Abstract
Porphyromonas gingivalis, a keystone periodontal pathogen, has emerged as a risk factor for systemic chronic diseases, including non-alcoholic fatty liver disease (NAFLD). To clarify the mechanism by which this pathogen induces such diseases, we simultaneously analyzed the transcriptome of intracellular P. gingivalis and infected host cells via dual RNA sequencing. Pathway analysis was also performed to determine the differentially expressed genes in the infected cells. Further, the infection-induced notable expression of P. gingivalis livk and livh genes, which participate in branched-chain amino acid (BCAA) transfer, was also analyzed. Furthermore, given that the results of recent studies have associated NAFLD progression with elevated serum BCAA levels, which reportedly, are upregulated by P. gingivalis, we hypothesized that this pathogen may induce increases in serum BCAA levels and exacerbate liver injury via livh/livk. To verify this hypothesis, we constructed P. gingivalis livh/livk-deficient strains (Δlivk, Δlivh) and established a high-fat diet (HFD)-fed murine model infected with P. gingivalis. Thereafter, the kinetic growth and exopolysaccharide (EPS) production rates as well as the invasion efficiency and in vivo colonization of the mutant strains were compared with those of the parental strain. The serum BCAA and fasting glucose levels of the mice infected with either the wild-type or mutant strains, as well as their liver function were also further investigated. It was observed that P. gingivalis infection enhanced serum BCAA levels and aggravated liver injury in the HFD-fed mice. Additionally, livh deletion had no effect on bacterial growth, EPS production, invasion efficiency, and in vivo colonization, whereas the Δlivk strain showed a slight decrease in invasion efficiency and in vivo colonization. More importantly, however, both the Δlivk and Δlivh strains showed impaired ability to upregulate serum BCAA levels or exacerbate liver injury in HFD-fed mice. Overall, these results suggested that P. gingivalis possibly aggravates NAFLD progression in HFD-fed mice by increasing serum BCAA levels, and this effect showed dependency on the bacterial BCAA transport system.
Highlights
Non-alcoholic fatty liver disease (NAFLD), with a prevalence of approximately 25%, is one of the most common liver diseases (Younossi et al, 2018), and reportedly, it has as one of its pathological features, the excessive accumulation of triglyceride in the liver, owing to metabolic alterations (Zhang et al, 2018a)
Further analysis of the expression levels of PGN_RS06330 (BCAA ATPbinding cassette (ABC) transporter permease, livh) and PGN_RS06345 (ABC-type Branched-chain amino acids (BCAAs) transport system periplasmic component, livk) via Quantitative Reverse Transcription PCR (qRT-PCR) confirmed that the expression levels of livh and livk increased significantly in intracellular P. gingivalis (Figure 1D)
It was observed that P. gingivalis traA, traI, traG, traN, traJ, traO, traQ, traM, and traP genes were significantly upregulated in human umbilical vein endothelial cells (HUVEC) compared with levels in the culture medium (Table S3)
Summary
Non-alcoholic fatty liver disease (NAFLD), with a prevalence of approximately 25%, is one of the most common liver diseases (Younossi et al, 2018), and reportedly, it has as one of its pathological features, the excessive accumulation of triglyceride in the liver, owing to metabolic alterations (Zhang et al, 2018a). It has been observed that this disease can develop into reversible steatosis and non-alcoholic steatohepatitis (NASH), which is a more serious form of NAFLD that shows potential progression to liver cirrhosis or cancer (Friedman et al, 2018). It has been suggested that several potential risk factors, such as diabetes mellitus and obesity, can lead to its aggravation (Buzzetti et al, 2016; Friedman et al, 2018). P. gingivalis possesses a plurality of virulence factors that invade periodontal tissues and subsequently enter blood circulation and disseminate into the whole body, increasing the risk of several systemic diseases, notably diabetes, cardiovascular diseases, rheumatoid arthritis, Alzheimer’s disease, and NAFLD (Potempa et al, 2017; Nakahara et al, 2018; Mei et al, 2020; Fitzsimonds et al, 2021). Previous studies have shown that P. gingivalis infection aggravates liver inflammation and fibrosis in NASH mouse
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