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Comparative Study
. 2021 Sep 27;17(9):e1009949.
doi: 10.1371/journal.ppat.1009949. eCollection 2021 Sep.

Comparison of transcriptional profiles of Treponema pallidum during experimental infection of rabbits and in vitro culture: Highly similar, yet different

Bridget D De Lay  1 , Todd A Cameron  2 , Nicholas R De Lay  2 , Steven J Norris  1   2 , Diane G Edmondson  1
Affiliations
Comparative Study

Comparison of transcriptional profiles of Treponema pallidum during experimental infection of rabbits and in vitro culture: Highly similar, yet different

Bridget D De Lay et al. PLoS Pathog. .

Abstract

Treponema pallidum ssp. pallidum, the causative agent of syphilis, can now be cultured continuously in vitro utilizing a tissue culture system, and the multiplication rates are similar to those obtained in experimental infection of rabbits. In this study, the RNA transcript profiles of the T. pallidum Nichols during in vitro culture and rabbit infection were compared to examine whether gene expression patterns differed in these two environments. To this end, RNA preparations were converted to cDNA and subjected to RNA-seq using high throughput Illumina sequencing; reverse transcriptase quantitative PCR was also performed on selected genes for validation of results. The transcript profiles in the in vivo and in vitro environments were remarkably similar, exhibiting a high degree of concordance overall. However, transcript levels of 94 genes (9%) out of the 1,063 predicted genes in the T. pallidum genome were significantly different during rabbit infection versus in vitro culture, varying by up to 8-fold in the two environments. Genes that exhibited significantly higher transcript levels during rabbit infection included those encoding multiple ribosomal proteins, several prominent membrane proteins, glycolysis-associated enzymes, replication initiator DnaA, rubredoxin, thioredoxin, two putative regulatory proteins, and proteins associated with solute transport. In vitro cultured T. pallidum had higher transcript levels of DNA repair proteins, cofactor synthesis enzymes, and several hypothetical proteins. The overall concordance of the transcript profiles may indicate that these environments are highly similar in terms of their effects on T. pallidum physiology and growth, and may also reflect a relatively low level of transcriptional regulation in this reduced genome organism.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schematic diagram of the methods used in this study.
Fig 2
Fig 2
High consistency of RNA-seq read coverage patterns among 8 independently processed RNA samples, as exemplified by the large ribosomal protein gene operon region of T. pallidum (dashed arrow at bottom). Coverage patterns are shown for the 6 in vitro culture specimens (in vitro sets 1A-1C and 2A-2C) and the specimens from infected rabbits 1 and 2. The read coverage graphic was prepared using the Sashimi plot feature of the Integrative Genomic Viewer (IGV) program.
Fig 3
Fig 3. Functional roles of the fifty T. pallidum genes most highly expressed in rabbits and in vitro.
(A) Functional roles of the fifty T. pallidum genes with the highest expression during rabbit infection. (B) Functional roles of the fifty T. pallidum genes most highly expressed during in vitro culture.
Fig 4
Fig 4. Similarity of T. pallidum transcript levels between samples collected from infected rabbits and from in vitro culture.
A) Scatter plot comparing average log2-transformed FPKM values of T. pallidum collected during rabbit infection and in vitro culture. B) Poisson distance matrix based on pairwise DESeq2 differential expression analysis showing that RNA samples taken from the two individual rabbits group together, while the six in vitro culture samples group separately. Darker blue squares indicate that samples are more closely related than those with lighter blue squares.
Fig 5
Fig 5. Identification of genes with significantly different transcript levels between T. pallidum during in vitro culture vs. rabbit infection.
The volcano plot indicates significantly differentially expressed genes between T. pallidum grown in vitro and in rabbits, as determined by DESeq2. Log2-fold difference values reflect the ratio of rabbit/culture transcript levels. Significantly differentially-expressed genes, with false discovery rate (FDR) adjusted p-value ≤ 0.05 and |log2-fold difference| of ≥ 1 are indicated in green. N.S. indicates the p value is not significant (>0.05).
Fig 6
Fig 6. Comparison of differentially expressed ribosomal protein genes between T. pallidum grown in rabbits and in vitro.
Log2-fold difference and adjusted p-values calculated by DESeq2. Values in green indicate significantly differentially expressed genes with |log2-fold difference| of ≥ 1 and p-adjusted of ≤0.05. Values in yellow indicate genes with a significant p-adjusted value of ≤0.05, but without a significant log2-fold difference. A) Comparison of genes in the large ribosomal protein operon. B) Comparison of additional ribosomal protein genes.
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