Cytosolic clearance of replication-deficient mutants revealsFrancisella tularensisinteractions with the autophagic pathway

Abstract

Cytosolic bacterial pathogens must evade intracellular innate immune recognition and clearance systems such as autophagy to ensure their survival and proliferation. The intracellular cycle of the bacterium Francisella tularensis is characterized by rapid phagosomal escape followed by extensive proliferation in the macrophage cytoplasm. Cytosolic replication, but not phagosomal escape, requires the locus FTT0369c, which encodes the dipA gene (deficient in intracellular replication A). Here, we show that a replication-deficient, ∆dipA mutant of the prototypical SchuS4 strain is eventually captured from the cytosol of murine and human macrophages into double-membrane vacuoles displaying the late endosomal marker, LAMP1, and the autophagy-associated protein, LC3, coinciding with a reduction in viable intracellular bacteria. Capture of SchuS4ΔdipA was not dipA-specific as other replication-deficient bacteria, such as chloramphenicol-treated SchuS4 and a purine auxotroph mutant SchuS4ΔpurMCD, were similarly targeted to autophagic vacuoles. Vacuoles containing replication-deficient bacteria were labeled with ubiquitin and the autophagy receptors SQSTM1/p62 and NBR1, and their formation was decreased in macrophages from either ATG5-, LC3B- or SQSTM1-deficient mice, indicating recognition by the ubiquitin-SQSTM1-LC3 pathway. While a fraction of both the wild-type and the replication-impaired strains were ubiquitinated and recruited SQSTM1, only the replication-defective strains progressed to autophagic capture, suggesting that wild-type Francisella interferes with the autophagic cascade. Survival of replication-deficient strains was not restored in autophagy-deficient macrophages, as these bacteria died in the cytosol prior to autophagic capture. Collectively, our results demonstrate that replication-impaired strains of Francisella are cleared by autophagy, while replication-competent bacteria seem to interfere with autophagic recognition, therefore ensuring survival and proliferation.

FTT0831c/FTL_0325 Contributes to Francisella tularensis Cell Division, Maintenance of Cell Shape, and Structural Integrity

“…Further, given that growth in macrophages resulted in similar altered bacterial morphology followed by subsequent intracellular destruction in late-forming LAMP-1-positive vacuoles, it is possible that autophagy maybe the principal clearance and or detection mechanism for structurally compromised F. tularensis Schu S4. This proposal is consistent with recent observations that nonviable mutants are captured within LAMP-1-positive autophagosomes as a clearance mechanism for damaged cytosolic Francisella (44). However, based on our data alone, we cannot exclude the possibility that this instead results from hypercytotoxicity of the Schu S4 ⌬0831 mutant toward macrophages in vitro and the release and subsequent secondary engulfment of gentamicin-killed bacteria.…”

“…The association of Schu S4 ⌬0831 bacteria with LAMP-1-positive vacuoles corresponded well with the appreciable loss of cell viability observed in gentamicin protection assays (compare Fig. 7A and C, far right panel) and since these same bacteria were previously cytosolic, must mean that the cells had reentered the endocytic pathway, possibly by autophagy (44), to be destroyed in association with LAMP-1-positive vacuoles. Taken as a whole, these results further substantiate the role of FTT0831c in the pathogenesis of virulent F. tularensis by suggesting an unusual intracellular fate resulting not from failure to escape the phagosome, but rather, altered growth and morphology and subsequent destruction of otherwise replicating intracytosolic bacteria by late-forming LAMP-1-positive vacuoles.…”

Intracellular Staphylococcus aureus eludes selective autophagy by activating a host cell kinase

“…15 However, diverse bacteria such as Listeria monocytogenes, Shigella flexneri and Francisella tularensis have evolved strategies to escape from the autophagic machinery. [16][17][18][19] Nevertheless, mechanisms of autophagic escape are still relatively unknown. 6,8 S. aureus has been connected to autophagy before, but the molecular mechanisms for autophagosome formation, autophagosomal escape and intracellular survival of S. aureus are controversial.…”

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

“…To add further complexity, there is recent evidence that manipulation of autophagy is used as a means by pathogens to acquire energy and nutrients. With regard to Francisella tularensis, indirect evidence implies that it uses autophagy to increase the cytosolic nutrient pool and thereby provides energy for its rapid cytosolic replication (20,21).…”