Chris van der Does

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Type IV secretion in Neisseria gonorrhoeae

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to deliver macromolecular substrates to target cells. These target cells can be other bacteria to which DNA is transferred, but also eukaryotic cells to which oncogenic DNA or effector proteins are transported. The spread of DNA between bacterial species via T4SSs is one of the major causes of the rapid spread of antibiotic resistance, a problem, confronting communities and nations worldwide. T4SS are also found in pathogenic bacteria like Agrobacterium tumefaciens, Bordetella pertussis, Helicobacter pylori, Brucella suis, Legionella pneumophila and Rickettsia spp. where these systems transport DNA and/or effector proteins into their eukaryotic host to cause diseases like e.g. crown gall tumor formation in plants and whooping cough, gastritis, brucellosis, pneumonia (legionnaires' disease) and typhus in humans. Within these organisms the T4SSs play an important role in pathogenicity.

T4SSs consist of multi-subunit structures, which span both inner and outer membrane. The complex can extend a pilus structure which is used to contact recipient cells. In the Escherichia coli F-plasmid conjugation system, the pilus can extend and retract to form a close mating-pair. Currently, the T4SSs of the F-plasmid (Tra) and the Agrobacterium tumefaciens Ti-plasmid (Vir) systems have been studied in most detail. Recruitment of DNA or effector proteins to the T4SSs takes place via a coupling protein (TraD/VirD4) which is located in the inner membrane. It has become clear that even for T4SSs in which DNA is transported, the DNA is first coupled to a protein, and then this DNA/protein complex is targeted to the coupling protein. The DNA binding protein, also called the relaxase binds to an origin-of-transfer (oriT) region on the DNA, thus forming the relaxosome. This is followed by DNA nicking and initiation of unwinding of the DNA. It has been shown that some relaxases and effector proteins are targeted to the coupling protein via a C-terminal targeting signal. Via the coupling protein the substrates are targeted to the T4SS complex. This complex consists of multiple components (11-13 core components), which span both membranes.

Our studies focus on a T4SS which was identified in the human pathogens Neisseria gonorrhoeae and Neisseria meningitides. This T4SS secretes DNA directly into the medium. The secreted DNA is rapidly taken up by the highly active competence system of Neisseria species and incorporated in the genome, so it could be involved in the exchange of genetic material. Remarkably, this system is also found more often in patients with a severe form of gonorrhea, the disseminated gonococcal infection, where the infection has spread to internal parts. This could suggest an interaction with the host organism.

Within our group, the research is focussed on:

• Analysis of interacting proteins and the assembly pathway of the T4SS complex.
• In vitro characterization of the targeting to and transport of the substrates to the inner membrane.
• Elucidation of membrane insertion, processing and assembly of the pilus.

We aim to acquire detailed insight in the mechanism of DNA export in N.gonorrhoeae, in the T4SSs involved in bacterial conjugation and pathogenicity and in protein secretion in general