Phylogenetic analysis of the bacterial Pro-Pro-endopeptidase domain reveals a diverse family including secreted and membrane anchored proteins

Abstract

Pro-Pro-endopeptidases (PPEP, EC 3.4.24.89) are secreted, zinc metalloproteases that have the unusual capacity to cleave a peptide bond between two prolines, a bond that is generally less sensitive to proteolytic cleavage. Two well studied members of the family are PPEP-1 and PPEP-2, produced by Clostridioides difficile, a human pathogen, and Paenibacillus alvei, a bee secondary invader, respectively. Both proteases seem to be involved in mediating bacterial adhesion by cleaving cell surface anchor proteins on the bacterium itself. By using basic alignment and phylogenetic profiling analysis, this work shows that the complete family of proteins that contain a PPEP domain includes proteins from more than 130 species spread over 9 genera. These analyses also suggest that the PPEP domain spread through horizontal gene transfer events between species within the Firmicutes' classes Bacilli and Clostridia. Bacterial species containing PPEP homologs are found in diverse habitats, varying from human pathogens and gut microbiota to free-living bacteria, which were isolated from various environments, including extreme conditions such as hot springs, desert soil and salt lakes. The phylogenetic tree reveals the relationships between family members and suggests that smaller subgroups could share cleavage specificity, substrates and functional similarity. Except for PPEP-1 and PPEP-2, no cleavage specificity, specific physiological target, or function has been assigned for any of the other PPEP-family members. Some PPEP proteins have acquired additional domains that recognize and bind noncovalently to various elements of the bacterial peptidoglycan cell-wall, anchoring these PPEPs. Secreted or anchored to the cell-wall surface PPEP proteins seem to perform various functions.

Keywords: Adhesion; Bacterial virulence; CBD, Cell-wall-binding-domain; Cell wall; Endopeptidases; FN3, Fibronectin type III domain; Metalloprotease; Metalloproteases; Motility; MucBP, Mucin binding protein; PPEP, Pro-pro-endopeptidases; Phylogeny; SCWP, Secondary cell-wall polysaccharide; SLH, surface layer homology; Secreted protease; TED, thioester domain; VMSP, VWFA-MucBP-SLH-protein; VWFA, Von Willebrand factor type A.

Graphical abstract

Fig. 1

Domain organization of Clostridioides difficile and Paenibacillus alvei adhesion proteins anchored to the peptidoglycan cell wall layer, and their PPEP-1 and PPEP-2 cleavage sites. Arrows indicate PPEP-1 and PPEP-2 cleavage sites with bold, underlined specific recognition sequence. Peptidoglycan cell wall layer (gray). L= LPXTG-like cell wall anchor, R = repetitive sequence, N = Amino terminal domain, Orange = region containing PPEP recognition sites. For other domain names see text.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

3D structure of PPEP-1 with and without substrate peptide and conserved amino acids of PPEP-family. A) without peptide, PDB 5A0P; B) with peptide (orange), PDB 6R57; Black Lines indicate atom-pair contacts with cut-off value of 4 Å (Aydlnkal et al., 2019). C) Ball and stick representation of peptide binding cleft; peptide in red (spheres), contacting residues (sticks) in light blue. D) Conserved residues in PPEP-family alignment (95% threshold) shown as sidechain sticks and indicated in the PPEP-1 structure (PDB 6R57) without peptide (Pichlo et al.2019). Substrate proline P1 contacting residues in green, substrate proline P1’ contacts shown in orange. Residues shown in red are involved in zinc coordination and catalytic activity.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Phylogenetic relationship of PPEP-like proteins. A rooted phylogenetic tree was constructed using an alignment of 146 PPEP homologs from 131 species. Tree was built by using the Neighbor-Joining method with the Jukes-Cantor genetic distance model. Major species groups are highlighted by different colors as indicated. Outgroup is M34 peptidase of Vibrio cholera O1 (WP_108257476). Bootstrap values of branches corresponding to indicated groups are all >50 except for the Clostridioides group. Furthest distance in the tree is between B. lentus and J. saudimassiliensis (32%, identities; 52%, similarity; BLAST e-value 7e-28). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 4

PPEP family proteins domain organization. Several members of the PPEP family contain an additional domain, SH3 (Bacillus group B), FN3 (Salinicoccus/Jeotgalicoccus) or SLH (Paenibacillus group B), which are predicted to be involved in anchoring to the Gram-positive bacterium cell wall (gray). S = secretion signal. For other domain names see text.

Fig. 5

Genomic conservation of regions flanking the PPEP homologs (putative PPEP, pPPEP, in red). A) Genomic conservation of regions flanking PPEP-1, PPEP-2, and homologs in C. difficile and Paenibacillus. Paenibacillus group A containing VMSP (orange) PPEP-2 substrate and group B lacking VMSP. Light blue triangle indicates c-di-GMP type I riboswitch. Top shows C.difficile with its substrate protein (CD2831) in dark orange. Small red triangle indicates c-di-GMP type II riboswitch. SLH, surface-layer homology, domain bind to secondary cell-wall polymers. Paenibacillus (P.); Clostridioides (C.). SLH-domain is predicted to bind surface layer. B) Bacillus group A. Bacillus (B.), Parageobacillus (Pa.), Geobacillus (G.) and Anoxybacillus (A.). C) Bacillus group B (bottom) and C (top). SH3b-domain is predicted to be a peptidoglycan cell-wall-binding-domain. D) Salinicoccus (S.) and Jeotgalicoccus (J.) group. FN3 domains is predicted to bind to cell wall carbohydrates. E) Clostridium species.(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 6

Comparison of genomic regions between closely related species with and without a PPEP homolog. Genome pairs with (top) and without PPEP gene (bottom). 1 Paenibacillus daejeonensis DSM 15491 (438541 – 442364 and Paenibacillus baekrokdamisoli CECT 8890_05 (172529–176227); 2 Bacillus firmus NBRC 15306_18 (2246-4072) and Bacillus sp. 1NLA3E (3111676- 3114991); 3 Bacillus megaterium CH447_03 (510523–515539) and Bacillus megaterium ATCC 14,581_04 (396657–400370). Black lines indicate DNA alignment with the coding sequences (arrow) above and below the respective genes.