{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T09:05:22Z","timestamp":1789376722323,"version":"build-2803163510"},"reference-count":43,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,12]],"date-time":"2026-09-12T00:00:00Z","timestamp":1789171200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Postdoctoral Research Start-up Project"},{"name":"the Postdoctoral Science Foundation Project"},{"name":"Ningxia Hui Autonomous Region Science and Technology Innovation Team Building Project","award":["2022BSB03107"],"award-info":[{"award-number":["2022BSB03107"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Mycoplasma synoviae (M. synoviae) is an avian pathogen responsible for respiratory disease and synovitis. The VlhA (variable lipoprotein hemagglutinin) family of surface adhesins plays a critical role in host cell attachment, yet the specific residues and structural determinants governing this interaction remain incompletely understood. In this study, we selected the C-terminal lipid-associated membrane hemagglutinin (LAM HA) domain within the VlhA family as the bait protein, based on its conserved C-terminal region. Yeast two-hybrid screening identified 18 LAM HA-interacting host proteins, and molecular docking pinpointed five residues (S83, R85, Y88, N124, K192) as the most frequent interaction hotspots. To assess their collective functional relevance, we constructed a combined deletion mutant lacking these five candidate residues. At the optimal pH of 6.0\u20136.5, wild-type LAM HA showed the highest titer (1:2), significantly exceeding that at pH 7.0\u20137.5 (no activity) and pH 5.0\u20135.5 (1:1) (p &lt; 0.05), whereas the deletion mutant displayed a reduced titer (from 2 to 1; p = 0.05). Secondary structure analysis at the same pH revealed decreased \u03b1-helix content (from 7.90% to 7.57%), along with increased \u03b2-sheet (from 38.92% to 38.83%) and random coil (from 10.83% to 10.44%). Molecular dynamics simulations revealed that the deletion mutant exhibited elevated RMSF (from 2.83 \u00b1 1.88 \u00c5 to 4.18 \u00b1 2.65 \u00c5) and Rg (from 40.48 \u00b1 1.31 \u00c5 to 54.16 \u00b1 4.47 \u00c5) at pH 6.0\u20136.5, while RMSD showed a slight decrease (11.17 \u00b1 1.33 \u00c5 vs. 12.14 \u00b1 0.21 \u00c5), collectively indicating compromised structural stability of the mutant. Collectively, these findings suggest that the LAM HA domain contributes to hemagglutination activity through pH-sensitive conformational stability and specific residue clusters\u2014a property that may be relevant to M. synoviae colonization in the acidic microenvironments of the respiratory tract and synovial fluid during infection.<\/jats:p>","DOI":"10.3390\/microorganisms14092031","type":"journal-article","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T08:10:23Z","timestamp":1789373423000},"page":"2031","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Residues Ser83, Arg85, Tyr88, Asn124, and Lys192 of C-Terminal Lipid-Associated Membrane Hemagglutinin Affect Mycoplasma synoviae Agglutination of Erythrocytes"],"prefix":"10.3390","volume":"14","author":[{"given":"Duoduo","family":"Si","sequence":"first","affiliation":[{"name":"Animal Infections Disease Laboratory, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7557-5069","authenticated-orcid":false,"given":"Shijun","family":"Bao","sequence":"additional","affiliation":[{"name":"Animal Infections Disease Laboratory, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lei","family":"Guo","sequence":"additional","affiliation":[{"name":"Clinical Veterinary Laboratory, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China"},{"name":"Ningxia Xiaoming Agriculture & Animal Husbandry Co., Ltd., Yinchuan 750011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiuhong","family":"Chen","sequence":"additional","affiliation":[{"name":"Ningxia Hui Autonomous Region Veterinary Drug Feed Supervision Institute, Yinchuan 750011, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fengqin","family":"Wen","sequence":"additional","affiliation":[{"name":"Animal Infections Disease Laboratory, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaoxiao","family":"He","sequence":"additional","affiliation":[{"name":"Animal Infections Disease Laboratory, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qing","family":"Wang","sequence":"additional","affiliation":[{"name":"Animal Infections Disease Laboratory, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuan","family":"Shi","sequence":"additional","affiliation":[{"name":"Animal Infections Disease Laboratory, College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shenghu","family":"He","sequence":"additional","affiliation":[{"name":"Clinical Veterinary Laboratory, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jidong","family":"Li","sequence":"additional","affiliation":[{"name":"Clinical Veterinary Laboratory, College of Animal Science and Technology, Ningxia University, Yinchuan 750021, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"277","DOI":"10.1007\/s00430-019-00644-3","article-title":"Interaction with the host: The role of fibronectin and extracellular matrix proteins in the adhesion of Gram-negative bacteria","volume":"209","author":"Vaca","year":"2020","journal-title":"Med. 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