Abstract
The eukaryotic-type serine/threonine kinase of Streptococcus suis serotype 2 (SS2) performs critical roles in bacterial pathogenesis. In this study, isobaric tags for relative and absolute quantification (iTRAQ) MS/MS were used to analyze the protein profiles of wild type strain SS2-1 and its isogenic STK deletion mutant (Δstk). A total of 281 significant differential proteins, including 147 up-regulated and 134 down-regulated proteins, were found in Δstk. Moreover, 69 virulence factors (VFs) among these 281 proteins were predicted by the Virulence Factor Database (VFDB), including 38 downregulated and 31 up-regulated proteins in Δstk, among which 15 down regulated VFs were known VFs of SS2. Among the down-regulated proteins, high temperature requirement A (HtrA), glutamine synthase (GlnA), ferrichrome ABC transporter substrate-binding protein FepB, and Zinc-binding protein AdcA are known to be involved in bacterial survival and/or nutrient and energy acquisition under adverse host conditions. Overall, our results indicate that STK regulates the expression of proteins involved in virulence of SS2 and its adaption to stress environments.
Avoid common mistakes on your manuscript.
Introduction
Streptococcus suis (S. suis) is a major swine pathogen that causes a wide range of diseases (Lun et al. 2007). In addition, S. suis is an important zoonotic agent responsible for severe human infections, including meningitis, endocarditis, and septic shock (Tang et al. 2006; Lun et al. 2007). Streptococcus suis serotype 2 (SS2), which is the serotype that is most virulent and most frequently isolated from diseased pigs, is most commonly involved in human infections (Tang et al. 2006). During the past few decades, more than 100 virulence factors of S. suis had been identified. These factors were classified into the following subgroups: surface/secreted elements, enzymes/proteases, transcription factors/regulatory systems and others (Fittipaldi et al. 2012). However, the mechanisms underlying the pathogenesis of the different virulence factors on SS2 has still not been entirely explained.
Bacteria use regulatory systems to sense and respond to environmental signals via regulation of specific gene expression. Two-component systems (TCSs), stand-alone regulators (SARs), regulator RNAs and unknown regulators constitute bacterial regulatory networks (Fittipaldi et al. 2012; Wu et al. 2014; Segura et al. 2017; Zheng et al. 2018a). Two-component systems such as SalK/SalR, VirR/VirS and VraSR, as well as the orphan response regulators RevS and CovR have been reported to contribute to bacterial adaptation to various environments and the expression of virulence factors of S. suis (Zheng et al. 2018a). In addition, several SARs have been reported to modulate S. suis virulence, including Rgg (Zheng et al. 2011), CcpA (Willenborg et al. 2011), CodY (Feng et al. 2016) and Rex (Zhu et al. 2018). Small RNAs also play a pivotal role in the pathogenicity of S. suis (**ao et al. 2017).
Several recent studies have shown that eukaryote-type serine/threonine kinases (ESTKs) and phosphatases (ESTPs) also play essential roles in sensing of external stimuli (Wright and Ulijasz 2014). Even though signaling systems composed of ESTKs/ESTPs do not have dedicated transcription factors, they are capable of affecting the expression of genes involved in cell growth and division, adherence to host cell, stress response, biofilm formation, and various metabolic, developmental and virulence processes (Burnside and Rajagopal 2011; Zhu et al. 2011, 2014; Wright and Ulijasz 2014; Manuse et al. 2016; Fang et al. 2017; Zhang et al. 2017).
The homologues of ESTK and ESTP in S. suis, which have been designated SsSTK and SsSTP, respectively, were found to contribute to bacterial adherence to host cells, survival in stress environments and virulence.(Zhu et al. 2011, 2014; Fang et al. 2017; Zhang et al. 2017). Comparative proteome analysis is a powerful method for elucidating gene expression patterns in microorganisms (Chen et al. 2011; Pian et al. 2015; Yu et al. 2018). Proteome and transcriptome profiles are not exactly the same because of differences in the post-transcriptional regulation that modulates the translation rate and half-lives of specific proteins or mRNAs, as well as their intracellular location and molecular association with other proteins (Chen et al. 2011; Shen et al. 2013). A previous comparative transcriptomic analysis have shown that SsSTK affects the transcription of a set of genes encoding functions involving in cell growth and division, glycolysis, carbohydrate metabolism, membrane transport and translation (Zhang et al. 2017). In the present study, the isobaric tag for relative and absolute quantitation (iTRAQ) and liquid chromatography tandem mass spectrometry (LC–MS/MS) were used to analyze the different protein expression profiles, especially those concerning the bacterial pathogencity, between the wild-type (WT) strain SS2-1 and its SsSTK mutant strain with the aim of revealing proteins involved in adaption to stress environments and virulence of S. suis.
Materials and methods
Bacterial strains and culture conditions
SS2 strain SS2-1, which was isolated from a diseased pig with septicemia in Jiangsu Province in 1998, has been confirmed as a highly virulent strain (Zhu et al. 2014). The SsSTK mutant of SS2-1 (Δstk) was constructed in a previous study (Zhu et al. 2014). For this study, SS2 strains were grown in Todd-Hewitt broth (THB, Becton, Dickinson and Company, USA) or plated on agar medium containing 10% fetal bovine serum at 37 °C.
Protein digestion and iTRAQ labelin
Protein samples were performed with a few modifications as described by previous descriptions (Shen et al. 2013; Yu et al. 2018). Briefly, the WT strain SS2-1 and the mutant strain Δstk were each cultured in THB in triplicate (three SS2-1 and three Δstk) and collected during the log-growth phase (OD600 = 0.7) (Shen et al. 2013; Yu et al. 2018). Cells were then centrifuged at 10,000 × g for 5 min at 4 °C, after which the pellets were washed twice with PBS (Shen et al. 2013). Comparative proteome analysis was subsequently performed at Wuhan GeneCreate Biological Engineering Co., Ltd. (Wuhan, GeneCreate, China). Protein digestion was performed as previously described (**g et al. 2008), with some modifications. Briefly, SS2 cell pellets were ground to powder in liquid nitrogen and then incubated in dissolution buffer (8 M urea/100 mM triethylammonium hydrogen carbonate buffer (TEAB), pH 8.0) containing 1 mM PMSF and 2 mM EDTA (final concentration) for 5 min, after which 10 mM DTT (final concentration) was added to the sample. Next, the suspension was sonicated for 15 min and then centrifuged at 4 °C at 14,000×g for 20 min. The supernatant was subsequently mixed with four volumes of precooled acetone at − 20 °C overnight. After another centrifugation, the resulting protein pellets were air-dried and resuspended in 8 M urea/100 mM TEAB (pH 8.0). Protein samples were then reduced with 10 mM DTT at 56 °C for 30 min and alkylated with 50 mM iodoacetamide (IAM) for 30 min in the dark. Next, the protein concentration was measured using a Bradford Protein Assay Kit (Beyotime, Shanghai, China). After being diluted 5 × with 100 mM TEAB, equal amounts of proteins from each sample were used for tryptic digestion. Trypsin was added at an enzyme protein ratio of 1:50 (w/w), after which samples were digested at 37 °C for 12–16 h. Following digestion, peptides were desalted using C18 columns and the resulting desalted peptides were dried under vacuum. The dried peptide powder was later re-dissolved with 0.5 M TEAB and processed according to the manufacturer’s instructions for the iTRAQ Reagent-8 plex Multiplex Kit (AB Sciex U.K. Limited). Three biological replicates of SS2-1 (1A, 1B, and 1C) were labeled with iTRAQ tags 113, 114, and 115, respectively, and three biological replicates of Δstk (20A, 20B, and 20C) were labeled with tags 116, 117, and 118, respectively. The peptide samples were then fractionated using a Durashell C18 column (5 µm, 100 Å, 4.6 × 250 mm) on an Ultimate 3000 HPLC system (Thermo DINOEX, USA) operating at 1 ml/min. Peptides were separated by increasing acetonitrile (ACN) concentrations under high pH (pH 10) conditions and fractions were collected at 1 ml intervals and pooled into 12 fractions. Each fraction was then dried under vacuum.
LC–ESI–MS/MS analysis
Peptide samples were dissolved in 2% acetonitrile/0.1% formic acid and then analyzed using a Triple TOF 5600+ mass spectrometer coupled with the Eksigent nanoLC System (SCIEX, USA) as previously described (Lin et al. 2015). The raw files collected from the Triple TOF 5600 were interpreted using ProteinPilot version 4.5 (July 2012, Applied Biosystems; Foster City, CA, USA). MS/MS spectra were searched against the Uniprot S. suis database (80,299 items, updated Jan 2017). For analysis, the instrument was set as TripleTOF 5600 plus with cysteine carbamidomethylation and 8 multiplex iTRAQ labeling was set as a fixed modification. In addition, methionine oxidation was used as a variable modification, and digestion by trypsin allowing for no more than one missed cleavage. The ratio of Δstk to SS2-1 represents the expression of proteins with a 1% false discovery rate for the protein identification confidence (Unwin et al. 2010). The differences in abundance were considered significant when whose criteria were met a ratio-fold change ≥ 1.5 or ≤ 0.67 (Yu et al. 2018).
Bioinformatics analysis
Gene Ontology (GO) analysis was conducted to classify differently expressed proteins (DEPs) in three categories (cell component, molecular function, and biological process) using the UniPort-GOA database (http://www.ebi.ac.uk/GOA/), InterProScan (http:// www.ebi.ac.uk/interpro/) and GO annotation (http://geneontology.org/). In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (http://www.genome.jp/kegg/) of DEPs were further categorized utilizing the same resource. Furthermore, the protein–protein interactions for these DEPs predicted by the Virulence Factor Database (VFDB) (Chen et al. 2005) in this study and the known VFs reported by others were analyzed using the Cytoscape software to construct a virulence network of S. suis and screen the novel DEPs for those that are connected with the known VFs. The protein–protein interaction network was obtained from the STRING database (http://string-db.org/newstring_cgi/show_input_page.pl), which defined a ‘confidence score’ to evaluate the interaction confidence. We obtained all interactions with a confidence score of at least 0.4 (Yu et al. 2018).
Western blot validation of comparative proteomic analysis
SS2-1 and Δstk were prepared for western blot analysis. Two proteins, OppA (putative oligopeptide-binding protein) and DnaJ (chaperone protein DnaJ), were chosen for validation of the comparative proteomic data. In the Δstk/SS2-1 comparison, OppA is a lower abundance protein and DnaJ is a higher abundance protein. EF-Tu was used as the loading control because its abundance is relatively constant. Equal amounts (30 µg for each lane) of whole cell proteins from the WT and mutant strains were separated on a 12% SDS-PAGE gel, then transferred onto polyvinylidene fluoride (PVDF) membranes (BioRad). The membranes were then incubated with a 1:500 dilution of the primary antibodies for OppA and EF-Tu (kindly provided by Prof. Wei Zhang, Nan**g Agricultural University) and DnaJ (kindly provided by Prof. Weihuan Fang, Zhejiang University), after which they were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at a 1:10,000 dilution. Signals were detected using enhanced chemiluminescence (ECL) substrate (ThermoFisher Scientific).
Statistical analysis
The means of groups were compared using the Student’s t test (unpaired, two-tailed) in GraphPad Prism 5 (San Diego, USA), with a P < 0.05 considered to be statistically significant.
Results
Comparative proteome analysis of SsSTK mutant strain and WT strain
iTRAQ coupled mass spectrometry identified a total of 1120 proteins from WT strain SS2-1 and its mutant strain Δstk. Among these, the expression levels of 281 proteins differed significantly (> 1.5-fold change or < 0.67-fold change, P- value < 0.05) in Δstk compared with its WT strain SS2-1, with 134 (47.7%) down-regulated and 147 (52.3%) up-regulated (Table S1).
Functional classification annotation analysis of DEPs
To gain insight into the functional categories of the 281 differentially expressed proteins (DEPs), GO analysis was performed to generate classification clusters based on biological process and molecular function. In the biological processes classification cluster, the five most enriched GO terms were biosynthetic processes (41 proteins [32.03%] upregulated; 54 proteins [46.96%] downregulated), nitrogen compound metabolic processes (49 proteins [38.28%] upregulated; 30 proteins [26.09%] downregulated), protein metabolic processes (26 proteins [20.31%] upregulated; 39 proteins [33.91%] downregulated), nucleotide and nucleic acid metabolic processes (41 proteins [32.03%] upregulated; 14 proteins [28.1%] downregulated), and carbohydrate metabolic processes (23 proteins [17.97%] upregulated; 21 proteins [18.26%] downregulated) (Fig. 1a). In the molecular function classification, the most enriched GO terms were nucleotide binding (51 proteins [39.84%] upregulated; 31 proteins [28.70%] downregulated), hydrolase activity (46 proteins [35.94%] upregulated; 25 proteins [23.15%] downregulated), transferase activity (43 proteins [31.25%] upregulated; 26 proteins [24.07%] downregulated), ATP binding (46 proteins [39.84%] upregulated; 25 proteins [20.37%] downregulated), and cation binding (28 proteins [21.88%] upregulated; 13 proteins [12.04%] downregulated) (Fig. 1b).
KEGG pathway analysis of DEPs
To reveal the roles of SsSTK in SS2, KEGG pathway analysis was performed (Fig. 2). The DEPs were mainly involved in metabolic pathways (60 proteins [68.97%] up-regulated; 43 proteins [55.13%] down-regulated), biosynthesis of secondary metabolites (33 proteins [37.93%] up-regulated; 21 proteins [26.92%] down-regulated) and microbial metabolism in diverse environments (20 proteins [22.92%] up-regulated; 11 proteins, [14.1%] down-regulated, Fig. 2a). The up-regulated proteins included those associated with purine metabolism (13 proteins, 14.94%), ABC transporters (11 proteins, 12.64%), fructose and mannose (10 proteins, 11.49%), propanoate (9 proteins, 10.34%), peptidoglycan biosynthesis (8 proteins, 9.2%), pyrimidine metabolism (8 proteins, 9.2%) and pyruvate metabolism (7 proteins, 8.97%). The down-regulated pathways were associated with ribosomes (26 proteins, 33.3%), ABC transporters (16 proteins, 20.51%), purine metabolism (10 proteins, 12.82%), pyrimidine metabolism (7 proteins, 8.97%), amino sugar and nucleotide sugar metabolism (6 proteins, 7.69%), the two-component system (5 proteins, 6.41%) and aminoacyl-tRNA biosynthesis (5 proteins, 6.41%) (Fig. 2b). In general, most of these DEPs are involved in key metabolic and pathways, which may contribute to the pathogenicity of SS2.
SsSTK regulates known virulence factors
The SsSTK deletion significantly reduced SS2 virulence. Among the 281 DEPs, there were 69 virulence factors (VFs) predicted by VFDB, including 38 down-regulated proteins (Table 1) and 31 up-regulated proteins (Table 2) in the Δstk, of which 26 were known VFs of SS2 (Fittipaldi et al. 2012). In addition, the following 16 VFs were down-regulated in Δstk: capsular polysaccharide biosynthesis locus genes CPS2A (regulation), CPS2B (chain length determination) and CPS2H (glycosyltransferase), sialic acid synthase (NeuB), UDP-N-acetylglucosamine 2-epimerase (NeuC), putative oligopeptide-binding protein (OppA), high-affinity zinc uptake system protein (ZnuA/TroA) and IgM protease (IdeSsuis) (Fittipaldi et al. 2012; Rungelrath et al. 2018). Additionally, the putative IgA-specific zinc metalloproteinase (ZmpC or IgA1) (Zhang et al. 2011; Dumesnil et al. 2018), translation initiation factor 2 (HP0272 or SadP) (Ferrando et al. 2017), chaperone protein DnaJ (Zhang et al. 2015), and sensor histidine kinase TCS VarS (Zheng et al. 2018a; Zhong et al. 2018) were significantly up-regulated in the mutant strain. Another 43 VFs that have been reported in other pathogens were identified as DEPs in the ∆stk, including putative 5'-nucleotidase (5NuC) (Zheng et al. 2015; Ma et al. 2017), trypsin-like serine protease (HtrA) (Backert et al. 2018) and metalloendopeptidas (PepO) (Agarwal et al. 2013, 2014). These VFs were mainly assigned into eight classes: (i) amino acid transport and metabolism (12 proteins); (ii) posttranslational modification, protein turnover, chaperones (10 proteins); (iii) nucleotide transport and metabolism (9 proteins); (iv) transcription (8 proteins); (v) inorganic ion transport and metabolism (6 proteins); (vi) cell wall/membrane/envelope biogenesis (6 proteins); (vii) general function prediction (3 proteins); and (viii) other proteins of unknown function (7 proteins) (Tables 1 and 2).
Interaction network analysis confirmed the roles of the newly identified DEPs in the known virulence factor system
For further insight into the roles of these newly identified DEPs in virulence, we visualized the network formed by the known VFs of SS2 and the novel identified VFs predicted by VFDB using the Cytoscape software. The protein–protein interaction network demonstrated 241 direct physical interactions among the 80 nodes (Table S2). Sixty-six of the interactions had a score higher than 0.70 (high confidence). The 36 newly identified DEPs (yellow nodes) were implicated in and complemented the virulence interaction network, with some playing an important role of bridging to link the known VFs (green nodes and red nodes) and forming important hub proteins. Overall, the results indicated that 37 of the 69 novel DEPs are involved in the known virulence network and may play a role in virulence (Fig. 3).
Confirmation of the proteomics results by western blot analysis
The up-regulated VF DnaJ (41kD) and down-regulated VF OppA (66kD) were selected for confirmation of the comparative proteomics analysis. The EF-Tu protein was used as an internal reference because its abundance is relatively constant in both groups. The western blot results support those of the proteomic analysis, as the levels of OppA was decreased and the levels of DnaJ were increased in Δstk, which indicating that the proteomics data and western blot results agreed (Fig. 4).
Western blot analysis of comparative proteomics data. Equal amounts (30 µg for each lane) of total bacterial cell proteins were separated on a 12% SDS-PAGE gel, then subjected to western blotting. From left to right the lanes were loaded with SS2-1 and Δstk samples. Differentially expressed OppA (66 kDa, the first line), DnaJ (41 kDa, the second line) and EF-Tu (44 kDa, the last line) proteins were analyzed using their respective antibodies. The CBB-R250-stained gel was used as a loading control. Protein bands were visualized using the ECL substrate
Discussion
Bacterial ESTKs have emerged as important regulation elements that are indispensable for pathogenesis (Burnside and Rajagopal 2011; Wright and Ulijasz 2014; Manuse et al. 2016). In Mycobacterium tuberculosis, two-dimensional gel electrophoresis was used to investigate the effects of the serine/threonine protein kinase (pknE) on the bacterial protein expression under nitric oxide stress conditions. In response to NO stress, ΔpknE had increased number of proteins involved in intermediary and lipid metabolism (Parandhaman et al. 2014). In S.pneumoniae, a mass-spectrometry based label-free quantitative (LFQ) approach was used to characterize and determine the impact of StkP on the protein expression profiles. Notable changes in the proteome of the kinase mutant ΔstkP in comparison to the WT strain have been observed especially in the cluster of amino acid metabolism, energy metabolism, regulatory fuction and transcription(Hirschfeld et al. 2019). In the present study, comparative proteomics approaches revealed that SsSTK can regulate the expression of proteins involved in bacterial central metabolism, stress response and virulence. These findings provide further support for the previous data that showed attenuated growth in vivo, reduced survival rate in various stress environments and virulence of Δstk (Zhu et al. 2014).
As previously reported, the deletion of stk in strain SS2 resulted in alteration of bacterial pathogenicity (Zhu et al. 2014; Zhang et al. 2017). This attenuation may result from the impaired growth of Δstk in vivo and because of direct effects on the expression of VFs. The transcriptomic profiles showed that 32 VFs were down-regulated in stk-deletion strain of SC-19, including 9 known VFs of SS2, such as subtilisin-like protease (SspA), DNA nuclease (SsnA), mannose-specific PTS (ManN), adenylosuccinate synthase (PurA) and phosphoribosylamine-glycine ligase (PurD) (Wilson et al. 2007; Fittipaldi et al. 2012; Zhang et al. 2017).Our iTRAQ analysis showed 38 VFs, including 26 known VFs and 12 novel identified VFs that were predicted by VFDB, with repressed expression in Δstk such as AtlA, IdeSsuis, OppA, HtrA, HtpsB, PurD and 5NuC (Fittipaldi et al. 2012). In SS2 virulent strain HA9801, the AtlA mutant strain (ΔatlA) exhibited a significant reduction in adherence to epithelial cells, biofilm formation and virulence (Ju et al. 2012). In SS2 virulent strain 10, IgM cleaving activity of IdeSsuis is important for bacterial survival in porcine blood and evasion of the classical complement pathway (Rungelrath et al. 2018). In Strptococci, HtrA involved in tissue invasion, chronic airway infections and secretion of VFs, such as in S. pyogenes (Lyon and Caparon 2004) and S. pneumonia (Ibrahim et al. 2004). The histidine triad-family protein Htps B, a homologue of internalin A (InlA) of Listeria monocytogenes, has been reported to be important in surface invasions of bacteria and to facilitate both bacterial attachment and internalization in cells that express its receptor of E-cadherin (Bergmann et al. 2002). In SS2 virulent strains, OppA is involved in cell growth, binding to host proteins and virulence (Zhang et al. 2014; Zheng et al. 2018b). In SS2 strain S735, purD insertion mutant strain obtained by the signature-tagged mutagenesis system was showed apathogenic in both mouse and caesarian-derived, colostrum-deprived (CDCD) pig models (Wilson et al. 2007). Recently transcriptome profiles and our proteome analysis both showed that PurD were down-regulated in stk deletion mutant strain (Zhang et al. 2017). The extracellular nucleases and or nucleotidase play important roles in degradation of the DNA backbone of neutrophil extracellular traps (NETs) and allow bacteria to evade the host immune system. In S. equi subsp. zooepidemicus ATCC35246, 5'-Nucleotidase (5NuC) was found to directly degrade the NET DNA backbone to deoxyadenosine, negatively influencing macrophage phagocytic activity, while the mutant strain Δ5nuc exhibited lower virulence and a weaker ability to spread from blood to organs than the WT strain (Ma et al. 2017).Similar findings were reported in S. pyogenes for 5'-nucleotidase A (S5nA)(Zheng et al. 2015). Therefore, our results are in agreement those of previous studies showing SsSTK mutant strains displayed reduced ability to adhere to epithelial cells, increased immune evasion and increased sensitivity to phagocytosis (Zhu et al. 2014; Zhang et al. 2017).
During infection, bacteria must often cope with stress in the form of oxidative conditions, excessive temperature, extreme osmolarity, low pH, and nutrient limitations. Our previous study showed that Δstk displayed defects in the ability to adapt to various environmental conditions (Zhu et al. 2014). These may be because of down-regulation of the following series of DEPs that are necessary for bacterial survival and/or nutrient and energy acquisition under adverse host conditions. (i) The general stress response protein (e.g., HtrA). The virulence features of bacterial HtrA, an important stress response regulation protein, have primarily been attributed to increased fitness of pathogens because of resistance against stress conditions during infection (Backert et al. 2018). (ii) Amino acid transport and metabolism (e.g., GlnA, IlvC, LivK and ASD). Glutamine synthase (GlnA), which converts glutamate and ammonia to glutamine, plays a central role in regulating the carbon/nitrogen balance in the metabolism and the pathogenicity of bacteria, such as in Salmonella enterica and SS2 (Si et al. 2009; Aurass et al. 2018). The ketol-acid reductoisomerase IlvC and branched-chain amino acid ABC transporter substrate-binding protein LivK are involved in the branched-chain amino acids (BCAAs; isoleucine, leucine, valine) biosynthesis pathway, which contributes to the virulence of pathogens (Ribardo and Hendrixson 2011; Kim et al. 2017). In S. pneumoniae strain D39, an ilvC deletion mutant (∆ilvC) diminished Ply and LytA virulence factor expression and showed a higher survival rate and lower bacterial burden in a mice infection model relative to the WT strain (Kim et al. 2017). Aspartate-semialdehyde dehydrogenase (ASD) is an essential enzyme for the biosynthesis of lysine, methionine, and threonine from aspartate. In Burkholderia pseudomallei, a deletion mutant strain Δasd exhibited attenuated intracellular infectivity and the mutation showed protection against acute inhalation melioidosis in mice (Norris et al. 2011). (iii) Inorganic ion transport and metabolism (e.g., AdcA, FepB and MgtA). Apart from the two DEPs mentioned above, these proteins can help pathogens overcome the hostile environments created by ion starvation. The Adc protein contributes zinc uptake and streptococcal virulence. In S. agalactiae, Adc and Lmb are involved in zinc acquisition and contribute to bacterial growth and survival (Moulin et al. 2016). In S. pneumoniae, AdcA enables zinc acquisition during growth in vitro and systemic virulence in vivo (Bayle et al. 2011; Plumptre et al. 2014). In Salmonella enterica serovar Typhimurium wild-type strain SL1344, deletion of fepB attenuated Salmonella replication and colonization within macrophages and mice (Nagy et al. 2013). Moreover, the transcriptional level of mgtA in Δstk was found to be decreased in previous studies (Zhang et al. 2017). Down-regulation of these VFs may hinder the acquisition of nutrients by bacteria and therefore decrease the adaptation of SS2 to various stress environments.
Conclusion
In summary, our comparative proteome analysis identified 38 down-regulated VFs in the mutant strain Δstk that were involved in adherence to host cells and adaption to and survival in the host environments during SS2 infection. Consistently, phenotypic assays in previous studies have confirmed that the Δstk mutant strain displayed deficient growth in various stress environments in vitro and in vivo and attenuated pathogenicity. Therefore, STK is important to cell growth, stress response, and virulence of SS2.
Data availability
All data during the study appear in the submitted article and the supplementary materials.
References
Agarwal V, Kuchipudi A, Fulde M, Riesbeck K, Bergmann S, Blom AM (2013) Streptococcus pneumoniae endopeptidase O (PepO) is a multifunctional plasminogen- and fibronectin-binding protein, facilitating evasion of innate immunity and invasion of host cells. J Biol Chem 288:6849–6863. https://doi.org/10.1074/jbc.M112.405530
Agarwal V, Sroka M, Fulde M, Bergmann S, Riesbeck K, Blom AM (2014) Binding of streptococcus pneumoniae endopeptidase O (PepO) to complement component C1q modulates the complement attack and promotes host cell adherence. J Biol Chem 289:15833–15844. https://doi.org/10.1074/jbc.M113.530212
Aurass P, Duvel J, Karste S, Nubel U, Rabsch W, Flieger A (2018) glnA truncation in salmonella enterica results in a small colony variant phenotype, attenuated host cell entry, and reduced expression of flagellin and SPI-1-associated effector genes. Appl Environ Microbiol. https://doi.org/10.1128/AEM.01838-17
Backert S, Bernegger S, Skorko-Glonek J, Wessler S (2018) Extracellular HtrA serine proteases: an emerging new strategy in bacterial pathogenesis. Cell Microbiol 20:e12845. https://doi.org/10.1111/cmi.12845
Bayle L, Chimalapati S, Schoehn G, Brown J, Vernet T, Durmort C (2011) Zinc uptake by streptococcus pneumoniae depends on both AdcA and AdcAII and is essential for normal bacterial morphology and virulence. Mol Microbiol 82:904–916. https://doi.org/10.1111/j.1365-2958.2011.07862.x
Bergmann B, Raffelsbauer D, Kuhn M, Goetz M, Hom S, Goebel W (2002) InlA- but not InlB-mediated internalization of Listeria monocytogenes by non-phagocytic mammalian cells needs the support of other internalins. Mol Microbiol 43:557–570
Burnside K, Rajagopal L (2011) Aspects of eukaryotic-like signaling in Gram-positive cocci: a focus on virulence. Future Microbiol 6:747–761. https://doi.org/10.2217/fmb.11.62
Chen B, Zhang A, Xu Z, Li R, Chen H, ** M (2011) Large-scale identification of bacteria-host crosstalk by affinity chromatography: capturing the interactions of Streptococcus suis proteins with host cells. J Proteome Res. https://doi.org/10.1021/pr200758q
Chen L et al (2005) VFDB: a reference database for bacterial virulence factors. Nucleic Acids Res 33:D325-328. https://doi.org/10.1093/nar/gki008
Dumesnil A et al (2018) Characterization of the zinc metalloprotease of Streptococcus suis serotype 2. Vet Res 49:109. https://doi.org/10.1186/s13567-018-0606-y
Fang L, Zhou J, Fan P, Yang Y, Shen H, Fang W (2017) A serine/threonine phosphatase 1 of Streptococcus suis type 2 is an important virulence factor. J Vet Sci 18:439–447. https://doi.org/10.4142/jvs.2017.18.4.439
Feng L et al (2016) The CodY regulator is essential for virulence in Streptococcus suis serotype 2. Sci Rep 6:21241. https://doi.org/10.1038/srep21241
Ferrando ML, Willemse N, Zaccaria E, Pannekoek Y, van der Ende A, Schultsz C (2017) Streptococcal Adhesin P (SadP) contributes to Streptococcus suis adhesion to the human intestinal epithelium. PLoS ONE 12:e0175639. https://doi.org/10.1371/journal.pone.0175639
Fittipaldi N, Segura M, Grenier D, Gottschalk M (2012) Virulence factors involved in the pathogenesis of the infection caused by the swine pathogen and zoonotic agent Streptococcus suis. Future Microbiol 7:259–279. https://doi.org/10.2217/fmb.11.149
Hirschfeld C et al (2019) Proteomic investigation uncovers potential targets and target sites of pneumococcal serine-threonine kinase StkP and phosphatase PhpP. Front Microbiol 10:3101. https://doi.org/10.3389/fmicb.2019.03101
Ibrahim YM, Kerr AR, McCluskey J, Mitchell TJ (2004) Role of HtrA in the virulence and competence of Streptococcus pneumoniae. Infect Immun 72:3584–3591. https://doi.org/10.1128/IAI.72.6.3584-3591.2004
**g HB et al (2008) Proteome analysis of Streptococcus suis serotype 2. Proteomics 8:333–349. https://doi.org/10.1002/pmic.200600930
Ju CX, Gu HW, Lu CP (2012) Characterization and functional analysis of atl, a novel gene encoding autolysin in Streptococcus suis. J Bacteriol 194:1464–1473. https://doi.org/10.1128/JB.06231-11
Kim GL et al (2017) Effect of decreased BCAA synthesis through disruption of ilvC gene on the virulence of Streptococcus pneumoniae. Arch Pharm Res 40:921–932. https://doi.org/10.1007/s12272-017-0931-0
Lin X et al (2015) An integrated quantitative and targeted proteomics reveals fitness mechanisms of Aeromonas hydrophila under oxytetracycline stress. J Proteome Res 14:1515–1525. https://doi.org/10.1021/pr501188g
Lun ZR, Wang QP, Chen XG, Li AX, Zhu XQ (2007) Streptococcus suis: an emerging zoonotic pathogen. Lancet Infect Dis 7:201–209. https://doi.org/10.1016/S1473-3099(07)70001-4
Lyon WR, Caparon MG (2004) Role for serine protease HtrA (DegP) of Streptococcus pyogenes in the biogenesis of virulence factors SpeB and the hemolysin streptolysin S. Infect Immun 72:1618–1625
Ma F, Guo X, Fan H (2017) Extracellular nucleases of Streptococcus equi subsp. zooepidemicus degrade neutrophil extracellular traps and impair macrophage activity of the host. Appl Environ Microbiol. https://doi.org/10.1128/AEM.02468-16
Manuse S, Fleurie A, Zucchini L, Lesterlin C, Grangeasse C (2016) Role of eukaryotic-like serine/threonine kinases in bacterial cell division and morphogenesis. FEMS Microbiol Rev 40:41–56. https://doi.org/10.1093/femsre/fuv041
Moulin P et al (2016) The Adc/Lmb system mediates zinc acquisition in streptococcus agalactiae and contributes to bacterial growth and survival. J Bacteriol 198:3265–3277. https://doi.org/10.1128/JB.00614-16
Nagy TA, Moreland SM, Andrews-Polymenis H, Detweiler CS (2013) The ferric enterobactin transporter Fep is required for persistent Salmonella enterica serovar typhimurium infection. Infect Immun 81:4063–4070. https://doi.org/10.1128/IAI.00412-13
Norris MH, Propst KL, Kang Y, Dow SW, Schweizer HP, Hoang TT (2011) The Burkholderia pseudomallei Deltaasd mutant exhibits attenuated intracellular infectivity and imparts protection against acute inhalation melioidosis in mice. Infect Immun 79:4010–4018. https://doi.org/10.1128/IAI.05044-11
Parandhaman DK, Sharma P, Bisht D, Narayanan S (2014) Proteome and phosphoproteome analysis of the serine/threonine protein kinase E mutant of Mycobacterium tuberculosis. Life Sci 109:116–126. https://doi.org/10.1016/j.lfs.2014.06.013
Pian Y et al (2015) Proteomics identification of novel fibrinogen-binding proteins of Streptococcus suis contributing to antiphagocytosis. Front Cell Infect Microbiol 5:19. https://doi.org/10.3389/fcimb.2015.00019
Plumptre CD et al (2014) AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae. Mol Microbiol 91:834–851. https://doi.org/10.1111/mmi.12504
Ribardo DA, Hendrixson DR (2011) Analysis of the LIV system of Campylobacter jejuni reveals alternative roles for LivJ and LivK in commensalism beyond branched-chain amino acid transport. J Bacteriol 193:6233–6243. https://doi.org/10.1128/JB.05473-11
Rungelrath V et al (2018) IgM cleavage by Streptococcus suis reduces IgM bound to the bacterial surface and is a novel complement evasion mechanism. Virulence. https://doi.org/10.1080/21505594.2018.1496778
Segura M, Fittipaldi N, Calzas C, Gottschalk M (2017) Critical Streptococcus suis virulence factors: are they all really critical? Trends Microbiol. https://doi.org/10.1016/j.tim.2017.02.005
Shen X et al (2013) Proteome analysis of the two-component SalK/SalR system in epidemic Streptococcus suis serotype 2. Curr Microbiol 67:118–122. https://doi.org/10.1007/s00284-013-0343-4
Si Y et al (2009) Contribution of glutamine synthetase to the virulence of Streptococcus suis serotype 2. Vet Microbiol 139:80–88. https://doi.org/10.1016/j.vetmic.2009.04.024
Tang J et al (2006) Streptococcal toxic shock syndrome caused by Streptococcus suis serotype 2. PLoS Med 3:e151. https://doi.org/10.1371/journal.pmed.0030151
Unwin RD, Griffiths JR, Whetton AD (2010) Simultaneous analysis of relative protein expression levels across multiple samples using iTRAQ isobaric tags with 2D nano LC-MS/MS. Nat Protoc 5:1574–1582. https://doi.org/10.1038/nprot.2010.123
Willenborg J et al (2011) Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis. Microbiology 157:1823–1833. https://doi.org/10.1099/mic.0.046417-0
Wilson TL et al (2007) A novel signature-tagged mutagenesis system for Streptococcus suis serotype 2. Vet Microbiol 122:135–145. https://doi.org/10.1016/j.vetmic.2006.12.025
Wright DP, Ulijasz AT (2014) Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens. Virulence 5:863–885. https://doi.org/10.4161/21505594.2014.983404
Wu Z et al (2014) The Streptococcus suis transcriptional landscape reveals adaptation mechanisms in pig blood and cerebrospinal fluid. RNA 20:882–898. https://doi.org/10.1261/rna.041822.113
**ao G et al (2017) Streptococcus suis small RNA rss04 contributes to the induction of meningitis by regulating capsule synthesis and by inducing biofilm formation in a mouse infection model. Vet Microbiol 199:111–119. https://doi.org/10.1016/j.vetmic.2016.12.034
Yu Y et al (2018) Infection and adaption-based proteomic changes of Streptococcus suis serotype 2 in a pig model. J Proteomics 180:41–52. https://doi.org/10.1016/j.jprot.2017.12.001
Zhang A, Mu X, Chen B, Han L, Chen H, ** M (2011) IgA1 protease contributes to the virulence of Streptococcus suis. Vet Microbiol 148:436–439. https://doi.org/10.1016/j.vetmic.2010.09.027
Zhang H et al (2014) The identification of six novel proteins with fibronectin or collagen type I binding activity from Streptococcus suis serotype 2. J Microbiol 52:963–969. https://doi.org/10.1007/s12275-014-4311-x
Zhang X et al (2015) DnaJ of streptococcus suis type 2 contributes to cell adhesion and thermotolerance. J Microbiol Biotechnol 25:771–781
Zhang C et al (2017) The eukaryote-like serine/threonine kinase STK regulates the growth and metabolism of zoonotic Streptococcus suis. Front Cell Infect Microbiol 7:66. https://doi.org/10.3389/fcimb.2017.00066
Zheng F et al (2011) Contribution of the Rgg transcription regulator to metabolism and virulence of Streptococcus suis serotype 2. Infect Immun 79:1319–1328. https://doi.org/10.1128/IAI.00193-10
Zheng L, Khemlani A, Lorenz N, Loh JM, Langley RJ, Proft T (2015) Streptococcal 5’-nucleotidase A (S5nA), a novel streptococcus pyogenes virulence factor that facilitates immune evasion. J Biol Chem 290:31126–31137. https://doi.org/10.1074/jbc.M115.677443
Zheng C et al (2018a) Role of two-component regulatory systems in the virulence of Streptococcus suis. Microbiol Res 214:123–128. https://doi.org/10.1016/j.micres.2018.07.002
Zheng F et al (2018b) Identification of oligopeptide-binding protein (OppA) and its role in the virulence of Streptococcus suis serotype 2. Microb Pathog 118:322–329. https://doi.org/10.1016/j.micpath.2018.03.061
Zhong X et al (2018) The two-component signaling system VraSRSS is critical for multidrug resistance and full virulence in Streptococcus suis serotype 2. Infect Immun. https://doi.org/10.1128/IAI.00096-18
Zhu H et al (2011) The novel virulence-related gene stp of Streptococcus suis serotype 9 strain contributes to a significant reduction in mouse mortality. Microb Pathog 51:442–453. https://doi.org/10.1016/j.micpath.2011.08.002
Zhu H et al (2014) Contribution of eukaryotic-type serine/threonine kinase to stress response and virulence of Streptococcus suis. PLoS ONE 9:e91971. https://doi.org/10.1371/journal.pone.0091971
Zhu H et al (2018) The redox-sensing regulator rex contributes to the virulence and oxidative stress response of Streptococcus suis serotype 2. Front Cell Infect Microbiol 8:317. https://doi.org/10.3389/fcimb.2018.00317
Acknowledgements
We thank Liwen Bianji, Edanz Group China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript.
Funding
This work was supported by National Key Research and Development Program (2018YFD0500101), National Natural Sciences Foundation of China (31302114), the Special Fund for Public Welfare Industry of Chinese Ministry of Agriculture (201303041), and the Innovation of Agricultural Sciences in Jiangsu province (CX(14)5042). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Comparative proteomics were performed with the help of Wuhan GeneCreate Biological Engineering Co., Ltd.
Author information
Authors and Affiliations
Contributions
Conceived and designed the experiments: HZ, YN and KH. Performed the experiments: HZ, JZ and DW. Analyzed the data: HZ and JZ. Contributed reagents/materials/analysis tools: ZY, BL, YN, and KH. Wrote the manuscript: HZ, YN and KH. All authors read, advised, and approved the final manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Additional information
Communicated by Erko Stackebrandt.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhu, H., Zhou, J., Wang, D. et al. Quantitative proteomic analysis reveals that serine/threonine kinase is involved in Streptococcus suis virulence and adaption to stress conditions. Arch Microbiol 203, 4715–4726 (2021). https://doi.org/10.1007/s00203-021-02369-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00203-021-02369-5