Differential expression and roles of Staphylococcus aureus virulence determinants during colonization and disease

Abstract

Staphylococcus aureus is a Gram-positive, commensal bacterium known to asymptomatically colonize the human skin, nares, and gastrointestinal tract. Colonized individuals are at increased risk for developing S. aureus infections, which range from mild skin and soft tissue infections to more severe diseases, such as endocarditis, bacteremia, sepsis, and osteomyelitis. Different virulence factors are required for S. aureus to infect different body sites. In this study, virulence gene expression was analyzed in two S. aureus isolates during nasal colonization, bacteremia and in the heart during sepsis. These models were chosen to represent the stepwise progression of S. aureus from an asymptomatic colonizer to an invasive pathogen. Expression of 23 putative S. aureus virulence determinants, representing protein and carbohydrate adhesins, secreted toxins, and proteins involved in metal cation acquisition and immune evasion were analyzed. Consistent upregulation of sdrC, fnbA, fhuD, sstD, and hla was observed in the shift between colonization and invasive pathogen, suggesting a prominent role for these genes in staphylococcal pathogenesis. Finally, gene expression data were correlated to the roles of the genes in pathogenesis by using knockout mutants in the animal models. These results provide insights into how S. aureus modifies virulence gene expression between commensal and invasive pathogens.

Importance: Many bacteria, such as Staphylococcus aureus, asymptomatically colonize human skin and nasal passages but can also cause invasive diseases, such as bacteremia, pneumonia, sepsis, and osteomyelitis. The goal of this study was to analyze differences in the expression of selected S. aureus genes during a commensal lifestyle and as an invasive pathogen to gain insight into the commensal-to-pathogen transition and how a bacterial pathogen adapts to different environments within a host (e.g., from nasal colonization to invasive pathogen). The gene expression data were also used to select genes for which to construct knockout mutants to assess the role of several proteins in nasal colonization and lethal bacteremia. These results not only provide insight into the factors involved in S. aureus disease pathogenesis but also provide potential therapeutic targets.

FIG 1

Protein and carbohydrate adhesin genes in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression in vitro (A) and the maximum expression in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P  < 0.05 (Student’s t test). clfB expression was not detected in vitro, and so calculations for clfB expression were based on an in vitro threshold cycle C_T value equal to the limit of detection (40 cycles).

FIG 2

Metal cation acquisition gene expression in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression level in vitro (A) and the maximum expression level in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P  < 0.05 (Student’s t test).

FIG 3

Immune evasion and exotoxin gene expression in a cotton rat nasal colonization model. Transcripts were assessed relative to 16S rRNA, and results from nasal colonization were compared to the minimum expression level in vitro (A) and the maximum expression level in vitro (B). In vitro growth conditions are described in Table S2 in the supplemental material. Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 12 animals/experiment. *, P < 0.05 (Student’s t test).

FIG 4

Differential gene expression between nasal colonization and early bacteremia. Transcripts were assessed relative to 16S rRNA, and results from the bloodstream were compared to the expression results from nasal colonization for protein and carbohydrate adhesins (A), metal cation acquisition genes (B), and immune evasion genes and exotoxins (C). Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 10 animals/experiment. *, P < 0.05 (Student’s t test). clfA, clfB, and sasG expression was not detected in the blood samples, and so calculations for clfA, clfB, and sasG expression were based on the threshold cycle (C_T) value equal to the limit of detection (40 cycles).

FIG 5

Differential gene expression in the transition from the bloodstream to heart tissue. Transcripts were assessed relative to the 16S rRNA, and results from the heart tissue were compared to the expression results from the bloodstream for protein and carbohydrate adhesins (A), metal cation acquisition genes (B), and immune evasion genes and exotoxins (C). Analysis was performed for two S. aureus strains, SF8300 (black bars) and ARC633 (blue bars). These results are the means of three independent experiments with 10 animals/experiment. *, P < 0.05 (Student’s t test). clfB and sasG expression was not detected in the blood samples or the heart samples; therefore, expression numbers are not reported for these genes.

FIG 6

CFU enumeration of SF8300 wild-type and mutant strains in a cotton rat nasal colonization model. Groups of 12 cotton rats were challenged intranasally with 5 × 10⁵ CFU S. aureus in 10 µl (5 µl per nostril). For ΔsdrC ΔclfB, P =0.0047 (Mann-Whitney test). Data shown are from a single replicate of three independent experiments.

FIG 7

CFU enumeration of SF8300 wild-type and mutant strains in a murine thromboembolic lesion model. Groups of 10 female BALB/c mice were challenged intravenously (i.v.) with 200 µl of S. aureus (5 × 10⁷ CFU) by tail vein injection. P values were as follows: Δhla, 0.0030; ΔclfA, <0.0001; ΔsdrC, 0.0050; ΔisdB, <0.0001 (Mann-Whitney test). Data shown are from a single replicate of three independent experiments.