L12. C. Diff S-layer Flashcards
Where is the S-layer found on a bacteria?
Outer most layer of the cell.
What structure is an S-layer?
> S-layers are a Paracrystalline array surrounding the cell.
> Paracrystalline meaning not perfectly crystalline (2D crystal that surrounds the cell).
What is an S-layer made up of in a) most bacteria b) C. diff
a) In most bacteria made up of glycosylated S-layer proteins.
b) In C. diff most strains the proteins are not glycosylated.
What bacteria a) contain b) don’t contain an S-layer?
a) Vast majority of bacterial species possess S-layers (gram positive or gram negative), even every archaea species has an S-layer too, one of the most ubiquitous protein structures present on the planet
b) S-layers are not found on many model bacteria, such as E.coli, Strep A, Strep pneumo (could be question asking to compare what structures are in the different types of gram positive bacteria in this lecture series).
How many protein subunits make up C. diff S-layers?
> Approx 600,000 protein subunits to make up a C. diff S-layer (as C. diff is a large cell).- the most highly expressed protein in C. diff (10% of all protein produced makes this structure)
> Very expensive structure to assemble on outside of cell (many amino acids)
Describe the 4 steps of S-layer biogenesis in C. diff.
- Signal Peptide for Secretion:
>During translation, the initial 30 amino acids of SlpA act as a signal peptide directing the secretion of the unfolded SlpA protein through the membrane. - Secretion Mechanism:
>Secretion energy is provided by the dedicated ATPase, SecA2:
>C. difficile contains two SecA proteins: SecA1 and SecA2. SecA1 is required for the secretion of all other secreted proteins in the cell, while SecA2 is dedicated exclusively to the S-layer protein.
>The ATPase binds to the polypeptide and, through sequential rounds of ATP binding and hydrolysis, conformational changes push the peptide through the membrane channel SecYEG. - Post-Secretion Cleavage:
>Once outside the cell, the SlpA is cleaved to form low molecular weight (LMW) SLP and high molecular weight (HMW) SLP.
>This cleavage event is mediated by a cell wall protein called Cwp84. - Assembly and Anchoring:
>The subunits self-assemble to form a high-affinity heterodimer complex, which is anchored to the cell wall by a polysaccharide called PS-II.
>In addition to the peptidoglycan cell wall, all Gram-positive bacteria have another mixture of sugars interwoven into the cell wall, providing additional functions (PS-II as an example).
>The exact nature of these sugars varies between species. For example, teichoic acids are secondary cell wall polymers found in only a small number of species.
What is SlpA and what is its unique ability among the cell wall proteins (Cwp) in Clostridium difficile?
SlpA is a member of a family of 29 proteins in C. difficile and is the only one that can form a 2D crystal, creating a true S-layer on the bacterial cell surface.
What common feature do all members of the Cwp family share?
All members of the Cwp family have 3 copies of the Cell wall binding domain 2 (grey), which interacts with PS-II to anchor the proteins to the cell surface.
What are the primary functions of the Cwp proteins in Clostridium difficile?
The Cwp proteins perform various functions, including acting as anchors, proteases, and enzymes that contribute to virulence and antibiotic resistance.
How does the Cwp family in C. difficile compare to sortase-anchored LPXTG proteins in other Gram-positive bacteria?
The Cwp family in C. difficile replaces the role of sortase-anchored LPXTG proteins found in other Gram-positive bacteria.
What is the function of Cwp84 and Cwp13 in the Cwp family?
Cwp84 is important for maturation of S-layer protein and Cwp13 we don’t know the function for. Both of which can break down other protein structures so probably involved in virulence for breaking down host proteins
What role does Cwp20 play in antibiotic resistance in Clostridium difficile?
Cwp20 is a β-lactamase, an enzyme that degrades β-lactam antibiotics, making C. difficile resistant to these antibiotics.
Describe the current model of the S-layer in Clostridium difficile.
> The S-layer is primarily composed of SlpA, which makes up 90% of the layer. The remaining 10% is composed of other Cwp proteins that add functionality to the cell surface, especially in defects where SlpA is missing.
> Every now in the 2D crystal there is a single copy of SlpA missing (defect in the crystal lattice), this space will be taken up by one of these other proteins instead which adds functionality to the cell surface.
How does the quantity of S-layer proteins in Clostridium difficile compare to other bacteria?
The quantity of S-layer proteins in C. difficile is comparable to the sortase LPXTG protein family in Staphylococcus aureus, with a similar size of 30 member proteins being used whether an S-layer is present or not.
What hampered studies on the function of the S-layer in C. diff?
In C. diff struggled to make mutant for gene SlpA, took 16 years to make an SlpA knockout in C. diff, now has been made due to CRISPR, but this was only a few days ago, so what have they done before this.
What is the classic molecular biology approach to study protein function mentioned in the notes?
The approach involves taking the wild-type (WT) bacterium, mutating genes so it can’t make a specific protein, complementing the mutant by reintroducing the gene, and then observing if the original phenotype is restored. This method follows molecular Koch’s postulates.
What has AvidBiotics developed to target Clostridium difficile?
AvidBiotics has developed R-type bacteriocins, also known as Avidocin, which are phage particles that act as therapeutics to kill different bacterial species, including C. difficile.
How do the R-type bacteriocins produced by Clostridium difficile work to kill other bacteria?
They resemble contractile bacteriophages but lack the capsid head containing DNA. They bind to the surface of the target cell using tail fibers, contract to draw a needle through the cell surface, and kill the cell by causing a loss of cations, ATP, and H+ ions.
