Seminario biomol Maria Clara Torres Ferrer

1. Maria Clara Torres Ferrer, estudiante de tercer semestre, facultad de medicina Universidad Pontificia Bolivariana

2. Generalities of Salmonella
La Salmonella y los alimentos [Internet]. Centros para el control y la prevención de enfermedades. 2020 [citado el 23 de febrero de 2022]. Disponible en:
https://www.cdc.gov/foodsafety/es/communication/salmonella-and-food-sp.html
• It is a genus of gram-negative bacilli that belongs to the Enterobacteriaceae
family.
• Salmonella enterica serotype Enteritidis and Salmonella enterica serotype
Typhimurium are the two most important serotypes of Salmonella
transmitted from animals to humans in most parts of the world.
• Salmonella can be found in various foods, such as chicken, beef, pork, eggs,
fruits, vegetables, and even processed foods.
• Food is not the only way Salmonella spreads to people.
• Certain people are more likely to get a serious Salmonella infection.
• Illness caused by Salmonella can be serious. Symptoms usually begin 6
hours to 6 days after infection. They include diarrhea that may be bloody,
fever, and stomach cramps. Most people recover in 4 to 7 days without
antibiotic treatment. But some people with severe diarrhea may need to be
hospitalized or take antibiotics.

3. Salmonella effector
proteins: SopD2 and PipB2
Bacterial effectors are proteins
secreted by pathogenic bacteria into
the cells of their host, usually using a
type 3 secretion system (TTSS/T3SS),
a type 4 secretion system
(TFSS/T4SS) or a type 6 secretion
system (T6SS).
Effector proteins usually help the
pathogen to invade host tissue,
suppress its immune system, or
otherwise help the pathogen to
survive.
SopD2 and PipB2 are two of
Salmonella’s effector proteins.
Both of them use the type three
secretion system: T3SS2.
SopD12, PipB2 originate from the
Salmonella pathogenicity island 2.
Salmonella pathogenicity islands
(SPIs) are large gene cassettes within
the Salmonella chromosome that
encode determinants responsible for
establishing specific interactions
with the host and are required for
bacterial virulence in a given animal
model.
Terence A. Agbor and Beth A. McCormick. Salmonella Effectors: Important players modulating host cell function during infection [Internet]. NCBI. 2011 [citado el 23 de febrero de
2022]. Disponible en: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3381885/#:~:text=Salmonella%20effectors%20mediate%20bacterial%20entry,as%20the%20intestinal%20epit

4. General objective: Identify vía quantitative
proteomic screen if annexin A2 is a host
target for Salmonella pathogenicity island-
2 effectors SopD2 and PipB2.

5. Methods
Immunofluorescence
Type of immunohistochemistry
technique that utilizes fluorophores
to visualize various cellular
antigens such as proteins.
Fluorophores are compounds that
emit light when exposed to a
certain wavelength of light. To
detect protein expression, the
biological sample of interest is
incubated with an antibody specific
to the protein of interest.
The antibody may be coupled to a
fluorophore (direct fluorescence)
or may be detected by a secondary
antibody conjugated to a
fluorophore (indirect
fluorescence).
The proteins or antigens can then
be visualized by examination under
a fluorescent microscope. This
technique is commonly used in
clinical practice as well as in
research applications to determine
protein expression and localization.
SDS PAGE
Stands for sodium dodecyl sulfate–
polyacrylamide gel electrophoresis and is
an electrophoresis method that allows protein
separation by mass.
SDS-PAGE is used in combination with
the western blot for the determination of the
presence of a specific protein in a mixture of
proteins.
In the present study, SDS-PAGE and Western blot
are used to determine the presence of SopD2 and
PipB2.
Kyuseok Im, Sergey Mareninov, M. Fernando Palma Diaz, and William H. Yong. An
introduction to Performing Immunofluorescence Staining [Internet]. NCBI. 2020 [citado el 20
de febrero de 2022]. Disponible en: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918834/
Introduction to SDS-PAGE [Internet]. Rice University. 2012 [citado el 22 de febrero de
2022]. Disponible en: https://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html

6. Methods continued…
Western Blot Mass spectrophotometry
Analytical tool useful for measuring the mass-to-charge ratio (m/z)
of one or more molecules present in a sample. It allows us to
identify a particular molecule because it gives us information about
it’s molecular mass and its components.
Brody LC. Western Blot [Internet]. National Human Genome Research Institute. [citado el 23 de febrero de 2022]. Disponible en: https://www.genome.gov/genetics-
glossary/Western-Blot#:~:text=Western%20blotting%20is%20a%20laboratory,the%20surface%20of%20a%20membrane.

7. Results
1. a, b and c:
Overexpression of the SsrB
regulator enables
identification of SPI-2
effectors during infection.

8. Results continued…
• 3. h: Representative images for select strains. White boxes
indicate zoomed-in region in inset. Arrowheads indicate
LAMP1+-tubules. Scale Bar = 10 µ
• 5. a: SopD2 targets AnxA2 in vitro.

9. Disicussion
Authors and title of study Point of view
Do the authors of the
present study agree or
disagree?
Elliott Jennings, Teresa L.M. Thurston, and
David W. Holden.
Salmonella SPI-2 Type III Secretion System
Effectors: Molecular Mechanisms And
Physiological Consequences
The authors consider that very little is still
known about the SPI2 effector proteins other
than they are vital to the pathogenicity of
Salmonella. They state that a “major
challenge is to understand how the functions
of the effectors that control vacuolar
membrane dynamics are coordinated with
each other and with the host adaptors,
regulators, and motors with which they
interact.”
Agree
Doris L. LaRock, Anu Chaudhary, and Samuel I.
Miller.
Salmonellae interactions with host processes
The authors give several examples supporting
their belief that a major function of the SPI-2
T3SS is the modification of the endosomal
membrane. Agree
George S. Niemann, Roslyn N. Brown, Jean K.
Gustin, Afke Stufkens, Afshan S. Shaikh-
Kidwai, Jie Li, Jason E. McDermott, Heather
M. Brewer, Athena Schepmoes, Richard D.
Smith, Joshua N. Adkins, and Fred Heffron.
Discovery of Novel Secreted Virulence Factors
from Salmonella enterica Serovar
Typhimurium by Proteomic Analysis of
Culture Supernatants
The authors confirmed their hypothesis that
the overexpression of plasmid encoded ssrB
might increase the probability of identifying
novel type III effectors because in their
findings, two novel effectors were exclusive
to SsrB overexpression
Agree
Jennings E, Thurston TLM, Holden DW. Salmonella SPI-2 Type III Secretion System Effectors: Molecular Mechanisms And Physiological Consequences. Cell Host Microbe. 2017 Aug 9;22(2):217-231. doi: 10.1016/j.chom.2017.07.009. PMID: 28799907.
LaRock DL, Chaudhary A, Miller SI. Salmonellae interactions with host processes. Nat Rev Microbiol. 2015 Apr;13(4):191-205. doi: 10.1038/nrmicro3420. Epub 2015 Mar 9. PMID: 25749450; PMCID: PMC5074537.
Niemann GS, Brown RN, Gustin JK, Stufkens A, Shaikh-Kidwai AS, Li J, McDermott JE, Brewer HM, Schepmoes A, Smith RD, Adkins JN, Heffron F. Discovery of novel secreted virulence factors from Salmonella enterica serovar Typhimurium by proteomic analysis of
culture supernatants. Infect Immun. 2011 Jan;79(1):33-43. doi: 10.1128/IAI.00771-10. Epub 2010 Oct 25. PMID: 20974834; PMCID: PMC3019877.

10. Conclusions
Molecular biology is a branch of biology that is concerned with
the study of biological processes from the deepest and most
elemental point of view: molecules. With that being said, its
relevance and great importance for medicine is more than
apparent.
Molecular biology has allowed us to learn about many aspects of
our genome and has provided us with numerous tools that serve
us both to carry out studies and, in the clinical scope, to make
diagnoses.
I found this study to be quite interest and valuable as it broadens
our knowledge about the pathogenic mechanisms of Salmonella,
a common disease that has the potential to be life-threatening.