Proteomic biomarkers associated with Streptococcus agalactiae invasive genogroups

Abstract

Group B streptococcus (GBS, Streptococcus agalactiae) is a leading cause of meningitis and sepsis in newborns and an etiological agent of meningitis, endocarditis, osteoarticular and soft tissue infections in adults. GBS isolates are routinely clustered in serotypes and in genotypes. At present one GBS sequence type (i.e. ST17) is considered to be closely associated with bacterial invasiveness and novel proteomic biomarkers could make a valuable contribution to currently available GBS typing data. For that purpose we analyzed the protein profiles of 170 genotyped GBS isolates by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI). Univariate statistical analysis of the SELDI profiles identified four protein biomarkers significantly discriminating ST17 isolates from those of the other sequence types. Two of these biomarkers (MW of 7878 Da and 12200 Da) were overexpressed and the other two (MW of 6258 Da and 10463 Da) were underexpressed in ST17. The four proteins were isolated by mass spectrometry-assisted purification and their tryptic peptides analyzed by LC-MS/MS. They were thereby identified as the small subunit of exodeoxyribonuclease VII, the 50S ribosomal protein L7/L12, a CsbD-like protein and thioredoxin, respectively. In conclusion, we identified four candidate biomarkers of ST17 by SELDI for high-throughput screening. These markers may serve as a basis for further studies on the pathophysiology of GBS infection, and for the development of novel vaccines.

Figure 1. Heatmap/hierarchical analysis of 164 protein clusters of 170 S. agalactiae isolates divided into five groups according to origin/clinical outcome: bovine mastitis (n = 23), endocarditis (n = 15), meningitis (n = 54), vaginal carriage (n = 54), and respiratory tract infections (n = 24).

The clusters were obtained by combining the results of the ProteinChip array conditions CM10 and Q10. The five isolate group names (upper line, in bold) and the 170 individual isolate names (lower line) are indicated above the image; the protein masses as detected on CM10 (red) and Q10 (blue) ProteinChip arrays are presented.

Figure 2. Expression levels of four biomarker proteins produced by S. agalactiae isolates clustered into five groups according to origin/clinical outcome.

X - axis: bovine mastitis (red), endocarditis (blue), meningitis (green), respiratory infections (magenta), vaginal carriage (cyan). Y- axis: protein abundance expressed as absolute intensity (µA/laser pulse). Each point represents the mean intensity of one sample tested in duplicate. P-values obtained with the Kruskal-Wallis H test: p6258 -<1.10⁻⁶; p7878 - 0.000059; p10464 - 0.09447; p12200 - 0.000004.

Figure 3. Expression levels of p6258, p7878, p10464 and p12200 in 149 S. agalactiae isolates genotyped by multilocus sequence typing (MLST) and represented as a dendrogram showing genetic relationships among the different sequence types (STs).

The dendrogram, based on MLST data, was constructed using BioNumerics 6.5 software (Applied Maths, Sint-Martens-Latem, Belgium). Cluster analysis was based on an unweighted pair group method using arithmetic averages (UPGMA). The nine main MLST groups (A to I) are indicated on the right of the dendrogram as vertical dotted lines. The allelic profiles corresponding to each sequence type (ST), the number of isolates belonging to each ST, the origin of the isolate, the serotype and the presence (+) or absence (−) of the biomarkers of interest are reported in detail. Within each ST, the presence of the biomarker is indicated as a ratio between the number of isolates with a detectable corresponding peak in SELDI and the number of all isolates in the ST. A grey scale also illustrates the prevalence of the biomarker (dark grey when the peak corresponding to the biomarker is detected in all isolates; light grey when the peak corresponding to the biomarker is partially detected).