EuroPneumo Special Issue / pneumonia 2015 Oct 21;7:I–72


Binding activities and anti-phagocytic properties of PclA, the

pneumococcal collagen-like protein A

Sandra Koch 1 , Elena Fuchs 1 , Tim J. Mitchell 2 , Andrea M. Mitchell 2 , Marcus Fulde 3 , Michael

Steinert 1 , Simone Bergmann 1


Technische Universität Braunschweig, Braunschweig, Germany; 2 University of Birmingham, Birmingham, UK, 3 University of Veterinary Medicine

Hannover, Hannover, Germany

The pneumococcal collagen-like protein A (PclA) is covalently linked to the surface of Streptococcus pneumoniae via

an LPxTG motif and harbours a variable number of repetitive amino acid sequences with high homologies to human

collagen. PclA is expressed in up to 45% of tested pneumococcal isolates and mediates pneumococcal adherence to

nasopharyngeal cells [1]. Molecular analyses indicated that the collagen-like sequence repeats are clustered in up to

3 collagen-like domains spanning a maximum of 6 repeated amino acid sequences per domain. The total number of

collagen-like repeats of PclA ranges from 2 repeats in a serotype 19F isolate, to up to 10 repeats in total in serotype 2

isolates, e.g. R6. Consequently, the molecular weight of PclA varies between 187 kDa and 289 kDa depending on the

numbers of collagen-like repeats. The effect of PclA on phagocytic neutralisation was analysed in phagocytic killing

assays with macrophage-like U937 cells and with neutrophils prepared from blood of two different human donors.

Phagocytosis of a pclA-deficient mutant by U937 cells was similar to the corresponding wild type strain. In contrast,

depending on the donor, the prepared neutrophils were less efficient in phagocytosis of pclA-deficient pneumococcal

mutants. Furthermore, we demonstrate for the first time specific binding activity of the collagen-like domain 1 of S.

pneumoniae strain D39 to human plasma fibronectin and von Willebrand factor. The interaction of the PclA domain with

host proteins indicates an important contribution of PclA to pneumococcal colonisation and virulence.

1. Paterson GK, Nieminen L, Jefferies JM, Mitchell TJ. PclA, a pneumococcal collagen-like protein with selected strain distribution,

contributes to adherence and invasion of host cells. FEMS Microbiol Lett 2008;285:170–6. PMID:18557785 http://dx.doi.



Delineating the interaction of pneumococcal surface proteins with the

human adhesive glycoprotein fibronectin

Sajida Kanwal 1 , Inga Jensch 1 , Thomas P. Kohler 1 , Roland Frank 2 , Werner Tegge 2 , Mark Brönstrup 2 ,

Sven Hammerschmidt 1


Department Genetics of Microorganisms, Interfaculty Institute for Genetics and Functional Genomics, Ernst Moritz Arndt University of Greifswald,

Greifswald, Germany; 2 Department of Chemical Biology, Helmholtz Centre for Infection Research, Braunschweig, Germany

The virulence potential of Streptococcus pneumoniae is closely associated with its expression and exposure of a

diverse repertoire of colonising and virulence factors. Several of these factors belong to microbial surface components

recognising adhesive matrix molecules (MSCRAMMs), which exploit eukaryotic host matricellular proteins such as

fibronectin, vitronectin, thrombospondin, collagen, or plasminogen as a molecular mediator to interact with host

cells. Pneumococcal adherence and virulence factor A (PavA) and B (PavB) are fibronectin-binding proteins (FnBPs),

representing a sub-class of MSCRAMMs. PavB is covalently anchored to the pneumococcal cell wall by sortase A,

whereas the PavA protein is a member of the so-called non-classical surface proteins (NCSPs) with an ambiguous

unknown mechanism of secretion and surface-exposure. In this study direct protein–protein interaction approaches have

been used to delineate pneumococcal PavA and PavB interactions with the human glycoprotein fibronectin (Fn). Flow

cytometric analysis showed that S. pneumoniae recruits fibronectin from human plasma in a dose-dependent manner.

Pneumococci interact with the C-terminal part of Fn, while other Gram-positive bacteria bind to the N-terminal Fn type

I repeats. In order to assess the underlying molecular mechanism of these PavA/PavB–fibronectin interactions, different

Fn fragments and type repeats were used in far western blots and surface plasmon resonance (SPR) studies. Far western

blots demonstrated binding of PavA and PavB to C-terminally located type III repeats. The interaction between type III

repeats and PavA or PavB was confirmed in our SPR analysis. Among the Fn type III repeats, Fn type III12-14 (heparin

binding domain) bound with highest affinity to PavA and PavB. In addition, peptide arrays are used to identify crucial

binding motifs in Fn type III repeats. This study shows that FNBPs preferentially bind to type III repeats of Fn molecules

pneumonia 2015 Volume 7


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