Bacterial type IV secretion systems are evolutionarily related to conjugation systems and play a pivotal role in infection by delivering numerous virulence factors into host cells. Using transmission electron microscopy, we report the native molecular structure of the core complex of the Dot/Icm type IV secretion system encoded by Legionella pneumophila, an intracellular human pathogen. The biochemically isolated core complex, composed of at least five proteins--DotC, DotD, DotF, DotG, and DotH--has a ring-shaped structure. Intriguingly, morphologically distinct premature complexes are formed in the absence of DotG or DotF. Our data suggest that DotG forms a central channel spanning inner and outer membranes. DotF, a component dispensable for type IV secretion, plays a role in efficient embedment of DotG into the functional core complex. These results highlight a common scheme for the biogenesis of transport machinery.

Campylobacter jejuni is a major cause of infectious diarrhoea worldwide but relatively little is known about its ecology. In this study, we examined its interactions with Acanthamoeba castellanii, a protozoan suspected to serve as a reservoir for bacterial pathogens. We observed rapid degradation of intracellular C.jejuni in A.castellanii 5 h post gentamicin treatment at 25°C. Conversely, we found that A.castellanii promoted the extracellular growth of C.jejuni in co-cultures at 37°C in aerobic conditions. This growth-promoting effect did not require amoebae - bacteria contact. The growth rates observed with or without contact with amoeba were similar to those observed when C.jejuni was grown in microaerophilic conditions. Preconditioned media prepared with live or dead amoebae cultivated with or without C.jejuni did not promote the growth of C.jejuni in aerobic conditions. Interestingly, the dissolved oxygen levels of co-cultures with or without amoebae - bacteria contact were much lower than those observed with culture media or with C.jejuni alone incubated in aerobic conditions, and were comparable with levels obtained after 24 h of growth of C.jejuni under microaerophilic conditions. Our studies identified the depletion of dissolved oxygen by A.castellanii as the major contributor for the observed amoeba-mediated growth enhancement.

The human pathogen Legionella pneumophila delivers a large array of the effector proteins into host cells using the Dot/Icm type IVB secretion system. Among the proteins composing the Dot/Icm system, an inner membrane protein DotI is known to be crucial for the secretion function but its structure and role in type IV secretion had not been elucidated. We report here the crystal structures of the periplasmic domains of DotI and its ortholog in the conjugation system of plasmid R64, TraM. These structures reveal a striking similarity to VirB8, a component of type IVA secretion systems, suggesting that DotI/TraM is the type IVB counterpart of VirB8. We further show that DotI and its partial paralog DotJ form a stable heterocomplex. R64 TraM, encoded by the conjugative plasmid lacking DotJ ortholog, forms a homo-hexamer. The DotI-DotJ complex is distinct from the core complex, which spans both inner and outer membranes to form a substrate conduit, and seems not to stably associate with the core complex. These results give insight into VirB8-family inner membrane proteins essential for type IV secretion and aid towards understanding the molecular basis of secretion systems essential for bacterial pathogenesis.

Bacterial pathogens like Salmonella and Legionella establish intracellular niches in host cells known as bacteria-containing vacuoles. In these vacuoles, bacteria can survive and replicate. Ubiquitin-dependent selective autophagy is a host defense mechanism to counteract infection by invading pathogens. The Legionella effector protein RavZ interferes with autophagy by irreversibly deconjugating LC3, an autophagy-related ubiquitin-like protein, from a phosphoglycolipid phosphatidylethanolamine. Using a co-infection system with Salmonella, we show here that Legionella RavZ interferes with ubiquitin recruitment to the Salmonella-containing vacuoles. The inhibitory activity is dependent on the same catalytic residue of RavZ that is involved in LC3 deconjugation. In semi-permeabilized cells infected with Salmonella, external addition of purified RavZ protein, but not of its catalytic mutant, induced removal of ubiquitin associated with Salmonella-containing vacuoles. The RavZ-mediated restriction of ubiquitin recruitment to Salmonella-containing vacuoles took place in the absence of the host system required for LC3 conjugation. These observations suggest the possibility that the targets of RavZ deconjugation activity include not only LC3, but also ubiquitin.