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. 2017 Oct 17;199(22):e00266-17.
doi: 10.1128/JB.00266-17. Print 2017 Nov 15.

Revisiting the Role of Csp Family Proteins in Regulating Clostridium difficile Spore Germination

Yuzo Kevorkian  1 , Aimee Shen  2
Affiliations

Revisiting the Role of Csp Family Proteins in Regulating Clostridium difficile Spore Germination

Yuzo Kevorkian et al. J Bacteriol. .

Abstract

Clostridium difficile causes considerable health care-associated gastrointestinal disease that is transmitted by its metabolically dormant spore form. Upon entering the gut, C. difficile spores germinate and outgrow to produce vegetative cells that release disease-causing toxins. C. difficile spore germination depends on the Csp family of (pseudo)proteases and the cortex hydrolase SleC. The CspC pseudoprotease functions as a bile salt germinant receptor that activates the protease CspB, which in turn proteolytically activates the SleC zymogen. Active SleC degrades the protective cortex layer, allowing spores to outgrow and resume metabolism. We previously showed that the CspA pseudoprotease domain, which is initially produced as a fusion to CspB, controls the incorporation of the CspC germinant receptor in mature spores. However, study of the individual Csp proteins has been complicated by the polar effects of TargeTron-based gene disruption on the cspBA-cspC operon. To overcome these limitations, we have used pyrE-based allelic exchange to create individual deletions of the regions encoding CspB, CspA, CspBA, and CspC in strain 630Δerm Our results indicate that stable CspA levels in sporulating cells depend on CspB and confirm that CspA maximizes CspC incorporation into spores. Interestingly, we observed that csp and sleC mutants spontaneously germinate more frequently in 630Δerm than equivalent mutants in the JIR8094 and UK1 strain backgrounds. Analyses of this phenomenon suggest that only a subpopulation of C. difficile 630Δerm spores can spontaneously germinate, in contrast with Bacillus subtilis spores. We also show that C. difficile clinical isolates that encode truncated CspBA variants have sequencing errors that actually produce full-length CspBA.IMPORTANCEClostridium difficile is a leading cause of health care-associated infections. Initiation of C. difficile infection depends on spore germination, a process controlled by Csp family (pseudo)proteases. The CspC pseudoprotease is a germinant receptor that senses bile salts and activates the CspB protease, which activates a hydrolase required for germination. Previous work implicated the CspA pseudoprotease in controlling CspC incorporation into spores but relied on plasmid-based overexpression. Here we have used allelic exchange to study the functions of CspB and CspA. We determined that CspA production and/or stability depends on CspB and confirmed that CspA maximizes CspC incorporation into spores. Our data also suggest that a subpopulation of C. difficile spores spontaneously germinates in the absence of bile salt germinants and/or Csp proteins.

Keywords: Clostridium difficile; Csp; germination; spontaneous germination; spores.

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Figures

FIG 1
FIG 1
Construction of clean deletions in the csp locus. (A) Schematic of the cspBAC locus and the indicated deletion strains created in C. difficile 630Δerm ΔpyrE. (B) Colony PCR verification of each csp mutant. The primers used to screen for the gene deletions are shown in blue.
FIG 2
FIG 2
CspA production and/or stability depends on CspB. Western blot analyses of sporulating cells (A) and purified spores (B) from the wild type (WT) and csp family mutants. Each mutant is complemented chromosomally by cspBA (BA), cspC (C), or cspBAC (BAC) at the pyrE locus. – indicates that the pyrE locus was restored in the parental csp ΔpyrE strains. The Δspo0A (Δ0A) mutant was used as a negative control for sporulating cells, since it is unable to sporulate (10). CspBA (130 kDa) is detected by both anti-CspB and anti-CspA antibodies. * indicates a nonspecific band detected by the CspA antibody. SpoIVA was used as a loading control, since it serves as an indicator of sporulation (54). For purified spores, the ΔsleC mutant was used as a negative control, since this mutant cannot hydrolyze cortex (30). The outer coat protein CotA (55) was used as a loading control (32). The heat resistance of sporulating cultures and germination efficiencies of purified spores for each strain are expressed relative to the wild type. The results are the means and standard deviations from at least three independent experiments; germination efficiencies are derived from two independent spore preparations for a given strain. The gray text indicates that these values are derived from counts taken after a 2-day incubation relative to a 1-day incubation for the values in black text. Statistical significance relative to the wild type was determined using a one-way analysis of variance and Tukey's test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
FIG 3
FIG 3
csp mutant spores do not appear to hydrolyze cortex. Change in optical density at 600 nm (OD600) of purified mutant (A) and complementation strain (B) spores following addition of 1% taurocholate is shown. The ΔsleC mutant serves as a negative control, since cortex hydrolysis depends on SleC (30). Individual csp mutants and the ΔsleC mutant exhibited statistically significant differences in OD600 over time (P < 0.0001). While spores from csp complementation strains exhibited OD600 changes that were significantly different compared to ΔsleC mutant, spores from cspBA complementation strains exhibited OD600 changes that were significantly different from the wild type (ΔcspBA/cspBA and ΔcspB/cspBA strains, P < 0.0001; ΔcspA/cspBA strain, P < 0.005). Results were analyzed using a two-way analysis of variance and Tukey's test.
FIG 4
FIG 4
Delayed colony formation by ΔcspBAC and ΔsleC spores on rich media. Purified spores of the C. difficile 630Δerm, ΔsleC, and ΔcspBAC strains were serially diluted, and replicates were plated onto BHIS supplemented with 0.1% taurocholate (top) or BHIS (bottom) and incubated at 37°C. Pictures were taken at the indicated time points after plating.
FIG 5
FIG 5
Comparison of spore germination on BHIS and CDDM. Purified spores of the C. difficile 630Δerm, ΔsleC, and ΔcspBAC strains were serially diluted and plated on BHIS and CDDM ± 0.1% taurocholate supplementation. The CFU produced by these spores 48 h after incubation are shown. The results are the means and standard deviations from three biological replicates from two independent spore preparations. Statistical significance was determined using a one-way analysis of variance and Tukey's test. No statistically significant difference was measured for the “spontaneous germination” of wild-type, ΔsleC, and ΔcspBAC spores plated on BHIS and CDDM. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; n.s., not significant.
FIG 6
FIG 6
Germination of spores over time on solid media. Purified wild-type, ΔsleC, ΔcspBAC (ΔBAC), and ΔcspBAC/BAC (ΔBAC/BAC) spores were spread onto 15 cm BHIS with and without 0.1% taurocholate and incubated for up to 9 days. The new CFU that arose on these plates were counted every 24 h. (A) The total number of CFU observed over the 9 days was determined for a given dilution, and the percentage of CFU that appeared for a given day is plotted. The results from three independent replicates on two independent spore preparations are shown. (B) The total number of CFU obtained over time (corrected for the dilution counted) is shown. The average and standard deviation of these values are plotted.
FIG 7
FIG 7
Aerobic killing of germinating spores. Purified wild-type, ΔsleC, ΔcspBAC (ΔBAC), and ΔcspBAC/BAC (ΔBAC/BAC) spores were plated on BHIS with and without 0.1% taurocholate, incubated aerobically for the indicated times at 37°C, and transferred to anaerobic conditions for 48 h at 37°C. CFU that arose from spores treated in this manner are shown. The first time point (−2 h) represents the CFU produced after spores were plated on media that had been prereduced anaerobically for 2 h at 37°C. The results are based on three biological replicates from a single spore preparation to minimize variation due to spore preparations. Similar results were obtained for an independent spore preparation (data not shown).
FIG 8
FIG 8
Csp levels and germination efficiency of clinical isolates predicted to be defective in CspBA production. (A) Western blot analyses of purified spores from strains 630Δerm, DA00232, CD70, CD42, CD160, and CD17 (31). 630Δerm ΔcspBAC (ΔBAC) was used as a negative control for analyzing Csp levels between clinical isolates. Germination efficiencies were determined from three biological replicates performed on two independent spore preparations for a given strain and expressed relative to 630Δerm. (B) Western blot analyses of sporulating cultures of the indicated strains. CspBA was detected using an antibody against CspB. 630Δerm Δspo0A was used as a negative control for sporulating cells, since it is unable to sporulate. (C) CFU produced by spores from the indicated strains plated on BHIS and BHIS supplemented with 0.1% taurocholate (TA). Statistical significance was determined using a one-way analysis of variance and Tukey's test. No statistically significant difference in CFU was observed between strains plated on BHIS, with the exception of DA00232 relative to CD42 (P < 0.01). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.
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