Clostridium Lecture 2024-1 PDF

Clostridial Diseases Glen Carter [email protected] Department of Microbiology & Immunology Peter Doherty Institute for Infection and Immunity Learning Objectives At the completion of this lecture students should be able to: Give examples of pathogenic...

Clostridial Diseases Glen Carter [email protected] Department of Microbiology & Immunology Peter Doherty Institute for Infection and Immunity Learning Objectives At the completion of this lecture students should be able to: Give examples of pathogenic Clostridium (Clostridiodes) species. Understand the importance of extracellular toxins in the diseases caused by the pathogenic clostridia. Understand the mechanism of action of several major clostridial toxins. Appreciate the importance of using molecular approaches to understand toxin function. The Genus Clostridium A very diverse genus. Clostridium are: Gram-positive. Obligate anaerobes. Form heat resistant endospores. Several important human and animal pathogens. Not all are pathogenic Important part of the normal microflora. The Pathogenic Clostridia Neurotoxic Clostridium tetani - tetanus Clostridium botulinum – botulism Enterotoxic Clostridium perfringens – food poisoning Clostridiodes difficile – pseudomembraneous colitis Histotoxic Clostridium perfringens – gas gangrene Clostridium septicum – atraumatic gas gangrene Clostridiodes difficile Leading cause of infectious diarrhoea in hospitals worldwide. Also a problem in the community and in animals (pigs, cattle, horses). Spores essential for transmission and persistence in hospitals. C. difficile colonises the gut when the normal microbiota is disrupted usually via antibiotics. C. difficile Infections (CDI) Causes a spectrum of diseases collectively referred to as CDI: Self limiting diarrhoea (Mild) Pseudomembraneous colitis (Severe) Toxic megacolon Sepsis Death More recently linked with multiple organ dysfunction syndrome (MODS) High mortality rate. Poorly understood. C. difficile is an urgent threat Associated with huge economic costs annually. One of the most prevalent nosocomial infections worldwide. 10x more infections than MRSA. Historical emergence of “hypervirulent” isolates around the year 2000 associated with explosion of case numbers.  Virulence factors of C. difficile Major virulence factors of C. difficile are two toxins: TcdA (aka Toxin A). TcdB (aka Toxin B). Both toxins are members of the large clostridial toxin (LCT) family. Disease is dependent on these toxins. Strains that do not produce these toxins are considered avirulent. Some strains also make a third toxin known as CDT. Role in disease is unclear. TcdA and TcdB of C. difficile TcdA and TcdB are encoded in the Pathogenicity Locus (PaLoc) TcdR is an alternative sigma factor. TcdC is an anti-sigma factor. TcdE is related to holin proteins (secretion???). Homologous regions identified in other LCT producing clostridia. TcdA/TcdB Structure - Function Both toxins are monoglucosyltransferases. Glucosylate Rho family GTPases. Disruption of actin cyctoskeleton and cell death. Toxin have a modular “ACDB” structure. Facilitates their uptake and activity A - Activity C - Cutting D - Delivery. B - Binding TcdA/TcdB Structure - Function Which is the major toxin? For >30 years TcdA was considered to be the major toxin. Only purified TcdA resulted in disease in hamster model. TcdA+TcdB- clinical isolates cause disease in humans. Studies with isogenic mutants showed TcdB was the major virulence factor. Demonstrates the power of using molecular studies. Highlights the importance of the “whole bacterial system”. TcdA-TcdB- Mock TcdA+TcdB- 1 TcdA+TcdB- 2 TcdA-TcdB+ 1 TcdA-TcdB+ 2 CDT- Wild type Carter et al. 2015 mBio Which is the major toxin? Why do we care? Design of therapeutics had focussed on TcdA. Explains several high profile drug failures: Actoxumab Tolevamer Targeting TcdA alone exacerbates disease! New Therapeutics now know to target TcdB or both toxins. Clostridium perfringens C. perfringens is an important pathogen of humans and animals. It causes an array of different diseases including: Clostridial myonecrosis (gas gangrene). Food poisoning. Historically typed according to the toxin profile of infecting strain (A-E). Only considered α, β, ε, ι toxins Many toxins on mobile elements! Produces up to 17 different toxins. Clostridium perfringens toxins Toxins are structurally and functionally unrelated. Toxins play varying roles in different disease manifestations. α-toxin is essential toxin in gas gangrene. Ingestion of CPE strains mediates human food poisoning. NetB is major toxin involved in necrotic enteritis in poultry. Other toxins considered accessory. Clostridial Myonecrosis Histotoxic infection of wounds or ischaemic tissue. C. Perfringens is the major cause of traumatic gas gangrene. Traumatic wound Contamination with C. perfringens (spores or vegetative cells) Rapid growth and toxin production (α-toxin and PfoA) Extensive necrosis of muscle tissue Clostridial myonecrosis (gas gangrene) α-toxin is essential in gas gangrene Molecular approach used to determine which toxin(s) were essential for clostridial myonecrosis. Isogenic mutants needed due to presence of multiple toxins. α-toxin gene (plc) was inactivated and then complemented. Only the wild type and complemented strain showed signs Wild type plc mutant plc mutant of myonecrosis. Independent plc mutants showed no signs of disease. plc mutant plc mutant + vector + plc+ plasmid α-toxin mechanism of action Phospholipase toxin. Phospholipase C activity. Cleaves cell membrane phosphatidylcholine to phosphorylcholine and diacylglycerol. Destablises cell membrane leading to cell death. C. perfringens Food Poisoning Major cause of food poisoning globally. Third most common cause of foodborne disease in US. Ingestion of C. perfringens enterotoxin (CPE) producing strains in under-cooked food. Unlikely to be caused by intoxication. CPE is produced during the early stages of sporulation and is released following mother cell lysis. Symptoms include abdominal cramping, diarrhoea and vomiting. Usually resolves in 24 hours. C. perfringens enterotoxin (CPE) CPE is a pore forming toxin of the aerolysin family. It can be located on plasmids or on the chromosome. Strains with chromosomal CPE most often cause FP. CPE binds to claudin receptors which induces oligomerisation and pore formation. Pores insert into membrane and interact with occludin. Leads to tight junction damage and disruption of epithelial permeability barrier. Onset of symptoms. C. botulinum C. botulinum is a neurotoxic Clostridium. Ubiquitous in soil. It is the causative agent of botulism. Production of botulinum toxin (BoNT) is responsible for disease symptoms. Targets the nervous system. Historically any species producing BoNT was considered C. botulinum. C. botulinum is very diverse group. At least five bacterial species known to produce BoNT. Botulism Three types of botulism: Foodborne botulism. Ingestion of preformed BoNT. Traditionally spoiled canned food. Wound botulism. Normally associated with injection drug users. Contamination of wound with C. botulinum. Followed by BoNT production. Infant botulism (floppy baby syndrome). Most common form of botulism in US. Infants under 1 year old. Ingestion of C. botulinum spores (honey). Colonisation of small intestine and BoNT production. Botulinum toxin (BoNT) The most potent biological toxin known. 1 teaspoon purified BoNT could kill every human on Earth. BoNT causes flaccid paralysis Blocks nerve function muscular & respiratory paralysis Death. There are 7 distinct BoNT types (A-G). Types A,B,E and rarely F associated with human disease. Structurally similar (light chain and heavy chain). Inactive protoxin is cleaved to active form (AB toxin). Antigenically and serologically distinct. Activity - Light chain Binding – Heavy chain BoNT Mechanism of Action BoNT enters neurones by endocytosis at presynaptic membrane. Endosome acidification leads to conformational change in BoNT. Light chain escapes endosome. Cleaves SNARE-family proteins by proteolysis. Blocks release of acetylcholine into synaptic cleft. BoNT Mechanism of Action Different BoNTs target different SNARE proteins: BoNTA/C/E cleave SNAP-25 BoNTC cleaves Syntaxin BoNTG/B/D/F/C cleave Synaptobrevin BoNTs block association of v-SNARES (VAMP/Synaptobrevin) and t-SNARES (SNAP-25 and Syntaxin). Disrupts SNARE complex formation. Prevents membrane fusion and acetylcholine release. Summary The clostridia are responsible for many devastating human diseases. Different clostridial species produce a vast array of different extracellular toxins. Enterotoxins (TcdA, TcdB, CPE). Histotoxins (α-toxin). Neurotoxins (BoNT). Extracellular toxins mediate many clostridial diseases. Toxin negative isolates are often avirulent. Molecular approaches have been critical for understanding the functional role of many clostridial toxins. QUESTIONS???