Expression regulation of Corynebacterium diphtheriae's toxin

Cortesi SaraMolecular Microbiology Classa.a. 2007-2008

Corynebacterium diphtheriae overview

KINGDOM Bacteria PHYLUM ActinobacteriaCLASS ActinobacteriaSUBCLASS Actinobacteridae ORDER ActinomycetalesSUBORDER Corynebacterineae;FAMILY Corynebacteriaceae GENUS CorynebacteriumSPECIES Corynebacterium diphtheriae

Biotyps: gravis, mitis, belfanti, intermedius

Διφθερα = diphthera

Gram positive Not mobile Asporigeno Facultative anaerobic

Todar, 2008

Disease is characterized by:• Fever;• Sore throat;• Ulcere della pelle• Epitelial necrosis• Septicemy• Septic arthritis• Endocarditis

Pseudomembranes made of fibrin and leucocytes involve:• Pharingis• Tonsils• Nasal cavity

Myocarditis Polineurite Sistemic damages Loss of sensibility Loss of motor control

Diphteria disease

BIODIC

A look at History...

400 A.C.: Hippocrates describes the disease for the first time.

1883: Klebs discover that the pathogenic agent is a bacterium.

1884: Loeffler identifies C. diphtheriae and imagine that the disease is due to a toxin

1900: Von Behring and Kitasato immunized guinea pigs with a serum extract and obtain the first anti-toxin (that will lead to a vaccin in 1913)

1909: Smith produces the first vaccine, but it is causes damaged dannoso

1913: Schick developes the first “skin test”.

1929: Ramon creates a vaccine from toxoid.

1951: Freeman describes the bacteria as lysogenized by phage beta

60's: Pappenheimer uncover the method of action of the toxin

Diagnosis and treatment

Criteria of analysis:- strain isolation from patient- hystopathological analysis

Clinical criteria:- upper breathing apparatus illness- fever;- adherent pseudomembrane

Treatment:- intubation or tracheotomy - antibiotics subministration (Erythromicin or procaine)

Diphteria diffusion

case rates report - WHO in 1997:- more than 100 - between 50 and 100- between 1 and 49i- none

Corynebacterium diphtheriae NCTC 13129

The genome of Corynebacterium diphtheriae

Lenght: 2,488,635 bpGC balance: 53% Codifying %: 87% Typology: circularGenes: 2389Codifying proteins: 2272Structural RNA: 69Pseudogenes: 48

Cerdeño-Tárraga et al., 2003

The first step of C. diphtheriae invasion: Adhesion

C. diphtheriae binds host cells' membrane by adhesive structures called pili.

Sortase, a transpeptidase, recognizes LPLTG (or NPQTG) domain in Spa subunits and helps in the assembly of the pili.

SpaA, SpaB and SpaC present a signal peptide to address them in the cytoplasm.

Pizarro-Cerdà and Cossart, 2006

DtxR: toxin gene repressor

Transcription is influenced by stress conditions. Research as demonstrated that even if iron is critical for toxin repressor action, Fe level does not affect transcription.

Tricistronic operon sigB-dtxR-galE:dtxR codifies for toxin promoter repressor:sigB codifies for a sigma70 factor galE codifies for UDP-galactose-4-epimerase

DtxR regulates transcription of toxP and other genes involved in oxidative stress, siderophores, iron assumption, iron carriers and sortases.

Oram et al., 2006

Structure of DtxR

DtxR has three different domains:

C-terminal helps binding to the ion

Ion binding and dimerisation domain

N-terminal binds DNA

Repressor needs a conformation change to the active form

A conformation change induce the transition of the repressor from the inactive (apo-DtxR) to the active form (holo-DtxR).

SH3 domain does no more bind the Pro rich sequence. Prolines now stabilize the dimerisation domain.

Rangachari et al., 2005

DtxR's action in toxin transcription regulation

Fe2+ is DtxR co-repressor.

In a low iron environment, DtxR does not repress toxin expression.

The repressor acts in a dimer by binding the palindromic sequences at the operator.

Operator binding sequence is a palindrome

Repressor recognize a 19 bp interrupted palindrome.

This sequence is maintained with few modifications in all the genes regulated by DtxR

Kunkle et al., 2005

Diphteria toxin structure

Diphteria toxin is a 535 aa AB toxin.

A domain is catalytic.

B domain is connected to A domain by a disulphuric bridge and a peptidic bond, and it binds cell receptor.

T domain is hydrophobic and binds the endoplasmic membrane.

Todar, 2008

Endocytosis of the toxin

Bafilocimycin A1 inhibits release of toxins in the cytoplasm by blocking their escape from endosomes.

It blocks ATPases, that normally acidify the endosomes. It also seems to block subunit A and B separation

Diphteria toxin bind HB-EGF (heparin binding epidermal growth factor) on cell surface.As far as it enters the cell via endocytosis the acidification of the endosome cause a conformational change in the toxin and it can translocate the catalytic domain in the cytoplasm.

Todar, 2008

Mechanism of action

Diphteric toxin blocks protein elongation by binding EF2 elongation factor

EF2 + NAD+ -------------> ADP-ribose-dipthamide-EF2 + Nicotinamide + H+

By removing EF2 from the intracellular environment, it blocks the transition between A site and P site in the ribosome.

Diphteria toxin has an extremely low lethal dose.

Diphteria toxin analysis in depth: a selected research study

SSCP analysis of toxin promoter

Only 2 strains (4 and 5) among the 81that produce the toxin show a different electrophoretic pathway.

This two strains has a mutation located in promoter region.

Base transitions T to C in -54 e -184 lead to an overproduction of diphteria toxin

Point mutation in promoter region

A mutation in -54 changes one base in the palindromic sequence. It corresponds to -10 sequence of tox promoter.

ATAATTAGG ---------> ACAATTAGG

-54 position is in tox promoter region

DtxR sequence analysis

C7: control strain, not lysogenic, which produce no toxin.

Strains that show -54 base transition.

Only few changes in aminoacid sequence of DtxR repressor occur.

Modifications in aminoacidic sequence

Conclusions

Point mutation in position -54 of the promoter alters recognizing sequence, producing a lower repressor affinity to tox promoter.

An altered binding of repressor to operator site end into hyperproduction of the toxin.

Mutation in -184 overlap the edge between operator and promoter.

By enhancing Fe concentration repression decreases, but still lasts.

Also some mutation in DtxR aminoacidic sequence could modulate interaction.

Bibliography

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