immunosuppressed such as those receiving chemotherapy, radiotherapy, or using immune suppressive drugs. Effective detection and treatment of high-risk populations are crucial to prevent most deaths (Bongomin et al., 2017; Pathakumari et al., 2020).

5. Conclusion

The recent outcome has encouraged us to take on a new molecular technique development for the identification and discrimination of other medically significant human fungal infections, in addition to Candida species. Species-specific primers were discovered to be a more efficient, rapid, and accurate molecular approach for the identification of yeast species, thereby eliminating the necessity for time-consuming and labour-intensive DNA sequencing methodologies. Species-specific PCR is a versatile tool for quickly identifying a broad range of pathogenic microorganisms at the species level with the ability to add newly developed primers to other medically important human pathogens.