Biology subjects

Fagan, R. P.

Publications and source records attributed to Fagan, R. P..

9 recordsLinked to original sources

bioRxiv2026

Protein architectures of the bacterial spore envelope - common principles of assembly

Bacterial spores are among the most durable of cell forms, protected by robust envelopes of layered protein assemblies. Remarkably, these envelopes often share similar architectures across distantly related bacteria despite extensive divergence in their molecular components. How evolution has converged on such similar highly ordered and resilient cellular structures is relatively unexplored. Here, we combine targeted mutagenesis, cryo-electron microscopy, atomic force microscopy and structure prediction to determine the organisation and assembly of the outer spore envelope of Clostridium sporogenes, a genetically tractable surrogate for Group I Clostridium botulinum. We reveal a hierarchy of protein structures, including a semi-permeable two-dimensional crystalline exosporium; CsxA forms the exosporium scaffold with an outer "hairy nap" partially composed of BclA. We observe a previously uncharacterised multilayered three-dimensional crystalline parasporal assembly within the interspace between exosporium and coat (CsxC). These distinct structures are built from related cysteine-rich SPOCS (SpoVID-CotE-SipL)-domain proteins that self-assemble into highly ordered lattices. Such crystalline organisation, combined with high symmetry, can provide a template for local enhancement of cysteine concentration, favouring cooperative disulphide cross-linking and the formation of exceptionally stable supramolecular structures. Unexpectedly, the three-dimensional CsxC structure grows through screw dislocations, a mechanism familiar from inorganic and synthetic crystal growth but rarely demonstrated in native biological assemblies, with the exception of some biomineralisation processes. Our observations now suggest that this classical crystal-growth mechanism can be exploited in both mineralised and proteinaceous biological materials. Related SPOCS-domain proteins are implicated in spore-envelope assembly across diverse Clostridia, where they have diversified to act as both structural components and morphogenetic organisers. Remarkably, distantly related Bacilli construct similarly highly symmetric crystalline, cysteine-rich and disulphide-stabilised spore layers using proteins with very different protein folds. Thus, crystallisation and cooperative disulphide formation appear to represent a convergent physicochemical strategy for building diverse self-assembling proteins into exceptionally robust cellular assemblies. Together these findings provide the first molecular framework for understanding the organisation and assembly of the Clostridium spore envelope and reveal previously unrecognised principles governing the evolution of protective proteinaceous structures across the Bacillota (Firmicutes).

microbiology↗

bioRxiv2025

A novel two-component system controls vancomycin resistance in epidemic Clostridioides difficile

The glycopeptide antibiotic vancomycin is the frontline treatment for C. difficile infection in the UK. There have been only sporadic reports of resistance in the clinic but testing is rare so the true resistance landscape is unclear. We have previously shown that resistance can emerge rapidly in vitro via distinct but complementary pathways. Strain Bc2, characterised here, is an experimentally evolved derivative of C. difficile strain R20291 that displays a 16-fold increase in vancomycin MIC over its parent. Bc2 has point mutations in dacS, bclA3, CDR20291_0794, CDR20291_1871 and CDR20291_3124 (vnrS). By genetically engineering a wild-type vancomycin susceptible strain, we demonstrated that a combination of just two mutations, dacSc.798A>T and vnrSc.692G>T, both of which encode two-component system histidine kinases, was sufficient to recapitulate Bc2 resistance. We have previously shown that mutations in dacS can confer low level resistance via increases in the expression of a D,D-carboxypeptidase DacJ. dacSc.798A>T also led to increased transcription of dacJ and led to a modest increase in vancomycin MIC. Surprisingly vnrSc.692G>T led to overexpression of the vanG cluster, which encodes all of the enzymes needed for resistance via substitution of the terminal D-Ala on peptidoglycan lipid II precursors with D-Ser. Genomic analysis of a large collection of European C. difficile strains showed that a three gene cluster, which includes vnrS, vnrR (encoding the cognate response regulator) and an adjacent gene CDR20291_3123, is unique to the phylogenetic branch that contains strains belonging to epidemic ribotypes 027 and 176. Transcriptomic analysis of the wider VnrS regulon also revealed an additional previously unknown role in regulating the expression of the flagellum, an important virulence factor in C. difficile. Analysis of the response to vancomycin exposure also revealed that dacJ is one of a small set of genes that are upregulated shortly after antibiotic stress, even in the absence of mutations that typically lead to its overexpression. Together these data reveal a new synergistic route, needing only two point mutations, by which epidemic lineages of C. difficile can attain vancomycin resistance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/671617v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@bba369org.highwire.dtl.DTLVardef@e562acorg.highwire.dtl.DTLVardef@1b112deorg.highwire.dtl.DTLVardef@4dcf8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2023

Multiple evolutionary pathways lead to vancomycin resistance in Clostridioides difficile

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 severe fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified two 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 trains. Moreover, our data provide a mutational roadmap to inform genomic surveillance.

microbiology↗

bioRxiv2023

Molecular mechanism of bacteriophage tail contraction- structure of an S-layer-penetrating bacteriophage

Viruses that infect bacteria (bacteriophages or phages) attach to the host cell envelope, inject their genetic material into the host cytosol and either persist as prophage or hijack the host machinery to produce progeny virions. Attachment is mediated through phage receptor binding proteins that are specific for different host cell surface molecules. A subset of phage, the myoviruses, possess contractile tails, the outer sheath of which contracts upon receptor binding, driving an inner tail tube through the cell envelope and delivering the phage genome into the host cytosol. The molecular details of phage tail contraction and mode of cell envelope penetration have remained poorly understood and were completely unknown for any phage infecting bacteria enveloped by a proteinaceous S-layer. Here we reveal the extended and contracted atomic structures of an intact contractile-tail phage that binds to and penetrates the protective S-layer of the Gram positive human pathogen Clostridioides difficile. Surprisingly, we find no evidence of the intrinsic enzymatic domains that other phages exploit in cell wall penetration, suggesting that sufficient energy is released upon tail contraction to penetrate the S-layer and the thick cell wall without enzymatic activity. However, it is also notable that the tail sheath subunits move less than those studied in related contractile injection systems such as the model phage T4. Instead, the unusually long tail length and flexibility upon contraction likely contribute towards the required free energy release for envelope penetration. Our results show that the principles of phage contraction and infection as determined in the model system of T4 are not universal. We anticipate that our structures will form a strong foundation to engineer C. difficile phages as therapeutics, and highlight important adaptations made in order to infect S-layer containing pathogens.

microbiology↗

bioRxiv2022

An intact S-layer is advantageous to Clostridioides difficile within the host.

Clostridioides difficile is responsible for substantial morbidity and mortality in antibiotically-treated, hospitalised, elderly patients, in which toxin production correlates with diarrhoeal disease. While the function of these toxins has been studied in detail, the contribution of other factors, including the paracrystalline surface layer (S-layer), to disease is less well known. Here, we highlight the essentiality of the S-layer in vivo by reporting the recovery of S-layer revertants, following infection with the S-layer-null strain, FM2.5. Sequencing of the slpA gene revealed either correction of the original point mutation or modification of the sequence upstream of the mutation, which restored the reading frame, and translation of slpA. Selection of these strains was rapid, with up to 90% of isolates identified as revertants 24 h post infection. Two revertant isolates, RvA and RvB, showed modification of 3 and 13 amino acids respectively, compared to wild type sequence. Structural determination of SlpA from RvB revealed a different orientation of its domains, resulting in a reorganisation of the lattice assembly and changes in interacting interfaces which might result in functional differences. These revertants showed differing patterns of disease in vivo; RvA causing equivalent severity to R20291 and RvB an attenuated FM2.5-like phenotype. Comparative RNA sequencing (RNA-Seq) analysis of in vitro grown isolates showed large changes in differentially expressed genes (DEGs) between R20291 and FM2.5 namely in TcdA/TcdB expression, in transcripts associated with sporulation and those linked to cell wall integrity, which may account for attenuation observed in vivo. In comparison, smaller differences were observed between RvA/R20291, and RvB/FM2.5 respectively, which correlated with observed disease severity in vivo. Cumulatively, these data highlight that the S-layer plays a role in C. difficile disease. Author SummaryThe S-layer of C. difficile is a paracrystalline array that covers the outer surface of the bacterial cell but its contribution to overall disease remains unclear. A previously described, spontaneous slpA-null mutant, FM2.5, with a point mutation in slpA offered an opportunity to study the role of the S-layer in vivo. Here, we confirm that this strain is less virulent in vivo despite effectively colonising the host and producing toxin. We also show in vivo selection for sequence modifications that restore slpA translation and produce an S-layer. While such modifications do not affect the overall 3D structure of individual SlpA (sub)domains, they can lead to altered orientation of the structural domains and subsequent S-layer assembly. Importantly, RNA-Seq analysis in vitro showed large differences in gene expression between FM2.5 and R20291. Detected differences in transcription of genes involved in toxin expression and sporulation suggests that the S-layer provides a selective survival advantage within the host, which contributes to disease severity.

microbiology↗

bioRxiv2022

The Clostridioides difficile S-Layer Protein A (SlpA) serves as a general phage receptor

Therapeutic bacteriophages (phages) are being considered as alternatives in the fight against Clostridioides difficile infections. To be efficient, phages should have a wide host range, but the lack of knowledge about the cell receptor used by C. difficile phages hampers the rational design of phage cocktails. Recent reports suggested that the C. difficile surface layer protein A (SlpA) is an important phage receptor, but clear and unambiguous experimental evidence is lacking. Here, using the epidemic R20291 strain and its FM2.5 mutant derivative lacking a functional S-layer, we show that the absence of SlpA renders cells completely resistant to infection by phiCD38-2, phiCDIII and phiCD146, which normally infect the parental strain. Complementation assays with 12 different Slayer Cassette Types (SLCTs) expressed from a plasmid revealed that SLCT-6 also allowed infection by phiCD111, and SLCT-11 enabled infection by phiCD38-2 and phiCD146. Of note, expression of SLCTs 1, 6, 8, 9, 10 or 12 conferred susceptibility to infection by 5 myophages that normally do not infect the R20291 strain, namely phiMMP02, phiMMP03, phiMMP04, phiCD506 and phiCD508. Adsorption assays showed that >50% adsorption was required for productive phage infection. Altogether, our data suggest that many phages use SlpA as their receptor and most importantly, morphologically distinct phages of the Siphoviridae and Myoviridae families target SlpA despite major differences in their tail structures. Our study therefore represents an important breakthrough in our understanding of the molecular interaction between C. difficile and its phages. IMPORTANCEPhage therapy represents an interesting alternative to treat Clostridioides difficile infections because contrary to antibiotics, most phages are highly species-specific, thereby sparing the beneficial gut microbes that protect from infection. However, currently available phages against C. difficile have a narrow host range and target members from only one or a few PCR ribotypes. Without a clear comprehension of the factors that define host specificity, and in particular the host receptor recognized by phages, it is hard to develop therapeutic cocktails in a rational manner. In our study, we provide clear and unambiguous experimental evidence that SlpA is a common receptor used by many siphophages and myophages. Although work is still needed to define how a particular phage RBP binds to a specific SLCT, identification of SlpA as a common receptor is a major keystone that will facilitate the rational design of therapeutic phage cocktails against clinically important strains.

microbiology↗

bioRxiv2020

Architecture and self-assembly of the Clostridium sporogenes/botulinum spore surface illustrate a general protective strategy across spore formers

Spores, the infectious agents of many Firmicutes, are remarkably resilient cell forms. Even distant relatives have similar spore architectures incorporating protective proteinaceous envelopes. We reveal 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 molecular filter. A cysteine-rich protein, CsxA, when expressed in E. coli, self-assembles into a highly thermally stable structure identical to native exosporium. Like exosporium, CsxA arrays require harsh reducing conditions for disassembly. We conclude that in vivo, CsxA self-organises into a highly resilient, disulphide cross-linked array decorated with additional protein appendages enveloping the forespore. This pattern is remarkably similar in Bacillus spores, despite lack of protein homology. In both cases, intracellular disulphide formation is favoured by the high lattice symmetry. We propose that cysteine-rich proteins identified in distantly related spore formers may adopt a similar strategy for intracellular assembly of robust protective structures.

microbiology↗

bioRxiv2019

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.

microbiology↗

bioRxiv2018

Spatial organization of Clostridium difficile S-layer biogenesis

Surface layers (S-layers) are protective protein coats which form around all archaea and most bacterial cells. Clostridium difficile is a Gram-positive bacterium with an S-layer covering its peptidoglycan cell wall. The S-layer in C. difficile is constructed mainly of S-layer protein A (SlpA), which is a key virulence factor and an absolute requirement for disease. S-layer biogenesis is a complex multi-step process, disruption of which has severe consequences for the bacterium. We examined the subcellular localization of SlpA secretion and S-layer growth; observing formation of S-layer at specific sites that coincide with cell wall synthesis, while the secretion of SlpA from the cell is relatively delocalized. We conclude that this delocalized secretion of SlpA leads to a pool of precursor in the cell wall which is available to repair openings in the S-layer formed during cell growth or following damage.

microbiology↗