Legionella pneumophila secretes a mitochondrial carrier protein during infection.

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

EspG of enteropathogenic and enterohemorrhagic E. coli binds the Golgi matrix protein GM130 and disrupts the Golgi structure and function.

The enteric pathogens enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Shigella flexneri all translocate at least one effector protein of the EspG protein family into host cells via a type III secretion system (T3SS). The EspG family comprises EspG, EspG2 and VirA. From a Y2H screen, we identified the Golgi matrix protein GM130 as a potential binding partner of EspG. We confirmed EspG:GM130 protein interaction by affinity co-purification. In co-immunoprecipitation experiments EspG was co-precipitated with GM130 while both GM130 and tubulins were co-precipitated with EspG. When expressed ectopically in HeLa cells, the EspG protein family all localized to the Golgi and induced fragmentation of the Golgi apparatus. All EspG family proteins were also able to disrupt protein secretion to a greater extent than the T3SS effector NleA/EspI, which has previously been shown to localize to the Golgi and interact with SEC24 to disrupt COPII vesicle formation. We hypothesize that EspG:GM130 interaction disrupts protein secretion either through direct disruption of GM130 function or through recruitment of other EspG interacting proteins to the Golgi.

Enteropathogenic and enterohaemorrhagic Escherichia coli: even more subversive elements.

The human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) share a unique mechanism of colonization that results from the concerted action of effector proteins translocated into the host cell by a type III secretion system (T3SS). EPEC and EHEC not only induce characteristic attaching and effacing (A/E) lesions, but also subvert multiple host cell signalling pathways during infection. Our understanding of the mechanisms by which A/E pathogens hijack host cell signalling has advanced dramatically in recent months with the identification of novel activities for many effectors. In addition to further characterization of established effectors (Tir, EspH and Map), new effectors have emerged as important mediators of virulence through activities such as mimicry of Rho guanine nucleotide exchange factors (Map and EspM), inhibition of apoptosis (NleH and NleD), interference with inflammatory signalling pathways (NleB, NleC, NleE and NleH) and phagocytosis (EspF, EspH and EspJ). The findings have highlighted the multifunctional nature of the effectors and their ability to participate in redundant, synergistic or antagonistic relationships, acting in a co-ordinated spatial and temporal manner on different host organelles and cellular pathways during infection.

Binding to Na(+) /H(+) exchanger regulatory factor 2 (NHERF2) affects trafficking and function of the enteropathogenic Escherichia coli type III secretion system effectors Map, EspI and NleH.

Enteropathogenic Escherichia coli (EPEC) strains are diarrhoeal pathogens that use a type III secretion system to translocate effector proteins into host cells in order to colonize and multiply in the human gut. Map, EspI and NleH1 are conserved EPEC effectors that possess a C-terminal class I PSD-95/Disc Large/ZO-1 (PDZ)-binding motif. Using a PDZ array screen we identified Na(+)/H(+) exchanger regulatory factor 2 (NHERF2), a scaffold protein involved in tethering and recycling ion channels in polarized epithelia that contains two PDZ domains, as a common target of Map, EspI and NleH1. Using recombinant proteins and co-immunoprecipitation we confirmed that NHERF2 binds each of the effectors. We generated a HeLa cell line stably expressing HA-tagged NHERF2 and found that Map, EspI and NleH1 colocalize and interact with intracellular NHERF2 via their C-terminal PDZ-binding motif. Overexpression of NHERF2 enhanced the formation and persistence of Map-induced filopodia, accelerated the trafficking of EspI to the Golgi and diminished the anti-apoptotic activity of NleH1. The binding of multiple T3SS effectors to a single scaffold protein is unique. Our data suggest that NHERF2 may act as a plasma membrane sorting site, providing a novel regulatory mechanism to control the intracellular spatial and temporal effector protein activity.

A C-terminal class I PDZ binding motif of EspI/NleA modulates the virulence of attaching and effacing Escherichia coli and Citrobacter rodentium.

Enteropathogenic Escherichia coli induces characteristic attaching-effacing (A/E) lesions on the intestinal mucosa during infection. The locus of enterocyte effacement is essential for A/E lesion formation and encodes a type III secretion system that translocates multiple effector proteins into the host cell. Following translocation, EspI/NleA localizes to the Golgi. Using the yeast two-hybrid system (Y2HS) and PSD-95/Disk-large/ZO-1 (PDZ)-domain protein array overlays, we identified 15 putative host-interacting partners of EspI. All but two of the target proteins contained PDZ domains. Examination of the EspI amino acid sequence revealed a C-terminal consensus class I PDZ binding motif. Deletion of the last 7 amino acids of EspI to generate EspI(DeltaC7) abrogated the Y2HS interaction between EspI and 5 of the 6 putative host cell target proteins tested. Deletion of the EspI PDZ binding motif also resulted in delayed trafficking of EspI to the Golgi. Using a mouse model of infection, we showed that Citrobacter rodentium expressing truncated EspI(DeltaC7) was attenuated when in competition with C. rodentium expressing full-length EspI. Overall, these results suggested that EspI may modulate the virulence of A/E pathogens by binding host PDZ-domain proteins.

The bacterial virulence factor NleA inhibits cellular protein secretion by disrupting mammalian COPII function.

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) maintain an extracellular lifestyle and use a type III secretion system to translocate effector proteins into the host cytosol. These effectors manipulate host pathways to favor bacterial replication and survival. NleA is an EHEC/EPEC- and related species-specific translocated effector protein that is essential for bacterial virulence. However, the mechanism by which NleA impacts virulence remains undetermined. Here we demonstrate that NleA compromises the Sec23/24 complex, a component of the mammalian COPII protein coat that shapes intracellular protein transport vesicles, by directly binding Sec24. Expression of an NleA-GFP fusion protein reduces the efficiency of cellular secretion by 50%, and secretion is inhibited in EPEC-infected cells. Direct biochemical experiments show that NleA inhibits COPII-dependent protein export from the endoplasmic reticulum. Collectively, these findings indicate that disruption of COPII function in host cells contributes to the virulence of EPEC and EHEC.