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Published on: October 30, 2016
Sequence and phylogenetic analysis of the gene for surface layer protein, slpA, from 14 PCR ribotypes of Clostridium
Insights
Clostridium difficile surface protein A (slpA) gene variations correlate with PCR ribotypes in elderly patients. Analysis revealed distinct slpA sequence groups, suggesting independent evolution with the secA gene.
Area of Science:
- Microbiology
- Genetics
- Molecular Biology
Background:
- Clostridium difficile is a leading cause of antibiotic-associated diarrhea, particularly in hospitalized elderly individuals.
- The organism's surface protein layer (S-layer) is crucial for its structure and is encoded by the slpA gene.
- slpA encodes a precursor protein cleaved into high molecular weight (HMW) and low molecular weight (LMW) peptides, with LMW being a key antigen for serotyping.
Purpose of the Study:
- To investigate the genetic diversity of the slpA gene in Clostridium difficile strains from elderly patients.
- To compare slpA gene sequences with PCR ribotyping profiles.
- To analyze the evolutionary relationship between slpA and the secA gene.
Main Methods:
- Sequencing of the slpA gene and flanking DNA from 14 Clostridium difficile ribotypes isolated from elderly patients.
- Comparison of obtained sequences with each other and with published sequences.
- Phylogenetic analysis using neighbor-joining trees for both SlpA and SecA amino acid sequences.
Main Results:
- Identical slpA sequences were found in ribotypes 046 and 092.
- Minor sequence differences (1-3 nucleotides) were observed in slpA between certain ribotype pairs.
- Nine distinct slpA sequence groups were identified, with significant variations in the LMW peptide region.
- A strong association was observed between specific ribotypes (012, 046/092, 017, 031, 094) for both SlpA and SecA.
- Independent evolution of slpA and secA was indicated by differing phylogenetic associations for some ribotypes.
Conclusions:
- The slpA gene exhibits significant sequence diversity among Clostridium difficile ribotypes, particularly in the LMW peptide region.
- The observed genetic variations in slpA correlate with PCR ribotyping, suggesting its potential utility in strain characterization.
- The independent evolution of slpA and secA implies different selective pressures acting on these genes.
Abstract:
Clostridium difficile is the commonest cause of antibiotic-associated diarrhoea, with the hospitalized elderly being at particular risk. The organism makes a crystalline surface protein layer (S-layer), encoded by the slpA gene, the product of which is cleaved to give two mature peptides which associate to form the layer. The larger peptide (high molecular weight; HMW), derived from the C-terminal portion of the precursor, is relatively conserved, whereas the smaller peptide (low molecular weight; LMW), derived from the N-terminal portion of the precursor, is a dominant antigen which substantially forms the basis for serotyping of isolates. PCR ribotyping is a more discriminatory typing method, based on the intergenic rRNA. We obtained the sequence for slpA and some flanking DNA from a collection of C. difficile strains of 14 ribotypes isolated from elderly patients. Sequences from different ribotypes were compared with one another and with published sequences. Sequences from C. difficile ribotypes 046 and 092 were identical. Sequences from ribotype pairs 005 and 054, 012 and 046/092, 014 and 066 and 031 and 094 differed by 1-3 nt in the slpA gene. There were ultimately nine ribotypes or groups of ribotypes with very different slpA sequences, particularly in the region encoding the LMW peptide. The sequence from ribotype 002 was very different from previously published sequences. The DNA segment sequenced included the 5' 315 bp of a secA homologue, encoding a putative transport protein required for peptide secretion across the plasma membrane. The amino acid sequences of the predicted HMW peptides were aligned and a neighbour-joining tree was produced using 10,000 bootstrap replicates. The predicted SecA N-terminal region was similarly analysed. For both SlpA and SecA, a strong association was found between ribotypes 012, 046/092, 017, 031 and 094. Ribotypes 001 and 078 formed part of this clade for SlpA but not SecA, indicating independent evolution for slpA and secA, presumably because they come under different selection pressures.
