Investigation of the host transcriptional response to intracellular bacterial infection using Dictyostelium discoideum as a host model

Background During infection by intracellular pathogens, a highly complex interplay occurs between the infected cell trying to degrade the invader and the pathogen which actively manipulates the host cell to enable survival and proliferation. Many intracellular pathogens pose important threats to human health and major efforts have been undertaken to better understand the host-pathogen interactions that eventually determine the outcome of the infection. Over the last decades, the unicellular eukaryote Dictyostelium discoideum has become an established infection model, serving as a surrogate macrophage that can be infected with a wide range of intracellular pathogens. In this study, we use high-throughput RNA-sequencing to analyze the transcriptional response of D. discoideum when infected with Mycobacterium marinum and Legionella pneumophila. The results were compared to available data from human macrophages. Results The majority of the transcriptional regulation triggered by the two pathogens was found to be unique for each bacterial challenge. Hallmark transcriptional signatures were identified for each infection, e.g. induction of endosomal sorting complexes required for transport (ESCRT) and autophagy genes in response to M. marinum and inhibition of genes associated with the translation machinery and energy metabolism in response to L. pneumophila. However, a common response to the pathogenic bacteria was also identified, which was not induced by non-pathogenic food bacteria. Finally, comparison with available data sets of regulation in human monocyte derived macrophages shows that the elicited response in D. discoideum is in many aspects similar to what has been observed in human immune cells in response to Mycobacterium tuberculosis and L. pneumophila. Conclusions Our study presents high-throughput characterization of D. discoideum transcriptional response to intracellular pathogens using RNA-seq. We demonstrate that the transcriptional response is in essence distinct to each pathogen and that in many cases, the corresponding regulation is recapitulated in human macrophages after infection by mycobacteria and L. pneumophila. This indicates that host-pathogen interactions are evolutionary conserved, derived from the early interactions between free-living phagocytic cells and bacteria. Taken together, our results strengthen the use of D. discoideum as a general infection model.

Comparison of gene regulation detected by microarray (p-value < 0.05 and log2(FC) > 1 or < -1) with the corresponding regulation determined with RNA-seq for L. pneumophila infected cells. Marked in red: significantly regulated genes according to both RNA-seq (FDR < 0.05) and microarray analyses; Marked in black: significantly regulated genes according to microarray but where the regulation detected by RNA-seq failed to meet the FDR cut off (0.05). Note that the ranges for the two axes differ.    Figure S5. Regulation of overlapping genes in D. discoideum in response to M. marinum, L. pneumophila and E. coli. In the scatterplots, each data point represents a gene differentially expressed in response to different bacterial challenges/hpi (x-and y-axes). Capital letters (A-K) in each graph header refer back to the overlapping sections of the Venn diagram in Figure 6. Glutathione-s-transferase X X X Down

D. discoideum response to M. marinum is enriched for genes involved in intracellular trafficking, autophagy and phagosome maturation.
GTP-binding proteins and actin GTP-binding proteins commonly belong to small GTPases of the Ras superfamily. Based on sequence and functional similarities, the Ras super family members are further divided into five families: Ras, Rho, Rab, Ran and Arf (reviewed in [1]). In our data, we detected upregulation of genes belonging to several different members of the Ras superfamily such as the Rab family e.g. rab1c and rab8b; the Ras family: rasY, rasZ and rasD and the Rho family: rac1B, racO, rac1C, and racA GTPases. GTPases of these families are often activated by extracellular stimuli, which in turn triggers regulation of e.g. gene expression within the cell [1]. In addition to the induction of small GTPases, the RNA-seq analysis showed that genes for dynamin GTPases, dymA and dymB, are up-regulated where DymA has been shown to associate with F-actin on the early D. discoideum phagosome [2]. The effect on actin dynamics was also reflected in the increased expression of e.g. hatB and comA (Additional file 2).
ESCRT and membranes GO-term enrichment analysis showed that genes connected to Endosomal Sorting Complexes Required for Transport (ESCRT) were up-regulated in response to M. marinum infection. The ESCRT machinery is composed of several complexes and associated proteins and has been connected to a wide range of biological processes [3]. The three main complexes, ESCRT-I -III are conserved in D. discoideum [4]. Although most of the genes associated to ESCRT-I and ESCRT-III were up-regulated in response to M. marinum infection, ESCRT-II genes were unaffected. ESCRT-II is not essential for the function of the ESCRT machinery as ESCRT-I and ESCRT-III can be bridged via the interaction of PDCD6/ALG2, PDCD6IP/Alix and ESCRT-I component TSG101 [5]. In line with this, both the D. discoideum orthologues of PDCD6/ALG2, pefA, and PDCD6IP/Alix, alxA, were up-regulated in our analysis. Furthermore, we detected up-regulation of the ESCRT-associated genes lipopolysaccharide induced tumor necrosis factor (litaf), involved in recruitment of ESCRT-I components to cytoplasmic membranes [6], as well as Vps4-Vta1 complex genes, vps4 and vta1. These findings are further strengthened by a recent study, which showed a recruitment of ESCRT-1 component Tsg101 as well as ESCRT-III components Vps32 and Vps4 to the MCV already 1.5 hpi in response to M. marinum infection in D. discoideum [7]. Furthermore, M. tuberculosis have been shown to interfere with the ESCRT machinery in macrophages, which in turn prevents normal phagosome maturation [8,9].

Autophagy
Many of the genes detected as up-regulated after M. marinum infection are involved in different aspects of autophagy and most of these have previously been characterized mainly by gene knockouts and microscopy [10][11][12][13]. In addition, transcriptional activation of some autophagy related genes, i.e. atg8a, atg8b and atg1, as well as the proposed autophagy receptor sqstm1/p62 has been shown by RT-qPCR [13]. We detect an induction of all these genes in the RNA-seq data, except atg1, which failed to meet the FDR cut off. Also, our data showed increased expression of atg5, atg12 and atg18. In D. discoideum. Atg5-Atg12 complex is associated with phagophore membrane elongation via regulation of the attachment of Atg8 to phosphatidylethanolamine (PE) in the phagophore membrane [14]. Three autophagy receptors have been proposed in D. discoideum; sqstm1/p62, CueA, CnrD [14]. As previously mentioned, sqstm1/p62 is up-regulated early in M. marinum infection. In addition, we found that cnrD is up-regulated while no regulation of cueA was detected.

Genes for transmembrane transporters are downregulated during M. marinum infection.
Compared to the up-regulated genes, a smaller fraction (9%) showed reduced expression. The majority if these were enriched for GO-terms associated with transmembrane transport (Fig.  3, Additional file 4). This set of down regulated genes included genes for three ABCG family transporters, abcG10, abcG12 and abcG17 and two iron transporters, nramp1and mcfF. Surprisingly, the gene coding for the putative copper transporter p80 [15] was down-regulated even though P80 has been shown to accumulate at the MCV [11].

L. pneumophila infection induces expression of genes related to reactive oxygen species ROS
In D. discoideum, ROS production relies on the Toll-Interleukin (TIR) receptor domaincontaining protein gene tirA and the NADPH oxidase genes noxA-C [16]. Of these genes, tirA and noxB were up-regulated both at one and six hours post infection (Additional file 3), corroborating previously reported up-regulation of tirA at one, four and six hours after L. pneumophila infection [17]. In addition, the RNA-seq analysis showed up-regulation of the superoxide dismutase gene sodB six hours post infection. Superoxide dismutase enzymes are involved in ROS production by converting superoxide to hydrogen peroxide [18].

Common transcriptional responses to M. marinum and L. pneumophila infection
In addition to the effect on small GTPases, iron transporters, and RNAi components, the common response to L. pneumophila and M. marinum included vacuolins, similar to flotillins, which are associated with late endosomes in mammalian cells [19]. The D. discoideum vacuolins are encoded by three genes, vacA-C [20]. One of the genes, vacA, was up-regulated in response to both pathogens, while vacB and vacC were up-regulated only in response to M. marinum. Two of them, vacA and vacB, have previously been studied during M. marinum infection in D. discoideum where depletion of vacB caused decreased proliferation of the pathogen while no difference was seen for cells lacking a functional vacA [11].

Only a minimal set of genes are commonly regulated in response to intracellular infection and food bacteria.
Altogether only 20 D. discoideum genes were differentially regulated in response to all three bacteria (L. pneumophila, M. marinum, and E. coli). Of these, nine responded in the same way, i.e. six were up-regulated and three were down-regulated. The products of all three down-regulated genes have proposed functions; two transporters, nramp1 and tmem144b, and one putative glutathione-S-transferase [20]. Only two of the up-regulated genes have suggested functions, the TatD-related DNase iliI and the orthologue to human PDCD6/ALG2 pefA (Additional file 6).