RAA enzyme is a new family of class A extended-spectrum β-lactamase from the Riemerella anatipestifer RCAD0122 strain

Whole genome sequencing of Riemerella anatipestifer isolate RCAD0122 revealed a chromosomally-located β-lactamases gene, bla RAA-1 , which encoded a novel class A extended-spectrum β-lactamases (ESBL), RAA-1. The RAA-1 shared ≤ 65% amino acid sequence identity with other characterized β-lactamases. The kinetic assay of native purified RAA-1 revealed ESBL-like hydrolysis activity. Furthermore, bla RAA-1 could be transferred to a homologous strain by natural transformation. However, the epidemiological study showed that the bla RAA-1 gene is not prevalent currently.

Duck Interleukin-22: Identification and Expression Analysis in Riemerella anatipestifer Infection

Riemerella anatipestifer is one of the most devastating pathogens affecting the global duck farms. Infection is involved in secretion of proinflammatory cytokines, including interleukin- (IL-) 17A. During the immune response to infection, IL-22 and IL-17A are often produced concurrently and at high levels in inflamed tissues. Little is known about duck IL-22 (duIL-22) during R. anatipestifer infection. We describe the characterization of duIL-22 and its mRNA expression analysis in splenic lymphocytes and macrophages treated with heat-killed R. anatipestifer and in the spleens and livers of R. anatipestifer-infected ducks. Full-length cDNA of duIL-22 encoded 197 amino acids. The deduced amino acid sequence of duIL-22 shared a 30.4–40.5% similarity with piscine counterparts, 57.4–60.1% with mammalian homologs, and 93.4% similarity to the chicken. Duck IL-22 mRNA expression level was relatively high in the skin of normal ducks. It was increased in mitogen-stimulated splenic lymphocytes and in killed R. anatipestifer-activated splenic lymphocytes and macrophages. Compared with healthy ducks, IL-22 transcript expression was significantly upregulated in the livers and spleens on days 1 and 4 postinfection, but not on day 7. IL-17A was significantly increased in the spleens only on day 4 postinfection and in the livers at all time points. When splenic lymphocytes were stimulated with heat-killed R. anatipestifer, CD4+ cells predominantly produced IL-22 while IL-17A was expressed both by CD4+ and CD4- cells. These results suggested that IL-22 and IL-17A are likely expressed in different cell types during R. anatipestifer infection.

Putative Riemerella anatipestifer Outer Membrane Protein H Affects Virulence

Riemerella anatipestifer causes serious contagious disease in ducks, geese, and other fowl. However, as a harmful pathogen causing significant economic losses in the poultry industry, R. anatipestifer is still poorly understood for its pathogenesis mechanisms. In a previous study, we developed an indirect ELISA method for detecting R. anatipestifer infection using B739_0832 protein, a putative outer membrane protein H (OmpH) that is conserved among different serotypes of R. anatipestifer. Although OmpH in some pathogenic bacteria, such as Pasteurella, has been reported as a virulence factor, it is still not clear whether B739_0832 protein contributes to the virulence of R. anatipestifer. In this study, we confirmed that B739_0832 protein in R. anatipestifer localizes to the outer membrane. We constructed a B739_0832 deletion mutant strain (ΔB739_0832) and assayed various effects from the deletion of B739_0832. ΔB739_0832 strain had a similar growth rate to wild-type R. anatipestifer CH-1. However, the survival rate of ducklings in 10 days after infection from ΔB739_0832 strain was 50%, whereas no ducklings survived from wild-type R. anatipestifer infection. Furthermore, the median lethal dose (LD50) of the ΔB739_0832 strain was approximately 150 times higher than that of the wild-type strain. Pathology examinations on infected ducklings found that, at 36 h after infection, bacterial loads in blood, liver, and brain tissues from ΔB739_0832-infected ducklings were considerably lower than those from wild-type infected ducklings. These results demonstrate that the B739_0832 protein contributes to the virulence of R. anatipestifer CH-1.

Identification of the Natural Transformation Genes in Riemerella anatipestifer by Random Transposon Mutagenesis

In our previous study, it was shown that Riemerella anatipestifer, a Gram-negative bacterium, is naturally competent, but the genes involved in the process of natural transformation remain largely unknown. In this study, a random transposon mutant library was constructed using the R. anatipestifer ATCC11845 strain to screen for the genes involved in natural transformation. Among the 3000 insertion mutants, nine mutants had completely lost the ability of natural transformation, and 14 mutants showed a significant decrease in natural transformation frequency. We found that the genes RA0C_RS04920, RA0C_RS04915, RA0C_RS02645, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, RA0C_RS09020, and RA0C_RS04870 are essential for the occurrence of natural transformation in R. anatipestifer ATCC11845. In particular, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, and RA0C_RS04870 were putatively annotated as ComEC, DprA, ComF, and RecA proteins, respectively, in the NCBI database. However, RA0C_RS02645, RA0C_RS04920, RA0C_RS04915, and RA0C_RS09020 were annotated as proteins with unknown function, with no homology to any well-characterized natural transformation machinery proteins. The homologs of these proteins are mainly distributed in the members of Flavobacteriaceae. Taken together, our results suggest that R. anatipestifer encodes a unique natural transformation machinery.

Effect of Nutritional Determinants and TonB on the Natural Transformation of Riemerella anatipestifer

Riemerella anatipestifer is a gram-negative bacterium that is the first naturally competent bacterium identified in the family Flavobacteriaceae. However, the determinants that influence the natural transformation and the underlying mechanism remain unknown. In this study, we evaluated the effects of various nutritional factors of the GCB medium [glucose, L-glutamine, vitamin B1, Fe (NO3)3, NaCl, phosphate, and peptone], on the natural transformation of R. anatipestifer ATCC 11845. Among the assayed nutrients, peptone and phosphate affected the natural transformation of R. anatipestifer ATCC 11845, and the transformation frequency was significantly decreased when phosphate or peptone was removed from the GCB medium. When the iron chelator 2,2′-dipyridyl (Dip) was added, the transformation frequency was decreased by approximately 100-fold and restored gradually when Fe (NO3)3 was added, suggesting that the natural transformation of R. anatipestifer ATCC 11845 requires iron. Given the importance of TonB in nutrient transportation, we further identified whether TonB is involved in the natural transformation of R. anatipestifer ATCC 11845. Mutation of tonBA or tonBB, but not tbfA, was shown to inhibit the natural transformation of R. anatipestifer ATCC 11845 in the GCB medium. In parallel, it was shown that the tonBB mutant, but not the tonBA mutant, decreased iron acquisition in the GCB medium. This result suggested that the tonBB mutant affects the natural transformation frequency due to the deficiency of iron utilization.

An exposed outer membrane haemin-binding protein facilitates haemin transport by a TonB-dependent receptor in Riemerella anatipestifer

Iron is an essential element for the replication of most bacteria, including Riemerella anatipestifer (R. anatipestifer, RA), a gram-negative bacterial pathogen of ducks and other birds. R. anatipestifer utilizes haemoglobin-derived haemin as an iron source; however, the mechanism by which this bacterium acquires haemin from haemoglobin is largely unknown. Here, RhuA disruption was shown to impair iron utilization from duck haemoglobin in R. anatipestifer CH-1. Moreover, the putative lipoprotein RhuA was identified as a surface-exposed, outer membrane haemin-binding protein, but it could not extract haemin from duck haemoglobin. Mutagenesis studies showed that recombinant RhuAY144A, RhuAY177A and RhuAH149A lost haemin-binding ability, suggesting that amino acid sites tyrosine 144 (Y144), Y177 and histidine 149 (H149) are crucial for haemin binding. Furthermore, RhuR, the gene adjacent to RhuA, encodes a TonB2-dependent haemin transporter. The function of RhuA in duck haemoglobin utilization was abolished in the RhuR mutant strain, and recombinant RhuA was able to bind the cell surface of R. anatipestifer CH-1ΔRhuA rather than R. anatipestifer CH-1ΔRhuRΔRhuA, indicating that RhuA associates with RhuR to function. The sequence of the RhuR-RhuA haemin utilization locus exhibits no similarity with those of characterized haemin transport systems. Thus, this locus is a novel haemin uptake locus with homologues distributed mainly in the Bacteroidetes phylum. IMPORTANCE In vertebrates, haemin from haemoglobin is an important iron source for infectious bacteria. Many bacteria can obtain haemin from haemoglobin, but the mechanisms of haemin acquisition from haemoglobin differ among bacteria. Moreover, most studies have focused on the mechanism of haemin acquisition from mammalian haemoglobin. In this study, we found that the RhuR-RhuA locus of R. anatipestifer CH-1, a duck pathogen, is involved in haemin acquisition from duck haemoglobin via a unique pathway. RhuA was identified as an exposed outer membrane haemin-binding protein, and RhuR was identified as a TonB2-dependent haemin transporter. Moreover, the function of RhuA in haemoglobin utilization is RhuR dependent, not vice versa. The homologues of RhuR and RhuA are widely distributed in bacteria in marine environments, animals, and plants, representing a novel haemin transportation system of gram-negative bacteria. This study not only was important for understanding haemin uptake in R. anatipestifer but also enriched the knowledge about the haemin transportation pathway in gram-negative bacteria.

XRE-type regulator BioX acts as a negative transcriptional factor of biotin metabolism in Riemerella anatipestifer

Biotin is essential for the growth and pathogenicity of microorganisms. Damage to biotin biosynthesis results in impaired bacterial growth and decreased virulence in vivo. However, the mechanisms of biotin biosynthesis in Riemerella anatipestifer remain unclear. In this study, two R. anatipestifer genes associated with biotin biosynthesis were identified. AS87_RS05840 encoded a BirA protein lacking the N-terminal winged helix-turn-helix DNA binding domain, identifying it as a Group I biotin protein ligase, and AS87_RS09325 encoded a BioX protein, which was in the helix-turn-helix xenobiotic response element family of transcription factors. Electrophoretic mobility shift assays demonstrated that BioX bound to the promoter region of bioF. In addition, the R. anatipestifer genes bioF (7-keto-8-aminopelargonic acid synthase), bioD (dethiobiotin synthase), and bioA (7,8-diaminopelargonic acid synthase) were in an operon and were regulated by BioX. Quantitative reverse-transcription PCR showed that transcription of the bioFDA operon increased in the mutant Yb2ΔbioX in the presence of excessive biotin, compared with that in the wild-type strain Yb2, suggesting that BioX acted as a repressor of biotin biosynthesis. Streptavidin blot analysis showed that BirA caused biotinylation of BioX, indicating that biotinylated BioX was involved in metabolic pathways. Moreover, as determined by the median lethal dose, the virulence of Yb2ΔbioX was attenuated a 500-fold compared with that of Yb2. To summarize, the genes bioA and bioX were identified in R. anatipestifer, and BioX was found to act as a repressor of the bioFDA operon involved in the biotin biosynthesis pathway and identified as a bacterial virulence factor. IMPORTANCE Riemerella anatipestifer is a causative agent of diseases in ducks, geese, turkeys, and various other domestic and wild birds. Our study reveals that biotin synthesis of R. anatipestifer is regulated by the BioX through binding to the promoter region of bioF gene to inhibit transcription of bioFDA operon. Moreover, bioX is required for R. anatipestifer pathogenicity, suggesting BioX is a potential target for treatment of the pathogen. R. anatipestifer BioX is thus identified as a novel negative regulator involved in biotin metabolism and associated with bacterial virulence in this study.

Isolation and Molecular Characterization of Riemerella anatipestifer from Domesticated Ducks of Assam, India

Background: Riemerella anatipestifer (R. anatipestifer) is a gram negative, microaerophilic, non-motile, bipolar bacteria. High genetic diversity and molecular differentiation were reported among field isolates. Although the bacterium causes one of the most economically important duck diseases in the north-eastern region of India, little work has been done on isolation, identification and molecular characterization of the bacteria. Hence, the present investigation was undertaken with a view to characterize the R. anatipestifer isolates from ducks of Assam.Methods: Phenotypic and molecular identification of R. anatipestifer isolates from domesticated ducks of Assam, India were carried out during the period from February, 2019 to January 2020. A total of 624 samples (Ocular swab, throat swab, liver, spleen, kidney, brain, heart, lung) from ducks comprising of apparently healthy, ailing and dead ducks were collected from five districts of Assam, India were processed to isolate and identify the bacteria. The tentative identification of the bacteria was done based on phenotypic characteristics viz., colony morphology, growth characteristics and biochemical reactions. All the phenotypically positive isolates were further subjected to molecular identification based on PCR assay targeting 16S rRNA gene and ERIC sequence.Result: The bacteria could be isolated from different field samples. The highest percentage of the samples that yielded the bacteria are from brain (76%) followed by spleen (74%) of dead ducks and less number of ocular swab (33%) from apparently healthy ducks were found positive. Sequencing of the amplified product of the selected R. anatipestifer isolates targeting 16S rRNA gene revealed homology percentage of 96.5-100%. Further, sequences representing five geographical locations were submitted to NCBI gene bank. Phylogenetic studies of the isolates indicated that there is prevalence of at least two genetically different strains of R. anatipestifer in the study area. The study suggested that the R. anatipestifer infection is endemic in Assam causing varying rate of morbidity (39%) and mortality (53%) and molecular based confirmation is necessary besides phenotypic identification.