Metabolomic Responses of Yersinia Enterocolitica Under Acid Stress

Survival of bacteria in acidic environments is critical for pathogens to successfully multiply and infect the gastrointestinal tract. In this study, a metabolomics approach was conducted to understand and explain the effect of pH-induced stress metabolites in Yersinia enterocolitica . Cells were cultured in tryptic soy broth with or without pH adjustment using HCl (pH 6.8 or pH 3, respectively). In this study, we employed untargeted proton nuclear magnetic resonance (1H-NMR) spectroscopy followed by statistical multivariate analysis was used to identify the metabolites and measure the metabolic profiles and pathways impacted by acid stress. Under conditions of low pH, increased branched-chain amino acid biosynthesis and protein acetylation were predominantly observed. There was also evidence of osmotic dysregulation during acid challenge. Modulation of these systems might confer bacteria with resistance to stressors encountered during infection of organisms or food contamination. The study presented here with other related researches may help to explain further, how pathogenic bacteria survive and tolerate the environmental stresses. Metabolomic Responses of Yersinia Enterocolitica Under Acid Stress.


Introduction
Foodborne pathogens such as Salmonella Typhimurium, Escherichia coli, Yersinia and other enteric bacteria must overcome several environmental stressors and barriers involved in food production, storage, and host defenses [1]. The enteropathogenic Yersiniae, Y. pseudotuberculosis and Y. enterocolitica, are fecaloral pathogens known to cause a range of invasive gastrointestinal diseases. Furthermore, the bacteria are known for their ability to grow at low temperatures and in acidic environments [2,3].
It has been reported that the pathogenic Yersinia species have also been studied as a model bacterium to understand bacteriahost cell interactions and active pathogen evolution [4][5][6]. Many foodborne pathogens have developed survival systems that protect them against acidic environments in the stomach, but how these pathogens survive in acidic conditions remains largely unknown [7].
Although transcriptomic and proteomic studies have provided insights into the stress responses of Yersinia, limited metabolomics data exists for some strains of this group [8]. There is a tremendous improvement in food safety and quality research, and application of metabolomics at a holistic overview of the food system, thus improving the overall objectives of ensuring food safety and quality [9]. The high-throughput technique of metabolomics is one of the most recently introduced 'omics' that aims to analyze metabolites in a given biological sample [10,11]. Applications of metabolomics include the study of small molecules in a biological system that participates in the metabolic reactions responsible for cell growth, survival, and other normal cellular functions [12][13][14]. The two leading analytical approaches to metabolomics are mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy.
NMR data are highly reproducible and quantitative over a wide dynamic range and are unmatched for determining structures of unknowns [15]. NMR spectroscopy is increasingly renowned for its efficacy, non-invasiveness (non-destructive), throughput and linearity [16]. Moreover, such spectroscopy also provides structural, chemical-kinetics and other information in multidimensional applications [17]. NMR spectroscopy-based metabolomics have been used to study a wide range of biological systems such as tissues [18,19], biofluids [20], mammalian cell cultures [21], plants [22], and bacteria [23,24]. Currently, NMR application has been broadly used and reported for untargeted metabolite profiling of complex food mixtures to address issues of food safety and quality [25,26].
Additionally, a novel NMR-based metabolomics strategy for rapid discrimination and identification of several bacterial species that relies on untargeted metabolicprofiling of supernatants from bacterial culture media [27]. The overall objective of this paper is to investigate the potential of proton nuclear magnetic resonance (1H-NMR)-based metabolomics for the characterization and assessment of Yersinia responses mechanisms to acid stress. Currently, there are limited reports on metabolomic studies examining Yersinia enterocolitica exposure to different environmental stressors including low pH levels. Therefore, in this study, the potential of 1H-NMR metabolomics approach to determine stress responses to acidity in Yersinia was examined to provide in-depth knowledge and application of how foodborne pathogens tolerate the external stresses.

Sampling and Metabolite Extraction
The metabolite extraction was performed as previously described [28] with slight modification. Briefly, after 3 h of incubation period, the culture was placed on ice and centrifuged seconds. The sonicated suspension was then centrifuged at 17,949 x g for 10min at room temperature to remove cellular debris.
The supernatant was collected and dried down using a vacuum centrifuge overnight at 30°C. The dry extract was then stored at -80°C until NMR analysis.

H-NMR Data Acquisition
Before the analysis, cell extract was reconstituted in 700μl of

Pre-processing and Data Analysis
The residual H 2 O 1H-NMR resonance between 4.6 and 5.0 ppm was excluded from analyses along with the extraction solvent peak (methanol, 3.35ppm). Each spectrum was referenced (aligned) and normalized to the TSP reference peak at 0.0ppm. Spectra were then stacked and binned from 0.5 to 10ppm. A

Results
Metabolic profiling of acid stressed and non-stressed Y.
enterocolitica revealed a few changes to the metabolome. Figure   1 shows a comparison of the representative spectral profiles for Y. enterocolitica cultures grown at neutral pH (6.8)     Ideal models will have R2 and Q2 values near 1, although in biological systems this is rarely observed due to their variability.
In general, a Q2 > 0.5 defined from the metabolomics data is considered good (Figure 4).     Figure 7: Impact of metabolic pathways for Y. enterocolitica cultures after exposure to acidic conditions (3-pH) as detected by the bioinformatics software, MetaboAnalyst 4.0 (Escherichia coli K-12 MG1655 metabolism from KEGG pathway database was used as a reference). Circle sizes indicate relative pathway impact on the organism's overall metabolism, while circle color represents relative significance of the pathway to metabolite changes found in the study.

Discussion and Conclusion
Bacteria sense a variety of physical and chemical signals in their environment and use this information to coordinate an adaptive response that promotes their growth and survival. These signals include nutrients such as sugars, lipids, metals, and amino acids, and their depletion triggers upregulation of high-affinity nutrient acquisition systems and/or biosynthesis pathways [30].
Gastrointestinal bacterial pathogens evolved elaborate mechanisms to cope with excursions into acidic environments. Acid survival mechanisms are remarkably different among different pathogens.
To cope with different degrees of acidic environmental stress, several acid survival systems have evolved in enteric bacteria [31,32]. The two gastrointestinal pathogens, Y. pseudotuberculosis and Y. enterocolitica, transmit to humans after the ingestion of contaminated water or food.
Like many foodborne pathogens, they have developed different survival mechanisms that protect them against acidic conditions for successful colonization and infection [31,32]. Metabolomics is on the verge of potentially making major impacts including in the areas of foodomics. Previous studies have characterized the metabolic response of different foodborne pathogens in acid environment bacteria [33][34][35]. It has been suggested that altered metabolites or metabolomics profiles could predict specific metabolic diseases with high accuracy and help to understand related fundamental mechanisms as well as affected metabolic pathways [36]. Untargeted metabolomics measures the levels of thousands of metabolite features in a single analysis, providing a snapshot of metabolism at the systems.
Several visualization techniques have become common in global metabolomic data analysis such as scores and loadings plots, heat maps, scatter plots, volcano plots and recently designed cloud plots level [37][38][39]. Metabolic analysis reveals evidence for branched chain amino acid catabolism crosstalk and the potential for improved treatment of organic aciduria. Deciphering regulatory circuits that rely on global metabolites enables intuitive understanding and monitoring of cellular decision-making processes [40] results showed that under conditions of low pH, increased branched-chain amino acid biosynthesis protein acetylation were predominantly observed. The paradigm for acetylation in bacteria involves the acetylation of acetyl-CoA synthetase, whose activity must be tightly regulated to maintain energy charge homeostasis [41].
The branched-chain amino acids represent important nutrients in bacterial physiology, with roles that range from supporting protein synthesis to signaling and fine-tuning the adaptation to amino acid starvation [9]. Leucine, isoleucine, and valine are essential amino acids termed branched-chain amino acids (BCAA) due to its aliphatic sidechain. In several pathological and physiological conditions, increased BCAA plasma concentrations have been described [42]. Bacteria synthesize BCAAs through a conserved pathway that is present in fungi and plants, but absent in mammals [43]. Thus, the importance of BCAAs for bacterial physiology stems from their integration with central metabolism, their requirement for protein synthesis, and their requirement for environmental adaptation via Branched-Chain Fatty Acids (BCFAs) synthesis in Gram-positive bacteria [44]. The role of carbohydrate metabolism in acid survival of enteric bacteria remains largely unknown [45].
More is known about the important connections between amino acid metabolism and acid survival in bacteria. Several acid resistance systems, such as glutamate, arginine, or lysine-dependent, have been described in E. coli [46]. In alignment to our results, it has been reported that the enzyme aspartate Ammonia Lyase Or Aspartase (AspA), which is involved in aspartate metabolism by catalyzing the deamination of L-aspartate to form fumarate and ammonia, plays a role in acid survival in Y. pseudotuberculosis [10]. AspA increases acid survivability of bacteria by producing ammonia from aspartate as demonstrated by mutational and in vitro enzyme activity studies.
Interestingly, this aspartate dependent acid survival pathway appears to exist in Y. enterocolitica and other foodborne pathogenic bacteria including E. coli O157:H7 and Salmonella enterica, given that the addition of aspartate into culture media alsoincreases their survivability at low pH [10].
The decrease level of aspartic acid and phenylalanine in our results may have been a consequence of both reduced viability and growth after 3 hours of exposure to an acidic environment. We also observed a significant decrease in the osmoprotectant compound betaine from the VIP scores plot. It has been previously indicated that the accumulation of betaine increases the volume of water in E. coli and increases the growth rate of osmotically stressed cells [47].
Likewise, exposure to lethal levels of pH seemed to have resulted in dysregulation of osmotic homeostasis of Yersinia from our study.
In some previous report, Glutamate has been indicated to play a central role in a wide range of metabolic processes in bacterial cells. The Glutamate Decarboxylase (GAD) system has been implicated in acid tolerance in several bacterial genera. This system facilitates intracellular pH homoeostasis by consuming protons in a decarboxylation reaction that produces γ-aminobutyrate (GABA) from glutamate [48].
Acid exposure induces specific metabolite accumulation in bacterial cells. There are reports of increased valine and isoleucine levels in Streptococcus mutans exposed to acidic conditions [49].
They showed that upregulation of biosynthetic genes such as ilvC and ilvE occurred under acid stress conditions. Additionally, these amino acids can be used as an energy source for bacteria, and they are precursors of branched-chain fatty acids, predominant membrane fatty acids [44]. Moreover, significant changes observed for branched-chain amino acids in this study suggest acid-related stress responses of Y. enterocolitica as key co-regulators of virulence. In addition to branched-chain amino acid metabolism, protein acetylation increased in response to acid stress after 3 hours. Protein acetylation is a widely used protein modification in prokaryotic organisms. Certain acetylases in E. coli and Salmonella have been found to be involved in basic physiological processes, such as primary metabolism, chemotaxis, and stress responses [50].
Protein acetylation impairment in cobB and yfiQ mutants greatly decreased bacterial tolerance to cold, hot, high-salt, and acidic environments for Y. pestis [50]. In alignment to this report, Y. enterocolitica may also upregulate the acetylation of proteins in response to low pH environments. Foodborne pathogens like Yersinia enterocolitica may encounter a wide range of pH environments as the pathogen pass through the oral cavity, gastric transit, and its survival and growth in food matrices. In conclusion, 1H-NMR