Pseudomonas aeruginosa O-antigen chain length is determined before ligation to lipid A core

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that infects immunocompromised patients and trauma victims and causes fatal lung infections in people with cystic fibrosis. This microorganism produces a number of virulence factors, one of which is lipopolysaccharide (LPS), which has been shown to mediate many biological effects including resistance to serum killing and phagocytosis. These biological activities have been correlated to the length of the O-polysaccharide and its distribution on the outer membrane. Wzz is responsible for regulation of the size distribution of the O-antigen. Wzz has been found to participate solely in the Wzy-dependent pathway for LPS biosynthesis, which produces heteropolymeric O-polysaccharide such as the B-band LPS of P. aeruginosa . Our laboratory has previously reported characterization of a Wzz protein encoded in the B-band O-antigen biosynthesis cluster of PAO1. The availability of the genome sequence of P. aeruginosa PAO1 has made it possible to identify a second functional Wzz protein (PA0938, Wzz(2)). Gene replacement was used to generate an unmarked wzz(2)Delta knock-out and a wzz(2)Delta/wzz(1)::Gm double knock-out. As expected, the wzz(2)Delta strain produced LPS with modal length imparted by Wzz(1), and the wzz(2)Delta/wzz(1)::Gm strain produced LPS O-antigen with a non-modal (random) length. Both wzz(1) and wzz(2) from P. aeruginosa PAO1 were cloned and expressed with an N-terminal His(6) tag. His(6)-Wzz(1) and His(6)-Wzz(2) were purified to near homogeneity by immobilized metal affinity chromatography (IMAC). These preparations were used to develop specific polyclonal antibodies against each of the proteins. In vivo protein cross-linking followed by Western immunoblotting indicated that Wzz(1) forms dimers whereas Wzz(2) forms octamers. By generation of a wzz(2)Delta/rmlC double mutant and analysis of the LPS, we have made the novel observation that polymerization of modal chain length-distributed O-antigen occurred before ligation to the lipid A core. We have shown an association between the Wzz proteins and O-antigen polymer chains using immunoprecipitation with anti-O5 O-antigen monoclonal antibody MF15-4. Both Wzz(1) and Wzz(2) could be co-precipitated with O5 polymer.