Bifidobacterium adolescentis L2-32 Lipoprotein and TLR stimulation Research

Research poster that was presented at the Undergraduate Exhibition.

Background:

Lipoproteins are important globular proteins that are involved with nutrient intake, signal transduction, antibiotic resistance and much more functions. Lipoproteins are found in both gram negative and positive bacteria. Lipoproteins structures are controlled by their N-acylation structures that are controlled by a group of enzymes. Enzymes like Lgt and Lst and found in almost all bacteria. Many common lipoprotein forms are found in the triacyl form, but some are diacyl and other forms. Lipoproteins act as Pathogen Assoicated Molecular Patterns (PAMPS) which are conserved structures that can activate the innate immune system Toll-Like Receptors  2 (TLR). TLR2 will form a heterodimer will TLR1 or TLR 6 depending on the acylation form, TLR2/1 for triacyl and TLR2/6 for diacyl form. When TLR receptors become activated they cause Nf-kB to become activate which then causes cytokines to be produce and cause an immune response and inflammation to occur. Lipoproteins are important and unknown field of structural proteins that can activate the immune system.

Bifidobacterium species are gram positive, strict anaerobic bacteria that are found in the human intestines. Bifidobacterium adolescentis is a common bacteria found in the human intestines that is found to colonize the gut shorty after birth. Bifidobacterium adolescentis has been found to be a commensal microbe that produces anti-inflammatory molecules. Bifidobacterium adolescentis L2-32 has an unknown lipoprotein structure and it was unknown if it could activate the immune system.

Methods and Results:

Research began with a protein blast search of all the lipoprotein enzymes because it was important to know if the bacteria will have a similar structure. Bifidobacterium adolescentis protein blast shown only Lgt and Lst sequences, which was expected because these enzymes are found in all bacteria.

In order to confirm that the bacteria was the actual Bifidobacterium adolescentis bacteria, Polymerase Chain Reaction (PCR) was performed. PCR had to be done because the original bacteria stocks come from third party sources. Also, while working in the anaerobic chamber, the chance of contamination is higher. The bacteria samples 16 sRNA ribosomal subunit was extracted, PCR was performed and Sanger Sequencing was done. Bifidobacterium adolescentis was confirmed at a 99.57% match.

After confirmation of the bacteria species, lipoprotein extraction was performed. Lipoprotein N-terminal acylation are made of of non-polar fatty acids so a non-ionic detergent like Triton-X114 was used. Triton allows the separation of compounds by first separating polar and non-polar molecules. Further separation of non-polar compounds is achieved by using a change in temperature where the lipoproteins and other membrane protein will be separated. After extraction was achieved, two SDS gels were run. One contained a supernatant of the first wash with triton and the lipoprotein extraction. The point of this was to see if the lipoprotein bands were present if the supernatant. If they were present, it would not be a good band to extract.

The second SDS gel that was ran only contained the lipoprotein extractions. These bands were then transferred to a nitrocelluose membrane for extraction. The first extraction that was performed only contained one cell pellet, which resulted in a too small of a yield. The second attempt involved three cell pellets being combined and lipoprotein concentrations been run at different amounts on the gel. The second attempt resulted in two bands being extracted for mass spectrometry.

TLR assay was performed using HEK-BluehTLR2 cells were used. These are human embryonic kidney cells that express TLR2/1/6 receptors. Three types of Staphylococcus aureus were used. The first type was the negative control, ΔLgt which contained no lipoproteins and is expected to not activate TLR receptors. The next was ΔLnsA/B, this form is a diacyl and would activate TLR receptors but only 2/6. The last was the positive control, the wildtype form which is triacyl. This form would activate TLR receptors, but only 2/1. Bifidobacterium adolescentis was also tested to see if it activated and if it had similar activation to any of the controls. The bacteria pellets were diluted by seven-fold to get a seven different cell concentrations. After testing on the cells, Bifidobacterium adolescentis was found to activate TLR cells and had similar activation to the ΔLnsA/B diacyl form. 

Conclusions

• Bifidobacterium adolescentis L2-32 can activate TLR2 receptors and cause Nf-kB and AP-1 activation
• Bifidobacterium adolescentis has similar activation levels as the S.aureus diacyl strain.

Future Research

• Determine N-terminal acylation structure from mass spectrometry
• Determine if structure is diacyl form
• Determine if activation is TLR2/6 or TLR2/1

Video of Research Presentation:

 

What has this experience done for Kyle?

Before Kyle gained this research experience, Kyle was afraid to do research and did not have the confidence to working in a research laboratory. Now, Kyle is able to work, communicate, gain valuable critical thinking skills, learned new laboratory techniques, equipment and much more. Working on lipoprotein characterization, Kyle was able to gain interest for future research ideas and helped him decide that he wants to become an expert in the field of microbial structures and their interacts with the immune system. The research is very important to have in the microbiology field because it allowed to have experience with isolating protein structures, have laboratory techniques, use laboratory equipment, and communicate the findings.