Article Figures & Data
Figures
- FIG. 1.
(A) Map of the construction of the oppA isogenic mutant showing the BclI restriction sites in the oppA locus. The predicted hybridization fragments are also shown. (B) Southern blot assay of genomic DNA digested with BclI and extracted from GAS A-20 (lane 1), SW552 (lane 2), and SW553 (lane 3). A λ HindIII marker was used as a molecular size standard (lane M). The 1.97-kb probe used was specific for the oppA gene and λ DNA. (C) Transcription of oppA in the wild-type strain (A-20) at various times and in its oppA isogenic mutant (SW552). The results of RT-PCR were amplified with oppA Fwd-2 and Rev-2 primers. A 100-bp marker was used as a molecular size standard (lane M).
- FIG. 2.
Variations in hemolytic activities observed in time course assays with wild-type strain A-20 and opp isogenic mutant SW552. The growth curve for A-20 is also shown. (A) SLO activities of A-20 and SW552. (B) SLS activities of A-20 and SW552. The error bars indicate the standard deviations. The mean activities of SLO and SLS were calculated in three independent experiments.
- FIG. 3.
Effects of Opp on streptococcal virulence factor expression. (A) RT-PCR analysis of speF, sagA, speX, and slo. (B) Northern hybridization analysis of speF, sagA, speX, and slo. Streptococcal virulence gene transcription among wild-type strain A-20, opp isogenic mutant SW552, opp revertant SW553, and opp complementation strain SW563 is shown.
- FIG. 4.
RT-PCR analysis of the expression of streptococcal regulators and virulence factors during various growth periods. RT-PCR-amplified products of sagB (A), pel (B), slo (C), rofA (D), and dppA (E) were made from cell harvested from strains A-20 and SW552 at 5, 7, 9, and 11 h. Time zero is defined as the time when the bacterial suspension was transferred to fresh medium. The transcript of 16S rRNA was used as an internal control. The extent of expression was normalized to the ratio of virulence gene RNA and 16S rRNA. The data represent the means and standard deviations obtained from at least three independent experiments.
- FIG. 5.
Survival of GAS-infected mice after inoculation in air pouches with 109 CFU of wild-type strain A-20 (n = 9), speB mutant SW507 (n = 9), opp mutant SW552 (n = 12), and opp complementation strain SW563 (n = 10). These mice were injected subcutaneously with about 1 ml of air to form an air pouch, and then a bacterial suspension (0.1 ml) was inoculated into the air pouch. The mortality rates were monitored every day during the experimental period. Bacterial numbers were determined by measuring the OD600 with a spectrophotometer.
Tables
- TABLE 1.
Specific PCR primer sets used in RT-PCR analysis
Gene Primera Sequence (5′→3′) Start site Reference or sourceb speB Fwd GGATCCATATGGATCAAACTTTGCTCGTAACG 81 12 Rev GAATTCGGATCCTAAGGTTTGATGCCTACAACAG 1175 speF Fwd ATGGCGCAAGCAAGTACCTA 977 31 Rev TTTGAGTAGGTGTACCTTAT 1624 speX Fwd TTTCTCGTCCTGTGTTTGGA 122 10 Rev TTGTGATATTAAAAATTTTCTCGCC 574 speG Fwd ACCCCATGCGATTATGAAAA 4797 8 Rev GGGAGACCAAAAACATCGAC 4966 speJ Fwd TCTTTCATGGGTACGGAAGTG 41 This study Rev AGCTCTCGACCTCAGAATCAA 238 sagA Fwd CTCGCGTTCTTATCAGTTAC 140 2 Rev ACCTGGCGTATAACTTCC 339 sagB Fwd TGTCCGCCAATAACTGTTGA 727 This study Rev CACCGTATTCCGCAAAATCT 1365 slo Fwd ACTCTGTCACTGATAGGACC 698 This study Rev GAGCTGCTTCAATCTGTG 998 csrS Fwd TGGGTTTTCCATGACACAAA 1400 23 Rev AGCGCCGCGTAGTAATTAAG 1594 csrR Fwd TGCAACATGAGGGGTATGAA 220 23 Rev TAAACCTGCAACCACATCCA 531 emm1 Fwd AACGGCTTCAGTAGCGGTAG 1082 11 Rev CTGCAACTTCCATTGCATTC 1281 sdaD (DNase) Fwd CACAACAGCCAGGGAATTTT 925 37 Rev GATGGTCTTGGTCCTCCTTG 1105 16S rRNA Fwd GCAGCAGTAGGGAATCTTCG 330 This study Rev CGCTCGGGACCTACGTATTA 530 oppA Fwd-1 ATGAAGAAAAGTAAATGGTT 1382 36 Fwd-2 GATCCACGGACCTACCTTGA 2901 Rev-1 TTATTTTTCAACGTGATCAG 3352 Rev-2 CAGCTGCCACAACATCCTTA 3003 dacA Fwd-1 AGGCGATGACCCACTAGGGA 1015 36 Fwd-2 TATTAGCTCCTAAAAATG 559 dppA Fwd GAAAACTGCAGTGACTGAGGCGCATTCGACA 1247 35 Rev GGCCGGAATTCTCAGAAGATGTCATTGCTT 2189 rofA Fwd ATCGCTCTGCTATATAGTAAG 520 This study Rev ACCGTCTCAGTGCTATCAA 2031 pel Fwd AGGAGGTAAACCTTATGTT 24 Rev GCTAAATAGATTATTTACCTG oppF Rev TTACAATTCTTTTTGATATT 7840 36 ↵ a Fwd, forward; Rev, reverse.
↵ b Primers for which the source is listed as this study were predicted according to Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi ) calculations. The accession numbers for speJ, sagB, slo, 16S rRNA, and rofA are AF321000 , AF067649 , A28468 , AB023575 , and U01312 , respectively, in the National Center for Biotechnology Information nucleotide database.
- TABLE 2.
SLO activities in oppA-positive and oppA-negative mutant strains of GAS in various atmospheres
Atmosphere Mean ± SEM % of SLO hemolysis ina: A-20 SW552 SW553 20% O2, 0.5% CO2 20.00 ± 1.1b 44.4 ± 1.1 24.2 ± 4.3 20% O2, 5% CO2 25.20 ± 3.8b 43.5 ± 1.4 25.2 ± 1.3 5% O2, 10% CO2 10.90 ± 0.5b 17.7 ± 1.4 12.2 ± 1.8
