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Alekseeva et al. Microbiome Res Rep 2023;2:10 https://dx.doi.org/10.20517/mrr.2023.06 Page 3 of 15

Figure 1. Diagram illustrating the PFNA operon of B. longum subsp. longum GT15, and the FN3 protein and its fragments ( FN3.1 protein,
2D FN3, CD FN3) used in our experiments aimed at testing the protein’s ability to bind the cytokine TNFα. The genes of the PFNA
operon are pkb2 (serine-threonine protein kinase Pkb2, BLGT_RS02820, AIW43409.1), fn3 (fibronectin type III do-main-containing
protein, BLGT_RS02815, AIW43408.1), aaa-atp (AAA-ATPase MoxR, BLGT_RS02810, AIW43407.1), duf58 (hypothetical protein with
DUF58 domain, BLGT_RS02805, AIW43406.1), tgm (transglutaminase, BLGT_RS02800, AIW43405.1), prpC (protein phosphatase,
BLGT_RS02795, AIW43404.1), hypothetical protein (BLGT_RS02790, AIW43403.1), fha (FHA do-main-containing protein,
BLGT_RS02785, AIW43402.1). SP: signal peptide; TM: transmembrane.

the MoxR Proper subfamily encoded by the aaa-atp gene is a chaperone that is actively phosphorylated by
the protein kinase Pkb2 . It was shown that the cultivation of the strain B. longum subsp. longum GT15 in
[22]
the presence of TNFα leads to a significant increase in the expression of genes making up the PFNA
operon .
[25]
Previously, we proposed a hypothetical scheme of interaction between proteins encoded by the PFNA
operon and the host immune system [Figure 2] [22,26] .

The present work is part of the study of the mechanism of interaction between Bifidobacterium and
elements of the host’s immune system and the role of the ∆FN3.1 protein fragment encoded by the unique
species-specific PFNA operon in this process. The most important part of the presented study is devoted to
determining the ability of fragments of the ∆FN3.1 protein [Figure 1] separately, namely the two FN3
domains (2D FN3) and the C-terminal domain (CD FN3), to bind the tumor necrosis factor TNFα. The
spatial structures of the studied protein fragments were also predicted to identify potential cytokine-binding
regions. In this study, we performed a bioinformatic analysis of the presence of FN3 domains in proteins of
bacteria of the genus Bifidobacterium, as well as the families Lactobacillaceae, Bacteroidaceae, and
Clostridioides, which are typical inhabitants of the human intestinal microbiota in order to detect proteins
potentially capable of interacting with elements of the host’s immune system.