The S-layer protein of a Clostridium difficile SLCT-11 strain displays a complex glycan required for normal cell growth and morphology

Abstract

Clostridium difficile is a bacterial pathogen that causes major health challenges worldwide. It has a well-characterized surface (S)-layer, a para-crystalline proteinaceous layer surrounding the cell wall. In many bacterial and archaeal species, the S-layer is glycosylated, but no such modifications have been demonstrated in C. difficile. Here, we show that a C. difficile strain of S-layer cassette type 11, Ox247, has a complex glycan attached via an O-linkage to Thr-38 of the S-layer low-molecular-weight subunit. Using MS and NMR, we fully characterized this glycan. We present evidence that it is composed of three domains: (i) a core peptide–linked tetrasaccharide with the sequence -4-α-Rha-3-α-Rha-3-α-Rha-3-β-Gal-peptide; (ii) a repeating pentasaccharide with the sequence -4-β-Rha-4-α-Glc-3-β-Rha-4-(α-Rib-3-)β-Rha-; and (iii) a nonreducing end–terminal 2,3 cyclophosphoryl-rhamnose attached to a ribose-branched sub-terminal rhamnose residue. The Ox247 genome contains a 24-kb locus containing genes for synthesis and protein attachment of this glycan. Mutations in genes within this locus altered or completely abrogated formation of this glycan, and their phenotypes suggested that this S-layer modification may affect sporulation, cell length, and biofilm formation of C. difficile. In summary, our findings indicate that the S-layer protein of SLCT-11 strains displays a complex glycan and suggest that this glycan is required for C. difficile sporulation and control of cell shape, a discovery with implications for the development of antimicrobials targeting the S-layer.

The recent emergence of a highly related virulent Clostridium difficile clade with unique characteristics

“…It has recently been shown that S-layer glycosylation is important for adherence to Caco-2 intestinal epithelial cells but not for biofilm formation [20]. Therefore, glycosylation of the S-layer in clade 3 may be important for colonization but not persistence, explaining a low level of carriage and recurrence of these strains.…”

“…3c). Strain Ox247 (RT005, clade 1) containing SLCT11 [20] along with S-layer preparations from three representative RT023 strains show an alternative pattern of SLPs. HMW SLP migrates at its expected molecular weight, but a band at 18 kDa for LMW SLP is absent.…”

Utilizing Ribose Compounds: How Bacteroides PUL It Off

“…In the study highlighted in this Preview, Glowacki et al (2020) identify and characterize a locus, designated rus, which is essential for utilization of ribose (Glowacki et al, 2020). In addition to being a component of the nucleotides of RNA, this pentose is also a constituent of diverse microbial glycans (Richards et al, 2018;Toukach, 2011) and other molecules such as NADH and ATP. In this study, the authors show that the type strain of Bacteroides thetaiotaomicron is able to use not only ribose, but also nucleosides, as a carbon and energy source in a ribose-utilization system (RUS)-dependent manner (Glowacki et al, 2020).…”

Immunostimulation by Lactobacillus kefiri S-layer proteins with distinct glycosylation patterns requires different lectin partners

“…Inter-strain differences in the glycosylation patterns of SLP were also seen some years ago in Thermoanaerobacter thermohydrosulfuricus and Thermoanaerobacterium thermosaccharolyticum (31). Moreover, in Clostridium difficile the presence of a glycosylated SLP was only demonstrated in the strain Ox247 which encodes a putative glycosylation locus (18), while normally most C. difficile strains do not possess a glycosylated S-layer (32). On the other hand, Anzengruber and coworkers reported that SlpB from L. buchneri CD034, SlpN from L. buchneri NRRL B-30929 and L. buchneri SlpB from 41021/251 showed identical O-glycans consisting of on average seven glucoses at four serine residues within the sequence S152-A-S154-S155-A-S157 without any evidence of the presence of N-glycosidic chains in those SLP (29).…”

“…Noteworthy, although the S-layer proteins were the first glycoproteins described in prokaryotes, the studies of the role of the glycan residues in the functional properties of these proteins are scarce and are usually focused on some archaea (16) or pathogenic bacteria (17,18). Regarding the genus Lactobacillus, there are some reports describing the involvement of the carbohydrate receptor DCspecific ICAM-3-grabbing nonintegrin (DC-SIGN) in the functional activity of the S-layer proteins from L. acidophilus NCFM (19), L. plantarum (20) and L. kefiri JCM 5818 (21).…”