pppL (SP-STP, SPy_1626) is the PPM/PP2C-type, divalent-metal-dependent protein serine/threonine phosphatase of group A Streptococcus, encoded immediately downstream of and co-transcribed with the eukaryote-type Ser/Thr kinase SP-STK (SPy_1625). Together the two enzymes form a one-component reversible-phosphorylation module: SP-STK phosphorylates threonine residues on the two-component response regulators WalR and CovR and on a histone-like protein, and SP-STP removes them. Through this cognate control SP-STP sets the expression of genes governing septum formation and chain length (via WalR and CdhA), capsule and hemolysin synthesis (via CovR), surface proteins and SpeB, and fatty acid biosynthesis (via the FabT repressor). The enzyme is largely cytoplasmic but is also a major secreted protein; secreted SP-STP enters human pharyngeal epithelial cells, crosses into the nucleus, dephosphorylates histone H1 at Thr146 and triggers caspase-dependent apoptosis, an activity that requires its intact catalytic and structural core. Loss of pppL leaves the bacterium viable but strongly attenuated in vivo, so the protein is both a central physiological regulator and a host-directed virulence effector.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000918 division septum site selection | IMP PMID:21917918 Role of serine/threonine phosphatase (SP-STP) in Streptococc... | MODIFY | Summary: The stp mutant phenotype is long chains with multiple asymmetric and parallel septa, which the authors interpret as regulation of septum formation acting through WalR phosphorylation and CdhA levels, not as participation in division-site selection. Reason: CACAO row. What the paper shows (Fig. 2A-D): Gram and fluorescent-vancomycin staining plus TEM of the non-polar M1DeltaSTP and M1T1DeltaSTP mutants give long chains, thickened walls and multiple asymmetric or parallel septa, reverted by complementation; the authors conclude a role in the regulation of septa formation and in turn cell division. The mechanism offered (Fig. 3) is indirect and regulatory: SP-STK/SP-STP reversibly phosphorylate WalR, and CdhA protein and transcript rise in the mutants. GO:0000918 is the wrong granularity and the wrong kind of claim, because it names the machinery that positions the division plane (Min/Noc/DivIVA-type activity), and nothing here shows SP-STP doing that work rather than tuning the expression of division genes. This is the phenotype-read-as-process shape, so MODIFY to the regulatory term the authors themselves assert, GO:0032955 regulation of division septum assembly, which also covers the misplaced-septum observation. Proposed replacements: regulation of division septum assembly Supporting Evidence: PMID:21917918 revealed unusual staining patterns with multiple asymmetric and parallel septa formation PMID:21917918 indicated that SP-STP plays a role in the regulation of septa formation and in turn cell division in GAS PMID:21917918 an increase in the level of CdhA protein in the culture supernatants of early log phase-grown mutant strains |
| GO:0004721 phosphoprotein phosphatase activity | IDA PMID:16487973 Identification and biochemical characterization of a eukaryo... | MODIFY | Summary: Direct biochemistry on recombinant SP-STP established Mn2+-dependent phosphatase activity whose substrate site is a threonine residue, so the specific child term GO:0004722 is what the assay supports. Reason: CACAO row. What the paper shows: SP-STK and SP-STP were purified and assayed with the endogenous histone-like substrate SP-HLP; activity of both enzymes was primarily Mn2+-dependent and the reversibly modified site was only threonine. The term is therefore correct but is the genus of what was measured. The gene already carries the specific child GO:0004722 (IDA from PMID:21917918, and IEA), and every mapped dephosphorylation event in this protein's literature (SP-HLP, WalR, CovR, host histone H1 Thr146) is on threonine. Per the refinement rule this is MODIFY to the supported descendant rather than a bare accept of the general term. The full text of this 2006 paper is not available (not in PMC), but the abstract states the assay result directly and the claim is independently confirmed, so no curator-deference problem arises. Proposed replacements: protein serine/threonine phosphatase activity Supporting Evidence: PMID:16487973 Enzyme activities of SP-STK and SP-STP primarily depended on Mn(2+). PMID:16487973 The site on the substrate for reversible phosphorylation by these enzymes was found to be only the threonine residue. |
| GO:0004722 protein serine/threonine phosphatase activity | IDA PMID:21917918 Role of serine/threonine phosphatase (SP-STP) in Streptococc... | ACCEPT | Summary: Recombinant SP-STP dephosphorylates SP-STK-phosphorylated WalR and CovR in vitro, on threonine residues; this is the core molecular function of the protein. Reason: CACAO row, and the best-supported one. What the paper shows (Fig. 3C-D, Fig. 4C-D): in vitro kinase and phosphatase assays with purified SP-STKK and SP-STP on the response regulators SP-WalR and SP-CovR; the kinase phosphorylates both specifically on threonine (TLC of acid hydrolysates) and the cognate phosphatase removes the label. The term matches the assay exactly, at the right granularity, and is reinforced by the PPM/PP2C family assignment (PROSITE PS51746, Pfam PF13672, EC 3.1.3.16) and by the independent 2006 biochemistry. Retained as the core function. Supporting Evidence: PMID:21917918 The SP-STP was able to dephosphorylate the SP-STK-phosphorylated SP-WalR PMID:21917918 The specificity of SP-STK mediated CovR phosphorylation was confirmed by its subsequent dephosphorylation by the cognate SP-STP PMID:21917918 The latter possesses conserved I-XI motifs belonging to a distinct class of metal-dependent PPM/PP2C-type serine/threonine phosphatase |
| GO:0004722 protein serine/threonine phosphatase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Automated assignment from the PP2C/PPM signature, agreeing with the experimental IDA row for the same term. Reason: The electronic assignment rests on InterPro IPR015655 (PP2C) and EC 3.1.3.16, and the UniProt record carries the PPM-type phosphatase domain over almost the whole protein (residues 2-240, PROSITE PS51746) plus Pfam PF13672. The inference is confirmed directly for this protein by the in vitro dephosphorylation of WalR and CovR (PMID:21917918) and by the 2006 enzymology, so this row is consistent with, and reinforces, the IDA row for the same term. Supporting Evidence: file:STRP1/pppL/pppL-uniprot.txt FT DOMAIN 2..240 PMID:21917918 The SP-STP was able to dephosphorylate the SP-STK-phosphorylated SP-WalR |
| GO:0035970 peptidyl-threonine dephosphorylation | IDA PMID:21917918 Role of serine/threonine phosphatase (SP-STP) in Streptococc... | ACCEPT | Summary: The phospho-acceptor removed by SP-STP was mapped to threonine on both response regulators, and in a later study to Thr146 of host histone H1. Reason: CACAO row, well chosen. What the paper shows (Fig. 3D, Fig. 4D): TLC of acid hydrolysates of SP-STK-phosphorylated WalR and CovR identifies threonine as the modified residue, and the same preparations are dephosphorylated by SP-STP, so the dephosphorylation event SP-STP catalyses is peptidyl-threonine. The 2006 paper reached the same conclusion for the SP-HLP substrate, and PMID:22262847 shows loss of the H1 Thr146 phospho-epitope in treated host nuclei. The term is the process counterpart of the accepted molecular function at exactly the right granularity; no serine site has been demonstrated for this enzyme, so this is the better-supported of the two possible residue-specific terms. Supporting Evidence: PMID:21917918 SP-STK was able to phosphorylate SP-WalR ( Fig. 3 C ) specifically at threonine residues, as is evident by TLC PMID:21917918 the TLC revealed that SP-STK mediates phosphorylation of CovR specifically at its threonine residues PMID:22262847 A significant reduction in the reactivity of anti-Pan H1-reacting 27-kDa protein in the nuclear extract obtained from the SP-STP-treated Detroit cells with anti- Thr(P)-146 H1 antibody |
| GO:0045717 negative regulation of fatty acid biosynthetic process | IMP PMID:21917918 Role of serine/threonine phosphatase (SP-STP) in Streptococc... | KEEP AS NON CORE | Summary: Deleting stp raises total saturated and unsaturated fatty acid content 2-3-fold and lowers the fabT repressor transcript, so the sign of the regulation is right, but the effect is transcriptional and indirect and is peripheral to the enzyme's core function. Reason: CACAO row. What the paper shows (Table 1 and Fig. 6C-D): gas-chromatographic fatty acid methyl ester analysis of M1SF370 versus M1DeltaSTP gives 2-3-fold higher saturated and unsaturated fatty acid and about 3-fold higher total lipid in the mutant, with loss of C15:0 anteiso and C17:0 cyclopropane species, alongside 3-6-fold down-regulation of the GAS fabT homolog (marR/SPy1755) and 4-10-fold down-regulation of fabH in both mutants. Loss of the phosphatase therefore increases fatty acid synthesis, which is the direction GO:0045717 asserts, and the paper notes the complementary finding that SP-STK positively regulates fatty acid biosynthesis. The term is thus supported in sign and at a reasonable granularity. It is kept as non-core rather than accepted because the link is indirect (the authors themselves only speculate that SP-STK phosphorylates the FabT repressor, and do not test it), the lipid measurement was made in one of the two strain backgrounds, and fatty acid output is one of many downstream readouts of this global regulator rather than part of what the protein does. Supporting Evidence: PMID:21917918 both saturated fatty acid (SFA) and unsaturated fatty acid (UFA) content in the mutant were 2β3-fold up-regulated PMID:21917918 we also observed down-regulation of the fabT homolog of GAS, marR / SPy1755 (3β6-fold), in both of the mutant strains |
| GO:0046872 metal ion binding | IEA GO_REF:0000104 | MODIFY | Summary: The PPM fold binds two divalent metal ions per subunit and SP-STP activity was shown experimentally to depend primarily on Mn2+, so the supported term is manganese ion binding. Reason: The UniRule-derived term is true but is the genus of what is known. UniProt's PROSITE-ProRule cofactor annotation states that the PPM-type domain binds two magnesium or manganese ions per subunit, and direct enzymology on purified SP-STP showed that its activity depends primarily on Mn2+ (PMID:16487973), which is also how the recombinant protein was assayed in the later work. Per the refinement rule the row should carry the supported descendant GO:0030145 manganese ion binding. Magnesium is left out of the replacement deliberately, being a fold-level expectation from the rule rather than a measured property of this protein; it can be added if a metal-substitution assay supports it. Proposed replacements: manganese ion binding Supporting Evidence: PMID:16487973 Enzyme activities of SP-STK and SP-STP primarily depended on Mn(2+). PMID:21917918 The latter possesses conserved I-XI motifs belonging to a distinct class of metal-dependent PPM/PP2C-type serine/threonine phosphatase |
| GO:0052151 symbiont-mediated activation of host apoptosis | IMP PMID:22262847 Serine/threonine phosphatase (SP-STP), secreted from Strepto... | ACCEPT | Summary: Wild-type GAS kills pharyngeal cells while the stp mutants barely do, complementation restores killing, purified SP-STP alone is sufficient and specific, the effect is caspase-dependent, and catalytically inactive protein is inert; this is a direct host-directed activity of the gene product. Reason: CACAO row, the strongest in the set. What the paper shows: MTT proliferation assays (Fig. 2A) give more than 90% inhibition by wild-type M1SF370 and M1T15448 but only 5% and 18% by M1DeltaSTP and M1T1DeltaSTP, with restoration in the stp-complemented strains; purified SP-STP alone inhibits Detroit 562 and A549 cells dose-dependently while the structurally homologous EF-STP does not (Fig. 2B, 3A); annexin-V staining, JC-1 mitochondrial depolarization, caspase-3, caspase-9 and PARP cleavage and blockade by caspase inhibitors establish apoptosis rather than necrosis (Figs. 3, 4 and 10); mouse lung TUNEL confirms it in vivo (Fig. 5); and the catalytically inactive D192A protein neither internalizes nor kills, including when expressed inside the cell (Figs. 7-9). SP-STP itself performs the work here, entering the host cell, reaching the nucleus and dephosphorylating host histone H1, so the participation requirement is met and the term is at the right granularity. One caveat recorded for completeness, that killing was seen in two of four human cell lines, makes the activity cell-type selective but does not weaken the claim. Supporting Evidence: PMID:22262847 significantly lower inhibition of proliferation of pharyngeal cells treated with GAS strains devoid of SP-STP (M1ΞSTP and M1T1ΞSTP, 5 and 18%, respectively) unlike the wild-type GAS strains (>90% inhibition) PMID:22262847 only SP-STP and not EF-STP displayed dose-dependent proliferation inhibition of Detroit pharyngeal cells PMID:22262847 a significant number of TUNEL-positive cells were found in the lungs of wild-type M1SF370-infected mice as compared with the lungs of M1ΞSTP-infected mice PMID:22262847 The programmed cell death induced by SP-STP, which requires active transcription and de novo protein synthesis, is also caspase-dependent |
| GO:0062085 positive regulation of capsule polysaccharide biosynthetic process | IMP PMID:16487973 Identification and biochemical characterization of a eukaryo... | KEEP AS NON CORE | Summary: SP-STP positively regulates hyaluronic acid capsule synthesis through CovR, but the quantified stp-mutant evidence for that direction is in PMID:21917918; the cited 2006 paper asserts capsule modulation by this kinase and phosphatase system while its isogenic mutants were kinase mutants. Reason: CACAO row. What the cited paper shows: the 2006 abstract reports that the three isogenic mutants constructed lacked the entire SP-STK or one of its domains, and states that these proteins modulate expression of the major virulence factors, the M protein and the capsule. The full text is not available (J Mol Biol, not in PMC), so what else was assayed for SP-STP cannot be read, and the curator-deference rule forbids calling the row mis-referenced on an abstract alone. The direction asserted by the term is independently and quantitatively established in the stp mutants themselves, with 3-4-fold less capsule and 14-100-fold lower has transcript in both M1DeltaSTP and M1T1DeltaSTP, restored by complementation, and CovR identified as the mediating response regulator (PMID:21917918, Fig. 4A). Noted for the project record is that capsule goes up in the SP-STK deletion, so the sign cannot be transferred from the kinase mutants of the cited paper. Kept as non-core rather than accepted because the regulation is a transcriptional consequence of CovR dephosphorylation, one of many outputs of this global regulator, rather than part of the enzyme's own activity. Supporting Evidence: PMID:16487973 the ability of these three proteins to modulate the expression of the major virulence factors, the M protein and the capsule PMID:21917918 In both of the STP mutants, we observed a 3β4-fold decrease in the capsule production |
| GO:0075294 positive regulation by symbiont of entry into host | IMP PMID:21917918 Role of serine/threonine phosphatase (SP-STP) in Streptococc... | MODIFY | Summary: The only entry readout in the paper goes the other way, the stp mutant being 6-fold more invasive, while the adherence decrease that motivated the term was seen in only one of two strain backgrounds, so the sign of the regulation is not established. Reason: CACAO row, and the clearest error in the set. What the paper shows (Fig. 5A-B): on Detroit 562 pharyngeal cells, adherence was significantly reduced for M1DeltaSTP but unaltered for M1T1DeltaSTP relative to their parents, and for internalization the mutant was more invasive, with the discussion stating that the adherent mutant strain was 6-fold more invasive. Entry into the host therefore increases, not decreases, on loss of stp, so positive regulation by symbiont of entry into host has the sign inverted; it appears to have been read off the adherence panel, which measures attachment rather than entry and is itself strain-specific (the authors attribute it to down-regulated pilin, scpA, sic and scl transcripts, and the increased invasion in part to about 14-fold speB up-regulation). Because SP-STP demonstrably modulates adherence and invasion but the direction for entry is not established, MODIFY to the sign-free parent GO:0052372 modulation by symbiont of entry into host. REMOVE is not used, because this is an experimental annotation whose underlying phenomenon is real and the defect is the sign, not the existence of the effect. Proposed replacements: modulation by symbiont of entry into host Supporting Evidence: PMID:21917918 The results showed a significantly reduced adherence pattern for the M1ΞSTP strain and unaltered adherence for the M1T1ΞSTP mutant in comparison with their corresponding wild-type strains PMID:21917918 the M1ΞSTP mutant was found to be more invasive than the M1T1ΞSTP mutant, reiterating the strain-specific STP-mediated regulation in GAS adherence and invasion PMID:21917918 although the M1ΞSTP mutant lost the ability to adhere to pharyngeal cells, the adherent mutant strain was 6-fold more invasive |
| GO:0042025 host cell nucleus | IDA PMID:22262847 Serine/threonine phosphatase (SP-STP), secreted from Strepto... | NEW | Summary: Secreted SP-STP added to pharyngeal cells reaches the nucleus of most cells, GFP-STP expressed in those cells is nuclear, and the host substrate it acts on, histone H1 Thr146, is nuclear. Reason: Proposed because the location where part of the accepted host-directed function is executed is missing from GOA. Fluorescently labelled SP-STP added exogenously is membrane-proximal at 3 h, cytoplasmic at 6 h and nuclear in more than 70% of cells by 12-24 h, confirmed by subcellular fractionation; ectopically expressed GFP-SP-STP is likewise nuclear; and the functional consequence read out in the nucleus is loss of the H1 Thr146 phospho-epitope. Two independent delivery routes (exogenous protein and ectopic expression) plus a nuclear enzymatic readout support the location, so this is not a single-assay localization. Supporting Evidence: PMID:22262847 More than 70% of the total cells displayed the labeled SP-STP within the nucleus PMID:22262847 The ectopically expressed wild-type SP-STP was found to be in the nucleus and conferred apoptosis of Detroit 562 pharyngeal cells PMID:22262847 A significant reduction in the reactivity of anti-Pan H1-reacting 27-kDa protein in the nuclear extract obtained from the SP-STP-treated Detroit cells with anti- Thr(P)-146 H1 antibody |
| GO:0005576 extracellular region | IDA PMID:16487973 Identification and biochemical characterization of a eukaryo... | NEW | Summary: SP-STP is a major secreted protein of GAS, detected with specific antibodies outside the cell, and secretion is what makes the host-directed apoptotic activity possible. Reason: Proposed because the secreted pool is a required part of the protein's biology and no cellular component row exists in GOA. Antibodies raised against SP-STP identified it as a major secretory protein of GAS (PMID:16487973), and immunoblotting of supernatants from GAS exposed to a pharyngeal cell extract detects the released 27-kDa protein, which is the form that enters host cells (PMID:22262847). The protein has no classical signal peptide, which the authors note explicitly, so the term asserts only where the protein is found and says nothing about a secretion mechanism. Supporting Evidence: PMID:16487973 SP-STP, primarily a cytoplasmic protein, was found to be a major secretory protein of GAS and essential for bacterial survival. PMID:22262847 the entry of SP-STP into the cytoplasm is dependent on its secondary structure as the catalytically inactive SP-STP with an altered structure is unable to internalize and cause apoptosis |
| GO:0005737 cytoplasm | IDA PMID:16487973 Identification and biochemical characterization of a eukaryo... | NEW | Summary: Antibody-based localization places SP-STP primarily in the bacterial cytoplasm, which is where its response-regulator substrates WalR and CovR are dephosphorylated. Reason: Proposed to record the compartment in which the core phosphatase function acts, since GOA has no cellular component row for this protein. The 2006 study used antibodies specific for SP-STK and SP-STP to localize them, reporting SP-STP as primarily cytoplasmic (in contrast to the membrane-associated kinase), and its substrates WalR and CovR are cytoplasmic response regulators. The same sentence's claim that stp is essential for survival was later corrected by the non-polar mutants of PMID:21917918; that correction concerns essentiality, not localization. Supporting Evidence: PMID:16487973 SP-STP, primarily a cytoplasmic protein, was found to be a major secretory protein of GAS and essential for bacterial survival. |
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Download this section (compressed HTML)Q: Does SP-STP dephosphorylate phosphoserine at all? Every mapped site in the literature (SP-HLP, WalR, CovR, host histone H1 Thr146) is a threonine, yet the protein is named and annotated as a serine/threonine phosphatase on the basis of the PPM fold and EC 3.1.3.16.
Q: What is the full substrate set of SP-STP in GAS, and how much of the pleiotropic stp mutant phenotype is explained by WalR and CovR alone versus additional direct substrates?
Q: By what route is a signal-peptide-less cytoplasmic phosphatase secreted, and how is the partition between the cytoplasmic regulatory pool and the secreted effector pool controlled during infection?
Q: How does SP-STP cross the host plasma membrane and the nuclear envelope, and is a host receptor responsible for the cell-type selectivity, with Detroit 562 and A549 responding while Cal-27 and HepG2 do not?
Q: Is host histone H1 Thr146 dephosphorylation causal for the apoptosis or a correlate of it, given that blocking the ERK-driven H3 phosphorylation did not prevent killing?
Q: Does loss of stp genuinely increase bacterial entry into epithelial cells relative to the wild type, independently of the SpeB up-regulation seen in M1SF370, and does the direction hold in the M1T1 background?
Experiment: Assay purified SP-STP against matched phosphopeptide pairs differing only in phospho-Ser versus phospho-Thr at the same position, including the WalR, CovR and H1 Thr146 contexts, measuring kinetic constants, and complement this with phosphoproteomics of wild-type versus stp-mutant GAS to see whether phospho-Ser sites respond to stp status at all.
Hypothesis: SP-STP is a threonine-specific rather than a dual serine/threonine phosphatase.
Type: enzyme kinetics with defined phosphopeptides plus differential phosphoproteomics
Experiment: Replace walR and covR with phosphoablative (Thr to Ala) and phosphomimetic alleles at the SP-STK target threonines in both M1 backgrounds, and test whether these alleles reproduce or suppress the chain and septum, has transcript, capsule and fatty acid phenotypes of the stp deletion.
Hypothesis: The division and capsule phenotypes of the stp mutant are mediated specifically by the phospho-threonine sites of WalR and CovR.
Type: allele-replacement epistasis analysis
Experiment: Repeat adherence and internalization assays with wild type, stp mutant and complemented strain side by side in both M1SF370 and M1T15448, normalizing invasion to the adherent population and using a speB deletion background to remove the confounding protease effect, so the sign of the stp effect on entry can be stated for each strain.
Hypothesis: Entry of GAS into pharyngeal cells is not positively regulated by SP-STP.
Type: quantitative adherence and gentamicin-protection invasion assay
Experiment: Express H1 variants that cannot be phosphorylated or that mimic phosphorylation at Thr146 in Detroit 562 cells and test whether SP-STP-induced caspase activation and killing is blocked or bypassed; in parallel, target catalytically active SP-STP to the cytoplasm only, by fusing a nuclear export signal, to separate nuclear from cytoplasmic contributions.
Hypothesis: Nuclear histone H1 dephosphorylation by SP-STP is required for host apoptosis.
Type: substrate-site mutagenesis with compartment-restricted effector
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