JB Accepts, published online ahead of print on 12 October 2007

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J. Bacteriol. doi:10.1128/JB.00436-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Burkholderia cenocepacia C5424 Produces a Pigment with Antioxidant Properties Using a Homogentisate Intermediate

Karen E. Keith, Lauren Killip, Panqing He, Graham R. Moran, and Miguel A. Valvano^*

Infectious Diseases Research Group, Siebens-Drake Research Institute, Departments of Microbiology and Immunology, and Medicine, University of Western Ontario, London, Ontario, N6A 5C1, Canada. Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N. Cramer Street, Milwaukee, Wisconsin, 53211

^* To whom correspondence should be addressed. Email: mvalvano{at}uwo.ca.

Burkholderia cenocepacia is a gram-negative opportunistic pathogen that belongs to the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly within phagocytic cells and some epidemic strains produce a brown melanin-like pigment that can scavenge free radicals, resulting in the attenuation of the host cell oxidative burst. In this work, we demonstrate that the brown pigment produced by B. cenocepacia C5424 is synthesized from a homogentisate (HGA) precursor. The disruption of BCAL0207 (hppD) by insertional inactivation resulted in loss of pigmentation. Steady-state kinetic analysis of the BCAL0207 gene product demonstrated that it has 4-hydroxyphenylpyruvic acid dioxygenase (HPPD) activity. Pigmentation could be restored by the addition of hppD in trans. The hppD mutant was resistant to paraquat challenge, but sensitive to H₂O₂ and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the non-pigmented bacteria co-localized in a dextran positive vacuole, suggesting that they are being trafficked to the lysosome. In contrast, the wild-type strain did not localize with dextran. Co-localization of the non-pigmented strain with dextran was reduced in the presence of the NADPH oxidase inhibitor, diphenyleneiodonium, and also the inducible nitric oxide inhibitor aminoguanidine. Together, these observations suggest that the brown pigment produced by B. cenocepacia C5424 is a pyomelanin synthesized from a HGA intermediate, which is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.

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