Date Published: December 3, 2015
Publisher: Public Library of Science
Author(s): Mateja Ozanic, Valentina Marecic, Yousef Abu Kwaik, Marina Santic, Virginia L. Miller.
Francisella tularensis is a gram-negative, facultative, intracellular bacterium that survives in mammals, arthropods, and amoebae; however, macrophages are considered the key cells in pathogenesis of tularemia in mammals. Understanding intracellular trafficking of F. tularensis within various host cells is indispensable to our understanding of bacterial ecology, intracellular adaptation to various hosts’ microenvironments, and subversion of host cell defenses. Within mammalian and arthropod-derived cells, F. tularensis transiently resides within an acidic vacuole prior to escaping to the cytosol, where the bacteria replicate. In contrast, F. tularensis resides and replicates within non-acidified, membrane-bound vacuoles within the trophozoites of amoebae. The Francisella pathogenicity island (FPI) genes encode a type VI Secretion System (T6SS), which is indispensable for phagosomal escape of F. tularensis within mammalian and arthropod cells and for intravacuolar growth within amoeba. In this review, we discuss the divergent F. tularensis intracellular lifestyle in different hosts and its role in pathogenic evolution and intracellular proliferation within diverse hosts.
Nonpathogenic bacteria are taken up by host cells into vacuoles or phagosomes that are processed through the endocytic pathway, through which the vacuoles mature and fuse to the lysosomes, in which the bacteria are degraded. To avoid this fate within phagocytic cells, intracellular pathogens have evolved different strategies to survive and evade phagosome–lysosome fusion . Understanding the mechanisms by which pathogens manipulate vesicle trafficking in different hosts is extremely important for understanding the ability of various pathogens to cause disease and is essential for designing novel and effective strategies for prevention and therapeutic intervention.
Understanding the virulence factors of Francisella is indispensable for elucidating various aspects of tularemia pathogenesis. Many studies have been focused on a genomic region called the Francisella pathogenicity island (FPI). The FPI is duplicated in F. tularensis subsp. tularensis and F. tularensis subsp. holarctica strains, but is a single copy in F. novicida. It has been shown that many of the FPI genes are essential for phagosome biogenesis and escape of the bacterium into the cytosol, which is the crucial event in the intracellular life cycle of Francisella .
F. tularensis survives and replicates within various cells, but macrophages are considered the important cells in developing tularemia . Francisella enters into macrophages by looping phagocytosis and binding to surface receptors, depending on the opsonization state . Upon entry into macrophages, F. tularensis recruits ”lipid rafts” (cholesterol-rich lipid domains) with caveolin-1 on the host cell membrane . Cholesterol and caveolin-1 are incorporated into the FCP membrane during the initial phase of biogenesis of the FCP .
Vector-borne transmission of tularemia to mammalian hosts has an important role in pathogenesis of the disease . However, little is known about the interaction of F. tularensis with the arthropod vectors at the molecular level. Drosophila melanogaster and D. melanogaster-derived S2 cells have been used for studying F. tularensis infection . It has been shown that F. tularensis infects and kills adult Drosophila flies in a dose-dependent manner . Similar to human macrophages, within arthropod cells, F. tularensis transiently resides within an acidified phagosome that matures to a late endosomal stage, followed by rapid bacterial escape into the cytosol, where the bacteria proliferate (Fig 2B) . The intracellular lifestyle of F. tularensis within human macrophages and arthropod-derived cells is very similar in terms of the virulence factors involved. Studies using a mosquito cell line, SualB, derived from Anopheles gambie, have shown that the FPI-encoded IglA, IglB, IglC, IglD, PdpA, and PdpB are necessary for efficient intracellular replication of F. novicida within this insect’s cells . However, the FPI proteins PdpC and PdpD are not required for replication within the SualB cell line . In addition, out of 394 mutants of F. novicida identified to be defective for proliferation within S2 cells, only 135 (including the FPI genes) are also defective for replication within human macrophages . F. novicida virulence factors have also been studied in adult flies compared to mice . Among 249 F. tularensis genes important for virulence in the mice model, only 49 genes were important in adult D. melanogaster, including 14 of the FPI genes . The FPI genes that are not required for virulence in the adult fly are pdpC, pdpE, pdpD, and anmK , which is consistent with findings in the mosquito cell line . Therefore, although F. tularensis uses similar molecular mechanisms of pathogenesis within arthropod and mammalian cells, some distinct virulence factors are differentially utilized for bacterial proliferation in the two evolutionarily distinct hosts .
Recent studies have shown that Francisella enters and multiplies within Acanthamoeba castellanii , Hartmannella vermiformis , and Dictyostelium discoideum cells (our unpublished results). In addition, F. tularensis subsp. holarctica is found within cysts of A. castellanii, suggesting a potential for long-term bacterial survival in nature . Studies have also shown intravacuolar replication of F. novicida within H. vermiformis (Figs 1B and 2C), which is a major difference from the cytosolic proliferation of this bacterium within mammalian and arthropod-derived cells. Interestingly, the FPI-encoded protein IglC and the MglA global regulator, which are essential for phagosomal escape and intramacrophage growth of F. tularensis, are also important for intravacuolar replication of F. tularensis within amoeba cells [36,38].
The ability to invade and replicate in a variety of host cells appears to be a major feature of the ecology and epidemiology of F. tularensis. Within both mammalian and arthropod-derived cells, the FCP transiently matures to an acidified late endosome, followed by rapid bacterial escape into the host cell cytosol where the replication occurs. However, within amoeba cells, the bacterium resides and replicates within non-acidified, membrane-bound vacuoles. It is possible that the virulence of Francisella for mammalian hosts may be higher after intra-amoebal growth. Many virulence factors of F. tularensis have been discovered and investigated, including the FPI proteins, which play a crucial role in patho-adaptation of the bacteria to different hosts. At the moment, some studies are focused on the FPI genes that encode the T6SS in Francisella. Future studies should elucidate the role of T6SS translocated proteins in disruption of the phagosome and intracellular replication of F. tularensis. The relevance of bacterial escape into the cytosol should be investigated from various perspectives and cells models. It is evident that the short transition of the bacterium in vacuoles within mammalian and arthropod cells plays an important role in pathogenesis of tularemia. The differences between the amoebal vacuoles and the mammalian and arthropod vacuoles where the bacterium permanently or shortly resides, respectively, have yet to be discovered. It is intriguing how IglC is required for phagosomal escape in mammalian and arthropod-derived cells but is indispensable for intravacuolar growth within amoeba. It is possible that the long-term evolution of F. tularensis within amoeba has facilitated its intravacuolar adaptation in the aquatic environment for long-term survival and for transmission to arthropod and mammalian hosts.