Francisella–Arthropod Vector Interaction and its Role in Patho-Adaptation to Infect Mammals

Francisella tularensis is a Gram-negative, intracellular, zoonotic bacterium, and is the causative agent of tularemia with a broad host range. Arthropods such as ticks, mosquitoes, and flies maintain F. tularensis in nature by transmitting the bacteria among small mammals. While the tick is largely believed to be a biological vector of F. tularensis, transmission by mosquitoes and flies is largely believed to be mechanical on the mouthpart through interrupted feedings. However, the mechanism of infection of the vectors by F. tularensis is not well understood. Since F. tularensis has not been localized in the salivary gland of the primary human biting ticks, it is thought that bacterial transmission by ticks is through mechanical inoculation of tick feces containing F. tularensis into the skin wound. Drosophila melanogaster is an established good arthropod model for arthropod vectors of tularemia, where F. tularensis infects hemocytes, and is found in hemolymph, as seen in ticks. In addition, phagosome biogenesis and robust intracellular proliferation of F. tularensis in arthropod-derived cells are similar to that in mammalian macrophages. Furthermore, bacterial factors required for infectivity of mammals are often required for infectivity of the fly by F. tularensis. Several host factors that contribute to F. tularensis intracellular pathogenesis in D. melanogaster have been identified, and F. tularensis targets some of the evolutionarily conserved eukaryotic processes to enable intracellular survival and proliferation in evolutionarily distant hosts

Host Factors Required for Modulation of Phagosome Biogenesis and Proliferation of Francisella tularensis within the Cytosol

Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of ∼21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals

The Moraxella catarrhalis Autotransporter McaP Is a Conserved Surface Protein That Mediates Adherence to Human Epithelial Cells through Its N-Terminal Passenger Domain

The protein McaP was previously shown to be an adhesin expressed by the Moraxella catarrhalis strain O35E, which also displays esterase and phospholipase B activities (J. M. Timpe et al., Infect. Immun. 71:4341-4350, 2003). In the present study, sequence analysis suggests that McaP is a conventional autotransporter protein that contains a 12-stranded β-barrel transporter module (amino acids [aa] 383 to 650) linked to a surface-exposed passenger domain exhibiting lipolytic activity (aa 62 to 330). An in-frame deletion removing most of this predicted N-terminal passenger domain was engineered, and Escherichia coli expressing the truncated McaP protein exhibited greatly reduced adherence to A549 human lung epithelial cells compared to E. coli expressing wild-type McaP. Site-directed mutagenesis of a serine residue at position 62 of McaP, predicted to be important for the lipolytic activity of the protein, resulted in loss of hydrolysis of p-nitrophenyl ester of caproate. E. coli expressing this mutated McaP, however, adhered to A549 monolayers at levels greater than recombinant bacteria expressing the wild-type adhesin. These results indicate that the predicted passenger domain of McaP is involved in both the binding and the lipolytic activity of the molecule and demonstrate that the adhesive properties of McaP do not require its lipolytic activity. Sequence analysis of mcaP from eight Moraxella catarrhalis strains revealed that the gene product is highly conserved at the amino acid level (98 to 100% identity), and Western blot analysis demonstrated that a panel of 16 isolates all express McaP. Flow cytometry experiments using antibodies raised against various portions of McaP indicated that its predicted passenger domain as well as transporter module contain surface-exposed epitopes. In addition to binding to the surface of intact bacteria, these antibodies were found to decrease adherence of M. catarrhalis to A549 human lung cells by up to 47% and to reduce binding of recombinant E. coli expressing McaP by 98%. These results suggest that McaP should be considered as a potential vaccine antigen