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Seminario Biologia Molecular
1. 1
Tercer semestre - Facultad de medicina - Universidad Pontificia Bolivariana
2. INTRODUCTION
2
It is an acid-fast, aerobic bacillus with a high cell
wall content of high-molecular-weight lipids.
It is the major causative pathogen of
tuberculosis
Genus : Mycobacterium
Species: M. tuberculosis
It is a facultative intracelular pathogen
3. Rv1075c
3
It is a conserved hypothetical
protein
It shares 24.16% identity
with another GDSL-like
lipase/acylhydrolase
Mutation of Rv1075c led to significantly
reduced bacterial growth in
macrophages and in mice.
4. OBJETIVE
They identified that Rv1075c amino acid
sequence shares similarities with other
bacterial lipase/esterases and they
demonstrated that it has esterase activity, with
preference for short-chain fatty acids,
particularly acetate, with highest activity at
45°C, pH 9.
6. 6
PCR
• Utilizada para realizar amplificación
enzimática de un material genético.
Sintetizar varias cadenas de
DNA mediante una cadena de
DNA polimerasa
Reacción en cadena de la polimerasa
7. Expresión del
gen Rv1075c
en condiciones
de estrés
7
La cepa de M. tuberculosis CDC1551
Se expuso a diferentes condiciones de estrés
Estrés por
temperaturaEstrés
nutricional
Estrés
oxidativo Estrés
acido
Esta técnica se utilizo para
determinar la expresión del gen
Rv1075c
8. Infección
del ratón
8
◌ Cepa de CDC1551
de M. tuberculosis -
expresa tdTomato.
Cepa mutante
Rv1075c
Cepa complementada
Rv1075c
Infectar
macrófagos
• Ratones hembra
C3HeB / FeJ
• 6 a 8 semanas
de edad
Fueron infectados con
M. tuberculosis salvaje
tipo Rv1075c mutante y
Rv1075c
complementada.
El estudio fue
realizado
durante 56 días
10. 10
Electroforesis en Gel
M: Marcador del peso molecular de
proteínas
1-2 : muestras de ADN
RESULTADO FINAL: el gen Rv1075c
purificado tenía una masa molecular
de aproximadamente 32 kDa
CARRILES:
3-4-5: Resultados según tamaño
Proteína recombinante de
Rv1075c extraída de la cepa
M. smegmatis diseñada que
expresa el gen Rv1075c.
12. 12
Cromatografía en
capa fina (TLC)
Resultado:
proteína Rv1075c tiene actividad
de triacilglicerol acil hidrolasa.
Triacetina Diacetina y Monoacetina
Tributirina Dibutirina y Monobutirina
13. DISCUSSION
13
Camus JC, Pryor
MJ, Medigue C,
Cole ST.
Genomic sequencing has predicted
that M. tuberculosis
possesses more than 250 genes
related to lipid metabolism
Flannagan RS,
Cosio G,
Grinstein S.
Their data demonstrate that M.
tuberculosis Rv1075c is an
esterase containing a GDST motif.
Its expression is induced by low
pH, which is one of the challenges
that M. tuberculosis faces when it
is in macrophage phagosomes
Wilhelm S,
Tommassen J,
Jaeger KE.
Based on the evolutionary
phylogenetic tree (Figure 1B),
Rv1075c is most closely related to
TesA of Pseudomonas aeruginosa
14. CONCLUSIONS
14
Rv1075c mutated could be used to decrease the
colony forming unit in the lung and prevent the
development of Tuberculosis complications
Rv1075c has characteristics of lipase/esterase that
allows the hydrolysis of fatty acids; Then M.
tuberculosis could enter and survive in an
environment where it can make changes into the
host.