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Page 12 of 15 Alekseeva et al. Microbiome Res Rep 2023;2:10 https://dx.doi.org/10.20517/mrr.2023.06

To date, there is no consensus on how FN3 domains originated. It is likely that bacterial FN3 domains were
acquired from animals and subsequently spread through horizontal transfer between closely related
[35]
bacteria . However, other studies have suggested that FN3 domains of eukaryotic and archaeal proteins
were acquired initially from bacteria [4,36] .

Of all the bacterial taxa that we studied, only bifidobacteria harbored proteins of the GH family 3 (beta-
glucosidase). In the human body, beta-glucosidase plays an important role in the degradation of
glycosphingolipids, which constitute a major part of the cell membrane of eukaryotic cells. It is noteworthy
that bifidobacteria [Supplementary Table 1] contained only GH family 3, in which FN3 domains lacked
MCRs. Of all the bacterial taxa that we analyzed, GH family 3 (beta-glucosidase) proteins were only present
in bifidobacteria. Thus, bifidobacteria, some of the Earth’s oldest inhabitants as well as of anaerobic body
cavities of animals, have evolved two types of domains in proteins: (1) FN3 domains encoded by one of the
genes of the PFNA operon, which are involved in the interaction with the host’s immune system; (2) FN3
domains embedded in proteins of the GH family 3 capable of cleaving glycosphingolipids (components of
the cytoplasmic membrane of human cells), thereby participating in intercellular interactions. Interestingly,
strains of the B. dentium species living in various teeth cavities of humans possess 12 different GH family 3,
which are capable of cleaving cellobiose and salicin . Bifidobacteria represent a unique model organism for
[37]
studying the role of FN3 domains in the interaction with components of the immune system (PFNA
operon) and adaptation to environmental niches (GH family 3).

Comparative analysis of the FN3 domains of GH family 3 and the FN3 domain of the protein encoded by
the fn3 gene of the PFNA operon did not reveal any sequence homology between them, which may be
grounded in their different evolutionary origins and their involvement in completely different functions: on
the one hand, adaptation and usage of various carbohydrate sources abundant in the intestinal microbiota;
on the other hand, adaptation to the interaction with components of the host immune system. It is possible
that the first group of FN3 domains in hydrolases first emerged in bacteria and then was passed on to
eukaryotes, while the second group of FN3 domains (harboring MCRs), such as the one found in the FN3
protein encoded by the fn3 gene of the bifidobacterial PFNA operon may have been acquired by
bifidobacteria from eukaryotes [4,29,30] . It is important to note that the PFNA operon in different species of
bifidobacteria is localized in different regions of the genome and possibly originated from a common
ancestor. The spread of the operon and the ability of the proteins encoded by its genes to interact with the
immune system of the host and promote the adaptation of bifidobacteria to various ecological niches led to
a high divergence of the operon genes and, possibly, contributed to the speciation of bifidobacteria, the
most ancient inhabitants of the Earth and the microbiota of animals.

Elucidation of how “feedback loops” between the human body and commensal microorganisms operate will
open up numerous possibilities for regulating the composition and activity of the human microflora, as well
as modulating the human immune system.

There is a great interest in the functional role of fibronectin domains containing MCRs in proteins in other
taxa of the gut microbiota inhabitants: Lactobacillacea, Bacteroidaceae, and Clostridioides. The possible
involvement of such FN3 proteins in the interaction of bacteria with components of the host immune
system is an interesting research direction. Type 3 fibronectin domains were found in the families of
Lactobaccilacea, Bacteroidaceae, and Clostridioides, mainly in glycoside hydrolases of various families (18,
20, 20b, 31, 43, 65, 92). Depending on the class of GH, FN3 domains were localized in the central part, N-
terminal or C-terminal end of the protein [Supplementary Tables 1 and 2]. The location of the FN3 domain
and the presence of MCRs may be important indicators of the protein’s function, namely, whether it