Page 46 - Read Online

Alekseeva et al. Microbiome Res Rep 2023;2:10 https://dx.doi.org/10.20517/mrr.2023.06 Page 13 of 15

participates in binding to cytokines and other elements of the immune system. In all analyzed groups of
bacteria, FN3 domains seemed to participate in cell adhesion to one or another substrate. In our case, we are
particularly interested in the proteins’ capability to interact with the components of the host immune
system. For this reason, we focused on proteins containing FN3 domains located in the C-terminal end of
the protein and containing MCRs.

MCRs were found in FN3 domains of the GH family 31 in the families Lactobacillaceae and Bacteroidaceae,
in FN3 domains of the GH family 43 in Bacteroidaceae, in FN3 domains of the GH family 18, and in FN3
domains of the GH family 20b in Clostridioides. Analysis of proteins identified in bacteria of the families
Lactobacillaceae, Bacteroidaceae, and Clostridioides revealed that only FN3 domains containing annotated
MCRs exhibited sequence homology with FN3 domains of human proteins [Supplementary Table 4].
Sequences of FN3 domains that lacked MCRs exhibited no homology with FN3 domains of eukaryotic
proteins. This may indicate the possible involvement of proteins harboring MCRs in interaction with
components of the human immune system, namely, by binding to cytokines.

Only the complete ∆FN3.1 protein of B. longum subsp. longum GT15 can selectively bind to TNFα. FN3
domains alone are not sufficient for binding to TNFα, a C-terminal region is also required. Analysis of 3D
models of the 2D FN3, CD FN3, and ΔFN3.1 proteins showed that only the ΔFN3.1 protein is potentially
capable of forming a pocket, apparently providing TNFα binding. Bifidobacteria have two types of
fibronectin domains - FN3 domains with MCRs, encoded by the PFNA operon, and FN3 domains without
MCRs, which can be found in the GHs family 3. Bioinformatics analysis of the genomes of bacteria of the
families Lactobacillaceae, Bacteroidaceae, and Clostridioides allowed us to identify proteins containing single
FN3 domains. Two groups of FN3 domains were identified in the detected proteins: those containing MCRs
and those not having MCRs. Comparative analysis showed that only FN3 domains containing MCRs exhibit
homology with FN3 domains of human proteins.

DECLARATIONS
Authors’ contributions
Made substantial contributions to the conception and design of the study as well as provided administrative,
technical, and material support: Danilenko VN
Made substantial contributions to the conception and design of the study and carrying out in vitro and in
silica investigation: Alekseeva MG, Dyakov IN
Made substantial contributions to carrying out the experiments: Mavletova DA, Bushkova KK,
Chernyshova IC, Masalitin IA
Made substantial contributions to writing, reviewing and editing the article: Danilenko VN, Alekseeva MG,
Nezametdinova VZ, Koshenko TA, Yunes RA

Availability of data and materials
Not applicable.

Financial support and sponsorship
This work was performed as part of the State Task (no. 0092-2022-003), the topic “Mechanisms of Genetic
Processes in Microorganisms, Plants, Animals, and Humans”: “Human Intestinal Microbiome:
Immunomodulatory and Antioxidant Potential”.

Conflicts of interest
All authors declared that there are no conflicts of interest.