Abstract
BackgroundLactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. The bacterium has great potential as food-grade cell factory and for in situ delivery of biomolecules. Since protein secretion is important both for probiotic activity and in biotechnological applications, we have carried out a genome-wide experimental study of signal peptide (SP) functionality.ResultsWe have constructed a library of 76 Sec-type signal peptides from L. plantarum WCFS1 that were predicted to be cleaved by signal peptidase I. SP functionality was studied using staphylococcal nuclease (NucA) as a reporter protein. 82% of the SPs gave significant extracellular NucA activity. Levels of secreted NucA varied by a dramatic 1800-fold and this variation was shown not to be the result of different mRNA levels. For the best-performing SPs all produced NucA was detected in the culture supernatant, but the secretion efficiency decreased for the less well performing SPs. Sequence analyses of the SPs and their cognate proteins revealed four properties that correlated positively with SP performance for NucA: high hydrophobicity, the presence of a transmembrane helix predicted by TMHMM, the absence of an anchoring motif in the cognate protein, and the length of the H+C domain. Analysis of a subset of SPs with a lactobacillal amylase (AmyA) showed large variation in production levels and secretion efficiencies. Importantly, there was no correlation between SP performance with NucA and the performance with AmyA.ConclusionThis is the first comprehensive experimental study showing that predicted SPs in the L. plantarum genome actually are capable of driving protein secretion. The results reveal considerable variation between the SPs that is at least in part dependent on the protein that is secreted. Several SPs stand out as promising candidates for efficient secretion of heterologous proteins in L. plantarum. The results for NucA provide some hints as to the sequence-based prediction of SP functionality, but the general conclusion is that such prediction is difficult. The vector library generated in this study is based on exchangeable cassettes and provides a powerful tool for rapid experimental screening of SPs.
Highlights
Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract
The two algorithms yielded the same conclusions for 78 signal peptide (SP) and these were selected for further studies
The present study shows that at least 82% of the tested putative signal peptidase I-dependent SPs in the genome of L. plantarum WCFS1 functions as a signal for secretion
Summary
Lactobacillus plantarum is a normal, potentially probiotic, inhabitant of the human gastrointestinal (GI) tract. Lactobacillus plantarum is a Gram-positive lactic acid bacterium (LAB) with a long tradition in food fermentation, and is Generally Regarded As Safe (GRAS status). L. plantarum is adapted to survive in the harsh conditions of the GI-tract, as has been illustrated by recent genome-wide gene expression studies of the response of the bacterium to (mouse) GI-tract conditions [8,9]. Both the potential probiotic effects of L. plantarum and the high survival rate during the passage of the GI-tract make this bacterium a promising candidate as a vehicle for in situ delivery of therapeutically interesting proteins [10]. Promising results have been obtained with LAB that secrete or anchor antigens to the cell (recently reviewed by Wells and Mercenier [12]; see [13])
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