Evolution of Toxins

Question 1

Clostridium difficile: TcdB target receptor

Answer 1

• binds Frizzled on basolateral surface of cells

Question 1

Clostridium sordellii

Answer 1

• anaerobic gram-positive bacterium

Question 2

where is C. sordellii found (3)

Answer 2

• soil
• gastrointestinal tracts
• vaginal tracts

Question 3

C. sordelli: virulence (2)

Answer 3

• most carriers are asymptomatic
• infections caused by pathogenic strains are rapid and highly lethal

Question 4

C. sordelli: pathogenic (2)

Answer 4

• almost all occur in women following child birth
• can be involved in medically induced abortion and toxic shock syndrome

Question 5

what is the primary source of high mortality in C. sordellii infections

Answer 5

• lethal toxin TcsL

Question 6

what is the LCT family (3)

Answer 6

• large clostridial toxin family
• highly similar at sequence level
• differ in tissue specificity and effects on cell morphology

Question 7

LCT family: mechanism of intoxication (6)

Answer 7

1. LCTs enter host cell by receptor-mediated endocytosis
2. enter acidified endosomes
3. pH-dependent pore formation occurs and
4. toxin is translocated to the cytosol
5. after cytosol processing, cytotoxic glucosyltransferase enzymes inactivate small Rho-family GTPases
6. host cell function is potently modulated

Question 8

TcsL vs TcdB: similarities

Answer 8

• TcsL is mot related to TcdB, sharing 90% sequence similarity

Question 9

TcsL vs TcdB: differences (2)

Answer 9

• TcdB receptor is Frizzled in colonic epithelium
• TcsL does not bind Frizzled and C. sordellii does not infect gut, suggesting that it uses a different receptor

Question 10

what strategy was used to identify possible TcsL receptors (3)

Answer 10

• single gene knock outs of non-essential host cell genes made using CRISPR
• host cell library treated with TcsL
• observation into 2 surviving clones

Question 11

TcsL receptor experiment: what genes were knocked out in the two surviving clones (2)

Answer 11

• SEMA6A
• UGP2

Question 12

UGP2 gene

Answer 12

• involved in UGP glucosylation

Question 13

what strategy was used to test is SEMA6A was the TcsL receptor in vivo (2)

Answer 13

• tested whether purified SEMA6A would prevent TcsL intoxication in mouse model (competition experiment)
• purified SEMA6A would bind TcsL and prevent it from binding to SEMA6A on the host, so no disease occuts

Question 14

TcsL receptor experiment: BSA (2)

Answer 14

• bovine serum albumin
• negative control protein for purified SEMA6A in vivo injection

Question 15

what strategy was used to observe the molecular compatibility of SEMA6A and TcsL (4)

Answer 15

• co-crystal structure of TcsL and SEMA6A were observed
• revealed compatible interface between toxin and receptor
• interface residues were mapped onto primary amino acid sequence for receptor and ligand
• sequence alignment with related receptors/toxins suggest receptor/ligand specificity

Question 16

what strategy was used to confirm the differences between TcsL and TcdB receptor-ligand specificity (3)

Answer 16

• 15 targeted amino acid mutations were made in TcsL to produce a more TcdB-like sequence
• recombinant TcsL protein unmutated only bound to SEMA6A
• recombinant TcsL protein mutated only bound to Frizzled receptor

Question 17

what did we learn from the TcsL vs TcdB receptor-ligand specificity experiment

Answer 17

• less conserved interface that confers specificity can be different by just 15 mutations from another toxin

Question 18

TcsL receptor experiment: what were the study highlights/takeaways (4)

Answer 18

• CRISPR screen identifies SEMA6A and SEMA6N as receptors for toxin
• soluble SEMA6A ectodomain protects mouse lungs from TcsL-induced edema
• cryo-EM structure of TcsL bound to SEMA6A reveals atomic details of interaction
• 15 mutations in TcsL receptor-binding interface rewire specificity from SEMA6 to Frizzed