contributed equally to this work Most strains of uropathogenic Escherichia coli (UPEC) encode ®lamentous adhesive organelles called type 1 pili. We have determined that the type 1 pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between a-actinin and vinculin were found to correlate with type 1 pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.

Strains of uropathogenic

SummaryRickettsia conorii, an obligate intracellular tickborne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non-phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70-rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70-dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB-mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3-kinase-dependent activities and microtubule stability. Small-interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c-Cbl, clathrin and caveolin-2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70-dependent invasion of mammalian cells and that clathrin-and caveolin-dependent endocytic events likely contribute to the internalization process.

Rickettsia conorii, a strictly intracellular and category C priority bacterial pathogen (NIAID), invades different mammalian cells. Although some signaling events involved in bacterial entry have been documented, the bacterial and host proteins mediating entry were not known. We report the identification of the Ku70 subunit of DNA-dependent protein kinase (DNA-PK) as a receptor involved in R. conorii internalization. Ku70 is recruited to R. conorii entry sites, and inhibition of Ku70 expression impairs R. conorii internalization. Bacterial invasion is dependent on the presence of cholesterol-enriched microdomains containing Ku70. R. conorii infection stimulates the ubiquitination of Ku70. In addition, the ubiquitin ligase c-Cbl is recruited to R. conorii entry foci, and downregulation of endogenous c-Cbl blocks bacterial invasion and Ku70 ubiquitination. An affinity chromatography approach identified the rickettsial protein rOmpB as a ligand for Ku70. This is the first report of a receptor-ligand interaction involved in the internalization of any rickettsial species.

Rickettsia conorii, the causative agent of Mediterranean spotted fever, is able to attach to and invade a variety of cell types both in vitro and in vivo. Although previous studies show that entry of R. conorii into non-phagocytic cells relies on actin polymerization, little else is known about the molecular details governing Rickettsia-host cell interactions and actin rearrangements. We determined that R. conorii recruits the Arp2/3 complex to the site of entry foci and that expression of an Arp 2/3 binding derivative of the WASP-family member, Scar, inhibited bacterial entry into Vero cells, establishing that Arp2/3 is an active component of this process. Using transient transfection with plasmids expressing dominant negative versions of small GTPases, we showed that Cdc42, but not Rac1 is involved in R. conorii invasion into Vero cells. Using pharmacological approaches, we show that this invasion is dependent on phosphoinositide (PI) 3-kinase and on protein tyrosine kinase (PTK) activities, in particular Src-family kinases. C-Src and its downstream target, p80/85 cortactin, colocalize at entry sites early in the infection process. R. conorii internalization correlated with the tyrosine phosphorylation of several other host proteins, including focal adhesion kinase (FAK), within minutes of R. conorii infection. Our results reveal that R. conorii entry into nonphagocytic cells is dependent on the Arp2/3 complex and that the interplay of pathways involving Cdc42, PI 3-kinase, c-Src, cortactin and tyrosine-phosphorylated proteins regulates Arp2/3 activation leading to the localized actin rearrangements observed during bacterial entry. This is the first report that documents the mechanism of entry of a rickettsial species into mammalian cells.

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X-linked adrenoleukodystrophy (X-ALD) is an inherited metabolic disorder of the nervous system characterized by axonopathy in spinal cords and/or cerebral demyelination, adrenal insufficiency and accumulation of very long-chain fatty acids (VLCFAs) in plasma and tissues. The disease is caused by malfunction of the ABCD1 gene, which encodes a peroxisomal transporter of VLCFAs or VLCFA-CoA. In the mouse, Abcd1 loss causes late onset axonal degeneration in the spinal cord, associated with locomotor disability resembling the most common phenotype in patients, adrenomyeloneuropathy. We have formerly shown that an excess of the VLCFA C26:0 induces oxidative damage, which underlies the axonal degeneration exhibited by the Abcd1(-) mice. In the present study, we sought to investigate the noxious effects of C26:0 on mitochondria function. Our data indicate that in X-ALD patients' fibroblasts, excess of C26:0 generates mtDNA oxidation and specifically impairs oxidative phosphorylation (OXPHOS) triggering mitochondrial ROS production from electron transport chain complexes. This correlates with impaired complex V phosphorylative activity, as visualized by high-resolution respirometry on spinal cord slices of Abcd1(-) mice. Further, we identified a marked oxidation of key OXPHOS system subunits in Abcd1(-) mouse spinal cords at presymptomatic stages. Altogether, our results illustrate some of the mechanistic intricacies by which the excess of a fatty acid targeted to peroxisomes activates a deleterious process of oxidative damage to mitochondria, leading to a multifaceted dysfunction of this organelle. These findings may be of relevance for patient management while unveiling novel therapeutic targets for X-ALD.

No abstract