Ulrika Ringdahl

Identification of a plasminogen‐binding motif in PAM, a bacterial surface protein

Surface‐associated plasmin(ogen) may contribute to the invasive properties of various cells. Analysis of plasmin(ogen)‐binding surface proteins is therefore of interest. The N‐terminal variable regions of M‐like (ML) proteins from five different group A streptococcal serotypes (33,41,52,53 and 56) exhibiting the plasminogen‐binding phenotype were cloned and expressed in Escherichia coli. The recombinant proteins all bound plasminogen with high affinity. The binding involved the kringle domains of plasminogen and was blocked by a lysine analogue, 6‐aminohexanoic acid, indicating that lysine residues in the M‐like proteins participate in the interaction. Sequence analysis revealed that the proteins contain common 13–16‐amino‐acid tandem repeats, each with a single central lysine residue. Experiments with fusion proteins and a 30‐amino‐acid synthetic peptide demonstrated that these repeats harbour the major plasminogen‐binding site in the ML53 protein, as well as a binding site for the tissue‐type plasminogen activator. Replacement of the lysine in the first repeat with alanine reduced the plasminogen‐binding capacity of the ML53 protein by 80%. The results precisely localize the binding domain in a plasminogen surface receptor, thereby providing a unique ligand for the analysis of interactions between kringles and proteins with internal kringle‐binding determinants.

A role for the fibrinogen-binding regions of streptococcal M proteins in phagocytosis resistance

All virulent group A streptococcal isolates bind fibrinogen, a property that is closely linked to expression of type‐specific antiphagocytic surface molecules designated M proteins. Here we show that although the M proteins from two different strains, M1 and M5, both bind fibrinogen with high affinity, they interact with different regions in the ligand. Moreover, mapping experiments demonstrated that the fibrinogen‐binding regions in the M1 and M5 proteins are quite dissimilar at the amino acid sequence level and that they bind to different regions in the plasma protein. In spite of these differences, the fibrinogen‐binding regions of M1 and M5 could both be shown to contribute to streptococcal survival in human blood, providing evidence for the distinct function of a plasma protein interaction in bacterial pathogenesis.

Plasminogen enhances virulence of group A streptococci by streptokinase-dependent and streptokinase-independent mechanisms

Interactions between host plasminogen (Plg) and streptokinase (SK) secreted by group A streptococci (GAS) have been hypothesized to promote bacterial invasion of tissues. The virulence of GAS strain UMAA2616, after being subcutaneously inoculated into mice, was studied. Skin lesions and mortality were observed after inoculation of 7x106 cfu. Coadministration of human Plg with UMAA2616 markedly increased virulence. SK-deficient UMAA2616 (UMAA2616-SK(-)) was generated. Mean skin-lesion area and mortality, after bacterial inoculation (3x105 cfu), were significantly greater with UMAA2616 in the presence of human Plg than with UMAA2616-SK(-) in the presence of human Plg (P=.0001). Human Plg also enhanced UMAA2616-SK(-) virulence. Exogenous human Plg enhanced the virulence of MGAS166, a human clinical isolate. These findings suggest that SK-Plg interactions are an important determinant of GAS invasiveness in vivo and that both SK and host Plg activators appear to promote virulence of GAS by catalyzing plasmin formation.

Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation

A common mammalian defense mechanism employed to prevent systemic dissemination of invasive bacteria involves occlusion of local microvasculature and encapsulation of bacteria within fibrin networks. Acquisition of plasmin activity at the bacterial cell surface circumvents this defense mechanism, allowing invasive disease initiation. To facilitate this process, S. pyogenes secretes streptokinase, a plasminogen‐activating protein. Streptokinase polymorphism exhibited by S. pyogenes isolates is well characterized. However, the functional differences displayed by these variants and the biological significance of this variation has not been elucidated. Phylogenetic analysis of ska sequences from 28 S. pyogenes isolates revealed 2 main sequence clusters (clusters 1 and 2). All strains secreted streptokinase, as determined by Western blotting, and were capable of acquiring cell surface plasmin activity after incubation in human plasma. Whereas culture supernatants from strains containing cluster 1 ska alleles also displayed soluble plasminogen activation activity, supernatants from strains containing cluster 2 ska alleles did not. Furthermore, plasminogen activation activity in culture supernatants from strains containing cluster 2 ska alleles could only be detected when plasminogen was prebound with fibrinogen. This study indicates that variant streptokinase proteins secreted by S. pyogenes isolates display differing plasminogen activation characteristics and may therefore play distinct roles in disease pathogenesis.—McArthur, J. D., McKay, F. C., Ramachandran, V., Shyam, P., Cork, A. J., Sanderson‐Smith, M. L., Cole, J. N., Ringdahl, U., Sjöbring, U., Ranson, M., Walker, M. J. Allelic variants of streptokinase from Streptococcus pyogenes display functional differences in plasminogen activation. FASEB J. 22, 3146–3153 (2008)

Modelling the benefits of early diagnosis of pancreatic cancer using a biomarker signature.

Pancreatic cancer (PC) has a poor prognosis, with a 5‐year survival of 3–4%. This is mainly due to late diagnosis because of diffuse symptoms, where 80–85% of the patients are inoperable. Consequently, early diagnosis would be of significant benefit, resulting in a potential 5‐year survival of 30–40%. However, new technologies must be carefully evaluated concerning effectiveness and healthcare costs. We have developed a framework for modelling cost and health effects from early detection of PC, which for the first time allowed us to analyse its cost‐effectiveness. A probabilistic cohort model for estimating costs and quality adjusted life‐years (QALY) arising from screening for PC, compared to a “wait‐and‐see”‐approach, was designed. The test accuracy, Swedish survival and costs by tumour stage, expected life gain from early detection and pretest probabilities in risk groups, were retrieved from previous investigations. In a cohort of newly diagnosed diabetic patient (incidence 0.71%) the incremental cost per QALY gained (ICER) was €13,500, which is considered cost‐effective in Europe. Results were mainly sensitive to the incidence with the ICER ranging from €315 to €204,000 (familial PC 35% and general population 0.046%, respectively). This is the first study focusing on clinical implementation of advanced testing and what is required for novel technologies in cancer care to be cost‐effective. The model clearly demonstrated the potential of multiplexed proteomic‐testing of PC and also identified the requirements for test accuracy. Consequently, it can serve as a model for assessing the possibilities to introduce advanced test platforms also for other cancer indications.

Plasma protein profiling in a stage defined pancreatic cancer cohort – Implications for early diagnosis

Pancreatic ductal adenocarcinoma (PDAC) is a disease where detection preceding clinical symptoms significantly increases the life expectancy of patients. In this study, a recombinant antibody microarray platform was used to analyze 213 Chinese plasma samples from PDAC patients and normal control (NC) individuals. The cohort was stratified according to disease stage, i.e. resectable disease (stage I/II), locally advanced (stage III) and metastatic disease (stage IV). Support vector machine analysis showed that all PDAC stages could be discriminated from controls and that the accuracy increased with disease progression, from stage I to IV. Patients with stage I/II PDAC could be discriminated from NC with high accuracy based on a plasma protein signature, indicating a possibility for early diagnosis and increased detection rate of surgically resectable tumors.