Characterization of RASA-1, a novel class A extended-spectrum beta-lactamase from Riemerella anatipestifer.

A novel chromosomally-located ß-lactamase gene, blaRASA-1, was identified in Riemerella anatipestifer RA-CH-1. The RASA-1, encoded by blaRASA-1, was a class A extended-spectrum ß-lactamase (ESBL), which shared 42.7% and 40.5% identities with the RAA-1 and CGA-1 ß-lactamase, respectively. Overexpression of RASA-1 in Escherichia coli confers broad resistance to ß-lactams and the purified native RASA-1 revealed ESBL-like hydrolysis activity. Blasting in GenBank showed that blaRASA-1 was exclusively detected in Riemerella anatipestifer. Moreover, sequence analysis revealed that this gene was located within the multi-resistance region of Riemerella anatipestifer genome.

A Riemerella anatipestifer Metallophosphoesterase That Displays Phosphatase Activity and Is Associated with Virulence.

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrated that bacterial virulence and secretion proteins of the type IX secretion system (T9SS) mutant strains Yb2ΔgldK and Yb2ΔgldM were significantly reduced, in comparison to those of wild-type strain Yb2. In this study, the T9SS secretion protein AS87_RS00980, which is absent from the secretion proteins of Yb2ΔgldK and Yb2ΔgldM, was investigated by construction of gene mutation and complementation strains. The virulence assessment showed >1,000-fold attenuated virulence and significantly reduced bacterial loads in the blood of ducks infected with Yb2Δ00980, the AS87_RS00980 gene deletion mutant strain. Bacterial virulence was recovered in complementation strain cYb2Δ00980 Further study indicated that the T9SS secretion protein AS87_RS00980 is a metallophosphoesterase (MPPE), which displayed phosphatase activity and was cytomembrane localized. Moreover, the optimal reactive pH and temperature were determined to be 7.0 and 60°C, respectively, and the Km and Vmax were determined to be 3.53 mM and 198.1 U/mg. The rMPPE activity was activated by Zn2+ and Cu2+ but inhibited by Fe3+, Fe2+, and EDTA. There are five conserved sites, namely, N267, H268 H351, H389, and H391, in the metallophosphatase domain. Mutant proteins Y267-rMPPE and Y268-rMPPE retained 29.30% and 19.81% relative activity, respectively, and mutant proteins Y351-rMPPE, Y389-rMPPE, and Y391-rMPPE lost almost all MPPE activity. Taken together, these results indicate that the R. anatipestiferAS87_RS00980 gene encodes an MPPE that is a secretion protein of T9SS that plays an important role in bacterial virulence.IMPORTANCERiemerella anatipestifer T9SS was recently discovered to be associated with bacterial gliding motility and secretion of virulence factors. Several T9SS genes have been identified, but no effector has been reported in R. anatipestifer to date. In this study, we identified the T9SS secretion protein AS87_RS00980 as an MPPE that displays phosphatase activity and is associated with bacterial virulence. The enzymatic activity of the rMPPE was determined, and the Km and Vmax were 3.53 mM and 198.1 U/mg, respectively. Five conserved sites were also identified. The AS87_RS00980 gene deletion mutant strain was attenuated >1,000-fold, indicating that MPPE is an important virulence factor. In summary, we identified that the R. anatipestiferAS87_RS00980 gene encodes an important T9SS effector, MPPE, which plays an important role in bacterial virulence.

Riemerella anatipestifer GldM is required for bacterial gliding motility, protein secretion, and virulence.

Riemerella anatipestifer is a major pathogenic agent of duck septicemic and exudative diseases. Genetic analyses suggest that this pathogen has a novel protein secretion system, known as the "type IX secretion system" (T9SS). We previously reported that deletion of the AS87_RS08465 gene significantly reduced the bacterial virulence of the R. anatipestifer strain Yb2, but the mechanism remained unclear. The AS87_RS08465 gene is predicted to encode the gliding motility protein GldM (GldM) protein, a key component of the T9SS complex. In this study, Western blotting analysis demonstrated that R. anatipestifer GldM was localized to the cytomembrane. Further study revealed that the adhesion and invasion capacities of the mutant strain RA2281 (designated Yb2ΔgldM) in Vero cells and the bacterial loads in the blood of infected ducks were significantly reduced. RNA-Seq and PCR analyses showed that six genes were upregulated and five genes were downregulated in the mutant strain Yb2ΔgldM and that these genes were mainly involved in the secretion of proteins. Yb2ΔgldM was also found to be defective in gliding motility and protein secretion. Liquid chromatography-tandem mass spectrometry analysis revealed that nine of the proteins had a conserved T9SS C-terminal domain and were differentially secreted by Yb2ΔgldM compared to Yb2. The complementation strain cYb2ΔgldM recovered the adhesion and invasion capacities in Vero cells and the bacterial loads in the blood of infected ducks as well as the bacterial gliding motility and most protein secretion in the mutant strain Yb2ΔgldM to the levels of the wild-type strain Yb2. Taken together, these results indicate that R. anatipestifer GldM is associated with T9SS and is important in bacterial virulence.

Riemerella anatipestifer gene AS87_08785 encodes a functional component, GldK, of the type IX secretion system.

Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrated that the deletion of the AS87_08785 gene significantly reduced the virulence of R. anatipestifer strain Yb2, but the mechanism remained unclear. In this study, R. anatipestifer strains with mutated or complemented AS87_08785 genes were constructed and characterized. A sequence analysis indicated that the AS87_08785 gene encoded a putative GldK protein, which localized to the membrane fraction in a western blotting analysis. The mutant strain Yb2ΔgldK displayed defective gliding motility on agar plates, reduced protease activity, and a reduced capacity for protein secretion. RNA sequencing and quantitative PCR analyses indicated that the transcription of 13 genes was downregulated in mutant Yb2ΔgldK. Animal experiments showed that the bacterial loads in the blood of Yb2ΔgldK-infected ducks were significantly reduced relative to those in wild-type strain Yb2 infected ducks. Most of the defective biological properties of the mutant were restored in complementation strain cYb2ΔgldK. Our results demonstrated that R. anatipestifer gene AS87_08785 encoded a component of the type IX secretion system, GldK, which functioned in bacterial gliding motility, protein secretion, and bacterial virulence.

Cas1 and Cas2 From the Type II-C CRISPR-Cas System of Riemerella anatipestifer Are Required for Spacer Acquisition.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide acquired genetic immunity against the entry of mobile genetic elements (MGEs). The immune defense provided by various subtypes of the CRISPR-Cas system has been confirmed and is closely associated with the formation of immunological memory in CRISPR arrays, called CRISPR adaptation or spacer acquisition. However, whether type II-C CRISPR-Cas systems are also involved in spacer acquisition remains largely unknown. This study explores and provides some definitive evidence regarding spacer acquisition of the type II-C CRISPR-Cas system from Riemerella anatipestifer (RA) CH-2 (RA-CH-2). Firstly, introducing an exogenous plasmid into RA-CH-2 triggered spacer acquisition of RA CRISPR-Cas system, and the acquisition of new spacers led to plasmid instability in RA-CH-2. Furthermore, deletion of cas1 or cas2 of RA-CH-2 abrogated spacer acquisition and subsequently stabilized the exogenous plasmid, suggesting that both Cas1 and Cas2 are required for spacer acquisition of RA-CH-2 CRISPR-Cas system, consistent with the reported role of Cas1 and Cas2 in type I-E and II-A systems. Finally, assays for studying Cas1 nuclease activity and the interaction of Cas1 with Cas2 contributed to a better understanding of the adaptation mechanism of RA CRISPR-Cas system. This is the first experimental identification of the naïve adaptation of type II-C CRISPR-Cas system.

ATPase activity of GroEL is dependent on GroES and it is response for environmental stress in Riemerella anatipestifer.

Riemerella anatipestifer (Ra) is a serious gram-negative pathogen of birds and can cause considerable economic losses. The survival mechanisms of R. anatipestifer in the host and environment remain largely unknown. Previous results have demonstrated that GroEL is a molecular chaperone and an important component of the response to various stresses in most bacteria. This study focused on whether GroEL is implicated in this process in R. anatipestifer. The 1629 bp groEL is highly conserved among other gram-negative bacteria (levels of sequence similarity > 60%). A structural analysis and ATPase activity assay revealed that RaGroEL had weak ATPase activity and that the enzyme activity was temperature and ion dependent. GroES partially enhanced the GroEL ATPase activity in the same temperature range. In addition, we studied the mRNA expression of groEL under abiotic stresses caused by heat shock, pH, salt and hydrogen peroxide. These stresses increased the transcription of groEL to varying degrees. In R. anatipestifer, the ATPase activity of GroEL is dependent on GroES and temperature. The expression of groEL was strongly induced by heat, pH, hydrogen peroxide and salt stress. This study is the first to show that GroEL in R. anatipestifer might play a major role in response to environmental stress.

Riemerella anatipestifer extracellular protease S blocks complement activation via the classical and lectin pathways.

Riemerella anatipestifer (RA) is the causative agent of infectious serositis in ducklings and other avian species. It is difficult to control the disease due to its 21 serotypes, poor cross-protection, and bacterial resistance to antimicrobial agents. The complement system is an important component of the innate immune system. However, bacterial pathogens exploit several strategies to evade detection by the complement system. Here, we purified and identified a 59-kDa RA extracellular protease S (EcpS) consisting of a gelatinase. In this study, we aimed to determine how EcpS interferes with complement activation and whether it could block complement-dependent neutrophil function. We found that EcpS potently blocked RA phagocytosis and killing by duck neutrophils. Furthermore, EcpS inhibited the opsonization of bacteria by complement 3b. EcpS specifically blocked complement 3b and complement 4b deposition via the classical and lectin pathways, whereas the alternative pathway was not affected. In summary, we show that RA can survive the bactericidal activity of the duck complement system. These results indicate that RA has evolved mechanisms to evade the duck complement system that may increase the efficiency by which this pathogen can gain access and colonize the inner tissues where it may cause severe infections.

DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.

Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared with canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as a DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e. the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strand separation and intense ATP hydrolysis.

Identification of ribosomal RNA methyltransferase gene ermF in Riemerella anatipestifer.

Riemerella anatipestifer is a major bacterial pathogen of waterfowl, globally responsible for avian septicaemic disease. As chemotherapy is the predominant method for the prevention and treatment of R. anatipestifer infection in poultry, the widespread use of antibiotics has favoured the emergence of antibiotic-resistant strains. However, little is known about R. anatipestifer susceptibility to macrolide antibiotics and its resistance mechanism. We report for the first time the identification of a macrolide resistance mechanism in R. anatipestifer that is mediated by the ribosomal RNA methyltransferase ermF. We identified the presence of the ermF gene in 64/206 (31%) R. anatipestifer isolates from different regions in China. An ermF deletion strain was constructed to investigate the function of the ermF gene on the resistance to high levels of macrolides. The ermF mutant strain showed significantly decreased resistance to macrolide and lincosamide, exhibiting 1024-, 1024-, 4- and >2048-fold reduction in the minimum inhibitory concentrations for erythromycin, azithromycin, tylosin and lincomycin, respectively. Furthermore, functional analysis of ermF expression in E. coli XL1-blue showed that the R. anatipestifer ermF gene was functional in E. coli XL1-blue and conferred resistance to high levels of erythromycin (100 µg/ml), supporting the hypothesis that the ermF gene is associated with high-level macrolide resistance. Our work suggests that ribosomal RNA modification mediated by the ermF methyltransferase is the predominant mechanism of resistance to erythromycin in R. anatipestifer isolates.

A homolog of glyceraldehyde-3-phosphate dehydrogenase from Riemerella anatipestifer is an extracellular protein and exhibits biological activity.

Riemerella anatipestifer is the causative agent of septicemia anserum exsudativa in ducks. Its pathogenesis and virulence factors are still unclear. The glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), an anchorless and multifunctional protein on the surface of several pathogenic microorganisms, is involved in virulence and adhesion. Whether homologs of GAPDH exist, and display similar characteristics in R. anatipestifer (RaGAPDH) has not been determined. In our research, the RaGAPDH activity from various R. anatipestifer isolates was confirmed. Twenty-two gapdh genes from genomic DNA of R. anatipestifer isolates were cloned and sequenced for phylogenetic analysis. The distribution of RaGAPDH in R. anatipestifer CZ2 strain was confirmed by antisera to recombinant RaGAPDH. The ability of purified RaGAPDH to bind host proteins was analyzed by solid-phase ligand-binding assay. Results revealed that all R. anatipestifer isolates showed different levels of GAPDH activity except four strains, which contained a gapdh-like gene. The gapdh of R. anatipestifer, which is located phylogenetically in the same branch as enterohemorrhagic Escherichia coli (EHEC), belonged to class I GAPDH, and encoded a 36.7-kDa protein. All RaGAPDH-encoding gene sequences from field isolates of R. anatipestifer displayed 100% homology. The RaGAPDH localized on the extracellular membrane of several R. anatipestifer strains. Further, it was released into the culture medium, and exhibited GAPDH enzyme activity. We also confirmed the binding of RaGAPDH to plasminogen and fibrinogen. These results demonstrated that GAPDH was present in R. anatipestifer, although not in all strains, and that RaGAPDH might contribute to the microorganism's virulence.