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Page 4 of 15 Alekseeva et al. Microbiome Res Rep 2023;2:10 https://dx.doi.org/10.20517/mrr.2023.06

Figure 2. Hypothetical schematic illustration of the interaction between proteins encoded by the PFNA operon of B. longum subsp.
longum GT15 and elements of the host’s immune system. The Pkb2 signaling system is activated by an unknown ligand, which is most
likely a component of the host’s immune system (possibly TNFα). Serine-threonine protein kinase Pkb2, following its activation by a
ligand, autophosphorylated [22,23] and phosphorylated other substrates, including the MoxR-ATPase protein encoded by the aaa-atp gene
of the PFNA operon [22] . MoxR-ATPase is a chaperone that is possibly involved in the folding of proteins, including the FN3 protein. FN3 is
a secreted protein possessing a transmembrane domain that is presumably attached to the outer surface of the bacterial cell membrane.
[24]
It has been experimentally shown that the ΔFN3.1 protein is capable of selective binding of the cytokine TNFα . The strain B. longum
[27]
subsp. longum GT15 also increases the expression of TNFα, IL8, and IL10 cytokines in human cells . Apparently, TNFα interacts with an
unknown signaling system in B. longum subsp. longum GT15 (possibly, Pkb2), upregulating the expression of the PFNA operon genes,
including the fn3 gene encoding the FN3 protein [25] , which is capable of binding TNFα [24] . Figure 2 offers a putative mechanism of
bidirectional interaction between Bifidobacterium and the human immune system.
METHODS
Bacterial strains, plasmid vectors, culture media and conditions
[28] -
In our study, we used the strains: B. longum subsp. longum GT15 , Escherichia (E.) coli DH5a (F , Ф 80 Δ
-
-
lacZΔM15, Δ(lacZYA-argF), U169) (Promega, USA) and E. coli BL21(DE3) [F , dcm, ompT,hsdS(rB mB ),
[29]
-
gal λ (DE3)] (Novagen, USA) and the expression vector pET16b (Novagen, USA) containing a His-Tag
[30]
linker in the N-terminal region for protein isolation and purification.
The model strain B. longum subsp. longum GT15 was used. B. longum subsp. longum GT15 belongs to the
most studied and widespread species: B. longum. This strain was isolated from the feces of a healthy adult in
our laboratory and deposited in the Russian National Collection of Industrial Microorganisms [VKPM,
Moscow, Russia, http://eng.genetika.ru/service-offer/vkpm/; VKPM is a member of the World Federation
for Culture Collections (WFCC) and European Culture Collections’ Organization (ECCO)]. The
international catalog number in VKPM: VKPM AC-1928. The genome of B. longum subsp. longum GT15
was sequenced (status complete, NZ_CP006741.1) . We conducted RNA-Seq analysis [25,32] . This strain is
[31]