Upload
aleskarmen
View
1.377
Download
0
Tags:
Embed Size (px)
Citation preview
Aplicación de la biología molecular al
diagnóstico de enfermedades
transmitidas por bacterias
Dra. Patricia Saravia, Ph.D
Departamento de Bioquímica
Facultad de CCQQ y Farmacia
USAC
WHICH ARE THE WHICH ARE THE
VIRULENCE FACTORS?VIRULENCE FACTORS?Flagellum
Pili
Plasmid
Capsule
Cell wall/outer
membrane (if present)
TOXINS
TOXINSTo
xinstox
ins
I. TOOLS FOR ADHESION I. TOOLS FOR ADHESION
AND SPREADAND SPREAD
PiliPili//fimbriaefimbriae
Other colonization factorsOther colonization factors (less (less well defined well defined
proteins on cell proteins on cell surfacesurface) )
ExoenzymesExoenzymes promote spread promote spread of of bacteriabacteria in in tissues tissues
((hyaluronidasehyaluronidase, , collagenasecollagenase, , streptokinasestreptokinase, , DNaseDNase))
II. TOOLS FOR INVASIONII. TOOLS FOR INVASION
Invasins induce cellular uptake Invasins induce cellular uptake ofof bacteriabacteria
Factors promoting intracellular survival Factors promoting intracellular survival
-- in in phagocytesphagocytes
-- in in epithelialepithelial cellscells
Transcytosis across epitheliaTranscytosis across epithelia
III. TOOLS TO DESTROY III. TOOLS TO DESTROY
CELLS AND TISSUESCELLS AND TISSUES
Exotoxins/enzymes
1.1. HemolysinsHemolysins//cytolysins cytolysins Induce Induce cell cell membrane damagemembrane damage
2.2. EnterotoxinsEnterotoxins Intestinal effectsIntestinal effects ((diarrhoeadiarrhoea))
3.3. NeurotoxinsNeurotoxins Affect nervousAffect nervous systemsystem
4.4. CytotoxinsCytotoxins KillKill cells by cells by effects other thaneffects other than
membrane damagemembrane damage
5.5. SuperantigenicSuperantigenic toxinstoxins OverstimulateOverstimulate immune systemimmune system
Non-toxic exoenzymes Hyaluronidase, Collagenase,
Lipase
EndotoxinEndotoxin
Acido Desoxiribonucleico (ADN)
Generalidades
• Almacena y transmite la
información genética
• A simple vista, la estructura
del ADN de un humano es
igual a la de una bacteria
• Está formado por bases
nitrogenadas, grupos fosfato y una pentosa
(desoxiribosa)
Secuencia nucleotídica
Puentes de
Hidrógeno
Estructura de azúcar-fosfato
Molécula de
fosfatoDesoxiribosa
Bases
Diagnóstico de Mycobacterium
tuberculosis utilizando la técnica
de PCR (polymerase chain
reaction)
Licda. Maria Eugenia Castellanos
Tesis de graduación, 2005
www.icampus.ucl.ac.be/. ../PCR/PCR.html
Pt8
Pt9
Pt9
TB290
Primers externos
Primers internos
PCR HEMIANIDADO
IS6110
DETECCION DEL PRODUCTO DE
AMPLIFICACION
9494ººC =C = DesnaturalizaciDesnaturalizacióónn
6565ººC =C = HibridaciHibridacióónn
7272ººC =C = ExtensiExtensióónn
www.pitt.edu/ ~heh1/PCR.jpg
DETECCION DEL PRODUCTO DE
AMPLIFICACION
Electroforesis en gel deagarosa al 2% en muestrasde esputo (por duplicado)mostrando producto amplificado (337 pb).
Pozo 1= H20
Pozo 2 = Control Neg.
ADN humano
Línea 5 = Control Pos.
M. tuberculosis
A2 y A9= Positivas
310 pb
Detección de genes que codifican por
exotoxinas bacterianas utilizando la técnica
de dot blot ó Southern blot
AdeninaAdenina
CitosinaCitosina
GuaninaGuanina
TiminaTimina
Sondas de ADN/ARN
ADN de cadena doble (duplex)
Desnaturalización
ADN de cadena simple
Sonda marcada
(ADN o ARN)
Hibridación a
secuencias
complementarias en
ADN