Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model
Creators: name: Nissen, Lorenzo <1977>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2008
Subjects: AGR/16 Microbiologia agraria

description: The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to
select the best Bifidobacterium longum strains suitable to set the basis of our study.
We were looking for strains with the abilities to colonize the intestinal mucosa and
with good adhesion capacities, so that we can test these strains to investigate their
ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are
the two process that we want to study to better understand the role of an adhesion
protein that we have previously identified and that have top scores homologies with
the recent serpin encoding gene identified in B. longum by Nestlè researchers.
Bifidobacterium longum is a probiotic, known for its beneficial effects to the human
gut and even for its immunomodulatory and antitumor activities. Recently, many
studies have stressed out the intimate relation between probiotic bacteria and the GIT
mucosa and their influence on human cellular homeostasis. We focused on the
apoptotic deletion of cancer cells induced by B. longum. This has been valued in
vitro, performing the incubation of three B.longum strains with enterocyte-like Caco-
2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains
tested were defined for their adhesion properties using adhesion and autoaggregation
assays. These features are considered necessary to select a probiotic strain. The three
strains named B12, B18 and B2990 resulted respectively: “strong adherent”,
“adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to
investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted
positive in DNA fragmentation test, only when adherent strains were used (B12 and
B18). These results indicate that the interaction with adherent B. longum can induce
apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the
gastrointestinal tract and in restoring the ecology of damaged colon tissues. These
results were used to keep on researching and the strains tested were used as recipient
of recombinant techniques aimed to originate new B.longum strains with enhanced
capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new
goal it was decided to clone the serpin encoding gene of B. longum, so that we can
understand its role in adhesion and apoptosis induction. Bifidobacterium longum has
immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell
line. It secretes a hypothetical eukaryotic type serpin protein, which could be
involved in this kind of deletion of damaged cells. We had previously characterised a
protein that has homologies with the hypothetical serpin of B. longum (DD087853).
In order to create Bifidobacterium serpin transformants, a B. longum cosmid library
was screened with a PCR protocol using specific primers for serpin gene. After
fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia
coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B.
longum transformation were performed and the best efficiency was obtained using
MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot
assay to detect antigens presence against anti-antitrypsin polyclonal antibody.
The best signal was produced by one starin that has been renamed B. longum BLKS
7. Our research study was aimed to generate transformants able to over express serpin
encoding gene, so that we can have the tools for a further study on bacterial apoptotic
induction of Caco-2 cell line.
After that we have originated new trasformants the next step to do was to test
transformants abilities when exposed to an intestinal cell model. In fact, this part of
the project was achieved in the Department of Biochemistry of the Medical Faculty
of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship
Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic
ability of some bacterial strains using intestinal cells from a 6 years old pig. The use
of intestinal mammalian cells is essential to study this symbiosis and a functional cell
model mimics a polarised epithelium in which enterocytes are separated by tight
junctions.
In this list of strains we have included the Bifidobacterium longum BKS7
transformant strain that we have previously originated; in order to compare its
abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight
Lactobacillus strains of different sources were co-cultured with porcine small
intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell
filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb.
plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian
coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess
reaction, H202 by tetramethylbenzidine reaction and O2
- by cytochrome C reduction.
Cytotoxic effect by crystal violet staining and induction on metabolic activity by
MTT cell proliferation assay were tested too. Transepithelial electrical resistance
(TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to
observe epithelium permeability, decrease during pathogenesis and tissue becomes
permeable to ion passive flow lowering epithelial barrier function. Probiotics can
prevent or restore increased permeability. Lastly, dot-blot was achieved against
Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI
C1 increased slightly after co-culture not affecting mitochondrial functions. No strain
was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as
measured by the release of NO2, H202 and O2
- by PoM2 and PSI C1. During coculture
TER of polarised PSI C1 was two-fold higher comparing with constant TER
(~3000 ) of untreated cells. TER raise generated by bacteria maintains a low
permeability of the epithelium. During treatment Interleukin-6 was detected in cell
supernatants at several time points, confirming immunostimulant activity. All results
were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as
controls. In conclusion we can state that both the list of putative probiotic bacteria
and our new transformant strain of B. longum are not harmful when exposed to
intestinal cells and could be selected as probiotics, because can strengthen epithelial
barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell
model. Indeed, we have found out that none of the strains tested that have good
adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains
tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we
have assayed even the capacity of producing certain citokynes that are correlated with
immune response. The detection of Interleukin-6 was assayed in all our samples,
including B.longum transformant BKS 7 strain, this result indicates that these bacteria
can induce a non specific immune response in the intestinal cells. In fact, when we
assayed the presence of Interferon-gamma in cells supernatant after bacterial
exposure, we have no positive signals, that means that there is no activation of a
specific immune response, thus confirming that these bacteria are not recognize as
pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The
most important result is the measure of Trans Epithelial Electric Resistance that have
shown how the intestinal barrier function get strengthen when cells are exposed to
bacteria, due to a reduction of the epithelium permeability. We have now a new strain
of B. longum that will be used for further studies above the mechanism of apoptotic
induction to “damaged cells” and above the process of “restoring ecology”. This
strain will be the basis to originate new transformant strains for Serpin encoding gene
that must have better performance and shall be used one day even in clinical cases as
in “gene therapy” for cancer treatment and prevention.