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

Abstract

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 complementation providing hppD in trans. The hppD mutant was resistant to paraquat challenge but sensitive to H 2 O 2 and to extracellularly generated superoxide anions. Infection experiments in RAW 264.7 murine macrophages showed that the nonpigmented bacteria colocalized 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. Colocalization of the nonpigmented 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 an HGA intermediate that is capable of protecting the organism from in vitro and in vivo sources of oxidative stress.

A Putative ABC Transporter, HatABCDE, Is among Molecular Determinants of Pyomelanin Production inPseudomonas aeruginosa

“…Pyomelanin-producing strains are generally considered to be less virulent, though more persistent, and it is thought that the presence of pyomelanin alone increases resistance to oxidative stress (28,49,68). It will be interesting to determine whether mutants impaired in melanogenesis, such as the hat mutant and others identified in our screen, might be competitively disadvantaged relative to melanogenic strains for long-term survival.…”

“…Proposed roles for pyomelanin include the enhancement of bacterial surface attachment (20), extracellular electron transfer (61), iron reduction/acquisition (8), induction of virulence factor expression (63), heavy metal binding (21), and protection from environmental stress (11,28,32,44,53,65). A protective role has also been proposed to occur in P. aeruginosa PA14, where pyomelanin was shown to contribute to the persistence of an overproducing strain in a chronic CF infection model in mice (49).…”

“…Notably, isolates of other bacterial species associated with chronic infections of the CF lung, Burkholderia cenocepacia and Stenotrophomonas maltophilia, can also be melanogenic (28,58), suggesting a possible role for this pigment in the establishment and/or persistence of infection. Some genera produce melanin under normal conditions via polyphenol oxidases or laccases, while others synthesize the pigment only in response to specific environmental conditions (17,35).…”

Melanins: Skin Pigments and Much More—Types, Structural Models, Biological Functions, and Formation Routes

“…Thus, homogentisic acid is subsequently oxidized and an ochre-colored polymer is formed. More recently, this type of melanin has also been characterized in Burkholderia cenocepacia, a gram-negative opportunistic pathogen that can survive within phagocytic cells, and the synthesis of this melanin is a defense mechanism against the host cell [149], similarly to some pathogenic fungus, such as Aspergillus fumigates [124]. Moreover, pyomelanin from HGA has some relevance since the situation is comparable to the human alkaptonuria [147,148], a rare disease due to mutations in the homogentisate dioxygenase human gene.…”

Comparative Metabolic Systems Analysis of Pathogenic Burkholderia

“…Ornibactin enables iron acquisition, and salicylate potentially acts as a siderophore and is also required for production of other siderophores; both compounds have been connected with enhanced virulence both clinically and in animal models (48). Homogentisate is a precursor of the melanin-like pigment produced by some Burkholderia species that provides protection from reactive oxygen and nitrogen species (66,67).…”