Cj1386 Is an Ankyrin-Containing Protein Involved in Heme Trafficking to Catalase in Campylobacter jejuni

Abstract

Campylobacter jejuni, a microaerophilic bacterium, is the most frequent cause of human bacterial gastroenteritis. C. jejuni is exposed to harmful reactive oxygen species (ROS) produced during its own normal metabolic processes and during infection from the host immune system and from host intestinal microbiota. These ROS will damage DNA and proteins and cause peroxidation of lipids. Consequently, identifying ROS defense mechanisms is important for understanding how Campylobacter survives this environmental stress during infection. Construction of a ⌬Cj1386 isogenic deletion mutant and phenotypic assays led to its discovery as a novel oxidative stress defense gene. The ⌬Cj1386 mutant has an increased sensitivity toward hydrogen peroxide. The Cj1386 gene is located directly downstream from katA (catalase) in the C. jejuni genome. A ⌬katA⌬ Cj1386 double deletion mutant was constructed and exhibited a sensitivity to hydrogen peroxide similar to that seen in the ⌬Cj1386 and ⌬katA single deletion mutants. This observation suggests that Cj1386 may be involved in the same detoxification pathway as catalase. Despite identical KatA abundances, catalase activity assays showed that the ⌬Cj1386 mutant had a reduced catalase activity relative to that of wild-type C. jejuni. Heme quantification of KatA protein from the ⌬Cj1386 mutant revealed a significant decrease in heme concentration. This indicates an important role for Cj1386 in heme trafficking to KatA within C. jejuni. Interestingly, the ⌬Cj1386 mutant had a reduced ability to colonize the ceca of chicks and was outcompeted by the wild-type strain for colonization of the gastrointestinal tract of neonate piglets. These results indicate an important role for Cj1386 in Campylobacter colonization and pathogenesis.

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

“…4C). Indeed, the sensitivity of the ⌬cj1386ϩcj1386 Y57A strain also was similar to that of the ⌬katA mutant strain (25.30 mm [7]; not significant at P Ͼ 0.05). This result highlights that loss of tyrosine 57 results in a severe inability of C. jejuni to resist H 2 O 2 at levels comparable to those in the absence of the catalase enzyme itself.…”

“…Specifically, KatA immunoprecipitated from a ⌬cj1386 mutant had a significant reduction in the hemin content associated with its KatA relative to KatA isolated from the wild-type strain. In turn, the decreased hemin content of KatA in the ⌬cj1386 mutant resulted in reduced catalase activity within the cell and, consequently, sensitivity to H 2 O 2 (7). In this report, we further characterize Cj1386 function to demonstrate its direct role in hemin trafficking within C. jejuni.…”

“…Furthermore, due to the low expression levels of Cj1386 in wild-type C. jejuni (see Fig. S3 in the supplemental material), we subsequently probed for a KatA-Cj1386 interaction in a ⌬perR mutant background in which cj1386 is derepressed (7). Detection of a KatA-Cj1386 protein-protein interaction was observed by coimmunoprecipitation of KatA from cross-linked proteins isolated from the ⌬perR mutant strain and subsequent probing for Cj1386 using Western blot analysis (Fig.…”

“…Recently, our laboratory has identified a novel protein, Cj1386, that plays an important role in catalase biogenesis and the hemin content of C. jejuni KatA (7). We demonstrated that Cj1386 is required for optimal H 2 O 2 detoxification within the cell and the presence of hemin within KatA.…”

Inactivation of the LysR regulator Cj1000 of Campylobacter jejuni affects host colonization and respiration

“…The upregulation of the tpx, katA and ahpC transcripts coincided with a peak in neutrophil numbers in the chamber indicating the importance of these genes for oxidant detoxification in vivo. Furthermore, both katA and ahpC mutants exhibited a reduced ability to colonize the chick caecum compared to the wild-type strain (Palyada et al, 2009), and the katA mutant was recently shown to be outcompeted by the wild-type strain in the piglet infectious model (Flint et al, 2012) Other upregulated or downregulated genes Transcriptome analysis of the cj1000 : : aph mutant revealed upregulation of genes involved in flagellum biogenesis (flaA, flaB, flgG2, flgH, fliD and fliS), the exceptions being flaC and flhB, which were downregulated (Table 1). Despite these changes in transcript levels, the motility in vitro of the cj1000 mutant is unchanged compared to the wild-type strain (data not shown).…”