Surface layer proteins from virulent Clostridium difficile ribotypes exhibit signatures of positive selection with consequences for innate immune response.
Clostridium difficile is a nosocomial pathogen prevalent in hospitals worldwide and increasingly common in the community. Sequence differences have been shown to be present in the Surface Layer Proteins (SLPs) from different C. difficile ribotypes (RT) however whether these differences influence severity of infection is still not clear.



We used a molecular evolutionary approach to analyse SLPs from twenty-six C. difficile RTs representing different slpA sequences. We demonstrate that SLPs from RT 027 and 078 exhibit evidence of positive selection (PS). We compared the effect of these SLPs to those purified from RT 001 and 014, which did not exhibit PS, and demonstrate that the presence of sites under positive selection correlates with ability to activate macrophages. SLPs from RTs 027 and 078 induced a more potent response in macrophages, with increased levels of IL-6, IL-12p40, IL-10, MIP-1α, MIP-2 production relative to RT 001 and 014. Furthermore, RTs 027 and 078 induced higher expression of CD40, CD80 and MHC II on macrophages with decreased ability to phagocytose relative to LPS.



These results tightly link sequence differences in C. difficile SLPs to disease susceptibility and severity, and suggest that positively selected sites in the SLPs may play a role in driving the emergence of hyper-virulent strains.
Immunomodulation Research Group, School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
Mark
M
Lynch
Bioinformatics and Molecular Evolution Group, School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
Thomas A
TA
Walsh
Immunomodulation Research Group, School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
Izabela
I
Marszalowska
Bioinformatics and Molecular Evolution Group, School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
Andrew E
AE
Webb
Department of Clinical Microbiology, Trinity College Dublin, St James Hospital Dublin, Dublin, Ireland.
Micheál
M
Mac Aogain
Department of Clinical Microbiology, Trinity College Dublin, St James Hospital Dublin, Dublin, Ireland.
Thomas R
TR
Rogers
Institute of Molecular Medicine, Trinity College Dublin, Dublin, Ireland.
Henry
H
Windle
Faculty of Medicine, Imperial College London, London, SW7 2AZ, UK.
Dermot
D
Kelleher
Bioinformatics and Molecular Evolution Group, School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland. m.oconnell@leeds.ac.uk.|Computational and Molecular Evolutionary Biology Research Group, School of Biology, Faculty of Biological Sciences, The University of Leeds, Leeds, LS2 9JT, UK. m.oconnell@leeds.ac.uk.
Mary J
MJ
O'Connell
Immunomodulation Research Group, School of Biotechnology, Dublin City University, Glasnevin, Dublin 9, Ireland.
Christine E
CE
Loscher
D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D016360:Clostridioides difficile / Q000145:classification / Q000276:immunology / Q000472:pathogenicity; D003015:Clostridium Infections / Q000276:immunology* / Q000382:microbiology; D006801:Humans; D007113:Immunity, Innate; D008264:Macrophages / Q000276:immunology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D010587:Phagocytosis; D010802:Phylogeny; D021521:Ribotyping
D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; C055764:S-layer proteins
Clostridium difficile; Positive selection; Protein evolution; Protein functional shift; Surface layer protein; Virulence