Sporulation Strategies and Potential Role of the Exosporium in Survival and Persistence of Clostridium botulinum

Abstract

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I-III. We propose two distinct sporulation strategies used by C. botulinum Groups I-III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum.

Keywords: Clostridium botulinum; exosporium; morphology; spore.

Figure 1

Electron micrographs of sporulating Clostridium botulinum cells. Images of thin sections of fixed, sporulating C. botulinum cultures were acquired to observe the ultrastructure of sporulating cells. The cells were categorized according to their morphological stages: VC, vegetative cell; Asy, asymmetric division; Eng, engulfment; CA, coat assembly; MF, mature forespore; MCL, mother cell lysis. Exosporia visible while the forespore was still in the mother cell are indicated with black arrows. Scale bars represent 500 nm. Enlarged versions of these images can be found in Figures S2 and S3.

Figure 2

Electron micrographs depicting the coat assembly in sporulating Clostridium botulinum cells. (A) ATCC 3502, (B) ATCC 19397, (C) UN1/10-7B, (D) ATCC 17841, (E) UN5/11-8, (F) Eklund 17B, (G) Beluga, (H) CB11/1-1, (I) Stockholm C, (J) BKT015925. Coat fragments are indicated with black arrows.

Figure 3

Morphology of purified Clostridium botulinum spores. (A) Typical morphologies of Group I strains and Group II Eklund 17B, (B) Group II Beluga, and (C) Group III strains, presenting: Cr, core; Cx, cortex; Co, coat; Exp, exosporium; and App, appendages. (D) Electron micrographs of thin sections of purified spores from ATCC 3502, ATCC 19397, UN1/10-7B, ATCC 17841, UN5/11-8, Eklund 17B, Beluga, Stockholm C, and BKT015925.

Figure 4

Morphology of non-purified spores of Clostridium botulinum CB11/1-1. CB11/1-1 spores show a morphology reminiscent of both Beluga and Eklund 17B morphologies. (A) CB11/1-1 spore displaying both appendages (white arrow) and exosporium (black arrow). (B) The morphologic scheme of CB11/1-1 spore: Cr, core; Cx, cortex; Co, coat; Exp, exosporium; and App, appendages.

Figure 5

Thickness of the exosporium in Clostridium botulinum spores. The exosporium thickness of 10 spores of each purifiable strain was measured in three projections per spore. (A) The average thickness and (B) the minimum measured thickness of the exosporia were plotted in a scatter plot and boxplot combination to show both data density and distribution. UN1/10-7B was the only strain significantly different from others (p < 0.01).

Figure 6

Autoaggregation of Clostridium botulinum spores. (A) Photographic screening of the rate of sedimentation in spore suspensions of each strain, (B) assessment of spore deposition by decrease in OD_600nm, columns representing the average of three replicates with standard deviation shown by the error bars. (C) Phase contrast microscopy of the initial suspension and bottom fraction after 24 h of deposition. Scale bars represent 2 µm.

Figure 7

Hydrophobicity of Clostridium botulinum spores. The capacity of the purified spores of each strain to adhere to hexadecane was tested in a BATH assay; bars represent the average of three technical replicates with standard deviation shown by the error bars.

Figure 8

Thermal destruction of Clostridium botulinum spores. Purified spore suspensions of OD_600nm~1 were subjected to (A) 98 °C for Group I strains, (B) 75 °C for Group II strains, (C) and 90 °C for Group III strains until a 3-log kill was achieved.