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Mycoplasma gallisepticum (HS strain) surface lipoprotein pMGA interacts with host apolipoprotein A-I during infection
Fuli Hu1, Chengcheng Zhao1, Dingren Bi2
1Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China.
Abstract:
The adhesin protein from Mycoplasma gallisepticum (HS strain), namely pMGA1.2, is required for M. gallisepticum (MG) infection in chicken. However, the host factor(s) that interact with pMGA1.2 is not known. In this study, we prepared the membrane fraction of trachea epithelial cells from chicken embryos. Using an improved virus overlay protein blot assay (VOPBA) and glutathione S-transferase (GST) pull-down assay, we found that pMGA1.2 specifically bound to a ∼30 kDa host protein. This host protein was further identified by mass spectrometry as chicken apolipoprotein A-I (ApoA-I). We expressed and purified the recombinant ApoA-I protein in Escherichia coli and confirmed that it bound to the purified pMGA1.2 protein in vitro. Transiently expressed pMGA1.2 and ApoA-I were colocalized in HeLa cells. Finally, we designed small interfering RNA (siRNA) molecules to knock down the expression of either ApoA-I or pMGA1.2, which inhibited the MG-induced cell cycle disruption in cells of chicken embryo fibroblast cell line (DF-1). Similarly, knockdown of ApoA-I inhibited the cilia loss and damage in chicken trachea cells in MG infection. In summary, ApoA-I may be an essential host factor in MG infection through interacting with pMGA1.2.
Insights
Mycoplasma gallisepticum (MG) infection in chickens involves the adhesin pMGA1.2 interacting with chicken apolipoprotein A-I (ApoA-I). This interaction is crucial for MG-induced cell damage and infection progression.
Area of Science:
- Veterinary Microbiology
- Infectious Diseases
- Host-Pathogen Interactions
Background:
- Mycoplasma gallisepticum (MG) causes significant respiratory disease in poultry.
- The adhesin protein pMGA1.2 is essential for MG infection, but its host cell receptor remains unidentified.
- Understanding host-pathogen interactions is key to developing effective control strategies.
Purpose of the Study:
- To identify the host protein(s) that interact with the MG adhesin pMGA1.2.
- To elucidate the role of this host-pathogen interaction in MG pathogenesis.
- To investigate potential therapeutic targets for MG infection.
Main Methods:
- Preparation of chicken embryo trachea epithelial cell membrane fractions.
- Virus overlay protein blot assay (VOPBA) and glutathione S-transferase (GST) pull-down assays to identify protein interactions.
- Mass spectrometry for host protein identification.
- Recombinant protein expression and purification (ApoA-I, pMGA1.2).
- Co-localization studies in HeLa cells.
- Small interfering RNA (siRNA) mediated gene knockdown in DF-1 cells and chicken trachea cells.
Main Results:
- pMGA1.2 specifically binds to a ~30 kDa host protein identified as chicken apolipoprotein A-I (ApoA-I).
- Direct binding between purified recombinant ApoA-I and pMGA1.2 was confirmed in vitro.
- pMGA1.2 and ApoA-I co-localized when transiently expressed in HeLa cells.
- Knockdown of ApoA-I or pMGA1.2 expression inhibited MG-induced cell cycle disruption in DF-1 cells.
- Knockdown of ApoA-I reduced cilia loss and damage in chicken trachea cells during MG infection.
Conclusions:
- Chicken apolipoprotein A-I (ApoA-I) is identified as a host factor interacting with the Mycoplasma gallisepticum adhesin pMGA1.2.
- The interaction between ApoA-I and pMGA1.2 plays a critical role in MG pathogenesis, including cell cycle disruption and cilia damage.
- ApoA-I represents a potential target for therapeutic interventions against MG infection.
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