RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium

Abstract

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.

Fig 1. RNA-seq–based strategy to identify promoters, transcribed regions, and small RNAs of S. Typhimurium active during macrophage infection.

S. Typhimurium strain 4/74 was grown within macrophages for 8 hours using the gentamicin protection assay, and bacterial RNA was isolated using TRIzol (Materials and Methods). The cDNA generated from total RNA was sequenced either directly for gene/sRNA expression analysis (RNA-seq) or after enrichment of primary transcripts (dRNA-seq), and compared with data from 4/74 grown to ESP [17]. The graphs show representations of sequence reads mapped uniquely against the 4/74 genome in different conditions. Transcript per Million (TPM) analysis was used to calculate gene expression values from the number of sequence reads mapped against the 4/74 genome. The promoter usage value (PUV) indicates the TPM value of the first 10 nucleotides from the transcription start sites (TSS) in the direction of transcription, and represents promoter strength. Each curved arrow indicates location of TSS upstream of the respective gene; the width and height of each curved arrow is proportional to TSS expression, based on relative PUV, macrophage versus ESP.

Fig 2. The primary transcriptome of intra-macrophage Salmonella.

(A) Classification of 3583 Salmonella TSS during intra-macrophage proliferation (Dataset 3 in S1 Table). The TSS were categorized based on their relative-PUV, macrophage versus ESP (Materials and Methods). The red and blue circles represent the TSS that are up/down-regulated in the ∆ssrA versus InSPI2 experiment, respectively (B) The STM0854 TSS (indicated by the dotted vertical line) is a representative of a TSS highly up-regulated in macrophages. Each horizontal arrow represents the gene in scale with the whole island. Each coloured track above the island represents RNA-seq/dRNA-seq reads mapped against the genome in the corresponding conditions, visualized in the IGB browser. Each curved arrow indicates the location of a TSS; the width and height of each curved arrow is proportional to the TSS expression, based on relative PUV, macrophage versus ESP (Dataset 3 in S1 Table) (Materials and Methods).

Fig 3. The transcriptional organization of SPI2 in intra-macrophage Salmonella.

Horizontal arrows represent individual SPI2 genes in scale with the whole Island. The ssrA-B gene products regulate SPI2 expression, the sseA-E genes encode effector proteins, the sscA-B genes encode chaperone proteins and the ssaB-E and ssaG-U loci encode components of the T3SS apparatus. The colour of each gene represents its relative expression, macrophage versus ESP (Dataset 4 in S1 Table) based on the colour scale given in Fig 4. Each track above and below the island shows the mapping of the RNA-seq or dRNA-seq reads against the plus or minus strand of the 4/74 chromosome visualized in IGB. Each curved arrow indicates the location of a TSS upstream of the respective gene; the width and height of each curved arrow is proportional to the TSS expression, based on relative PUV, macrophage versus ESP (Dataset 3 in S1 Table) (Materials and Methods). The red hash on PssaR indicates that the TSS was reported previously [17], and confirmed in this study by 5’RACE (S1 Fig). The blue hashes indicate that the location of promoters PssaB, PsseA, PssaG, PssaM and PssrB that have been confirmed independently by 5’RACE [107] (S1 Fig).

Fig 4. The relative intra-macrophage expression of the different pathogenicity islands of S. Typhimurium.

Each horizontal arrow represents individual genes to scale within each SPI Island; the different islands are not scaled against each other. The colour of each arrow represents relative gene expression, macrophage versus ESP (Datasets 4 and 5 in S1 Table) based on the colour scale at the bottom of the figure. SPI2 expression is shown in Fig 3.

Fig 5. The intra-macrophage expression of Salmonella effector genes.

(A) A comparison of absolute expression levels of different S. Typhimurium effector genes within macrophages (Dataset 4 in S1 Table) and in 20 in vitro infection-related conditions [17]. The heatmap colours represent the absolute expression levels (log₁₀ TPM values) based on the colour bar, ranging from TPM values of 49 to 706. (B) The relative expression levels (macrophage versus ESP) of each gene are defined in the colour bar to the right. The genes prgH and ssaG are included to show the SPI1-like and SPI2-like patterns of expression, respectively. The T3SS-independent genes were described by Kidwai et al. (2013) [108].

Fig 6. Relative intra-macrophage expression of Salmonella transcription factors and selected target genes.

The expression of individual genes is shown as fold change, intra-macrophage versus ESP. Transcription factors are shown in bold. Target genes controlled by individual transcription factors are shown in the same row (A and C). Expression of transcription factors that regulate SPI2-related genes (A). The regulation of SPI1 genes is controlled by a hierarchy, and the transcription factors are depicted as co-regulators, with their combined target genes (B). Relative expression of 13 metabolic systems (C). Relative expression of up-regulated alternative sigma factors and transcription factors that control oxidative stress, and iron and zinc homeostasis (D). ^ǂNo dedicated transcription factor for glucose metabolism was assigned.

Fig 7. Salmonella genes that are specifically up-regulated inside macrophages.

(A) Heatmap showing expression of 31 S. Typhimurium genes that are specifically up-regulated during infection of macrophages, compared to 20 in vitro conditions [17] (Dataset 7 in S1 Table) (Materials and Methods). The heatmap colours represent the absolute expression levels (log₁₀ TPM values) based on the colour bar below. (B) The relative expression level of each gene is the fold-change of macrophage versus [expression in the in vitro condition where the gene in maximally expressed], based on the colour bar to the right. (C) The SPI13 operon (lgl [37] and ripABC [25], or STM3117 and STM3118-STM3120, respectively) is highly induced within macrophages.

Fig 8. Intra-macrophage expression profile of Salmonella sRNAs.

(A) Total number of sRNAs that are up or down-regulated in macrophages, based on the relative TPM value (>2-fold macrophage versus ESP; Dataset 9 in S1 Table). (B) Histograms representing the total number of Hfq-associated sRNAs [109] that are up or down-regulated within macrophages. (C) The relative expression of S. Typhimurium sRNAs in macrophages (TPM values; macrophage versus ESP). The red histograms depict the 34 macrophage up-regulated sRNAs (>2-fold). The blue histograms show sRNAs down-regulated >6-fold within macrophage (Dataset 9 in S1 Table). The orange circles indicate Hfq-bound sRNAs [17]. The names of sRNAs shown as magenta bold text are specific to the Salmonella genus (Dataset 10 in S1 Table).