The incidence of antibiotic resistance is still increasing and poses serious limitations to antibacterial therapeutic strategies. The search for new classes of antibiotics is difficult and in principle there is a great risk that a new antibiotic will also be prone for resistance. A set-back of antibiotic therapy is that commensal organisms are also affected which makes the patient more vulnerable to secondary infections. Moreover, most bacteria reside in biofilms which are intrinsically more resistant to the host’s immune system as well as to antibiotics.
It is therefore relevant to develop alternative strategies for treating bacterial infections. Inhibition of virulence factors can be considered as a specific and direct approach. This project addresses the question whether interference with specific virulence factors could lead to the identification of novel antibacterial therapies. More specifically, focus will be on the inhibition of proteolytic processes used by bacteria to degrade extracellular matrix components of the host. Pathogenic bacteria produce proteases themselves (often reported as virulence factors) but can also activate proteolytic cascades of the host (e.g. matrix metallo-proteinases and components of the fibrinolytic pathway).
Inhibition of collagen degradation as a target for new bacterial virulence inhibitors is the basic principle of an innovative in vitro evaluation model. The Gram-negative, anaerobic bacterium Porphyromonas gingivalis was chosen as the model organism (cfr. infra). This germ is present in dental plaque (= biofilm) and can cause caries, gingivitis and periodontitis leading to tooth loss. In case of periodontitis, bacteria may spread into the blood circulation to the different organs. Epidemiological studies indicate that patients with periodontitis have a higher risk for cardiovascular disease, such as atherosclerosis, heart valve failure and embolisms in the heart and brain. Oral P. gingivalis infection accelerated early atherosclerosis in an ApoE deficient mouse model. Moreover, it is now generally accepted that periodontitis is a complicating factor in preeclampsia and premature birth.
The pathogenicity of P. gingivalis relies on the following virulence factors: a) its ability to adhere and invade periodontal and other tissues, b) the presence of proteases and endotoxins, and c) its immunomodulating properties. Bacterial fimbriae are involved in binding of the bacteria to epithelial cells, fibronectin, fibrinogen and proline-rich saliva proteins. Following adhesion, the bacteria invade the epithelial cells in a process associated with cytoskeletal rearrangements in the cells and dependent upon P. gingivalis protease activity. Bacterial proteases are important for the initiation and progression of tissue destruction. They are however not always able to directly degrade tissue, but rather activate cellular matrix metalloproteinases (MMPs). These MMPs are produced by inflammatory cells of the host and are activated by e.g. bacterial gingipains. Apart from a relatively homologous tripeptidyl peptidase and several arginine and lysine specific cysteine proteases, P. gingivalis produces the serine protease dipeptidyl peptidase 4 (DPP4). DPP4 stimulates collagen degradation by active MMPs using a mechanism that is not yet completely understood on the molecular level. DPP4 is a protease that cleaves dipeptides off the N-terminal end of peptides, preferably after proline or alanine on the penultimate position. DPP4 is widely expressed by eukaryotes but a homologous enzyme is also found in bacteria such as P. gingivalis, Streptococcus gordonii, Streptococcus thermophilus en Chryseobacterium meningosepticum. Kumagai and coworkers demonstrated, using a mouse abscess model, that DPP4-deficient mutants of P. gingivalis have reduced virulence (decreased abscess formation, lower lethality and improved recovery) compared to the wild type strains. Reduction of the pathogenicity of P. gingivalis by inhibitors of MMP activation was also shown for tetracyclins.
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