Abstract
Listeriolysin O (LLO) is a cholesterol-dependent cytolysin that mediates escape of L. monocytogenes from phagosomes and enables the bacteria to grow within the host. LLO is a versatile tool allowing Listeria to trigger several cellular responses. In this study, rapid phosphorylation of ERK1/2 on Caco-2 cells caused by Listeria infection was demonstrated to be highly dependent on LLO activity. The effect could be strongly induced by adding purified recombinant LLO alone and could be inhibited by exogenous cholesterol. Lack of the PEST sequence, known to tightly control cytotoxicity of LLO, did not affect ERK1/2 activation. However, the recombinant non-cytolytic LLOT515AL516A, with mutations in the cholesterol-binding motif, was unable to trigger this response. Recombinant LLON478AV479A, which lacks most of the cytolytic activity, also failed to activate ERK1/2 phosphorylation, and this effect could be rescued when the protein concentration reached a cytolytic level. Infection with an LLO-deficient mutant (Δhly) or the mutant complementing LLOT515AL516A abrogated the capacity of the bacteria to activate ERK1/2. However, infection with the Δhly mutant complementing LLON478AV479A, which retained partial pore-forming ability and could grow intracellularly, was capable of triggering ERK1/2 phosphorylation. Collectively, these data suggest that ERK1/2 activation by L. monocytogenes depends on the permeabilization activity of LLO and more importantly correlates with the cholesterol-binding motif of LLO.
Highlights
Listeria monocytogenes, a facultative intracellular pathogen that has the capacity to actively invade and multiply within mammalian cells, is the causative agent of listeriosis, which affects immunocompromised individuals, as well as pregnant women and elderly people [1, 2]
Caco-2 cells incubated with as little as ∼1.5 hemolytic units (5 nM, according to the hemolytic activity assay in this study) of the purified Listeriolysin O (LLO) showed a more rapid phosphorylated extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation starting at 5 min incubation with a maximum at 15 and 30 min, and decreasing as the incubation time continued (Figure 1C)
Previous studies have documented that pretreatment of LLO with cholesterol inhibits hemolytic activity but does not interfere with the initial binding step of LLO to natural membranes [32, 33], we demonstrated that inhibition of ERK1/2 phosphorylation by pre-incubation of LLO with cholesterol was definitely due to loss of cytolytic activity but our data do not rule out an important role for cholesterol in the binding of LLO to the cell membrane
Summary
A facultative intracellular pathogen that has the capacity to actively invade and multiply within mammalian cells, is the causative agent of listeriosis, which affects immunocompromised individuals, as well as pregnant women and elderly people [1, 2]. LLO is a phagosome-specific cytolysin that forms pores in host membranes and is continuously expressed throughout the intracellular lifecycle of L. monocytogenes. Uncontrolled expression of LLO could lead to perforation of organelles and the host plasma membrane from the inside of the cell, causing cell death and destruction of the intracellular niche of L. monocytogenes, and thereby exposing the bacteria to the host immune system [5, 7, 8]. L. monocytogenes tightly controls synthesis and activity of LLO to disrupt vacuolar membranes without killing host cells. It is well-established that L. monocytogenes mutants with increased LLO expression or activity efficiently escape from vacuoles but are less virulent because they over-toxic to host cells, thereby destroying their intracellular niche [9]. The PEST-like sequence of LLO near its N-terminus that reduces the intracellular toxicity of this cytolysin is necessary for L. monocytogenes to better survive intracellularly following escape from phagocytic vacuoles [6, 11]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
