Evaluation of capsular and acapsular strains of S. aureus in an experimental brain abscess model

Abstract

Brain abscesses are mainly caused by either direct or indirect inoculation of gram positive bacteria including Stapylococcus aureus (S. aureus) or Streptococcus species into the central nervous system. In the present study, we aimed to compare potential changes in brain abscess pathogenesis induced by two different strains of S. aureus, namely the laboratory strain RN6390 and the clinical isolate Reynolds. Although the Reynolds strain was expected to be more resistant to eradication by the host, due to the existence of a polysaccharide capsule, and subsequently to be more virulent, instead we found parenchymal damage and mortality rates to be more prominent following RN6390 infection. In contrast, the Reynolds strain proliferated faster and induced early expression of the chemokine CXCL2, matrix metalloproteinase-9 (MMP-9), and complement 3a and C5. Furthermore, there were early and more abundant infiltration of PMNs, T cells and erythrocyte extravasation in brain abscesses induced by the Reynolds strain. However, several immune parameters were not different between the two strains during the later stages of the disease. These results suggest that capsular S. aureus can modulate innate immunity and complement system activation differently than the acapsular strain RN6390, and the early changes induced by Reynolds strain may have an important impact on survival.

Fig. 1

S. aureus Reynolds is proliferated faster. Mice (n=4–6 mice per group/time point) were injected with either agarose-laden S. aureus strain RN6390- or S. aureus strain Reynolds-beads (10⁴CFU) as described in the Materials and methods. Animals were euthanized at the indicated time points and the number of viable organisms associated with brain abscesses was determined by quantitative culture. Titers are expressed as the mean log₁₀ CFU per milliliter of brain abscess homogenate (A). (B) mortality rates are determined as the percentages of dead mice to surviving mice at the indicated time points (n=20–25 mice per group). Significant differences in mortality rates between S. aureus RN6390- and Reynolds-infected mice are denoted with asterisks (**, p<0.001). Results are the mean (±SEM) of three independent experiments.

Fig. 2

Following Reynolds infection the early damage to surrounding tissue is controlled better. Mice (n=4–6 mice per group/time point) were injected with either agarose-laden S. aureus strain RN6390- or S. aureus strain Reynolds-beads (10⁴CFU) as described in the Materials and methods. Animals were euthanized at the indicated time points, whereupon brain tissues were flash frozen on dry ice for subsequent cryostat sectioning. Serial sections were prepared throughout the entire abscess to ensure that the maximal cross-sectional area was identified and stained with H & E to demarcate the histological appearance of lesions. (A) Stained slices were imaged under 1.25× objective to visualize the whole section. Lesions from 3 individual animals/group are presented to demonstrate the extent of abscess formation. (B) Abscess area (mm²; mean±SEM) was quantitated using the MetaMorph image analysis program by measuring the two largest lesion sizes for each tissue specimen. Significant differences in brain abscess size between S. aureus RN6390- and Reynolds-infected mice are denoted with asterisks (*, p<0.05). Results are mean of three independent experiments.

Fig. 3

Strain Reynolds induces more proinflammatory, but less anti-inflammatory mediator earlier than RN6390. Brain abscess homogenates from S. aureus strain RN6390- or Reynolds-infected animals (n=4 to 6 per group for each time point), as well as sterile bead injected mice (n=2 per group for each time point) were prepared at the indicated time points and analyzed for TNF-α (A), CXCL2 (B), IL-10 (C), and IL-12p40 (D) protein expression by ELISA. Abscess-associated cytokine levels were normalized to the amount of total protein recovered to correct for differences in tissue sampling size and reported as the mean values of cytokine (picograms) per milligram of protein. The results are the mean (±SEM) of 3 independent experiments. Significant differences between S. aureus strain RN6390- and Reynolds-infected mice are denoted with asterisks (*, p<0.05), while the pound sign represents the differences in the same group among different time points (^#, p<0.05).

Fig. 4

Reynolds induces early infiltration of PMNs into brain abscesses. Abscess-associated cells were recovered from mice (3–4 animal/group/time point) at days 1, 3 and 7 following S. aureus strain RN6390 or Reynolds infection using a Percoll gradient method and analyzed by FACS. Results are presented as the percent positive cells for each population pooled from three independent experiments (mean±SEM). Significant differences between S. aureus strain RN6390- and Reynolds-infected mice are denoted with asterisks (*, p<0.05).

Fig. 5

More T cells subsets infiltrate into brain abscesses following Reynolds infection. Abscess-associated cells were recovered from mice (3–4 animal/group/time point) at days 1, 3 and 7 following S. aureus strain RN6390 or Reynolds infection using a Percoll gradient method and analyzed by FACS. Results are presented as the percent positive cells for each population pooled from three independent experiments (mean±SEM). Significant differences between S. aureus strain RN6390- and Reynolds-infected mice are denoted with asterisks (*, p<0.05, **, p<0.001).

Fig. 6

The expression of MMP-9 is augmented earlier in Reynolds-infected mice. Mice infected with either S. aureus strain RN6390- or S. aureus strain Reynolds- encapsulated agarose beads (10⁴CFU) as well as sterile beads as described in Materials and methods, whereupon brain abscess homogenates were prepared (n=4 to 6 animals per group for each time point) at the indicated intervals and analyzed for MMP-9 mRNA and protein expression by qRT-PCR (A) and Western blotting (B), respectively. (A) MMP-9 gene expression was normalized to the house keeping gene GAPDH expression for each tissue and then normalized to that of naïve brain mRNA. The results represent the mean (±SEM) of three independent experiments. Significant differences between S. aureus strain RN6390- and Reynolds-infected mice are denoted with asterisks (*, p<0.05), while the pound sign represents the difference in the same group from other time points (^#, p<0.05). (B) Western blots were stripped and reprobed with an antibody specific for actin to verify the uniformity in gel loading. Results are presented from two or three individual mice per group for each time point and representative of three independent experiments.

Fig. 7

Reynolds infection induces significant hemolysis in brain tissue. Brain abscess homogenates from S. aureus strain RN6390- or Reynolds-infected animals (n=4 to 6 per group for each time point) as well as sterile bead injected mice (n=2 per group for each time point) were prepared at the indicated time points and analyzed for IgG (A) protein expression by ELISA and colorimetric hemoglobin (Hb) quantitation (B). Abscess-associated IgG levels were normalized to the amount of total protein recovered to correct for differences in tissue sampling size and reported as the mean values of IgG (picograms) per milligram of protein. Raw OD readings at 630 nm are used directly without any further normalization since all tissues were perfused prior to homogenization. The results represent the mean (±SEM) of three independent experiments. Significant differences between S. aureus strain RN6390- and Reynolds-infected mice are denoted with asterisks (*, p<0.05, **, p<0.001).

Fig. 8

S. aureus regulates C3a induction in experimental brain abscess. Mice infected with either agarose-laden S. aureus strain RN6390- or S. aureus strain Reynolds-beads (10⁴CFU) as described in Materials and methods, whereupon brain abscess protein extracts (40 μg per sample) were prepared at the indicated days post-infection and analyzed for C3 expression by Western blotting as described in Materials and methods. To compare with the baseline expression of C3 in the brain, naïve brain tissue was also included. Blots were stripped and reprobed with an antibody specific for actin to verify uniformity in gel loading. Results are presented from three individual animals per group and are representative of three independent experiments.

Fig. 9

S. aureus strain Reynolds induce C5 levels prominently in experimental brain abscess. Mice infected with either agarose-laden S. aureus strain RN6390- or S. aureus strain Reynolds-beads (10⁴CFU) as described in Materials and methods, whereupon brain abscess protein extracts (40 μg per sample) were prepared at the indicated days post-infection and analyzed for C5 expression by Western blotting as described in Materials and methods. To compare with the baseline expression of C5 in the brain, naïve brain tissue was also included. Blots were stripped and reprobed with an antibody specific for actin to verify uniformity in gel loading. Results are presented from two individual animals per group and are representative of three independent experiments.

Fig. 10

Fibronectin deposition along the developing brain abscess wall become prominent at day 7. Mice (n=4–6 mice per group/time point) were injected with either S. aureus strain RN6390- or S. aureus strain Reynolds- encapsulated agarose beads (10⁴CFU) as described in the Materials and methods. Animals were euthanized at the indicated time points, whereupon brain tissues were flash frozen on dry ice for subsequent cryostat sectioning. Serial 10 μm thick sections were prepared throughout the entire abscess, subjected to immunofluorescence staining for fibronectin (red), and imaged using a 40× oil immersion objective lens by confocal microscopy. Nuclei were visualized using Hoechst 33342 (blue). Fibronectin immunoreactivity is shown along the peri-abscess area in the vicinity of the developing wall, where the dark areas represent necrotic regions. Results are representative of two independent experiments.

Fig. 11

Mice (n=4–6 mice per group/time point) were injected with either S. aureus strain RN6390- or S. aureus strain Reynolds-beads (10⁴CFU) as described in the Materials and methods. Animals were euthanized at the indicated time points, whereupon brain tissues were flash frozen on dry ice for subsequent cryostat sectioning. Serial 10 μm thick sections were prepared throughout the entire abscess, subjected to immunofluorescence staining for tryptase (red), and imaged by confocal microscopy (magnification 63×). Nuclei were visualized using Hoechst 33342 (blue). Tryptase immunoreactivity was prominent at day 7 in the abscess area. Results are representative of two independent experiments.