Proteomic analysis at the sites of clinical infection with invasive Streptococcus pyogenes

Abstract

Invasive Streptococcus pyogenes infections are rare, with often-unexplained severity. Prompt diagnosis is desirable, as deaths can occur rapidly following onset and there is an increased, but preventable, risk to contacts. Here, proteomic analyses of clinical samples from invasive human S. pyogenes infections were undertaken to determine if novel diagnostic targets could be detected, and to augment our understanding of disease pathogenesis. Fluid samples from 17 patients with confirmed invasive S. pyogenes infection (empyema, septic arthritis, necrotising fasciitis) were analysed by proteomics for streptococcal and human proteins; 16/17 samples had detectable S. pyogenes DNA. Nineteen unique S. pyogenes proteins were identified in just 6/17 samples, and 15 of these were found in a single pleural fluid sample including streptococcal inhibitor of complement, trigger factor, and phosphoglycerate kinase. In contrast, 469 human proteins were detected in patient fluids, 177 (38%) of which could be identified as neutrophil proteins, including alpha enolase and lactotransferrin which, together, were found in all 17 samples. Our data suggest that streptococcal proteins are difficult to detect in infected fluid samples. A vast array of human proteins associated with leukocyte activity are, however, present in samples that deserve further evaluation as potential biomarkers of infection.

Figure 1

SDS-polyacrylamide gel separation of clinical fluid sample proteins. Clinical fluids F1–F17 were separated by electrophoresis along with a molecular weight ladder (MWL). Following staining with coomassie blue each lane of the gel was cut into 22 sections, as indicated by the arrows on the lefthand side, in preparation for proteomic analysis as described in the Methods section. For the purpose of clarity the image shown has been cropped to show the lanes corresponding to the clinical fluid samples. The uncropped image is shown as Supplementary Figure S1.

Figure 2

Immunoblotting of clinical fluid samples with CTAbs. The expression of PGK, enolase, SIC, and SpeB in fluid samples in clinical fluid samples was confirmed using antibodies against each of these antigens. Clinical fluid samples F2 and F8 along with corresponding cultured supernatant samples, S2 and S8, were prepared and analysed as described in the Methods section along with purified preparation of SIC and SpeB. The blots were developed with both a CTAb antiserum and corresponding pre-immune serum in each case to ensure that overdevelopment was avoided and to provide evidence that the observed immunoreactivity was antigen-specific. Blots developed using different antibodies are shown as clearly separate panels surrounded by white space; full length images of relevant lanes are shown.

Figure 3

Comparative composition of human proteins in clinical fluid samples. Human proteins in each of the fluid samples were categorised as originating from neutrophils, plasma or neither of these sources (other). Categories were based on previous proteomic identifications and do not indicate that such proteins are limited to these sources. (a) The total number of proteins and the contribution from each of these groups is indicated. The order of the fluids have been arranged to highlight the variation in the number neutrophil proteins detected. (b–d) Pie charts showing the relative number of commonly found proteins in each category. Each figure represents groups of the number of proteins found to be common amongst the 17 fluids, with values ranging from 1 (proteins found in just one fluid sample) to 17 (proteins found in all 17 fluid samples).

Figure 4

Distributions of the most commonly found human proteins in clinical fluid samples. Each coloured cell represents the presence of the protein indicated in clinical fluid samples F1–F17 (ordered so that those fluids with the greatest number of neutrophil proteins are to the left). The nature of the infection is indicated as necrotising fasciitis (NF), septic arthritis (SA), or empyema (E). Proteins identified in the clinical fluid samples were classified as either from neutrophil, plasma, or neither of these groups. Note that classifications do not indicate that such proteins are limited to these sources. Some proteins appeared with a lower mass than expected and these are indicated (yellow). Within each group the proteins are ordered with those proteins represented most frequently in the fluid samples placed at the top.