Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood-brain barrier damage in ducks during Riemerella anatipestifer infection.

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

Iron efflux by IetA enhances ß-lactam aztreonam resistance and is linked to oxidative stress through cellular respiration in Riemerella anatipestifer.

BACKGROUND: Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown. OBJECTIVES: To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance. METHODS: In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence. RESULTS: The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the ß-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT. CONCLUSION: Iron efflux is critical to alleviate oxidative stress damage and ß-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.

Functional characterization of two TolC in the resistance to drugs and metals and in the virulence of Riemerella anatipestifer.

IMPORTANCE: Riemerella anatipestifer (RA) is a notorious duck pathogen, characterized by a multitude of serotypes that exhibit no cross-reaction with one another. Moreover, RA is resistant to various antibacterial agents. Consequently, understanding the mechanisms behind resistance and identifying potential targets for drug development have become pressing needs. In this study, we show that the two TolC proteins play a role in the resistance to different drugs and metals and in the virulence. The results suggest that TolCA has a wider range of efflux substrates than TolCB. Except for gentamicin, neither TolCA nor TolCB was involved in the efflux of the other tested antibiotics. Strikingly, TolCA but not TolCB enhanced the frequency of resistance-conferring mutations. Moreover, TolCA was involved in RA virulence. Given its conservation in RA, TolCA has potential as a drug target for the development of therapeutics against RA infections.

Manganese Efflux Achieved by MetA and MetB Affects Oxidative Stress Resistance and Iron Homeostasis in Riemerella anatipestifer.

In bacteria, manganese homeostasis is controlled by import, regulation, and efflux. Here, we identified 2 Mn exporters, MetA and MetB (manganese efflux transporters A and B), in Riemerella anatipestifer CH-1, encoding a putative cation diffusion facilitator (CDF) protein and putative resistance-nodulation-division (RND) efflux pump, respectively. Compared with the wild type (WT), ΔmetA, ΔmetB, and ΔmetAΔmetB exhibited sensitivity to manganese, since they accumulated more intracellular Mn2+ than the WT under excess manganese conditions, while the amount of iron in the mutants was decreased. Moreover, ΔmetA, ΔmetB, and ΔmetAΔmetB were more sensitive to the oxidant NaOCl than the WT. Further study showed that supplementation with iron sources could alleviate manganese toxicity and that excess manganese inhibited bacterial cell division. RNA-Seq showed that manganese stress resulted in the perturbation of iron metabolism genes, further demonstrating that manganese efflux is critical for iron homeostasis. metA transcription was upregulated under excess manganese but was not activated by MetR, a DtxR family protein, although MetR was also involved in manganese detoxification, while metB transcription was downregulated under iron depletion conditions and in fur mutants. Finally, homologues of MetA and MetB were found to be mainly distributed in members of Flavobacteriaceae. Specifically, MetB represents a novel manganese exporter in Gram-negative bacteria. IMPORTANCE Manganese is required for the function of many proteins in bacteria, but in excess, manganese can mediate toxicity. Therefore, the intracellular levels of manganese must be tightly controlled. Manganese efflux transporters have been characterized in some other bacteria; however, their homologues could not be found in the genome of Riemerella anatipestifer through sequence comparison. This indicated that other types of manganese efflux transporters likely exist. In this study, we characterized 2 transporters, MetA and MetB, that mediate manganese efflux in R. anatipestifer in response to manganese overload. MetA encodes a putative cation diffusion facilitator (CDF) protein, which has been characterized as a manganese transporter in other bacteria, while this is the first observation of a putative resistance-nodulation-division (RND) transporter contributing to manganese export in Gram-negative bacteria. In addition, the mechanism of manganese toxicity was studied by observing morphological changes and by transcriptome sequencing. Taken together, these results are important for expanding our understanding of manganese transporters and revealing the mechanism of manganese toxicity.

DEAD Box Protein DhR1 Is a Global Regulator Involved in the Bacterial Fitness and Virulence of Riemerella anatipestifer.

DEAD box proteins perform diverse cellular functions in bacteria. Our group previously reported that the transposon Tn4531 insertion in Riean_0395 (designated dhR1), which encodes a putative DEAD box helicase, attenuated the virulence of R. anatipestifer strain YZb1. Here, we show that, compared to the wild-type (WT) R. anatipestifer strain Yb2, the growth or survival of the ΔdhR1 mutant in tryptic soy broth (TSB) was significantly decreased in response to cold, pH, osmotic stress, ethanol, Triton X-100, and oxidative stress, and the dhR1 deletion significantly reduced biofilm formation and the adhesion capacity to Vero cells, whereas the growth of ΔdhR1 was less impaired in iron-limited TSB. Moreover, the virulence of ΔdhR1 in ducklings was attenuated by about 80-fold, compared to the WT. In addition, a transcriptome analysis showed that the dhR1 deletion in the strain Yb2 affected the expression of 58 upregulated genes and 98 downregulated genes that are responsible for various functions. Overall, our work reveals that the deletion of DhR1 results in a broad effect on the bacterial fitness, biofilm formation, iron utilization, and virulence of R. anatipestifer, which makes it a global regulator. IMPORTANCE R. anatipestifer infection has been a continued and serious problem in many duck farms, but little is known about the mechanism underlying the pathogenesis of R. anatipestifer and how R. anatipestifer adapts to the external environment and thereby persists in duck farms. The results of this study demonstrate that the DEAD box protein DhR1 is required for the tolerance of R. anatipestifer to cold, pH, and other stresses, and it is also necessary for biofilm formation, iron utilization, and virulence in ducklings, demonstrating multiple functions of DhR1.

Riemerella anatipestifer AS87_RS02955 Acts as a Virulence Factor and Displays Endonuclease Activity.

Riemerella anatipestifer is an important bacterial pathogen in the global duck industry and causes heavy economic losses. In our previous study, we demonstrated that R. anatipestifer type IX secretion system components GldK and GldM, and the secretion protein metallophosphoesterase, acted as virulence factors. In this study, R. anatipestifer AS87_RS02955 was investigated for virulence and enzymatic activity properties. We constructed AS87_RS02955 mutation and complementation strains to assess bacterial virulence. In vivo bacterial loads showed a significantly reduced bacterial loads in the blood of ducks infected with mutant strain Yb2Δ02955, which was recovered in the blood of ducks infected with the complementation strain cYb2Δ02955, demonstrating that AS87_RS02955 was associated with virulence. Further studies showed AS87_RS02955 was a novel nonspecific endonuclease with no functionally conserved domain, but enzymatic activity toward DNA and RNA was indicated. DNase activity was activated by Zn2+, Cu2+, Mg2+, Ca2+, and Mn2+ ions but inhibited by ethylenediaminetetraacetic acid. RNase activity was independent of metal cations, but stimulated by Mg2+, Ca2+, and Mn2+. RAS87_RS02955 enzymatic activity was active across a broad pH and temperature range. Moreover, we identified four sites in rAS87_RS02955, F39, F92, I134, and F145, which were critical for enzymatic activity. In summary, we showed that R. anatipestifer AS87_RS02955 encoded a novel endonuclease with important roles in bacterial virulence. IMPORTANCE R. anatipestifer AS87_RS02955 was identified as a novel T9SS effector and displayed a nonspecific endonuclease activity in this study. The protein did not contain a conserved His-Asn-His motif structure, which is similar to the endonuclease from Prevotella sp. Its mutant strain Yb2Δ02955 demonstrated significantly attenuated virulence, suggesting AS87_RS02955 is an important virulence factor. Moreover, AS87_RS02955 displayed nonspecific endonuclease activity to cleave λ DNA and MS2 RNA, while four protein sites were critical for endonuclease activity. In conclusion, R. anatipestifer AS87_RS02955 plays important roles in bacterial virulence.

Genome-Wide Analysis and Characterization of the Riemerella anatipestifer Putative T9SS Secretory Proteins with a Conserved C-Terminal Domain.

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) acts as a crucial virulence factor. We previously identified two T9SS component proteins, GldK and GldM, and one T9SS effector metallophosphoesterase, which play important roles in bacterial virulence. In this study, 19 T9SS-secreted proteins that contained a conserved T9SS C-terminal domain (CTD) were predicted in R. anatipestifer strain Yb2 by searching for CTD-encoding sequences in the whole genome. The proteins were confirmed with a liquid chromatography-tandem mass spectrometry analysis of the bacterial culture supernatant. Nine of them were reported in our previous study. We generated recombinant proteins and mouse antisera for the 19 predicted proteins to confirm their expression in the bacterial culture supernatant and in bacterial cells. Western blotting indicated that the levels of 14 proteins were significantly reduced in the T9SS mutant Yb2ΔgldM culture medium but were increased in the bacterial cells. RT-qPCR indicated that the expression of these genes did not differ between the wild-type strain Yb2 and the T9SS mutant Yb2ΔgldM. Nineteen mutant strains were successfully constructed to determine their virulence and proteolytic activity, which indicated that seven proteins are associated with bacterial virulence, and two proteins, AS87_RS04190 and AS87_RS07295, are protease-activity-associated virulence factors. In summary, we have identified at least 19 genes encoding T9SS-secreted proteins in the R. anatipestifer strain Yb2 genome, which encode multiple functions associated with the bacterium's virulence and proteolytic activity. IMPORTANCE Riemerella anatipestifer T9SS plays an important role in bacterial virulence. We have previously reported nine R. anatipestifer T9SS-secreted proteins and clarified the function of the metallophosphoesterase. In this study, we identified 10 more secreted proteins associated with the R. anatipestifer T9SS, in addition to the nine previously reported. Of these, 14 proteins showed significantly reduced secretion into the bacterial culture medium but increased expression in the bacterial cells of the T9SS mutant Yb2ΔgldM; seven proteins were shown to be associated with bacterial virulence; and two proteins, AS87_RS04190 and AS87_RS07295, were shown to be protease-activity-associated virulence factors. Thus, we have demonstrated that multiple R. anatipestifer T9SS-secreted proteins function in virulence and proteolytic activity.

Riemerella anatipestifer T9SS Effector SspA Functions in Bacterial Virulence and Defending Natural Host Immunity.

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Recent studies have shown that the R. anatipestifer type IX secretion system (T9SS) is a crucial factor in bacterial virulence. The AS87_RS04190 protein was obviously missing from the secreted proteins of the T9SS mutant strain Yb2ΔgldM. A bioinformatic analysis indicated that the AS87_RS04190 protein contains a T9SS C-terminal domain sequence and encodes a putative subtilisin-like serine protease (SspA). To determine the role of the putative SspA protein in R. anatipestifer pathogenesis and proteolysis, we constructed two strains with an sspA mutation and complementation, respectively, and determined their median lethal doses, their bacterial loads in infected duck blood, and their adherence to and invasion of cells. Our results demonstrate that the SspA protein functions in bacterial virulence. It is also associated with the bacterial protease activity and has a conserved catalytic triad structure (Asp126, His158, and Ser410), which is necessary for protein function. The optimal reactive pH and temperature were determined to be 7.0 and 50°C, respectively, and Km and Vmax were determined to be 10.15 mM and 246.96 U/mg, respectively. The enzymatic activity of SspA is activated by Ca2+, Mg2+, and Mn2+ and inhibited by Cu2+ and EDTA. SspA degrades gelatin, fibrinogen, and bacitracin LL-37. These results demonstrate that SspA is an effector protein of T9SS and functions in R. anatipestifer virulence and its proteolysis of gelatin, fibrinogen, and bacitracin LL-37. IMPORTANCE In recent years, Riemerella anatipestifer T9SS has been reported to act as a virulence factor. However, the functions of the proteins secreted by R. anatipestifer T9SS are not entirely clear. In this study, a secreted subtilisin-like serine protease SspA was shown to be associated with R. anatipestifer virulence, host complement evasion, and degradation of gelatin, fibrinogen, and LL-37. The enzymatic activity of recombinant SspA was determined, and its Km and Vmax were 10.15 mM and 246.96 U/mg, respectively. Three conserved sites (Asp126, His158, and Ser410) are necessary for the protein's function. The median lethal dose of the sspA-deleted mutant strain was reduced >10,000-fold, indicating that SspA is an important virulence factor. In summary, we demonstrate that the R. anatipestifer AS87_RS04190 gene encodes an important T9SS effector, SspA, which plays an important role in bacterial virulence.

RAA Enzyme Is a New Family of Class A Extended-Spectrum ß-Lactamase from Riemerella anatipestifer Strain RCAD0122.

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

Source Tracking and Global Distribution of the Tigecycline Non-Susceptible tet(X).

The emergence of tet(X) genes has compromised the clinical use of the last-line antibiotic tigecycline. We identified 322 (1.21%) tet(X) positive samples from 12,829 human microbiome samples distributed in four continents (Asia, Europe, North America, and South America) using retrospective data from worldwide. These tet(X) genes were dominated by tet(X2)-like orthologs but we also identified 12 samples carrying novel tet(X) genes, designed tet(X45), tet(X46), and tet(X47), were resistant to tigecycline. The metagenomic analysis indicated these tet(X) genes distributed in anaerobes dominated by Bacteroidaceae (78.89%) of human-gut origin. Two mobile elements ISBf11 and IS4351 were most likely to promote the transmission of these tet(X2)-like orthologs between Bacteroidaceae and Riemerella anatipestifer. tet(X2)-like orthologs was also developed during transmission by mutation to high-level tigecycline resistant genes tet(X45), tet(X46), and tet(X47). Further tracing these tet(X) in single bacterial isolate from public repository indicated tet(X) genes were present as early as 1960s in R. anatipestifer that was the primary tet(X) carrier at early stage (before 2000). The tet(X2) and non-tet(X2) orthologs were primarily distributed in humans and food animals respectively, and non-tet(X2) were dominated by tet(X3) and tet(X4). Genomic comparison indicated these tet(X) genes were likely to be generated during tet(X) transmission between Flavobacteriaceae and E. coli/Acinetobacter spp., and ISCR2 played a key role in the transmission. These results suggest R. anatipestifer was the potential ancestral source of tet(X). In addition, Bacteroidaceae of human-gut origin was an important hidden reservoir and mutational incubator for the mobile tet(X) genes that enabled spread to facultative anaerobes and aerobes. IMPORTANCE The emergence of the tigecycline resistance gene tet(X) has posed a severe threat to public health. However, reports of its origin and distribution in human remain rare. Here, we explore the origin and distribution of tet(X) from large-scale metagenomic data of human-gut origin and public repository. This study revealed the emergency of tet(X) gene in 1960s, which has refreshed a previous standpoint that the earliest presence of tet(X) was in 1980s. The metagenomic analysis from data mining covered the unculturable bacteria, which has overcome the traditional bacteria isolating and purificating technologies, and the analysis indicated that the Bacteroidaceae of human-gut origin was an important hidden reservoir for tet(X) that enabled spread to facultative anaerobes and aerobes. The continuous monitoring of mobile tigecycline resistance determinants from both culturable and unculturable microorganisms is imperative for understanding and tackling the dissemination of tet(X) genes in both the health care and agricultural sectors.

Duck Complement Factor H Binds to Outer Membrane Protein Omp24 of Riemerella anatipestifer.

The resistance to serum complement-mediated killing is a vital virulence property of microbial pathogens. Complement factor H (FH) is a key negative regulator of the complement alternative pathway (AP) that prevents formation and accelerates the decay of AP C3 convertase and acts as a cofactor in the inactivation of C3b. Pathogens can recruit host FH through their surface proteins to escape the clearance of the complement system. Riemerella anatipestifer could also evade the complement system attack to achieve host infection, but the mechanism is still unclear. In this study, the R. anatipestifer proteins that could interact with FH in host serum were screened and analyzed, and the functions were determined. Affinity chromatography with a Ni-nitrilotriacetic acid Sefinose column and mass spectrometry identified three outer membrane proteins (Omp) of R. anatipestifer, Omp54, Omp53, and Omp24, as potential FH-binding proteins. We then successfully conducted the prokaryotic expression and polyclonal antibody preparation of three candidate proteins. Indirect immunofluorescence assay showed that three candidate proteins were all present in R. anatipestifer. The affinity blotting assay, anti-serum-inhibiting assay, and serum bactericidal assay presented evidence that Omp24 could bind FH. Moreover, FH bound to Omp24 was associated with resistance to the alternative pathway and functional for R. anatipestifer survival in the normal duck serum. These results suggested that R. anatipestifer Omp24 was a FH-binding protein and the interaction with FH blocked the alternative pathway. Recruitment of complement regulatory proteins may facilitate better R. anatipestifer resistance to this vital line of host defense.

Artículo regular­El factor H del complemento de pato se une a la proteína de la membrana externa Omp24 de Riemerella anatipestifer La resistencia a la destrucción mediada por el complemento sérico es una propiedad vital para la virulencia de los patógenos microbianos. El factor de complemento H (FH) es un regulador negativo clave de la vía alterna del complemento (AP) que previene la formación y acelera la descomposición de la C3 convertasa de la vía alterna y actúa como cofactor en la inactivación de C3b. Los patógenos pueden reclutar factor H del huésped a través de sus proteínas de superficie para escapar de la destrucción por el sistema del complemento. Riemerella anatipestifer también pudo evadir el ataque del sistema del complemento para lograr la infección del huésped, pero el mecanismo aún no está claro. En este estudio, se seleccionaron y analizaron las proteínas de R. anatipestifer que podrían interactuar con el factor H en el suero del huésped y se determinaron las funciones. La cromatografía de afinidad con una columna de sefinosa de Ni-NTA y la espectrometría de masas identificaron tres proteínas de la membrana externa de R. anatipestifer, Omp54, Omp53 y Omp24, como posibles proteínas que se unen al factor H. Posteriormente, se llevó a cabo con éxito la expresión procariota y la preparación de anticuerpos policlonales de las tres proteínas candidatas. El ensayo de inmunofluorescencia indirecta mostró que las tres proteínas candidatas estaban presentes en R. anatipestifer. El ensayo de transferencia para afinidad, el ensayo anti-inhibidor del suero y el ensayo bactericida sérico presentaron evidencia de que la proteína Omp24 podría unirse al factor H. Además, el factor H unido a la proteína Omp24 se asoció con resistencia a la vía alterna y funcional para la supervivencia de R. anatipestifer en el suero de pato normal. Estos resultados sugirieron que la proteína Omp24 de R. anatipestifer era una proteína de unión al factor H y que la interacción con este factor bloqueaba la vía alterna del complemento. El reclutamiento de proteínas reguladoras del complemento puede facilitar una mejor resistencia de R. anatipestifer a esta línea vital de defensa del huésped.

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 (encoding 7-keto-8-aminopelargonic acid synthase), bioD (encoding dethiobiotin synthase), and bioA (encoding 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 500-fold compared with that of Yb2. To summarize, the genes birA 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 the bioF gene to inhibit transcription of the bioFDA operon. Moreover, bioX is required for R. anatipestifer pathogenicity, suggesting that BioX is a potential target for treatment of the pathogen. R. anatipestifer BioX has thus been identified as a novel negative regulator involved in biotin metabolism and associated with bacterial virulence in this study.

An Exposed Outer Membrane Hemin-Binding Protein Facilitates Hemin Transport by a TonB-Dependent Receptor in Riemerella anatipestifer.

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

Functional characterization of Fur in iron metabolism, oxidative stress resistance and virulence of Riemerella anatipestifer.

Iron is essential for most bacteria to survive, but excessive iron leads to damage by the Fenton reaction. Therefore, the concentration of intracellular free iron must be strictly controlled in bacteria. Riemerella anatipestifer (R. anatipestifer), a Gram-negative bacterium, encodes the iron uptake system. However, the iron homeostasis mechanism remains largely unknown. In this study, it was shown that compared with the wild type R. anatipestifer CH-1, R. anatipestifer CH-1Δfur was more sensitive to streptonigrin, and this effect was alleviated when the bacteria were cultured in iron-depleted medium, suggesting that the fur mutant led to excess iron accumulation inside cells. Similarly, compared with R. anatipestifer CH-1∆recA, R. anatipestifer CH-1∆recAΔfur was more sensitive to H2O2-induced oxidative stress when the bacteria were grown in iron-rich medium rather than iron-depleted medium. Accordingly, it was shown that R. anatipestifer CH-1∆recAΔfur produced more intracellular ROS than R. anatipestifer CH-1∆recA in iron-rich medium. Electrophoretic mobility shift assays showed that R. anatipestifer CH-1 Fur suppressed the transcription of putative iron uptake genes through binding to their promoter regions. Finally, it was shown that compared with the wild type, R. anatipestifer CH-1Δfur was significantly attenuated in ducklings and that the colonization ability of R. anatipestifer CH-1Δfur in various tissues or organs was decreased. All these results suggested that Fur is important for iron homeostasis in R. anatipestifer and its pathogenic mechanism.

Comparative genomics and metabolomics analysis of Riemerella anatipestifer strain CH-1 and CH-2.

Riemerella anatipestifer is a major pathogenic microorganism in poultry causing serositis with significant mortality. Serotype 1 and 2 were most pathogenic, prevalent, and liable over the world. In this study, the intracellular metabolites in R. anatipestifer strains RA-CH-1 (serotype 1) and RA-CH-2 (serotype 2) were identified by gas chromatography-mass spectrometer (GC-MS). The metabolic profiles were performed using hierarchical clustering and partial least squares discriminant analysis (PLS-DA). The results of hierarchical cluster analysis showed that the amounts of the detected metabolites were more abundant in RA-CH-2. RA-CH-1 and RA-CH-2 were separated by the PLS-DA model. 24 potential biomarkers participated in nine metabolisms were contributed predominantly to the separation. Based on the complete genome sequence database and metabolite data, the first large-scale metabolic models of iJL463 (RA-CH-1) and iDZ470 (RA-CH-2) were reconstructed. In addition, we explained the change of purine metabolism combined with the transcriptome and metabolomics data. The study showed that it is possible to detect and differentiate between these two organisms based on their intracellular metabolites using GC-MS. The present research fills a gap in the metabolomics characteristics of R. anatipestifer.

Characterization of RaeE-RaeF-RopN, a putative RND efflux pump system in Riemerella anatipestifer.

Resistance-nodulation-division (RND) efflux systems are ubiquitous in Gram-negative bacteria and play a predominant role in antimicrobial resistance and other diverse phenotypes, but the knowledges of RND efflux systems are poorly understood so far in Riemerella anatipestifer. According to the sequence annotation, RIA_1117-RIA_1118-RIA_1119 operon in RA-GD strain encodes a putative tripartite RND efflux system. RIA_1117, RIA_1118 and RIA_1119 genes encode an outer member protein (OMP), an inner membrane pump protein (pump transporter), and a periplasmic membrane fusion protein (MFP), respectively. Furthermore, RIA_1119 protein is annotated as a MexE component. In this work, the biological functions of RIA_1117-RIA_1118-RIA_1119 proteins were studied. The antibiotic susceptibility testing showed that the inactivation of RIA_1117, RIA_1118 and RIA_1119 genes all raised susceptibility to amikacin, streptomycin and SDS. By induction with the above antimicrobial agents, the transcription levels of RIA_1117 and RIA_1118 genes were up-regulated significantly using qRT-PCR detection, but no significance difference was observed for the transcription level of RIA_1119 gene. CCCP inhibitor assay confirmed that RIA_1117, RIA_1118 and RIA_1119 proteins mediated amikacin, streptomycin and SDS resistance depending on proton motive force (PMF). Spot assay and streptomycin accumulation assay confirmed that RIA_1117, RIA_1118 and RIA_1119 proteins contributed to export streptomycin, and CCCP increased the accumulation of streptomycin. Furthermore, RIA_1117, RIA_1118 and RIA_1119 proteins also were involved in the fitness and virulence of RA-GD strain. These results showed that RIA_1117-RIA_1118-RIA_1119 operon encoded a RND efflux system, which has the substrate specificity for streptomycin, amikacin and SDS and contributed to the growth and virulence of RA-GD. RIA_1117-RIA_1118-RIA_1119 was designated RaeE-RaeF-RopN efflux system. Based on the above results and structural analysis, RIA_1117, RIA_1118 and RIA_1119 proteins corresponded to RopN (OMP), RaeF (pump transporter) and RaeE (MFP), respectively.

[Cloning and expression of duck C4BPα and verification of its interaction with Riemerella anatipestifer].

To study the interaction between C4b-binding protein (C4BP) and Riemerella anatipestifer (RA), we cloned duck C4BPα, conducted prokaryotic expression and prepared the polyclonal antibody by immunizing mice. Then indirect immunofluorescence assay and dot blotting hybridization assay were used to verify the interaction between C4BP and RA. The full length of duck C4BPα nucleotide sequence was 1 230 bp, with the highest similarity to chicken C4BPα (82.1%). Phylogenetic tree analysis showed that duck C4BPα and chicken C4BPα were on the same phylogenetic tree branch and the genetic evolution relationship between them was the closest. C4BPα was efficiently expressed in Escherichia coli BL21 (DE3). The recombinant proteins existed in intracellular soluble form. The titer of polyclonal antibody was more than 1:10 000 and polyclonal antibodies could specifically recognize the recombinant proteins. The results of indirect immunofluorescence assay and dot blot hybridization assay showed that RA could interact with duck C4BP. The results provide a basis to further reveal the pathogenesis of RA.

Characterization of the hemolytic activity of Riemerella anatipestifer.

Riemerella anatipestifer infection causes serious economic losses in the duck industry worldwide. Acute septicemia and high blood bacterial loading in R. anatipestifer infected ducks indicate that R. anatipestifer may be able to obtain iron and other nutrients by lysing duck erythrocytes to support its rapid growth and proliferation in the blood. However, so far, little is known about the hemolytic activity of R. anatipestifer to duck erythrocytes. In this study, 29 of 52 R. anatipestifer strains showed hemolytic activity on duck blood agar, whereas all the tested dba+ (with hemolytic activity on duck blood agar) and dba- strains created pores in the duck red blood cells, with 4.35-9.03% hemolytic activity in a liquid hemolysis assay after incubation for 24 h. The concentrated culture supernatants of all the tested R. anatipestifer strains and the extracted outer membrane proteins (OMPs) from dba+R. anatipestifer strains showed hemolytic activity on duck blood agar. These results, together with the median lethal dose (LD50) of some dba+ and dba-R. anatipestifer strains in ducklings, suggested that there was no direct relationship between the hemolytic capacity of R. anatipestifer on duck blood agar and its virulence.

The Riemerella anatipestifer M949_RS01035 gene is involved in bacterial lipopolysaccharide biosynthesis.

In this study, the Riemerella anatipestifer mutant strain RA1062 was obtained by screening a random Tn4351 transposon mutant library. The mutant strain was unreactive with the anti-CH3 lipopolysaccharide monoclonal antibody, as demonstrated with an enzyme-linked immunosorbent assay, and its M949_RS01035 gene was inactivated. When cultured in trypticase soy broth, the late stage growth of the mutant RA1062 was significantly decreased. The mutant RA1062 was stained with crystal violet and presented a rough lipopolysaccharide phenotype, which differed from that of the wild-type strain CH3, suggesting that deletion of the M949_RS01035 gene resulted in defective lipopolysaccharide. Silver staining and Western blot analyses further confirmed that the RA1062 lipopolysaccharide had a deficiency in ladder-like binding pattern, as compared to lipopolysaccharide of the wild-type CH3 strain. In addition, the mutant RA1062 showed a higher susceptibility to complement-dependent killing, increased bacterial adhesion and invasion capacities to Vero cells, decreased blood bacterial loads, and attenuated virulence in infected ducks, when compared to the wild-type strain CH3. Moreover, RNA-Seq and real-time polymerase chain reaction analyses indicated that two genes were up-regulated and two were down-regulated in the mutant RA1062 genome. Furthermore, an animal protection experiment showed that immunization of ducks with inactivated RA1062 bacterin conferred effective cross-protection against challenge with the virulent R. anatipestifer serotypes 1, 2, and 10. This study presents evidence that the M949_RS01035 gene is involved in bacterial phenotype, virulence, and gene regulation in R. anatipestifer. The mutant strain RA1062 could be used as a cross-protective vaccine candidate.

Roles of B739_1343 in iron acquisition and pathogenesis in Riemerella anatipestifer CH-1 and evaluation of the RA-CH-1ΔB739_1343 mutant as an attenuated vaccine.

Iron is one of the most important elements for bacterial survival and pathogenicity. The iron uptake mechanism of Riemerella anatipestifer (R. anatipestifer, RA), a major pathogen that causes septicemia and polyserositis in ducks, is largely unknown. Here, the functions of the putative TonB-dependent iron transporter of RA-CH-1, B739_1343, in iron utilization and pathogenicity were investigated. Under iron-starved conditions, the mutant strain RA-CH-1ΔB739_1343 exhibited more seriously impaired growth than the wild-type strain RA-CH-1, and the expression of B739_1343 in the mutant strain restored growth. qRT-PCR results showed that the transcription of B739_1343 was not regulated by iron conditions. In an animal model, the median lethal dose (LD50) of the mutant strain RA-CH-1ΔB739_1343 increased more than 104-fold (1.6×1012 CFU) compared to that of the wild-type strain RA-CH-1 (1.43×108 CFU). In a duck co-infection model, the mutant strain RA-CH-1ΔB739_1343 was outcompeted by the wild-type RA-CH-1 in the blood, liver and brain of infected ducks, indicating that B739_1343 is a virulence factor of RA-CH-1. Finally, immunization with live bacteria of the mutant strain RA-CH-1ΔB739_1343 protected 83.33% of ducks against a high-dose (100-fold LD50) challenge with the wild-type strain RA-CH-1, suggesting that the mutant strain RA-CH-1ΔB739_1343 could be further developed as a potential live attenuated vaccine candidate for the duck industry.