Two novel functions of hyaluronidase from Streptococcus agalactiae are enhanced intracellular survival and inhibition of proinflammatory cytokine expression - PubMed (original) (raw)
Two novel functions of hyaluronidase from Streptococcus agalactiae are enhanced intracellular survival and inhibition of proinflammatory cytokine expression
Zhaofei Wang et al. Infect Immun. 2014 Jun.
Abstract
Streptococcus agalactiae is the causative agent of septicemia and meningitis in fish. Previous studies have shown that hyaluronidase (Hyl) is an important virulence factor in many Gram-positive bacteria. To investigate the role of S. agalactiae Hyl during interaction with macrophages, we inactivated the gene encoding extracellular hyaluronidase, hylB, in a clinical Hyl(+) isolate. The isogenic hylb mutant (Δhylb) displayed reduced survival in macrophages compared to the wild type and stimulated a significantly higher release of proinflammatory cytokines, such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α), than the wild type in macrophages as well as in mice. Furthermore, only Hyl(+) strains could grow utilizing hyaluronic acid (HA) as the sole carbon source, suggesting that Hyl permits the organism to utilize host HA as an energy source. Fifty percent lethal dose (LD50) determinations in zebrafish demonstrated that the hylb mutant was highly attenuated relative to the wild-type strain. Experimental infection of BALB/c mice revealed that bacterial loads in the blood, spleen, and brain at 16 h postinfection were significantly reduced in the ΔhylB mutant compared to those in wild-type-infected mice. In conclusion, hyaluronidase has a strong influence on the intracellular survival of S. agalactiae and proinflammatory cytokine expression, suggesting that it plays a key role in S. agalactiae pathogenicity.
Figures
FIG 1
Confirmation of the S. agalactiae GD201008-001 hylB mutant and complemented strains. (A) Schematic of the strategy for producing the hylB mutant by allelic exchange mutagenesis. (B) Multiple-PCR analysis of the wild-type (WT), mutant (Δ_hylB_), and complemented (CΔ_hylB_) strains. The primer combinations used in the PCR were as follows: lanes 1, 2, and 3, _hylB_-LF/_hylB_-RR; lanes 4, 5, and 6, hylB_-F/hylB_-R. Genomic DNA from the following strains was used as the template: the wild-type strain (lanes 1 and 4), the Δ_hylB mutant strain (lanes 2 and 5), and the CΔ_hylB complemented strain (lanes 3 and 6). The 5-kb DNA ladder marker (M) is shown on the left. (C) mRNA expression levels of hylB upstream, downstream, and hylB self ORF in the S. agalactiae GD201008-001 wild-type, mutant, and complemented strains in vitro. The values of the hylB_-associated genes in the wild-type strain were normalized to 1.0. The relative changes in gene expression ratios of selected genes were normalized to the expression of a single housekeeping gene (16S rRNA gene) and calculated as described by the 2−ΔΔ_CT method. Error bars indicate SEM from three independent experiments.
FIG 2
Growth assays of the wild-type (WT), hylB mutant (Δ_hylB_), and complemented (CΔ_hylB_) strains. Strains were grown in chemically defined medium (CDM) supplemented with 5 mg/ml hyaluronic acid (HA) or 12 mM glucose. OD600 values are the means ± SEM from three independent experiments.
FIG 3
Variation in the ability of the wild-type (WT), hylB mutant (Δ_hylB_), and complemented (CΔ_hylB_) strains of S. agalactiae to degrade hyaluronic acid. Degradation of the hyaluronic acid was detected as a clear zone (labeled areas) resulting from acetic acid precipitation of a complex of albumin and nondegraded hyaluronic acid. Assays were conducted at least twice.
FIG 4
Bacterial loads in different tissues from mice infected intraperitoneally with the S. agalactiae GD201008-001 wild-type (WT), mutant (Δ_hylB_), and complemented (CΔ_hylb_) strains. Bacterial loads in blood (A) are expressed as CFU/ml, and those in the brain (B) and spleen (C) are expressed as CFU/g of tissue. Results are expressed as means ± SEM from at least three infected mice at 16 h postinfection. Asterisks indicate significant differences (P < 0.05) in the numbers of CFU observed between the mutant and wild-type strains.
FIG 5
Intracellular growth of S. agalactiae in RAW264.7 macrophages. (A) Intracellular survival of wild-type S. agalactiae GD201008-001 in RAW264.7 macrophages compared to that of the Δ_hylB_ mutant and complemented strains infected at an MOI of 1. Phagocytosis was allowed to proceed for 1 h before the addition of antibiotics for 1 h. This initial antibiotic treatment was extended for different times up to 24 h. The relative number of CFU (rCFU) was estimated by plating out the lysates of infected macrophages and counting the number of CFU at each time point. Asterisks indicate the time points when the intracellular bacteria survival rates elicited by the Δ_hylB_ mutant were significantly lower (P < 0.05) than those produced by wild-type infection. (B) RAW264.7 macrophages were infected with different numbers of cells of the S. agalactiae GD201008-001 wild-type (WT), mutant (Δ_hylB_), and complemented (CΔ_hylb_) strains. At 1 h postinfection, cells were extensively washed to remove extracellular bacteria after antibiotic treatment and were lysed; CFU were then measured. The data shown are means ± SEM from three independent experiments.
FIG 6
Effect of bacterial cell-mediated cytotoxicity as measured by LDH cytotoxicity assays. RAW264.7 macrophages were infected with S. agalactiae GD201008-001 wild-type (WT), mutant (Δ_hylB_), and complemented (CΔ_hylb_) strains at the indicated multiplicities of infection (MOI) for 4 h to measure the LDH release into the supernatant. “Control” corresponds to maximum lysis achieved using 2% Triton X-100. The data shown are means ± SEM from three independent experiments.
FIG 7
Intracellular S. agalactiae-induced cytokine expression in RAW264.7 macrophages. RAW264.7 macrophages were infected with S. agalactiae GD201008-001 wild-type (WT), mutant (Δ_hylB_), and complemented (CΔ_hylB_) strains at an MOI of 10:1 at 1 h. Extracellular bacteria were killed by antibiotics, and cells were harvested at different time points. The expression levels of IL-1β, IL-6, and TNF-α were measured by quantitative RT-PCR. Uninfected cells served as the control. Levels of IL-1β, IL-6, and TNF-α mRNA were normalized to mRNA levels of β-actin and then were expressed as n_-fold increases with respect to the control. The data shown are means ± SEM from no less than six independent experiments. Asterisks indicate the time points when the cytokine levels elicited by the Δ_hylB mutant were significantly greater (P < 0.05) than those produced by wild-type infection.
FIG 8
Cytokine expression in the spleens of BALB/c mice injected intraperitoneally with 5 × 102 CFU of S. agalactiae GD201008-001 wild-type (WT), mutant (Δ_hylB_), and complemented (CΔ_hylB_) strains. Supernatants from spleen homogenates were collected at different time points after infection and assayed for IL-1β (A), IL-6 (B), and TNF-α (C) by quantitative RT-PCR. Uninfected mice served as the control. Five mice per group were sacrificed at each time point. Levels of IL-1β, IL-6, and TNF-α mRNA were normalized to mRNA levels of β-actin and then were expressed as n_-fold increases with respect to the control. The data shown are means ± SEM from no less than six independent experiments. Asterisks indicate the time points when the cytokine levels elicited by the Δ_hylB mutant were significantly greater (P < 0.05) than those produced by wild-type infection.
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