Cold Tolerance Regulated by the Pyruvate Metabolism in Vibrio parahaemolyticus

Vibrio parahaemolyticus is a common foodborne pathogen found in seafood, and represents a major threat to human health worldwide. Low-temperature storage is an important seafood processing method, but is not sufficient to completely eliminate the bacteria and avoid foodborne illness. To determine the mechanisms behind such cold tolerance, RNA-seq and iTRAQ analyses were first performed to obtain the global transcriptomic and proteomic patterns of frozen squid and clinical V. parahaemolyticus isolates under cold conditions. The integrated analysis revealed the modulation of multiple pathways such as the co-occurrence of down-regulated pyruvate metabolism and up-regulated fatty acid biosynthesis, which likely contribute to V. parahaemolyticus cold tolerance. Furthermore, we found that increasing concentrations of pyruvate can reduce the fatty acid content to influence V. parahaemolyticus growth in cold conditions. Thus, regulation of pyruvate concentration may be an effective method to control this seafood-borne pathogen.


INTRODUCTION
The causative agent of diarrheal disease, Vibrio parahaemolyticus, is the major pathogen responsible for seafood associated gastroenteritis in humans worldwide (Samoǐlenko et al., 2003). Seafood is very popular in China, and its higher consumption is correlated with an increase in the overall living standard of the population. However, this high rate of seafood consumption is also accompanied by high rates of foodborne illness. In the city of Sanya, China alone, there were 29 outbreaks caused by V. parahaemolyticus resulting in 499 illnesses from 2010 to 2016, accounting for about half of all cases of microbiological food poisoning (Deng et al., 2017). Raw seafood is generally subject to lowtemperature storage as an important food preservation method, which can effectively limit bacterial growth and metabolism (Graumann and Marahiel, 1996). However, V. parahaemolyticus has still been isolated from seafood that has gone through the low-temperature treatment to ultimately cause foodborne illness (Wang et al., 2016) suggesting a specific mechanism of cold tolerance that has yet to be elucidated.
Previous studies have indicated various adaptations of microorganisms to low temperature, including reduced affinity of enzymes for their substrates, decreased thermal energy and reaction rates, and increased aqueous viscosity (D'Amico et al., 2006). Complex cold shock responses, including cold shock protein (CSP) production, DNA supercoiling modifications, and membrane fluidity maintenance, can help a bacterium survive under detrimental cold conditions (Horn et al., 2007). Nevertheless, different microorganisms have evolved unique strategies in response to low temperatures. For example, unsaturated fatty acids were shown to be effective coldtolerant factors in Pseudomonas sp. (Garba et al., 2016). Another four proteins (ATP-dependent, ClpP, pyruvate kinase and a putative glycoprotein endopeptidase) appear to play important roles in cold adaptation for Lactobacillus acidophilus. Despite this basic information on bacterial cold adaptation, the complete strategies, pathways, and signals that compensate for low-temperature metabolism in V. parahaemolyticus remain poorly understood (Amato and Christner, 2009).
High-throughput sequencing technology has now made it possible to obtain detailed transcriptomic profiles and improve our understanding of the genetic variation involved in pathogen infection and virulence (Jiang et al., 2016;Sun et al., 2016). The proteome, a term coined by Wilkins in 1992, refers to all of the proteins expressed by the genome, which includes those assessed at the cellular or organismal level (Wasinger et al., 1995). Isobaric tags for relative and absolute quantitation (iTRAQ) is a relatively advanced quantitative proteomic technique that enables relative protein measurements simultaneously with determination of the absolute levels of a target protein using synthetic isobaric peptide standards (Ross et al., 2004), To date, analysis of the V. parahaemolyticus proteome has mostly been based on the results of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and two-dimensional electrophoresis methods (Chen et al., 2016), but a comprehensive understanding of the components involved in the activation, regulation, and tolerance of low-temperature remains incomplete. The recent advent of sequencing technologies has now made it possible to obtain detailed transcriptomic and proteome profiles of this pathogen in different environments. Indeed, microarray analysis helped to elucidate the effects of salt and acid stress on the V. parahaemolyticus transcriptome (Yang et al., 2010;Sun et al., 2014).
To further illustrate the molecular mechanisms driving the low-temperature adaptation of V. parahaemolyticus, we employed RNA-sequencing (RNA-seq) and iTRAQ methods to obtain the global transcriptome and proteome patterns of V. parahaemolyticus under low-temperature, respectively. Analysis of these transcriptional and proteome profiles is expected to broaden our understanding of the pathways induced in V. parahaemolyticus to facilitate survival under lowtemperature, which can help to identify potential targets for developing food processing strategies to achieve more effective control of this pathogen and prevent widespread outbreaks of foodborne illness.

Culture and Incubation Conditions
Vibrio parahaemolyticus strains V82 (isolated from frozen squid) and VL8 (clinical isolate, Xie et al., 2017) were grown overnight at 37 • C in tryptic soytone broth (TSB) medium (HuanKai Microbial, Guangzhou, China). Dilutions of each culture were prepared up to 1:10 3 dilution (TSB medium), further incubated at 37 • C for 12 h, and then transferred to 4 • C for 12 h. The control group was cultured at 37 • C for 24 h continuously. Growth status was measured spectrophotometrically based on the optical density at 600 nm. According to the growth curve, stable phase strains were selected for RNA-seq and iTRAQ analysis (Supplementary Figure S1).

RNA-Seq Analysis
The total RNA was extracted using Bacterial RNA Kit (Omega Bio-Tek, Salt Lake City, UT, United States) according to the manufacturer's instructions. The complementary DNA (cDNA) libraries from two replicates of all samples were constructed and sequenced by GENE DENOVO, Ltd. (Guangzhou, China) on the Illumina sequencing platform (HiSeq TM 2500), producing 150-bp single-end reads. All clean reads were aligned to the reference genome (GCF_001558495.1) of V. parahaemolyticus using Tophat2, and the fragments per kilobases per million values for the genes were estimated by Cufflinks software (Trapnell et al., 2012). Based on the threshold of log 2 fold change ≥ 1 and false discovery rate (FDR) ≤ 0.05, the differentially expressed genes (DEGs) in each comparison were determined by the Cuffdiff module. All DEGs were annotated to the Gene Ontology database 1 and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (Kanehisa et al., 2008). The functional enrichment was determined by comparison to the annotation of reference transcripts (FDR ≤ 0.05). To support this analysis, 20 DEGs were randomly selected for measurement of expression levels in each sample by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR).

Quantitative Real-Time PCR (qRT-PCR) Analysis
The total RNA was extracted using Bacterial RNA Kit (Omega Bio-Tek) according to the manufacturer's instructions. RNA degradation and contamination were monitored on 1% agarose  gels and the RNA concentration was quantified cDNA was synthesized using M-MLV First Strand cDNA Synthesis Kit (Omega Bio-Tek) and was quantified using Perfectstart SYBR Green qPCR master mix (Omega Bio-Tek). qRT-PCR was performed on the QuanStudio TM Real-Time PCR system (Applied Biosystems, Foster City, CA, United States). The mRNA level of each gene was normalized to that of 16S rRNA. A list of all primers used in this study is provided in Supplementary  Table S2.

Fatty Acids Detection
Vibrio parahaemolyticus strain V82 was grown in TSB at 37 • C, and then transferred to 4 • C for the low-temperature treatment. The cultures were harvested by centrifugation (5000 × g, 4 • C) and the fatty acid compositions were determined as described by Zhu et al. (2005) and the National Food Safety Standards of China guideline GB5009.168-2016 for examination of fatty acids.

Detection of V. parahaemolyticus by Confocal Laser Scanning Microscopy
Confocal laser scanning microscopy (CLSM) (Zeiss, Berlin, Germany) analysis was performed using a LIVE/DEAD R BacLight TM Bacterial Viability and Counting Kit (Invitrogen, Carlsbad, CA, United States) with an appropriate mixture of the SYTO 9 and propidium iodide (PI).

Statistical Analysis
Integrated analysis of the transcriptome and proteome data was conducted using locally designed Perl scripts. For the statistical analysis of qRT-PCR, the relative mRNA levels and − CT values were calculated from the obtained cycle threshold values of three biological replicates (Livak and Schmittgen, 2012). The results of fatty acids detection are indicated as mean standard error of the mean (n = 3), and analyzed using significant range tests. Differences between groups were considered significant at P < 0.05.

Transcriptome Profiles for Strains VL8 and V82 Under Cold Stress
RNA-seq analysis was conducted to determine global changes at the transcript level for V. parahaemolyticus strains VL8 and V82 under cold stress, which revealed numerous DEGs for both strains cultured at 4 and 37 • C. There was also obvious variation in V. parahaemolyticus gene expression profiles between the pathogenic strain VL8 and environmental strain V82 ( Figure 1A, R 2 = 0.2498). The cold stress elicited 513 DEGs in VL8, representing approximately 11.01% of all genes in V. parahaemolyticus. Of these, 182 genes were significantly up-regulated and 331 genes were significantly down-regulated ( Figure 1B and Supplementary Table S3). The DEGs in V82 were enriched in functional categories of multiple catabolic processes, phosphate ion transport, and oxidoreductase activity ( Figure 1C and Supplementary Table S4).
In contrast, 118 genes, which accounted for only 2.53% of all genes in the bacterium, showed significantly differential expression in V82, including 42 up-regulated and 76 downregulated genes that were enriched in the functional categories of glutamine and arginine metabolic processes, and oxidoreductase activity ( Figure 1D). Interestingly, most of the DEGs in V82 overlapped with those in VL8 (66.67% of up-regulated genes and 80.26% of down-regulated genes, Figure 1B), reflecting that most of the responses to cold stress in V82 should be similar to those in VL8.
The annotation of KEGG pathways also demonstrated the differences and similarities between the responses of strains VL8 and V82 to cold stress. There were more DEGs annotated to the pathway of the ATP-binding cassette (ABC) transporter in VL8 (Figure 2A), while more DEGs in V82 were annotated to the pathway of the biosynthesis of amino acids (Figure 2B). In addition, the DEGs identified in these two strains were commonly enriched in the pathways of carbon metabolism, arginine biosynthesis, pyruvate metabolism, and butanoate metabolism.
Moreover, the randomly selected DEGs showed significantly differential expression levels by qRT-PCR analysis that were consistent with the results of RNA-seq analysis, suggesting the reliability of the RNA-seq results (Supplementary Figure S2).

Global Changes at the Protein Levels in VL8 and V82 Under the Cold Stress and Their Correlation With Transcript Levels
The iTRAQ mass spectrometric analysis was performed on three replicates of the cold-treated VL8 and V82 experiments to identify the differences in protein levels. In total, 1539 proteins were verified with quantitative information in all three replicates. The cold stress resulted in 20 up-accumulated   Tables S5, S6). Cold stress induced distinct changes in carbon metabolism and the pentose phosphate pathway for both strains (Figures 3A,B). We also observed changes in protein clusters of nucleotide excision repair ( Figure 3A) and ribosome (Figure 3B), which might indicate a response to prevent the misfolding of proteins under cold stress.
We then performed an integrated analysis of the iTRAQ and RNA-seq data sets. Overall, 38 and 42 overlapping pathways were found between the RNA-seq and iTRAQ data in VL8 and V82, respectively ( Figure 4A). Moreover, a total of 22 pathways were found to be affected in all data sets, including some important metabolic pathways such as carbon metabolism, biosynthesis of amino acids, and fatty acid metabolism (Figure 4B and Supplementary Table S7). Interestingly, the numbers of affected genes were relatively high in the pathways of pyruvate metabolism and ABC transporters, reflecting their important roles in the acclimation to cold of V. parahaemolyticus.

Decrease in Pyruvate Metabolism Confers Resistance to Cold Stress in V. parahaemolyticus
Consistent with the significant change detected in the pyruvate metabolic pathway in both VL8 and V82 under cold stress based on the integrated analysis, the KEGG mapping results demonstrated that the biosynthesis and utilization of pyruvate were significantly decreased (Supplementary Figure S3). Consequently, tricarboxylic acid (TCA) cycle pathway was also down-regulated ( Figure 5B). Interestingly, the lower metabolism of pyruvate led to the promotion of fatty acid biosynthesis, resulting in suppression of fatty acid degradation (Supplementary Figure S3).
To clarify whether the up-regulation of fatty acid biosynthesis was caused by a decrease in pyruvate metabolism, different concentrations of pyruvate were added to the culture medium inoculated and maintained at 4 • C. Compared with the cultures without pyruvate supplement, the fatty acid contents in almost all pyruvate supplement cultures (except for those supplemented with 5 g/L pyruvate) were significantly reduced ( Figure 5A). We then measured the growth curves of cultures supplemented with three concentrations of pyruvate (2.5, 10, and 20 g/L) at 4 • C, which were compared to those of cultures without pyruvate supplement at both 4 and 37 • C. At physiological temperature (37 • C), the bacteria grew gradually whereas growth was markedly suppressed at low temperature (4 • C) (Figure 5B). The cultures without pyruvate supplement could overcome the cold stress over time and slowly resumed normal growth. However, the pyruvate-supplemented populations could barely recover growth throughout the experiment.
Quantitative reverse transcription-polymerase chain reaction analysis was conducted to further determine the changes in the expression of key genes involved in the corresponding pathways identified to be enriched under cold stress. Three genes in the pyruvate metabolism pathway, gene1140 (fumarate reductase), gene1772 (acetaldehyde dehydrogenase), and gene2848 (formate C-acetyltransferase), were significantly down-regulated under 4 • C ( Figure 5C). Notably, the OD600 values decreased for cultures at low temperature during the first 1 h. Confocal laser scanning microscopy images indicated that most bacteria died at 4 • C compared to the culture at 37 • C (Supplementary Figure S4). Thus, we inferred that the decrease in OD600 values were mainly attributed to cellular lysis of dead bacterial. However, the addition of pyruvate disrupted the down-regulation of these genes, and even elevated their mRNA levels. Similarly, the up-regulation of a key gene (gene2332, hydroxydecanoyl-ACP dehydratase) in the fatty acid biosynthesis pathway was also prevented to a certain degree by the pyruvate treatment. Collectively, these results suggested that cold stress induced the accumulation of fatty acids by reducing pyruvate metabolism, but that supplementation of exogenous pyruvate would disrupt this process (Figure 6).

DISCUSSION
In parallel with improvements in the standard of life, consumers are increasingly concerned with both the taste and nutritive value of their food. To meet this consumer demand, food FIGURE 6 | The possible action mode mediated by pyruvate metabolism that underlie low-temperature adaptation of V. parahaemolyticus.
Frontiers in Microbiology | www.frontiersin.org producers must continuously adjust processing methods and storage conditions to maintain the original taste and quality of foods (Ma, 2016). Post-harvest processing is particularly important for maintaining the quality and taste of seafood, and low-temperature storage is an essential aspect of this process so as to challenge the survival of V. parahaemolyticus given that its virulence decreases in a low-temperature environment, including a reduction in hemolysin and protease production to induce a cytotoxic effect (Mahoney et al., 2010). Indeed, microorganism fitness (growth and survival) has been shown to be significantly linked with temperature (Boonyawantang et al., 2012). Thus, to survive and adapt to changes of the outside environment, microorganisms including foodborne pathogens can undergo a series of changes in gene regulation to control relevant metabolic pathways to combat the negative effects of stress conditions (Azizoglu et al., 2009). Given that V. parahaemolyticus can clearly survive under the low-temperature storage conditions in seafood and cause foodborne illness, we assessed the global changes in transcript and protein levels of a clinical and a frozen food V. parahaemolyticus isolate at low temperature to determine the adaptation mechanism. This integrated analysis revealed several stimulus-response pathways that were induced under cold stress in both strains, including the commonly affected pathways of ABC transporters, pyruvate metabolism, and fatty acid biosynthesis. Although common patterns were detected in both isolates, there were many more unique DEGs identified in VL8, suggesting that the gene expression profile in the environmental strain was relatively more stable under cold stress, while the pathogenic strain would need to mobilize more gene resources to protect itself from the cold stress.
The ABC transporter proteins, also known as highly conserved in ATP-binding sites proteins (Higgins et al., 1986), are found in all domains of life, and are mainly involved in the uptake of nutrients and micronutrients, extrusion of building blocks, and drug resistance by exporting certain toxic substances outside of the cell (Cuthbertson et al., 2010;Locher, 2016). Similar to the present findings, ABC transporters were found to be significantly inhibited in V. parahaemolyticus isolates with a mutation in the CSP gene cspA to compensate for the deficiency and protect the bacterium from low temperature induced damage (Zhu et al., 2017). Another report showed that V. parahaemolyticus ABC transporters demonstrated notable variation in response to acid and toxicity stress (Nydam et al., 2014;Sun et al., 2014). Thus, our results confirm an important role of ABC transporter pathways in response to cold stress at both the transcriptome and proteome levels in V. parahaemolyticus, which have been shown to play an important role in low-temperature adaptation of many other bacteria (Allen et al., 2009).
Interestingly, the down-regulation of pyruvate metabolism and the up-regulation of fatty acid biosynthesis co-occurred in this bacterium under cold stress. Fatty acids are crucial metabolites that are present in almost all living organisms, which play essential roles in maintaining membrane integrity with a direct effect on some cellular processes such as environmental adaptation, cellular differentiation, DNA replication, and cell death (Mansilla et al., 2008;Garba et al., 2016). The accumulation of fatty acids has also been considered to confer an advantage to bacteria for growth under low temperature (Suutari and Laakso, 1994), and Mansilla et al. (2004) showed that bacteria could stringently control the production and modulation of fatty acids within varying temperatures to maintain membrane lipids in the correct physical state. Thus, changing the fatty acid composition is a common strategy for bacteria to overcome low-temperature pressure. For example, different kinds of fatty acids were shown to respond to cold stress in Shewanella piezotolerans strains (Wang et al., 2009), and multiple genes related to fatty acid metabolism were induced under cold exposure over time in a previous study of V. parahaemolyticus (Yang et al., 2009). Changes of lipids and lipid-like molecules in V. parahaemolyticus at low temperature were also observed at the metabolome level (Feng et al., 2016). Pyruvate, a small molecule that plays a central role in metabolism, is the final product of glycolysis and the starting substrate for the TCA cycle (Fink, 2003), also acting as a potent and effective reactive oxygen species scavenger to protect bacteria from oxidative stress (Karabeyoglu et al., 2008). Pyruvic acid affects the metabolic rate of fatty acids, which in turn influences the TCA cycle (de Groot et al., 1995;Zhang et al., 2010).
Besides demonstrating a link to pyruvate metabolism and fatty acid biosynthesis in terms of gene and protein expression changes under cold stress, we found that direct supplementation of exogenous pyruvate to the culture medium could reduce the fatty acid content to suppress V. parahaemolyticus growth under low temperature. Based on these results, we inferred that reduction of pyruvate synthesis can decrease the energy metabolism in V. parahaemolyticus in the face of cold stress. However, the addition of pyruvate may transmit an error signal to activate the TCA cycle and intensify the utilization rate of fatty acids. Since the accumulation of fatty acids is a significant microbial cold tolerance adaptation, The downregulation of fatty acid synthesis caused by exogenous pyruvate is a strategy that could induce the death of V. parahaemolyticus cells growth at low temperature and improve food safety. These findings provide a new method to control the growth of this pathogen on frozen aquatic products during transport by adjusting the concentration of pyruvic acid to decrease the survival rate of V. parahaemolyticus and reduce the potential of contamination.

CONCLUSION
This study represents the first investigation of foodborne and clinical V. parahaemolyticus isolates in response to low temperature using an integrated transcriptome and proteome analysis. We detected distinct global-level gene expression and protein accumulation profiles, revealing several potential key mechanisms for coping with cold stress, such as modulating the pathways of ABC transporters, pyruvate metabolism, and fatty acid biosynthesis. Moreover, pyruvate addition reduced the fatty acid biosynthesis to suppress V. parahaemolyticus growth at low temperature. Our results should facilitate further in-depth research of the cold stress resistance of V. parahaemolyticus to provide a potential strategy of controlling this seafood-borne pathogen.

AUTHOR CONTRIBUTIONS
TX and RP are the common first authors, finish the article experiment, and write the article together. QW give the idea and experiments support. JZ, TL, YL, JW, YD, MC, and JB help to finish the experiment on article.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2019.00178/full#supplementary-material FIGURE S1 | The growth curve of V82, VL8 at different temperature stages. FIGURE S2 | Differentially expressed genes validated by qRT-PCR analysis.
FIGURE S3 | The important pathways affected by cold stress in V. parahaemolyticus, including pyruvate metabolism pathway (A), citrate cycle (TCA cycle) pathway (B), fatty acid biosynthesis pathway (C), and fatty acid degradation pathway (D). Red indicates that the gene is up-regulated at low temperature, while green indicates that the gene is down-regulated at low temperature.