Characterization of Lactobacillus coryniformis DSM 20001 T Surface Protein Cpf Mediating Coaggregation with and Aggregation among Pathogens

Abstract

Phenotypic characterization of aggregation phenotypes of Lactobacillus coryniformis revealed that strain DSM 20001^T coaggregated with Escherichia coli K88, Campylobacter coli, and Campylobacter jejuni but not with other human pathogens. In addition, cells of these pathogens aggregated in the presence of the spent culture supernatant (SCS) of strain DSM 20001^T. Cells of E. coli K88 remained viable in the coaggregates and aggregates for up to 24 h. Both coaggregation and aggregation (co/aggregation) occurred at pH 3.5 to 7.5 and was sensitive to heat (85°C for 15 min) and proteinase K. The co/aggregation-promoting factor (Cpf) was purified, and the gene was identified by PCR with degenerate primers derived from internal amino acid sequences. The cpf gene encoded a 19.9-kDa preprotein with a sec-dependent leader and an isoelectric point of 4.4. The amino acid sequence had no significant similarity to proteins with known functions. Northern analysis revealed not only major transcription from the promoter of cpf but also major transcription from the promoter of the preceding insertion element, ISLco1 belonging to the IS3 family. Recombinant Cpf produced in E. coli mediated aggregation of pathogens comparable to the aggregation obtained with purified Cpf or SCS of strain DSM 20001^T. Cpf could be removed from cells of strain DSM 20001^T by treatment with 5 M LiCl and could be subsequently reattached to the cell surface by using SCS or recombinant Cpf, which resulted in restoration of the co/aggregation property. These results together with those of the amino acid sequence analysis suggest that Cpf is a novel surface protein of L. coryniformis that mediates co/aggregation of some pathogens.