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RopB represses the transcription of speB in the absence of SIP in group A Streptococcus

View ORCID ProfileChuan Chiang-Ni  Correspondence email, Yan-Wen Chen, Kai-Lin Chen, Jian-Xian Jiang, View ORCID ProfileYong-An Shi, Chih-Yun Hsu, View ORCID ProfileYi-Ywan M Chen, Chih-Ho Lai, Cheng-Hsun Chiu
Chuan Chiang-Ni
1https://ror.org/00d80zx46Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Taoyuan, Taiwan
2https://ror.org/00d80zx46Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
3https://ror.org/02dnn6q67Department of Orthopedic Surgery, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan
4https://ror.org/02dnn6q67Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan
Roles: Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing
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  • For correspondence: entchuan@gap.cgu.edu.tw
Yan-Wen Chen
1https://ror.org/00d80zx46Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Taoyuan, Taiwan
Roles: Data curation, Formal analysis, Investigation
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Kai-Lin Chen
3https://ror.org/02dnn6q67Department of Orthopedic Surgery, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan
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Jian-Xian Jiang
2https://ror.org/00d80zx46Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
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Yong-An Shi
2https://ror.org/00d80zx46Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
Roles: Data curation, Formal analysis, Validation
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Chih-Yun Hsu
1https://ror.org/00d80zx46Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Taoyuan, Taiwan
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Yi-Ywan M Chen
1https://ror.org/00d80zx46Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Taoyuan, Taiwan
2https://ror.org/00d80zx46Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
4https://ror.org/02dnn6q67Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan
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Chih-Ho Lai
1https://ror.org/00d80zx46Department of Microbiology and Immunology, College of Medicine, Chang Gung University, Taoyuan, Taiwan
2https://ror.org/00d80zx46Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
4https://ror.org/02dnn6q67Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan
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Cheng-Hsun Chiu
2https://ror.org/00d80zx46Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan
4https://ror.org/02dnn6q67Molecular Infectious Disease Research Center, Chang Gung Memorial Hospital at Linkou, Taoyuan, Taiwan
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Published 31 March 2023. DOI: 10.26508/lsa.202201809
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Abstract

RopB is a quorum-sensing regulator that binds to the SpeB-inducing peptide (SIP) under acidic conditions. SIP is known to be degraded by the endopeptidase PepO, whose transcription is repressed by the CovR/CovS two-component regulatory system. Both SIP-bound RopB (RopB-SIP) and SIP-free RopB (apo-RopB) can bind to the speB promoter; however, only RopB-SIP activates speB transcription. In this study, we found that the SpeB expression was higher in the ropB mutant than in the SIP-inactivated (SIP*) mutant. Furthermore, the deletion of ropB in the SIP* mutant derepressed speB expression, suggesting that apo-RopB is a transcriptional repressor of speB. Up-regulation of PepO in the covS mutant degraded SIP, resulting in the down-regulation of speB. We demonstrate that deleting ropB in the covS mutant derepressed the speB expression, suggesting that the speB repression in this mutant was mediated not only by PepO-dependent SIP degradation but also by apo-RopB. These findings reveal a crosstalk between the CovR/CovS and RopB-SIP systems and redefine the role of RopB in regulating speB expression in group A Streptococcus.

Introduction

Streptococcus pyogenes (group A Streptococcus, GAS) is a gram-positive bacterial pathogen that causes various diseases, including pharyngitis, scarlet fever, cellulitis, necrotizing fasciitis, and toxic shock syndrome (Cunningham, 2008). CovR/CovS (control of virulence), previously designated CsrR/CsrS (Levin & Wessels, 1998), is a two-component regulatory system in GAS (Federle et al, 1999). CovS phosphorylates intracellular CovR, and the phosphorylated CovR primarily acts as a transcriptional repressor (Miller et al, 2001; Dalton & Scott, 2004; Gusa et al, 2006; Churchward, 2007). Spontaneous mutations in covS, which result in a functional loss in its capacity to phosphorylate CovR, derepress the expression of one group of virulence factors (streptolysin O, streptokinase, and hyaluronic acid capsule) but repress the transcription of a second group of genes (speB, grab, and spd3) (Sumby et al, 2006; Trevino et al, 2009; Ikebe et al, 2010; Friaes et al, 2015). Specifically, the expression of SpeB protease is down-regulated in the covS mutant compared with that in the wild-type strain (Sumby et al, 2006; Trevino et al, 2009; Tran-Winkler et al, 2011; Chiang-Ni et al, 2019a), suggesting that phosphorylated CovR can transcriptionally activate speB. Furthermore, Finn et al (Finn et al, 2021) showed that non-phosphorylated CovR can bind to the speB promoter and repress speB expression. These results suggest that the expression of speB is activated by phosphorylated CovR but repressed by non-phosphorylated CovR; however, the deletion of covR in the wild-type strain and the covS mutant results in the derepression of speB (Chiang-Ni et al, 2016). Therefore, the phosphorylated and non-phosphorylated CovR-mediated regulatory mechanisms of speB expression require further investigation.

The SpeB cysteine protease is secreted as a zymogen (42 kD), and its protease activity is essential for the autocatalysis of the zymogen to the mature SpeB protease (28 kD) (Doran et al, 1999; Chen et al, 2003). SpeB degrades or cleaves both host proteins (fibrin, fibronectin, vitronectin, immunoglobulins, and complement proteins) and bacterial surface and virulence-associated proteins (Rasmussen & Bjorck, 2002). Therefore, SpeB is considered an important virulence factor, and its expression is tightly regulated in GAS. RopB (Regulator of protease B) is an Rgg-like regulator identified as a transcriptional activator of speB (Lyon et al, 1998). Both speB and ropB are located adjacent to one another on the chromosome but are transcribed in opposite directions (Neely et al, 2003). Two promoters of speB are located within the ropB–speB intergenic region, and the P1 promoter adjacent to ropB is the principal promoter for RopB binding and speB transcription (Neely et al, 2003). As a quorum-sensing protein, RopB binds to an eight-amino acid leaderless SpeB-inducing peptide (SIP) to induce speB expression (Do & Kumaraswami, 2016; Perez-Pascual et al, 2016; Do et al, 2017). Do et al (Do et al, 2019) showed that RopB binds to SIP under acidic conditions, suggesting that SIP mediates the growth phase-and pH-dependent speB expression. The intracellular SIP concentration is modulated by the endopeptidase PepO. A study showed that the up-regulation of pepO in the covR mutant mediates SIP degradation, thereby disrupting the RopB-SIP quorum-sensing pathway (Shi et al, 2022). Interestingly, although SIP-bound RopB (RopB-SIP) is required to activate speB transcription, RopB-SIP and SIP-free RopB (apo-RopB) have similar DNA-binding activities to the P1 promoter of speB (Do et al, 2017). Therefore, the role of apo-RopB in regulating speB transcription remains unclear.

RopB is a positive regulator of speB and essential for inducing speB transcription. In this study, we demonstrate that in the absence of SIP, RopB acts as a transcriptional repressor of speB. Therefore, the non-phosphorylated CovR-mediated down-regulation of speB in the covS mutant is mediated by apo-RopB. These results redefine the current understanding of RopB-mediated regulation of speB and reveal a new interaction between the CovR/CovS and RopB-SIP systems in GAS.

Results

RopB represses speB transcription in the absence of SIP

Our previous study showed that the up-regulation of PepO in the covR mutant mediates the degradation of SIP and the down-regulation of speB (Shi et al, 2022). The expression of speB in the pepO mutant was higher than that in the wild-type A20 strain (Fig 1A), suggesting that PepO in the wild-type strain mediates SIP degradation. In this study, we constructed a pepO mutant in an SIP-inactivated background to verify the role of PepO in degrading the exogenous supplemented SIP. The start codon of SIP (ATG) in the wild-type A20 strain was substituted with TAG to inactivate SIP translationally. This strain was designated as the SIP* mutant. The expression of speB in the SIP* mutant was repressed compared with that in the wild-type A20 strain (Fig 1A). The open reading frame of SIP is located in the ropB–speB intergenic region (Do et al, 2017). SpeB was up-regulated in the SIP* mutant complemented with the ropB–speB intergenic region (PSIP) and ropB with its native promoter [PropB (SIP+)] compared with that in the vector-control strain (Vec) (Fig 1B), indicating that there are no other undefined factors related to the down-regulation of speB in the SIP* mutant. In the exogenous SIP-supplementation conditions, lower levels of SpeB were observed in the SIP* mutant compared with its pepO isogenic mutant (SIP*/∆pepO) under the same concentration of SIP treatments (Fig 1C–E), indicating that PepO mediates SIP degradation in the wild-type strain. Furthermore, to verify that the expression of SpeB is induced by RopB under SIP stimuli, the ropB gene was deleted in the SIP* mutant (SIP*/∆ropB), and the expression of SpeB in this mutant under SIP and the scrambled peptide (SCRA) treatments were analyzed. No difference was observed in SpeB expression in the SIP*/∆ropB mutant under treatment with 0–1.5 μM SIP (Fig 1F). Less than a 1.2fold increase was found in the RNA level (Fig 1C), suggesting that SIP induces speB expression in a RopB-dependent manner.

Figure 1.
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Figure 1. The expression of speB in the wild-type strain (Wt), ∆ropB mutant, SIP* mutant, ∆pepO mutant, SIP/∆pepO mutant, SIP*/∆ropB mutant, and the SIP and ropB trans-complementary strains treated with different concentrations of the synthetic SIP and scramble peptide (SCRA).

(A) The transcription of speB in the wild-type strain and its pepO (∆pepO) and SIP-inactivated (SIP*) mutants. (B) The expression of speB in the SIP mutant [with the empty vector (Vec)] and its SIP (PSIP) and ropB with its native promoter [PropB (SIP+)] trans-complementary strains. (C, D, E) The transcription of speB and the expression of SpeB in the SIP* mutant, SIP*/∆pepO mutant, and SIP*/∆ropB mutant under SIP and SCRA treatments. (F) The expression of SpeB in the SIP* mutant and the SIP*/∆ropB mutant in the treatment of different concentrations of SIP and SCRA. (G) The transcription of speB in the wild-type strain, the ropB isogenic mutant (∆ropB), and the ropB trans-complementary strain [PropB (SIP+)]. (H, I) The expression of SpeB and the transcription of speB in the wild-type strain, ∆ropB mutant, SIP* mutant, and SIP*/∆ropB mutant after 6–7 h of incubation. Culture supernatant was used for Western blot analysis. zSpeB, zymogen form of SpeB; mSpeB, mature form of SpeB. Bacterial RNA was extracted for real-time quantitative PCR (RT–qPCR) analysis. The expression of speB was normalized to that of gyrA. *P < 0.05.

Source data are available for this figure.

Source Data for Figure 1.1[LSA-2022-01809_SdataF1.1_F2.1_F3.1_FS4.pdf]

Source Data for Figure 1.2[LSA-2022-01809_SdataF1.2_F2.2_F3.2_F4_F5_FS2_FS3.pdf]

We also observed that the expression of speB was down-regulated in the ropB mutant compared with that in the wild-type and ropB-complementary strains (Fig 1G), suggesting that RopB is the transcriptional activator of speB (Lyon et al, 1998; Neely et al, 2003). Nonetheless, in comparison with the SIP* mutant, the deletion of ropB in the SIP* mutant (SIP*/∆ropB) resulted in the up-regulation of speB (Fig 1C and F). Also, the SIP*/∆ropB mutant (without SIP treatments) showed a significant elevation in SpeB expression compared with that of the SIP* mutant under the 1.5 μM-SIP treatments (Fig 1F). These results suggest that RopB represses the transcription of speB in the SIP* mutant.

To elucidate the role of RopB in the regulation of speB in the presence and absence of SIP, we performed Western blotting and analyzed the levels of SpeB in the wild-type A20, SIP* mutant, and ropB mutant strains. SpeB expression in the ropB mutant was down-regulated compared with that in the wild-type A20 strain (Fig 1H), suggesting that RopB positively regulates speB transcription in the presence of SIP. Compared with the wild-type strain, SpeB expression was down-regulated in the SIP* mutant; notably, SpeB expression in the SIP* mutant was lower than that in the ropB isogenic mutant (Fig 1H). Furthermore, the SpeB expression in the SIP*/∆ropB mutant increased to a level similar to that in the ropB isogenic mutant (Fig 1H). Consistent with results from the Western blot analysis, the transcription level of speB in the ropB isogenic mutant and SIP*/∆ropB mutant was similar (Fig 1I). In addition, speB transcription was down-regulated in the SIP* mutant compared with that in the SIP*/∆ropB mutant (Fig 1I). These results suggest that in the SIP-inactivated background, RopB acts as a transcriptional repressor of speB.

RopB inhibits the growth-phase-dependent SpeB expression in the SIP-inactivated covR mutant

Compared with that in the wild-type strain, the expression of speB was up-regulated in the covR and ∆covR/∆pepO mutants (Fig 2A), suggesting that CovR also has roles in regulating speB expression. To exclude the effects of CovR, the role of RopB in regulating SpeB expression in the presence and absence of SIP was further analyzed in the covR mutant. As expected, the deletion of ropB in the covR mutant (∆covR/∆ropB) down-regulated speB transcription in the stationary phase compared with the covR mutant (6–7 h, Fig 2B and C). Noticeably, the increase in SpeB expression was still observed in the ∆covR/∆ropB mutant after 7 h of incubation (Fig 2B and C), indicating that the growth-phase-dependent SpeB expression was not completely abolished in the absence of RopB. Although the RopB was present, the expression of SpeB both transcriptionally and translationally in the SIP-inactivated covR mutant (SIP*/∆covR) was repressed in comparison to that in the covR and the ∆covR/∆ropB mutants (Fig 2B and C). To elucidate the role of RopB in regulating SpeB expression in the SIP*/∆covR mutant, the expression of SpeB in the SIP*/∆covR mutant and its isogenic ropB mutant (SIP*/∆covR/∆ropB) were compared. The ∆covR/∆ropB mutant and SIP*/∆covR/∆ropB mutant showed a similar level of speB transcription after 5 h of incubation (Fig 2D). At the protein level, inactivation of SIP translation in the ∆covR/∆ropB mutant (SIP*/∆covR/∆ropB) had a minor effect on SpeB expression compared with that in the ∆covR/∆ropB mutant (Fig 2E), indicating that SIP-mediated SpeB expression is primarily through RopB. Furthermore, we found that SpeB expression was derepressed in the SIP*/∆covR/∆ropB mutant compared with that in the SIP*/∆covR mutant (Fig 2D and E), suggesting that, in the absence of SIP, RopB inhibits speB transcription in the covR mutant.

Figure 2.
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Figure 2. Expression of SpeB in the wild-type strain, pepO mutant (∆pepO), covR mutant (∆covR), ∆covR/∆pepO mutant, SIP*/∆covR mutant, ∆covR/∆ropB mutant, and SIP*/∆covR/∆ropB mutant.

(A) The expression of speB in the wild-type strain and its pepO mutant, covR mutant, and ∆covR/∆pepO mutant. (B, C) Transcription of speB and the expression of SpeB in the covR mutant, ∆covR/∆ropB mutant, and SIP*/∆covR mutant. (D, E) Transcription of speB and expression of SpeB in the ∆covR/∆ropB mutant, SIP*/∆covR mutant, and SIP*/∆covR/∆ropB mutant. Culture supernatant was used for Western blot analysis. zSpeB, zymogen form of SpeB; mSpeB, mature form of SpeB. Bacterial RNA was extracted for real-time quantitative PCR (qPCR) analysis. The expression of speB was normalized to that of gyrA. *P < 0.05.

Source data are available for this figure.

Source Data for Figure 2.1[LSA-2022-01809_SdataF1.1_F2.1_F3.1_FS4.pdf]

Source Data for Figure 2.2[LSA-2022-01809_SdataF1.2_F2.2_F3.2_F4_F5_FS2_FS3.pdf]

RopB represses SpeB expression in the CovS kinase-inactivated mutant

Unlike the covR mutant, the CovS-inactivated [the covS-deletion (∆covS) and the kinase-inactivated (CovSH280A)] mutants still produce the non-phosphorylated CovR protein (Fig 3A) that represses ropB transcription (Chiang-Ni et al, 2019a; Finn et al, 2021; Horstmann et al, 2022). Therefore, speB is derepressed in the covR mutant but repressed in the covS mutant (Sumby et al, 2006; Chiang-Ni et al, 2016, 2019a; Finn et al, 2021; Horstmann et al, 2022). Similar to the covR mutant, Western blot analysis showed that PepO expression was higher in the covS mutant than in the wild-type strain (Fig 3B). We also found that the pepO-deleted covS mutant (∆covS/∆pepO) expressed a higher level of speB than the covS mutant under the same concentration of SIP treatments (Fig 3C). These results indicate that PepO is involved in abrogating the SIP-induced speB expression. Therefore, the up-regulation of PepO may have contributed to the low SIP concentration in the covS mutant.

Figure 3.
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Figure 3. The expression of PepO in the covS mutant (∆covS) and the expressions of speB and ropB in the covS mutants, the CovR D53A substitution mutant (CovRD53A), their ropB mutants, and the ∆covS/∆pepO mutant.

(A) The phosphorylation level of CovR in the wild-type strain (Wt), CovS kinase-inactivated (CovSH280A) mutant, and CovS phosphatase-inactivated (CovST284A) mutant. The ∆covS mutant (the mutant that cannot phosphorylate CovR) and the covR mutant (∆covR) were used as experimental controls. The total protein is used as the internal loading control. (B) The expression of PepO in the wild-type strain, its pepO mutant (∆pepO), ∆covS mutant, and the ∆covS/∆pepO mutant. The lower panel shows the total protein as the internal loading control. (C) The transcription of speB in the ∆covS mutant and ∆covS/∆pepO mutant under the synthetic SIP and scramble peptide (SCRA) treatments. (D) The transcription of ropB in the wild-type strain, CovSH280A mutant, and CovST284A mutant. (E) The expression of SpeB in the wild-type strain, CovSH280A mutant, CovST284A mutant, and their ropB mutants. (F) The expression of speB in the CovSH280A mutant and its ropB mutant (CovSH280A/∆ropB). (G) The transcription of speB in the CovR D53A substitution mutant (CovRD53A) and its ropB mutant (CovRD53A/∆ropB). Culture supernatant was used for Western blot analysis. zSpeB, zymogen form of SpeB; mSpeB, mature form of SpeB. Bacterial RNA was extracted for real-time quantitative PCR (qPCR) analysis. The expression of ropB and speB was normalized to that of gyrA. *P < 0.05.

Source data are available for this figure.

Source Data for Figure 3.1[LSA-2022-01809_SdataF1.1_F2.1_F3.1_FS4.pdf]

Source Data for Figure 3.2[LSA-2022-01809_SdataF1.2_F2.2_F3.2_F4_F5_FS2_FS3.pdf]

Furthermore, the role of RopB in regulating speB expression was analyzed in the CovS kinase-inactivated mutant (CovSH280A) and a CovS phosphatase-inactivated mutant (CovST284A). Consistent with our previous study (Chiang-Ni et al, 2019a), CovR phosphorylation was inactivated in the CovSH280A mutant but slightly increased in the CovST284A mutant (Fig 3A). In addition, the transcription of ropB was repressed in the CovSH280A mutant compared with the wild-type strain and the CovST284A mutant (Fig 3D). Next, the expression of SpeB in the wild-type strain, CovSH280A mutant, CovST284A mutant, and their ropB mutants (CovSH280A/∆ropB and CovST284A/∆ropB) were evaluated via Western blotting. As expected, SpeB expression in the ropB isogenic mutant (∆ropB) and CovST284A/∆ropB mutant were down-regulated compared with that in their parental strains (Fig 3E). The CovSH280A mutant showed low levels of ropB transcription (Fig 3D); however, SpeB expression was completely repressed (Fig 3E). Notably, the expression of SpeB in the CovSH280A/∆ropB mutant was increased to a level similar to that in the ropB mutant and CovST284A/∆ropB mutant (Fig 3E). At the transcriptional level, the expression of speB was significantly up-regulated in the CovSH280A/∆ropB mutant compared with that in the CovSH280A mutant (Fig 3F). These results indicate that SpeB expression in the CovSH280A mutant was inhibited by RopB.

CovS phosphorylates the D53 residue of CovR (Dalton & Scott, 2004). Similar to the covS mutant, speB expression is repressed in the CovR D53A substituted (CovRD53A) mutant (Chiang-Ni et al, 2019a). To demonstrate the role of RopB in regulating speB expression in the CovR non-phosphorylated mutant, the expression of speB in the CovRD53A mutant and its ropB isogenic mutant (CovRD53A/∆ropB) was compared. The results showed that the expression of speB in the CovRD53A/∆ropB mutant was derepressed compared with that in the CovRD53A mutant (Fig 3G), suggesting that the transcription of speB was inhibited by RopB in the CovRD53A mutant.

Apo-RopB represses the expression of speB and its co-transcripts in the GAS transcriptome

To elucidate the role of apo-RopB in the GAS transcriptome, RNA was extracted from the wild-type A20 strain, its SIP* mutant, and the SIP*/∆ropB mutant and analyzed by RNA sequencing. In comparison with the wild-type strain, only three genes, speB, spi, and M5005_Spy1733, were significantly (q value < 0.05) down-regulated in the SIP* mutant (closed points in Fig S1 and Table S1). Furthermore, in the SIP*/∆ropB mutant, the expression of speB and spi was significantly down-regulated (q value < 0.05) compared with the wild-type strain (Fig 4A and Table S2) but up-regulated when compared with that in the SIP* mutant (Fig 4B and Table S3), indicating that apo-RopB and RopB-SIP would act differently on regulating speB and spi expression. spi and M5005_Spy1733 are downstream of speB, and M5005_Spy1733 has been annotated as a hypothetical protein in the emm1-type MGAS5005 strain (open arrow, Fig 4C; NCBI Accession: CP000017.2). However, in the emm1-type SF370 strain, the prsA gene was annotated instead of M5005_Spy1733 (gray arrow; Fig 4C; NCBI Accession: NC_002737.2). Furthermore, Ma et al (2006) showed that the prsA gene is transcribed by its promoter (1.2 kb) or co-transcribed with speB and spi (speB-spi-prsA, 3.2–3.8 kb; Fig 4C) by the speB promoter. We used primers targeting speB, the intergenic regions of M5005_Spy1734 (spi), M5005_Spy1733, and prsA to verify whether apo-RopB represses speB-spi-prsA and prsA transcription. RT–qPCR analysis showed that the transcription of speB and speB-spi-prsA was down-regulated in the ropB mutant compared with that in the wild-type strain (Fig 4D). Further, in support of the RNA-Seq results, the expression of these genes was up-regulated in the SIP*/∆ropB mutant compared with that in the SIP* mutant (Fig 4D). Noticeably, the expression of speB and speB-spi-prsA in the SIP*/∆ropB mutant was up-regulated by ∼415-fold and 57-fold, respectively, compared with the SIP* mutant. Although prsA is co-transcribed with speB and spi (Ma et al, 2006), the expression of prsA was increased by only ∼threefold in the SIP*/∆ropB mutant compared with that in the SIP* mutant (Fig 4D). These results suggested that apo-RopB plays a minor role in regulating prsA expression and represses the expression of only speB and its co-transcripts in the GAS transcriptome.

Figure S1.
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Figure S1. RNA sequencing analysis of the wild-type strain (Wt) and SIP* mutant.

The volcano plot visualizes the genes that were differentially expressed in the SIP* mutant versus the wild-type strain. Blue circles and red circles indicate the down-regulated and up-regulated genes, respectively, in the SIP* mutant compared with the wild-type strain (P < 0.05). The solid circles indicate that the expression difference is statistically significant (adjusted P-value, q value < 0.05).

Table S1. Significantly up-regulated and down-regulated genes (q value < 0.05) in the SIP* mutant compared with those in the wild-type A20 strain.

Figure 4.
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Figure 4. RNA-sequencing analysis of the wild-type strain (Wt), SIP* mutant, and SIP*/∆ropB mutant, and the expression of speB and its co-transcripts in these strains.

(A, B) The genes those were differentially expressed in (A) SIP*/∆ropB mutant versus the wild-type strain (Wt) and (B) SIP*/∆ropB mutant versus the SIP* mutant are visualized by the volcano plot. Blue circles and red circles indicate the down-regulated and up-regulated genes, respectively, in the (A) SIP*/∆ropB mutant compared with that of the wild-type strain and (B) SIP*/∆ropB mutant compared with that of the SIP* mutant (P < 0.05). The solid circles indicate that the expression difference is statistically significant (adjusted P-value, q value < 0.05). (C) Schematic representation of the speB, spi, and prsA genes (arrows). The speB and its co-transcripts (dashed lines) and the location of primers (Primer-1–Primer-3) used for detecting speB and its co-transcripts are indicated. The genes and their annotations are indicated according to MGAS5005 (NCBI Accession: CP000017.2; the open arrows) and SF370 (NCBI Accession: NC_002737.2; the gray arrow). (D) The expression of speB and its co-transcripts in the wild-type strain, the ropB mutant, SIP* mutant, and SIP*/∆ropB mutants detected by Primer-1–Primer-3. Bacterial RNA was extracted for sequencing and real-time quantitative PCR (qPCR) analyses. The expression of the target transcript was normalized to that of gyrA. *P < 0.05.

Source data are available for this figure.

Source Data for Figure 4[LSA-2022-01809_SdataF1.2_F2.2_F3.2_F4_F5_FS2_FS3.pdf]

Table S2. The significantly up-regulated and down-regulated genes (q value < 0.05) in the SIP*/∆ropB mutant compared with those in the SIP* mutant.

Table S3. The significantly up-regulated and down-regulated genes (q value < 0.05) in the SIP*/∆ropB mutant compared with those in the wild-type strain.

SIP-mediated quorum-sensing regulation acts predominantly on the speB operon

The results of the transcriptomic analysis suggest that SIP could be a signal that explicitly controls the expression of speB and its co-transcripts. To test this, the role of SIP in regulating RopB-regulated genes was analyzed. The expression of M5005_Spy1176 and six phage-related gene mutants was down-regulated and up-regulated in the wild-type A20 strain, respectively, compared with that in the SIP*/∆ropB mutant (Fig 4A and B and Tables S2 and S3). In line with the RNA-Seq results (Fig 5A, the upper panel), the RT–qPCR analysis showed that the expression of M5005_Spy1176 was down-regulated in the wild-type and the SIP* mutant strains compared with that in the SIP*/∆ropB mutant (Fig 5A, the lower panel), and the expression of M5005_Spy1416 and M5005_Spy1426 was undetectable in the SIP*/∆ropB mutant (Fig 5B, the lower panel), indicating that the expression of these genes was regulated by RopB. Noticeably, the inactivation of SIP had a minor impact on the expression of these genes (the fold change in expression was less than twofold, Fig 5A and B, lower panels).

Figure 5.
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Figure 5. Expression of RopB-SIP-regulated genes in the wild-type A20 strain, its ropB mutant (∆rpoB), SIP* mutant, and SIP*/∆ropB mutant in the early stationary phase and under the neutral and acidic conditions.

(A, B) Expression of (A) M5005_Spy1176 (negatively regulated by RopB-SIP) and (B) M5005_1416 and M5005_Spy1426 (positively regulated by RopB-SIP) in A20, the SIP* mutant, and SIP*/∆ropB mutant in the early stationary phase of growth (O.D.600 = 1.0). The upper and lower panels of (A, B) show the results from RNA-seq analysis and real-time quantitative PCR (qPCR) analysis, respectively. (C, D, E) Expression of (C) ropB and speB, (D) M5005_Spy1176, and (E) M5005_1416 and M5005_Spy1426 in A20, the SIP* mutant, and SIP*/∆ropB mutant under neutral (pH 7.5) and acidic (pH 6.0) conditions. RNAs were extracted for qPCR analysis. The expression of target genes was normalized to that of gyrA. bdl, below detection limit. *P < 0.05.

Source data are available for this figure.

Source Data for Figure 5[LSA-2022-01809_SdataF1.2_F2.2_F3.2_F4_F5_FS2_FS3.pdf]

RopB binds to SIP under acidic pH conditions (Do et al, 2019). To evaluate the role of SIP in regulating RopB-regulated genes, the expressions of ropB, speB, M5005_Spy1176, M5005_Spy1416, and M5005_Spy1426 in the wild-type strain, SIP* mutant, and their ropB mutants were checked under neutral (pH 7.5) and acidic (pH 6.0) conditions. The expression levels of ropB in the wild-type A20 strain and the SIP* mutant were similar under neutral and acidic conditions (Fig 5C), and this acted as an experimental control. The speB expression was only induced in the wild-type strain but not in the ropB and SIP* mutants (Fig 5C), indicating that speB expression under acidic conditions is activated upon the binding of RopB to SIP. Upon comparing the wild-type A20 strain, the ropB mutant, and the SIP* mutant, the expression of M5005_Spy1176 was found to be up-regulated, whereas that of M5005_Spy1416/Spy1426 was down-regulated in the SIP*/∆ropB mutant (Fig 5D and E). These results suggest that RopB-SIP has a crucial role in regulating the expression of these genes. However, SIP was not involved in regulating the expression of these genes under neutral and acidic conditions (Fig 5D and E). We also examined the expression of other RopB-SIP-regulated genes, including M5005_Spy1189, adh2, and M5005_Spy0023, by RT–qPCR. We found that SIP did not play a role in regulating the expression of these genes under neutral and acidic conditions (Fig S2).

Figure S2.
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Figure S2. Expression of RopB-SIP-regulated genes in the early stationary phase and under neutral and acidic conditions in the wild-type A20 strain (Wt), its ropB mutant (∆rpoB), SIP* mutant, and SIP*/∆ropB mutant.

(A, B) Expression of (A) M5005_Spy1189 (negatively regulated by RopB-SIP) and (B) adh2 and M5005_Spy0023 (positively regulated by RopB-SIP) in A20, the SIP* mutant, and SIP*/∆ropB mutant in the early stationary phase of growth (O.D.600 = 1.0). The upper panels and lower panels of (A, B) show the results from RNA-seq analysis and real-time quantitative PCR (qPCR) analysis, respectively. (C) Expression of M5005_Spy1189, adh2 and M5005_Spy0023 in the wild-type strain, SIP* mutant, and SIP*/∆ropB mutant under neutral (pH 7.5) and acidic (pH 6.0) conditions. RNA was extracted for RT–qPCR analysis. The expression of target genes was normalized to that of gyrA. *P < 0.05.

Source data are available for this figure.

Source Data for Figure S2[LSA-2022-01809_SdataF1.2_F2.2_F3.2_F4_F5_FS2_FS3.pdf]

Discussion

RopB is a quorum-sensing protein that binds to SIP under acidic conditions to activate speB transcription (Do et al, 2017, 2019). Finn et al (Finn et al, 2021) suggested that CovR might regulate speB expression indirectly through RopB. Our previous study showed that SIP could be degraded by the CovR/CovS-controlled endopeptidase PepO (Shi et al, 2022). Therefore, increased PepO expression in covR and covS mutants could down-regulate SIP-induced SpeB expression. Transcription of ropB and speB is repressed by both phosphorylated and non-phosphorylated CovR (Miller et al, 2001; Chiang-Ni et al, 2019a). In the covR mutant, the effect of PepO-mediated SIP degradation was compensated by the derepression of ropB and speB, resulting in the up-regulation of speB in the stationary phase of growth (Figs 6 and S3) (Shi et al, 2022). In the covS mutant, the transcription of ropB is repressed by non-phosphorylated CovR (Chiang-Ni et al, 2019a; Finn et al, 2021). In this study, we further demonstrated that RopB functions as a transcriptional repressor of speB in the absence of SIP. Therefore, the repression of speB transcription in the covS mutant is not only mediated by the down-regulation of ropB transcription but also by RopB-dependent transcriptional repression (Fig 6).

Figure 6.
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Figure 6. Hypothetical models of speB regulation in the covR and covS mutants.

The expression of ropB and pepO are repressed by CovR. Although the up-regulated PepO would degrade SIP in the covR mutant, the effect of PepO degradation could be compensated by the derepression of ropB and SIP, and the SIP-bound RopB (RopB-SIP) could activate speB transcription. In the covS mutant, the expression of pepO is up-regulated, whereas that of ropB is repressed by the non-phosphorylated CovR. Therefore, the repression of speB in the covS could be mediated by the PepO-dependent SIP degradation and the SIP-free RopB (apo-RopB)-dependent transcriptional repression.

Figure S3.
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Figure S3. Expression of speB and ropB in the wild-type strain (Wt) and the covR mutant (∆covR).

(A, B) The expression of SpeB and ropB in the wild-type strain and covR mutant. (A) The upper panel of (A) shows the expression of speB, and the lower panel shows the SpeB protein in the bacterial culture supernatant. Bacterial strains were grown to O.D.600 = 1.0. RNA was extracted for real-time quantitative PCR (RT–qPCR) analysis, and culture supernatants were collected for Western blot hybridization. The expression of speB and ropB was normalized to that of gyrA. *P < 0.05.

Source data are available for this figure.

Source Data for Figure S3[LSA-2022-01809_SdataF1.2_F2.2_F3.2_F4_F5_FS2_FS3.pdf]

RopB binds to the speB promoter, and this interaction has been considered essential for activating speB transcription (Lyon et al, 1998; Neely et al, 2003; Makthal et al, 2016). Do et al (2017) proposed that in the log phase of growth, the inhibition peptide Vfr binds to RopB (Shelburne et al, 2011) to inhibit the RopB–DNA interaction and abolishes speB transcription. Our study showed that the deletion of ropB in the SIP* mutant derepressed the transcription of speB, indicating that the interaction between RopB and the speB promoter is not essential for speB transcription. We also show that disrupting the interaction between RopB and SIP or decreasing the intracellular concentration of SIP mediates an apo-RopB-dependent down-regulation of speB transcription. The speB transcription is increased dramatically under acidic stimuli or in the stationary phase of growth compared with that in the neutral pH or log phase of growth (Loughman & Caparon, 2006; Chiang-Ni et al, 2012). In addition, SpeB is the most abundant protein secreted in the GAS culture supernatants. The results of this study suggest that RopB would not only augment the speB expression under acidic culture and stationary phase growth conditions but also play a critical role in preserving energy by preventing the transcriptional leakage of speB under neutral pH and log phase growth conditions in an SIP-dependent manner.

RopB engages in high-affinity interactions with SIP under acidic conditions, suggesting that the pH- and growth-phase dependence of speB expression is because of the influence of pH on the association between RopB and SIP (Unnikrishnan et al, 1999; Loughman & Caparon, 2006; Chiang-Ni et al, 2012; Do et al, 2017, 2019). The RNA-seq and RT–qPCR analyses in this study show that under regular and acidic culture conditions, SIP-mediated regulation acts predominantly on the transcription of speB and its co-transcripts. Do et al (2017) showed that in the SIP*-inactivated mutant, the expression of speB (SpyM3_1742) and its downstream proteins spi (SpyM3_1741) and M3_1743 (SpyM3_1743) were down-regulated by over 1,000fold compared with that in the wild-type MGAS10870 strain. The fold change of other identified genes in their RNA-seq analysis was between 4.4 and 2.0 (Do et al, 2017), supporting that the SIP signal would have the most significant impact on controlling the speB transcription. However, the SIP-regulated genes in MGAS10870 (emm3) and A20 (emm1) were not identical. Lynskey et al (2015) demonstrated that a premature stop codon in the rocA gene was found in the M3 serotype strains, including MGAS10870 (Jain et al, 2017). RocA is an accessory protein that inhibits the phosphatase activity of CovS (Chiang-Ni et al, 2020). Furthermore, the study also showed that the M1 serotype GAS strains had high levels of phosphorylated CovR compared with that of the M3 serotype strains (Horstmann et al, 2015). CovR/CovS can modulate the regulatory activity of RopB by controlling pepO transcription. Therefore, the inconsistent RNA-sequencing results from the M1 and M3 type strains could be related to different levels of phosphorylated CovR. These results also reveal complicated interactions between the two-component CovR/CovS system and the RopB-SIP quorum-sensing system in the GAS regulatory network.

The expression of speB, spd3, and grab were repressed in the covS mutant compared with that in the wild-type strain, suggesting that CovS phosphorylates CovR to activate the expression of these genes (Trevino et al, 2009; Tran-Winkler et al, 2011). Horstmann et al (2022) suggested that in contrast to the transcriptional repression of phosphorylated CovR, predominantly mediated by a direct mechanism, phosphorylated CovR-mediated transcriptional activation is indirect and could be complex. This study further showed that the repression of speB in the covS mutant was mediated by apo-RopB, indicating that non-phosphorylated CovR-mediated speB repression is a consequence of the interaction between the CovR/CovS and RopB-SIP systems. Nonetheless, the repression of spd3 and grab in the covS mutant was mediated by a RopB-independent mechanism (data not shown), suggesting that multiple regulatory pathways are involved in non-phosphorylated CovR-mediated transcriptional regulation (Finn et al, 2021).

This study showed that RopB functions as a transcriptional repressor of speB in the absence of SIP, revealing unidentified roles of RopB in regulating speB expression. Do et al (2017) showed that purified apo-RopB forms a homodimer and can bind the speB promoter with activity similar to that of RopB-SIP in vitro. Therefore, we suggest that the RopB dimer could form different structures with the speB promoter in the presence or absence of SIP in vivo, and these RopB-DNA structures are crucial for modulating speB transcription. Unfortunately, this hypothesis cannot be further verified because modification of the ropB-speB intergenic region abolishes speB transcription (Fig S4). The underlying mechanisms by which apo-RopB and RopB-SIP act differentially to control speB expression remain to be investigated.

Figure S4.
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Figure S4. Expression of SpeB in the wild-type A20 strain (Wt), its ropB mutant (∆ropB), and the ropB–speB intergenic region deletion mutants.

(A) Schematic representation of the ropB–speB intergenic region. The transcriptional start sites of speB (P1 and P2) are in bold and marked by the bent arrow above the sequence. The numbers below the nucleotides and at the left of the sequence indicate nucleotide position relative to the translation start codon of speB. The closed arrows on the sequence show the locations of inverted repeats. P_del-1, P_del-2, P_del-3, and P2-del indicate the deletion regions in the ropB-speB intergenic region. (B) Expression of SpeB in the wild-type strain, ropB mutant, and the ropB–speB intergenic region deletion mutants. Bacteria were grown to O.D.600 = 1.0 and culture supernatants were collected for Western blot analysis. zSpeB, zymogen form of SpeB; mSpeB, mature form of SpeB.

Source data are available for this figure.

Source Data for Figure S4[LSA-2022-01809_SdataF1.1_F2.1_F3.1_FS4.pdf]

Materials and Methods

Bacterial strains and culture conditions

GAS A20 (emm1-type) bacteria were isolated and cultured as described previously (Chiang-Ni et al, 2009). Strain AP3 is an animal passage isolate of A20 with a frameshift deletion in the covS gene (Chiang-Ni et al, 2016). GAS strains were cultured on trypticase soy agar containing 5% sheep blood or in tryptic soy broth (Becton Dickinson and Company) supplemented with 0.5% yeast extract (TSBY). Escherichia coli DH5α was purchased from Yeastern (Yeastern Biotech Co., Ltd.) and was cultured in lysogeny broth (LB) at 37°C with vigorous aeration. SpeB-inducing peptide (SIP; MWLLLLFL; purity: 94.469%) and scrambled control peptide (SCRA, LLFLWLLM; purity: 92.822%) (Do et al, 2017) were purchased from Leadgene Biomedical Inc. These synthetic peptides were suspended in 100% DMSO to prepare a 10 mM stock solution and stored at −20°C until use. Working solutions were prepared by diluting the stock solution with 25% DMSO. SIP- and SCRA-supplemented culture conditions have been described previously (Shi et al, 2022). Briefly, GAS strains were grown to O.D.600 = 0.8 in TSBY broth. Bacterial pellets were collected and incubated in an acidic TSBY broth (pH 6.0) supplemented with different concentrations of SIP and SCRA for 1 h. To treat bacteria with neutral and acidic broth, bacterial pellets were collected (O.D.600 = 0.4), resuspended in either pH 7.5 or 6.0 broths, and cultured for another 4 h. The bacterial strains and plasmids used in this study are listed in Table 1. When appropriate, the antibiotics chloramphenicol (25 μg/ml for E. coli and 3 μg/ml for GAS) and spectinomycin (100 μg/ml) were used for selection.

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Table 1.

Plasmids and strains used in this study.

DNA and RNA manipulations

Bacterial genomic DNA and RNA extractions and reverse transcription were performed as previously described (Wang et al, 2013). Real-time PCR was performed in a 20 μl reaction mixture containing 1 μl of cDNA, 0.8 μl of primers (10 μM), and 10 μl of SensiFAST SYBR Lo-ROX pre-mixture (Bioline Ltd.) according to the instructions of the manufacturer. Biological replicates were performed using two to three independent RNA preparations in duplicate. The expression level of each target gene was normalized to gyrA and analyzed using the ∆∆Ct method (QuantStudio 3 System; Thermo Fisher Scientific Inc.). All values of the control and experimental groups were divided by the mean of the control samples before statistical analysis (Valcu & Valcu, 2011). Primers used for real-time PCR analysis (Table S4) were designed using Primer3 (v.0.4.0, http://frodo.wi.mit.edu) based on the MGAS5005 sequence (NCBI accession number: CP000017.2). RNA samples were analyzed by RNA-sequencing (Welgene Biotech). SureSelect XT HS2 mRNA library preparation kit (Agilent) was used for library construction, followed by size selection using AMPure XP beads (Beckman Coulter). The sequence was determined using Illumina sequencing-by-synthesis technology (Illumina). Sequencing data (FASTQ reads) were generated using Welgene Biotech’s pipeline based on the Illumina base-calling program bcl2fastq v2.20. The adjusted P-value (q-value) cut off to 0.05 (DESeq with non-grouped sample using blind mode) was set for discovering differentially expressed genes.

Table S4. Primers used in this study.

Construction of the ropB-deletion, pepO-deletion, and SIP-inactivation mutants

To construct the ropB mutant, the ropB gene with its upstream (485 bp) and downstream (490 bp) regions was amplified using the primers ropB-F-5 and ropB-R-4 (Table S4). The PCR amplicon was digested with restriction enzyme (SphI) and ligated into the temperature-sensitive vector pCN143 (Chiang-Ni et al, 2016). The ropB gene was removed via inverted PCR using the primers ropB-EcoRV-F and ropB-EcoRV-R (Table S4) and replaced with the chloramphenicol cassette from Vector 78 (Chiang-Ni et al, 2012) to generate pCN146 (Table 1). Plasmids used for constructing pepO-deletion mutants (pCN210) and SIP-inactivation mutants (pCN215) have been described previously (Shi et al, 2022). These plasmids were transformed into GAS strains via electroporation, and the transformants were selected as described previously (Chiang-Ni et al, 2016). Deletions of ropB and pepO and replacement of TAG in the SIP open reading frame were confirmed by Sanger sequencing.

Construction of SIP and ropB trans-complementary strains

The ropB trans-complementary strain was constructed using a method described previously (Chiang-Ni et al, 2016). The open reading frame of SIP is located in the intergenic region between ropB and speB (Do et al, 2017). To construct the SIP trans-complementary strain, the intergenic region of ropB and speB was amplified using the primers PspeB-SacI-F-2 and PspeB-SacI-R-2 (Table S4), and the PCR product (956 bp) was ligated into pDL278 (Table 1). The constructed plasmid was designated pCN228 and transformed into SIP* mutants via electroporation.

Western blot and Phos-tag Western blot

To detect phosphorylated CovR, bacteria were cultured in TSBY broth for 6 h, and then the bacterial cells were disrupted using a bead beater (Mini-Beadbeater; BioSpec Products Inc.). The bacterial cell lysate was centrifuged, and the supernatant was collected for analysis. Total protein (10 μg) was mixed with 6× protein loading dye, boiled for 5 min, and subjected to 12% SDS–PAGE. For Phos-tag Western blot analysis, the bacterial proteins were mixed with 6× protein loading dye (without boiling) and loaded into a 10% SDS–PAGE containing 10 μM of Phos-tag (Wako Pure Chemical Industries Ltd.) and 0.5 μM MnCl2 (Chiang-Ni et al, 2016). To detect SpeB, the filtered (0.22 μm membrane filter; Millipore) culture supernatants were collected and subjected to 10% SDS–PAGE. Separated proteins were transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk in PBST buffer (PBS containing 0.2% vol/vol Tween-20) at 37°C for 1 h. CovR protein was detected using anti-CovR serum (Chiang-Ni et al, 2016), PepO was detected using a polyclonal anti-PepO antibody (Shi et al, 2022), and SpeB was detected using an anti-SpeB antibody (Toxin Technology, Inc.). After hybridization, the membrane was washed with PBST buffer and hybridized with a peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Cell Signaling Technology, Inc.) at room temperature (25–28°C) for 1 h. The blots were developed using Pierce ECL Western blotting substrate (Thermo Fisher Scientific Inc.), and the signals were detected using a Gel Doc XR+ system (Bio-Rad Laboratories, Inc.).

Statistical analysis

Statistical analyses were performed using Prism software version 5 (GraphPad Software, Inc.). Significant differences between multiple groups were determined using ANOVA. Post hoc tests for ANOVA were performed using Tukey’s honest significance difference test. Statistical significance was set at P < 0.05. For RNA-sequencing analysis, the hypergeometric P-value was calculated as the probability of randomly drawing. The P-value was adjusted by false discovery rate for significance discovering (q-value). Differential gene expression with P-value and q-value < 0.05 was taken as significant.

Acknowledgements

We appreciate the assistance from Prof. Shuying Wang and Miss Yu-Tzu Chao (National Cheng Kung University, Taiwan) for EMSA analysis. This work was supported by a grant from the Chang Gung Memorial Hospital at Linkou, Taiwan (BMRPD19) and Ministry of Science and Technology, Taiwan (MOST 110-2628-B-182-012 and 111-2628-B-182-005).

Author Contributions

  • C Chiang-Ni: conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, project administration, and writing—original draft, review, and editing.

  • Y-W Chen: data curation, formal analysis, and investigation.

  • K-L Chen: data curation, formal analysis, and investigation.

  • J-X Jiang: data curation, formal analysis, and investigation.

  • Y-A Shi: data curation, formal analysis, and validation.

  • C-Y Hsu: data curation, formal analysis, and validation.

  • Y-YM Chen: conceptualization and supervision.

  • C-H Lai: conceptualization and supervision.

  • C-H Chiu: conceptualization, supervision, and funding acquisition.

Conflict of Interest Statement

The authors declare that they have no conflict of interest.

  • Received November 8, 2022.
  • Revision received March 23, 2023.
  • Accepted March 23, 2023.
  • © 2023 Chiang-Ni et al.
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This article is available under a Creative Commons License (Attribution 4.0 International, as described at https://creativecommons.org/licenses/by/4.0/).

References

  1. ↵
    1. Chen CY,
    2. Luo SC,
    3. Kuo CF,
    4. Lin YS,
    5. Wu JJ,
    6. Lin MT,
    7. Liu CC,
    8. Jeng WY,
    9. Chuang WJ
    (2003) Maturation processing and characterization of streptopain. J Biol Chem 278: 17336–17343. doi:10.1074/jbc.M209038200
    OpenUrlAbstract/FREE Full Text
  2. ↵
    1. Chiang-Ni C,
    2. Zheng PX,
    3. Ho YR,
    4. Wu HM,
    5. Chuang WJ,
    6. Lin YS,
    7. Lin MT,
    8. Liu CC,
    9. Wu JJ
    (2009) emm1/sequence type 28 strains of group A streptococci that express covR at early stationary phase are associated with increased growth and earlier SpeB secretion. J Clin Microbiol 47: 3161–3169. doi:10.1128/JCM.00202-09
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Chiang-Ni C,
    2. Zheng PX,
    3. Tsai PJ,
    4. Chuang WJ,
    5. Lin YS,
    6. Liu CC,
    7. Wu JJ
    (2012) Environmental pH changes, but not the LuxS signalling pathway, regulate SpeB expression in M1 group A streptococci. J Med Microbiol 61: 16–22. doi:10.1099/jmm.0.036012-0
    OpenUrlCrossRefPubMed
  4. ↵
    1. Chiang-Ni C,
    2. Chu TP,
    3. Wu JJ,
    4. Chiu CH
    (2016) Repression of Rgg but not upregulation of LacD.1 in emm1-type covS mutant mediates the SpeB repression in group A Streptococcus. Front Microbiol 7: 1935. doi:10.3389/fmicb.2016.01935
    OpenUrlCrossRef
    1. Chiang-Ni C,
    2. Tseng HC,
    3. Hung CH,
    4. Chiu CH
    (2017) Acidic stress enhances CovR/S-dependent gene repression through activation of the covR/S promoter in emm1-type group A Streptococcus. Int J Med Microbiol 307: 329–339. doi:10.1016/j.ijmm.2017.06.002
    OpenUrlCrossRef
  5. ↵
    1. Chiang-Ni C,
    2. Kao CY,
    3. Hsu CY,
    4. Chiu CH
    (2019a) Phosphorylation at the D53 but not the T65 residue of CovR determines the repression of rgg and speB transcription in emm1- and emm49-type group A streptococci. J Bacteriol 201: e00681-18. doi:10.1128/JB.00681-18
    OpenUrlCrossRef
    1. Chiang-Ni C,
    2. Tseng HC,
    3. Shi YA,
    4. Chiu CH
    (2019b) Effect of phosphatase activity of the control of virulence sensor (CovS) on clindamycin-mediated streptolysin O production in group A Streptococcus. Infect Immun 87: e00583-19. doi:10.1128/IAI.00583-19
    OpenUrlAbstract/FREE Full Text
  6. ↵
    1. Chiang-Ni C,
    2. Chiou HJ,
    3. Tseng HC,
    4. Hsu CY,
    5. Chiu CH
    (2020) RocA regulates phosphatase activity of virulence sensor CovS of group A Streptococcus in growth phase- and pH-dependent manners. mSphere 5: e00361-20. doi:10.1128/mSphere.00361-20
    OpenUrlCrossRef
  7. ↵
    1. Churchward G
    (2007) The two faces of Janus: Virulence gene regulation by CovR/S in group A streptococci. Mol Microbiol 64: 34–41. doi:10.1111/j.1365-2958.2007.05649.x
    OpenUrlCrossRefPubMed
  8. ↵
    1. Cunningham MW
    (2008) Pathogenesis of group A streptococcal infections and their sequelae. Adv Exp Med Biol 609: 29–42. doi:10.1007/978-0-387-73960-1_3
    OpenUrlCrossRefPubMed
  9. ↵
    1. Dalton TL,
    2. Scott JR
    (2004) CovS inactivates CovR and is required for growth under conditions of general stress in Streptococcus pyogenes. J Bacteriol 186: 3928–3937. doi:10.1128/JB.186.12.3928-3937.2004
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Do H,
    2. Kumaraswami M
    (2016) Structural mechanisms of peptide recognition and allosteric modulation of gene regulation by the RRNPP family of quorum-sensing regulators. J Mol Biol 428: 2793–2804. doi:10.1016/j.jmb.2016.05.026
    OpenUrlCrossRef
  11. ↵
    1. Do H,
    2. Makthal N,
    3. VanderWal AR,
    4. Rettel M,
    5. Savitski MM,
    6. Peschek N,
    7. Papenfort K,
    8. Olsen RJ,
    9. Musser JM,
    10. Kumaraswami M
    (2017) Leaderless secreted peptide signaling molecule alters global gene expression and increases virulence of a human bacterial pathogen. Proc Natl Acad Sci U S A 114: E8498–E8507. doi:10.1073/pnas.1705972114
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Do H,
    2. Makthal N,
    3. VanderWal AR,
    4. Saavedra MO,
    5. Olsen RJ,
    6. Musser JM,
    7. Kumaraswami M
    (2019) Environmental pH and peptide signaling control virulence of Streptococcus pyogenes via a quorum-sensing pathway. Nat Commun 10: 2586. doi:10.1038/s41467-019-10556-8
    OpenUrlCrossRef
    1. Doran JD,
    2. Nomizu M,
    3. Takebe S,
    4. Menard R,
    5. Griffith D,
    6. Ziomek E
    (1999) Autocatalytic processing of the streptococcal cysteine protease zymogen: Processing mechanism and characterization of the autoproteolytic cleavage sites. Eur J Biochem 263: 145–151. doi:10.1046/j.1432-1327.1999.00473.x
    OpenUrlCrossRefPubMed
  13. ↵
    1. Federle MJ,
    2. McIver KS,
    3. Scott JR
    (1999) A response regulator that represses transcription of several virulence operons in the group A Streptococcus. J Bacteriol 181: 3649–3657. doi:10.1128/jb.181.12.3649-3657.1999
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Finn MB,
    2. Ramsey KM,
    3. Dove SL,
    4. Wessels MR
    (2021) Identification of group A Streptococcus genes directly regulated by CsrRS and novel intermediate regulators. mBio 12: e0164221. doi:10.1128/mBio.01642-21
    OpenUrlCrossRef
  15. ↵
    1. Friaes A,
    2. Pato C,
    3. Melo-Cristino J,
    4. Ramirez M
    (2015) Consequences of the variability of the CovRS and RopB regulators among Streptococcus pyogenes causing human infections. Sci Rep 5: 12057. doi:10.1038/srep12057
    OpenUrlCrossRef
  16. ↵
    1. Gusa AA,
    2. Gao J,
    3. Stringer V,
    4. Churchward G,
    5. Scott JR
    (2006) Phosphorylation of the group A streptococcal CovR response regulator causes dimerization and promoter-specific recruitment by RNA polymerase. J Bacteriol 188: 4620–4626. doi:10.1128/JB.00198-06
    OpenUrlAbstract/FREE Full Text
  17. ↵
    1. Horstmann N,
    2. Myers KS,
    3. Tran CN,
    4. Flores AR,
    5. Shelburne SA III.
    (2022) CovS inactivation reduces CovR promoter binding at diverse virulence factor encoding genes in group A Streptococcus. PLoS Pathog 18: e1010341. doi:10.1371/journal.ppat.1010341
    OpenUrlCrossRef
  18. ↵
    1. Horstmann N,
    2. Sahasrabhojane P,
    3. Saldana M,
    4. Ajami NJ,
    5. Flores AR,
    6. Sumby P,
    7. Liu CG,
    8. Yao H,
    9. Su X,
    10. Thompson E, et al.
    (2015) Characterization of the effect of the histidine kinase CovS on response regulator phosphorylation in group A Streptococcus. Infect Immun 83: 1068–1077. doi:10.1128/IAI.02659-14
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Ikebe T,
    2. Ato M,
    3. Matsumura T,
    4. Hasegawa H,
    5. Sata T,
    6. Kobayashi K,
    7. Watanabe H
    (2010) Highly frequent mutations in negative regulators of multiple virulence genes in group A streptococcal toxic shock syndrome isolates. PLoS Pathog 6: e1000832. doi:10.1371/journal.ppat.1000832
    OpenUrlCrossRefPubMed
  20. ↵
    1. Jain I,
    2. Miller EW,
    3. Danger JL,
    4. Pflughoeft KJ,
    5. Sumby P
    (2017) RocA is an accessory protein to the virulence-regulating CovRS two-component system in group A Streptococcus. Infect Immun 85: e00274-17. doi:10.1128/IAI.00274-17
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Levin JC,
    2. Wessels MR
    (1998) Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus. Mol Microbiol 30: 209–219. doi:10.1046/j.1365-2958.1998.01057.x
    OpenUrlCrossRefPubMed
  22. ↵
    1. Loughman JA,
    2. Caparon M
    (2006) Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: A model for in vivo gene expression. J Bacteriol 188: 399–408. doi:10.1128/JB.188.2.399-408.2006
    OpenUrlAbstract/FREE Full Text
  23. ↵
    1. Lynskey NN,
    2. Turner CE,
    3. Heng LS,
    4. Sriskandan S
    (2015) A truncation in the regulator RocA underlies heightened capsule expression in serotype M3 group A streptococci. Infect Immun 83: 1732–1733. doi:10.1128/IAI.02892-14
    OpenUrlFREE Full Text
  24. ↵
    1. Lyon WR,
    2. Gibson CM,
    3. Caparon MG
    (1998) A role for trigger factor and an rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes. EMBO J 17: 6263–6275. doi:10.1093/emboj/17.21.6263
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Ma Y,
    2. Bryant AE,
    3. Salmi DB,
    4. Hayes-Schroer SM,
    5. McIndoo E,
    6. Aldape MJ,
    7. Stevens DL
    (2006) Identification and characterization of bicistronic speB and prsA gene expression in the group A Streptococcus. J Bacteriol 188: 7626–7634. doi:10.1128/JB.01059-06
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Makthal N,
    2. Gavagan M,
    3. Do H,
    4. Olsen RJ,
    5. Musser JM,
    6. Kumaraswami M
    (2016) Structural and functional analysis of RopB: A major virulence regulator in Streptococcus pyogenes. Mol Microbiol 99: 1119–1133. doi:10.1111/mmi.13294
    OpenUrlCrossRefPubMed
  27. ↵
    1. Miller AA,
    2. Engleberg NC,
    3. DiRita VJ
    (2001) Repression of virulence genes by phosphorylation-dependent oligomerization of CsrR at target promoters in S. pyogenes. Mol Microbiol 40: 976–990. doi:10.1046/j.1365-2958.2001.02441.x
    OpenUrlCrossRefPubMed
  28. ↵
    1. Neely MN,
    2. Lyon WR,
    3. Runft DL,
    4. Caparon M
    (2003) Role of RopB in growth phase expression of the SpeB cysteine protease of Streptococcus pyogenes. J Bacteriol 185: 5166–5174. doi:10.1128/jb.185.17.5166-5174.2003
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Perez-Pascual D,
    2. Monnet V,
    3. Gardan R
    (2016) Bacterial cell-cell communication in the host via RRNPP peptide-binding regulators. Front Microbiol 7: 706. doi:10.3389/fmicb.2016.00706
    OpenUrlCrossRef
  30. ↵
    1. Rasmussen M,
    2. Bjorck L
    (2002) Proteolysis and its regulation at the surface of Streptococcus pyogenes. Mol Microbiol 43: 537–544. doi:10.1046/j.1365-2958.2002.02766.x
    OpenUrlCrossRefPubMed
  31. ↵
    1. Shelburne SA III.,
    2. Olsen RJ,
    3. Makthal N,
    4. Brown NG,
    5. Sahasrabhojane P,
    6. Watkins EM,
    7. Palzkill T,
    8. Musser JM,
    9. Kumaraswami M
    (2011) An amino-terminal signal peptide of Vfr protein negatively influences RopB-dependent SpeB expression and attenuates virulence in Streptococcus pyogenes. Mol Microbiol 82: 1481–1495. doi:10.1111/j.1365-2958.2011.07902.x
    OpenUrlCrossRefPubMed
  32. ↵
    1. Shi YA,
    2. Chen TC,
    3. Chen YW,
    4. Liu YS,
    5. Chen YYM,
    6. Lai CH,
    7. Chiu CH,
    8. Chiang-Ni C
    (2022) The bacterial markers of identification of invasive CovR/CovS-inactivated group A Streptococcus. Microbiol Spectr 10: e0203322. doi:10.1128/spectrum.02033-22
    OpenUrlCrossRef
  33. ↵
    1. Sumby P,
    2. Whitney AR,
    3. Graviss EA,
    4. DeLeo FR,
    5. Musser JM
    (2006) Genome-wide analysis of group A streptococci reveals a mutation that modulates global phenotype and disease specificity. PLoS Pathog 2: e5. doi:10.1371/journal.ppat.0020005
    OpenUrlCrossRefPubMed
  34. ↵
    1. Tran-Winkler HJ,
    2. Love JF,
    3. Gryllos I,
    4. Wessels MR
    (2011) Signal transduction through CsrRS confers an invasive phenotype in group A Streptococcus. PLoS Pathog 7: e1002361. doi:10.1371/journal.ppat.1002361
    OpenUrlCrossRefPubMed
  35. ↵
    1. Trevino J,
    2. Perez N,
    3. Ramirez-Pena E,
    4. Liu Z,
    5. Shelburne SA,
    6. Musser JM,
    7. Sumby P
    (2009) CovS simultaneously activates and inhibits the CovR-mediated repression of distinct subsets of group A Streptococcus virulence factor-encoding genes. Infect Immun 77: 3141–3149. doi:10.1128/IAI.01560-08
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Unnikrishnan M,
    2. Cohen J,
    3. Sriskandan S
    (1999) Growth-phase-dependent expression of virulence factors in an M1T1 clinical isolate of Streptococcus pyogenes. Infect Immun 67: 5495–5499. doi:10.1128/iai.67.10.5495-5499.1999
    OpenUrlAbstract/FREE Full Text
  37. ↵
    1. Valcu M,
    2. Valcu CM
    (2011) Data transformation practices in biomedical sciences. Nat Methods 8: 104–105. doi:10.1038/nmeth0211-104
    OpenUrlCrossRefPubMed
  38. ↵
    1. Wang CH,
    2. Chiang-Ni C,
    3. Kuo HT,
    4. Zheng PX,
    5. Tsou CC,
    6. Wang S,
    7. Tsai PJ,
    8. Chuang WJ,
    9. Lin YS,
    10. Liu CC, et al.
    (2013) Peroxide responsive regulator PerR of group A Streptococcus is required for the expression of phage-associated DNase Sda1 under oxidative stress. PLoS One 8: e81882. doi:10.1371/journal.pone.0081882
    OpenUrlCrossRefPubMed
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Apo-RopB represses speB transcription
Chuan Chiang-Ni, Yan-Wen Chen, Kai-Lin Chen, Jian-Xian Jiang, Yong-An Shi, Chih-Yun Hsu, Yi-Ywan M Chen, Chih-Ho Lai, Cheng-Hsun Chiu
Life Science Alliance Mar 2023, 6 (6) e202201809; DOI: 10.26508/lsa.202201809

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Apo-RopB represses speB transcription
Chuan Chiang-Ni, Yan-Wen Chen, Kai-Lin Chen, Jian-Xian Jiang, Yong-An Shi, Chih-Yun Hsu, Yi-Ywan M Chen, Chih-Ho Lai, Cheng-Hsun Chiu
Life Science Alliance Mar 2023, 6 (6) e202201809; DOI: 10.26508/lsa.202201809
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