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Germination and Sporulation of Clostridioides difficile Derek TanMarch 10, 2020MMIC 7150
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Presentation Overview• History
• Lifecycle
• Spores
Function and Structure
• Germination
• Sporulation
• Infection Prevention
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Discovery
• First isolated and described in 1935 by Ivan Hall and Elizabeth O’Toole
“… hitherto undescribed obligate anaerobic pathogen …”
“Morphologically, it is a large Gram-positive rod with elongate subterminal spores of about the same width as the rods”
Carr J et al. CDC. 2004Hall I, O'Toole E. Am J Dis Child. 1935
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Hall I, O'Toole E. Am J Dis Child. 1935 4
Nomenclature
Bacillus difficilus (1935)
Clostridium difficile (1978)
Clostridioides difficile (2016)
5Hall I, O'Toole E. Am J Dis Child. 1935George RH et al. Br Med J. 1978
Lifecycle
Seekatz A et al. J Clin Invest. 2014
Lifecycle
Rao K et al. Inflamm Bowel Dis. 2016
Purpose of Spores • Survival mechanism
Can tolerate many different types of stresses
• Produced in response to stresses
8Ray K. Nat Rev Gas & Hep. 2019
Oxygen Tolerance
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Oxygen Concentrations0% 1% 2% 3% 5%
Giordano N. Path and Dis. 2018.
Spore Structure
10Kochan TJ et al. J Bacteriol. 2018
Core
• Houses bacterial DNA, transcription factors and other proteins• DNA supercoiled and bound to small acid-soluble proteins (SASP)
• Very low water content • Rich in calcium dipicolinic acid (Ca-DPA)
11Kochan TJ et al. J Bacteriol. 2018
Inner Membrane
• Layer of phospholipid proteins • Protects the core• Low permeability
12Kochan TJ et al. J Bacteriol. 2018
Germ Cell Wall
• Protection • Derived from mother cell’s cell wall• Incorporated into germinated cell’s cell wall
13Kochan TJ et al. J Bacteriol. 2018
Cortex
• Protection• Thick layer of modified peptidoglycan
• ½ N-acetylmuramic acid is converted to muramic-δ-lactam (MAL)
14Kochan TJ et al. J Bacteriol. 2018Paredes-Dabja. Trends Micriboil. 2015
Outer Membrane
• Protection• Layer of peptidoglycan
15Kochan TJ et al. J Bacteriol. 2018
Spore Coat
• Protection• Dense layer of protein
16Kochan TJ et al. J Bacteriol. 2018
Exosporium
• Protection• Loose fitting layer• Composed of highly permeable carbohydrates
17Kochan TJ et al. J Bacteriol. 2018
Spore Structure• CR: Core
• GCW: Germ cell wall
• CS: Cortex
• OM: Outer membrane
• CT: Spore coat
• Ex: Exosporium
18Permpoonpattana P et al. J Bacteriol. 2011.
Germination• Signals of germination
Environmental based
Nutrient based
• Antibiotics
• Bile salts
• Amino acids
19Shen A et al. J of Bacteriol. 2017
Bile Salts• Cholate-derived bile acids are exclusively produced exclusively in the
mammalian gut
• Taurocholate (secondary bile acid) most effectively promotes germination
Increased levels in dysbiotics gut during antibiotic treatment
Not sufficient alone to trigger germination
• Chenodeoxycholate is an efficient competitive inhibitor
One of the two primary bile acids synthesized by the liver
20Shen A et al. J of Bacteriol. 2017Kochan TJ et al. J Bacteriol. 2018
Bile Salts + Amino Acids • Unknown pathway
• Glycine works the best
• Other amino acids can work
L-Alanine or D-Alanine
L-Histidine
L-Serine or D-Serine
Glycine Alanine
21Yamakawa K et al. J Med Microbiol. 1994
Shen A et al. J of Bacteriol. 2017Kochan TJ et al. J Bacteriol. 2018
Bile Salts + Divalent Cation• Unknown pathway
• Calcium and magnesium can substitute the need for amino acids
• Synergy exists between the amino acid and divalent cation pathways
Concentrations can be reduced 10-fold when in combination
22Shen A et al. J of Bacteriol. 2017Kochan TJ et al. J Bacteriol. 2018
Bile Salts and Germination
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Bile
Acid
CspC
CspBPro-SleC
CspB
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Germination Theories
Adams CM et al. PLoS Pathog. 2013 Francis MB et al. PLoS Pathog. 2013
Kochan TJ et al. J Bacteriol. 2018
Other Proteins• GerG
Highly expressed lipoprotein during sporulation
Mutant strains are unable to initiate cortex hydrolysis
Thought to modify the cortex during germination
• GerS
Mutant strains are unable to initiate cortex hydrolysis
Thought to transport Csp proteins to the cortex during sporulation
• CD630_32980
Mutants strains respond to bile salts and amino acids but not to divalent cations
Thought to transport Ca-DPA across the outer membrane
25Shen A et al. J of Bacteriol. 2017Kochan TJ et al. J Bacteriol. 2018
Sporulation
26Shen A et al. J of Bacteriol. 2017
Spo0A Phosphorylation • Spo0A phosphorylation initiates sporulation
• CD1579 and CD2492
Phosphorylate Spo0A
Regulated by sigma factor H (σH)
Knockout decreases spore production by 3-fold
• CD1492
Dephosphorylates Spo0A
Knockout increases spore production by 4-fold
Regulated by sigma factor B (σB)
27Shen A et al. J of Bacteriol. 2017Underwood S. J Bacteriol. 2009
Spo0A Regulation
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• RstA (Regulator of sporulation and toxins A)
Knockout decreases spore production by 20-fold
Shen A et al. J of Bacteriol. 2017
LIST K: EPA's Registered Antimicrobial Products Effective against Clostridiumdifficile Spores
29EPA. 2018.
Prevention
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References• Carr J, Wiggs S. ID#: 6252. CDC. 2004• Hall IC, O'Toole E. Intestional flora in new-born infants. Am J Dis Child. 1935 Feb 1;49(2):390.• Lance George W, Goldstein EJC, Sutter VL, Ludwig SL, Finegold SM. Aetiology of antimicrobial-agent-
associated colitis. Lancet. 1978 Apr 15;311(8068):802–3. • Seekatz AM, Young VB. Clostridium difficile and the microbiota. J Clin Investig. 2014; 124: 4182-
4189. • Rao K, Higgins PD. Epidemiology, diagnosis, and management of Clostridium difficile infection in
patients with inflammatory bowel disease. Inflamm Bowel Dis. 2016 Jul;22(7):1744-54• Ray K. Closing in on C. difficile infection. Nat Rev Gas & Hep. 2019;16:581.• Giordano N, Hastie JL, Carlson PE. Transcriptomic profiling of Clostridium difficile grown under
microaerophillic conditions. Path and Dis. 2018;76:10. • Kochan TJ, Foley MH, Shoshiev MS, Somers MJ, Carlson PE, Hanna PC. Updates to Clostridium
difficile spore germination. J Bacteriol. 2018;200(16):1–12.• Permpoonpattana P, Tolls EH, Nadem R, Tan S, Brisson A, Cutting SM. Surface layers of Clostridium
difficile endospores. J Bacteriol. 2011 Dec;193(23):6461–70. • Shen A, Edwards AN, Sarker MR, Paredes-Sabja D. Sporulation and germination in Clostridial
pathogens. Microbiol Spectr. 2019;7(6):1–30. • Yamakawa K, Kamiya S, Meng XQ, Karasawa T, Nakamura S. Toxin production by Clostridium difficile
in a defined medium with limited amino acids. J Med Microbiol. 1994;41(5):319–23. • Underwood S, Guan S, Vijayasubhash V, Baines SD, Graham L, Lewis RJ, Wilcox MH, Stephenson K.
2009. Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. J Bacteriol. 191:7296–7305.
• Adams CM, Eckenroth BE, Putnam EE, Doublié S, Shen A. Structural and functional analysis of the CspB protease required for Clostridium spore germination. PLoS Pathog. 2013;9(2).
• Francis MB, Allen CA, Shrestha R, Sorg JA. Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog. 22013;9(5).
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