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. 2005 Sep;73(9):5743-53.
doi: 10.1128/IAI.73.9.5743-5753.2005.

Characterization of MtsR, a new metal regulator in group A streptococcus, involved in iron acquisition and virulence

Christopher S Bates  1 , Chadia Toukoki, Melody N Neely, Zehava Eichenbaum
Affiliations

Characterization of MtsR, a new metal regulator in group A streptococcus, involved in iron acquisition and virulence

"VSports在线直播" Christopher S Bates et al. Infect Immun. 2005 Sep.

Abstract

Group A streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces capable of producing a variety of diseases. In this study, we investigated regulation of iron uptake in GAS and the role of a putative transcriptional regulator named MtsR (for Mts repressor) with homology to the DtxR family of metal-dependent regulatory proteins. An mtsR mutant was constructed in NZ131 (M49 serotype) and analyzed. Western blot and RNA analysis showed that mtsR inactivation results in constitutive transcription of the sia (streptococcal iron acquisition) operon, which was negatively regulated by iron in the parent strain VSports手机版. A recombinant MtsR with C-terminal His(6) tag fusion (rMtsR) was cloned and purified. Electrophoretic mobility gel shift assays demonstrated that rMtsR specifically binds to the sia promoter region in an iron- and manganese-dependent manner. Together, these observations indicate that MtsR directly represses the sia operon during cell growth under conditions of high metal levels. Consistent with deregulation of iron uptake, the mtsR mutant is hypersensitive to streptonigrin and hydrogen peroxide, and (55)Fe uptake assays demonstrate that it accumulates 80% +/- 22. 5% more iron than the wild-type strain during growth in complete medium. Studies with a zebrafish infection model revealed that the mtsR mutant is attenuated for virulence in both the intramuscular and the intraperitoneal routes. In conclusion, MtsR, a new regulatory protein in GAS, controls iron homeostasis and has a role in disease production. .

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"VSports手机版" Figures

FIG. 1.
FIG. 1.
The streptococcal metal transport repressor (MtsR). (A) Schematic presentation of the mtsR (spy0450) chromosomal locus in the wild-type strain NZ131. The stem-loop structures represent transcriptional terminators. The genes are represented by their names. The mts genes are annotated as such in the S. pyogenes SF370 genome, while mtsR is annotated as spy0450. (B) CLUSTALW alignment of MtsR (Spy0450) with SloR of S. mutans. White letters on a black background indicate identical residues; white letters on a gray background indicate similar residues. The residues overscored with a single line indicate the helix-turn-helix domain, and those with a double line indicate the FeoA domain. Ancillary metal binding residues (metal site 1) are indicated by the number 1 over the white or black letters, and the primary metalloregulatory residues (metal site 2) are indicated by the number 2. (C) Schematic presentation of the mtsR mutation in ZE491 strain. KmR represents aphA-3, the kanamycin resistance gene, flanked by the Omega transcriptional termination signals (43); mtsRΔ indicates the 5′ portion of mtsR remaining in the ZE491 mutant.
FIG. 2.
FIG. 2.
MtsR controls the expression of the sia operon. GAS cells were used to inoculate complete (ZTH; black symbols) or iron-limiting (NTA; empty symbols) medium and incubated at 37°C, and cell growth was monitored over time. Culture samples were taken in the exponential-growth phase, and total proteins and RNA were prepared. (A) Growth curves. Cell growth is expressed as Klett units. Squares indicate the wild-type NZ131, and circles represent the mtsR mutant (ZE491). (B) Western blot analysis of Shr and SiaA proteins. Proteins, standardized based on cell number, were separated by SDS-PAGE and were reacted with rabbit antibody to Shr (top panel) or SiaA (bottom panel). (C) RT-PCR analysis of sia genes. cDNA synthesized from 1 μg of total RNA by use of gene-specific primers was amplified by PCR and separated on an agarose gel. PCRs are shown for the housekeeping gene recA (top panel) and the shr gene (bottom panel).
FIG. 3.
FIG. 3.
MtsR directly binds to the promoter region of shr. (A) Schematic presentation of the sia operon's promoter/operator region (Pshr). The positions of the putative −35 and −10 regions of shr, the sia operon's first gene, are indicated. (B). The EMSA was done with the 32P-labeled promoter fragment and purified MtsR-His protein in a range of concentrations from 0 to 22.5 ng as indicated above the lanes. The DNA-protein complexes C1 and C2 are indicated.
FIG. 4.
FIG. 4.
MtsR binding to the sia promoter region is specific and metal dependent. A. Binding of the purified rMtsR to Pshr fragment. All EMSAs were done with the 32P-labeled promoter fragment. Additional components of the different binding reactions are indicated above the lanes. Lane 1 from the left, [32P]Pshr fragment only. Lane 2, [32P]Pshr fragment but with 7.5 ng of purified MtsR-His6. Lane 3, the same as in lane 2 but with 250 μM EDTA. Lane 4, the same as in lane 3 but with 25 μM FeSO4. Lane 5, the same as in lane 3 but with 75 μM FeSO4. Lane 6, the same as in lane 3 but with 50 μM FeSO4. Lane 7, the same as in lane 3 but with 100 μM FeSO4. Lane 8, the same as in lane 2 but with a 10-fold increase in the level of unlabeled (cold) Pshr fragment. Lane 9, the same as in lane 2 but with a 10-fold increase in the level of unlabeled recA fragment. Lane 10, [32P]Pshr fragment and 100 ng of SiaA-His6 (4). B. Iron restores binding of EDTA-treated rMtsR to DNA. Lane 1, [32P]Pshr fragment only. Lane 2, the same as in lane 1 but with 10 ng of rMtsR pretreated with EDTA. Lane 3, the same as in lane 1 but with 10 ng of untreated rMtsR. Lane 4 the same as in lane 2 but with 25 μM FeSO4. Lane 5, the same as in lane 2 but with 75 μM FeSO4. Lane 6, the same as in lane 2 but with 150 μM FeSO4. C. Manganese restores binding of EDTA-treated rMtsR to DNA. Lane 1, [32P]Pshr fragment only. Lane 2, the same as in lane 1 but with 10 ng of rMtsR pretreated with EDTA. Lanes 3 and 4, the same as in lane 2 but with 25 and 50 μM MnCl2, respectively.
FIG. 5.
FIG. 5.
MtsR inactivation results in hypersensitivity to streptonigrin and hydrogen peroxide. (A) Sensitivity of the wild-type (NZ131) and mtsR mutant (ZE491) to streptonigrin. Bacteria were inoculated into fresh THY medium containing 0.36 μM streptonigrin. The culture optical density (OD600) was determined after overnight growth (∼20 h). The data are presented as the ratio of the OD600 obtained in overnight cultures grown in THY containing the drug to that obtained in THY alone over the same time period. (B) Sensitivity of NZ131 and ZE491 to hydrogen peroxide. Bacteria were inoculated into fresh THY containing 1 mM hydrogen peroxide, and overnight growth was determined and presented as described for panel A. In both panel A and panel B, error bars represent the standard deviation of the mean (n = 3).
FIG. 6.
FIG. 6.
ZE491 is attenuated in the zebrafish infection model. GAS cells harvested at the logarithmic growth phase were injected into groups of zebrafish (Danio rerio) (5 × 105 cells per fish) either intramuscularly (IM) or intraperitoneally (IP) as previously described (38). The average number of dead fish at 48 h postinjection is shown for each infection route; error bars represent the standard error of the mean (n = 5).
FIG. 7.
FIG. 7.
Phylogenetic tree of Fur and DtxR homologs. A. Phylogenetic analysis of Fur and DtxR metalloregulators. B. Phylogenetic analysis of the corresponding RecA proteins from the organisms used in panel A. Trees were generated using ClustalW 1.75 software as described in Materials and Methods. Phylograms with weighted branch lengths are shown. S. gordonii and R. equi RecA proteins are not included, as their sequences are incomplete or not available. Accession numbers for all proteins in panels A and B are listed in Materials and Methods.
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