A sporulation signature protease is required for assembly of the spore surface layers, germination and host colonization in Clostridioides difficile

Abstract

A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.

Fig 1. The yabG region of the C. difficile chromosome and organization of YabG.

A: Schematic representation of the yabG region of the C. difficile chromosome. A σ^E/K-dependent promoter in front of the yabG gene is represented. The lines below the gene represent the regions coding to distinct regions of the YabG protein (as in panel C). B: The alignments show highly conserved or invariant residues (grey) in the vicinity of Cys residues at positions 119 (blue) and 207 of YabG (brown) and around His161 and Asp162 (brown). C: An AlphaFold2 model of C. difficile YabG. The model highlights a N-terminal SH3-like domain (A, orange; residues 1–57) and a C-terminal CheY-like domain (B, green; residues 99–286) connected by a linker (L, blue, residues 58–98). A putative active site cleft is indicated at the interface between A and B. D: Expansion of the active site region to show the relative positions of Cys207, His161, Asp162 and Cys119 with estimated distances shown in Å. E and F: The WT and the indicated forms of YabG and CspBA were produced in E. coli. In F, the indicated forms of YabG were produced together with or in the absence of CspBA The proteins in whole cell extracts were resolved by SDS-PAGE and the gels stained with Coomassie or subject to immunoblotting (in F) with anti-CspB antibodies. In E and F, the black arrowheads show the position of YabG, YabG^C207A or CspBA. The green arrowhead in F shows the position of CspB released from CspBA, independently of YabG, while the red arrowhead points to CspB released from CspBA through the action of YabG. The asterisk refers to an unspecific band.

Fig 2. yabG mutations affect the extractability of coat and exosporium proteins and result in abnormal spore surface layers.

A: Spores of the WT, ΔyabG, yabG^C (complementation strain) and yabG^C207A strains were purified on density gradients, fractionated [107] and the cortex/coat/exosporium and cortex/core proteins extracted. The proteins were resolved by SDS-PAGE and the gels stained with Coomassie (top panel) or subjected to immunoblot analysis (lower panels) with anti-YabG, anti-SleC, anti-GPR, anti-CdeC, anti-CotA and anti-CotM antibodies, as indicated. The red arrowheads indicate proteins that appear to be more extractable from ΔyabG and yabG^C207A spores while black arrowheads show proteins with reduced extractability. The position of YabG^C207A is indicated by a green arrowhead; asterisks show the position of non-specific species. Proteins in the indicated bands in the Coomassie-stained gel were identified by mass spectrometry. B: Spores of the indicated strains imaged by scanning electron microscopy. The yellow arrowheads point to the polar regions in yabG and yabG^C207A spores. The red arrowhead points to material that seems to peel-off the pole of a yabG^C207A spore. C: Spores of the WT (630Δerm) and yabG^C207A mutant were analysed by thin sectioning transmission electron microscopy (TEM). The regions within the red and yellow circles in the two left panels are magnified on the right panels. In WT spores, the yellow arrowheads point to the coat region and the blue arrowheads to the exosporium region. In yabG^C207A spores, the red arrowheads point to the lamellae seen in the appendage region, the green arrowheads to electron dense coat or exosporium material loosely attached to yabG^C207A spores and the black arrowheads to unstructured material present between the cortex and the coat layers. Also note the material peeling off from the appendage (brown arrowheads). Cr, spore core; Cx, cortex; Ap, appendage region. The numbers refer to the percentage of spores in which the coat is detached from the cortex (red) or with a long polar appendage with a lamellar structure (blue); 60–95 spores were counted for each strain. Scale bars: 3 μm in B; 500 nm (left column) or 100 nm (all other panels) in C. In B and C, the spores were purified on density gradients. See also S12 Fig.

Fig 3. YabG affects the expression of genes coding for coat and exosporium components.

A and B: Coomassie stained gel and immunoblotting analysis of sporulating cells of the WT, ΔyabG, yabG^C and yabG^C207A 14 (A) and 20 hours (B) after inoculation in 70:30 agar plates [53]. Proteins in whole cell extracts were resolved by SDS-PAGE and the gels subjected to immunoblotting with anti-CotA, anti-CdeC, and anti-CdeM antibodies. The red arrowheads point to the various forms of CotA, CdeC and CdeM. C: Quantification of the expression of the indicated genes (cdeC, cdeM and cotA) by qRT-PCR. Total RNA was extracted from C. difficile 630Δerm and ΔyabG strains grown in 70:30 agar plates for 14 and 20 hours. The graph shows the fold-change in the expression of cdeC, cdeM and cotA between the ΔyabG and the WT. Error bars correspond to the standard deviation derived from three biological replicates. Statistical analysis used a Student’s t-test: * p<0.01; **p<0.001.

Fig 4. YabG-independent expression of cotA and cdeM restores assembly of CotA and CdeM to ΔyabG and yabG^C207A spores.

Coomassie stained SDS-PAGE gel of the proteins extracted from purified spores of the WT, ΔyabG and yabG^C207A mutants and derivatives expressing P_cotE- cdeM (A) or P_cotE- cotA (B). In A, the bottom panel shows the immunoblot analysis of the corresponding gel using an anti-CotA antibody. The position of the main forms of CdeM (in A) or CotA (in B) is shown by red arrowheads. C: spores produced by the yabG^C207A mutant and by derivatives expressing P_cotE-cdeM or P_cotE-cotA were imaged by TEM. The polar region of yabG^C207A/P_cotE-cotA and yabG^C207A/P_cotE-cdeM spores is magnified in the panels to the right. Cr, core; Cx, cortex; Ap, spore appendage region. Green arrowheads, coat and exosporium material peeling off the spore; blue arrowheads, regions with an exposed cortex. Scale bars: 100 nm for the magnified images, 500 nm for all other panels.

Fig 5. Localization of YabG-SNAP^Cd in sporulating cells.

A: Localization of YabG^WT-SNAP^Cd and YabG^C207A-SNAP^Cd in C. difficile 630Δerm (WT) and ΔyabG strains. Cells were collected after 20h of growth in 70:30 agar plates [53], stained with the SNAP substrate TMR-Star and examined by phase contrast and fluorescence microscopy (red channel for TMR signal and green channel for autofluorescence signal). The numbers refer to the percentage of cells at the represented stage showing SNAP fluorescence. Yellow and white arrowheads point to the position of the forespore and the mother cell respectively. At least 150 cells were analysed for each strain, in three independent experiments. Scale bar, 1 μm. B: Accumulation of YabG-SNAP^Cd and YabG^C207A-SNAP^Cd in sporulating cells of strains 630Δerm (WT) and ΔyabG at 20h of growth in 70:30. Proteins in whole cell extracts were resolved by SDS-PAGE and the gel subject to immunoblot analysis with anti-SNAP antibodies. Samples collected from the WT and the ΔyabG mutant bearing no SNAP^Cd fusion were used to control for antibody specificity. The Coomassie-stained gel is included as a loading control. Red arrowheads point to the position of YabG^C207A-SNAP (52 kDa). Asterisks denote possible degradation products that include the SNAP moiety (~19.4 kDa). The black arrowhead shows the position of a cross-reactive species.

Fig 6. Localization of YabG^WT- or YabG^C207A-SNAP^Cd in mature spores.

A: Localization of YabG^WT- or YabG^C207A-SNAP^Cd in mature spores using SR-SIM, in either the WT or ΔyabG backgrounds. The spores were stained with the membrane dye MTG (green) and with TMR-Star (red) prior to imaging. The blue arrows point to the SNAP signal at the spore poles and the white arrows to the signal along the side of the spore (see also S15 Fig). The distribution of the fluorescence signal (in arbitrary units, AU) in three dimensional intensity graphs is shown below the microscopy images. Scale bar, 500 nm. B: Coomassie stained SDS-PAGE gel of the proteins extracted from the cortex/coat/exosporium and core/cortex fractions of purified spores of the WT strain, the ΔyabG mutant, and of strains producing YabG^WT-SNAP^Cd or YabG^C207A-SNAP^Cd in either the WT or ΔyabG backgrounds. The gel was subjected to immunoblot analysis with anti-SNAP, anti-YabG, anti-SleC, anti-CdeC, anti-CotA and anti-CdeM antibodies. The arrowheads points to the position of the relevant proteins. YabG-SNAP denotes the position of either full-length WT or the C207A variant fused to the SNAP tag. Asterisks denote possible degradation products or cross-reactive species.

Fig 7. YabG is required for colonization of the hamster colon.

A: Kaplan-Meier curve for hamster challenged with spores of the WT (red) and ΔyabG mutant (blue). Syrian hamsters were first gavaged with clindamycin to induce susceptibility to infection and challenged with 10³ WT or ΔyabG spores five days after. Hamsters showing signs of disease were euthanized. B: Enumeration of C. difficile cells and spores in faecal material following gavage of hamsters with spores produced by the WT (pink panel) and ΔyabG mutant (light blue panel) (as in B). Faecal samples were collected daily, and both C. difficile vegetative cells and spores were enumerated by plating. The data was set to the median values and the limit of detection (dashed line) is shown.

Fig 8. Model for the role of YabG in the assembly of the spore surface layers.

A: yabG expression is first detected in the mother cell compartment after asymmetric division under the control of σ^E but persists in this cell under σ^K control [30]. σ^K also directs transcription of cdeC (which additionally requires SpoIIID); transcription of cdeC and the σ^K-dependent period of yabG transcription begins coincidently with the appearance of phase-dark forespores and CdeC is probably recruited to the forespore surface at this stage. The assembly of YabG, however, is auto-regulatory and self-limiting in that YabG undergoes self-degradation in either the mother cell cytoplasm or at the spore surface. The stability of YabG increases when the spore turns phase-bright, and may require production of the YabG substrates and/or increased production of the protease. At this stage, YabG accumulates at the spore surface and is also required in the mother cell cytoplasm for the degradation of an as yet unidentified repressor (R) or for the activation of a activator (A) of transcription of a class of σ^K-dependent genes that includes cdeM and cotA. The encoded proteins are produced and assembled when repression is relieved or activation is triggered. Full-length YabG is represented, as it unknown whether domain A is removed. B: YabG is involved in processing of CspBA and SleC^FL to produce CspB and Pro-SleC. and possibly also of CotE, to separate the N-terminal chitinase (CotE^N) and the C-terminal peroxiredoxin (CotE^C) domains. YabG is also required for the degradation of SpoIVA, and possibly of CdeC. It is not known where are these proteins processed. Both the role of YabG in enforcing the proper structure of the spore and in germination are thought to contribute to host colonization. Finally, YabG is required for the transcription of at least cdeM and cotA (red arrow).