Abstract
We examined two variants of the genome-sequenced strain, Campylobacter jejuni NCTC11168, which show marked differences in their virulence properties including colonization of poultry, invasion of Caco-2 cells, and motility. Transcript profiles obtained from whole genome DNA microarrays and proteome analyses demonstrated that these differences are reflected in late flagellar structural components and in virulence factors including those involved in flagellar glycosylation and cytolethal distending toxin production. We identified putative sigma(28) and sigma(54) promoters for many of the affected genes and found that greater differences in expression were observed for sigma(28)-controlled genes. Inactivation of the gene encoding sigma(28), fliA, resulted in an unexpected increase in transcripts with sigma(54) promoters, as well as decreased transcription of sigma(28)-regulated genes. This was unlike the transcription profile observed for the attenuated C. jejuni variant, suggesting that the reduced virulence of this organism was not entirely due to impaired function of sigma(28). However, inactivation of flhA, an important component of the flagellar export apparatus, resulted in expression patterns similar to that of the attenuated variant. These findings indicate that the flagellar regulatory system plays an important role in campylobacter pathogenesis and that flhA is a key element involved in the coordinate regulation of late flagellar genes and of virulence factors in C. jejuni.
Highlights
Campylobacter jejuni is a significant food- and water-borne pathogen [1, 2]
Identified in the genome sequence of C. jejuni NCTC11168 [11, 12], suggesting that certain pathways in this organism may be coordinately regulated. rpoD encodes 70, which is involved in regulating expression of housekeeping genes in C. jejuni [13], whereas a number of flagellar genes are regulated by the alternative sigma factors, rpoN (54) and fliA (28) (14 –17)
In Gram-negative bacteria, flagellar biosynthesis is regulated in a hierarchical cascade, with genes expressed in the order in which they are required for the assembly of the flagellum
Summary
Bacterial Strains and Growth Conditions—C. jejuni NCTC11168 (HS:2) was originally isolated from a case of human enteritis in 1977 [32] and later sequenced by Parkhill et al [12]. Isolation and Labeling of Total RNA and Genomic DNA for Microarray Analysis—C. jejuni cells were harvested after 15 h of growth and homogenized in Trizol (Invitrogen) by passing the mixture through a syringe. Quantitative Analysis of Gene Expression by Real Time PCR—cDNA was synthesized as described above from RNA samples used in microarray experiments, except RNA was DNase-treated (1 unit of DNase; Ambion) and aminoallyl-dUTP was replaced with dTTP. Real time PCR analysis was performed using an iCycler iQ detection system (Bio-Rad), with a PCR condition consisting of 2 min at 95 °C and 40 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 60 s (primer sequences available upon request).
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