Stability Assessment of Housekeeping Genes for qRT-PCR in Yersinia enterocolitica Cultured at 22℃ and 37℃

doi:10.21203/rs.3.rs-4205397/v1

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Stability Assessment of Housekeeping Genes for qRT-PCR in Yersinia enterocolitica Cultured at 22℃ and 37℃

https://doi.org/10.21203/rs.3.rs-4205397/v1

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Background:

Yersinia enterocolitica, a species within the genus Yersinia, thrives optimally at 22–25°C but can also grow at mammalian core body temperature of 37°C. This dual temperature adaptability necessitates the establishment of both temperature conditions in research to examine the effects on various biological inquiries. In quantitative real-time PCR (qRT-PCR) assays, the selection of appropriate housekeeping genes is vital for data accuracy. Nevertheless, the paucity of alternatives and information frequently leads to the default use of the 16s rRNA gene, despite potential limitations.

Methodology:

This investigation sourced 16 potential reference genes through a comprehensive review of the literature and transcriptome sequencing data analysis. We validated the expression stability of these genes via qRT-PCR across 12 Y. enterocolitica strains, representing the four prevalent serotypes O:3, O:5,27, O:8, and O:9, isolated from diarrheal patient stool samples. This approach aimed to minimize the impact of serotype heterogeneity. Post-acquisition of Cq values, gene stability was evaluated using four established algorithms—∆Cq, geNorm, NormFinder, and BestKeeper—and subsequently synthesized into a consolidated ranking through the Robust Rank Aggregation (RRA) method.

Conclusion:

Our study suggests that the genes glnS, nuoB, glmS, gyrB, dnaK, and thrS maintain consistent expression across varying culture temperatures, endorsing them as robust housekeeping gene candidates. We advise against the exclusive use of 16s rRNA for this purpose. Should tradition prevail in its utilization, it must be employed with discernment, preferably alongside one or two of the housekeeping genes identified in this study as internal controls.

Yersinia enterocolitica is a human pathogen belonging to the Yersiniaceae family, which is widely found in both environment and animal reservoirs. It is a zoonotic bacterium that spreads through the fecal-oral route, generally lead to mild self-limiting enterocolitis, terminal ileitis or adenitis, and occasionally followed by chronic inflammatory diseases such as arthritis and erythema nodosum. Y. enterocolitica can adapt to various temperature ranges, but specific temperatures can influence their behavior and characteristics. Its optimal growth temperature was 22°C, at which it was a motile single cell with high lipopolysaccharide (LPS) O-polysaccharide (OPS) expression and had the highest growth rate. When transferred to the mammal host or raise the incubation temperature to 37°C, it switched to a non-motile and autoagglutinating form and increased expression level of virulence proteins such as invasin (Inv), Yersinia adhesin A (YadA), and plasmid-encoded Yersinia exoproteins (Yops). Therefore, culture temperature is an important aspect to consider or pay attention to in studying Y. enterocolitica, as well as Y. pestis and Y. pseudotuberculosis. In most studies of these bacteria, researchers typically divide their experiments into two groups based on temperature conditions: room temperature (approximately 22–25°C) and host temperature (37°C). Subsequent experiments are then conducted to observe and analyze the results obtained. This approach allows for a comprehensive understanding of the bacterial behavior and responses under different temperature environments.

Quantitative RT-PCR (qRT-PCR) is a popular technique for the quantification and comparative analysis of gene expression levels, prized for its rapidity, precision, and cost-efficiency. Housekeeping genes are indispensable internal controls in this methodology, utilized to normalize gene expression data and reduce experimental variation. Ideally, genes that exhibit stable expression throughout the experimental conditions should be selected as housekeeping genes. However, the process of choosing housekeeping genes often lacks careful consideration. Researchers frequently simply rely on housekeeping genes reported in previous literature, with 16s rRNA being the most commonly selected. The appropriateness of this conventional approach merits careful evaluation.

In this study, we leverage the differential gene expression characteristics of Y. enterocolitica at 22°C and 37°C as a starting point, aiming to carefully evaluate the stability of six "traditional housekeeping genes" including 16s rRNA that have been reported in existing literature, as well as ten novel "candidate housekeeping genes" identified from RNA-seq data downloaded from the Sequence Read Archive (SRA) database (https://www.ncbi.nlm.nih.gov/sra). The experimental validation was conducted using twelve Y. enterocolitica strains isolated from patients with diarrhea, with the strains' serotypes encompassing as comprehensively as possible the most common serotypes of this bacterium, namely O:3; O:5,27; O:8; and O:9, in order to eliminate heterogeneity between different serotypes.

Sample collection

Y.enterocolitica strains are collected from Chinese Pathogen Identification Net (China PIN) project[1] (Table 1). All strains were isolated from fecal specimens of diarrhea patients. Strain identification was performed using VITEK® 2 Compact (bioMérieux, France). The O-serogroup of the isolates was determined by slide agglutination test (Denka Seiken, Japan).

RNA-Seq datasets and transcript abundances analysis

Y. enterocolitica RNA-Seq datasets (BioProject: PRJNA264525 [2] and BioProject: PRJNA277186 [3, 4]), which uploaded by a research team from University of Helsinki, were obtained from the Sequence Read Archive (SRA) database. Both studies were used Y. enterocolitica Y11 (serotype: O3) as the wild-type strain, all strains were incubated at two temperatures, 22°C and 37°C, respectively, before sequencing. To improve the robustness of the screening outcomes, RNA-Seq data from genetically engineered mutant strains in these studies were also incorporated.

Raw FASTQ-files were extracted by using prefetch and fasterq-dump tool from SRA Toolkit 3.02, low quality reads and adapters were trimmed by fastp 0.23.2 with default settings[5]. Y.enterocolitica Y11 (RefSeq: GCF_000253175.1) genome were firstly annotated by Bakta (v1.8.1) and then used to build transcriptome index. Transcript abundances were quantified using Kallisto v0.48.0 to get transcripts per million (TPM) value[6].

Identification of novel candidate housekeeping genes from transcriptomes

Candidate reference genes that were stably expressed between 22 / 37℃ were identified using the method provided by Eisenberg and Levanon, with minor modifications [7]. Briefly, Transcripts Per Kilobase Million (TPM) was used instead of Reads Per Kilobase Million (RPKM) to represent gene expression levels, and the following four principles were used in the screening process of candidate housekeeping genes: (1) low variance between samples cultured at 22 / 37℃: Standard Deviation (SD) of log2(TPM) < 0.5; (2) no exceptional expression in any single sample: Max |log₂(TPM) – mean [log₂(TPM)]| < 1; (3) relatively high expression levels: Mean [log₂(TPM)] > 5; and (4) highly conserved in the Y. enterocolitica genome. To assess the gene conservation, genomic data of Y. enterocolitica were obtained from the NCBI using the ncbi-genome-download tool (v0.3.1), and the conservation of candidate housekeeping genes was subsequently assessed by blastn (v2.12.0) comparison, with the filtering condition of being able to have a %identity > 95 match in at least 90% of the genome. Genes that met all of the above criteria were ranked in descending order of CV value, and top 10 genes were selected for qRT-PCR validation. The scope of target genes was limited to coding sequences (CDS) and ribosomal RNA (rRNA), with non-coding RNAs (ncRNAs), small open reading frames (sORFs), transfer RNAs (tRNAs), and transfer-messenger RNAs (tmRNAs) being specifically excluded from the study.

Validation the expression stability of target genes

The expression stability of housekeeping genes reported from previous literature (6 in total) as well as the candidate housekeeping genes screened in this study (10 in total) were verified by qRT-PCR (Table 2). Primers were designed using Primer3Plus website tool[8]. Total RNA was extracted using Spin Column Bacteria Total RNA Purification Kit (Sangon, B518655) and RNase-Free DNase Set (Sangon, B618253). RNA concentration was measured using NanoDrop Lite Spectrophotometer (Thermo), the RNA purity was distinguished by the ratio of OD260 / 280. qRT-PCR was conducted in a 96-well plate using HiScript II One Step qRT-PCR SYBR Green Kit (Vazyme) and BioRad CFX system, each 20 µl reaction contained 10 µl 2 × One Step SYBR Green Mix, 1 µl One-Step SYBR Green Enzyme Mix, 0.4 µl each of 10 µM primers and total of 1 ng RNA. Reactions were run using cycling parameters of 50℃ for 3min for generate cDNA, 95°C for 30 s, 40 cycles of 95°C for 10 s and 60°C for 30 s, followed by a single cycle at 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s for melt curve analysis. Each qRT-PCR analysis was performed in triplicate, and melting curve were analyzed to assess whether the assays have produced single, specific products.

The stability of the expression of the selected genes was assessed using RefFinder, a web-base tool for evaluating the stability and reliability of reference genes, by integrating the result of major computational programs including geNorm [9], NormFinder [10], BestKeeper [11] and comparative delta-Ct method [12]. The comprehensive ranking was conducted using the Robust Rank Aggregation (RRA) package in R [13]。

1. Selection of Novel Candidate Housekeeping Genes Based on Transcriptome Sequencing Data

By analyzing RNA-Seq data, we acquired transcripts per million (TPM) values for all genes in Y. enterocolitica Y11. We computed both the log2(TPM) values and the maximum absolute deviation for each gene's log2(TPM). These values were then plotted in a scatter plot (Fig. 1). In this plot, genes positioned closer to the bottom-left corner are indicative of minimal expression variability due to temperature and inter-strain differences. To identify novel candidate housekeeping genes, we delineated a stringent criterion (marked by the red box in Fig. 1) and selected 10 genes that exhibited sufficient sequence conservation, relatively high expression levels, and the lowest coefficient of variation (CV) for their log2(TPM) values. The genes identified are: rpoD, thrS, ftsY, rlmL, rpoN, dnaX, glmS, lepA, argS, and glnS. To provide a clear visual comparison of the expression stability at 22°C and 37°C for the six housekeeping genes previously reported in the literature and the 10 newly identified candidate housekeeping genes, we constructed Fig. 2. The proximity between the red and blue circles represents the stability of gene expression across the two temperatures: the closer the circles, the more stable the gene expression at 22°C and 37°C.

2. Acquiring Cq Values of Housekeeping Genes in Clinical Strains Cultured at 22°C / 37°C

The subsequent step in our research was to simulate a realistic laboratory scenario by validating the expression stability of 16 genes identified in previous work at temperatures of 22°C and 37°C, using Y. enterocolitica strains. The primer sequences for these experiments are detailed in Table 2. These primers have been confirmed to effectively amplify the target genes, and their melting curves exhibit a single peak, indicating the specificity of amplification (data not shown). To lend more credibility to our experimental outcomes, we refrained from utilizing standard strains and instead employed clinical samples isolated from the feces of patients with diarrhea. Moreover, to reduce the impact of heterogeneity among different serotypes, we made a concerted effort to collect a comprehensive set of strains from four serotypes: O:3, O5:27, O:8, and O:9, securing three strains from each serotype. This resulted in a total of 12 bacterial strains for qRT-PCR validation.

After obtaining the Cq values for each strain and gene, we initially computed the sum of the ∆Cq values at 22°C and 37°C for each gene (Fidure 3). It is tentatively concluded that genes with smaller sums of ∆Cq values are more stable in their expression levels. When ranking the sum of ∆Cq values for each gene in ascending order, the four genes (top 25%) with the smallest values were rpoB, nuoB, rpoD, and glmS. However, when evaluating the stability within each of the four serotypes separately, a total of nine unique genes emerged. Among these, nuoB, rpoB, and rpoD were identified three times, glmS twice, and argS, dnaK, gyrB, lepA, and 16s rRNA each appeared once.

3. Analysis of gene expression stability by different statistical algorithms

Next, the expression stability of the 16 target candidate housekeeping genes was assessed by three dedicated analytical tools: geNorm[9], NormFinder[10], and BestKeeper[11]algorithms.

GeNorm is the most commonly used algorithm for assessing the stability of candidate housekeeping genes. It calculates a gene-stability measure value (M) by evaluating the average pairwise variation of each candidate gene in comparison to other genes. A lower M value indicates greater stability. After calculating M values for all genes, the two genes with the lowest scores are recommended as housekeeping genes. The experimental bacterial strains were categorized into four groups based on their serotypes. The M values for each gene within the three strains in each group were computed and arranged in ascending order according to their average values (Fig. 4). The top 25% genes with the smallest M values from each serotype group (four genes per group) were selected, resulting in a total of nine unique genes. Among them, glnS, rlmL, and thrS appeared three times each, rpoN appeard twice, and remaining five genes ftsY, glmS gyrB, lepA and nuoB appeared only once.

NormFinder is another popular algorithm for evaluating gene expression stability. It adopts a model-based approach to assess stability and calculates a stability value for each gene by considering both the overall variation and the variation attributed to specific sample groups. Similarly, a lower score from this algorithm indicates higher stability of the gene. The top 25% of genes with the smallest NormFinder scores were selected within each serotype group, resulting in 16 genes, out of which 10 were unique. Among them, glnS and gyrB appeared three times, glmS and ropN appeared twice, while the remaining six genes ftsY, lepA, nuoB, rlmL, rpoD and thrS appeared once.

The BestKeeper algorithm calculates the stability of candidate genes by analyzing their standard deviation (SD) of Cq values, as well as the coefficient of variance (CV), correlation coefficient (r), and p-value (p), which are also important parameters. It separately calculates the top 25% most stable genes in each serotype group, resulting in a total of 16 genes, out of which 8 are unique. Among them, rpoD is counted 4 times, ropB is counted 3 times, ftsY, glmS, and nuoB are each counted 2 times, and argS, gyrB, and lepA are each counted 1 time.

4. Comprehensive Ranking of Housekeeping Gene Stability Using the Robust Rank Aggregation Algorithm

It is evident that even for the same set of qRT-PCR data, different algorithms may yield rankings that are not entirely consistent. To obtain a composite ranking, we employed the RRA algorithm to integrate 48 ranking results from 12 clinical bacterial strains obtained through four different evaluation methods. This method led us to a comprehensive ranking (see Fig. 8). The results show that the composite rankings of the 16 candidate housekeeping genes can generally be divided into three tiers. The first tier includes glnS, nuoB, glmS, gyrB, dnaK, and thrS, which have the lowest RRA scores, indicating their consistently high rankings and good stability across various algorithms. glnS stands out in particular, with an RRA score of only 0.01, placing it in the foremost position. The second tier genes, rlmL, rpoD, and rpoB, display moderate overall ranking performance. The third tier comprises lepA, ftsY, 16s rRNA, rpoN, argS, gmk, and dnaX, which suggests that their rankings fluctuate significantly across different algorithms or they tend to rank lower more consistently.

In recent years, with the proliferation of technology, an increasing number of studies have begun to employ transcriptomic and even proteomic approaches to observe differences in bacterial protein expression. However, qRT-PCR, as a traditional and mature technology, remains one of the most commonly used and reliable techniques in microbial laboratories. This is due to its low technical threshold, widespread availability of equipment, low reagent costs, good reproducibility, and particularly because its results are intuitive and easy to analyze.

When conducting relative quantification experiments using this technique, it is necessary to designate a "housekeeping gene" as an "internal control" and use it as a "baseline" to assess changes in the expression levels of other genes. The selection of an appropriate housekeeping gene has always been a topic of interest. In mammalian studies, commonly used housekeeping genes include GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), ACTB (β-actin), B2M (β-2 microglobulin), etc., which either encode cytoskeletal proteins or are involved in basic biological functions and are expressed relatively stably and consistently across various cell types. However, further research has revealed that the expression levels of these commonly used housekeeping genes can fluctuate in different organs or under specific types of external disturbances [14]. Therefore, a consensus has been reached that housekeeping genes should be carefully evaluated and selected according to specific experimental conditions to minimize the generation of experimental errors as much as possible [7].

In microbial research, although some studies have assessed the stability of housekeeping genes in different types of bacteria under various environmental conditions and offered selection recommendations, researchers often rely on consulting published literature when choosing housekeeping genes. Particularly, 16s rRNA, due to its presence and relative conservation in all bacteria, as well as its prominence in microbial classification and identification, is often the first choice as a housekeeping gene in most qRT-PCR experiments. Although in some conditions, 16S rRNA can fulfill the role of a housekeeping gene[15] [16], in reality, no single gene, including 16S rRNA, can achieve absolute stability under ideal conditions. After careful evaluation based on experimental conditions, more scientific choices often emerge[17] [18] [19] [20].

To effectively adapt to the internal environments of humans and other mammals, the optimal growth temperature for most human pathogens is 37°C. However, Yersinia, a genus of bacteria that includes "psychrophilic" organisms, can grow at 0–4°C, with an optimal temperature of 22–28°C; at 37°C, the body temperature of humans and other mammals, these bacteria can also effectively infect and proliferate[21]. Since temperature changes significantly affect some of the bacterium's physiological characteristics, including virulence, morphology, phage cleavage, lipopolysaccharide structure, etc., researchers studying these bacteria often set both 22–25°C and 37°C as cultivation temperatures to observe and understand the effects of temperature changes on their scientific questions of interest [22–24]. However, little research has focused on how the expression levels of housekeeping genes change under different temperatures.

Based on the background mentioned above, this study attempts to organize the stability of housekeeping genes in Y. enterocolitica at two cultivation temperatures, 22°C and 37°C. We identified a list of genes for testing through both existing literature and transcriptome sequencing data analysis; collected 12 clinical strains covering the four most common serotypes for qRT-PCR validation; and after obtaining Cq values, assessed them using four classic algorithms, integrating the assessment results to provide a comprehensive ranking. Our results show that glnS, nuoB, glmS, gyrB, dnaK, and thrS exhibit minor differences in expression levels when the cultivation temperature changes, thus recommending them as housekeeping genes. We advise against solely designating 16s rRNA as a housekeeping gene. However, if it is still preferred by habit, it should be treated with caution, and one or two of the housekeeping genes recommended in this study should be selected to serve as the "internal reference" for the research.

Our study indicates that simply selecting housekeeping genes based on reference literature is inappropriate in bacterial research. The designation of 16s rRNA as a housekeeping gene requires careful consideration. In Yersinia enterocolitica, for its most common experimental variable — the two cultivation temperatures of 22°C / 37°C, the expression levels of glnS, nuoB, glmS, gyrB, dnaK, and thrS show minor differences, and we recommend designating one or more of them as housekeeping genes in qRT-PCR experiments.

Funding

This work was supported by the National Natural Science Foundation of China (81902124 and 82002205), the General Financial Grant from the China Postdoctoral Science Foundation (2019M651802).

Author Contribution

C.Li. led the research design, conducted the experiments, and drafted the manuscript. L.Zhu contributed to overseeing the research process L.Zou. and J.L. were responsible for data analysis. X.M. provided software support and validated the data. D.W. assisted in conducting experiments and collecting data. M.H. contributed to manuscript review and editing. L.M. managed the project and offered guidance throughout. H.S. and C.Liu. conceived the study idea, secured funding, and supervised the entire research process.

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Table 1 and 2 are available in the Supplementary Files section.

No competing interests reported.

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You are reading this latest preprint version

Stability Assessment of Housekeeping Genes for qRT-PCR in Yersinia enterocolitica Cultured at 22℃ and 37℃

Status:

Version 1

Abstract

Background:

Methodology:

Conclusion:

Figures

Background

Method

Sample collection

RNA-Seq datasets and transcript abundances analysis

Identification of novel candidate housekeeping genes from transcriptomes

Validation the expression stability of target genes

Results

1. Selection of Novel Candidate Housekeeping Genes Based on Transcriptome Sequencing Data

2. Acquiring Cq Values of Housekeeping Genes in Clinical Strains Cultured at 22°C / 37°C

3. Analysis of gene expression stability by different statistical algorithms

Discussion

Conclusion

Declarations

Funding

Author Contribution

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1