Intracellular fate of Francisella tularensis within arthropod‐derived cells

Abstract

Since transmission of Francisella tularensis into the mammalian host occurs via arthropod vectors such as ticks, mosquitoes, horseflies and deerflies, recent studies have established Drosophila melanogaster as an arthropod vector model system. Nothing is known about the intracellular fate of F. tularensis within arthropod-derived cells, and the role of this host-parasite adaptation in the evolution of this pathogen to infect mammals. In this report, we explored intracellular trafficking of F. tularensis ssp. novicida in D. melanogaster-derived S2 cells. First, we show that similar to the F. tularensis ssp. holarctica-derived LVS strain, F. tularensis ssp. novicida is highly infectious, replicates exponentially within S2 cells and within adult flies, and is fatal to adult fruit flies in a dose-dependent manner, while the iglC, iglD and mglA mutants are defective. Using electron and fluorescence microscopy-based phagosome integrity assays, we show that the wild-type strain escapes into the cytosol of S2 cells within 30-60 min post infection and by 6 h, 90% were cytosolic. In contrast, approximately 40-50% of the iglC and iglD mutants escape into the cytosol by 6 h while the other subpopulation becomes enclosed within multilamellar vesicles (MLVs). Pre-treatment of S2 cells with the autophagy inhibitor methyl adenine blocks formation of the MLVs and all the vacuolar subpopulation of the iglC and iglD mutant bacteria become enclosed within single membrane-surrounded vacuoles. Endocytic trafficking studies of F. tularensis within S2 cells show transient colocalization of the bacterial phagosome with D. melanogaster LAMP2-GFP fusion but not with lysosomes pre-loaded with fluorescent dextran. Our data show that MLVs harbouring the iglC mutant acquire Lamp2 and dextran while MLVs harbouring the iglD mutant exclude these late endosomal and lysosomal markers. Our data indicate crucial differences in the role of the pathogenicity island-encoded proteins in modulating intracellular trafficking within human macrophages and arthropod vector-derived cells.

Dermacentor andersoni Transmission of Francisella tularensis subsp. novicida Reflects Bacterial Colonization, Dissemination, and Replication Coordinated with Tick Feeding

“…novicida infection in a tick host, a recognized biological vector of Francisella spp., via a natural route of acquisition, whereas previous F. tularensis subsp. novicida-arthropod studies have utilized direct infection of fruit flies or insect (e.g., mosquito and fruit fly) cell lines (1,9,14,16). The ability of F. tularensis subsp.…”

Prison Break: Pathogens' Strategies To Egress from Host Cells

“…In the case of Francisella, it is well documented that the bacterium first escapes its vacuole, a phagosome with features of a late endosomal compartment, within the first 30 min of infection (124). It then multiplies in the cytosol of its host macrophage, of mammalian or arthropod origin (122), and is able to trigger pyroptosis and cell lysis, possibly as a primary strategy for dissemination (123,125). However, at least in murine macrophages, it is subsequently taken up again into a membranous compartment by autophagy.…”

A Phenotype at Last: Essential Role for the Yersinia enterocolitica Ysa Type III Secretion System in a Drosophila melanogaster S2 Cell Model

“…S2 cells are a macrophage-like cell line and have been demonstrated as a suitable model for the study of host-pathogen interactions, replicating many phenotypes observed in mouse and human cell lines (10)(11)(12). The genetic and cellular tools available for use with these cells are plentiful, and numerous approaches to RNA interference (RNAi) screens have been employed to examine host-pathogen interactions (13)(14)(15)(16)(17)(18)(19)(20).…”