Cj1386, an Atypical Hemin-Binding Protein, Mediates Hemin Trafficking to KatA in Campylobacter jejuni

Abstract

. While the structure and biochemical properties of catalase enzymes have been well characterized over many decades of research, it remained unclear how catalases acquire hemin. We have previously reported that Cj1386 is essential for ensuring proper hemin content in Campylobacter jejuni catalase (KatA) (A. Flint, Y. Q. Sun, and A. Stintzi, J Bacteriol 194:334 -345, 2012). In this report, an in-depth molecular characterization of Cj1386 was performed to elucidate the mechanistic details of this association. Coimmunoprecipitation assays revealed that KatA-Cj1386 transiently interact in vivo, and UV-visible spectroscopy demonstrated that purified Cj1386 protein binds hemin. Furthermore, hemin titration experiments determined that hemin binds to Cj1386 in a 1:1 ratio with hexacoordinate hemin binding. Mutagenesis of potential hemin-coordinating residues in Cj1386 showed that tyrosine 57 was essential for hemin coordination when Cj1386 was overexpressed in Escherichia coli. The importance of tyrosine 57 in hemin trafficking in vivo was confirmed by introducing the cj1386 Y57A allele into a C. jejuni ⌬cj1386 mutant background. The cj1386 Y57A mutation resulted in increased sensitivity toward H 2 O 2 relative to the wild type, suggesting that KatA was not functional in this strain. In support of this finding, KatA immunoprecipitated from the ⌬cj1386؉cj1386 Y57A mutant had significantly reduced hemin content compared to that of the cj1386 WT background. Overall, these findings indicate that Cj1386 is involved in directly trafficking hemin to KatA and that tyrosine 57 plays a key role in this function.

The adaptive transcriptional response of pathogenicLeptospirato peroxide reveals new defenses against infection-related oxidative stress

“…The immediate upstream ORF (LIMLP_10150), encoding an ankyrin repeat-containing protein, was also up-regulated with a comparable fold. In bacteria such as Pseudomonas aeruginosa and Campylobacter jejuni, protein with ankyrin repeats were found to be required for the catalase activity, probably by allowing heme binding (19,20). In L. interrogans, katE and ank were organized as an operon (S1 Fig) and significant up-regulation of the ank-katE operon upon exposure to sublethal dose of H 2 O 2 was confirmed by RT-qPCR (Table 1 and S1 Fig).…”

PerR controls oxidative stress defence and aerotolerance but not motility-associated phenotypes of Campylobacter jejuni

“…Exposure of C. jejuni perR mutants to hydrogen peroxide on discs revealed that the level of peroxide resistance was considerably higher than previously reported, where 3 % hydrogen peroxide was used (Baillon et al, 1999;Flint & Stintzi, 2015;Palyada et al, 2009;van Vliet et al, 1999). Wild-type C. jejuni were sensitive to 5 % H 2 O 2 (Fig.…”

“…More importantly, the high levels of, especially, catalase resulted in a significant increase in resistance against hydrogen peroxide, with the perR mutant able to grow close to discs infused with 30 % (8.8 M) hydrogen peroxide, which is the highest commercially available concentration of hydrogen peroxide. This concentration is much higher than previously tested, where either 3 % hydrogen peroxide was used (Baillon et al, 1999;Flint & Stintzi, 2015;Palyada et al, 2009;van Vliet et al, 1999), or concentrations ranging from 1-5 mM (Huergo et al, 2013;Hwang et al, 2011a, b), and highlights the very high levels of catalase expression in the perR mutant. A similar phenotype (albeit not as striking) was observed (Chaudhuri et al, 2011).…”

Oxidative and nitrosative stress defences ofHelicobacterandCampylobacterspecies that counteract mammalian immunity

“…Characterisation of the Cj1386 protein revealed that it binds haem b at a 1:1 ratio and that it interacts with KatA (Flint and Stintzi 2015). Similarly to H. pylori KapA, Cj1386 did not show catalase activity, but was found to indirectly contribute towards optimal catalase activity within cells (Flint, Sun and Stintzi 2012).…”

“…Furthermore, these catalase-negative strains lack homologues of the haem-trafficking protein, cj1386 (Flint and Stintzi 2015). Recently, Koolman et al have investigated the presence of sodB and katA by PCR in 24 Campylobacter isolates obtained from poultry or human origin belonging to the C. jejuni, C. coli and C. lari species.…”