Clostridium perfringens spore germination: characterization of germinants and their receptors

Abstract

Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of L-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, L-asparagine triggered spore germination in C-cpe isolates only; and (ii) L-alanine or L-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with L-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.

FIG. 1.

Germination of C. perfringens spores with various germinants. Spores of strain SM101 (wild type) were heat activated and germinated at 30°C in 25 mM sodium phosphate buffer (pH 7.0) with no germinant (▵) or with 100 mM l-alanine (+), l-asparagine (▴), KCl (□), AK (▪), or AGFK (•), and the OD₆₀₀ was measured as described in Materials and Methods.

FIG. 2.

KCl concentration dependence of C. perfringens spore germination. Heat-activated SM101 spores (wild type) were germinated with various KCl concentrations. The maximum rate of germination was calculated as described in Materials and Methods.

FIG. 3.

Effects of temperature (A and B) and pH (C and D) on germination of C. perfringens spores. Heat-activated spores of strains SM101 (A and C) and NB16 (B and D) were germinated with 100 mM AK (•), 100 mM KCl (○), or 100 mM l-alanine (□). The maximum rate of germination was calculated as described in Materials and Methods.

FIG. 4.

Analysis of genes encoding nutrient germinant receptors in C. perfringens. (A) Comparison of genes encoding nutrient germinant receptor proteins in B. subtilis and C. perfringens. Data were obtained from the Entrez Genome website (http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi?view=1). (B) Percent amino acid sequence similarities between nutrient germinant receptor protein homologues from B. subtilis and C. perfringens. (C) RT-PCR analysis of C. perfringens genes encoding germinant receptor homologues. RNAs from sporulating cells of strains SM101 (wild type) and DPS101 (gerK) were subjected to RT-PCR analysis using gerKA-, gerKC-, and gerAA-specific internal primers. Lanes labeled “wt-RT” and “mt-RT” contain RT-PCR products obtained from RNAs from strains SM101 and DPS101, respectively. Lanes labeled “PCR” contain PCR products obtained from SM101 DNA, using gerAA-, gerKA-, and gerKC-specific internal primers. The PCR- and RT-PCR-amplified products were analyzed by agarose (1%) gel electrophoresis and photographed under UV light. The presence of RT-PCR products cannot be explained by amplification from contaminated DNA because no PCR product was obtained from RNA in the absence of reverse transcriptase (data not shown).

FIG. 5.

Germination of C. perfringens wild-type and gerK spores with various germinants. Heat-activated spores of strains SM101 (wild type) (□) and DPS101 (gerK) (▪) were germinated with 100 mM KCl (A), 100 mM l-asparagine plus 100 mM KCl (B), and 100 mM l-asparagine (C) as described in Materials and Methods. The control germination (○) corresponds to heat-activated spores incubated in 25 mM sodium phosphate buffer (pH 7.0); no difference between SM101 and DPS101 spores was seen.

FIG. 6.

Germination of C. perfringens wild-type and gerAA spores with various germinants. Heat-activated spores of strains SM101 (wild type) (□) and DPS103 (gerAA) (▴) were germinated with 100 mM KCl (A), 100 mM l-asparagine and 100 mM KCl (B), 100 mM l-asparagine (C), 10 mM KCl (D), 10 mM l-asparagine and 10 mM KCl (E), and 10 mM l-asparagine (F) as described in Materials and Methods. The control germination (○) was heat-activated spores incubated in 25 mM sodium phosphate buffer (pH 7.0), and no difference between spores of SM101 and DPS103 was observed.

FIG. 7.

DPA release during germination of C. perfringens spores. Heat-activated spores of strains SM101 (wild type) (□), DPS101 (gerK) (▪), and DPS103 (gerAA) (▴) were germinated in 25 mM sodium phosphate buffer (pH 7.0) with 5 mM KCl (A) or 100 mM l-asparagine (B). At various times, DPA release was measured as described in Materials and Methods.

FIG. 8.

Germination of spores of C. perfringens strains in BHI broth. Heat-activated spores of strains SM101 (wild type) (□), DPS101 (gerK) (▪), and DPS103 (gerAA) (▴) were incubated at 40°C with BHI broth, and the OD₆₀₀ was measured as described in Materials and Methods.

FIG. 9.

Ca-DPA germination of spores of C. perfringens strains. Heat-activated spores of strains SM101 (wild type), DPS101 (gerK), and DPS103 (gerAA) were germinated with 50 mM Ca-DPA (pH 8.0) at 40°C for 60 min, and changes in the OD₆₀₀ of the culture (A) and the amount of DPA remaining in the spores (B) were measured as described in Materials and Methods. The values shown are averages for two experiments with two independent spore preparations. Error bars show 1 standard deviation from the mean.

FIG. 10.

Dodecylamine germination of spores of C. perfringens strains. Spores of strains SM101 (wild type) (□), DPS101 (gerK) (▪), and DPS103 (gerAA) (▴) were incubated at 60°C with 1 mM dodecylamine (pH 7.4), and DPA release was measured as described in Materials and Methods.

FIG. 11.

Putative model for nutrient and nonnutrient germination of C. perfringens spores. Nutrients activate germinant receptors, resulting in Ca-DPA release from the core, which triggers activation of SCLEs. External Ca-DPA induces germination through a mechanism that requires the GerK receptor to fully activate downstream germination events. Dodecylamine triggers DPA release by ultimately opening a DPA channel (composed of SpoVA proteins, by analogy with B. subtilis spores) in the spore's inner membrane. Since dodecylamine germination is unaffected by gerAA mutation but is reduced by loss of GerKA and GerKC, dodecylamine presumably acts on both the GerK receptor, to indirectly open a DPA channel, and the DPA channel itself. SCLEs are then activated by the Ca-DPA release triggered by dodecylamine, and SCLEs then promote cortex hydrolysis and completion of spore germination.