Legionella pneumophilaDotU and IcmF Are Required for Stability of the Dot/Icm Complex

Abstract

Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ⌬dotU ⌬icmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ⌬dotU ⌬icmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system

“…In L. pneumophila, these genes are nonessential components of the type IV secretion system (T4SS) required for cytotoxicity of L. pneumophila toward mammalian and D. discoideum cells (11,12). IcmF and IcmH (DotU) are thought to be accessory proteins that work in concert to improve the efficiency of T4SS translocation of bacterial effector proteins into the cytosol of eukaryotic target cells (13,14 (17), the V52 genome does not encode a T3SS other than the typical one required for flagella biosynthesis. Thus, it is also notable that no Dictyostelium-attenuated mutants were found in any gene known to be involved in flagellar biosynthesis.…”

“…2) show a high degree of sequence conservation with L. pneumophila icmF and icmH (dotU) (13,14), whereas an adjacent gene (VCA0116) has homology to the ClpB family of AAA proteins (27), which have recently been implicated in the ATPdependent export of proteins by the T3SS pathway (28). These three genes, together with several other closely linked genes, constitute a region that Das and Chaudhuri (6) designated the IAHP gene cluster, which can be found in one or more copies in many pathogenic and commensal Gram-negative bacterial species, including Yersinia pestis, P. aeruginosa, E. coli O157, S. enterica, Agrobacterium tumefaciens, E. ictaluri, and R. leguminosarum (6,8,19,20,29).…”

In vivo expression technology identifies a type VI secretion system locus in Burkholderia pseudomallei that is induced upon invasion of macrophages

“…S. Thomas, unpublished). In the L. pneumophila T4SSB, IcmF and DotU (IcmH) are proposed to form an inner membrane complex that serves to stabilize or optimize the function of the secretion machinery (Sexton et al, 2004;VanRheenen et al, 2004). Due to the similarity of TssL to DotU, TssL and TssM are likely to form a similar complex.…”

“…Recently, TssH has been hypothesized to provide the force for the translocation of exported proteins by the T6SS (Mougous et al, 2006). The IcmF-like TssM orthologues contain an N-terminal domain that is similar to members of the small GTPase superfamily present in eukaryotes, and includes a Walker box A nucleotide-binding site (Sexton et al, 2004;Zusman et al, 2004;M. S. Thomas, unpublished).…”

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems

“…These two components are required for the optimal function of the T4bSS in Legionella pneumophila and Coxiella burnetii (33,34). The TssL protein and its T4bSS IcmH/DotU counterpart are inner membrane proteins (32,(35)(36)(37)(38). The membrane topology of the TssL has been determined; TssL has a single trans-membrane segment (TMS) located at the C terminus of the protein (36).…”