Evolution of Toxins Flashcards
Clostridium difficile: TcdB target receptor
- binds Frizzled on basolateral surface of cells
Clostridium sordellii
- anaerobic gram-positive bacterium
where is C. sordellii found (3)
- soil
- gastrointestinal tracts
- vaginal tracts
C. sordelli: virulence (2)
- most carriers are asymptomatic
- infections caused by pathogenic strains are rapid and highly lethal
C. sordelli: pathogenic (2)
- almost all occur in women following child birth
- can be involved in medically induced abortion and toxic shock syndrome
what is the primary source of high mortality in C. sordellii infections
- lethal toxin TcsL
what is the LCT family (3)
- large clostridial toxin family
- highly similar at sequence level
- differ in tissue specificity and effects on cell morphology
LCT family: mechanism of intoxication (6)
- LCTs enter host cell by receptor-mediated endocytosis
- enter acidified endosomes
- pH-dependent pore formation occurs and
- toxin is translocated to the cytosol
- after cytosol processing, cytotoxic glucosyltransferase enzymes inactivate small Rho-family GTPases
- host cell function is potently modulated
TcsL vs TcdB: similarities
- TcsL is mot related to TcdB, sharing 90% sequence similarity
TcsL vs TcdB: differences (2)
- 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
what strategy was used to identify possible TcsL receptors (3)
- single gene knock outs of non-essential host cell genes made using CRISPR
- host cell library treated with TcsL
- observation into 2 surviving clones
TcsL receptor experiment: what genes were knocked out in the two surviving clones (2)
- SEMA6A
- UGP2
UGP2 gene
- involved in UGP glucosylation
what strategy was used to test is SEMA6A was the TcsL receptor in vivo (2)
- 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
TcsL receptor experiment: BSA (2)
- bovine serum albumin
- negative control protein for purified SEMA6A in vivo injection
what strategy was used to observe the molecular compatibility of SEMA6A and TcsL (4)
- 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
what strategy was used to confirm the differences between TcsL and TcdB receptor-ligand specificity (3)
- 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
what did we learn from the TcsL vs TcdB receptor-ligand specificity experiment
- less conserved interface that confers specificity can be different by just 15 mutations from another toxin
TcsL receptor experiment: what were the study highlights/takeaways (4)
- 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
