Global profiling of lysine acetylation in human histoplasmosis pathogen Histoplasma capsulatum
Introduction
Histoplasma capsulatum, the causative agent of human histoplasmosis, is a major cause of respiratory and systemic mycosis, especially in immune-compromised individuals (Graybill, 1988). Histoplasmosis typically manifests as a benign respiratory infection and even entirely asymptomatic. In some individuals, especially immunocompromised persons, H. capsulatum readily disseminates, causing significant morbidity and mortality (Kauffman, 2008). Histoplasmosis is a common endemic mycosis throughout most of the world, which is endemic in the mid-western United States (such as in the Mississippi and Ohio River valleys) and Central America (Kauffman, 2007). Sporadic cases of autochthonous histoplasmosis have been found in China, which was traditionally considered non-endemic for H. capsulatum. It is noteworthy that most cases of histoplasmosis occurred in regions through with the Yangtze River flows (Cao et al., 2010, Ge et al., 2010, Pan et al., 2013). H. capsulatum thrives in soil in mycelial (mold) form, as with most other dimorphic fungal pathogens, conversion to a unicellular haploid yeast form occurs following inhalation and exposure to the warmer temperature of the respiratory tract (Eissenberg and Goldman, 1991).
Recent genetic approaches have begun to reveal the regulatory machinery that governs the conversion of Histoplasma to the yeast form and expression of the gene set involved in pathogenesis with the assumption that virulence factors are components unique to the yeast-phase (Batanghari and Goldman, 1997, Colonna-Romano et al., 1998, Hwang et al., 2003). Some virulence factors of H. capsulatum yeasts were identified to subvert or avoid activating macrophage antifungal defenses (Holbrook and Rappleye, 2008). Histoplasma HSP60 localized to the cell surface of yeast cells and functions as adhesin for macrophage binding and phagocytosis (Habich et al., 2006). The β-glucan polysaccharides of all fungal cell walls were related to macrophages recognition, reactive oxygen compounds production and proinflammatory cytokines secretion (Robinson et al., 2006, Willment and Brown, 2008). Histoplasma yeasts effectively conceal their immunostimulatory β-glucan signatures underneath a cell wall layer composed of α-glucan which was involved in the virulence of Histoplasma, as genetic loss of the α-(1,3)-glucan covering through mutation or RNAi of α-glucan synthase (AGS1) severely attenuates Histoplasma virulence (Rappleye et al., 2004). Secreted factor YPS3 and calcium-binding protein CBP play an important role in the virulence of Histoplasma yeast (Bohse and Woods, 2007), as YPS3 can decrease the pathogen burden in the internal organs of infected mice and CBP can impair the intracellular growth of Histoplasma yeast and attenuate the ability to colonize the lung (Sebghati et al., 2000). However, the pathogenesis of H. capsulatum remains largely unknown.
The reversible lysine acetylation in proteins is now recognized as a common posttranslational modification (PTM) in both prokaryotes and eukaryotes (Thao and Escalante-Semerena, 2011, Xie et al., 2012). Since the lysine acetylome revealed in mammalian cells (Kim et al., 2006), global acetylation in eukaryotes has been reported and several biochemical studies showed that lysine acetylation may influence various cellular processes including metabolic pathway and transcriptional regulation (Rardin et al., 2013, Still et al., 2013, Weinert et al., 2011). Recent advances in antibody-based affinity enrichment and high sensitive MS-based proteomics have made contributions to the global analysis of lysine acetylation in bacteria including Mycobacteria tuberculosis (Liu et al., 2014, Xie et al., 2015), Escherichia coli (Yu et al., 2008), Salmonella enteric (Wang et al., 2010b), Bacillus subtilis (Kim et al., 2013), Geobacillus kaustophilus (Lee et al., 2013), Erwinia amylovora (Wu et al., 2013), and Thermus thermophilus (Okanishi et al., 2013), Saccharopolyspora erythraea (Huang et al., 2015), Streptomyces roseosporus (Liao et al., 2014), Pseudomonas aeruginosa (Ouidir et al., 2015). Despite the popular studies of lysine acetylation in bacteria, the progress of lysine acetylome in fungus is relative limited and only one fungus species Saccharomyces cerevisiae have been examined.
In this study, we investigated the first acetylproteome of the H. capsulatum using a high-resolution mass spectrometry-based proteomics approach. Combining the affinity immuno-separation of acetylated peptides with nano-HPLC−MS/MS analysis, we identified a total of 775 unique lysine acetylation sites on 456 proteins. Bioinformatics analysis showed that lysine-acetylated proteins are mainly involved in metabolic processes. Moreover, several sequence motifs including KacY, KacF, KacH, Kac*F, YKac and Kac***R were identified. Furthermore, a total of 116 acetylation sites on 86 proteins were also found to be succinylated, suggesting extensive overlap between acetylation and succinylation in this fungus. This first global acetyaltion profiling of H. capsulatum provides a basis for future interrogation of the roles of these acetylated proteins.
Section snippets
Strain culture and protein extraction
The total steps including protein extraction, peptides digestion, acetylated peptides enrichment, MS identification, data processing and bioinformatics analysis are described as our previous article (Xie et al., 2015). The Histoplasma capsulatum NAm1 cultured cells were harvested and washed twice with cold phosphate-buffered saline (PBS), then lysed in 8 M urea supplemented with 1 mM DTT, 2 mM EDTA, protease inhibitor cocktail (Protease Inhibitor Cocktail Set III; Calbiochem), and HDAC inhibitor
Establishment of the histoplasma acetylome
Lysine acetylation, one conserved PTM in both prokaryotic and eukaryotic cells, has been thought to play critical roles in regulating protein function in diverse ways. However, this PTM in Histoplasma capsulatum NAm1 has not yet been reported. The genome database of H. capsulatum has been established several year ago, which can promote the global analysis of the lysine acetylated sites and proteins in this species. To gain insights into the large-scale dataset of lysine acetylation in H.
Concluding remarks
Combining high-affinity enrichment of lysine acetylated peptides with high-sensitivity mass spectrometry and bio-informatics tools, we presented the first lysine-acetylated proteome dataset for a pathogenic fungus H. capsulatum, identifying 775 lysine acetylation sites in 456 acetylated proteins including 5 histones and 451 non-histone proteins. In addition, with the extensive characterization of the acetylation, we found that acetylation occurred on a large number of Histoplasma proteins
Supporting Information
Table S1 showing the identified acetylated protein annotation summary. Table S2 showing the functional analysis of biological process, molecular function and domain. Table S3 showing the GO enrichment analysis of biological process, molecular function, cellular component, pathway enrichment, and protein domain. Table S4 showing the detail of protein-protein interaction (PPI) network of all the lysine acetylated proteins. Table S5 showing the overlap between acetylation and succinylation in
Conflict of Interest
The authors have declared no conflict of interest.
Acknowledgements
The work was supported by the National Basic Research Program of China (2013CB531606), National Science and Technology Major Projects of China Grant (2013ZX10004612001), National Natural Science Fund (31270180, 81371851,) and Shanghai Key Laboratory of Molecular Medical Mycology Fund (14DZ2272900).
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2018, Journal of ProteomicsCitation Excerpt :Notably, the functions of acetylated proteins with low abundance or related to bacterial virulence should also be emphasized. It has been reported that lysine acetylation targets the bacterial virulence-associated proteins involved in the biosynthesis of lipopolysaccharide or CPS, biofilm formation, adherence, invasion and the secretion system [11,12,19,69–72]. These studies suggested that lysine acetylation may regulate the virulence of pathogens.
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2017, Journal of ProteomicsCitation Excerpt :It is likely that the catalog of succinylated proteins in H. capsulatum will continue to expand with the development of technology. However, our study represents the first lysine succinylome dataset for H. capsulatum, which not only increases the known PTM types of H. capsulatum (the first global file of PTM-lysine acetylation in Histoplasma was recently reported [45]) but also greatly expands the knowledge of lysine succinylation sites and succinylated proteins in this fungus. During comparison of the H. capsulatum succinylome with the previously reported succinylome of E. coli, we found that the number of succinylated proteins in H. capsulatum is smaller than that in E. coli.
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Equal contribution, co-first author.