Virulence determinants associated with the Asian community-associated methicillin-resistant Staphylococcus aureus lineage ST59

Abstract

Understanding virulence is vital for the development of novel therapeutics to target infections with community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), which cause an ongoing epidemic in the United States and are on a global rise. However, what defines virulence particularly of global CA-MRSA lineages is poorly understood. Threatening a vast population, the predominant Asian CA-MRSA lineage ST59 is of major epidemiological importance. However, there have been no molecular analyses using defined virulence gene deletion mutants in that lineage as of yet. Here, we compared virulence in skin, lung, and blood infection models of ST59 CA-MRSA isolates with geographically matched hospital-associated MRSA isolates. We selected a representative ST59 CA-MRSA isolate based on toxin expression and virulence characteristics, and produced isogenic gene deletion mutants of important CA-MRSA virulence determinants (α-toxin, PSM α, Agr) in that isolate for in-vitro and in-vivo analyses. Our results demonstrate strongly enhanced virulence of ST59 CA-MRSA over hospital-associated lineages, supporting the notion that enhanced virulence is characteristic for CA-MRSA. Furthermore, they show strong and significant contribution of Agr, α-toxin, and PSMα to pathogenesis of ST59 CA-MRSA skin, lung, and blood infection, emphasizing the value of drug development efforts targeted toward those virulence determinants.

Figure 1. Selection of representative strains by determination of PSMα3 and δ-toxin production levels.

(a) Relative PSMα3 and δ-toxin amounts in all ST59 CA-MRSA, ST5 HA-MRSA, and ST239 HA-MRSA isolates from skin and lung infections obtained from Renji hospital in 2014. Isolates selected for further study are shown with filled symbols. The peptide toxins were measured in cultures grown in TSB for 8 h by reversed-phase high-pressure chromatography/ion spray mass spectrometry. Comparisons are by 1-way ANOVA with Dunnett’s post-test versus ST59. ****p < 0.0001. Error bars show the standard error of the mean (±SEM). (b) Growth curves in TSB of all selected strains.

Figure 2. Comparison of ST59 CA-MRSA strains with geographically matched HA-MRSA and reference strains in an abscess infection model.

(a) Mice were injected subcutaneously with ~10⁷ CFU of 8 selected, representative isolates each of ST59, ST5 and ST239; 3 U.S. ST59 SSTI isolates (one mouse per isolate) and abscess areas were measured at day 2 after infection. Control animals (n = 3) received only sterile PBS. Values for USA300 are based on two mice infected with strain LAC. The data point corresponding to the strain selected for deletion mutant construction is represented in orange color. Comparisons are by 1-way ANOVA with Dunnett’s post-test versus ST59. *p < 0.05; ****p < 0.0001. Error bars show ±SEM. (b) Representative abscesses and histological results (H&E stain). Scale bars are 50 μm. Note pronounced infiltration of leukocytes (purple) in ST59 and USA300-infected tissue. The skin surface is located toward the left side of the pictures.

Figure 3. Comparison of ST59 CA-MRSA strains with geographically matched HA-MRSA and reference strains in a lung infection model.

(a,b) 4 × 10⁹ CFU was pipetted into the nares of mice (4 selected, representative isolates each of ST59, ST5 and ST239; Values for USA300 are based on three mice infected with strain LAC). Control animals (n = 3) received only sterile PBS. Lung weight, body weight (a), and CFU (b) were measured after euthanizing mice 48 h after infection. Comparisons are by 1-way ANOVA with Dunnett’s post-test versus ST59. *p < 0.05; **p < 0.01; ****p < 0.0001. Error bars show ±SEM. (c) Representative lungs and histological results (H&E stain) from infected animals. Scale bars are 50 μm. Note lung enlargement and hyperemia, infiltration of erythrocytes (orange) and leukocytes (purple), in ST59 and USA300-infected lungs.

Figure 4. Expression of psmα operon (encoding PSMα peptides), hla (α-toxin), and agr.

Expression levels were determined by qRT-PCR at stationary growth phase (8 h) during growth in TSB. Rel. exp., relative expression. ***p < 0.001; ****p < 0.0001. Comparisons are between all SSTI strains (ST59, ST5, ST239, USA300, ST59 U.S.) or between all lung infection isolates (ST59, ST5, ST239). *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (1-way ANOVA with Dunnett’s post-test vs. ST59). NS, not significant (p ≥ 0.05). Error bars show ±SEM.

Figure 5. Agr controls hla and psmα expression in ST59 CA-MRSA.

Shown are qRT-PCR measurements at 4 h of growth in TSB of wild-type ST59 CA-MRSA and isogenic hla and psmα deletion mutants. Comparisons are by unpaired t-tests. **p < 0.01; ***p < 0.001. Error bars show ±SEM.

Figure 6. PSMα, α-toxin and Agr impact the capacity of ST59 CA-MRSA to lyse human red and white blood cells.

(a) Lysis of human neutrophils by bacteria. Bacteria were grown to late logarithmic growth phase and neutrophils were incubated at a 10:1 ratio. (b) Lysis of human erythrocytes by culture filtrates of bacterial cultures at increasing dilutions. (a,b) *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; for two groups comparisons are by unpaired t-tests (for comparison of the plasmid-based expression versus with the corresponding strain harboring plasmid control); for more groups comparisons are by 1-way or 2-way ANOVA with Dunnett’s post-test. In (b), only statistics for the 1:1 dilution are shown, but differences were significant down to a dilution of 1:20 (for Δhla and Δpsmα vs. WT) or 1:40 (for Δpsmα pTX_Δpsmα vs. Δpsmα pTX_Δ16). (a,b) Error bars show ±SEM.

Figure 7. Impact of psmα, hla and agr on virulence of ST59 CA-MRSA in skin infection.

(a) Skin infection model. Ten mice per group were injected subcutaneously with ~10⁷ CFU and abscess areas were measured at day 2 after infection. ****p < 0.0001 (1-way ANOVA with Dunnett’s post-test vs. WT). Error bars show the standard error of the mean (SEM). (b) Representative abscesses.

Figure 8. Impact of psmα, hla and agr on virulence of ST59 CA-MRSA in blood infection.

(a) Ten mice per group were injected intro the retro-orbital vein with ~10⁷ CFU and CFU in kidney tissue were measured at day 4 after infection. ****p < 0.0001 (1-way ANOVA with Dunnett’s post-test vs. WT). Error bars show ±SEM. (b,c) Histological evaluation of kidneys and livers. Pictures show representative microscopic images of kidney and liver tissue at lower (left, scale bar 200 μm) and higher (right, scale bar 50 μm) magnification. In kidney samples, note abscess formation (arrow top left) and excessive infiltration of inflammatory cells (arrow top right) in mice infected with the WT strain, while these were largely absent from control mice and mice infected with the isogenic agr, hla, and psmα mutant strains. In liver samples, note necrosis and excessive infiltration of inflammatory cells of mice infected with the WT strain, while these were largely absent from control mice and mice infected with the mutant strains.

Figure 9. Impact of psmα, hla and agr deletion on virulence of ST59 CA-MRSA in lung infection.

(a,b) 4 × 10⁹ CFU was pipetted into the nares of mice (n = 7). Control animals (n = 3) received only PBS. One mouse in the WT group died 24 h after infection. The lung weight, body weight, and CFU of all other mice were measured after euthanizing the mice 48 h after infection. *p < 0.05; **p < 0.01; ****p < 0.0001 (1-way ANOVA with Dunnett’s post-test vs. WT). Error bars show ±SEM. (c) Macroscopic and histological (H&E stain) examination of lungs from representative infected animals. Scale bars are 50 μm. Note the reduction of enlargement and hyperemia, and infiltration of erythrocytes (orange) and leukocytes (purple), in the mutant versus WT-infected lungs.