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

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

Pathogenicity and virulence of Clostridioides difficile

Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhoea, and is responsible for a spectrum of diseases characterised by high levels of recurrence, morbidity and mortality. Treatment is complex, since antibiotics constitute both the main treatment and the major risk factor for infection. Worryingly, resistance to multiple antibiotics is becoming increasingly widespread, leading to classification of this pathogen as an urgent threat to global health. As a consummate opportunist, C. difficile is well-equipped for promoting disease, owing to its arsenal of virulence factors: transmission of this anaerobe is highly efficient due to formation of robust endospores, and an array of adhesins promote gut colonisation. C. difficile produces multiple toxins acting upon gut epithelia, resulting in manifestations typical of diarrhoeal disease, and severe inflammation in a subset of patients. This review focuses on such virulence factors, as well as the importance of antimicrobial resistance and genome plasticity in enabling pathogenesis and persistence of this important pathogen

Snap denaturation reveals dimerization by AraC-like protein Rns

Here we show that the Rns regulator of Escherichia coli dimerises in vivo and in vitro. Furthermore, we demonstrate that Rns forms aggregates in vitro and describe a methodology to ameliorate aggregation thus permitting the analysis of Rns by cross-linking

Clostridium difficile surface proteins are anchored to the cell wall using CWB2 motifs that recognise the anionic polymer PSII

Gram-positive surface proteins can be covalently or non-covalently anchored to the cell wall and can impart important properties on the bacterium in respect of cell envelope organisation and interaction with the environment. We describe here a mechanism of protein anchoring involving tandem CWB2 motifs found in a large number of cell wall proteins in the Firmicutes. In the Clostridium difficile cell wall protein family, we show the three tandem repeats of the CWB2 motif are essential for correct anchoring to the cell wall. CWB2 repeats are non-identical and cannot substitute for each other, as shown by the secretion into the culture supernatant of proteins containing variations in the patterns of repeats. A conserved Ile Leu Leu sequence within the CWB2 repeats is essential for correct anchoring, although a preceding proline residue is dispensable. We propose a likely genetic locus encoding synthesis of the anionic polymer PSII and, using RNA knock-down of key genes, reveal subtle effects on cell wall composition. We show that the anionic polymer PSII binds two cell wall proteins, SlpA and Cwp2, and these interactions require the CWB2 repeats, defining a new mechanism of protein anchoring in Gram-positive bacteria

A cortex-specific PBP contributes to cephalosporin resistance in Clostridium difficile

Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridium difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We have identified and characterised a penicillin binding protein (PBP) that is required for cortex synthesis in C. difficile. Surprisingly this PBP was also found to contribute to cephalosporin resistance, indicating an additional role in the synthesis of vegetative cell wall. This is the first description of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen

Architecture and self-assembly of clostridium sporogenes and clostridium botulinum spore surfaces illustrate a general protective strategy across spore formers

Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives can have similar spore architectures although some display unique features; they all incorporate protective proteinaceous envelopes. We previously found that Bacillus spores can achieve these protective properties through extensive disulfide cross-linking of self-assembled arrays of cysteine-rich proteins. We predicted that this could be a mechanism employed by spore formers in general, even those from other genera. Here, we tested this by revealing in nanometer detail how the outer envelope (exosporium) in Clostridium sporogenes (surrogate for C. botulinum group I), and in other clostridial relatives, forms a hexagonally symmetric semipermeable array. A cysteine-rich protein, CsxA, when expressed in Escherichia coli, self-assembles into a highly thermally stable structure identical to that of the native exosporium. Like the exosporium, CsxA arrays require harsh “reducing” conditions for disassembly. We conclude that in vivo, CsxA self-organizes into a highly resilient, disulfide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar to that in Bacillus spores, despite a lack of protein homology. In both cases, intracellular disulfide formation is favored by the high lattice symmetry. We have identified cysteine-rich proteins in many distantly related spore formers and propose that they may adopt a similar strategy for intracellular assembly of robust protective structures

Potential role of the host-derived cell-wall binding domain of endolysin CD16/50L as a molecular anchor in preservation of uninfected clostridioides difficile for new rounds of phage infection

Endolysin is a phage-encoded cell-wall hydrolase which degrades the peptidoglycan layer of the bacterial cell wall. The enzyme is often expressed at the late stage of the phage lytic cycle and is required for progeny escape. Endolysins of bacteriophage that infect Gram-positive bacteria often comprises two domains: a peptidoglycan hydrolase and a cell-wall binding domain (CBD). Although the catalytic domain of endolysin is relatively well-studied, the precise role of CBD is ambiguous and remains controversial. Here, we focus on the function of endolysin CBD from a recently isolated Clostridioides difficile phage. We found that the CBD is not required for lytic activity, which is strongly prevented by the surface layer of C. difficile. Intriguingly, hidden Markov model analysis suggested that the endolysin CBD is likely derived from the CWB2 motif of C. difficile cell-wall proteins but possesses a higher binding affinity to bacterial cell-wall polysaccharides. Moreover, the CBD forms a homodimer, formation of which is necessary for interaction with the surface saccharides. Importantly, endolysin diffusion and sequential cytolytic assays showed that CBD of endolysin is required for the enzyme to be anchored to post-lytic cell-wall remnants, suggesting its physiological roles in limiting diffusion of the enzyme, preserving neighboring host cells, and thereby enabling the phage progeny to initiate new rounds of infection. Taken together, this study provides an insight into regulation of endolysin through CBD and can potentially be applied for endolysin treatment against C. difficile infection. IMPORTANCE Endolysin is a peptidoglycan hydrolase encoded in a phage genome. The enzyme is attractive due to its potential use as antibacterial treatment. To utilize endolysin for the therapeutic propose, understanding of the fundamental role of endolysin becomes important. Here, we investigate the function of cell-wall binding domain (CBD) of an endolysin from a C. difficile phage. The domain is homologous to a cell-wall associating module of bacterial cell-wall proteins, likely acquired during phage-host coevolution. The interaction of CBD to bacterial cell walls reduces enzyme diffusion and thereby limits cell lysis of the neighboring bacteria. Our findings indicate that the endolysin is trapped to the cell-wall residuals through CBD and might serve as an advantage for phage replication. Thus, employing a CBD-less endolysin might be a feasible strategy for using endolysin for the treatment of C. difficile infection

Identification of pathways to high-level vancomycin resistance in Clostridioides difficile that incur high fitness costs in key pathogenicity traits

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance