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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