Inteins are invasive genetic elements that occur as intervening sequences in conserved coding host genes. They are transcribed and translated with the flanking host protein sequences and then self-excised by protein splicing. The flanking protein sequences (exteins) are joined by a peptide bond, constituting the functional protein (Chong et al., 1996; Perler, 2005).

Over the past three decades, inteins have been detected mainly in unicellular microorganisms in the three domains of life and in viruses (Perler, 2002). Among the Eukarya domain, inteins are found mostly in fungi, some green algae and other basal eukaryotes (Liu, 2000; Butler et al., 2001, 2006). In a recent review, 2729 genomes of bacteria, 345 of archaea and 6648 of eukarya were analyzed (Topilina et al., 2015b) and 24, 47, and 1.1% of these genomes, respectively, presented at least one intein.

Inteins are usually found at conserved sites of housekeeping proteins that have vital functions in the cell, such as DNA and RNA polymerases, aminoacyl tRNA synthetases, recombinases, topoisomerases, helicases and essential components of the spliceosome (Novikova et al., 2014). Some hypothesis for this distribution already exist and are in part supported by some evolutionary scenarios, including probable horizontal transfer, as well as genetic mobility of inteins by homing endonuclease, which means that the spread and increase of this element in populations is due to a gene conversion process by homologous recombination, rather than any selective advantage (Nagasaki et al., 2005; Gogarten and Hilario, 2006; Swithers et al., 2009). This idea rendered to inteins the title of “parasitic” genetic elements during the past 25 years, although some domesticated inteins, such as HO gene of Saccharomyces cerevisiae showed to have “gained” a function in the cell biology. In this specific case, the HO gene encodes for an endonuclease responsible for mating type conversion in yeast and its splicing domain is not active anymore (Koufopanou and Burt, 2005).

Nevertheless, recent works have evidenced a possible function for inteins in the post-translational regulation of gene expression. For instance, the splicing of SufB intein of Mycobacterium tuberculosis showed to be inhibited by reactive oxygen and nitrogen species (ROS and RNS) when expressed in Escherichia coli. These stressful conditions are also experimented by M. tuberculosis inside the macrophage (Topilina et al., 2015a). Other evidence came from the splicing modulation of RadA intein from the hyperthermophilic archaeon Pyrococcus horikoshii according to the temperature, solution conditions and remote extein point mutations. So, RadA intein might function as an environmental sensor, releasing the intein for full activity only at optimal growth conditions for the native organism (high temperatures), while sparing ATP consumption under cold-shock. The authors observed intein splicing at low temperature only after adding a combination of the detergent SDS and the ionic liquid 1-butyl-3-methylimidazolium chloride, showing that, besides temperature, other factors may also interfere in RadA intein splicing (Topilina et al., 2015b).

Although the experimental evidence for the intein’s role in modulation of gene expression is based on a non-native context (different extein and different host cell), it is an important clue for the actual functionality of these elements in nature. Their presence in particular conserved motifs might be explained by an adaptive process (Novikova et al., 2014). Novikova et al. (2015) observed that inteins have a certain “preference” for specific functional domains of related housekeeping proteins, like ATPase domains for example, and this does not entirely fit to the models describing inteins as merely mobile parasitic elements. The authors argue that this intein distribution might be a result of a selective retention of these elements, which might be beneficial under certain environmental stresses. So, the sporadic nature of intein in closely related species could be explained by different environmental stresses. If there is no strong selective pressure for intein maintenance in a certain subpopulation and its presence reduces the adaptive value of the host microorganism, the intein-free alleles will increase in this population by means of natural selection (Gogarten and Hilario, 2006).

Nevertheless, we are far from a global understanding about the reason for intein persistence in different housekeeping host proteins, only in unicellular organisms, over millions of years. It seems consensual that their maintenance may be due to either their parasitic nature, since they are invasive elements, as well as to a possible role in specific physiological conditions of the host cell. Yet, among the mobile elements, inteins are the least studied, so that few inteins have already had their activity tested under different conditions; and therefore their real function is still a puzzle. Regarding that most of the produced knowledge on genome organization during the last years has been changing the status of many mobile genes, mainly retrotransposable elements, which are intensively studied, from parasitic entities to dynamic elements involved in genome evolution and gene expression (Mita and Boeke, 2016), it seems plausible that intein’s function in cell deserves more scientific investigation. The more we understand about inteins, the better we can explore their biotechnological applicability.

Few fungal inteins have been described, most of them are located in PRP8 (precursor mRNA protein) and VMA (vacuolar ATPase) coding genes, others can be observed in some RNA Polymerase subunits, GLT1 (glutamate synthase), CHS2 (chitin synthase) and THRRS (threonyl-tRNA synthetase) coding genes (Poulter et al., 2007). Most fungal inteins are present in the PRP8 and VMA genes. The PRP8 intein is widely dispersed in Fungi Kingdom (fungi containing this intein are found in different phyla), being present in many important human pathogens, such as Cryptococcus neoformans, Cryptococcus gattii, Histoplasma capsulatum, Emmonsia spp., Paracoccidioides spp., Blastomyces dermatitidis, Aspergillus nidulans, Aspergillus fumigatus (Liu and Yang, 2004; Butler and Poulter, 2005; Butler et al., 2006; Theodoro et al., 2010). The VMA intein, on the other hand, is specifically distributed among Saccharomycetes, such as Saccharomyces, Zygosacchromyces and some Candida.

Here we updated the information on the intein distribution among Candida species and explored their potential as a source of phylogenetic information and, therefore, as species-specific diagnostic tool. Besides, the possible impact of the presence of these elements in housekeeping genes in Candida species life style was discussed raising their promising application as drug targets for most of the medically relevant non-albicans emergent Candida species.

Inteins come in three configurations: full-length, mini and split (Figure 1A). The full-length inteins have a homing endonuclease domain (HE) splitting the splicing domain in N and C-Spl terminals. When active, the HE recognizes a cognate allele, without the intein, performs a double strand break (DSB) and, by homologous recombination, copies the intein using the intein-containing allele as template for DNA repair. Mini-inteins lack the HE domain and have a continuous splicing domain, while split-inteins are mini-inteins, whose N and C-Spl terminals are transcribed and translated with different exteins. When the N and C-Spl terminals are assembled, the intein suffers a trans-splicing reaction, ligating the different exteins (Wood et al., 1999; Volkmann and Iwaı, 2010).

Intein mobility is triggered by its HE domain which recognizes long, and therefore specific, DNA sequences (14–40 bp) lacking the intein, producing a DSB. The host repair system is then activated and the template containing the intein is “copied” to the recipient chromosome by a gene conversion event (Figure 1B), distorting the Mendelian segregation, causing a Super-Mendelian inheritance or a rapid dissemination and fixation of the intein in population. When all alleles of a certain population are occupied by the intein there is no selection pressure for functional HE, because both, functional and non-functional HEs will be equally maintained in population simply by cellular propagation and the HE domain is “free” to degenerate (Figure 1C). That is why the HE domain is expected to have more sequence variation, including its complete degeneration and/or deletion, than the splicing domain (Burt and Koufopanou, 2004; Koufopanou and Burt, 2005; Gogarten and Hilario, 2006).

Inteins are particularly interesting as a source of phylogenetic information because they present the well-preserved splicing domain (N and C terminals), with the conserved motifs A, B (for the N-splicing) and F, G (for the C-splicing), flanking the central and more variable HE domain, in the full-length inteins. The presence of the motifs C, D, E, and H, as well as the two aspartic acid residues, one in C and other in E, in HEs from LAGLIDADG family (the most common in fungal inteins), indicate that the HE domain is probably active (Gimble and Thorner, 1992; Liu, 2000; Posey et al., 2004). In mini-inteins, HE domains are supposed to be completely deleted. In addition, their location in highly conserved genes facilitates the design of primers for their amplification (Butler and Poulter, 2005; Theodoro and Bagagli, 2009; Prandini et al., 2013).

Besides its potential as molecular markers, inteins have been largely used for biotechnological purposes, accordingly to their functional mechanisms. The splicing domain, for example, is explored for purification of recombinant proteins, cyclization of proteins, protein labeling, production of selenoproteins (Miraula et al., 2015) and, for pathogenic microorganisms, it has been studied as a drug target (Paulus, 2007; Zhang et al., 2010). The HE domain has also been intensively studied as a biotechnological tool, in this case for genome editing, since these enzymes can be reprogrammed to specifically cut a genome site and deliver a gene of interest by homologous recombination. The applications of this technology are numerous for different social-economic fields, such as gene therapy for monogenic diseases, insect vector control (already developed for malaria mosquitoes) and gene delivery for transgenic plants, such as maize (Belfort and Bonocora, 2014).

Candida species belong to Ascomycota phylum, Hemiascomycetes class and Saccharomycetales order. These species are distributed in three different clades making Candida a non-monophyletic group (Diezmann et al., 2004). With the exception of C. glabrata, which is in clade 3, together with C. castelli, C. norvegica, Saccharomyces cerevisiae and other biotechnologically important yeast species, the most relevant clinical species of Candida are clustered in clade 1, which includes C. albicans, C. parapsilosis, C. orthopsilosis, C. metapsilosis, C. tropicalis, and C. viswanathii, as well as two non-pathogenic species, Candida maltosa and Lodderomyces elongisporus. C. guilliermondii, C. intermedia, C. famata, and C. zeylanoides are clustered in clade 2. The clades 1 and 2 are also referred as CUG group by distinctly translate this codon as serine instead of the standard leucine. Whether this attribute may play a hole in pathogenicity is still unknown (Diezmann et al., 2004).

Candida spp. can colonize human tissues in different ways, varying from a normal microbiota in the intestinal mucosa, contiguously to the vaginal mucosa, to a true pathogen. The transition to pathogenicity in Candida species, mainly in C. albicans, occurs generally due to the host immunological condition, leading to a microbiota unbalance, which associated with important virulence factors, such as biofilm formation and hydrolytic enzymes production, promotes fungal dissemination to other sites and organs. The clinical manifestations vary from a localized mucosa or skin infection to disseminated disease (Lagunes and Rello, 2016).

Although less common, disseminated candidiasis has significantly increased over the last 15 years, affecting mainly patients under debilitating and immunosuppressive conditions. Data from Centers for Disease Control and Prevention (CDC) and the National Healthcare Safety Network, show that Candida species are the fifth pathogen related to nosocomial diseases and fourth pathogen among bloodstream infections – BSI (Wisplinghoff et al., 2004; Pfaller and Diekema, 2007). These nosocomial Candida infections are related to increment of invasive procedures and also to the intensive use of broad-spectrum antimicrobials, particularly in patients admitted in Intensive Care Units – ICU (Colombo et al., 2013).

About 95% of diagnosed fungal infections are related to Candida spp., mainly involving the species C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. lusitaniae, C. dubliniensis, C. pelliculosa, C. kefyr, C. lipolytica, C. famata, C. inconspicua, C. rugosa, and C. norvegensis, but C. albicans is still the most frequent species causing infection (Yapar, 2014). However, Candida infections caused by non-albicans Candida spp. have been increasing in the last few years (Richardson and Lass-Flörl, 2008). In a recent epidemiological analysis, non-albicans species accounted for more than 50% of all cases of invasive candidiasis in patients enrolled in the Prospective Antifungal Therapy Alliance, a sentinel surveillance network comprising 23 medical centers in the US and two in Canada. The non-albicans species identified were C. glabrata (46.4%), C. parapsilosis (24.7%), C. tropicalis (13.9%), C. krusei (5.5%), C. lusitaniae (1.6%), C. dubliniensis (1.5%) and C. guilliermondii (0.4%) (Pfaller et al., 2014). While C. albicans infections have been progressively replaced by C. glabrata infections in North America and in many European countries, C. parapsilosis or C. tropicalis are the most frequent non-albicans species in Latin America (Nucci et al., 2010; Cleveland et al., 2012; Lockhart et al., 2012). The central factor for the emergence of these non-albicans Candida spp. seems to be the undiscriminating use of antifungal drugs (Yapar, 2014).

The treatment of Candida infections, mainly candidemias, is based on antifungal agents that interfere on different metabolic pathways being fungicide, such as polyenes, azoles and equinocandis, or fungistatic, such as nucleoside analogs (Lopez-Martinez, 2010; Maubon et al., 2014). The polyenes act on the ergosterol, increasing the permeability of cell membrane by producing aqueous pores; the azoles inhibit the ergosterol biosynthesis and the echinocandins inhibit the synthesis of 1,3 β-glucan, a polysaccharide responsible for the cell wall integrity; and the nucleoside analogs inhibit DNA synthesis being used in combination with amphotericin B or fluconazole (Spampinato and Leonardi, 2013; Paramythiotou et al., 2014), which are the usual chosen drugs for treatment of Candida infections (Paramythiotou et al., 2014). The antifungal susceptibility can be easily accessed by Clinical and Laboratory Standards Institute (CLSI) reference microdilution method and also by commercially available antifungal susceptibility testing systems, such as the Sensititre YeastOne colorimetric panel (TREK Diagnostic Systems, Cleveland, OH, USA) (Pfaller et al., 2012).

Monitoring changes in antifungal drug resistance is as important as determining the incidence of candidemia by different Candida species. A recent report of surveillance for candidemia in Atlanta, GA, USA and Baltimore, MD, USA over a 5-year period, showed a shift in the species distribution among causative organisms, with a significant increase in C. glabrata, as well as in its resistance to echinocandin and fluconazole (Wang et al., 2012; Cleveland et al., 2015). This emergence of antifungal resistance, due to inappropriate prescriptions (Sardi et al., 2013), and the usual toxic side effects of some of these drugs strengthen the demand for new therapeutic targets.

The fungal systemic disease caused by Candida is an important cause of mortality, mainly in nosocomial infections and, since the antifungal susceptibility may vary among the different species (Bassetti et al., 2015), the correct diagnosis at species level is necessary. This can be accomplished by classical phenotypic methods, biochemical and physiological tests, which have automated versions, such as Vitek 2 ID – YST (bioMerieux), (Higashi et al., 2015). However, molecular epidemiological studies using different molecular markers (25S group I intron, rDNA D1/D2 region and ITS1-5.8S-ITS2) have revealed intraspecific variation in C. albicans and non-albicans Candida species and its correlation to antifungal susceptibility (Miletti and Leibowitz, 2000; Karahan et al., 2004; Steuer et al., 2004; Fahami et al., 2010; Gurbuz and Kaleli, 2010; Merseguel et al., 2015).

Besides intraspecific variation, molecular markers have also pointed out the existence of cryptic species in C. parapsilosis, actually composed by three species: C. parapsilosis, C. metapsilosis and C. orthopsilosis (Tavanti et al., 2005), which present differences in virulence and antifungal response. C. parapsilosis and C. orthopsilosis are more virulent in reconstituted human tissues models and the minimal inhibitory concentration values (MIC) of amphotericin B, caspofungin, anidulafungin and micafungin for C. orthopsilosis and C. metapsilosis isolates were significantly lower than those for C. parapsilosis (Gacser et al., 2007; Lockhart et al., 2008).

The correct recognition of close fungal genotypes or cryptic species would improve the treatment; however, this is not achieved by the usual biochemical methods available in most routine laboratories, mainly in developing countries. The distinction of these very close Candida species requires molecular techniques such as PCR- RFLP of SADH gene (Tavanti et al., 2005), quantitative PCR (qPCR) (Hays et al., 2011; Souza et al., 2012), pyrosequencing (Borman et al., 2009), microsatellite analysis (Lasker et al., 2006) or matrix-assisted laser desorption ionization–time (MALDI-TOF MS analysis) (Quiles-Melero et al., 2012). This last technique is based on the mass spectrum analysis of crude protein cell extract and requires validated databases for the achievement of rapid and reliable pathogen identification. Recent researches have created databases for the identification of bloodstream yeasts and showed practically 100% accuracy in distinguishing medically important species, such as C. tropicalis, C. parapsilosis, C. pelliculosa, C. orthopsilosis, C. albicans, C. rugosa, C. guilliermondii, C. lipolytica, C. metapsilosis, C. nivariensis (De Carolis et al., 2014; Ghosh et al., 2015). The main apparent disadvantage of this technology is the up-front cost of purchasing a MALDI-TOF MS instrument; however, it is offset in about 3 years, providing a noteworthy long-term cost saving for the laboratory (Tran et al., 2015). Some authors have also pointed out the relatively low analytical sensitivity of the method, as well as the few advances for distinguishing filamentous fungi, when compared to the yeast databases (Bailey et al., 2013).

In this scenario, intein research is particularly interesting because they might be a valuable additional or alternative molecular markers for species identification, such as observed for Cryptococcus spp., Paracoccidioides spp., Histoplasma capsulatum, and Candida spp. (Butler and Poulter, 2005; Theodoro et al., 2008; Prandini et al., 2013; Theodoro et al., 2013; Satish Kumar and Ramesh, 2014) and also, since they are usually present in housekeeping genes and absent in multicellular eukaryotes, they can be explored as drug targets, because protein splicing inhibition would make the host protein non-functional (Paulus, 2003; Liu and Yang, 2004). However, drug screening for the inhibition of intein splicing has only been carried out for bacterial pathogens, such as M. tuberculosis. The intein MtuRecA was inserted in the GFP (green fluorescent protein) coding sequence and its splicing efficiency was evaluated according to the fluorescence emission. More than 85 thousand compounds were tested. Some electrophilic compounds, such as cisplatin inhibited the intein MtuRecA splicing by blocking the intein’s first N-Spl cystein residue (Paulus, 2007; Zhang et al., 2010).

Three host proteins, considered essential for cell physiology, have been observed containing inteins in Saccharomycetales species: vacuolar ATPase (VMA), threonyl-tRNAsynthetase (ThrRS) and glutamate synthase (GLT1) (Poulter et al., 2007).

In order to update the distribution of these inteins in Candida, we carried out a search for inteins sequences in 81 sequenced genomes of 30 Candida species (Supplementary Table 1). Public Contigs deposited into MycoCosmosDB and NCBI WGS Databases were downloaded. By using GetORF program, all genomes were converted into six frames amino-acids sequences, for their ORFs deduction. Using those sequences, a local database was created, using NCBI Local BLAST+ (v.3.1.4) program in a UNIX system. Finally, sequences of VMA, ThrRS, GLT1 inteins were blasted to this database. This analysis revealed inteins in VMA, ThrRS and GLT1 proteins in Candida species, in which they have not been described before.

The VMA, ThrRS and GLT1 inteins are present in 14, 6, and 3 Candida species, respectively. The differences among Candida species concerning intein presence and/or size polymorphism (Table 1) can be easily accessed as molecular markers for species differentiation.

Some Candida species have more than one intein in their genome. The species C. maltosa, C. metapsilosis and C. orthopsilosis present both VMA and ThrRS inteins and C. famata presents both VMA and GLT1 inteins (Table 1). No inteins were found in C. albicans, C. arabinofermentans, C. auris, C. boidinii, C. dubliniensis, C. succiphila, C. tanzawaensis, C. tenuis, C. infanticola, C. krusei, C. lusitaniae, and C. ciferri.

Inteins are not exclusively found in clinically relevant Candida species, some of the intein containing species listed on Table 1 are frequently isolated from environmental sources. For instance, C. apicola is found in wine and cachaça fermentation processes (Vega-Alvarado et al., 2015), C. homilentoma is commonly associated to insects (Yun et al., 2015), C. sorboxylosa was originally found in fruits and described as close related to C. krusei (Nakase, 1971) and C. sojae can be isolated from water-soluble substances of defatted soybean flakes (Nakase et al., 1994).

Some environmental species presenting inteins, listed on Table 1, have been rarely reported in invasive candidemia in human. This is the case of C. intermedia, isolated from soil, beer, grapes and also present on skin, throat, or animal feces (Ruan et al., 2010), as well as C. famata (Debaryomyces hansenii), which is commonly found in natural substrates and in various types of cheese and accounts for 0.08–0.5% of isolates recovered during invasive candidiasis. C. famata is sometimes misidentified as C. guilliermondii, which has a variety of environmental sources and is a common constituent of the normal human microbiota, being associated with only 1–2% of candidemias (Desnos-Ollivier et al., 2008). The species C. castellii, C. nivariensis and C. bracarensis belong to Nakaseomyces genus (Kurtzman, 2003), being closely related to C. glabrata. The high genetic proximity between C. nivariensis or C. bracarensis and C. glabrata may have caused their misidentification as C. glabrata (Alcoba-flórez et al., 2005; Correia et al., 2006; Bishop et al., 2008), so that their emergent character may have been due to recent advances in molecular epidemiology tools. The ecological niches of these species are still poorly understood. C. nivariensis has been isolated from flowering plants in Australia, indicating a possible environmental source for human infections (Lachance et al., 2001). Despite their environmental aspect, C. nivariensis and C. bracarensis show an expansion in EPA genes, a family of glycosylphosphatydylinositol (GPI)-anchored cell-wall protein, considered an important virulence factor for emergent pathogens, such as C. glabrata (Gabaldón et al., 2013).

In this review, the list of intein containing Candida species was updated, as well as their sequence features and phylogenetic relationships. The horizontally transferred nature of VMA inteins was corroborated by our phylogeny. The possible lateral transfer of inteins from C. reinhardtii (RPB2 gene) and P. anserina (CHS2 gene) to THRRS and GLT1 genes, respectively, of Candida species, might be very ancient, since no significant deviation of RSCU was observed between these inteins and their respective exteins. Besides the peculiar evolutionary history of the inteins, their presence or absence, as well as their polymorphic sizes, should also be explored as molecular markers for species or cryptic species recognition, assisting the diagnosis and the therapeutic choices, since different species diverge in their clinical and antifungal susceptibility aspects.

The recent discussions about whether inteins are parasitic genetic elements or post-translational expression modulators gives rise to important questions: Can the splicing efficiency of an intein be altered under different life-style conditions of opportunistic or pathogenic fungal species? Can inteins regulate gene expression in different ways during infection? Here we reviewed the importance of the VMA, ThrRS and GLT1 genes in the yeast cell physiology. Besides acidifying vacuoles, the VMA protein can also be important for fungal filamentation, an important virulence feature in C. albicans. Can the presence of an intein in the VMA protein modulate its function in the intein-containing Candida species, interfering in their filamentation capacity? The ThrRS protein is essential for protein synthesis in cell; the splicing inefficiency of its intein would prevent cell proliferation and maintenance. This protein is inhibited by some drugs and the presence of an intein in some species would add another way to target it, by inhibiting its intein splicing. The GOGAT protein, coding by GLT1 gene, on the other hand, is not considered so essential because it is part of one out of three possible pathways to synthetize glutamate, however, since this pathway seems to be largely used in some Candida species, the GLT1 intein could also constitute an important therapeutic target for those non-albicans Candida species that contain it.

In order to address the aforementioned questions many experimental researches must be done, clarifying the actual role of inteins in fungal pathogens and possibly opening new prospects for antifungal drugs researches. The use of inteins as a new drug target is, clearly, limited to the species in which they are present in at least one protein, while their absence/presence should also be explored as a molecular marker. The merit of this discussion can be extended for other pathogenic fungi, besides Candida spp., that contain inteins in housekeeping genes, and may open a new study field in medical mycology concerning invasive genetic elements as tools for drug screening and diagnosis.

JF, TP, and RT conceived, designed, did the literature review, provided and wrote the manuscript. JF carried out the search for inteins in Candida genomes and the RSCU analysis. TA, MC, and JG reviewed the taxonomy and epidemiology of Candida species. TA, MC, JG, and EB assisted in the preparation, final review, and co-wrote the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.