Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion

Javed, Muhammad Asif; Grant, A.; Bagnall, Mary C.; Maskell, Duncan J.; Newell, Diane G.; Manning, Georgina

doi:10.1099/mic.0.033399-0

Cited by 28 publications

(32 citation statements)

References 45 publications

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“…However, it may be possible that Fuc ϩ strains of C. jejuni have a competitive advantage in another host species due to variation in nutrient availability (e.g., amino acids), microbiome, and structural differences of host mucin. Interestingly, transposon mutagenesis identified fucP as an invasion determinant, since transposon insertion in this gene led to reduced invasion of cultured cells (22). How fucP contributes to the invasion process is unknown, but the finding illustrates the functional diversity of this gene.…”

Section: Discussionmentioning

confidence: 99%

Phenotypic and Genotypic Evidence for l -Fucose Utilization by Campylobacter jejuni

2011

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Campylobacter jejuni remains among the leading causes of bacterial food-borne illness. The current understanding of Campylobacter physiology suggests that it is asaccharolytic and is unable to catabolize exogenous carbohydrates. Contrary to this paradigm, we provide evidence for L-fucose utilization by C. jejuni. The fucose phenotype, shown in chemically defined medium, is strain specific and linked to an 11-open reading frame (ORF) plasticity region of the bacterial chromosome. By constructing a mutation in fucP (encoding a putative fucose permease), one of the genes in the plasticity region, we found that this locus is required for fucose utilization. Consistent with their function in fucose utilization, transcription of the genes in the locus is highly inducible by fucose. PCR screening revealed a broad distribution of this genetic locus in strains derived from various host species, and the presence of this locus was consistently associated with fucose utilization. Birds inoculated with the fucP mutant strain alone were colonized at a level comparable to that by the wild-type strain; however, in cocolonization experiments, the mutant was significantly outcompeted by the wild-type strain when birds were inoculated with a low dose (10 5 CFU per bird). This advantage was not observed when birds were inoculated at a higher inoculum dose (10 8 CFU per bird). These results demonstrated a previously undescribed substrate that supports growth of C. jejuni and identified the genetic locus associated with the utilization of this substrate. These findings substantially enhance our understanding of the metabolic repertoire of C. jejuni and the role of metabolic diversity in Campylobacter pathobiology.

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Section: Discussionmentioning

confidence: 99%

Phenotypic and Genotypic Evidence for l -Fucose Utilization by Campylobacter jejuni

2011

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“…Signature-tagged transposon mutagenesis has shown Campylobacter colonization in chickens to be complex and dynamic (Grant et al, 2005), and furthermore, screening of a transposon-based mutant library has identified C. jejuni genes that are related to polymyxin resistance and to acid adaptation of the pathogen (Lin et al, 2009;Reid et al, 2008). Additionally, transposon mutagenesis in a clinical isolate of C. jejuni has detected 26 mutants with a clearly reduced invasion capacity (Javed et al, 2010).…”

Section: Discussionmentioning

confidence: 99%

Campylobacter jejuni proteins Cj0952c and Cj0951c affect chemotactic behaviour towards formic acid and are important for invasion of host cells

et al. 2010

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Campylobacter jejuni, an important food-borne bacterial pathogen in industrialized countries and in the developing world, is one of the major causes of bacterial diarrhoea. To identify genes which are important for the invasion of host cells by the pathogen, we screened altogether 660 clones of a transposon-generated mutant library based on the clinical C. jejuni isolate B2. Thereby, we identified a clone with a transposon insertion in gene cj0952c. As in the well-characterized C. jejuni strain NCTC 11168, the corresponding protein together with the gene product of the adjacent gene cj0951c consists of two transmembrane domains, a HAMP domain and a putative MCP domain, which together are thought to act as a chemoreceptor, designated Tlp7. In this report we show that genes cj0952c and cj0951c (i) are important for the host cell invasion of the pathogen, (ii) are not translated as one protein in C. jejuni isolate B2, contradicting the idea of a postulated read-through mechanism, (iii) affect the motility of C. jejuni, (iv) alter the chemotactic behaviour of the pathogen towards formic acid, and (v) are not related to the utilization of formic acid by formate dehydrogenase.

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“…In vitro invasion and adhesion assay. The adhesion and invasion potentials of all strains were determined using in vitro invasion and adhesion assays as described earlier (19). Briefly, Caco-2 cell monolayers were grown in two 24-well cell culture plates.…”

Section: Methodsmentioning

confidence: 99%

“…Motility assays were undertaken using Mueller-Hinton broth supplemented with 0.4% agar as described earlier (19).…”

Section: Methodsmentioning

confidence: 99%

“…One of the mutants identified in a screen for reduced invasion of human intestinal epithelial cells harbored a transposon insertion into a gene homologous to cj1136 of C. jejuni NCTC11168 (19). cj1136 is part of the LOS biosynthesis locus (cj1132c to cj1152c) in C. jejuni NCTC11168 and shares homology with the genes for many bacterial ␤-1,3-galactosyltransferases (GalTs), but the function of the encoded protein and its importance in C. jejuni pathogenesis, host colonization, and LOS biosynthesis is unknown.…”

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Cj1136 Is Required for Lipooligosaccharide Biosynthesis, Hyperinvasion, and Chick Colonization by Campylobacter jejuni

et al. 2012

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ABSTRACTCampylobacter jejuniis a major cause of bacterial food-borne enteritis worldwide, and invasion into intestinal epithelial cells is an important virulence mechanism. Recently we reported the identification of hyperinvasiveC. jejunistrains and created a number of transposon mutants of one of these strains, some of which exhibited reduced invasion into INT-407 and Caco-2 cells. In one such mutant the transposon had inserted into a homologue ofcj1136, which encodes a putative galactosyltransferase according to the annotation of theC. jejuniNCTC11168 genome. In the current study, we investigated the role ofcj1136inC. jejunivirulence, lipooligosaccharide (LOS) biosynthesis, and host colonization by targeted mutagenesis and complementation of the mutation. Thecj1136mutant showed a significant reduction in invasion into human intestinal epithelial cells compared to the wild-type strain 01/51. Invasion levels were partially restored on complementing the mutation. The inactivation ofcj1136resulted in the production of truncated LOS, while biosynthesis of a full-length LOS molecule was restored in the complemented strain. Thecj1136mutant showed an increase in sensitivity to the bile salts sodium taurocholate and sodium deoxycholate and significantly increased sensitivity to polymyxin B compared to the parental strain. Importantly, the ability of the mutant to colonize 1-day-old chicks was also significantly impaired. This study confirms that a putative galactosyltransferase encoded bycj1136is involved in LOS biosynthesis and is important forC. jejunivirulence, as disruption of this gene and the resultant truncation of LOS affect both colonizationin vivoand invasivenessin vitro.

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Transposon mutagenesis in a hyper-invasive clinical isolate of Campylobacter jejuni reveals a number of genes with potential roles in invasion

Cited by 28 publications

References 45 publications

Phenotypic and Genotypic Evidence for l -Fucose Utilization by Campylobacter jejuni

Phenotypic and Genotypic Evidence for l -Fucose Utilization by Campylobacter jejuni

Campylobacter jejuni proteins Cj0952c and Cj0951c affect chemotactic behaviour towards formic acid and are important for invasion of host cells

Cj1136 Is Required for Lipooligosaccharide Biosynthesis, Hyperinvasion, and Chick Colonization by Campylobacter jejuni

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