Expression regulation of Corynebacterium diphtheriae's toxin Cortesi Sara Molecular Microbiology Class a.a. 2007-2008
Corynebacterium diphtheriae overview KINGDOM Bacteria PHYLUM Actinobacteria CLASS Actinobacteria SUBCLASS Actinobacteridae ORDER Actinomycetales SUBORDER Corynebacterineae; FAMILY Corynebacteriaceae GENUS Corynebacterium SPECIES Corynebacterium diphtheriae Biotyps: gravis, mitis, belfanti, intermedius Διφθερα = diphthera <ul><li>Gram positive </li></ul><ul><li>Not mobile </li></ul><ul><li>Asporigeno </li></ul><ul><li>Facultative anaerobic </li></ul>Todar , 2008
<ul><li>Disease is characterized by: </li></ul><ul><li>Fever; </li></ul><ul><li>Sore throat; </li></ul><ul><li>Ulcere della pelle </li></ul><ul><li>Epitelial necrosis </li></ul><ul><li>Septicemy </li></ul><ul><li>Septic arthritis </li></ul><ul><li>Endocarditis </li></ul><ul><li>Pseudomembranes made of fibrin and leucocytes involve: </li></ul><ul><li>Pharingis </li></ul><ul><li>Tonsils </li></ul><ul><li>Nasal cavity </li></ul><ul><li>Myocarditis </li></ul><ul><li>Polineurite </li></ul><ul><li>Sistemic damages </li></ul><ul><li>Loss of sensibility </li></ul><ul><li>Loss of motor control </li></ul>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 bp GC balance: 53% Codifying %: 87% Typology: circular Genes: 2389 Codifying proteins: 2272 Structural RNA: 69 Pseudogenes: 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 sigma 70 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 Fe 2+ 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.
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