N-terminal region of Mannheimia haemolytica leukotoxin serves as a mitochondrial targeting signal in mammalian cells

Abstract

SummaryMannheimia haemolytica leukotoxin (LktA) is a member of the RTX toxin family that specifically kills ruminant leukocytes. Previous studies have shown that LktA induces apoptosis in susceptible cells via a caspase-9-dependent pathway that involves binding of LktA to mitochondria. In this study, using the bioinformatics tool MitoProt II we identified an N-terminal amino acid sequence of LktA that represents a mitochondrial targeting signal (MTS). We show that expression of this sequence, as a GFP fusion protein within mammalian cells, directs GFP to mitochondria. By immunoprecipitation we demonstrate that LktA interacts with the Tom22 and Tom40 components of the translocase of the outer mitochondrial membrane (TOM), which suggests that import of this toxin into mitochondria involves a classical import pathway for endogenous proteins. We also analysed the amino acid sequences of other RTX toxins and found a MTS in the N-terminal region of Actinobacillus pleuropneumoniae ApxII and enterohaemorrhagic Escherichia coli EhxA, but not in A. pleuropneumoniae ApxI, ApxIII, Aggregatibacter actinomycetemcomitans LtxA or the haemolysin (HlyA) from uropathogenic strains of E. coli. These findings provide a new evidence for the importance of the N-terminal region in addressing certain RTX toxins to mitochondria.

Enterohemorrhagic Escherichia coli Hemolysin Employs Outer Membrane Vesicles to Target Mitochondria and Cause Endothelial and Epithelial Apoptosis

“…A putative mitochondrial targeting signal (MTS) was recently identified in the N-terminal region of EHEC-Hly sequence [50], though the ability of the toxin to target mitochondria was not investigated. MTSs are also present in N-terminal regions of secreted proteins EspF [47], [68] and Map [48], [49] of enteropathogenic E. coli (EPEC), leukotoxin of Mannheimia hemolytica [50], and ApxII exotoxin of Actinobacillus pleuropneumoniae [50] and direct these proteins to mitochondria [47], [48], [50] likely exploiting the endogenous import machinery for nuclear-encoded mitochondrial proteins [42], [43], [69]. The presence of MTS in the EHEC-Hly molecule thus provides a mechanistical basis for our evidence of the toxin's mitochondria-targeting activity.…”

“…Because the TOM machinery is the most common pathway for mitochondrial delivery of MTS-possessing proteins [42], [69], the presence of MTS in the EHEC-Hly molecule suggests that EHEC-Hly might be imported in mitochondria via the TOM complex. Moreover, the presence of a putative cleavage site in the EHEC-Hly MTS [50], which is also present in the EspP and Map MTSs [47]–[49] and is indicative of proteins' import across the mitochondrial membranes [69] suggests that EHEC-Hly may be transported into the mitochondrial matrix [50]. However, as for many other bacterial virulence factors that target mitochondria [42], the mitochondrial sorting route of EHEC-Hly and the mechanism of the mitochondrial injury are not yet determined.…”

Structure and function of RTX toxins

“…Further studies showed that LktA is able to enter into and traffic to the mitochondria of BL-3 cells, where it interacts with the translocase of the outer membrane of mitochondria (TOM) and cyclophilin D, a member of the mitochondria permeability transition pore [384,385].…”

RTX Toxins of Animal Pathogens and Their Role as Antigens in Vaccines and Diagnostics

“…This causes the collapse of the mitochondrial membrane potential and release of cytochrome C. They demonstrated that this process occurred through internalization of LktA and binding to the mitochondrial matrix protein cyclophilin D, assumedly by dynamin-2-triggered targeting [27,58,59]. Targeting LktA to the mitochondrial membrane of BL-3 cells requires the amino-terminal 31 amino acids of LktA [60]. It is interesting to note that mitochondrial targeting motifs were found in the first 54 amino-terminal residues in several RTX toxins, including LktA, ApxII, and the enterohemorrhagic E. coli hemolysin EhxA, but not in ApxI, ApxIII, the E. coli hemolysin HlyA, and the leukotoxin LtxA of the human pathogen Aggregibacter actinomycetemcomitans [60].…”

“…Targeting LktA to the mitochondrial membrane of BL-3 cells requires the amino-terminal 31 amino acids of LktA [60]. It is interesting to note that mitochondrial targeting motifs were found in the first 54 amino-terminal residues in several RTX toxins, including LktA, ApxII, and the enterohemorrhagic E. coli hemolysin EhxA, but not in ApxI, ApxIII, the E. coli hemolysin HlyA, and the leukotoxin LtxA of the human pathogen Aggregibacter actinomycetemcomitans [60]. This amino terminus of RTX toxins that forms an amphipathic helix (Figure 1), is the most divergent domain, suggesting variation in their capacity to interact with specific lipid membranes in their target cells.…”