Molecular genetic identification of yeast isolate MF22_1

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MOLECULAR GENETIC IDENTIFICATION OF YEAST ISOLATE MF22_1

M.O. Fomina, O.Ya. Filipishena, L.V. Polishchuk

D.K. Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine

Microscopic fungi including yeasts are important part of human microbiota. Among them, yeasts of genus Candida are very common and often the most predominant part of mycobiota. Candida spp., including Candida albicans and non- Candida albicans species, can be opportunistic pathogens affecting people with depressed immune system. The problem with some non- Candida albicans species, e.g. C. glabrata and C. krusei, is that they are highly resistant to antifungal therapy and, at the same time, difficult to identify accurately at the species level by widely clinically used conventional biochemical methods. The aim of this work was precise molecular genetic identification of human sputum yeast isolate MF22_1, allegedly belonging to Candida spp., by using multiple genetic markers. We used four fragments of its chromosomal DNA, which were sequenced and deposited by us in GenBank NCBI database: 1) ITS (GenBank OM479428, 548 bp), 2) 28S rRNA (OM479513, 607 bp), 3) 18S rRNA (OM4794321, 737 bp), and 4) RNA polymerase II gene -- RPB2 (OM524388, 1,217 bp). The part of Nucleotide collection database [http://www.ncbi.nlm.nih.gov/nuccore/] containing information on the nucleotide structure of fungal DNA (taxid: 4751) was studied using BLASTN [http://blast.ncbi.nlm.nih.gov/Blast.cgi]. The BLASTN analysis established that all four sequenced genetic markers: ITS, 28S rRNA, 18S rRNA, and RPB2 of the studied isolate MF22_1 had the maximum similarity to the corresponding sequences of the type strains of Pichia kudriavzevii species. Therefore, it was confirmed that the isolate belonged to species Pichia kudriavzevii, which anamorph (or non-ascosporic state) is Candida krusei. This ubiquitous in the environment species is a common clinical isolate responsible for about 2 % incidences of yeast infections caused by Candida species in humans. Comparative analysis of a primary structure of ITS region demonstrated the great similarity of the ITS sequence of Pichia kudriavzevii MF22_1 to the majority of other Pichia kudriavzevii clinical isolates from human sputum, stool, blood etc., which are preserved in different culture collection of the institutions specializing in medical studies of yeasts around the world. For these clinical isolates of Pichia kudriavzevii, no clear correlation was observed between the similarity of ITS sequences and the type of biomaterial sampled from humans.

Key words: Candida spp., yeast, identification, ITS region, 18S rRNA, 28S rRNA, RPB2.

molecular genetic identification human sputum yeast isolate сandida

The fungal, particularly yeast microbiota of humans and mammalian animals has been largely understudied compared to the bacterial component of microbiome [1]. However, there is growing evidence that fungi can play a crucial role in modulation of host immunity and consequently in health or disease of a macro-organism [1, 2]. From the very moment of their birth macro-organisms are exposed to the colonization by mycobiota originated, firstly, from maternal microbiome (vertical transmission), and then, throughout life, from environment and available foods. The most abundant and diverse fungal microbiota is characteristic for the mammalian gut where the most predominant members of fungal communities usually belong to genus Candida [1, 2]. Many representatives of Candida spp., including the major fungal species in the human gut Candida albicans, are typically regarded as opportunistic pathogens meaning that they only affect patients with depressed immune system. At the same time C. albicans is the most frequently detected fungus in faeces of healthy humans, and therefore, is considered a normal component of the human gut microbiota [1]. The pathogenesis caused by yeasts is thought to be largely associated with the disbalance of fungal microbiota which is in its turn tightly connected to bacterial dysbiosis.

Apart of intestine, such yeasts as C. albicans can also colonize other body sites including mouth, skin, and vagina. Candida spp. yeasts often colonize the upper respiratory tract in healthy individuals, and this oropharyngeal colonization is reflected in finding such yeasts in sputum at microbiological examination. However, Candida spp. can cause thrush or even pneumonia in severely immunocompromised patients [3, 4]. It was also found that Candida spp. never caused pneumonia alone but contributed to the pathological process in association with bacterial pathogens or opportunistic members of normal respiratory bacterial microbiota [3]. That is why the microbe-microbe interactions within the oral microbiome including inter-kingdom fungal-bacterial signalling affecting microbial pathogenicity have attracted increasing attention [4].

In recent years there has been a significant increase in the incidence of Candida infections related to high morbidity and mortality as well as the growing number of infections caused by non-Candida albicans yeasts belonging to genus Candida [5]. Some non- Candida albicans yeasts are known to possess higher intrinsic antifungal resistance compared to C. albicans. Such epidemiological shift of Candida pathogens, when the infection incidents, caused by both Candida albicans and non-Candida albicans yeasts, made up approximately 50 % each, together with the increased incidence of these infections required rapid and precise methods of identification of Candida species [5, 6]. The comparative assessment of biochemical and molecular genetic methods of taxonomic identification of Candida species demonstrated that the latter are more accurate and faster than biochemical approaches [5]. For example, the Candida species with the least level of correct identification by biochemical methods (Auxacolor and Vitek 2) were C. glabrata and C. krusei, which are usually associated with high levels of antifungal resistance.

Thus, the rapid and precise identification of yeast pathogens may become critical for the start of treatment and antifungal therapy strategy [5, 6]. And the molecular genetic approaches, particularly PCR-based assays which often use rRNA genes and spacers as the genetic markers, may provide rapid, sensitive and accurate diagnostics of fungal pathogens.

The aim of our work was precise molecular genetic identification of human sputum yeast isolate MF22_1, allegedly belonging to Candida spp., by using multiple genetic markers including ribosomal operon regions.

Materials and methods

Yeast strain MF22_1 from the collection of D.K. Zabolotny Institute of Microbiology and Virology of National Academy of Sciences of Ukraine was originally isolated from the sputum sample during routine bacteriological examinations of a hospitalized cardiac patient in Kyiv, Ukraine in 2019. Initial biochemical and morphological examination of the isolate on site suggested that it belonged to Candida spp., however failed to provide its exact identification at the level of the species. In order to detail the taxonomic position of this yeast strain we performed its molecular genetic identification using four fragments of its chromosomal DNA, which were sequenced and deposited by us in GenBank NCBI database: 1) internal transcribed spacer -- ITS (GenBank OM479428, 548 bp), 2) 28S rRNA or large subunit -- LSU (OM479513, 607 bp), 3) 18S rRNA or small subunit -- SSU (OM4794321, 737 bp), and 4) RNA polymerase II gene -- RPB2 (OM524388, 1,217 bp). The primary structure of ITS fragment of the strain MF22_1 was used in our study as a reference sequence #1; combined ribosomal cluster sequences [ITS + LSU + 18S rRNA, 2814 bp] served as a reference sequence #2; and RPB2 sequence was a reference sequence #3.

The commonly available Internet database of the NCBI server: «Nucleotide collection» [http://www.ncbi.nlm.nih.gov/nuccore/] was used in this work. The part of the Nucleotide collection database containing information on the nucleotide structure of fungal DNA (taxid: 4751) was analyzed. Sequence alignment was performed using the software package of the same BLASTN server [http://blast.ncbi.nlm.nih.gov/Blast.cgi]. We used the basic settings of the BLASTN program without changes.

Results and discussion

Molecular genetic identification of yeast strain MF22_1 in this study was based on the analysis of the sequences of four genetic markers: ITS, LSU, 18S rRNA, and RPB2.

The ITS fragment of ribosomal operon is regarded as a genetic marker with the highest probability of successful identification for the broadest range of fungi, providing the most clearly defined barcode gap between inter- and intraspecific variation [7--9]. Thus, over decade, ITS region has been used as a universal DNA barcode marker for Fungi, the second largest kingdom of eukaryotic life [8]. In fungi, the existence of teleomorph and anamorph stages in their life cycles presents the fundamental problem of fungal taxonomy. Teleomorphic and anamorphic stages usually differ greatly in their phenotype, making it difficult to directly associate an asexual anamorph with a sexual teleomorph. Moreover, different strains of the same fungal species may manifest such differences in their phenotype, such as micro- and macro-morphology, or nutrients utilization and other physiological characteristics, often leading to their description as different species, ultimately leading to long lists of synonyms, which is very common, for example, for yeasts [10--12].

In addition to ITS region, other genetic markers from ribosomal ope- ron (large subunit -- LSU or 28S rRNA, and small subunit -- SSU or 18S rRNA) are also commonly used for fungal identification [8, 13, 14]. Thus, the nuclear ribosomal LSU is a popular phylogenetic marker in certain groups of microscopic fungi which had superior species resolution in some taxonomic groups (e.g. the early diverging lineages and the ascomycetous yeasts). However, this marker was otherwise slightly inferior to the ITS. Regarding the nuclear ribosomal SSU, it has poor resolution on the species and intra-species level in fungi.

ITS use as bar coding of fungal DNA helps to identify and link the anamorph and teleomorph stages of fungi, and thus reduce the multiple names of fungi. The ITS sequence includes 5'-end of 18S rRNA (or SSU) gene, ITS1, 5.8S rRNA gene, ITS2, and 3'-end of 28S rRNA (or LSU) gene, and in this work for strain MF22_1 is referred to reference sequence #1.

As a result of analysis of the GenBank Internet database (Nucleotide collection; Fungi) using the BLASTN (megablast) program we found over 5,000 available sequences of ITS region for genomic DNA fragments of representatives of fungi, which were similar to the reference sequence #1 for strain MF22_1. These sequences similarity ranged from 100 % to 77 %, and they were found in the fragments varying in molecular size from 136 bp up to 11848804 bp. It should be noted that some of the detected sequences similar in primary structure to reference sequence #1 for strain MF22_1 are not defined by species but only by higher taxa of the kingdom Fungi: classes, orders, families or genera (Fig. 1).

Fig. 1. Diagram of the taxonomic organization of 5,000 fungal ITS-sequences similar to the ITS sequence of yeast strain MF22_1 (reference sequence #1)

More than half (53 %) of all detected sequences, 2,642 sequences were found to be similar to ITS sequences belonging to fungi from 28 genera of the Pichiaceae family. Of these, 2,114 sequences (43 %) belonged to the strains of species Pichia kudriavzevii (synonym Issatchenkia orientalis) and its anamorphs, including the most famous anamorph Candida krusei (see Fig. 1). Thus, according ITS data, it can be suggested that the studied strain MF22_1 belongs to species Pichia kudriavzevii.

Pichia kudriavzevii and its anamorph Candida krusei are ubiquitous yeasts widely distributed in the environment [12, 15, 16]. They are often found in soil, fruits and various agricultural products, natural fermentations and foods. However it is not regarded as a food spoilage species, in contrary it is used as a cheese flavouring and ripening agent in the handcrafted fermented food production [15, 16]. Moreover, the strains of this species under the names Pichia kudriavzevii and its former synonyms Issatchenkia orientalis and Candida glycerinogenes are used in the biotechnology for industrial production of glycerol and succinate [12]. At the same time this species is of clinical importance being often isolated from humans and animals [12, 16].

In our study over two thousands yeasts in GenBank database, identified by ITS fragments as species P. kudriavzevii (see Fig. 1), were isolated from various sources: plants, soil, food, beverages, animal feed, industrial equipment, human and animal tissues, blood and secretions, from around the world, different regions and countries (e.g. Poland, Hungary, Finland, France, USA, Mexico, Brazil, Argentina, Nigeria, Tunisia, Ghana, Egypt, China, Japan, South Korea, Vietnam, Indonesia, Kazakhstan, and Australia).

Among the 50 strains with the most similar ITS primary structure (Qc = 100 %, I > 90 %), there were 20 type (permanent reference) strains of Pichia kudriavzevii from the collection of CBS (The Convention of Biological Diversity, Montreal, Canada). The localization of substitutions in ITS sequence of P. kudriavzevii MF22_1 was detected using the program BLASTN (MSA viewer). It was shown that most of substitutions are located outside the 5.8S rRNA gene locus.

If compared to other P. kudriavzevii strains, the changes in ITS sequence of the studied strain P. kudriavzevii MF22_1 were found in a number of nucleotide bases (17--18, 79, 110, 443 and 538). As mentioned above, many P. kudriavzevii strains with DNA and RNA sequences added to GenBank were clinical isolates e.g., from human secretions and blood.

For clinical specimens of human excretions, it was reported that Candida species in general were more frequently isolated from: 1) urine, 2) sputum/bronchial wash/bronchoalveolar lavage, and 3) vaginal swabs, comprising 39 %, 25 % and 11 %, respectively [5]. The greatest variety of Candida species that also included C. krusei was observed for the second kind of specimens.

The studied yeast culture MF22_1 was also isolated from human sputum. It was of interest to establish strain MF22_1 genetic relatedness with other isolated from humans P. kudriavzevii strains based on the similarity of a primary structure of ITS region. Among analysed GenBank ITS sequences of P. kudriavzevii, we found that 11.5 % sequences belonged to clinical isolates from the collections of the research departments specializing in medical studies of yeasts. That is P. kudriavzevii strains isolated from human stool samples were from the collection of Pasteur Institute of Tunis, Tunisia; isolates from human sputum samples were from the collection of Al-Neelain University, Sudan; human blood isolates were from the collection of Federal University of Sao Paulo, Brazil; and isolates from human bio-specimens were from the collection of Laboratorium Parasitologi, Universitas Indonesia, Jacarta, Indonesia.

ITS-fragment BLASTN alignment for studied isolate MF22_1 and other human-isolated P. kudriavzevii strains revealed that their structures can vary ranging from complete sequence identity to dozens of differences such as mismatches, gaps, unalignment regions. The typical results of this alignment are illustrated using some selected strains in Fig. 2. Such differences may depend on the collection where certain P. kudriavzevii clinical isolates are preserved. For example many P. kudriavzevii strains from the collections of Pasteur Institute of Tunis, Al-Neelain University and Federal University of Sao Paulo demonstrated complete identity (I = 100 %) of their ITS sequences to those of strain MF22_1 (reference sequence #1) and the genetically well-studied strain P. kudriavzevii CY902, isolated from grape skin in China (see Fig. 2).

At the same time, the collection of the University of Jakarta included the strains, whose primary ITS structures had the greatest number of differences compared to reference sequence. According to GenBank information, the strains of this Indonesian collection were identified as P. kudriavzevii using both ITS and LSU as genetic markers. For some strains (e.g. CK8, CK9, CK10, CK12) localization of the differences in ITS fragment structures were observed within probable 5.8S rRNA gene region which is known to contain a highly conserved DNA sequence (see Fig. 2). In general, most of the differences in the ITS structure of P. kudriavzevii strains of Indonesian collection were localized within sequences of ITS1 and ITS2 regions. The structural differences of the analyzed ITS of P. kudriavzevii strains from University of Jakarta collection significantly differed from those for not only studied strain MF22_1 (reference sequence #1), but also for P. kudriavzevii type strains and P. kudriavzevii strains isolated from various sources.

Fig. 2. Differences in ITS nucleotide sequences of Pichia kudriavzevii clinical isolates from different collections compared to (1) the studied strain P. kudriavzevii MF22_1 (reference sequence #1) and (2) isolated from grape skin strain P. kudriavzevii CY902

Based on the available data on P. kudriavzevii clinical isolates from analysed collections, it was not possible to draw the clear conclusion whether an actual source of isolation (blood, stool, sputum etc.) had an impact on the differences in the ITS region of these strains.

It should be noted that one of the reasons that the regions of riboso- mal operon are particularly attractive targets in phylogenetic studies on microorganisms is because multiple copies are present in each genome, and the genes have both conserved regions for designing broad-range primers and more variable regions for identification of fungi and bacteria [6, 17]. The presence of multiple ITS regions has been also revealed in genome of some P. kudriavzevii strains. For example, for the mentioned above strain P. kudriavzevii CY902, it was found the existence of 14 copies of ITS regions located on three chromosomes (Table). Chromosome III contains 10 copies of rRNA cluster. One of these copies (84432--84961 bp) differs from all other in tandem with 2 mismatches and 20 gaps (Qc = 96 %, I = 94 %).

Information on GenBank database Pichia kudriavzevii strains with highly similar SSU, ITS and LSU primary structure-to-the P. kudriavzevii MF22_1 reference sequence #2

The complete sequence of 18S rRNA gene (SSU, 1737 bp) and the partial sequence of 28S rRNA gene (LSU, 607 bp) were also obtained for the studied strain P. kudriavzevii MF22_1. Hence, along with P. kudriav- zevii MF22_1 ITS region sequence, they were compared to the sequences of entire ribosome cluster or its large fragment of other P. kudriavzevii strains in the analysis of their phylogenetic relationships. In total, the integrated P. kudriavzevii MF22_1 sequence of the ribosomal cluster contained 2814 bp (reference sequence #2). It should be considered that the data of databases may vary, and contain sequences of different molecular size, such as the fragments of individual genes (e.g. LSU, RPB2), the whole genes (e.g. 18S rRNA), the fragments containing sequences of several genes (e.g. ITS region) or whole chromosomes (e.g. P. kudriavzevii CBS573 chromosome IV: CP028776.1). A BLASTN analysis of the GenBank database (Nucleotide collection, Fungi) was carried out to search for the yeast strains with genomes containing the most similar sequences to the structure of the reference sequence #2 of P. kudriavzevii MF22_1. This database contains tens of thousands of P. kudriavzevii sequences of the fragments of different rRNA gene clusters. On the one hand, there are deposited small fragments of genes, such as 18S rRNA gene sequences of P. kudriavzevii strains DIC (JF715173.1, 559 bp) and Y4 (JF715171.1, 556 bp). On the other hand, only for a small number of strains in the database, there are the sequences of both analyzed genes SSU and LSU, as well as ITS region, which are presented as the sets of three separate fragments and also as a part of a complete chromosome sequence for some P. kudriavzevii strains (see Table).

Of the thousands of identified DNA sequences of the representatives of P. kudriavzevii species (or its synonyms Issatchenkia orientalis or Candida krusei) in the database, only 25 fragments of the rRNA clusters of 10 chromosomes had the Query coverage 100 % (see Table). The similarity of their primary structures to the reference sequence #2 ranged from 99.89 % to 99.79 %. In eukaryotes, rRNA genes clusters are known to exist as tandem multicopy repeats. The database sequences contain mostly 1--2 copies, but chromosome I of P. kudriavzevii CY902 contains a tandem of 10 copies (see Table).

A number of P. kudriavzevii strains, that demonstrated high similarity to the reference sequence #2, are the type strains of this species from the largest in the world culture collections such as CBS (Westerdijk Fungal Biodiversity Institute, Netherlands) and ATCC (American Type Culture Collection, USA).

It should also be noted that the sequences of individual copies on one chromosome differ in the number of discrepancies with the reference sequence #2 of P. kudriavzevii MF22_1. For example, one copy (83845-- 86640 bp) out of ten copies on chromosome II of P. kudriavzevii strain CY902 has a lower similarity with reference sequence #2 (99.3 %) compared to other copies (99.9 %).

In addition to the traditional analysis of ribosomal cluster sequences, a structure of nuclear gene RPB2, which encodes the second largest subunit of RNA polymerase II, is also used in phylogenetic studies of fungi. The reasons are that this is a single-copy gene of large molecular size, and is characterized by a low rate of evolutionary changes, which is a prerequisite for providing a better understanding of the evolution of fungi, and a more precise definition of these microorganisms taxonomic relationships [18-20].

In our study, the obtained sequence of DNA-dependent RNA polymerase II for isolate P. kudriavzevii MF22_1 served as reference sequence #3 for RPB2 analysis. BLASTN analysis of the Internet database (Fungi) revealed 191 fungal sequences with only 21 sequences of strains that belong to family Pichiaceae. Of them, only six sequences of representatives of the species P. kudriavzevii from different collections were found. These six RPB2 sequences manifested the highest similarity to the reference sequence #3 (Qc = 100 %, I = 99.59-98.77 %), including one sequence belonging to the type culture of the species P. kudriavzevii CBS573 (Qc = 100 %, I = 99.59 %).

It was established that there are just a few differences in the primary structure of the RPB2 gene of P. kudriavzevii MF22_1 from the sequences of such gene of the representatives of P. kudriavzevii species (Fig. 3). At the same time, the number of differences was significantly greater when RPB2 sequence for P. kudriavzevii MF22_1 was aligned with RPB2 sequences of strains of other Pichia species (see Fig. 3).

Fig. 3. Localization of differences in a nucleotide sequence of RPB2 gene of a number of selected strains of family Pichiaceae, if compared to the reference sequence #3 for (1) RPB2 of P. kudriavzevii MF22_1; (2) P. kudriavzevii CBS573; (3) P. kudriavzevii CY902; (4) P. kudriavzevii CW-CM; (5) P. kudriavzevii CBS5147; (6) P. kudriavzevii SJP; (7) P. norvegensis CBS 8403; (8) Candida sorboxylosa NRRL Y-17669; (9) Kregervanrija fluxu- um NRRL YB-4273

Thus, the BLASTN analysis established that all four sequenced genetic markers: ITS, SSU, LSU, and RPB2 of the studied isolate MF22_1 had the maximum similarity to the corresponding sequences of type strains of P. kudriavzevii species and consequently the minimum number of nucleotide bases which are distinct from them.

Currently both names of this species: teleomorph P. kudriavzevii and anamorph C. krusei are still used in modern scientific literature mainly because name Candida krusei is still widely used in clinical routine and medical research. Presented by polyphyletic group of species, the genus Candida has served as a «dumping ground» for ascomycetous budding yeasts not forming ascospores [21]. After introduction of molecular genetic methods of yeasts identification, almost all clades of teleomorphic ascomycetous yeasts include representatives of Candida spp. As soon as an ascosporic state of Candida species is discovered, this species should be recognized as a member of a teleomorphic genus and named appropriately; and in our case C. krusei is recommended to be named P. kudriavzevii [12, 21].

Candida krusei is a common clinical isolate responsible for about 2 % of yeast infections caused by Candida species in humans. However, this species is characterised by a combination of, on one side, resistance of this species to azoles with, on the other side, difficulties with its precise taxonomic identification by conventional biochemical and physiological approaches (e.g. Vitek) [5, 12]. Phylogenetic analysis did not distinguish clinical and environmental isolates into separate clades, meaning that P. kudriavzevii/ C. krusei infections can be easily acquired from the environment [12]. Therefore, the special caution may be needed in the wide use of drug-resistant P. kudriavzevii strains in biotechnology and food applications.

Thus, the use of multiple genetic markers including ITS (OM479428), 28S rRNA (OM479513) and 18S rRNA (OM4794321) of ribosomal ope- ron, as well as RNA polymerase II gene -- RPB2 (OM524388) allowed to perform precise taxonomic identification of yeast isolate MF22_1 from human sputum at the species level. It was established that the isolate MF22_1 belonged to Pichia kudriavzevii (anamorph Candida krusei) which is considered as clinically important species. Comparative analysis of a primary structure of ITS region demonstrated the great similarity of the ITS sequence of Pichia kudriavzevii MF22_1 to the majority of other Pichia kudriavzevii clinical isolates from human sputum, stool, blood etc. in different culture collection around the world. There was no clear correlation between the ITS sequences of Pichia kudriavzevii clinical isolates and the type of material sampled from humans.

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МОЛЕКУЛЯРНО-ГЕНЕТИЧНА ІДЕНТИФІКАЦІЯ ІЗОЛЯТУ ДРІЖДЖІВ MF22_1

М.О. Фоміна, О.Я. Філіпішена, Л.В. Поліщук

Інститут мікробіології і вірусології імені Д.К. Заболотного Національної академії наук України

Мікроскопічні гриби, включно дріжджі, є важливою частиною мікробіоти людини. Серед них дріжджі роду Candida дуже поширені і часто становлять переважну частину мікобіоти. Candida spp., включно Candida albicans і види кандид non-albicans, можуть бути умовно-патогенними мікроорганізмами, що уражують людей із пригніченою імунною системою. Проблема з деякими видами кандид non-albicans, наприклад C. glabrata і C. krusei, полягає в тому, що вони є високорезистентними до протигрибкової терапії та, разом з тим, їх складно точно ідентифікувати на видовому рівні загальноприйнятими в клініці біохімічними методами. Метою цієї роботи була точна молекулярно-генетична ідентифікація за допомогою кількох генетичних маркерів дріжджового ізоляту MF22_1 з мокротиння людини, який за попередніми даними імовірно належав до Candida spp. Ми використали чотири фрагменти його хромосомної ДНК, секвеновані та депоновані нами в базі даних GenBank NCBI: 1) ITS (GenBank OM479428, 548 п.н.), 2) 28S рРНК (OM479513, 607 п.н.), 3) 18S рРНК (OM4794321, 737 п.н.) і 4) ген РНК-полімерази II - RPB2 (OM524388, 1217 п.н.). Частину бази даних колекції нуклеотидів [http://www.ncbi.nlm.nih.gov/nuccore/], що містить інформацію про нуклеотидну структуру ДНК грибів (таксид: 4751), було досліджено за допомогою BLASTN [http://blast.ncbi.nlm.nih.gov/Blast.cgi]. BLASTN аналіз показав, що всі чотири секвеновані генетичні маркери: ITS, 28S рРНК, 18S рРНК і RPB2 досліджуваного ізоляту MF22_1 мають максимальну схожість з відповідними послідовностями типових штамів виду Pichia kudriavzevii. Таким чином, було встановлено, що ізолят належить до виду Pichia kudriavzevii, анаморфою якого (або його неаскоспоровим станом) є Candida krusei. Тей всюдисущий у навколишньому середовищі вид є звичайним клінічним ізолятом, відповідальним за приблизно 2 % випадків дріжджових інфекцій, спричинених видами роду Candida у людей. Порівняльний аналіз первинної структури ділянки ITS продемонстрував велику подібність послідовності ITS Pichia kudriavzevii MF22_1 до більшості інших клінічних ізолятів Pichia kudriavzevii з мокротиння, калу, крові тощо, які зберігаються в різних колекціях культур установ, що спеціалізуються на медичних дослідженнях дріжджів, у всьому світі. Для цих клінічних ізолятів Pichia kudriavzevii не спостерігалося чіткої кореляції між послідовностями ITS і типом біоматеріалу, відібраного у людей.

Ключові слова: Candida spp., дріжджі, ідентифікація, ділянка ITS, 18S rRNA, 28S rRNA, RPB2.

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