Abstract

   Like eukaryotes, bacteria express one or more serine/threonine kinases
   (STKs) that initiate diverse signaling networks. The STK from
   Streptococcus suis is encoded by a single-copy stk gene, which is
   crucial in stress response and virulence. To further understand the
   regulatory mechanism of STK in S. suis, a stk deletion strain (Δstk)
   and its complementary strain (CΔstk) were constructed to systematically
   decode STK characteristics by applying whole transcriptome RNA
   sequencing (RNA-Seq) and phosphoproteomic analysis. Numerous genes were
   differentially expressed in Δstk compared with the wild-type parental
   strain SC-19, including 320 up-regulated and 219 down-regulated genes.
   Particularly, 32 virulence-associated genes (VAGs) were significantly
   down-regulated in Δstk. Seven metabolic pathways relevant to bacterial
   central metabolism and translation are significantly repressed in Δstk.
   Phosphoproteomic analysis further identified 12 phosphoproteins that
   exhibit differential phosphorylation in Δstk. These proteins are
   associated with cell growth and division, glycolysis, and translation.
   Consistently, phenotypic assays confirmed that the Δstk strain
   displayed deficient growth and attenuated pathogenicity. Thus, STK is a
   central regulator that plays an important role in cell growth and
   division, as well as S. suis metabolism.

   Keywords: Streptococcus suis, eukaryote-like serine/threonine kinase,
   phosphorylation, RNA-Seq, phosphoproteome, growth, virulence,
   metabolism

Introduction

   Upon sensing external stimuli, protein kinases, together with their
   cognate phosphatases, play a central role in signal transduction to
   quickly respond and adapt to constantly changing environments in both
   prokaryotes and eukaryotes. Reversible phosphorylation occurs on
   specific amino acid residues, most commonly serine (Ser), threonine
   (Thr), tyrosine (Tyr), histidine (His), and aspartate (Asp) (Pereira et
   al., [39]2011). Earlier classifications of prokaryote, kinases have
   been assumed to target only residues His and Asp, which are involved in
   two-component systems (TCS; Stock et al., [40]1990; Hoch, [41]2000).
   Increasing attention has been paid to the Ser/Thr kinases and their
   partner phosphatases. Some bacterial Ser/Thr kinases, which show
   conservation in their catalytic domains compared with eukaryotic
   Ser/Thr kinases, are designated as “eukaryote-like Ser/Thr kinases
   (eSTK)” (Pereira et al., [42]2011). Pkn1 of Myxococcus xanthus is the
   first characterized eSTK in bacteria (Muñoz-Dorado et al., [43]1991);
   subsequently a second eSTK, Pkn2, has been identified in M. xanthus
   (Udo et al., [44]1995). Furthermore, numerous bacterial eSTKs have been
   identified based on genome sequence databases (Galperin et al.,
   [45]2010). Multiple eSTKs exist in most bacteria; therefore,
   comprehensively characterizing their essentiality and identifying their
   specific substrates are difficult. For example, 11 eSTKs exist in
   Mycobacterium tuberculosis that has functional redundancy and/or
   substrate promiscuity (Boitel et al., [46]2003; Sajid et al.,
   [47]2015).

   The eSTKs have been widely studied for their roles in diverse
   biological processes, including development (Zhang, [48]1993; Nádvorník
   et al., [49]1999; Inouye and Nariya, [50]2008), cell competence
   (Hussain et al., [51]2006), cell division, and cell wall synthesis
   (Deol et al., [52]2005; Fernandez et al., [53]2006; Ruggiero et al.,
   [54]2012), central and secondary metabolism (Lee et al., [55]2002;
   Sawai et al., [56]2004), biofilm formation (Hussain et al., [57]2006;
   Liu et al., [58]2011), stress response (Neu et al., [59]2002;
   Mata-Cabana et al., [60]2012), and virulence (Madec et al., [61]2002;
   Rajagopal et al., [62]2003; Echenique et al., [63]2004). Gene
   expression profiles have proven their global regulatory roles in
   cellular processes (Sasková et al., [64]2007; Donat et al., [65]2009).
   Moreover, both phosphoproteomic analyses and kinase assays have
   identified eSTK substrates in Streptococcus pyogenes (Jin and Pancholi,
   [66]2006), Streptococcus pneumoniae (Nováková et al., [67]2005,
   [68]2010), Streptococcus agalactiae (Silvestroni et al., [69]2009),
   Staphylococcus aureus (Lomas-Lopez et al., [70]2007; Truong-Bolduc et
   al., [71]2008), Listeria monocytogenes (Archambaud et al., [72]2005),
   and M. tuberculosis (Arora et al., [73]2010). Most identified
   substrates are involved in cell growth/division and central metabolism
   of bacteria. Various microorganisms have been studied, but the profound
   effects of eSTKs and posttranslational modification on their targets
   remain poorly understood.

   Streptococcus suis is a zoonotic Gram-positive pathogen that causes
   lethal infections in pigs and humans (Lun et al., [74]2007). Two large
   outbreaks of human S. suis infection have been reported in 1998 and
   2005 in China, resulting in 229 infections and 52 deaths (Lun et al.,
   [75]2007). Among the 33 S. suis serotypes, serotype 2 (SS2) is the most
   virulent and prevalent serotype isolated from diseased pigs (Smith et
   al., [76]1999). Moreover, SS2 is the prominent agent that caused adult
   human meningitis in Vietnam and Hong Kong (Wertheim et al., [77]2009).
   Numerous virulence-associated factors of S. suis have been identified
   over the past decade, such as capsular polysaccharide,
   muramidase-released protein, suilysin, extracellular factor,
   fibrinonectin- and fibrinogen-binding proteins, enolase, arginine
   deiminase system, glyceraldehyde-3-phosphate dehydrogenase (GAPDH),
   inosine 5-monophosphate dehydrogenase (IMPDH), secreted nuclease A
   (SsnA), subtilisin-like protease A (Fittipaldi et al., [78]2012), H
   binding protein (Fhb; Pian et al., [79]2012), and so on. Compared with
   other Gram-positive bacteria, only a single-copy stk is present in the
   S. suis genome (Zhu et al., [80]2014). The STK of S. suis is involved
   in stress response and virulence. The disruption of stk in S. suis
   enables increased chain-length, reduced tolerance to high temperature,
   low acidic pH, oxidative stress, and decreased virulence (Zhu et al.,
   [81]2014).

   To further understand the regulatory mechanism of STK in S. suis, we
   constructed a stk-deletion mutant (Δstk) and investigated its
   biological characterizations using “-omics” approaches. By comparing
   the transcriptomic profiles, we identified differentially expressed
   genes (DEGs) between the Δstk strain and the wild-type parental strain,
   SC-19. Using phosphoproteome analyses, phosphorylation level of
   protein-coding sequences were systematically estimated. The analyses of
   both transcriptomic and phosphoproteomic provide functional context
   that STK can regulate cell growth and division, as well as metabolism
   of S. suis.

Materials and methods

Bacterial strains, plasmids, and culture conditions

   The bacterial strains and plasmids used in this study are listed in
   Table [82]1. The virulent S. suis strain SC-19 was isolated from a
   diseased pig during the 2005 outbreak in Sichuan, China (Li et al.,
   [83]2009). Since the genome of SC-19 has not been sequenced, the genome
   sequence of the strain S. suis 05ZYH33 (GenBank accession number
   [84]CP000407) was used as reference for gene clone, transcriptomic, and
   phosphoproteomic analysis. S. suis 05ZYH33 was isolated from an
   infected human during the same outbreak in Sichuan (Lun et al.,
   [85]2007). Both of these two isolates are serotype 2. Bacteria were
   grown in TODD-Hewitt broth (THB; OXOID, England) medium or plated on
   THB Agar (THA; OXOID) with 5% (v/v) sheep blood at 37°C. Erythromycin
   (90 μg/ml) was added to screen the mutant strain and erythromycin (90
   μg/ml) and spectinomycin (100 μg/ml) were added to select for a
   complementary strain.

Table 1.

   Bacterial strains and plasmids used in this study.
   Stains and plasmids Revelant characteristics and genotype[86]^* Sources
   or references