Investigating the Role of FlhF Identifies Novel Interactions With Genes Involved in Flagellar Synthesis in Campylobacter jejuni

Li, Xiaofei; Ren, Fangzhe; Cai, Guoqiang; Huang, Pingyu; Chai, Qinwen; Gundogdu, Ozan; Jiao, Xinan; Huang, Jinlin

doi:10.3389/fmicb.2020.00460

Cited by 15 publications

(18 citation statements)

References 69 publications

(73 reference statements)

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“…Finally, a number of other gene products have been implicated in flagellar motility, but their molecular roles in the process are unknown. These include a predicted transmembrane protein Cj 0390 ( Colegio et al, 2001 ; Coward et al, 2006 ), the GTPase FlhF, that contributes to the activity of σ54 via FlgR/FlgS ( Balaban et al, 2009 ; Li et al, 2020 ), and a protein that is potentially secreted from the cytoplasm, PflA ( Yao et al, 1994 ).…”

Section: Introductionmentioning

confidence: 99%

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction

et al. 2020

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Evolution experiments in the laboratory have focused heavily on model organisms, often to the exclusion of clinically relevant pathogens. The foodborne bacterial pathogen Campylobacter jejuni belongs to a genus whose genomes are small compared to those of its closest genomic relative, the free-living genus Sulfurospirillum, suggesting genome reduction during the course of evolution to host association. In an in vitro experiment, C. jejuni serially passaged in rich medium in the laboratory exhibited loss of flagellar motility-an essential function for host colonization. At early time points the motility defect was often reversible, but after 35 days of serial culture, motility was irreversibly lost in most cells in 5 independently evolved populations. Population re-sequencing revealed disruptive mutations to genes in the flagellar transcriptional cascade, rpoN (σ54)-therefore disrupting the expression of the genes σ54 regulates-coupled with deletion of rpoN in all evolved lines. Additional mutations were detected in virulencerelated loci. In separate in vivo experiments, we demonstrate that a phase variable (reversible) motility mutant carrying an adenine deletion within a homopolymeric tract resulting in truncation of the flagellar biosynthesis gene fliR was deficient for colonization in a C57BL/6 IL-10 −/− mouse disease model. Re-insertion of an adenine residue partially restored motility and ability to colonize mice. Thus, a pathogenic C. jejuni strain was rapidly attenuated by experimental laboratory evolution and demonstrated genomic instability during this evolutionary process. The changes observed suggest C. jejuni is able to evolve in a novel environment through genome reduction as well as transition, transversion, and slip-strand mutations.

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Section: Introductionmentioning

confidence: 99%

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction

et al. 2020

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“…The results also showed a significant decrease in flaB expression. As a flagella Class II gene, flaB expression is driven by σ 54 , whose transcriptional activity is regulated by the FlgSR system and FlhF [34,39]. This indicates that the decrease in flgR expression could have resulted in a decrease in flagella Class II gene expression (including flaB) through this pathway.…”

Section: Discussionmentioning

confidence: 99%

“…Firstly, motility is a key determinant of virulence in C. jejuni [ 36 , 37 ], and therefore the flagella biosynthesis pathway is a heavily researched area within the field. Despite this, gaps still exist in the understanding of the regulation of flagella biosynthesis[ 38 , 39 ]. If CRISPRi-based repression of genes involved in this pathway could be achieved, this would offer a useful tool to explore regulation of flagellar assembly in greater depth.…”

Section: Introductionmentioning

confidence: 99%

Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni

et al. 2022

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Background Campylobacter spp. are the leading cause of bacterial food-borne illness in humans worldwide, with Campylobacter jejuni responsible for 80% of these infections. There is an urgent need to understand fundamental C. jejuni biology for the development of new strategies to prevent and treat infections. The range of molecular tools available to regulate gene expression in C. jejuni is limited, which in turn constrains our ability to interrogate the function of essential and conditionally essential genes. We have addressed this by developing and utilising a CRISPR-based interference system known as CRISPRi in C. jejuni to control gene expression. To achieve this, a catalytically inactive (“dead”) cas9 and sgRNA backbone from the Streptococcus pyogenes CRISPRi system was combined with C. jejuni-derived promoters of predetermined expression activities to develop a CRISPRi-based repression tool in C. jejuni strains M1Cam and 81–176. Results The CRISPRi tool was validated through successful repression of the arylsulphatase-encoding gene astA using a range of sgRNA target sequences spanning the astA gene. The tool was also applied to target astA in an M1Cam CRISPR-Cas9 deletion strain, which showed that the presence of an endogenous CRISPR-Cas9 system did not affect the activity of the CRISPRi-based repression tool. The tool was further validated against the hippicurase-encoding gene hipO. Following this, the flagella genes flgR, flaA, flaB and both flaA and flaB were targeted for CRISPRi-based repression, which resulted in varying levels of motility reduction and flagella phenotypes as determined by phenotypical assays and transmission electron microscopy (TEM). Conclusions This is the first report of a CRISPRi-based tool in C. jejuni, which will provide a valuable resource to the Campylobacter community.

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“…The deletion mutant strains were constructed as previously described [ 37 ]. Genes selected for the deletion mutants were related to the transmembrane transport, which was significantly downregulated during the DATS treatment, including RS07815, RS07825, RS07990, RS06500, and RS05850 ( Table 2 ).…”

Section: Methodsmentioning

confidence: 99%

Antimicrobial Effect and the Mechanism of Diallyl Trisulfide against Campylobacter jejuni

Tang

et al. 2021

Antibiotics

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Campylobacter jejuni is an important foodborne pathogen causing campylobacteriosis. It can infect humans through the consumption of contaminated chicken products or via the direct handling of animals. Diallyl trisulfide (DATS) is a trisulfide compound from garlic extracts that has a potential antimicrobial effect on foodborne pathogens. This study investigated the antimicrobial activity of DATS on C. jejuni by evaluating the minimal inhibitory concentrations (MICs) of C. jejuni 81-168, and fourteen C. jejuni isolates from chicken carcasses. Thirteen of 14 C. jejuni isolates and 81-176 had MICs ≤ 32 μg/mL, while one isolate had MIC of 64 μg/mL. Scanning electron microscopy (SEM) analysis showed the disruption and shrink of C. jejuni bacterial cell membrane after the DATS treatment. A time-killing analysis further showed that DATS had a dose-dependent in vitro antimicrobial effect on C. jejuni during the 24 h treatment period. In addition, DATS also showed an antimicrobial effect in chicken through the decrease of C. jejuni colony count by 1.5 log CFU/g (cloacal sample) during the seven-day DATS treatment period. The transcriptional analysis of C. jejuni with 16 μg/mL (0.5× MIC) showed 210 differentially expression genes (DEGs), which were mainly related to the metabolism, bacterial membrane transporter system and the secretion system. Fourteen ABC transporter-related genes responsible for bacterial cell homeostasis and oxidative stress were downregulated, indicating that DATS could decrease the bacterial ability to against environmental stress. We further constructed five ABC transporter deletion mutants according to the RNA-seq analysis, and all five mutants proved less tolerant to the DATS treatment compared to the wild type by MIC test. This study elucidated the antimicrobial activity of DATS on C. jejuni and suggested that DATS could be used as a potential antimicrobial compound in the feed and food industry.

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Investigating the Role of FlhF Identifies Novel Interactions With Genes Involved in Flagellar Synthesis in Campylobacter jejuni

Cited by 15 publications

References 69 publications

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction

Experimental Evolution of Campylobacter jejuni Leads to Loss of Motility, rpoN (σ54) Deletion and Genome Reduction

Development and validation of a CRISPR interference system for gene regulation in Campylobacter jejuni

Antimicrobial Effect and the Mechanism of Diallyl Trisulfide against Campylobacter jejuni

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