The biology and pathogenicity of Clostridium perfringens type F: a common human enteropathogen with a new(ish) name

Abstract

SUMMARYIn the 2018-revised Clostridium perfringens typing classification system, isolates carrying the enterotoxin (cpe) and alpha toxin genes but no other typing toxin genes are now designated as type F. Type F isolates cause food poisoning and nonfoodborne human gastrointestinal (GI) diseases, which most commonly involve type F isolates carrying, respectivefooly, a chromosomal or plasmid-borne cpe gene. Compared to spores of other C. perfringens isolates, spores of type F chromosomal cpe isolates often exhibit greater resistance to food environment stresses, likely facilitating their survival in improperly prepared or stored foods. Multiple factors contribute to this spore resistance phenotype, including the production of a variant small acid-soluble protein-4. The pathogenicity of type F isolates involves sporulation-dependent C. perfringens enterotoxin (CPE) production. C. perfringens sporulation is initiated by orphan histidine kinases and sporulation-associated sigma factors that drive cpe transcription. CPE-induced cytotoxicity starts when CPE binds to claudin receptors to form a small complex (which also includes nonreceptor claudins). Approximately six small complexes oligomerize on the host cell plasma membrane surface to form a prepore. CPE molecules in that prepore apparently extend β-hairpin loops to form a β-barrel pore, allowing a Ca²⁺ influx that activates calpain. With low-dose CPE treatment, caspase-3-dependent apoptosis develops, while high-CPE dose treatment induces necroptosis. Those effects cause histologic damage along with fluid and electrolyte losses from the colon and small intestine. Sialidases likely contribute to type F disease by enhancing CPE action and, for NanI-producing nonfoodborne human GI disease isolates, increasing intestinal growth and colonization.

Keywords: Clostridium perfringens; enterotoxin; germination; orphan histidine kinases; small acid-soluble proteins; sporulation.

Fig 1

Model of C. perfringens sporulation and CPE production. Unidentified signals activating orphan histidine kinases (most importantly CPR1953 and CPR1954 and also other histidine kinases in certain environmental conditions) directly or indirectly initiate the production and phosphorylation of Spo0A. A number of other factors, as shown, can also modulate this sporulation initiation. Spo0A-P then leads to the production of the alternative sigma factor SigF, which in turn turns on the production of three additional sporulation-associated alternative sigma factors named SigE, SigG, and SigK (48, 49). SigE and SigK direct RNA polymerase to promoters upstream of the cpe open reading frame (ORF), leading to the transcription of the cpe gene and then CPE production (54). Once the endospore is mature, the mother cell lyses, which results in the release of its mature endospore as well as CPE toxin into the environment. Modified from references (47, 55) using new information from references (52, 53, 56).

Fig 2

Organization of cpe loci located on the chromosome vs plasmids among different cpe-positive C. perfringens isolates. Each box represents an ORF; arrows under the boxes show the orientation of each ORF. (A) Organization of chromosome cpe loci in various type F isolates, including the classical chromosomal cpe-locus in NCTC8239, a type F food poisoning isolate (103), and seven other chromosomal cpe-loci variants in type F strains recovered from foodborne disease outbreaks in France (37). (B) Organization of the plasmid-cpe loci in types C, D, E, and F strains. The asterisk indicates the silent cpe gene in the type E NCIB10748 isolate. Panels (A and B) compiled and modified from references (37, 98, 103, 105).

Fig 3

Structure of the CPE protein. (A) CPE has two domains. Receptor-binding activity maps to the CPE C-terminal domain. The N-terminal domain of CPE mediates oligomerization and contains the TM1 region, which is centered around the alpha-helical region of the N-terminal domain that is thought to unwind into a β-hairpin involved in β-barrel pore formation. Modified from reference (125) with permission. (B) Close-up view of the C-terminal domain of CPE, showing the claudin binding pocket. Modified from reference (127) (published under a Creative Commons license).

Fig 4

The cytotoxic mechanism of CPE. CPE (red) binds to receptors claudin (green boxes) present on the apical surface of host cells, forming a small complex (~90 kDa) that also contains nonreceptor claudin (gray boxes). Approximately six small complexes interact to oligomerize and form a prepore on the host cell plasma membrane surface. The β-hairpin loops of approximately six CPE molecules present in the prepore then interact to form a β-barrel that inserts into membranes and forms an active pore named CH-1 (~450 kDa). With low CPE dose treatment, few pores form, allowing only a modest calcium influx that causes some cytoplasmic calpain activation and caspase-3-mediated apoptosis. At high-CPE dose treatments, many pores form so a strong calcium influx occurs that results in substantial cytoplasmic calpain activation to induce cell death via necroptosis. Eventually, morphological damage develops in CPE-treated cells, which exposes the basolateral cell surface to permit additional binding of the toxin to additional claudins, forming an even larger (~600 kDa) complex named CH-2 that also contains the tight junction protein occludin (gold box). Modified from Gram-Positive Pathogens, Chapter 57 (128), with updated information from reference (129).

Fig 5

Involvement of RIPK1, RIPK3, and mixed-lineage kinase domain-like pseudokinase (MLKL) in CPE-induced host cell death. CPE-mediated cell death pathways in Caco-2 cells. (Left pathway) With low CPE doses, there is mild calpain activation resulting from limited Ca²⁺ influx. This low-level calpain activation either directly or indirectly affects a complex containing RIPK1, which results in caspase-3 activation to cause apoptosis. RIPK3 contributions to apoptosis are unknown. (Right pathway) With high-CPE dose treatment, calpain is greatly activated due to substantial Ca²⁺ influx. This strong calpain activation then either affects the necrosome, or acts after necrosome formation, to cause MLKL oligomerization; this MLKL oligomerization then either directly or indirectly results in necroptosis. Necrostatin-1s (Nec-1s) and GSK′963 are RIPK1-specific inhibitors, GSK′872 and GSK′842 are RIPK3-specific inhibitors, and necrosulfonamide (NSA) is an MLKL inhibitor. ? indicates the possibility that CPE-induced strong calpain activation might directly affect MLKL oligomerization, although this has not yet been assessed. Modified from reference (129).

Fig 6

Models for the pathway for sialic acid metabolism and the regulation of sialidase expression in C. perfringens. (A) In p-cpe type F isolates and most other C. perfringens isolates other than type F c-cpe isolates, both NanJ and NanI are secreted from C. perfringens to generate free sialic acids from mucus or host cell surfaces. Free sialic acid is then taken up by C. perfringens and metabolized for growth. VirS/VirR, RevR, ReeS, NanR, and CodY have been shown to directly or indirectly affect sialidase production. NanI can enhance the binding and action of CPE (192). Modified from reference (182) (published under a Creative Commons license). (B) In c-cpe type F food poisoning strains, NanR regulates NanH (upper panel) in SM101 or NanJ (lower panel) in 01E809 (193). In SM101, NanH is only produced in sporulating conditions and requires Spo0A production (71). Both NanH and NanJ have been shown to enhance CPE binding and cytotoxicity for host cells (71, 193). Green lines indicate positive regulation while red lines indicate negative regulation.

Fig 7

Updated models for C. perfringens type F human GI diseases. (Left) C. perfringens type F food poisoning. Type F c-cpe food poisoning isolates are ingested in large amounts, multiply, and then sporulate in vivo to produce CPE. Because these strains do not produce NanI or the EA pathway [C. perfringens can use ethanolamine for growth in infected tissues (45)], they do not colonize the intestines well, so they are rapidly cleared from the intestines by diarrhea. (Right) C. perfringens type F nonfoodborne human GI diseases. Type F nonfoodborne isolates use NanI and the EA pathway to promote their persistent intestinal growth and colonization, particularly in the antibiotic-dysregulated GI tract, chronic disease develops [modified from reference (182), published under a Creative Commons license].