Vibrio vulnificus is a Gram-negative marine bacterium that is known as a significant
opportunistic human pathogen. V. vulnificus mediated food-borne diseases are normally occurred by ingestion of the contaminated oyster and seafood. Typically, V. vulnificus entered the small intestine and invades the bloodstream causing severe septicemia (Gulig et al., 2005).
The extracellular secreted mucus and the cell surface glycocalyx prevent infection by the vast numbers of microorganisms that live in the healthy gut. Mucin glycoproteins are the major component of these barriers. However, successful enteric pathogens have evolved strategies to circumvent these barriers (McGuckin et al., 2011). The mucous layer contains mucins that are highly glycosylated
polymorphic glycoproteins and composes with water, salts, lipid related compounds (Allen, 1981). To be a successful pathogen, V. vulnificus must be able to use nutrients existed in the intestinal mucus layer. Mucin utilizing by V. vulnificus must be important step for the first stage of infection. Therefore, it is very significant to understand the V. vulnificus metabolism when they utilize mucin as a nutrient. I tried to provide insight into mucin utilization of V. vulnificus in order to find out control target for V. vulnificus initial stage of infection.
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The metabolome is much more sensitive to perturbations in the environment than either the transcriptome or the proteome because the metabolites within a cell are an integrated reflection of the cellular phenotype (Sellick et al., 2009). However, different from the other ‘omics’, metabolomics includes methodological problems derived from heterogeneity in chemical properties (Ohashi et al., 2008). In this reason, metabolome sampling is one of the most important factors determine the quality of metabolomics data (Kim et al., 2013). Here, I used cold methanol quenching method for in vitro experiment (Fig. 2) and fast filtration method for ex vivo experiment (Fig. 8) respectively. Even though when cold methanol quenching
was applied, serious losses of intracellular metabolites due to cell leakage were observed in bacteria (Shin et al., 2010b), still this method is widely used for Gram-negative and Gram-positive bacterial quenching. In bacteria, as an alternative to cold methanol quenching, the fast filtration method was developed and shown to be effective in minimizing the losses of intracellular metabolites both in Gram-negative and Gram-positive bacteria (Bolten et al., 2007). In the case of in vitro experiment, mucin polymer stuck the membrane filter that hindered the filtration thus that experiment was conducted by cold methanol quenching.
From the in vitro experiment, it was verified that V. vulnificus survived and grew using porcine gastric mucin as the sole source of nutrient (Fig. 1). Also, the principal component analysis revealed clear separations between the metabolite profiles of cells grown with mucin and glucose (Fig. 3). The discriminative metabolites between each conditions were 13 lipid related compounds, 8 amine
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related compounds, 17 amino acids, 8 sugars and 4 organic acids (Fig. 4).
Glycolysis intermediates such as glucose-6-p and fructose-6-p were decreased in the cells grown with mucin. However, most of metabolites were highly detected in the V. vulnificus metabolized mucin. Especially, silaic acid (Neu5Ac) pathway intermediates such as ManNAc-6-P and GlcNAc-6-P were more detected (Fig. 5).
N-acetylneuraminate lyase (NanA) initiates the catabolism of Neu5Ac by cleaving it into pyruvate and N-acetylmannosamine (ManNAc) in most bacteria ( Almagro-Moreno and Boyd, 2009). The nanA mutant was defective for intestinal colonization and significantly diminished in virulence in a mouse model (Jeong et al., 2009).
These results suggested that the catabolic utilization of Neu5Ac is essential for the pathogenesis of the bacteria by ensuring growth and survival during infection.
Therefore, this pathway related genes might be the important control target to V.
vulnificus initial infection.
In the experiment of ex vivo, V. vulnificus survival and growth ability when infected to mucin secreting human carcinoma HT29-MTX cell line were confirmed (Fig. 7).
It was known theat the interactions between enteric pathogens and mucins, and the mechanisms that these pathogens use to disrupt and avoid mucosal barriers (McGuckin et al., 2011). In addition to the principal component analysis showed clear separations between the metabolites between V. vulnificus infecting HT29-MTX which means mucin metabolized bacteria and medium control (Fig. 9). The discriminative metabolites between each conditions were including 3 lipid related
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compounds, 1 amine related compounds, 8 amino acids, 3 sugars and 1 organic acids (Fig. 10).
Analysis of both experiment collectively, the amounts of amino acids, fatty acids and TCA cycle intermediates were increased in V. vulnificus grown with mucin and infecting V. vulnificus. These suggest that V. vulnificus preferentially activates the catabolisms of amino acids and fatty acids to produce TCA cycle intermediates in the utilization of mucin. The major fatty acid species existing in the V. vulnificus are palmitic acid (16:0), palmitoleic acid (16:1), oleic acid (18:1). Those fatty acids increased in the result from mucin metabolism. The primary role of bacterial fatty acid is to act as the hydrophobic component of the membrane lipids and as components of storage lipids (Cronan and Thomas. 2009). The envelope plays a crucial role in facilitating response to environmental changes (Linder and Oliver.
1989). In this aspects, increased amounts of fatty acid by metabolizing mucin means important factors to stabilize cell membrane for V. vulnificus.
If we can prevent the metabolic pathway related to mucin utilization, we can control the V. vulnificus initial stage of infection. This study is expected to give important clues in understanding the initial infection stage of V. vulnificus metabolism by using mucin. However, it should be additionally studied deeper pathway analysis about each metabolites highly detected in the mucin utilizing V. vulnificus. Also, it would be better to try systematical interpretation with transcriptomic analysis in
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order to figure out more concise understanding of the state of V. vulnificus at initial infection stage.
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차이는 보이는 물질을 질량분석기 데이터 베이스와 표준물질의 크로마토그램 결과를 통해 확인한 결과, 다양한 지방산, 아미노산, TCA 회로의 중간 물질, 시알산 분해 경로의 중간 물질 등이 뮤신을 대사한 결과 증가함을 확인하였다. 이를 통해 초기감염 시, 패혈증 비브리오균이 뮤신을 이용하여 대사함을 확인하였고, 이를 통해 활성화 되는 대사경로 역시 알 수 있었다. 뮤신 이용 시 활성화되는 대사 경로를 비브리오 균을 제어하는 타겟으로 삼아, 비브리오 균의 초기감염을 막는 저해제를 찾을 수 있을 것이다.