Channel Formation by LktA of Mannheimia (Pasteurella) haemolytica in Lipid Bilayer Membranes and Comparison of Channel Properties with Other RTX-Cytolysins.

Roland Benz,Claudio Piselli,Andrew Potter

doi:10.3390/toxins11100604

Roland Benz, Claudio Piselli + Show 1 more

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https://doi.org/10.3390/toxins11100604

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Journal: Toxins	Publication Date: Oct 17, 2019
Citations: 1	License type: CC BY 4.0

Affiliation: Constructor University, University of Saskatchewan

Abstract

Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes. Higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. In this study we investigated channel formation by LktA in artificial lipid bilayer membranes made of different lipids. LktA purified from culture supernatants by polyethylene glycol 4000 precipitation and lyophilization had to be activated to frequently form channels by solution in 6 M urea. The LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of adenylate cyclase toxin of Bordetella pertussis. The LktA channels are highly cation-selective caused by negative net charges. The theoretical treatment of the conductance of LktA as a function of the bulk aqueous concentration allowed a rough estimate of the channel diameter, which is around 1.5 nm. The size of the LktA channel is discussed with respect to channels formed by other RTX-toxins. We present here the first investigation of LktA in a reconstituted system.

Highlights

Mannheimia haemolytica is a Gram-negative, facultative anaerobic bacterium of the family Pasteurellaceae within the class Gammaproteobacteria [1]
Key Contribution: This study demonstrates that the primary effect of the RTX–toxin LktA of Mannheimia haemolytica on ovine leukocytes is the formation of cation-selective channels that lead to the dissipation of ionic gradients across the cytoplasmic membranes followed by cell lysis and apoptosis
We extended the single-channel analysis of LktA to other conditions and changed the KClconcentration and the composition of the aqueous salts

Summary

Introduction

Mannheimia (formerly Pasteurella) haemolytica is a Gram-negative, facultative anaerobic bacterium of the family Pasteurellaceae within the class Gammaproteobacteria [1]. This pathogenic bacterium is responsible for the economically important loss of cattle and sheep during transport that can account for up to 1 million US$ per year [2,3]. Different strains of M. haemolytica have been characterized for the presence of pathogenicity factors [8] Most of these factors need further evaluation of their role in pasteurellosis, but it is quite clear that lipopolysaccharide (LPS) and leukotoxin (LktA) represent the major pathogenicity factors of

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