monocytogenes infection because a number of other fission-inducing agents (the protonophore FCCP, the potassium ionophore valinomycin, and the detergent digitonin) did not show a decrease in mitochondria-associated Drp1 ( Fig. monocytogenes infection were characterized by a decrease in Drp1 puncta associated with mitochondria ( Fig. (Scale bars: 10 µm.) Mitochondrial Drp1 Oligomers Dissociate upon Infection or LLO Treatment.įission-inducing agents such as uncouplers or the apoptosis inducer staurosporine appear to act by recruiting Drp1 to fragmenting mitochondria ( Fig. For C and D, the white box indicates a 2× enlarged region, which is shown below. monocytogenes induces mitochondrial fragmentation and loss of mitochondria-associated Drp1, whereas infection with the Δhly mutant does not overtly affect Drp1. monocytogenes or L.monocytogenes Δhly (LLO −) at an MOI of 50 and stained for Drp1 (green), mitochondria (CoxIV red), and nuclei/bacteria with DAPI (blue). ( D) HeLa cells were infected for 1 h with L. Staining reveals that mitochondrial Drp1 oligomers are lost upon LLO treatment (decrease in green puncta) but are retained upon CCCP or staurosporine treatment. Drp1 is shown in green (Drp1NT), mitochondria in red (Tom20), and nuclei in blue (DAPI). ( C) Immunofluorescence labeling of the same cells analyzed in B. ( B) HeLa cells were treated for 30 min with 10 µM CCCP, for 1.5 h with 1.2 µM staurosporine (sts), or with 6 nM LLO for the indicated timepoints, and total cell lysates are analyzed by Western blotting, showing no apparent decrease in total Drp1 levels upon LLO or drug treatment.
( A) Western blot of HeLa cells treated for 1, 5, or 10 min with 6 nM LLO or for 10 min with 10 µM CCCP and probed for Opa1, which is readily processed upon CCCP, but not LLO treatment. Analysis of Opa1 and Drp1 upon LLO-induced mitochondrial fragmentation. monocytogenes causes fragmentation of the host mitochondrial network by action of its pore-forming toxin LLO before bacterial entry ( 15).įig. For example, we have shown that infection with L. More recently, LLO has been found to have several intracellular and extracellular roles that extend beyond phagosomal escape. CDCs oligomerize on cholesterol-containing membranes to form nonselective ion-permeable pores of variable sizes ( 14) that act in concert with bacterial phospholipases to allow bacterial escape from the phagosome. LLO belongs to the family of cholesterol-dependent cytolysins (CDCs), most of which are produced by extracellular bacteria such as Streptococci or Clostridia. One of the most important virulence factors is listeriolysin O (LLO), a highly regulated secreted pore-forming toxin (reviewed in ref. monocytogenes correlates with the expression of several virulence genes ( 12). Listeria monocytogenes is a foodborne pathogen capable of invading nonphagocytic cells, where it can replicate and spread. We show that the ER marks LLO-induced mitochondrial fragmentation sites even in the absence of functional Drp1, demonstrating that the ER activity in regulating mitochondrial fission can be induced by exogenous agents and that the ER appears to regulate fission by a mechanism independent of the canonical mitochondrial fission machinery. LLO does not cause processing of the fusion protein optic atrophy protein 1 (Opa1), despite inducing a decrease in the mitochondrial membrane potential, suggesting a unique Drp1- and Opa1-independent fission mechanism distinct from that triggered by uncouplers or the apoptosis inducer staurosporine. Strikingly, LLO-induced mitochondrial fragmentation does not require the traditional fission machinery, as Drp1 oligomers are absent from fragmented mitochondria following Listeria infection or LLO treatment, as the dynamin-like protein 1 (Drp1) receptor Mff is rapidly degraded, and as fragmentation proceeds efficiently in cells with impaired Drp1 function. Here, we examine factors involved in canonical fusion and fission. We recently showed that infection by Listeria monocytogenes causes mitochondrial network fragmentation through the secreted pore-forming toxin listeriolysin O (LLO).