The Clostridium perfringens germinant receptor protein GerKC is located in the spore inner membrane and is crucial for spore germination

Abstract

The Gram-positive, anaerobic, spore-forming bacterium Clostridium perfringens causes a variety of diseases in both humans and animals, and spore germination is thought to be the first stage of C. perfringens infection. Previous studies have indicated that the germinant receptor (GR) proteins encoded by the bicistronic gerKA-gerKC operon as well as the proteins encoded by the gerKB and gerAA genes are required for normal germination of C. perfringens spores. We now report the individual role of these GR proteins by analyzing the germination of strains carrying mutations in gerKA, gerKC, or both gerKB and gerAA. Western blot analysis was also used to determine the location and numbers of GerKC proteins in spores. Conclusions from this work include the following: (i) gerKC mutant spores germinate extremely poorly with KCl, l-asparagine, a mixture of asparagine and KCl, or NaPi; (ii) gerKC spores germinate significantly more slowly than wild-type and other GR mutant spores with a 1:1 chelate of Ca(2+) and dipicolinic acid and very slightly more slowly with dodecylamine; (iii) the germination defects in gerKC spores are largely restored by expressing the wild-type gerKA-gerKC operon in trans; (iv) GerKC is required for the spores' viability, almost certainly because of the gerKC spores' poor germination; and (v) GerKC is located in the spores' inner membrane, with ∼250 molecules/spore. Collectively, these results indicate that GerKC is the main GR protein required for nutrient and nonnutrient germination of spores of C. perfringens food-poisoning isolates.

Fig 1

C. perfringens GR mutants. (A) Arrangement of GR genes in various C. perfringens mutant strains. (B) Analysis of GerKC levels in total lysates of C. perfringens SM101 (wild-type [WT]) and DPS119 (ΔgerKA) spores. Spores were decoated and lysed, and various amounts of protein in the total lysates from the spores of the two strains were run on SDS-polyacrylamide gels, followed by Western blotting using GerKC antibody, as described in Materials and Methods. The intensity of the GerKC band in the DPS119 lysate was compared to the intensities of bands given by various amounts of GerKC in the SM101 lysate (1× corresponds to the same amount of spores, ∼1.5 × 10⁷, in the two fractions). Analysis of the various band intensities by ImageJ gave a value for the GerKC level in DPS119 spores that was 12% of that in SM101 spores. The two bands above the GerKC band are likely bands that reacted nonspecifically, since their intensities were greatly increased relative to the intensity of the GerKC band in the DPS119 spores.

Fig 2

Germination of C. perfringens spores with various germinants. Heat-activated spores of strains SM101 (wild type) (◆), DPS101 (gerKA-KC) (●), DPS119 (gerKA) (▲), DPS122 (gerKC) (□), DPS124 (gerKB gerAA) (×), and DPS122(pSB18) (gerKC mutant complemented with wild-type gerKA-gerKC) (△) were incubated at 40°C with KCl (A), l-Asn (B), AK (C), or NaP_i (pH 6.0) (D), and germination was measured by determination of the OD₆₀₀, as described in Materials and Methods. The control germination (♢) was heat activation of spores, which were incubated in 25 mM Tris-HCl buffer (pH 7.0) at 40°C, and no germination difference was observed between spores of SM101 and the GR mutant strains.

Fig 3

DPA release by spores of C. perfringens strains during germination with KCl. Heat-activated spores of strains SM101 (wild-type) (◆), DPS119 (gerKA) (▲), DPS122 (gerKC) (□), DPS124 (gerKB gerAA) (●), and DPS122(pSB18) (gerKC mutant complemented with wild-type gerKA-gerKC) (△) were germinated with KCl (pH 7.0), and DPA release was measured as described in Materials and Methods. Error bars represent standard deviations.

Fig 4

Germination of C. perfringens wild-type and mutant spores with Ca-DPA (A) and dodecylamine (B). Heat-activated spores of strains SM101 (wild-type) (◆), DPS119 (gerKA) (▲), DPS122 (gerKC) (□), DPS124 (gerKB gerAA) (●), and DPS122(pSB18) (gerKC mutant complemented with wild-type gerKA-gerKC) (△) were germinated with Ca-DPA and dodecylamine. For Ca-DPA, changes in OD₆₀₀ were measured as described in Materials and Methods, and for dodecylamine, germination was monitored by measurement of DPA release, as described in Materials and Methods. Error bars represent standard deviations.

Fig 5

Germination of spores of C. perfringens strains in BHI broth. Heat-activated spores of strains SM101 (wild-type) (◆), DPS119 (gerKA) (▲), DPS122 (gerKC) (□), DPS124 (gerKB gerAA) (×), and DPS122(pSB18) (gerKC mutant complemented with wild-type gerKA-gerKC) (△) were germinated at 40°C in BHI broth, and the OD₆₀₀ was measured as described in Materials and Methods.

Fig 6

Localization of GerKC in C. perfringens spores. Dormant spores of C. perfringens strains SM101 (wild type) and DPS122 (gerKC) were decoated, disrupted with glass beads and sonication after lysozyme treatment, and centrifuged at 14,000 × g for 10 min, and the supernatant was centrifuged at 100,000 × g for 1 h, as described in Materials and Methods. Aliquots of the P100 pellet IM fractions (lanes 1 and 2) from wild-type and gerKC spores and the S100 supernatant (Sup.) fraction from wild-type spores (lane 3) were run on SDA-polyacrylamide gels, followed by Western blotting using GerKC antibody, as described in Materials and Methods. The arrow on the left indicates the size of GerKC, ∼40 kDa. The amounts of sample run in lanes 1 to 3 are given above the lanes as the percentage of the total IM protein or supernatant fraction protein. The S100 supernatant fraction from gerKC spores had no detectable GerKC band (data not shown). Lane 4, 0.5 ng purified GerKC antigen. The numbered lines to the left of the figure denote the migration positions of molecular mass markers (in kDa). All lanes are from the same gel, but an intervening region between lanes 3 and 4 was removed for clarity.

Fig 7

Quantitation of the GerKC level in spores of C. perfringens strain SM101. Various amounts of P100 pellet IM fractions (lanes 1 to 3) and pellet fractions of decoated, disrupted spores obtained by centrifugation at 14,000 × g (lanes 4 and 5) and various amounts of purified GerKC antigen (lanes 6 to 8) were run on SDS-polyacrylamide gels, followed by Western blot analysis using anti-GerKC antibody. The amounts of samples run are shown above the lanes as either the percentage of the total membrane protein or pellet fraction protein from 8.4 × 10⁹ spores (lanes 1 to 5) or the amount (in ng) of GerKC antigen (lanes 6 to 8). The numbered lines to the left of the figure denote the migration positions of molecular mass markers (in kDa). All lanes are from the same gel, but lanes 4 and 5 were moved from their original position to the right of the GerKC antigen lanes.

Fig 8

Analysis of the level of biotinylation of GerKC in decoated dormant and germinated spores of C. perfringens wild-type strain SM101. Biotinylation was carried out on decoated dormant spores and intact germinated spores as described in Materials and Methods. Spores were disrupted and fractionated, and the levels of various germination proteins in (i) the total IM fraction (T), (ii) the IM fraction that did not adsorb to the NeutrAvidin beads (F), and (iii) the eluate from the NeutrAvidin beads (E) were assayed by Western blotting using anti-GerKC antiserum, as described in Materials and Methods. Aliquots in the T, F, and E samples were from equal amounts of spores. The F_(50%) sample (lane 4) from dormant spores was also the protein that did not adsorb to the NeutrAvidin beads but was from 1/2 the amount of spores as in the other lanes. The E_(50%) sample (lane 8) from germinated spores was also the eluate from the NeutrAvidin beads but was from 1/2 the amount of spores as in the other lanes. Analysis of this Western blot by the program ImageJ indicated that ∼33% of GerKC was biotinylated in dormant spores, with ∼50% biotinylated in germinated spores.