Link between intraphagosomal biotin and rapid phagosomal escape in Francisella

Abstract

Cytosolic bacterial pathogens require extensive metabolic adaptations within the host to replicate intracellularly and cause disease. In phagocytic cells such as macrophages, these pathogens must respond rapidly to nutrient limitation within the harsh environment of the phagosome. Many cytosolic pathogens escape the phagosome quickly (15-60 min) and thereby subvert this host defense, reaching the cytosol where they can replicate. Although a great deal of research has focused on strategies used by bacteria to resist antimicrobial phagosomal defenses and transiently pass through this compartment, the metabolic requirements of bacteria in the phagosome are largely uncharacterized. We previously identified a Francisella protein, FTN_0818, as being essential for intracellular replication and involved in virulence in vivo. We now show that FTN_0818 is involved in biotin biosynthesis and required for rapid escape from the Francisella-containing phagosome (FCP). Addition of biotin complemented the phagosomal escape defect of the FTN_0818 mutant, demonstrating that biotin is critical for promoting rapid escape during the short time that the bacteria are in the phagosome. Biotin also rescued the attenuation of the FTN_0818 mutant during infection in vitro and in vivo, highlighting the importance of this process. The key role of biotin in phagosomal escape implies biotin may be a limiting factor during infection. We demonstrate that a bacterial metabolite is required for phagosomal escape of an intracellular pathogen, providing insight into the link between bacterial metabolism and virulence, likely serving as a paradigm for other cytosolic pathogens. S ubversion of the hostile phagosomal environment is required for the survival of intracellular bacteria. Although bacterial strategies to resist antimicrobial phagosomal defenses have been studied in great detail (1, 2), the ways in which bacteria counter phagosomal nutrient limitation are largely unknown. This is especially true for cytosolic pathogens that are often in the phagosome for a very limited time (15-60 min), before escaping this compartment to reach their replicative niche in the cytoplasm. During this brief and dynamic time, it is unclear if cytosolic pathogens require sequestration of nutrients or synthesis of de novo metabolites to promote their virulence strategies and escape the toxic phagosome.Francisella tularensis is a cytosolic intracellular Gram-negative bacterial pathogen that uses a multitude of mechanisms to evade phagosomal host defenses (3). This pathogen is highly virulent and causes the potentially fatal disease tularemia. Francisella novicida U112 and Francisella holarctica LVS (live vaccine strain) are less virulent yet highly related strains that are often used as models to study F. tularensis. Like other cytosolic bacterial pathogens, after initial contact with the host macrophage, Francisella spp. are taken up into a phagosome and rapidly escape (30-60 min) this compartment to reach and replicate within the cytosol (3-5). The mechanism by w...

Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

“…In contrast to the results in Histoplasma, both Mycobacterium tuberculosis and Candida biotin auxotrophs are impaired in intracellular replication (11,67,68,72). Furthermore, initial escape from the phagosome for Francisella also requires biotin biosynthesis (73). On the other hand, Salmonella biotin and pantothenate auxotrophs are not attenuated (74).…”

Microinjection of Francisella tularensis and Listeria monocytogenes Reveals the Importance of Bacterial and Host Factors for Successful Replication

“…Our findings imply, in addition, that the metabolic adaptation of intracytosolic microbes is a necessary, but alone not sufficient, factor for their replication and that an intricate host-bacterium interaction is required for the intracytosolic replication. In fact, using the microinjection technique, we previously demonstrated its utility to directly elucidate metabolic requirements for cytosolic replication, since it was demonstrated that a biotin biosynthesis mutant of Francisella novicida was incapable of intracytosolic growth unless biotin was added to the culture medium (19).…”

“…Therefore, it was hypothesized that bacteria which successfully replicate in the cytosol harbor a metabolic machinery that is adapted to this niche in order to utilize available nutrients (14). However, there is accumulating evidence that the metabolic requirements may be similar for bacteria residing within the eukaryotic cytosol and bacteria residing extracellularly (15)(16)(17)(18)(19), thus indicating that modulation of the cytosolic composition, e.g., by deprivation of the availability of metabolites, may be an important factor to control replication of intracellular bacteria. To add further complexity, there is recent evidence that manipulation of autophagy is used as a means by pathogens to acquire energy and nutrients.…”

PanG, a New Ketopantoate Reductase Involved in Pantothenate Synthesis

“…Several genetic screens for replication-defective mutants have implicated biosynthetic pathways as contributing factors to the pathogenesis of Francisella species (16). Interruption in genes involved in purine, aromatic amino acid, and biotin biosynthesis results in attenuation of F. tularensis within a mouse model of tularemia (17)(18)(19)(20). Collectively, these studies highlight the contribution of de novo biosynthetic pathways to the ability of F. tularensis to survive within the host environment.…”