Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system

Abstract

SummaryType IV secretion systems (T4SS) are utilized by a wide range of Gram negative bacteria to deliver protein and DNA substrates to recipient cells. The best characterized T4SS are the type IVA systems, which exhibit extensive similarity to the Agrobacterium VirB T4SS. In contrast, type IVB secretion systems share almost no sequence homology to the type IVA systems, are composed of approximately twice as many proteins, and remain largely uncharacterized. Type IVB systems include the Dot/ Icm systems found in the pathogens Legionella and Coxiella and the conjugative apparatus of IncI plasmids. Here we report the first extensive characterization of a type IVB system, the Legionella Dot/Icm secretion apparatus. Based on biochemical and genetic analysis, we discerned the existence of a critical five-protein subassembly that spans both bacterial membranes and comprises the core of the secretion complex. This transmembrane connection is mediated by protein dimer pairs consisting of two inner membrane proteins, DotF and DotG, which are able to independently associate with DotH/DotC/DotD in the outer membrane. The Legionella core subcomplex appears to be functionally analogous to the Agrobacterium VirB7-10 subcomplex, suggesting a remarkable conservation of the core subassembly in these evolutionarily distant type IV secretion machines.

Legionella oakridgensis ATCC 33761 genome sequence and phenotypic characterization reveals its replication capacity in amoebae

“…oakridgensis seems to have a very limited set of hosts and replicates significantly slower compared to L. pneumophila Corby. This may be in accordance to the low amount of effector proteins and eukaryote-like proteins (total ~80) identified, when compared to what is known for L. pneumophila (>300 effector proteins) (Vogel et al, 1998;Vincent et al, 2006;Burstein et al, 2009;Hubber et al, 2010). A recent in silico analysis of genome sequences revealed a core set of approximately 107 effector proteins present in L. pneumophila strains (Schroeder et al, 2010).…”

Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii

“…However, a study by Vincent et al (15,37) indicated that other Dot/Icm component proteins, such as T4SS chaperones IcmS and IcmW, may be equally promising baits for this type of screen. Our bioinformatics approach achieved significant success in predicting C. burnetii type IV substrates, as 26 of 42 predicted candidates were confirmed as Dot/Icm substrates (Table S3).…”

“…To obtain a more complete inventory of substrates transferred by the Dot/Icm system of C. burnetii, we initiated a study to identify such proteins by dual strategies that combined genetic screenings and bioinformatics analyses. Because a subset of Legionella Dot/ Icm substrates specifically interacts with DotF (5), an important component of the T4SS that localizes to the inner membrane of the bacterium (15). We hypothesized that similar interactions occur between Coxiella T4SS substrates and its DotF protein.…”

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors

“…The remainder was PE-up and included seven extracellular PE-on proteins (ABC transporter periplasmic-binding protein ArtJ, hypothetical protein Lpg0264, flagellar hook protein FlgK, phospholipase C PlcB, protease/elastase LasB, polysaccharide deacetylase Lpg1993, Lpg2443) (70,(92)(93)(94). The further PE-up extr majorly included 8 proteins without assigned function (Lpg2220, Lpg1647, Lpg1645, Lpg0957, Lpg0301 Lpg1318, Lpg2206, Lpg2246), 5 T2SS substrates (chitinase ChiA, phospholipase A/acyltransferase PlaC, hypothetical protein Lpg0956, Lpg0189, Lpg0264), three Dot/Icm effectors (LegP, Lpg1667, Lpg2443) and Dot/Icm apparatus core complex protein DotC, as well as further (in addition to FlgK) motility-related proteins (FliC, FliD) (26,70,(95)(96)(97). The phase-stable extracellular proteome (42 proteins) included 13 T2SS substrates (phospholipase C PlcA, NttC, endoglucanase CelA, lysophospholipase A PlaA, T2 ribonuclease SrnA, NttB, Lpg1832, SclB, aminopeptidases LapB, LapA, zinc metalloprotease ProA, major acid phosphatase Map, Lpg0873), five Dot/Icm effectors (LegY/GamA which was also detected as T2SS substrate (25), LirB, MavL, LegS, Lpg0041), three proteins involved in carbon and energy metabolism (enolase Eno, thioredoxin reductase TrxB1, xylanase-like protein YjeA), as well as nine functionally unassigned proteins (Fig.…”