Characterization of Lactobacillus coryniformis DSM 20001T surface protein Cpf mediating coaggregation with and aggregation among pathogens - PubMed (original) (raw)

Characterization of Lactobacillus coryniformis DSM 20001T surface protein Cpf mediating coaggregation with and aggregation among pathogens

Martina Schachtsiek et al. Appl Environ Microbiol. 2004 Dec.

Abstract

Phenotypic characterization of aggregation phenotypes of Lactobacillus coryniformis revealed that strain DSM 20001T 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 20001T. 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 degrees 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 20001T. Cpf could be removed from cells of strain DSM 20001T 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.

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Figures

FIG. 1.

Macro- and microscopic manifestation of the co/aggregation property of L. coryniformis DSM 20001T. Coaggregation of cells of strain DSM 20001T with E. coli LTH 1577 resulted in the formation of coaggregates (C) which sedimented to the bottom of reaction tubes (A). Addition of SCS of DSM 20001T to cells of E. coli LTH 1577 led to clumping of the cells (D) into sedimenting aggregates (B).

FIG. 2.

Schematic representation of the cpf gene of L. coryniformis DSM 20001T and its flanking regions. The boxes indicate the insertion element IS_Lco1_, the open reading frames of cpf, and incomplete orfX. The localization of promoters (P) and transcriptional terminators (T) is shown. Transcripts of IS_Lco1_ and cpf identified by Northern hybridization analysis are indicated by arrows.

FIG. 3.

Mapping of the 5′ end of mRNA of cpf by primer extension. Transcripts were generated from total RNA isolated from L. coryniformis DSM 20001T (lane 1). The arrow indicates the position of the product obtained; the asterisk indicates the transcription start site.

FIG. 4.

Northern hybridization analysis of RNA isolated from L. coryniformis DSM 20001T. The analysis was performed with a _cpf_-specific probe (A) and an IS_Lco1_-specific probe (B). The sizes of the marker fragments are indicated on the left.

FIG. 5.

Characterization of the Cpf activity of L. coryniformis DSM 20001T by quantification of co/aggregation. (A to C) Aggregation of E. coli LTH 1577 (A), C. coli DSM 4689T (B), and C. jejuni DSM 4688T (C) in the presence of recombinant Cpf (▪) or SCS (▴) of L. coryniformis DSM 20001T, as well as coaggregation of L. coryniformis DSM 20001T (▾) with the pathogens. The controls (•) consisted of cells of the corresponding pathogens only. (D) Coaggregation of E. coli LTH 1577 with cells of L. coryniformis DSM 20001T extracted with 5 M LiCl (•) and subsequently incubated in SCS (▴) or a solution containing recombinant Cpf (▪). Coaggregation of E. coli LTH 1577 with cells of L. curvatus LTH 684 incubated in SCS of strain DSM 20001T (▴) was also determined. The control (▾) consisted of lactobacillus cells that were treated with only water.

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Characterization of Lactobacillus coryniformis DSM 20001T surface protein Cpf mediating coaggregation with and aggregation among pathogens - PubMed (original) (raw)