Virulence gene expression in human community-acquired Staphylococcus aureus infection

Abstract

Isolates of methicillin-resistant Staphylococcus aureus (MRSA) were once linked uniformly with hospital-associated infections; however, community-acquired MRSA (CA-MRSA) now represents an emerging threat worldwide. To examine the association of differential virulence gene expression with outcomes of human infection, we measured transcript levels of target staphylococcal genes directly in clinical samples from children with active known or suspected CA-MRSA infections. Virulence genes encoding secreted toxins, including Panton-Valentine leukocidin, were highly expressed during superficial and invasive CA-MRSA infections. In contrast, increased expression of surface-associated protein A was linked only with invasive disease. Comparisons with laboratory-grown corresponding clinical isolates revealed that tissue-specific expression profiles reflect the activity of the staphylococcal accessory gene regulator during human infection. These results represent the first demonstration of staphylococcal gene expression and regulation directly in human tissue. Such analysis will help to unravel the complex interactions between CA-MRSA and its host environmental niches during disease development.

Figure 1. Expression of virulence genes in human cutaneous infection

Expression of putative virulence genes during growth in human tissue and in laboratory broth culture was determined by TaqMan real-time PCR. Data represent the fold-change in normalized transcript abundance for each gene in material collected from a cutaneous abscess relative to the corresponding S. aureus strain grown aerobically to early stationary phase in tryptic soy broth. Data for each of the 40 strains described in Table 1 are represented, and the target gene is specified below the panel; MRSA () and MSSA () strains are shown, and the geometric mean for each gene is indicated with a bar. Error bars were generally obscured by the symbol and were omitted for clarity. Differences in transcript abundance between these two groups were not statistically significant. The difference between the cutaneous samples and the stationary samples were statistically significant (P<0.01) for all genes except spa.

Figure 2. Comparison of gene expression profiles between bacteria taken from human cutaneous infection, stationary phase in vitro culture, and in vitro PMN encounter

Expression of putative virulence genes during growth in human tissue and in vitro was determined by TaqMan real-time PCR. (A) Data represent the fold-change in transcript abundance for each gene in a subset of the S. aureus clinical isolates shown in Figure 1 grown aerobically to early stationary phase in tryptic soy broth () relative to abundance in bacteria grown aerobically to mid-exponential phase in tryptic soy broth. Ten representative strains were chosen for this analysis, and target genes are specified below each panel. The geometric mean for each gene is indicated with a bar. The difference between the stationary sample and the exponential sample was statistically significant (P<0.002) for all genes. (B) The log-transformed average fold-change of target gene expression for the strains shown in panel A grown to stationary phase (x-axis) or in a human cutaneous abscess (y-axis) relative to mid-exponential phase expression is shown. There is a significant correlation (R=0.89; P<0.003) between the tissue and stationary-phase expression profiles. (C) The cutaneous abscess expression [; Abscess] or expression in the corresponding strain during in vitro encounter with human PMN (▼; PMN) is shown relative to expression of each gene in a media-only control condition (RPMI; see Materials and Methods for details). Five representative strains were chosen for this analysis, and the geometric mean for each gene is indicated with a bar. The majority of the genes showed a similar direction of differential regulation (denoted by asterisks), but there was a poor correlation (R=0.44) between the tissue and PMN expression profiles.

Figure 3. Expression of virulence genes during invasive human infection

Expression of putative virulence genes during growth in human tissue and in vitro was determined by TaqMan real-time PCR. (A) Data represent the fold-change in transcript abundance for each gene in material collected from an invasive human subperiosteal abscess (striped bars) or the corresponding S. aureus strain grown aerobically to early stationary phase in tryptic soy broth (solid bars) relative to the strain grown aerobically to mid-exponential phase in tryptic soy broth. For each target gene indicated below the panel, each of three strains is represented by a different shaded bar, and an asterisk indicates that the gene is not present in that strain. The difference between the invasive abscess samples and the stationary phase samples was statistically significant (P<0.05) for all genes except RNAIII. (B) The invasive abscess expression (striped bars) or expression of the corresponding strain during an in vitro encounter with human PMN (solid bars) is shown relative to expression of each gene in a media-only control condition (RPMI; see Materials and Methods for details). Axis and symbols are as described for panel A. As indicated in the text there was a significant correlation between the invasive abscess profile and the PMN profile for all three strains (R=0.86, 0.79, 0.90; P<0.01).

Figure 4

A schematic representation of the relationship between growth phase and disease progression with regard to virulence gene expression is shown. The paradigm of regulation in broth culture is well established; our data provide in vivo support for a proposed correspondence [2] between this in vitro paradigm and the regulation of virulence factor expression within the human host.