Component Identification and Functional Analysis of Outer Membrane Vesicles Released by Avibacterium paragallinarum

Abstract

Avibacterium paragallinarum, the causative agent of infectious coryza, is known to release outer membrane vesicles (OMVs). In the present study, we investigated the composition, bioactivities, and functional properties of the OMVs of A. paragallinarum. Following extraction and purification, the OMVs were observed to be spherical in shape, with diameters ranging from 20 to 300 nm. The vesicles contained endotoxin as well as genomic DNA. The molecular weights of the OMV-contained protein fragments were mostly concentrated at 65 and 15 kDa. The components of the OMV proteins were mainly various functional enzymes (e.g., ATP-dependent RNA helicase), structural components (e.g., streptomycin B receptor and membrane protein), and some hypothetical proteins with unknown functions. The expression levels of inflammation-related factors, such as interleukin (IL)-2, IL-6, IL-1β, IL-10, and inducible nitric oxide synthase (iNOs), were significantly upregulated in chicken macrophage cells HD11 incubated with OMVs. Serum IgG antibodies were measured after two intramuscular injections of an OMV-based vaccine into specific pathogen-free (SPF) chickens. The vaccinated chickens were then challenged by A. paragallinarum of homologous and heterologous serovars. It was noted that the vaccinated chickens produced immunoglobulin G (IgG) antibodies against A. paragallinarum. The OMVs conferred an acceptable level of protection (70%), defined as an absence of colonization and of clinical signs, against the homologous strain (serovar A), while the cross-protection against heterologous challenge with serovars B and C was much weaker. However, the OMVS did provide significant protection against clinical signs for all three serovars. Overall, this study laid a foundation for further unraveling the functional roles of OMVs released by A. paragallinarum.

The Outer Membrane Vesicles of Salmonella enterica Serovar Typhimurium Activate Chicken Immune Cells through Lipopolysaccharides and Membrane Proteins

“…In our study, Salmonella OMVs promoted the expression of LITAF, IL-6, and iNOS mRNA in HD11 cells ( Figure 3 ), demonstrating that OMVs from chicken S. Typhimurium CVCC542, could promote the activation of chicken antigen-presenting cells HD11, accompanied by the expression of LITAF, IL-6, and iNOS mRNA. Our results were also supported by Mei et al [ 28 ], who observed that the OMVs from the avian bacillus H. paragallinarum could stimulate the expression levels of IL-1β, IL-2, IL-6, IL-10, and iNOS in HD11.…”

“…Similarly, Chu et al [ 34 ] showed that incubating HD11 cells with LPS for 24 h significantly increased the phagocytosis of Chlamydia psittaci by macrophages, supporting the notion that LPS is important for the immunostimulatory effect of OMVs. LPS could stimulate the expression of IL-1β, IL-2, IL-6, IL-10, and iNOS in HD11 cells [ 28 ]. We also observed that OMVs from Salmonella entered the cell ( Figure 4 ), and LPS within the cell can activate inflammasomes and cause the production of IL-1β, as reported by Vanaja et al [ 35 ], who observed that E. coli OMVs could carry LPS into the cell to activate the activation of inflammasomes and cause the production of IL-1β.…”

Outer Membrane Vesicle Mediated Multidrug Resistance Gene Transfer in Avibacterium Paragallinarum

“…Both pathogenic and non-pathogenic gram-negative bacilli secrete vesicles [9]. The fact that bacterial OMVs contain DNA (plasmid, chromosomal, and/or phage-associated) has been admitted [9, 18, 26]. Whether OMVs have complete genome as their parent cells do and if OMVs contain all the genetic information of the bacterial genome are unclear.…”

“…DNA was extracted as previously discribed [18]. Since the nucleic acid sequence contained in OMVs is a small molecule sequence, and nanopore third-generation sequencing characterized by long-read sequencing, the genome of OMVs was sequenced through second-generation sequencing.…”

“…The isolation and purification steps of omv were modified on the basis of previous literature [18]. A. paragallinarum strain P4chr1 was inoculated in tryptic soy broth (TSB) containing 10% (v/v) chicken serum and 0.0025% (w/v) nicotinamide adenine dinucleotide (NAD), and cultured at 37 ℃ at 180rpm for 1:50, and cultured under the same conditions for 12-16h to make its OD600 reach 1.0.…”