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GidA, a tRNA Modification Enzyme, Contributes to the Growth, and Virulence of Streptococcus suis Serotype 2.

Gao T, Tan M, Liu W, Zhang C, Zhang T, Zheng L, Zhu J, Li L, Zhou R - Front Cell Infect Microbiol (2016)

Bottom Line: Here, we report a GidA homolog from a Chinese isolate SC-19 of the zoonotic Streptococcus suis serotype 2 (SS2). gidA disruption led to a defective growth, increased capsule thickness, and reduced hemolytic activity.This is consistent with the phenotypes of the mutant.Our findings provide new insight into the regulatory function of GidA in bacterial pathogens.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural UniversityWuhan, China; Veterinary Medicine Laboratory, Institute of Animal Husbandry and Veterinary Science, Hubei Academy of Agricultural ScienceWuhan, China; Wuhan Chopper Biology Co., Ltd.Wuhan, China.

ABSTRACT
Glucose-inhibited division protein (GidA), is a tRNA modification enzyme functioning together with MnmE in the addition of a carboxymethylaminomethyl group to position 5 of the anticodon wobble uridine of tRNA. Here, we report a GidA homolog from a Chinese isolate SC-19 of the zoonotic Streptococcus suis serotype 2 (SS2). gidA disruption led to a defective growth, increased capsule thickness, and reduced hemolytic activity. Moreover, the gidA deletion mutant (ΔgidA) displayed reduced mortality and bacterial loads in mice, reduced ability of adhesion to and invasion in epithelial cells, and increased sensitivity to phagocytosis. The iTRAQ analysis identified 372 differentially expressed (182 up- and 190 down-regulated) proteins in ΔgidA and SC-19. Numerous DNA replication, cell division, and virulence associated proteins were downregulated, whereas many capsule synthesis enzymes were upregulated by gidA disruption. This is consistent with the phenotypes of the mutant. Thus, GidA is a translational regulator that plays an important role in the growth, cell division, capsule biosynthesis, and virulence of SS2. Our findings provide new insight into the regulatory function of GidA in bacterial pathogens.

No MeSH data available.


Related in: MedlinePlus

Confirmation of the isogenic mutant ΔgidA. (A) Combined PCR analyses of the ΔgidA mutant. Lanes 1 and 4 represent the amplification of the upstream border of gidA using the primer set Gup-F and Gup-R. Lanes 2 and 5 represent the amplification of gidA using the primer set GidA-F and GidA-R. Lanes 3 and 6 represent the amplification of the downstream border of gidA using the primer set Gdown-F and Gdown-R. Lanes 1–3 use genomic DNA of SC-19 as templates, whereas Lanes 4–6 use genomic DNA of ΔgidA as templates. (B) Confirmation of the ΔgidA mutant by RT-PCR. Lanes 1 and 4 represent the amplification of downstream gene of gidA using the primer set 2162-F and 2162-R. Lanes 2 and 5 represent the amplification of gidA using primer set GidA-F and GidA-R. Lanes 3 and 6 represent the amplification of upstream gene of gidA using the primer set 2164-F and 2164-R. Lanes 1–3 use cDNA of SC-19 as templates, whereas Lanes 4–6 use cDNA of ΔgidA as templates. (C) Confirmation of the ΔgidA mutant by Western blot analysis. The supernatant of cell lysate from SC-19 and ΔgidA was disposed for immunoblot analysis with GidA or PGK polyclonal antibodies. An antibody directed against PGK was used as loading control.
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Figure 1: Confirmation of the isogenic mutant ΔgidA. (A) Combined PCR analyses of the ΔgidA mutant. Lanes 1 and 4 represent the amplification of the upstream border of gidA using the primer set Gup-F and Gup-R. Lanes 2 and 5 represent the amplification of gidA using the primer set GidA-F and GidA-R. Lanes 3 and 6 represent the amplification of the downstream border of gidA using the primer set Gdown-F and Gdown-R. Lanes 1–3 use genomic DNA of SC-19 as templates, whereas Lanes 4–6 use genomic DNA of ΔgidA as templates. (B) Confirmation of the ΔgidA mutant by RT-PCR. Lanes 1 and 4 represent the amplification of downstream gene of gidA using the primer set 2162-F and 2162-R. Lanes 2 and 5 represent the amplification of gidA using primer set GidA-F and GidA-R. Lanes 3 and 6 represent the amplification of upstream gene of gidA using the primer set 2164-F and 2164-R. Lanes 1–3 use cDNA of SC-19 as templates, whereas Lanes 4–6 use cDNA of ΔgidA as templates. (C) Confirmation of the ΔgidA mutant by Western blot analysis. The supernatant of cell lysate from SC-19 and ΔgidA was disposed for immunoblot analysis with GidA or PGK polyclonal antibodies. An antibody directed against PGK was used as loading control.

Mentions: The colonies sensitive to spectinomycin and resistant to erythromycin were selected as candidates of gidA deletion mutants, which were confirmed by PCR (Figure 1A), RT-PCR (Figure 1B), and Western blot analysis (Figure 1C). The colonies of ΔgidA appeared smaller than those of SC-19 when cultured on TSA plates overnight (Figure 2A). The growth curves showed that ΔgidA grew slower in the CDM than SC-19 (Figure 2B). However, no obvious difference in CFU counts was observed during the initial 3 h of growth. TEM revealed that the mean capsule was significantly thicker in ΔgidA (118 ± 5 nm) than in SC-19 (54 ± 3 nm; p < 0.001; Figure 2C).


GidA, a tRNA Modification Enzyme, Contributes to the Growth, and Virulence of Streptococcus suis Serotype 2.

Gao T, Tan M, Liu W, Zhang C, Zhang T, Zheng L, Zhu J, Li L, Zhou R - Front Cell Infect Microbiol (2016)

Confirmation of the isogenic mutant ΔgidA. (A) Combined PCR analyses of the ΔgidA mutant. Lanes 1 and 4 represent the amplification of the upstream border of gidA using the primer set Gup-F and Gup-R. Lanes 2 and 5 represent the amplification of gidA using the primer set GidA-F and GidA-R. Lanes 3 and 6 represent the amplification of the downstream border of gidA using the primer set Gdown-F and Gdown-R. Lanes 1–3 use genomic DNA of SC-19 as templates, whereas Lanes 4–6 use genomic DNA of ΔgidA as templates. (B) Confirmation of the ΔgidA mutant by RT-PCR. Lanes 1 and 4 represent the amplification of downstream gene of gidA using the primer set 2162-F and 2162-R. Lanes 2 and 5 represent the amplification of gidA using primer set GidA-F and GidA-R. Lanes 3 and 6 represent the amplification of upstream gene of gidA using the primer set 2164-F and 2164-R. Lanes 1–3 use cDNA of SC-19 as templates, whereas Lanes 4–6 use cDNA of ΔgidA as templates. (C) Confirmation of the ΔgidA mutant by Western blot analysis. The supernatant of cell lysate from SC-19 and ΔgidA was disposed for immunoblot analysis with GidA or PGK polyclonal antibodies. An antibody directed against PGK was used as loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835480&req=5

Figure 1: Confirmation of the isogenic mutant ΔgidA. (A) Combined PCR analyses of the ΔgidA mutant. Lanes 1 and 4 represent the amplification of the upstream border of gidA using the primer set Gup-F and Gup-R. Lanes 2 and 5 represent the amplification of gidA using the primer set GidA-F and GidA-R. Lanes 3 and 6 represent the amplification of the downstream border of gidA using the primer set Gdown-F and Gdown-R. Lanes 1–3 use genomic DNA of SC-19 as templates, whereas Lanes 4–6 use genomic DNA of ΔgidA as templates. (B) Confirmation of the ΔgidA mutant by RT-PCR. Lanes 1 and 4 represent the amplification of downstream gene of gidA using the primer set 2162-F and 2162-R. Lanes 2 and 5 represent the amplification of gidA using primer set GidA-F and GidA-R. Lanes 3 and 6 represent the amplification of upstream gene of gidA using the primer set 2164-F and 2164-R. Lanes 1–3 use cDNA of SC-19 as templates, whereas Lanes 4–6 use cDNA of ΔgidA as templates. (C) Confirmation of the ΔgidA mutant by Western blot analysis. The supernatant of cell lysate from SC-19 and ΔgidA was disposed for immunoblot analysis with GidA or PGK polyclonal antibodies. An antibody directed against PGK was used as loading control.
Mentions: The colonies sensitive to spectinomycin and resistant to erythromycin were selected as candidates of gidA deletion mutants, which were confirmed by PCR (Figure 1A), RT-PCR (Figure 1B), and Western blot analysis (Figure 1C). The colonies of ΔgidA appeared smaller than those of SC-19 when cultured on TSA plates overnight (Figure 2A). The growth curves showed that ΔgidA grew slower in the CDM than SC-19 (Figure 2B). However, no obvious difference in CFU counts was observed during the initial 3 h of growth. TEM revealed that the mean capsule was significantly thicker in ΔgidA (118 ± 5 nm) than in SC-19 (54 ± 3 nm; p < 0.001; Figure 2C).

Bottom Line: Here, we report a GidA homolog from a Chinese isolate SC-19 of the zoonotic Streptococcus suis serotype 2 (SS2). gidA disruption led to a defective growth, increased capsule thickness, and reduced hemolytic activity.This is consistent with the phenotypes of the mutant.Our findings provide new insight into the regulatory function of GidA in bacterial pathogens.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural UniversityWuhan, China; Veterinary Medicine Laboratory, Institute of Animal Husbandry and Veterinary Science, Hubei Academy of Agricultural ScienceWuhan, China; Wuhan Chopper Biology Co., Ltd.Wuhan, China.

ABSTRACT
Glucose-inhibited division protein (GidA), is a tRNA modification enzyme functioning together with MnmE in the addition of a carboxymethylaminomethyl group to position 5 of the anticodon wobble uridine of tRNA. Here, we report a GidA homolog from a Chinese isolate SC-19 of the zoonotic Streptococcus suis serotype 2 (SS2). gidA disruption led to a defective growth, increased capsule thickness, and reduced hemolytic activity. Moreover, the gidA deletion mutant (ΔgidA) displayed reduced mortality and bacterial loads in mice, reduced ability of adhesion to and invasion in epithelial cells, and increased sensitivity to phagocytosis. The iTRAQ analysis identified 372 differentially expressed (182 up- and 190 down-regulated) proteins in ΔgidA and SC-19. Numerous DNA replication, cell division, and virulence associated proteins were downregulated, whereas many capsule synthesis enzymes were upregulated by gidA disruption. This is consistent with the phenotypes of the mutant. Thus, GidA is a translational regulator that plays an important role in the growth, cell division, capsule biosynthesis, and virulence of SS2. Our findings provide new insight into the regulatory function of GidA in bacterial pathogens.

No MeSH data available.


Related in: MedlinePlus