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

Helicobacter Pseudomonas

Staphylococcus

BACTERIABACTERIA

Bacterial classification

based on Gram

staining

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

STAPHYLOCOCCAL CELL WALL STRUCTURESTAPHYLOCOCCAL CELL WALL STRUCTURE

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)

Superenrollamiento del ADN

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

MYCOBACTERIAL CELL WALL STRUCTUREMYCOBACTERIAL CELL WALL STRUCTURE

REACCION EN CADENA DE LA POLIMERASA

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

MODE OF ACTION OF CHOLERA TOXIN (CT)an ADP-ribosyltransferase

EnterotoxinsEnterotoxins

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

Dot blot

Southern blot

Gracias por su

atención !!!