Analysis of the Peptidoglycan Hydrolase Complement of Lactobacillus casei and Characterization of the Major γ-D-Glutamyl-L-Lysyl-Endopeptidase

Krzysztof Regulski,Ingmar J J Claes,Marie-Pierre Chapot-Chartier,Pascal Courtin,Alain Guillot,Sarah Lebeer,Mickael Meyrand,Pascal Hols,Jos Vanderleyden

doi:10.1371/journal.pone.0032301

Krzysztof Regulski, Ingmar J J Claes + Show 7 more

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https://doi.org/10.1371/journal.pone.0032301

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Peptidoglycan (PG) is the major component of Gram positive bacteria cell wall and is essential for bacterial integrity and shape. Bacteria synthesize PG hydrolases (PGHs) which are able to cleave bonds in their own PG and play major roles in PG remodelling required for bacterial growth and division. Our aim was to identify the main PGHs in Lactobacillus casei BL23, a lactic acid bacterium with probiotic properties.The PGH complement was first identified in silico by amino acid sequence similarity searches of the BL23 genome sequence. Thirteen PGHs were detected with different predicted hydrolytic specificities. Transcription of the genes was confirmed by RT-PCR. A proteomic analysis combining the use of SDS-PAGE and LC-MS/MS revealed the main seven PGHs synthesized during growth of L. casei BL23. Among these PGHs, LCABL_02770 (renamed Lc-p75) was identified as the major one. This protein is the homolog of p75 (Msp1) major secreted protein of Lactobacillus rhamnosus GG, which was shown to promote survival and growth of intestinal epithelial cells. We identified its hydrolytic specificity on PG and showed that it is a γ-D-glutamyl-L-lysyl-endopeptidase. It has a marked specificity towards PG tetrapeptide chains versus tripeptide chains and for oligomers rather than monomers. Immunofluorescence experiments demonstrated that Lc-p75 localizes at cell septa in agreement with its role in daughter cell separation. It is also secreted under an active form as detected in zymogram. Comparison of the muropeptide profiles of wild type and Lc-p75-negative mutant revealed a decrease of the amount of disaccharide-dipeptide in the mutant PG in agreement with Lc-p75 activity. As a conclusion, Lc-p75 is the major L. casei BL23 PGH with endopeptidase specificity and a key role in daughter cell separation. Further studies will aim at investigating the role of Lc-p75 in the anti-inflammatory potential of L. casei BL23.

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Peptidoglycan (PG) is the major cell wall component in Gram positive bacteria, and plays a key role to guarantee bacterial cell integrity and shape
In silico search of the PG hydrolases (PGHs) complement of L. casei BL23 The PGH complement of L. casei BL23 was identified in silico in the available BL23 genome sequence [10] by amino acid sequence similarity search with well known PGHs from other Gram positive species [3]
Identification of the main PGHs expressed during growth of L. casei BL23

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Peptidoglycan (PG) is the major cell wall component in Gram positive bacteria, and plays a key role to guarantee bacterial cell integrity and shape. The cell wall PG is the target of specific PG hydrolases (PGHs) ( named autolysins), synthesized by the bacteria themselves [2,3]. D-Ala-carboxypeptidases are usually present but are not able to provoke bacterial lysis on their own they are not classified as autolysins. These PGHs are involved in different cellular functions in bacteria, which require cell wall remodelling during growth and division. In conditions leading to growth arrest, their PG hydrolysing activity is susceptible to provoke bacterial lysis

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