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 28 and 54 promoters for many of the affected genes and found that greater differences in expression were observed for 28 -controlled genes. Inactivation of the gene encoding 28 , fliA, resulted in an unexpected increase in transcripts with 54 promoters, as well as decreased transcription of 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 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.

Analysis of the genomes of two distantly related bird species, chicken and zebra finch (divergence of about 100 million years), indicate that there are ten avian toll-like receptors and that five of these, TLR2a, 2b, 3, 4, 5 and 7, are clear orthologs to TLRs found in mammals. Duplication of genes has led to TLR1La and 1Lb, TLR2a and 2b, and two TLR7s in the zebra finch. Avian TLR21 may be orthologous to TLR21 found in fish and amphibians, and avian TLR15, which is phylogenetically related to the TLR2 family, appears to be unique to avian species. While TLR2 is conserved between mammalian and avian species, the other TLR2 family members evolved independently. Dimerization between either of the two avian TLR2 species and TLR1La or 1Lb permits recognition of the same broad range of molecules as recognized by mammalian TLR2 dimerized with either TLR1, 6 and 10. Similarly, while TLR9 has been lost from the avian genome, DNA high in unmethylated CpG motifs is immunostimulatory through avian TLR21 which is absent in mammals. Thus, while some TLR members were commonly retained in both mammals and birds, others were separately lost or gained, or diverged independently; but broadly speaking the TLRs of the two classes of vertebrates evolved to recognize very similar spectra of microbial products. Components of downstream TLR signaling are also mostly conserved but with some losses in avian species; notably, TRAM is absent in avian genomes and, hence, the TRIF/TRAM-dependent signaling pathway utilized by mammals in LPS activation appears to be absent in birds.

In eukaryotes, N-linked protein glycosylation is a universal modification involving addition of preformed oligosaccharides to select Asn-Xaa-Ser/Thr motifs and influencing multiple biological events. We recently demonstrated that Campylobacter jejuni is the first member of the Bacteria to possess an N-linked glycan pathway. In this study, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) was applied to probe and quantitate C. jejuni N-glycan biosynthesis in vivo. To confirm HR-MAS NMR findings, glycosylation mutants were screened for chicken colonization potential, and glycoproteins were examined by mass spectrometry and lectin blotting. Consistent with the mechanism in eukaryotes, the combined data indicate that bacterial glycans are assembled en bloc, emphasizing the evolutionary conservation of protein N glycosylation. We also show that under the conditions examined, PglG plays no role in glycan biosynthesis, PglI is the glucosyltransferase and the putative ABC transporter, and WlaB (renamed PglK) is required for glycan assembly. These studies underpin the mechanism of N-linked protein glycosylation in Bacteria and provide a simple model system for investigating protein glycosylation and for exploitation in glycoengineering.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, relatively little is known about C. jejuni's precise pathogenesis mechanisms, particularly in comparison to other well-studied enteric organisms such as Escherichia coli and Salmonella spp. Altered expression of phosphate genes in a C. jejuni stringent response mutant, together with known correlations between the stringent response, polyphosphate (poly-P), and virulence in other bacteria, led us to investigate the role of poly-P in C. jejuni stress survival and pathogenesis. All sequenced C. jejuni strains harbor a conserved putative polyphosphate kinase 1 predicted to be principally responsible for poly-P synthesis. We generated a targeted ppk1 deletion mutant (⌬ppk1) in C. jejuni strain 81-176 and found that ⌬ppk1, as well as the ⌬spoT stringent response mutant, exhibited low levels of poly-P at all growth stages. In contrast, wild-type C. jejuni poly-P levels increased significantly as the bacteria transitioned from log to stationary phase. Phenotypic analyses revealed that the ⌬ppk1 mutant was defective for survival during osmotic shock and low-nutrient stress. However, certain phenotypes associated with ppk1 deletion in other bacteria (i.e., motility and oxidative stress) were unaffected in the C. jejuni ⌬ppk1 mutant, which also displayed an unexpected increase in biofilm formation. The C. jejuni ⌬ppk1 mutant was also defective for the virulence-associated phenotype of intraepithelial cell survival in a tissue culture infection model and exhibited a striking, dose-dependent chick colonization defect. These results indicate that poly-P utilization and accumulation contribute significantly to C. jejuni pathogenesis and affect its ability to adapt to specific stresses and stringencies. Furthermore, our study demonstrates that poly-P likely plays both similar and unique roles in C. jejuni compared to its roles in other bacteria and that poly-P metabolism is linked to stringent response mechanisms in C. jejuni.

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