Characterization of Two Novel Lipopolysaccharide Phosphoethanolamine Transferases in Pasteurella multocida and Their Role in Resistance to Cathelicidin-2

Abstract

The lipopolysaccharide (LPS) produced by the Gram-negative bacterial pathogen <i>Pasteurella multocida</i> has phosphoethanolamine (PEtn) residues attached to lipid A, 3-deoxy-d-manno-octulosonic acid (Kdo), heptose, and galactose. In this report, we show that PEtn is transferred to lipid A by the <i>P. multocida</i> EptA homologue, PetL, and is transferred to galactose by a novel PEtn transferase that is unique to <i>P. multocida</i> called PetG. Transcriptomic analyses indicated that <i>petL</i> expression was positively regulated by the global regulator Fis and negatively regulated by an Hfq-dependent small RNA. Importantly, we have identified a novel PEtn transferase called PetK that is responsible for PEtn addition to the single Kdo molecule (Kdo₁), directly linked to lipid A in the <i>P. multocida</i> glycoform A LPS. <i>In vitro</i> assays showed that the presence of a functional <i>petL</i> and <i>petK</i>, and therefore the presence of PEtn on lipid A and Kdo₁, was essential for resistance to the cationic, antimicrobial peptide cathelicidin-2. The importance of PEtn on Kdo₁ and the identification of the transferase responsible for this addition have not previously been shown. Phylogenetic analysis revealed that PetK is the first representative of a new family of predicted PEtn transferases. The PetK family consists of uncharacterized proteins from a range of Gram-negative bacteria that produce LPS glycoforms with only one Kdo molecule, including pathogenic species within the genera <i>Vibrio</i>, <i>Bordetella</i>, and <i>Haemophilus</i> We predict that many of these bacteria will require the addition of PEtn to Kdo for maximum protection against host antimicrobial peptides.