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Cry4Aa exhibits a characteristic architecture comprising three domains (I, II, and III) and functions as a pore-forming toxin, consistent with many previously reported insecticidal Cry toxins. However, the mechanism preceding pore formation by Cry4Aa, including receptor recognition, remains to be elucidated. The present study examined the potential involvement of sugar moieties in the Cry4Aa mode of action, with a particular focus on the process of pore formation. Cry4Aa and the sugar\n                      <jats:italic toggle=\"yes\">N<\/jats:italic>\n                      -acetylgalactosamine exhibited considerably increased binding affinity. Furthermore,\n                      <jats:italic toggle=\"yes\">N<\/jats:italic>\n                      -acetylgalactosamine treatment significantly enhanced the toxicity of Cry4Aa against\n                      <jats:italic toggle=\"yes\">Culex pipiens<\/jats:italic>\n                      mosquito larvae. In the presence of\n                      <jats:italic toggle=\"yes\">N<\/jats:italic>\n                      -acetylgalactosamine, the binding of Cry4Aa to phosphatidylcholine liposomes increased by 1.4-fold. The presence of this sugar did not affect the single-channel conductance of Cry4Aa pores formed in artificial lipid bilayers; however, it did appear to increase the number of Cry4Aa pores per designated area.\n                      <jats:italic toggle=\"yes\">In silico<\/jats:italic>\n                      molecular docking analysis revealed promising binding sites within the cavity, surrounded by the Cry4Aa domains. These results suggest that the interaction of Cry4Aa with\n                      <jats:italic toggle=\"yes\">N<\/jats:italic>\n                      -acetylgalactosamine moieties is a vital step in the process of pore formation. This interaction may induce a conformational change in the Cry4Aa molecule that facilitates its insertion into the target membrane.\n                    <\/jats:p>\n                    <jats:sec>\n                      <jats:title>IMPORTANCE<\/jats:title>\n                      <jats:p>\n                        Cry toxins produced by the soil bacterium\n                        <jats:italic toggle=\"yes\">Bacillus thuringiensis<\/jats:italic>\n                        are used as an environmentally friendly biopesticide. Cry toxins are generally considered pore-forming toxins; however, the mechanism preceding pore formation appears to be complex and is not yet fully elucidated. This study examined the interaction between mosquito-larvicidal Cry4Aa toxin and sugar moieties, determining considerably higher binding affinity with\n                        <jats:italic toggle=\"yes\">N<\/jats:italic>\n                        -acetylgalactosamine. Furthermore, treatment with this sugar significantly increased the toxicity of Cry4Aa toward\n                        <jats:italic toggle=\"yes\">Culex pipiens<\/jats:italic>\n                        mosquito larvae. Subsequent analyses revealed that treatment with this sugar increases the binding of Cry4Aa to phosphatidylcholine bilayers, thereby facilitating pore formation. The findings of this study enhance our understanding of the mechanism underlying the insecticidal activity of Cry toxins.\n                      <\/jats:p>\n                    <\/jats:sec>\n                  <\/jats:sec>","DOI":"10.1128\/aem.00714-26","type":"journal-article","created":{"date-parts":[[2026,9,15]],"date-time":"2026-09-15T13:01:07Z","timestamp":1789477267000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":0,"title":["Interaction with the\n                    <i>N<\/i>\n                    -acetylgalactosamine moiety facilitates pore formation by Cry4Aa toxin, thereby increasing its toxicity"],"prefix":"10.1128","volume":"92","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1727-7623","authenticated-orcid":false,"given":"Mohammad Tofazzal Hossain","family":"Howlader","sequence":"first","affiliation":[{"id":[{"id":"https:\/\/ror.org\/03k5zb271","id-type":"ROR","asserted-by":"publisher"}],"name":"Department of Entomology, Bangladesh Agricultural University","place":["Mymensingh, Bangladesh"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taiyo","family":"Tsutsui","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02pc6pc55","id-type":"ROR","asserted-by":"publisher"}],"name":"Applied Chemistry and Biotechnology Program, School of Engineering, Okayama University","place":["Okayama, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kouichi","family":"Saito","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02pc6pc55","id-type":"ROR","asserted-by":"publisher"}],"name":"Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University","place":["Okayama, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2453-8150","authenticated-orcid":true,"given":"Tohru","family":"Hayakawa","sequence":"additional","affiliation":[{"id":[{"id":"https:\/\/ror.org\/02pc6pc55","id-type":"ROR","asserted-by":"publisher"}],"name":"Applied Chemistry and Biotechnology Program, School of Engineering, Okayama University","place":["Okayama, Japan"]},{"id":[{"id":"https:\/\/ror.org\/02pc6pc55","id-type":"ROR","asserted-by":"publisher"}],"name":"Graduate School of Interdisciplinary Science and Engineering in Health Systems, Okayama University","place":["Okayama, Japan"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","first-page":"355","article-title":"A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti, and Culex pipiens","volume":"37","author":"Goldberg LJ","year":"1977","unstructured":"Goldberg LJ, Margalit J. 1977. 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