KAROLINSKA INSTITUTET Microbiology & Tumor Biology Center • MTC Clinical Microbiology - KS

Background
One of the most important steps in bacterial infections is the initial binding to host tissue molecules. This step is considered as a prerequisite for bacterial survival and for the spread of the bacteria in the target tissues and for spreading into deeper tissues. A number of bacterial interactions with host molecules have so far been characterized and the research field is growing rapidly. One important goal is to identify molecular interactions between bacteria and host. Such knowledge might ultimately lead to new drugs or vaccines towards pathogenic bacteria.

My projects is focused on bacterial surface proteins which bind to host proteins such as plasminogen/plasmin, fibronectin, collagen, and vitronectin. The bacteria we study right now is the common human pathogen Helicobacter pylori, which is carried by about half of the human population and is considered to cause gastritis and gastric cancer.

Techniques
In order to identify bacteral proteins involved in the binding to a host protein we use a technique called phage display. The technique involves cloning and protein expression of fragmented bacterial chromosomal DNA in bacterial phages, which express the cloned gene products on the surface of the bacteriophage. Since the phages are easily amplified, and therefore also the cloned gene fragments, the gene phage library will contain billons of clones per milliliter. The phage library is added to wells containing a purified human protein such as fibronectin or another one of the above mentioned proteins attached to the walls. After several rounds of washes the remaining binding phages are eluted and the gene fragment of the eluted phages are isolated and DNA sequenced. Since the whole Helicobacter pylori chromosome is sequenced we can immediately identify which gene we have found.

For many of the Helicobacter pylori genes there is a function known. However, for most genes there is so far no biological function known. When a gene has been identified and sequenced we make in vitro mutations of the gene and transfer the mutated gene into Helicobacter pylori where it will be inserted into the chromosome by recombination. The mutated Helicobacter pylori strain is then tested for its virulence in a mouse model and compared to the native Helicobacter. We are also studying in more detail the identified bacterial gene products with surface plasmon resonance tecnique, using a BIAcore instrument. With this instrument we can get an idea how fast and how strong the binding is and which epitopes are involved in the binding.

  See article: Molecular cloning and characterization of two Helicobacter pylori genes coding for plasminogen-binding proteins

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