Deletion of the <i>Riemerella anatipestifer</i> type IX secretion system gene <i>sprA</i> results in differential expression of outer membrane proteins and virulence

Hu, Di; Guo, Yunqing; Guo, Jie; Wang, Ying; Pan, Zhe; Xiao, Yufei; Wang, Xiliang; Hu, Sishun; Liu, Mei; Li, Zili; Bi, Dingren; Zhou, Zutao

doi:10.1080/03079457.2019.1566594

Cited by 13 publications

(4 citation statements)

References 35 publications

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“…Riemerella anatipestifer total membrane, inner membrane, and outer membrane were extracted and separated based on methods previously described by Hu D. et al (2019) , Thein et al (2010) , and Osborn and Munson (1974) . Cells were grown in 1 L TSB to OD 600 ≈ 3 at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Putative Riemerella anatipestifer Outer Membrane Protein H Affects Virulence

Gao

Wang

et al. 2021

Front. Microbiol.

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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.

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Section: Methodsmentioning

confidence: 99%

Putative Riemerella anatipestifer Outer Membrane Protein H Affects Virulence

Gao

Wang

et al. 2021

Front. Microbiol.

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“…OMPs of R. anatipestifer RA-YM were extracted and separated based on methods described previously [ 34 ]. Far-western blotting was used to identify protein–protein interactions.…”

Section: Western Blottingmentioning

confidence: 99%

“…R. anatipestifer RA-YM and R. anatipestifer RA-YMΔOMP76 were grown to log phase (OD 600 = 0.6–0.8) in TSB medium, harvested, washed three times with sterile PBS, resuspended, and diluted to 5 × 10 9 , 5 × 10 8 , 5 × 10 7 , 5 × 10 6 , and 5 × 10 5 CFU/mL. Determination of bacterial virulence was performed as previously described [ 34 ]. Tissue lesions were was scored as: 0 (Normal), no obvious lesions were found in all tissues; 1 (Mild), the capsules of the heart, liver, spleen and brain were thickened, the area with cellulous exudation and fatty degeneration of cells was <25%, the area around blood vessels was oedematous, the area with inflammatory cell infiltration was <25%, and the area with neurolysis was <25%; 2 (Moderate), the area of lesions in each tissue as >25% and <50%; 3 (Severe), the area of lesions in each tissue was >50% and <75%; and, 4 (Very Severe), the area of lesions in each tissue was >75% and the total score was 16.…”

Section: Pathogenicity Analysis Of R Anatipestifer ...mentioning

confidence: 99%

Outer membrane protein OMP76 of Riemerella anatipestifer contributes to complement evasion and virulence by binding to duck complement factor vitronectin

Wang

Yang

et al. 2023

Virulence

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Riemerella anatipestifer is an important bacterial pathogen in poultry. Pathogenic bacteria recruit host complement factors to resist the bactericidal effect of serum complement. Vitronectin (Vn) is a complementary regulatory protein that inhibits the formation of the membrane attack complex (MAC). Microbes use outer membrane proteins (OMPs) to hijack Vn for complement evasion. However, the mechanism by which R. anatipestifer achieves evasion is unclear. This study aimed to characterise OMPs of R. anatipestifer which interact with duck Vn (dVn) during complement evasion. Far-western assays and comparison of wild-type and mutant strains that were treated with dVn and duck serum demonstrated particularly strong binding of OMP76 to dVn. These data were confirmed with Escherichia coli strains expressing and not expressing OMP76. Combining tertiary structure analysis and homology modelling, truncated and knocked-out fragments of OMP76 showed that a cluster of critical amino acids in an extracellular loop of OMP76 mediate the interaction with dVn. Moreover, binding of dVn to R. anatipestifer inhibited MAC deposition on the bacterial surface thereby enhancing survival in duck serum. Virulence of the mutant strain ΔOMP76 was attenuated significantly relative to the wild-type strain. Furthermore, adhesion and invasion abilities of ΔOMP76 decreased, and histopathological changes showed that ΔOMP76 was less virulent in ducklings. Thus, OMP76 is a key virulence factor of R. anatipestifer . The identification of OMP76-mediated evasion of complement by recruitment of dVn contributes significantly to the understanding of the molecular mechanism by which R. anatipestifer escapes host innate immunity and provides a new target for the development of subunit vaccines.

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“…Infected ducks show clinical signs of lethargy, diarrhea, and respiratory and nervous symptoms, all of which cause serious economic losses in the duck industry (4). An increasing number of virulence factors, i.e., CAMP cohemolysin, OmpA, nicotinamidase PncA, iron acquisition protein SprA, and glycosyltransferase VapD, etc., have been identified in R. anatipestifer (5)(6)(7)(8)(9)(10)(11)(12).…”

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confidence: 99%

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

Niu

Chen

Ren

et al. 2021

Appl Environ Microbiol

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Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our previous study, we demonstrate that bacterial virulence and secretion proteins of the T9SS mutant strains Yb2ΔgldK and Yb2ΔgldM were significantly reduced, compare to the wild-type strain Yb2. In this study, the T9SS secretion protein AS87_RS00980, which absent in the secretion proteins of Yb2ΔgldK and Yb2ΔgldM, was investigated by construction of the gene mutation and complementation strains. The virulence assessment showed a more than 1,000-folds attenuated virulence, and significantly reduced bacterial loads in blood of infected ducks for the mutant strain Yb2Δ00980. The bacterial virulence was recovered in the complementation strain cYb2Δ00980. Further study indicated that the AS87_RS00980 gene encodes a secretion protein metallophosphatase (MPPE), which displayed phosphatase activity and cytomembrane localized. Moreover, the optimal reactive pH and temperature were determined as 7.0 and 60°C, respectively; and the Km and Vmax were determined as 3.53 mM and 198.1U/mg. The MPPE activity was activated by Zn2+, Cu2+, but inhibited by Fe3+, Fe2+, and EDTA. There are five conserved sites of N267, H268 H351, H389, and H391 in the MEEP domain. The mutant proteins of 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 the MPPE activity. Taken together, these results indicated that R. anatipestifer AS87_RS00980 gene encodes a MPPE, which is a secretion protein of T9SS and plays an important role in bacterial virulence. Importance Riemerella anatipestifer T9SS is recently discovered to be associated with bacterial gliding motility and secretion of virulence factors. Several T9SS genes are identified, but no effector is reported in the R. anatipestifer up to date. In this study, we identified the T9SS secretion protein AS87_RS00980 is a metallophosphoesterase that displays phosphatases activity and associated with bacterial virulence. Enzymatic activity of the metallophosphoesterase was determined, and the Km and Vmax were 3.53 mM and 198.1U/mg respectively. Five conserved sites were also identified. The AS87_RS00980 gene deletion mutant strain was attenuated over 1000-fold, indicating it is an important virulence factor. In summary, we identified that R. anatipestifer AS87_RS00980 gene encodes an important T9SS effector MPPE, which plays an important role in bacterial virulence.

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Deletion of the Riemerella anatipestifer type IX secretion system gene sprA results in differential expression of outer membrane proteins and virulence

Cited by 13 publications

References 35 publications

Putative Riemerella anatipestifer Outer Membrane Protein H Affects Virulence

Putative Riemerella anatipestifer Outer Membrane Protein H Affects Virulence

Outer membrane protein OMP76 of Riemerella anatipestifer contributes to complement evasion and virulence by binding to duck complement factor vitronectin

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

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