Cellular microbiota: an inherent inhabitant of cells
Authors: sun xiaowei , zhang hua , zhang xiao , gao wenmin , zhou caiyun , kou xuanxuan , deng jingxin , zhang Jiangang 
Published: October 2022

Aim
The cell is the basic unit of life. It is composed of organelles and various organic and inorganic biomolecules. Recent 16S rRNA gene sequencing studies have revealed the existence of tissue microbiota, in both tumor tissues and normal tissues. Recently, we found that liver microbiota resided in hepatocytes. Here, we further report on cellular microbiota in parenchymal cells of visceral organs as inherent inhabitants.
Methods
The 16S rRNA gene sequencing was performed on visceral organs of male adult Sprague-Dawley (SD) rats (n = 6), pregnant rats (n = 6), newborn rats (n = 6, from the same litter), and fetuses and placentas (n = 6). Fluorescence in situ hybridization and immunofluorescence were performed in situ in visceral organs of male adult SD rats. Western blotting was performed on nuclear and cytoplasmic extractions of visceral organs of SD rats and cell lines HepG2, Huh-7, Hepa1-6, and HSC-T6.
Results
(1) A high abundance of 16S rRNA gene were detected in the visceral organs of male adult, pregnant, newborn, and fetal rats as well as their placentas. (2) In male adult rats, (i) the number of operational taxonomic units (OTUs) of visceral bacteria (1432.00 ± 39.87) was higher than that of the feces and ileum bacteria (1009.00 ± 5.66), P < 0.05. Nearly all the OTUs were shared across the organs and intestinal contents but the dominant bacteria varied. The alpha diversity of the visceral microbiota was significantly higher than that of the intestinal microbiota, P < 0.05. The similarity of visceral bacteria was significantly higher than that of the visceral organs to the intestine or the distance between intestinal microbiota, P < 0.05. (ii) Among 613 annotated genera visceral bacteria, 404 types of bacteria were shared across organs, including the top 10 abundant bacteria. Other bacteria were shared between a subset of organs, and very few bacteria were exclusive to only one organ. In an individual, about 15–40% (23.23% ± 7.91%) of visceral bacteria were shared and 8–11% (9.56% ± 1.37%) were exclusive to a specific organ. In each type of organ, the abundance of dominant bacteria varied among individuals. Each organ had exclusive bacteria with a low abundance (from 1 to 672). (iii) According to a KEGG analysis, the functional composition of visceral bacteria genomes were the same but differed from that of gut bacteria. (3) Bacterial 16S rRNA, LPS, and LTA were found in the parenchymal cells of visceral organs and in HepG2, Huh-7, HSC-T6, and Hepa1-6 cells. LPS consistently appeared in the nucleus of cells, while LTA was mainly found in the cytoplasm. (4) The visceral bacteria of newborn rats were shared with bacteria of skin tissue and maternal milk clots. The species richness of skin tissue and milk clots were the same, but were higher than that of intestinal tissues (including the contents), visceral organs, and skeletal muscle, P < 0.05. The species evenness of skin tissue was higher than that of the visceral organs and skeletal muscle. (5) The fetuses and placentas showed nearly the same species richness and evenness as the visceral organs of pregnant rats, but the distribution in the fetuses and placentas differed.
Conclusions
Cellular microbiota are the intrinsic components of cells. Gram-negative bacteria are located in the nucleus, and gram-positive bacteria are located in the cytoplasm. This differs from the gut microbiota and may be inherited.