A cortex-specific penicillin-binding protein contributes to heat resistance in Clostridioides difficile spores.

BACKGROUND: 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. Clostridioides 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 of which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the enzymes required for synthesis of cortex peptidoglycan in C. difficile. METHODS: Bioinformatic analysis of the C. difficile genome to identify putative homologues of Bacillus subtilis spoVD was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity. RESULTS: Deletion of CDR20291_2544 (SpoVDCd) abrogated spore formation and this phenotype was completely restored by complementation in cis. Analysis of SpoVDCd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVDCd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVDCd was also seen to localise to the developing spore by super-resolution confocal microscopy. CONCLUSIONS: We have identified and characterised a cortex specific PBP in C. difficile. This is the first characterisation of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen.

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.

Dynamic action of the Sec machinery during initiation, protein translocation and termination.

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

Two-way communication between SecY and SecA suggests a Brownian ratchet mechanism for protein translocation.

The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.

Polycystin-2 induces a conformational change in polycystin-1.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in the genes encoding either polycystin-1 (PC1) or polycystin-2 (PC2). PC2 acts as a nonselective cation channel and together with PC1 plays a role in intracellular Ca(2+) signaling. Using atomic force microscopy (AFM) imaging, we have shown previously that the N and C termini of PC1 appear as unequally sized particles connected by a "string" largely composed of tandem immunoglobulin-like, polycystic kidney disease (PKD) domains. Here, we show that coexpression of PC1 and PC2 causes an elongation of the PC1 string and a corresponding reduction in the size of the larger (C-terminal) particle. This change in the conformation of PC1 does not depend on its delivery to the plasma membrane. In addition, the use of the L3040H PC1 mutant showed that the conformational change does not require GPS cleavage. Coexpression of PC1 with PC2 mutants revealed that the conformational change in PC1 does not require either a stable interaction between PC1 and PC2 or PC2 channel function. Finally, we show that the tandem PKD repeats and to a lesser extent the receptor for egg jelly (REJ) domain both contribute to the extension of the PC1 string in the presence of PC2. We propose that the PKD repeats detach from the C-terminal fragment in response to PC2 activity. The resulting remodeling of PC1 may be responsible for enhancing GPS cleavage of PC1 and the separation of the PC1 N-terminal fragment from the C terminus during its maturation.

Atomic force microscopy imaging reveals the domain structure of polycystin-1.

Mutation of polycystin-1 (PC1) is the major cause of autosomal dominant polycystic kidney disease. PC1 has a predicted molecular mass of ~460 kDa comprising a long multidomain extracellular N-terminal region, 11 transmembrane regions, and a short C-terminal region. Because of its size, PC1 has proven difficult to handle biochemically, and structural information is consequently sparse. Here we have isolated wild-type PC1, and several mutants, from transfected cells by immunoaffinity chromatography and visualized individual molecules using atomic force microscopy (AFM) imaging. Full-length PC1 appeared as two unequally sized blobs connected by a 35 nm string. The relative sizes of the two blobs suggested that the smaller one represents the N-terminus, including the leucine-rich repeats, the first polycystic kidney disease (PKD) domain, and the C-type lectin motif, while the larger one is the C-terminus, including the receptor for egg jelly (REJ) domain, all transmembrane domains, and the cytoplasmic tail. The intervening string would then consist of a series of tandem PKD domains. The structures of the various PC1 mutants were all consistent with this model. Our results represent the first direct visualization of the structure of PC1, and reveal the architecture of the protein, with intriguing implications for its function.