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Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence.

ABSTRACT: Analysis of the complete flagellin glycosylation locus of Campylobacter jejuni strain 81-176 revealed a less complex genomic organization than the corresponding region in the genome strain, C. jejuni NCTC 11168. Twenty-four of the 45 genes found between Cj1293 and Cj1337 in NCTC 11168 are missing in 81-176. Mutation of six new genes, in addition to three previously reported, resulted in a non-motile phenotype, consistent with a role in synthesis of pseudaminic acid (PseAc) or transfer of PseAc to flagellin. Mutation of Cj1316c or pseA had been shown to result in loss of the acetamidino form of pseudaminic acid (PseAm). Mutation of a second gene also resulted in loss of PseAm, as well as a minor modification that appears to be PseAm extended with N-acetyl-glutamic acid. Previously described mutants in C. jejuni 81-176 and Campylobacter coli VC167 that produced flagella lacking PseAm or PseAc failed to autoagglutinate. This suggests that interactions between modifications on adjacent flagella filaments are required for autoagglutination. Mutants (81-176) defective in autoagglutination showed a modest reduction in adherence and invasion of INT407 cells. However, there was a qualitative difference in binding patterns to INT407 cells using GFP-labelled 81-176 and mutants lacking PseAm. A mutant lacking PseAm was attenuated in the ferret diarrhoeal disease model.

SUBMITTER: Guerry P

PROVIDER: S-EPMC1424674 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence.

Guerry Patricia P Ewing Cheryl P CP Schirm Michael M Lorenzo Maria M Kelly John J Pattarini Dawn D Majam Gary G Thibault Pierre P Logan Susan S

Molecular microbiology 20060401 2

Analysis of the complete flagellin glycosylation locus of Campylobacter jejuni strain 81-176 revealed a less complex genomic organization than the corresponding region in the genome strain, C. jejuni NCTC 11168. Twenty-four of the 45 genes found between Cj1293 and Cj1337 in NCTC 11168 are missing in 81-176. Mutation of six new genes, in addition to three previously reported, resulted in a non-motile phenotype, consistent with a role in synthesis of pseudaminic acid (PseAc) or transfer of PseAc t ...[more]

PMID: 16573682

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Project description:UNLABELLED:Burkholderia cenocepacia causes opportunistic infections in plants, insects, animals, and humans, suggesting that "virulence" depends on the host and its innate susceptibility to infection. We hypothesized that modifications in key bacterial molecules recognized by the innate immune system modulate host responses to B. cenocepacia. Indeed, modification of lipopolysaccharide (LPS) with 4-amino-4-deoxy-L-arabinose and flagellin glycosylation attenuates B. cenocepacia infection in Arabidopsis thaliana and Galleria mellonella insect larvae. However, B. cenocepacia LPS and flagellin triggered rapid bursts of nitric oxide and reactive oxygen species in A. thaliana leading to activation of the PR-1 defense gene. These responses were drastically reduced in plants with fls2 (flagellin FLS2 host receptor kinase), Atnoa1 (nitric oxide-associated protein 1), and dnd1-1 (reduced production of nitric oxide) null mutations. Together, our results indicate that LPS modification and flagellin glycosylation do not affect recognition by plant receptors but are required for bacteria to establish overt infection. IMPORTANCE:Virulence and pathogenicity are properties ascribed to microbes, which actually require careful consideration of the host. Using the term "pathogen" to define a microbe without considering its host has recently been debated, since the microbe's capacity to establish a niche in a given host is a critical feature associated with infection. Opportunistic bacteria are a perfect example of microbes whose ability to cause disease is intimately related to the host's ability to recognize and respond to the infection. Here, we use the opportunistic bacterium Burkholderia cenocepacia and the host plant Arabidopsis thaliana to investigate the role of bacterial surface molecules, namely, lipopolysaccharide and flagellin, in contributing to infection and also in eliciting a host response. We reveal that both molecules can be modified by glycosylation, and although the modifications are critical for the bacteria to establish an infection, they do not impact the host's ability to recognize the pathogen.

Project description:Flagellin glycosylation plays a crucial role in flagellar assembly, motility and virulence in several pathogenic bacteria. However, little is known about the genetic determinants and biological functions of flagellin glycosylation in Xanthomonas oryzae pv. oryzae (Xoo), the causal pathogen of bacterial blight of rice. Here, the structure, regulation and functions of a ten-gene cluster gigX (glycosylation island genes of Xoo), which was embedded in a flagellar regulon, were characterized. gigX1 to gigX10 encoded putative enzymes or proteins involved in glycan biosynthesis and transfer, including a nucleotide sugar transaminase, an acyl-carrier protein (ACP), a 3-oxoacyl-ACP synthase, a 3-oxoacyl-ACP reductase, a dehydrogenase, an acetyltransferase, a ring hydroxylating dioxygenase, a hypothetical protein, a methyltransferanse and a glycosyltransferase, respectively. The gigX genes were co-transcribed in an operon and up-regulated by the upstream σ54 factor RpoN2 and transcriptional activator FleQ. In-frame deletion of each gigX gene affected flagellin glycosylation modification, meaning that the unglycosylated flagellin of the mutants was smaller than the glycosylated flagellin of the wild-type. No significant changes in flagellar filament and motility were observed in the ΔgigX mutants, among which only ΔgigX6 displayed increased swimming ability. Importantly, all mutants, except ΔgigX9, showed significantly increased virulence and bacterial growth in the susceptible rice cultivar IR24, and ΔgigX1 and ΔgigX10 showed enhanced type III secretion system (T3SS)-related gene expression. Moreover, the glycosylated flagellin of the wild-type induced higher H2 O2 levels in rice leaves than did the unglycosylated flagellins of ΔgigX1 or ΔgigX10. Taken together, this study reveals that the gigX cluster determines flagellin glycosylation, and implicates the regulatory role of post-translational modification with the glycosylation, acetylation and methylation of flagellin in the regulation of motility and virulence of Xoo.

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Project description:Campylobacter fetus is commonly associated with venereal disease and abortions in cattle and sheep, and can also cause intestinal or systemic infections in humans that are immunocompromised, elderly, or exposed to infected livestock. It is also believed that C. fetus infection can result from the consumption or handling of contaminated food products, but C. fetus is rarely detected in food since isolation methods are not suited for its detection and the physiology of the organism makes culturing difficult. In the related species, Campylobacter jejuni, the ability to colonize the host has been linked to N-linked protein glycosylation with quantitative proteomics demonstrating that glycosylation is interconnected with cell physiology. Using label-free quantitative (LFQ) proteomics, we found more than 100 proteins significantly altered in expression in two C. fetus subsp. fetus protein glycosylation (pgl) mutants (pglX and pglJ) compared to the wild-type. Significant increases in the expression of the (NiFe)-hydrogenase HynABC, catalyzing H2-oxidation for energy harvesting, correlated with significantly increased levels of cellular nickel, improved growth in H2 and increased hydrogenase activity, suggesting that N-glycosylation in C. fetus is involved in regulating the HynABC hydrogenase and nickel homeostasis. To further elucidate the function of the C. fetus pgl pathway and its enzymes, heterologous expression in Escherichia coli followed by mutational and functional analyses revealed that PglX and PglY are novel glycosyltransferases involved in extending the C. fetus hexasaccharide beyond the conserved core, while PglJ and PglA have similar activities to their homologs in C. jejuni. In addition, the pgl mutants displayed decreased motility and ethidium bromide efflux and showed an increased sensitivity to antibiotics. This work not only provides insight into the unique protein N-glycosylation pathway of C. fetus, but also expands our knowledge on the influence of protein N-glycosylation on Campylobacter cell physiology.

Dataset Information

Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence.

Publications

Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence.

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