Dataset Information

Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown.

ABSTRACT: Riemerella anatipestifer is a duck pathogen that has caused serious economic losses to the duck industry worldwide. Despite this, there are few reported studies of the physiological and pathogenic mechanisms of Riemerella anatipestifer infection. In previous study, we have shown that TonB1 and TonB2 were involved in hemin uptake. TonB family protein (TbfA) was not investigated, since knockout of this gene was not successful at that time. Here, we used a plasmid based gene over-expression and knockdown to investigate its function. First, we constructed three Escherichia-Riemerella anatipestifer shuttle vectors containing three different native Riemerella anatipestifer promoters. The shuttle plasmids were introduced into Riemerella anatipestifer ATCC11845 by conjugation at an efficiency of 5 × 10^-5 antibiotic-resistant transconjugants per recipient cell. Based on the high-expression shuttle vector pLMF03, a method for gene knockdown was established. Knockdown of TbfA in Riemerella anatipestifer ATCC11845 decreased the organism's growth ability in TSB medium but did not affect its hemin utilization. In contrast, over-expression of TbfA in Riemerella anatipestifer ATCC11845ΔtonB1ΔtonB2. Significantly promoted the organism's growth in TSB medium but significantly inhibited its hemin utilization. Collectively, these findings suggest that TbfA is not involved in hemin utilization by Riemerella anatipestifer.

SUBMITTER: Liu M

PROVIDER: S-EPMC5109031 | biostudies-literature | 2016 Nov

REPOSITORIES: biostudies-literature

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Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown.

Liu MaFeng M Wang MengYi M Zhu DeKang D Wang MingShu M Jia RenYong R Chen Shun S Sun KunFeng K Yang Qiao Q Wu Ying Y Chen XiaoYue X Biville Francis F Cheng AnChun A

Scientific reports 20161115

Riemerella anatipestifer is a duck pathogen that has caused serious economic losses to the duck industry worldwide. Despite this, there are few reported studies of the physiological and pathogenic mechanisms of Riemerella anatipestifer infection. In previous study, we have shown that TonB1 and TonB2 were involved in hemin uptake. TonB family protein (TbfA) was not investigated, since knockout of this gene was not successful at that time. Here, we used a plasmid based gene over-expression and kno ...[more]

PMID: 27845444

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Project description:Riemerella anatipestifer is an important pathogen in waterfowl, and is generally multidrug resistant. This study assessed the current status of Riemerella anatipestifer antibiotic resistance and antibiotic-resistance genes (ARGs), compared the results of different detection methods, and evaluated a new method of studying the association between antibiotic resistance and ARGs in Riemerella anatipestifer. In this study, 51 strains of Riemerella anatipestifer were isolated from ducks on several farms, their resistance to 28 antibiotics was assessed, and the isolates were subjected to whole-genome sequencing. The number of ARGs carried by Riemerella anatipestifer was predicted, compared, and analyzed, and the consistency between ARGs and antibiotic-resistance phenotypes was assessed. The potential for loss of resistance genes during the sequencing and assembly of genome-wide framework map was assessed, and a new ARG detection method was pilot tested. The 51 strains of Riemerella anatipestifer were multidrug resistant (MDR) and had high level of resistance to aminoglycosides, trimethoprim, lincosamides, polypeptides, and macrolides. Based on the genome-wide framework map of the 51 strains, 3 local databases of ABRicate software and 1 online database of CARD website were used to detect ARGs, and a mean of 4 to 5 ARGs were identified per isolate. Although the detection results differed according to the database used, the general performance was consistent. The online website detected more types of ARGs than the ABRicate software. The association between ARGs and antibiotic-resistance phenotypes was assessed, and the ermF gene was identified as a possible key ARGs regulating macrolide resistance of Riemerella anatipestifer. The method used to investigate and detect Riemerella anatipestifer ARGs was convenient and rapid, and had strong accuracy and pertinence. The ARGs detection method reported here combined the advantages of PCR and genome detection, and could greatly reduce workload and detect ARGs more precisely.

Project description:UnlabelledRiemerella anatipestifer is a major bacterial pathogen that causes septicemic and exudative diseases in domestic ducks. In our previous study, we found that deletion of the AS87_01735 gene significantly decreased the bacterial virulence of R. anatipestifer strain Yb2 (mutant RA625). The AS87_01735 gene was predicted to encode a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, the AS87_01735 gene was expressed and identified as the PncA-encoding gene, using an enzymatic assay. Western blot analysis demonstrated that R. anatipestifer PncA was localized to the cytoplasm. The mutant strain RA625 (named Yb2ΔpncA in this study) showed a similar growth rate but decreased NAD(+) quantities in both the exponential and stationary phases in tryptic soy broth culture, compared with the wild-type strain Yb2. In addition, Yb2ΔpncA-infected ducks showed much lower bacterial loads in their blood, and no visible histological changes were observed in the heart, liver, and spleen. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Our results suggest that the R. anatipestifer AS87_01735 gene encodes PncA, which is an important virulence factor, and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate.ImportanceRiemerella anatipestifer is reported worldwide as a cause of septicemic and exudative diseases of domestic ducks. The pncA gene encodes a nicotinamidase (PncA), a key enzyme that catalyzes the conversion of nicotinamide to nicotinic acid, which is an important reaction in the NAD(+) salvage pathway. In this study, we identified and characterized the pncA-homologous gene AS87_01735 in R. anatipestifer strain Yb2. R. anatipestifer PncA is a cytoplasmic protein that possesses similar PncA activity, compared with other organisms. Generation of the pncA mutant Yb2ΔpncA led to a decrease in the NAD(+) content, which was associated with decreased capacity for invasion and attenuated virulence in ducks. Furthermore, Yb2ΔpncA immunization of ducks conferred effective protection against challenge with the virulent wild-type strain Yb2. Altogether, these results suggest that PncA contributes to the virulence of R. anatipestifer and that the Yb2ΔpncA mutant can be used as a novel live vaccine candidate.

Project description:Resistance to trimethoprim is mainly mediated by the acquisition of mobile dfrA genes, and most of them were discovered in Enterobacteriales. A total of 139 Riemerella anatipestifer isolates were collected from different farms in China during 2014 to 2020. Whole genome sequencing (WGS) and genome analysis of R. anatipestifer isolates revealed a 504-bp open reading frame (ORF) encoding a putative dfrA gene. This DfrA variant shared 66.47% amino acid sequence identity with DfrA36 and shared ≤51.20% identity with any other previously identified DfrA proteins. The novel dfrA gene, designated dfrA49, conferred trimethoprim (TMP) resistance when cloned into Escherichia coli BL21(DE3). Thirty dfrA49-positive isolates were identified from Jiangsu and Guangdong province (5/38, 13.16%, and 25/101, 24.75%, respectively). Five of the 38 isolates had obtained the complete genome sequences. Genomic analysis showed that the dfrA49 gene was located on chromosomes or a plasmid (four of them were on chromosomes and one was located on a plasmid). The plasmid p20190305E2-2_2 carried dfrA49, catB, ermF, ereD, blaOXA (88.36% identity with blaOXA-209), Δarr, and tet(X18). Further research indicated that dfrA49 usually coexisted with catB in R. anatipestifer. In this study, a novel trimethoprim resistance gene, dfrA49, was identified and characterized in chromosome and plasmid sequences from R. anatipestifer using WGS and bioinformatic methods. It further expands knowledge about the pool of mobile dfrA genes that confer resistance to trimethoprim and provides information about antibiotic resistance genes in R. anatipestifer, where the resistance gene pool circulating is not well understood. IMPORTANCE Trimethoprim is a synthetic antimicrobial agent inhibiting dihydrofolate reductase (DHFR), which is encoded by the folA gene. Acquired genes that confer trimethoprim resistance due to mutations in the folA gene are designated dfr and divided into two main families including dfrA and dfrB. Resistance to trimethoprim is mainly mediated by the acquisition of mobile dfrA genes, and most of them were discovered in Enterobacteriales. R. anatipestifer belongs to the Flavobacteriaceae family, and the reservoir of dfrA resistance genes in R. anatipestifer has not been fully investigated. A novel trimethoprim resistance gene, dfrA49, which was identified and characterized in chromosome and plasmid sequences in this study, increased the MIC of TMP (>256-fold) in E. coli BL21(DE3). Our study expands knowledge about the pool of mobile dfrA genes that confer resistance to trimethoprim and broadens the understanding of the host spectrum of dfrA family genes.

Dataset Information

Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown.

Publications

Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown.

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