Role of methionine sulfoxide reductases A and B of Enterococcus faecalis in oxidative stress and virulence - PubMed (original) (raw)
Role of methionine sulfoxide reductases A and B of Enterococcus faecalis in oxidative stress and virulence
Chen Zhao et al. Infect Immun. 2010 Sep.
Abstract
Methionine sulfoxide reductases A and B are antioxidant repair enzymes that reduce the S- and R-diastereomers of methionine sulfoxides back to methionine, respectively. Enterococcus faecalis, an important nosocomial pathogen, has one msrA gene and one msrB gene situated in different parts of the chromosome. Promoters have been mapped and mutants have been constructed in two E. faecalis strains (strains JH2-2 and V583) and characterized. For both backgrounds, the mutants are more sensitive than the wild-type parents to exposure to H2O2, and in combination the mutations seem to be additive. The virulence of the mutants has been analyzed in four different models. Survival of the mutants inside mouse peritoneal macrophages stimulated with recombinant gamma interferon plus lipopolysaccharide but not in naïve phagocytes is significantly affected. The msrA mutant is attenuated in the Galleria mellonella insect model. Deficiency in either Msr enzyme reduced the level of virulence in a systemic and urinary tract infection model. Virulence was reconstituted in the complemented strains. The combined results show that Msr repair enzymes are important for the oxidative stress response, macrophage survival, and persistent infection with E. faecalis.
Figures
FIG. 1.
(A) Genetic context of the msrA gene of E. faecalis. The open reading frames are represented by open arrows, and their orientation indicates the transcriptional direction. Numbers above the intergenic regions indicate the gene distances. The numbers below the genes indicate the sizes of the gene products in amino acids (AA). The size of the 5′ UTR is given in bp. The nucleotide sequences of the putative rho_-independent terminators, located 4 and 8 nucleotides downstream of the ef1680 and ef1684 stop codons, respectively, are shown. P_msrA and P_degV_ indicate the positions of two promoters mapped by 5′ RACE-PCR (see panel D) (B) Positions of hybridization of 5 oligonucleotide pairs (I to V) used for RT-PCR. The expected amplimer sizes are indicated in bp. The results of the RT-PCRs with the five primer pairs are shown in the electropherogram. Lanes 1, control PCR with chromosomal DNA; lanes 2, RT-PCR; lanes 3, PCR with RNA extraction without prior reverse transcriptase reaction (negative control); lanes M, molecular size standard, with sizes given at the left of the gel. (C) Northern blot analysis using RNA extracted from exponentially growing cells. Hybridization was performed with an [α-32P]dATP-labeled single-strand probe complementary to the msrA mRNA. The size of the transcript was estimated by comparison with the sizes on an RNA ladder. (D) Mapping of promoters P_msrA_ and P_degV_ by 5′ RACE-PCR. The transcriptional initiation sites (+1) are indicated, the putative −35 and −10 motifs are underlined, and the distances between the two boxes are given in bp. The putative ribosome binding sites are overlined, and the start codons are boxed.
FIG. 2.
(A) Genetic context of the msrB gene of E. faecalis. For detailed information, see the legend for Fig. 1A. TT, position of a putative _rho_-independent transcriptional terminator located 8 nucleotides downstream of the ef3163 stop codon. (B) The transcription start site of the msrB operon has been mapped using 5′ RACE-PCR (for details, refer to the legend for Fig. 1D).
FIG. 3.
Virulence test of msr mutants using the Galleria mellonella insect model. About 5 × 108 CFU was injected into 1 caterpillar, and 15 caterpillars were used for each strain. Viable caterpillars infected with the JH2-2 wild-type strain (⋄), the Δ_msrA_ mutant (□), the Δ_msrB_ mutant (▵), the Δ_msrA_ Δ_msrB_ double mutant (○), and the Δ_msrA_ (▪) complemented strain were counted after 15, 17, 19, 21, and 23 h after infection.
FIG. 4.
Survival of the E. faecalis JH2-2 wild type (⋄); its isogenic Δ_msrA_ (□), Δ_msrB_ (▵), and Δ_msrA_ Δ_msrB_ (○) mutant strains; and the Δ_msrA_ (▪) and Δ_msrB_ (▴) complemented strains in mouse peritoneal macrophages derived from an in vitro/in vivo infection model (A) or isolated from the animals and cultured in vitro with medium alone (B) or with rIFN-γ plus LPS (C) before infection. The data are expressed as means ± standard deviations for the number of viable intracellular bacteria per 105 macrophages in at least three different experiments.
FIG. 5.
Enterococcal tissue burdens in kidneys (A) and livers (B) of BALB/c mice infected intravenously with 5 × 108 cells of the E. faecalis JH2-2 wild type (⋄); its isogenic Δ_msrA_ (□), Δ_msrB_ (▵), and Δ_msrA_ Δ_msrB_ (○) mutant strains; and the Δ_msrA_ (▪) and Δ_msrB_ (▴) complemented strains. Groups of 10 mice were killed and necropsied at day 7 postinfection. (C) Enterococcal burdens of the kidneys of BALB/c mice infected transurethrally with 104 cells of the E. faecalis JH2-2 wild type (⋄); its isogenic Δ_msrA_ (□), Δ_msrB_ (▵), and Δ_msrAB_ (○) mutant strains; and the msrA (▪) and msrB (▴) complemented strains. Kidney pair homogenates were obtained from groups of 15 mice that were killed and necropsied 48 h after the transurethral challenge. The results, expressed as log10 CFU per gram of tissue, represent the values recorded separately for each mouse. Horizontal bars represent the geometric means. A value of 0 was assigned to uninfected kidneys.
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