1.6.19
MECHANISMS OF BACTERIAL RESISTANCE TO HOST DEFENCE PROTEINS

E LOPEZ-SOLANILLA, F GARCIA-OLMEDO and P RODRIGUEZ-PALENZUELA

Department of Biotechnology, ETS Ingenieros Agronomos, UPM Av. Complutense, 28040 Madrid, Spain

Background and objectives
An effective defence mechanism against invading pathogens, shared by plants and animals, is mediated by small cationic proteins such as cecropins, magainins and defensins from animals, and thionins and snakins from plants. These proteins exert a toxic action against bacteria, possibly through the alteration of membrane permeability. The role of antimicrobial peptides in pathogenesis has been highlighted by the observation of increased susceptibility to infection in Drosophila mutants affected in their synthesis, but no such evidence is available with respect to plant peptides, because appropriate plant mutants with decreased peptide levels have not yet been obtained. An alternative line of evidence about the role of antimicrobial peptides is based in the production of peptide-sensitive mutants of the pathogen. The hypothesis that the peptides are involved in defence would be supported by a decrease of virulence in this type of mutants. Indeed, in the animal pathogen Salmonella typhimurium, both rough lipopolysaccharide (LPS) mutants and sapA-F mutants, which show increased sensitivity to antimicrobial peptides (sap stands for sensitive to antimicrobial peptides), have reduced virulence, suggesting that resistance to host peptides has a direct role in Salmonella pathogenesis [1]. Similarly, we have previously found that thionin- and LTP-sensitive mutants of Ralstonia (Pseudomonas) solanacearum were both altered in their LPS structure and are avirulent in tobacco [2].

Erwinia chrysanthemi is an economically important phytopathogenic bacterium that causes soft-rot diseases in a wide range of crops. Little is known about the mechanisms that enable E. chrysanthemi to resist the action of antimicrobial agents from the plant host. Although S. typhimurium and E. chrysanthemi have very different pathogenic behaviour, the facts that they are phylogenetically related and that antimicrobial peptides occur in their respective animal and plant hosts lead us to investigate the possible role of the Sap system in plant-pathogen interactions.

Results and conclusions
The sapA-F operon from the pathogenic bacterium E. chrysanthemi has been characterized. It has five ORFs that are closely related (71% overall amino acid identity) and are in the same order as those of the sapA-F operon from S. typhimurium. An E. chrysanthemi sap mutant strain, BT105, was obtained by marker-exchange. Mutant BT105 was more sensitive than the wild-type to wheat alpha-thionin and to snakin-1, the most abundant antimicrobial peptide from potato tubers. Mutant BT105 was less virulent than the wild type in potato tubers: lesion area was 37% of control and growth rate was two orders of magnitude lower. The magnitude of the effect of sapA-F inactivation on virulence in potato tubers and chicory leaves was greater than the effects of mutations affecting the pel operon and the Hrp system in the same pathogen. These results indicate that the interaction of antimicrobial peptides from the host with the sapA-Foperon from the pathogen plays a similar role in animal and in plant bacterial pathogenesis. They also show that this operon is important for the pathogenicity of E. chrysanthemi.

References
1. Parra-Lopez C, Baer MT, Groisman EA, 1993. EMBO Journal 12, 4053-4062.
2. Titarenko E, Lopez-Solanilla E, Garcia-Olmedo F, Rodriguez-Palenzuela P, 1997. Journal of Bacteriology 179, 6699-6704.