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Two alkaline motifs in the Lactobacillus salivarius Lv72 OppA surface are important to its adhesin function

In: Beneficial Microbes
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C. Martín Área de Microbiología, Universidad de Oviedo, Julián Clavería 6, 33006 Oviedo, Spain.
Instituto Universitario Fernández- Vega, Universidad de Oviedo, Av. Doctores Fernández Vega, 34, 33012 Oviedo, Spain.

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S. Escobedo Área de Microbiología, Universidad de Oviedo, Julián Clavería 6, 33006 Oviedo, Spain.
Instituto Universitario de Biotecnología, Universidad de Oviedo, Doctor Fernando Bongera s/n, 33006 Oviedo, Spain.

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G. Pérez-Martínez Laboratorio de Bacterias Lácticas y Probióticos, Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Catedràtic Agustín Escardino Benlloch, 7, 46980 Valencia, Spain.

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J.M. Coll-Marqués Laboratorio de Bacterias Lácticas y Probióticos, Departamento de Biotecnología, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Catedràtic Agustín Escardino Benlloch, 7, 46980 Valencia, Spain.

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R. Martín National Institute of Agricultural Research, Commensals and Probiotics- Host Interactions Laboratory, Micalis Institute, AgroParisTech, Paris-Sud University, Allée de Vilvert, 78352 Jouy-en- Josas, France.

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J.E. Suárez Área de Microbiología, Universidad de Oviedo, Julián Clavería 6, 33006 Oviedo, Spain.
Instituto Universitario de Biotecnología, Universidad de Oviedo, Doctor Fernando Bongera s/n, 33006 Oviedo, Spain.
Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Río Linares, s/n, 33300 Villaviciosa, Spain.

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L.M. Quirós Área de Microbiología, Universidad de Oviedo, Julián Clavería 6, 33006 Oviedo, Spain.
Instituto Universitario Fernández- Vega, Universidad de Oviedo, Av. Doctores Fernández Vega, 34, 33012 Oviedo, Spain.

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Glycosaminoglycans are involved in the attachment of Lactobacillus salivarius Lv72, a strain of vaginal origin, to HeLa cell cultures, indicating that they play a fundamental role in the attachment of mutualistic bacteria to the epithelium lining cavities where the normal microbiota thrives. The bacterial OppA protein has been proposed as an adhesin involved in this adherence since, once purified, it significantly interferes with attachment of the lactobacilli to HeLa cell cultures. In this article, the role of OppA is confirmed through the determination of its location at the cell surface and its ability to promote Lactobacillus casei and Lactococcus lactis adherence to eukaryotic cell cultures upon cloning and expression of oppA in these bacteria. The OppA sequence showed five potential domains for glycosaminoglycan-binding, and structural modelling of the protein showed that two of them were located in the vicinity of an OppA superficial groove whose width approached the diameter of the helical form of heparin in solution. Their involvement in the binding was demonstrated through substitution of critical basic amino acids by acidic ones, which resulted in loss of affinity for heparan sulphate and chondroitin sulphate depending on the domain mutated, suggesting that there might be a certain degree of specialisation. In addition, circular dichroism analysis showed that the spectrum changes induced by OppA-heparan sulphate binding were attenuated by the variant proteins, indicating that these motifs are the OppA recognition domains for the eukaryotic cell surface.

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