Heterogeneity of S-layer proteins from aggregating and non-aggregating Lactobacillus kefir strains

Abstract

Since the presence of S-layer protein conditioned the autoaggregation capacity of some strains of Lactobacillus kefir, S-layer proteins from aggregating and non-aggregating L. kefir strains were characterized by immunochemical reactivity, MALDI-TOF spectrometry and glycosylation analysis. Two anti-S-layer monoclonal antibodies (Mab5F8 and Mab1F8) were produced; in an indirect enzyme-linked immunosorbent assay Mab1F8 recognized S-layer proteins from all L. kefir tested while Mab5F8 recognized only S-layer proteins from aggregating strains. Periodic Acid-Schiff staining of proteins after polyacrylamide gel electrophoresis under denaturing conditions revealed that all L. kefir S-layer proteins tested were glycosylated. Growth of bacteria in the presence of the N-glycosylation inhibitor tunicamycin suggested the presence of glycosydic chains O-linked to the protein backbone. MALDI-TOF peptide map fingerprint for S-layer proteins from 12 L. kefir strains showed very similar patterns for the aggregating strains, different from those for the non-aggregating ones. No positive match with other protein spectra in MSDB Database was found. Our results revealed a high heterogeneity among S-layer proteins from different L. kefir strains but also suggested a correlation between the structure of these S-layer glycoproteins and the aggregation properties of whole bacterial cells.

N-Linked glycoengineering for human therapeutic proteins in bacteria

“…The S-layer glycoprotein of Halobacterium halobium (salinarum) was the first published example of a prokaryotic glycoprotein (Mescher et al 1974). This led to a plethora of reports about S-layer glycoproteins in non-eukaryotes (Schaffer and Messner 2004) and recently, Mobili et al (2009) used periodic acidSchiff staining of polyacrylamide gel fractionated proteins to reveal that all extracted Lactobacillus kefir S-layer proteins were glycosylated (Mobili et al 2009). In addition to S-layer glycoproteins in bacteria, the pili and flagelli glycoproteins have also received a lot of attention.…”

The Major Autolysin Acm2 from Lactobacillus plantarum Undergoes Cytoplasmic O-Glycosylation

“…In Grampositive pathogens, such as streptococci and staphylococci, several serine-rich fimbria-like adhesins undergo glycosylation (4,33,41). In lactobacilli, the only glycoproteins characterized to date are S-layer proteins, in addition to a peptide bacteriocin (27,29,44).…”

Mutants ofLactobacillus plantarumML11-11 Deficient in Co-Aggregation with Yeast Exhibited Reduced Activities of Mixed-Species Biofilm Formation

“…Cell surface proteins extraction from LAB cells was performed using 5 M LiCl, as described in previous reports. 24,25) The cells removed surface protein were used in co-aggregation assay. The protein profile in the cell surface layer extracts was analyzed by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE), as described below.…”

“…9) There are other reports on the co-aggregation of LAB including Lactobacillus and yeasts including Saccharomyces. [10][11][12][13] Pretzer et al found that L. plantarum WCFS1 co-aggregates with yeast S. cerevisiae.…”