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The heme transporter HtsABC of group a Streptococcus contributes to virulence and innate immune evasion in murine skin infections

Frontiers in Microbiology (2018) 9(MAY)
DOI: 10.3389/fmicb.2018.01105
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Abstract

Group A Streptococcus (GAS) requires iron for growth, and heme is an important source of iron for GAS. Streptococcus heme transporter A (HtsA) is the lipoprotein component of the GAS heme-specific ABC transporter (HtsABC). The objective of this study is to examine the contribution of HtsABC to virulence and host interaction of hypervirulent M1T1 GAS using an isogenic htsA deletion mutant (ΔhtsA). The htsA deletion exhibited a significantly increased survival rate, reduced skin lesion size, and reduced systemic GAS dissemination in comparison to the wild type strain. The htsA deletion also decreased the GAS adhesion rate to Hep-2 cells, the survival in human blood and rat neutrophils, and increased the production of cytokine IL-1β, IL-6, and TNF-α levels in air pouch exudate of a mouse model of subcutaneous infection. Complementation of ΔhtsA restored the wild type phenotype. These findings support that the htsA gene is required for GAS virulence and that the htsA deletion augments host innate immune responses.

Figures

  • FIGURE 1 | Construction of a 1htsA mutant and 1htsA-htsA revertant. (A) The arrangement of the shr, shp, and htsABC genes in the operon. (B) PCR analysis of fragments amplified from chromosomes of GAS strains. The PCR fragment detected in the 1htsA mutant was 303-bp smaller than that in the wild type, and the PCR fragment detected in the 1htsA-htsA revertant was identical to that in wild type. (C) Western blotting analysis of GAS strains. HtsA protein was expressed in wild type and 1htsA-htsA strains but not in the 1htsA mutant. Shp was used as a loading control.
  • FIGURE 2 | Deletion of htsA attenuated GAS virulence in mice. (A) Kaplan–Meier survival curves showing the relative rates of mouse mortality caused by wild type, 1htsA and 1htsA-htsA strains. Groups of mice (n = 18) were injected subcutaneously with ∼2.0 × 108 CFU of GAS strains, and survival rates were observed daily for 15 days. Compared to wild type and 1htsA-htsA, the survival rate in mice infected with 1htsA was significantly higher. (B) Skin lesion sizes caused by wild type, 1htsA and 1htsA-htsA strains. Groups of mice (n = 7) were injected subcutaneously with ∼2.0 × 108 CFU of GAS strains, and skin lesion sizes were calculated by measuring the length and width at the longest point of the lesion (length by width) at 24 h postinoculation. Compared to wild type and 1htsA-htsA, the average area of skin lesion sizes in mice infected with 1htsA was significantly smaller. ∗∗P < 0.01, ∗∗∗P < 0.001.
  • FIGURE 3 | Deletion of htsA reduced GAS systemic dissemination in mice. Groups of mice (n = 7) were infected with ∼2 × 108 CFU of GAS strains, and then the GAS load in heparinized blood and tissue homogenates from spleen, liver, kidneys, and lungs was determined at 24 h postinoculation. The GAS load in samples was expressed as log10 CFU. GAS log10 CFU values in the spleen, liver, and kidneys of mice infected with 1htsA were significantly lower than those observed in mice infected with wild type, and 1htsA-htsA could restore the virulence of GAS. ∗P < 0.05, ∗∗P < 0.01.
  • FIGURE 4 | Deletion of htsA impeded GAS adhesion to and invasion into human laryngeal epithelial (Hep-2) cells. (A) GAS adhesion to Hep-2 cells. Adhesion of GAS strains to endothelial cells was assayed after 2 h of incubation. The data shown represent the percentages of adherent cells in relation to the initial bacterial inoculum. The mean adhesion rate to Hep-2 cells for 1htsA was significantly lower than that for wild type and 1htsA-htsA. (B) GAS invasion into Hep-2 cells. Invasion of GAS strains into endothelial cells was determined by an antibiotic protection assay. The data shown represent the percentages of invasive cells in relation to the initial bacterial inoculum. There was a tendency toward a reduced invasion rate in 1htsA compared to that in wild type and 1htsA-htsA. ∗P < 0.05.
  • FIGURE 5 | Deletion of htsA decreased GAS resistance to phagocytosis. (A) Deletion of htsA decreased survival in blood. Wild type, 1htsA, and 1htsA-htsA strains were incubated with heparinized blood, and bacterial CFU were determined at 0 min, 30 min, 60 min, 120 min, and 180 min postincubation. After 60 min of incubation, the growth of 1htsA in human blood was significantly slower than that of wild type and 1htsA-htsA; (B) deletion of htsA decreased survival in rat neutrophils. Wild type, 1htsA, and 1htsA-htsA strains were incubated with purified rat neutrophils, and bacterial CFU values were determined at 0 min, 30 min, 60 min, 120 min, and 180 min postincubation. At 120 min and 180 min of incubation, the growth of 1htsA in rat neutrophils was significantly slower than that of wild type and 1htsA-htsA.
  • FIGURE 6 | Deletion of htsA induced higher neutrophil recruitment at skin infection sites of mice. Groups of mice were subcutaneously injected with wild type, 1htsA, or 1htsA-htsA strains at a dose of ∼2.0 × 108 CFU, and the skin containing the infection area was excised and homogenized at 24 h postinfection. (A) MPO activity at skin infection sites of mice. The MPO activity (U/total) of mice infected with 1htsA was higher than that of mice infected with wild type or 1htsA-htsA. (B) The total bacterial CFU at skin infection sites of mice. There were no statistically significant differences in total bacterial CFU among the three GAS strains. (C) Microscopic pictures of Gram staining at a skin infection site. Finflammatory cells, Nbacteria. ∗∗P < 0.01.
  • FIGURE 7 | Deletion of htsA induced high production of cytokines in mice. Groups of mice (n = 7) were subcutaneously injected with 2 ml of air to form an air pouch and inoculated with ∼2 × 108 CFU of bacterial strains. At 24 h postinfection, air pouch exudate was collected, and the cytokine levels of IL-1β, IL-6, and TNF-α in the exudate were determined by ELISA assay. The result showed that cytokine levels of IL-1β, IL-6, and TNF-α in the mice infected with 1htsA were higher than those in mice infected with wild type or 1htsA-htsA. ∗P < 0.05, ∗∗P < 0.01.

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Song, Y., Zhang, X., Cai, M., Lv, C., Zhao, Y., Wei, D., & Zhu, H. (2018). The heme transporter HtsABC of group a Streptococcus contributes to virulence and innate immune evasion in murine skin infections. Frontiers in Microbiology, 9(MAY). https://doi.org/10.3389/fmicb.2018.01105

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