Characteristics of the Clostridium difficile cell envelope and its importance in therapeutics

Abstract

Clostridium difficile infection (CDI) is a challenging threat to human health. Infections occur after disruption of the normal microbiota, most commonly through the use of antibiotics. Current treatment for CDI largely relies on the broad-spectrum antibiotics vancomycin and metronidazole that further disrupt the microbiota resulting in frequent recurrence, highlighting the need for C. difficile-specific antimicrobials. The cell surface of C. difficile represents a promising target for the development of new drugs. C. difficile possesses a highly deacetylated peptidoglycan cell wall containing unique secondary cell wall polymers. Bound to the cell wall is an essential S-layer, formed of SlpA and decorated with an additional 28 related proteins. In addition to the S-layer, many other cell surface proteins have been identified, including several with roles in host colonization. This review aims to summarize our current understanding of these different C. difficile cell surface components and their viability as therapeutic targets.

Figure 1

Structure of the conserved cell wall polymers of Clostridium difficile. PG: C. difficile produces a peptidoglycan characterized by a very high degree of N‐acetylglucosamine deacetylation (up to 93%), the stem peptide l‐Ala‐d‐Glu‐A₂pm‐d‐Ala‐d‐Ala and an unusually high degree (73%) of 3‐3 cross‐links. PS‐II: a conserved cell wall polysaccharide polymer with a core hexasaccharide repeating unit of [→6)‐β‐d‐Glcp‐(1→3)‐β‐d‐Galp NAc‐(1→4)‐α‐d‐Glcp‐(1→4)‐[β‐d‐Glcp‐ (1→3]‐β‐d‐Galp NAc‐(1→3)‐α‐d‐Manp‐(1→P→]. PS‐III/LTA: a conserved lipid‐anchored cell wall polysaccharide in the extended lipoteichoic acid family with a core repeating unit of [→6)‐α‐d‐Glcp NAc‐(1→3)‐[→P‐6]‐α‐d‐Glcp NAc‐(1→2)‐d‐GroA]. This repeat unit is linked to →6)‐β‐d‐Glcp‐(1→6)‐β‐d‐Glcp‐(1→6)‐β‐d‐Glcp‐(1→1)‐Gro, with the terminal glycerol esterified with C₁₄, C₁₆, or C₁₈ saturated or mono‐unsaturated fatty acids.

Figure 2

The S‐layer locus. A. Clostridium difficile strain 630 encodes 29 cell wall proteins that use the CWB2 (PF04122) motif for non‐covalent anchoring to the cell wall. Twelve of these, including the S‐layer precursor SlpA, are encoded within a single genomic locus (green arrows) that also encodes the S‐layer secretion ATPase SecA2 (red arrow) and five unrelated proteins (black arrows). The core variable S‐layer cassette region is highlighted. An extensive glycan synthesis cluster is located immediately downstream of cwp7. It is believed that the proteins encoded in this cluster are responsible for the synthesis of PS‐II (Willing et al., 2015). B. One of the 12 identified S‐layer cassettes (cassette type 11) has a 23.8 kb insertion that includes 19 putative ORFs (Dingle et al., 2013). Functional predictions of each of the encoded proteins identified all of the activities necessary for the synthesis of a complex glycan and transfer to a substrate. In cassette type 11, the cwp2 gene is missing and the order of cwp66 and cd2790 is reversed.

Figure 3

Organization of the Clostridium difficile cell envelope. A. C. difficile has a normal Gram positive cell envelope with a surface exposed proteinaceous S‐layer on the outer surface. The S‐layer is decorated and functionalized by members of the CWP family; shown are the putative adhesin CwpV and cysteine protease Cwp84. Secretion of the S‐layer precursor SlpA and CwpV are dependent on the accessory ATPase SecA2. Following secretion, SlpA is cleaved by Cwp84 (green arrow), generating the LMW and HMW SLPs. These SLPs form a high‐affinity heterodimer that represents the basic subunit of the S‐layer. CwpV also undergoes post‐secretion processing via an enzyme‐independent auto‐proteolytic mechanism. In addition to the S‐layer and associated CWPs, C. difficile possesses numerous other cell surface proteins. The mechanism of secretion and cell wall anchoring of GroEL and Fbp68 (FbpA) is unclear but both can be detected on the cell surface. The lipoprotein CD0873 and sortase‐anchored proteins CbpA and CD2831 are likely secreted via the canonical Sec pathway. Following secretion, CD0873 is attached to the cell membrane via its lipid anchor and the sortase substrates are covalently linked to the peptidoglycan (Thr‐mDap) by the sortase enzyme CD2718. B. Domain organization of the proteins shown in A. N‐terminal secretion signals are shown as black boxes, the CD0873 lipobox is shown in grey and the (lipoprotein) signal peptidase cleavage sites are indicated with white arrows. Post‐secretion cleavage sites are indicated with black arrows. Functional domains demonstrated experimentally or identified using the Pfam database (Finn et al., 2016) are also highlighted. The sequence and location of sorting motifs are shown above CbpA and CD2831.