The doticm Type IVB Secretion System

The dot/icm type IV secretion system of L. pneumophila is probably its most important secretion system for infection as it is involved in many different stages of the intracellular life cycle. This macromolecular complex is encoded by 25 genes located on two genomic regions: region I contains seven genes (icmV, W, X, dotA, B, C, D) and region II is composed of 18 genes (icmT, S, R, Q, P, O, N, M, L, K, E, G, C, D, J, B, F, H). This system is conserved and present in the same chromosomal location in strains Paris, Lens, and Philadelphia 1. Recently, comparison of this region in 18 L. pneumophila and 17 other Legionella species by sequence and hybridization analysis showed that they are present in all strains sequenced and that their organization is conserved (Morozova et al. 2004). The nucleotide sequence conservation of the dot/icm genes is high (96-98%) among the different L. pneumophila strains, but among different Legionella species the nucleotide sequence similarity is less pronounced (62-79%) (Morozova et al. 2004). In general, locus II genes are more conserved than locus I genes. The biological implication of these sequence variations is not yet known. In the icm/dot system of L. longbeachae and L. micdadei the global organization is also conserved but, interestingly, in region II in L. micdadei the gene icmR is replaced by two genes, migA and migB, which do not show any homology to icmR. The same gene is replaced in L. longbeachae by a gene called ligB (Feldman and Segal 2004).

Given the central role of the dot/icm system in Legionella pathogenesis, many recent studies have aimed at identifying and characterizing its substrates. The first characterized effector was RalF, required for localization of the host protein ARF-1, a key regulator of vesicle trafficking from the endoplasmic reticulum to the phagosomes (Nagai et al. 2002). RalF is conserved in all three sequenced strains, like LidA, another substrate involved in recruitment of vesicles during vacuole biogenesis and in maintaining integrity of the dot/icm complex (Conover et al. 2003), and LepA and LepB, which are involved in egress of Legionella from the vacuole during amoeba infection (Chen et al. 2004). More recently, a number of candidate effector proteins named SidA-G were identified in the Philadelphia 1 strain by a two-hybrid screen with IcmG/DotF as bait followed by a screen of pro teins transferred interbacterially with a Cre/loxP-based protein translocation assay (Luo and Isberg 2004). SidA, SidB, SidC, SidE, and SidF are conserved in strains Paris and Lens; in contrast, SidG is missing in strain Lens, SidD is missing in strains Paris and Lens, and SidH is interrupted by two insertion sequence (IS) elements in strain Paris and missing in strain Lens. All Sid proteins except SidD contain a coiled-coil domain, a protein motif known to be involved in protein-protein interactions.

Until recently the translocation signal for the type IV secretion effectors was not known. Nagai and colleagues, who investigated the mechanism of translocation of RalF (Nagai et al. 2005), identified a 20-amino-acid C-terminal region of the RalF protein as necessary and sufficient for translocation. In particular, a hydrophobic residue at the C-terminal -3 position is critical for secretion of RalF, as a substitution to hydrophilic residues resulted in a severe defect in translocation. Comparison with other Dot/Icm substrates identified in most of them a hydrophobic residue or a proline residue at the -3 or -4 position, supporting the idea that these residues are critical for secretion by the type IV system.

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