Abstract Background Dermatophytes, the most common cause of fungal infections, affect millions of individuals worldwide. They pose a major threat to public health because of the severity and longevity of infections caused by dermatophytes and their refractivity to therapy. Trichophyton rubrum (T. rubrum), the most common dermatophyte species, is a promising model organism for dermatophyte research. Post-translational modifications (PTMs) have been shown to be essential for many biological processes, particularly in the regulation of key cellular processes that contribute to pathogenicity. Although PTMs have important roles, little is known about their roles in T. rubrum and other dermatophytes. Succinylation is a new PTM that has recently been identified. In this study, we assessed the proteome-wide succinylation profile of T. rubrum. This study sought to systematically identify the succinylated sites and proteins in T. rubrum and to reveal the roles of succinylated proteins in various cellular processes as well as the differences in the succinylation profiles in different growth stages of the T. rubrum life cycle. Results A total of 569 succinylated lysine sites were identified in 284 proteins. These succinylated proteins are involved in various cellular processes, such as metabolism, translation and epigenetic regulation. Additionally, 24 proteins related to pathogenicity were found to be succinylated. Comparison of the succinylome at the conidia and mycelia stages revealed that most of the succinylated proteins and sites were growth-stage specific. In addition, the succinylation modifications on histone and ribosomal proteins were significantly different between these two growth stages. Moreover, the sequence features surrounding the succinylated sites were different in the two stages, thus indicating the specific recognition of succinyltransferases in each growth phase. Conclusions In this study, we explored the first T. rubrum succinylome, which is also the first PTM analysis of dermatophytes reported to date. These results revealed the major roles of the succinylated proteins involved in T. rubrum and the differences in the succinylomes between the two major growth stages. These findings should improve understanding of the physiological and pathogenic properties of dermatophytes and facilitate future development of novel drugs and therapeutics for treating superficial fungal infections. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3977-y) contains supplementary material, which is available to authorized users. Keywords: Post-translational modification (PTM), Lysine succinylation (Ksucc), Dermatophyte, Trichophyton rubrum (T. rubrum) Background Post-translational modifications (PTMs) are an efficient strategy to extend the diversity of protein functions, and they play important roles in regulating multiple cellular events in both prokaryotic and eukaryotic organisms [[39]1, [40]2]. Lysine is a frequently modified amino acid residue because it is important in protein folding. The modification of lysine promotes the transformation of the spatial structures and chemical properties of proteins, hence affecting protein functions [[41]3, [42]4]. Lysine succinylation (Ksucc) was first identified in E. coli in 2011 [[43]4]. Ksucc-modified proteins have been reported to participate in a wide range of important cellular processes, such as the regulation of metabolism [[44]5]. Dermatophytes are a group of filamentous fungi that cause infections and are among the most common causes of human diseases, affecting nearly 20% of the global population [[45]6]. Examples of dermatophyte-caused infections are tinea pedis and tinea capitis. Furthermore, deep dermatophytosis caused by dermatophyte infections has also been reported, in which the infection penetrates the skin barrier and reaches internal tissues and organs [[46]7]. Although dermatophyte-caused infections rarely cause death, their prevalence, high incidence, difficulty to treat and contribution to morbidity represent a significant unsolved global public health problem [[47]8]. T. rubrum is the major causative agent of dermatomycoses, which accounts for more than 60% of dermatophyte infections [[48]9]. T. rubrum causes widespread infections because of its good vitality, that it remains viable for more than 6 months in the environment [[49]10]. T. rubrum has a non-sexual life-cycle, comprising a tear-shaped conidia stage and a hyaline septate mycelia stage. Conidia protect the T. rubrum genome during adverse environmental conditions, and infection is triggered when the conidia adhere to the corneum of the skin and form mycelia. The longitudinally growing mycelia penetrate deep into the corneum and cause skin damage [[50]11, [51]12]. Because of its growth features and prevalence, T. rubrum has been considered a good model system to use in the study of human pathogenic filamentous fungi [[52]11, [53]13]. Although a PTM analysis has not been conducted for T. rubrum and other dermatophytes, accumulating evidence has suggested that PTMs are essential for the growth and development of fungi, particularly in the regulation of key cellular processes that contribute to fungal pathogenicity. For example, the role of N-glycosylation in fungal pathogenesis has been validated. The α-1, 6-mannosyltransferase Och1 initiates the formation of a distinct branch on the N-glycan core, which allows for the subsequent addition of mannosylated outer chains. Och1 mutants in C. albicans display major cell wall defects and decreased virulence [[54]14]. In another case, protein O-mannosyltransferases (PMTs) initiate O-mannosyl glycan biosynthesis in the endoplasmic reticulum (ER). Triple PMT mutants are lethal in S. cerevisiae [[55]15], and the loss of PMTs leads to attenuated virulence in both C. albicans and C. neoformans [[56]16–[57]19]. In addition, the regulation of ubiquitination in fungal pathogens is involved in stress adaptation, metabolism, morphogenesis and other developmental processes. The pathways regulated by ubiquitination are fundamentally essential processes that are important for fungal virulence [[58]20]. Therefore, PTM analysis in T. rubrum should improve understanding of the regulatory strategies used in essential physical processes and their contributions to infections. This information should be informative for the development of drugs to treat infections caused by these clinically important fungi. This study sought to systematically identify the succinylated sites and proteins in T. rubrum and to reveal the roles of the succinylated proteins involved in cellular processes and the differences in the succinylation profiles in different growth stages of the T. rubrum life cycle. A total of 569 succinylated sites in 284 proteins were identified. These succinylated proteins are primarily involved in metabolism and translation, especially in pathogenicity-related processes. In addition, most of the succinylated proteins and sites have been shown to be growth-stage specific. Our study provides the first reported T. rubrum succinylome and is also the first PTM analysis of dermatophytes reported to date. Thus, this work represents great progress in research on these medically important fungi. Results and discussion Proteome-wide identification of lysine succinylation in T. rubrum Western blotting analysis showed that a large number of proteins were modified by succinylation and the succinylated proteins were more abundant in the mycelia stage than in the conidia stage (Fig. [59]1a). In the subsequent LC-MS/MS analysis, 569 succinylated sites in 284 proteins were identified in the two stages of the T. rubrum life cycle. Two hundred and twelve succinylated sites in 140 proteins were identified in the conidia stage, and 431 succinylated sites in 207 proteins were identified in the mycelia stage (Fig. [60]1b). The scores of all the identified peptides were above 40. The mass error for most peptides was <4 ppm (Fig. [61]1c), thus indicating that the mass accuracy of the MS data was sufficient for further analyses [[62]21]. The succinylated proteins and sites identified in each replicate for the conidia and mycelia stages are shown in Additional file [63]1: Figure S1 and Additional file [64]2: Table S1. In the three biological repeats, many more succinylated proteins and sites were identified, notably some low-abundance succinylated proteins and sites. In order to extensively investigate the function of the succinylated proteins identified in T. rubrum, the succinylated proteins and sites identified in any replicate were subjected to further study. Most of the identified proteins (175 proteins) possessed only one succinylated site, whereas 58 proteins possessed two sites, and 51 proteins possessed three or more succinylated sites (Fig. [65]1d). Fig. 1. Fig. 1 [66]Open in a new tab The identification of lysine succinylation in T. rubrum. a Western blot analysis. b A Venn diagram of the succinylated sites and proteins. c The distribution of mass error. d The number of succinylated sites per protein plotted against the number of proteins Functional annotation and subcellular localization of the succinylated proteins We performed a functional classification of all identified succinylated proteins by using GO analysis (Additional file [67]3: Table S2). As shown in the classification of the biological processes represented by the succinylated proteins (Fig. [68]2a), the three largest protein groups were involved in metabolism (38%), followed by cellular processes (28%) and single-organism processes (25%). As shown in the molecular function classification (Fig. [69]2b), the two largest protein groups were catalytic activity (48%) and binding (35%), which were consistent with the classification of the biological processes mentioned above. Fig. 2. Fig. 2 [70]Open in a new tab The classification of the succinylated proteins. a The GO characterization of the identified succinylated proteins on the basis of biological processes. b The GO characterization of the identified succinylated proteins on the basis of molecular function. c The distribution of the subcellular localization of the succinylated proteins predicted with [71]WoLF PSORT According to the results of the subcellular localization analysis (Fig. [72]2c), most succinylated proteins were localized to the mitochondria (40%) (Additional file [73]4: Table S3). This percentage was higher than the percentage in yeast (8%). However, in HeLa cells and mouse liver, these ratios were much higher, 45 and 70%, respectively [[74]22]. Succinylation tends to occur in the mitochondria because the mitochondria are the major provenance for succinyl-CoA and succinate from the tricarboxylic acid (TCA) cycle or odd-numbered fatty acid oxidation [[75]5]. In addition, in mouse liver, mitochondrial proteins are more frequently succinylated at multiple sites than non-mitochondria proteins [[76]22]. Our data showed the same bias, in that 47% of mitochondrial proteins contained multiple succinylated sites (≥ 2 succinylated sites) compared with 33% of non-mitochondrial proteins (Additional file [77]5: Table S4). However, the small succinyl metabolites (like succinyl-CoA and succinate) can traverse the mitochondrial membrane or be formed outside of mitochondria [[78]22]. For example, succinate could be formed in the cytoplasm as a side product by ketoglutarate-dependent enzymes [[79]23]. It has been suggested that in S. cerevisiae, H. sapiens and M. musculus succinyl metabolites drive succinylation in the cytoplasm and nucleus [[80]22]. In T. rubrum, 32% of the succinylated proteins are located in the cytosol, and 13% of succinylated proteins were found to be located in the nucleus. Enrichment analysis of the succinylated proteins We performed GO and KEGG enrichment analyses to further elucidate the types of proteins that are targets for succinylation. On the basis of GO enrichment for biological processes, the succinylated proteins were primarily involved in small molecule metabolic processes, oxoacid metabolic processes, carboxylic acid metabolic processes and organic acid metabolic processes, as shown in Fig. [81]3a. For the molecular function enrichment analysis, the structural constituents of the ribosome, structural molecule activity, cofactor binding and oxidoreductase activity were significantly enriched. Thus, a large percentage of the succinylated proteins participate in metabolic and translational relating roles. The enrichment analysis of the cellular components showed that the ribosome and ribonucleoprotein complex were significantly enriched, thus suggesting that succinylated proteins are closely related to translation and supporting the above conclusion. (Additional file [82]6: Table S5). Fig. 3. Fig. 3 [83]Open in a new tab The enrichment analysis of the succinylated proteins. a The GO enrichment based on the biological processes (p < 10^−7), molecular functions (p < 10^−5) and cellular components (p < 10^−7) of the proteins. b KEGG pathway enrichment (p < 0.01) On the basis of KEGG pathway enrichment analysis (Additional file [84]7: Table S6), 121 succinylated proteins were classified as being involved in metabolic pathways. As shown in Fig. [85]3b, proteins associated with processes such as the TCA cycle, carbon metabolism, oxidative phosphorylation, the ribosome, glycolysis/gluconeogenesis were significantly enriched. The TCA cycle is present in a wide variety of aerobic organisms. In our study, nearly every enzyme in the TCA cycle was succinylated, and most of these enzymes were succinylated at multiple sites (Fig. [86]4). The succinylated enzymes and modified sites are listed in Additional file [87]8: Table S7. For example, the enzyme aconitate hydratase (ACO), which catalyzes the conversion of citrate to isocitrate, is succinylated at 9 different sites. Another enzyme, isocitrate dehydrogenase (IDH), catalyzes the conversion of isocitrate to 2-oxoglutarate, the rate-limiting step of the TCA cycle. In our study, we identified 7 succinylated sites in IDH. In turn, lysine succinylation is also affected by the enzymes involved in the TCA cycle. For example, succinyl-CoA ligase utilizes succinate and CoA to form succinyl-CoA, which non-enzymatically catalyzes protein succinylation. In yeast, loss of α-ketoglutarate dehydrogenase (Kgd1) and succinyl-CoA ligase (LSC1) has been suggested to affect global succinylation levels [[88]22]. In T. rubrum, two α-ketoglutarate dehydrogenases (SucA and SucB) and two succinyl-CoA ligases (LSC1 and SucD) were succinylated, thus suggesting a potential effect of succinylation on the functions of these enzymes. Fig. 4. Fig. 4 [89]Open in a new tab The succinylated proteins involved in the TCA cycle. The succinylated proteins are highlighted in red Oxidative phosphorylation (OPP) is another pathway that occurs in the mitochondria of most eukaryotes. OPP is a highly efficient method for releasing the energy that is used to reform ATP. During OPP, electrons are transferred from electron donors to electron acceptors in redox reactions. In eukaryotes, five main protein complexes are involved in these redox reactions. In our study, succinylation was observed on several subunits of every protein complex involved in OPP (Additional file [90]1: Figure S2). For example, eight subunits of F-type ATPase (complex V) were succinylated, most of which were succinylated at multiple sites. The F-type ATPase alpha subunit (ATP1) contained 14 succinylated sites, the most of any protein. Three succinylated sites in ATP1 (K164, K233, and K430) were conserved in M. musculus (mouse) [[91]24]. In previous studies, ATP1 have been found to be heavily modified at lysine residues, and the most common types of modifications were acetylation and ubiquitination [[92]25]. These results suggest that succinylation and other types of modifications may work together to affect ATP1 function. The succinylated enzymes involved in OPP and the modified sites are listed in Additional file [93]9: Table S8. Succinylated proteins related to pathogenicity We identified 24 succinylated proteins related to pathogenicity in T. rubrum or homologous proteins involved in virulence in other fungi (Table [94]1). Secreted proteases, which digest hard keratin tissues during infection, are important in the virulence of dermatophytes. Eight secreted proteases were identified to be succinylated, including aminopeptidase, aspartic endopeptidase Pep2, leucine aminopeptidase 1, leucine aminopeptidase 2, subtilisin-like protease, Peptidase S41 family protein, tripeptidyl-peptidase SED2 and carboxypeptidase S1. In addition to secreted proteases, the mdr2-encoded ABC multidrug transporter (succinylated at K361 and K368) and AcuE-encoded malate synthase (succinylated at K161, K319, K483, K486 and K501) are also involved in dermatophyte infection [[95]26–[96]28]. Moreover, two Rho-type GTPases identified in our study were succinylated, Rho GTPase Rho1 and Rho-GDP dissociation inhibitor (Rho-GDI). Rho-type GTPases regulate many fundamental growth processes, such as cytoskeletal arrangement, vesicle trafficking, cell wall biosynthesis and polarized growth, and they have also been implicated in fungal infection [[97]29–[98]31]. Furthermore, heat shock proteins (Hsps) have also been implicated in fungal pathogenicity. In addition to their roles as chaperone proteins, Hsps have specific roles in fungi, such as dimorphic transition, drug resistance and virulence. For example, Hsp90, which is involved in morphogenesis, antifungal resistance and fungal pathogenicity, is considered a potential target for antifungal therapy [[99]32]. The four Hsps identified in our study, Hsp31, Hsp60, Hsp70 and Hsp90, were heavily succinylated. These key proteins that are critical for pathogenicity were identified as succinylation targets. Further experiments are needed to investigate whether succinylation plays a role in T. rubrum pathogenicity. Table 1. The identified succinylated proteins related to pathogenicity in fungi Protein names Species^a Protein accessions Succinylated lysine sites 14–3-3 family protein C. albicans TERG_01614T0 K51 TERG_01614T1 K28 TERG_06816T0 K49, K117, K122 ABC multidrug transporter Mdr2 T. rubrum, C. albicans, A. nidulans TERG_06399T0 K361, K368 ABC transporter C. albicans, A. fumigatus, C. neoformans, etc. TERG_04224T0 K12 Aminopeptidase T. rubrum, A. benhamiae, M. canis, A. fumigatus TERG_06767T0 K645 TERG_06767T2 K272, K518 TERG_12154T0 K680, K946 Hsp60-like protein C. albicans, H. capsulatum TERG_04141T0 K48, K75, K89, K130, K277, K282, K430, K437 AhpC/TSA family thioredoxin peroxidase A. fumigatus, C. albicans, C. neoformans TERG_05504T0 K66, K142 Aspartic endopeptidase Pep2 A. benhamiae, M. canis, C. albicans, A. fumigatus TERG_06704T2 K163 Calnexin A. fumigatus TERG_07527T0 K155, K203 Catalase A. nidulans, C. albicans, C. neoformans TERG_02005T0 K491 Glutathione S-transferase GstA A. fumigatus TERG_00370T0 K202 G-protein complex beta subunit CpcB C. heterostrophus, V. dahliae TERG_00783T0 K56 Heat shock protein 70 (Hsp70) C. albicans, H. capsulatum, C. neoformans TERG_03206T1 K37, K58, K137, K165, K222, K223, K276, K302, K331, K528, K545, K571 TERG_06505T0 K91, K157, K244, K326, K422, K511 TERG_06505T2 K422 TERG_03037T0 K134, K102, K131, K323, K367 TERG_01002T0 K482 TERG_01883T0 K361 Leucine aminopeptidase 1 T. rubrum, A. benhamiae, M. canis, A. oryzae, A. fumigatus TERG_05652T0 K116 Leucine aminopeptidase 2 T. rubrum, A. benhamiae, M. canis, A. oryzae, A. fumigatus TERG_08405T1 K17, K46, K380, K416 Subtilisin-like protease A. fumigatus, T. rubrum, A. benhamiae, M. canis and other dermatophyte species TERG_12591T0 K255 Malate synthase AcuE C. albicans, A. benhamiae TERG_01281T0 K161, K319, K483, K486, K501 Molecular chaperone Mod-E/Hsp90 F. graminearum, C. albicans, A. fumigatus TERG_06963T0 K171, K382, K385, K436, K479, K515, K550, K559, K565 Peptidase S41 family protein A. fumigatus, T. rubrum, A. benhamiae, M. canis TERG_08195T1 K490 Peptidyl-prolyl cis-trans isomerase C. albicans, C. neoformans TERG_01573T0 K36, K75, K120, K113 TERG_06858T0 K67, K75, K88, K126 Probable chaperone protein Hsp31 homologue, putative C. albicans TERG_00228T0 K159 Rho GTPase Rho1 C. albicans, C. neoformans TERG_07578T0 K155 Rho-gdp dissociation inhibitor C. neoformans TERG_05090T3 K125 TERG_05090T4 K122 TERG_05090T5 K125 Tripeptidyl peptidase SED2 A. fumigatus, T. rubrum TERG_00619T0 K340 carboxypeptidase S1, putative T. rubrum, A. benhamiae, M. canis, A. fumigatus TERG_08255T1 K490 [100]Open in a new tab ^aThe column “Species” indicates the organisms in which the proteins involved in pathogenicity have been reported Secondary structure properties of succinylated lysine We predicted the secondary structure features of the Ksucc sites by using NetSurfP. The secondary structures of succinylated lysines and all lysines were compared (Fig. [101]5). In our study, succinylated lysine residues were more frequently located on α-helix, and less frequently located on β-strand and coil, as compared with all lysine residues. The similar preferences were observed in rat that the