Cj1411c Encodes for a Cytochrome P450 Involved in Campylobacter jejuni 81-176 Pathogenicity

Cytochrome P450s are b-heme-containing enzymes that are able to introduce oxygen atoms into a wide variety of organic substrates. They are extremely widespread in nature having diverse functions at both biochemical and physiological level. The genome of C. jejuni 81-176 encodes a single cytochrome P450 (Cj1411c) that has no close homologues. Cj1411c is unusual in its genomic location within a cluster involved in the biosynthesis of outer surface structures. Here we show that E. coli expressed and affinity-purified C. jejuni cytochrome P450 is lipophilic, containing one equivalent Cys-ligated heme. Immunoblotting confirmed the association of cytochrome P450 with membrane fractions. A Cj1411c deletion mutant had significantly reduced ability to infect human cells and was less able to survive following exposure to human serum when compared to the wild type strain. Phenotypically following staining with Alcian blue, we show that a Cj1411c deletion mutant produces significantly less capsular polysaccharide. This study describes the first known membrane-bound bacterial cytochrome P450 and its involvement in Campylobacter virulence

The effect of <i>Cj1411c</i> gene deletion on CPS production.

<p>Alcian blue stained CPS of <i>C. jejuni</i> 81-176 wild type (lane 2) was compared with the CPS extracted <i>C. jejuni</i> 81-176 Δ<i>Cj1411c</i> (lane 3) and <i>C. jejuni</i> 81-176 Δ<i>Cj1411c</i>::<i>Cj1411c</i> (lane 4).</p

CYP1411c spectral change following incubation with the inner membrane fraction.

<p>IMP o/n 1-5 represent successive spectral recordings following incubation of purified CYP1411c protein with <i>C. jejuni</i> 81-176 inner membrane proteins. Spectra were recorded at 1 min intervals.</p

CYP1411c cellular localization.

<p>(A) Anti-P450 immunoblotting on bacterial fractions (cytosolic fraction, Cyt; inner membrane protein, IMP and outer membrane protein fraction, OMP). Recombinant CYP1411c protein (purified) was used as a positive control. Fractions were validated with an antibody recognizing the cytosolic Fur protein (B) and the outer membrane protein CadF (C).</p

Virulence and serum resitance.

<p>(a) adhesion to HCT-8 cells, (b) invasion of HCT-8 cells of <i>C. jejuni</i> 81-176 wild type, <i>C. jejuni</i> 81-176 Δ<i>Cj1411c</i> and <i>C. jejuni</i> 81-176 Δ<i>Cj1411c</i>::<i>Cj1411c</i>. (c) serum resistance - the survival rate is defined as the number of <i>C. jejuni</i> 81-176 colonies isolated following exposure to human serum divided by the number of colonies surviving in heat inactivated serum, expressed as a percentage. Statistical significance (Student’s <i>t</i> test) relative to the level of wild type strain is indicated. *P=0.001. The experiments were done in triplicate and on three separate occasions. The error bars represent standard deviations for six separate wells.</p

qRT-PCR analysis of <i>Cj1411c</i> gene expression during infection.

<p><i>Cj1411c</i> expression analysis in <i>C. jejuni</i> 81-176 following infection of HCT-8 cells. The gene expression level for 1.5h in RPMI was set to 1 as the basal level. All other levels are expressed as fold change over the basal gene expression. Error bars represent ±S.D. of 3 independent experiments. The corresponding protein expression levels assessed by Western blot with the anti-P450 antibody are shown.</p