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. 2001 Oct;69(10):6225-30.
doi: 10.1128/IAI.69.10.6225-6230.2001.

Ferrochelatase is present in Brucella abortus and is critical for its intracellular survival and virulence

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Free PMC article

Ferrochelatase is present in Brucella abortus and is critical for its intracellular survival and virulence

M Almirón et al. Infect Immun. 2001 Oct.
Free PMC article

Abstract

Brucella spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established, Brucella spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of Brucella abortus. During a B. abortus genomic sequencing project, a clone coding a putative gene homologous to hemH was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type hemH. These data demonstrate that B. abortus synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of B. abortus and thus for the establishment of chronic disease as well.

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Figures

FIG. 1
FIG. 1
Hemin auxotrophy of B. abortus 2308HM and genetic complementation of the phenotype by hemH. Five microliters from an overnight culture of 2308 (1), 2308HM (2), or 2308HMC (3) was spotted onto BB agar (A) or BB agar supplemented with hemoglobin (B), fetal bovine serum (C), hemin (D), iron citrate (E), or iron chloride (F). The six-well plate was incubated for 5 days at 37°C. The hemH mutant (spot 2) grew only in the presence of hemin and showed prototrophy when complemented with pBBRhemH (spot 3).
FIG. 2
FIG. 2
Intracellular survival of B. abortus strains in nonprofessional (A) and professional (B) phagocytes. (A) HeLa cells (105 cells/well) were infected with B. abortus 2308 (open circles), 2308HM (closed circles), and 2308HMC (open triangles) as described in Materials and Methods. At different times p.i., the cells were lysed, and the numbers of viable intracellular bacteria (CFU/milliliter) were determined. (B) J774 cells were infected at an MOI of 50 with the same strains as those used in panel A as indicated in Materials and Methods. formula image, below the experimental threshold of detection. Data represent means and standard deviations from one experiment performed in duplicate and are representative of four independent experiments.

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