Introduction
to bacterial type IV secretion systems Introduction
to bacterial type IV secretion systems
Pathogenicity in gram-negative bacteria is critically dependent upon secretion machineries which mediate the transport and injection of toxic molecules into target cells (Finlay and Falkow, 1997; Thanassi and Hultgren, 2000). These secretion systems are classified into four types (I to IV). Our structural genomics program focuses on type IV secretion systems and aims at gaining structural insights for all components of this type of secretion machinery.
A type IV secretion system is an apparatus of about 11 proteins (Akopyants et al., 1998; Censini et al., 1996) which have sequence homology with the components of the machineries responsible for bacterial conjugation, i.e. the transfer of DNA from one bacteria to another, and with T-DNA transfer in Agrobacterium tumefaciens responsible for transfer of T-DNA in plants and subsequent formation of plant tumors (Akopyants et al., 1998; Covacci et al., 1999; Winans et al., 1996). In fact, the type IV secretion system of A. tumefaciens has served as the original model system for this type of secretion apparatus (Winans et al., 1996).
Here, we give a brief summary of what is known of the proteins that forms the type IV secretion apparatus of A. tumefaciens.
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Virulence in A. tumefaciens is caused by the transfer into plant cell of the genetic material contained in a region of the Ti plasmid called the T-DNA (Zupan and Zambryski, 1995). The Ti plasmid contains at least one region, virB, which encodes virulence proteins which forms the so-called type IV secretion system (Figure on the right) (Kado, 1993; Winans et al., 1996). This apparatus actively transfers the T-DNA of the Ti plasmid into plant cells through a pilus appendage. VirB2 is the structural subunit of the pilus (Eisenbrandt et al., 1999). VirB7 and VirB9 interact through a disulfide bridge and formation of a VirB7-VirB9 complex appears to be important for stablization of many VirB proteins, notably VirB10, within the machinery (Anderson et al., 1996; Das et al., 1997; Fernandez et al., 1996; Spudich et al., 1996). VirB10 appears to self-associate within the membrane to form larger complexes (Beaupre et al., 1997). VirB4 and VirB11 have adenosine triphosphatase (ATPase) activities (Berger and Christie, 1993; Okamoto et al., 1991; Stephens et al., 1995). VirB4 is tightly associated with the inner membrane, while VirB11 is mostly cytoplasmic and associates weakly with the inner membrane (Christie et al., 1989; Krause et al., 2000a; Krause et al., 2000b; Rashkova et al., 1997)
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Many components of the type IV secretion system of Agrobacterium tumefaciens are homologous to proteins implicated in conjugative transfer of plasmid or phage DNAs, or in transport of toxic or transforming virulence proteins produced by bacterial pathogens targeting humans. For example, conjugative transfer of the E.coli plasmid pKM101 is carried out by a secretion apparatus, the components of which are encoded by the tra gene cluster. As shown in the figure above, many of the tra genes are homologous to the A. tumefaciens virB genes. Another example is the transport of the transforming protein CagA from Helicobacter pylori into the gastric epithelium by a secretion apparatus encoded by the cag gene cluster (see figure above). Here also, a number of the cag genes products share homology with some of the VirB proteins of Agrobacterium's type IV secretion system. These observations suggests that the transport apparatus for conjugative transfer of DNA or for protein transport may consist of an Agrobacterium-like type IV secretion system.
In an attempt to crystallize all components of the type IV secretion system, we have initiated a targeted structural genomics program which aims at cloning and expressing genes encoding protein homologues of the type IV secretion system across species. In the past, crystallographers have "jumped" species very often in order to find a homologue of the protein under investigation that would yield usable crystals. Hence, when the protein under investigation does not yield crystals or yields crystals of poor quality, it has been a common practice to renew similar attempts using a homologue of that protein using another species as a source. We applied this strategy on a small scale for a component of the type IV secretion system, the VirB11 homologue. The VirB11 homologue of H. pylori is called HP0525, while the VirB11 homologue in the plasmid RP4 conjugation system is called TrbB. While A. tumefaciens VirB11 is insoluble, HP0525 and TrbB yielded soluble and monodispersed protein preparations. However, only HP0525 crystallized. We will expand on this strategy by cloning all VirB gene homologues across 8 systems: A. tumefaciens, E. coli plasmid RP4 and pKM101 conjugal systems, H. pylori (associated with gastric and duodenal ulcers and gastric cancer), Bordetella pertussis (responsible for causing the whopping cough in infants), Rickettsia prowazeki (the causative agent for epidemic typhus), Legionella pneumophila (responsible for Legionnaires' pneumonia), and Brucella suis (the causative agent of Brucellosis in mammals).
References
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Last modification : 12/22/2000 by Thierry
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