Host-inherent variability influences the transcriptional response of Staphylococcus aureus during in vivo infection.

ARTICLE Received 4 Jul 2016 |Accepted 14 Dec 2016 |Published 3 Feb 2017 Host-inherent variability influences the transcriptional response of Staphylococcus aureus during in vivo infection Robert Tha ¨nert1, Oliver Goldmann1, Andreas Beineke2& Eva Medina1 The rise of antibiotic resistance calls for alternative strategies to treat bacterial infections. One attractive strategy is to directly target bacterial virulence factors with anti-virulence drugs. The expression of virulence traits by pathogens is, however, not constitutive but rather induced by the level of stress encountered within the host. Here we use dual RNA sequencing (RNA-seq) to show that intrinsic variability in the level of host resistance greatly affects the pathogen’s transcriptome in vivo. Through analysis of the transcriptional profiles of host and pathogen during Staphylococcus aureus infection of two mouse strains, shown to be susceptible (A/J) or resistant (C57BL/6) to the pathogen, we demonstrate that the expression of virulence factors is dependent on the encountered host resistance. We furthermore provide evidence that this dependence strongly influences the efficacy of anti-virulence strategies, highlighting a potential limitation for the implementation of these strategies. DOI: 10.1038/ncomms14268 OPEN 1Infection Immunology Research Group, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124 Braunschweig, Germany. 2Institute for Pathology, University of Veterinary Medicine Hannover, Bu ¨nteweg 17, 30559 Hannover, Germany. Correspondence and requests for materials should be addressed to E.M. (email: [email protected]). NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications 1 The rise of antimicrobial resistance is one of the most challenging problems in modern medicine, causing an increase in morbidity and mortality associated with common bacterial infections1. While available antibiotics are loosing their effectiveness, the introduction of novel bactericidal or bacteriostatic antibiotics cannot be considered a long-term solution because it is eventually followed by the emergence of resistant bacterial clones that become increasingly prevalent under selective drug pressure. Consequently, there is a pressing need for new anti-infective agents that do not impose similar levels of selection pressure on pathogens as classical antibiotics. In this regard, alternative approaches based on attenuating bacterial pathogenesis by targeting bacterial virulence, the so-called ‘anti-virulence’ strategies, are emerging as promising tools for the treatment of infections2. Bacterial pathogens express a large repertoire of different virulence factors to survive under the adverse conditions imposed by the host environment. Thus, anti-virulence strategies have been proposed that specifically target bacterial toxins produced by the pathogen to evade host defenses3, bacterial factors mediating adhesion to the host4, secretion systems5as well as regulatory systems6and quorum-sensing signalling7. The key feature of anti-virulence drugs is the attenuation of the pathogen’s virulence to aid clearance by the host’s immune defenses2. These drugs seem attractive, because it is believed that not killing the pathogen directly exerts less selective pressure for the development of resistance2. However, such an approach will only confer therapeutic benefit if the targeted virulence factor(s) are actually expressed by the bacterium during infection and if the natural defense mechanisms of the host are strong enough to clear the pathogen, weakened by the anti-virulence treatment. Bacterial pathogenesis, on the other hand, is strongly influenced by the strength of the host immune defense. For example, avirulent microorganisms can be pathogenic for immunocompromised hosts, whereas virulent microorganisms can be nonpathogenic in immune hosts8. This situation is further complicated by the fact that, in addition to the immune status, inherent characteristics of the host, such as the genetic background, significantly influence the capability of the immune system to overcome invading pathogens. Thus, the response to a specific pathogen can range from weak in susceptible hosts, causing severe infections, to strong in more resistant individuals, resulting in milder diseases. These differences imply that pathogens will encounter stronger immune pressure in resistant than in susceptible hosts and virulence factors that are essential for counteracting a weak immune response may not be the same as those required under stronger immune pressure in resistant hosts. Therefore, the dependence of virulence factor expression on host resistance is a potential limitation for the effectiveness of anti-virulence drugs. Here we investigate how intrinsic variability of host resistance to a pathogen affects the expression of virulence factors needed to successfully infect the host. We use Staphylococcus aureus,a human pathogen that can cause severe invasive infections9and is notorious for its capacity to develop antibiotic resistances. These characteristics make S. aureus one of the most dangerous and intractable infectious pathogens worldwide10. Despite numerous attempts to develop a vaccine that can prevent S. aureus infections, none of the vaccine candidates tested in clinical trials has succeeded so far11. This failure, in combination with the increase of antibiotic resistance, has lead to an intensification of efforts to search for alternative treatment approaches in recent years. In this regards, anti-virulence strategies targeting crucial pathogenicity factors produced by S. aureus during infection have been proposed as an attractive therapeutic option12,13. However, since the outcome of S. aureus infection is strongly influenced by the host factors such as racial origin, age and genetic makeup14,15, the search for anti-virulence targets in S. aureus has to consider the inherent variability of the host responses to infection. Similar to humans, variability in the host response to S. aureus has been also observed among different inbred strains of mice16. While some mouse strains (for example, A/J and DBA/2) are very susceptible to S. aureus infection, C57BL/6 mice are highly resistant and survive a bacterial dose that rapidly kills mice from susceptible strains16. These differences in the capacity to control S. aureus infection provide a unique experimental system to explore the extent to which intrinsic host variability affects the expression of bacterial virulence factors during infection. In this study, we use dual RNA sequencing (RNA-seq), and two mouse strains previously shown to display differential susceptibility to S. aureus infection (strain A/J is susceptible and strain C57BL/6 is resistant)16,17, to investigate how the intrinsic variability of host resistance to a pathogen affects the expression of virulence factors. Dual RNA-seq enables the simultaneous determination of the transcriptional response of the host and pathogen during infection and does not require physical separation of prokaryotic and eukaryotic RNA since the sequencing reads can be assigned to the host or to the pathogen genomes by in silico analysis18. Although dual RNA-seq has been successfully applied to characterize the transcriptional signature of bacteria and host in several in vitro infection systems14,15,19,20, the study presented here is one of the first using this technology in an in vivo system. Our results demonstrate the impact of the host genetic background on the transcriptional response of S. aureus during infection, and provide experimental evidence that host-dependent bacterial expression of virulence factors is a potential limitation for the efficacy of anti-virulence therapies. Results C57BL/6 and A/J mice differ in their resistance to S. aureus. For this study, we selected two strains of mice that differ in their susceptibility to S. aureus infection16,17. Whereas A/J mice are very vulnerable to S. aureus, resulting in a significantly increased bacterial growth in the kidneys (Fig. 1a) and liver (Fig. 1b) and fatal infection outcome (Fig. 1c), C57BL/6 mice exhibit greater resistance to S. aureus and were capable to significantly restrict bacterial growth in the kidneys (Fig. 1a) and liver (Fig. 1b) with all mice surviving (Fig. 1c). Therefore, these two mouse strains provide an excellent tool for investigating the extent to which variability in the host response to infection affects the transcriptional response of S. aureus within the host. Dual RNA-seq analysis of S. aureus and infected host tissue.A dual RNA-seq approach that enables simultaneous transcriptional profiling of bacteria and host tissue was used to characterize the host response to infection and investigate the impact of different levels of host resistance on the transcriptional response of S. aureus. The schematic representation of the experimental design is shown in Fig. 1d. Total RNA including host and pathogen RNA, was isolated from the kidneys of A/J and C57BL/ 6 mice at 48 h of infection and analysed by Illumina deep sequencing. Between 9 and 21 millions reads were uniquely aligned to the reference genome of Mus musculus assembly GRCm38.p3 (GCA_000001635.5), while between 32,228 and 5 millions reads were uniquely mapped to the revised reference genome of S. aureus strain 8325-4 (ref. 21). Transcriptome analysis of S. aureus-infected mice. Hierarchical clustering (Supplementary Fig. 1a) and principal component analysis (PCA) (Supplementary Fig. 1b) of gene expression datasets from infected A/J and C57BL/6 mice, as well as of ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14268 2NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications tissue from uninfected control mice, demonstrated high withingroup reproducibility and substantial between-group differences. Pairwise comparison by permutational multivariate analysis of variance (PERMANOVA) revealed that the differences between all groups were statistically significant (Po0.05, Supplementary Table 1). Differential gene expression analysis using DESeq2 (ref. 22) identified a total of 5,540 differentially expressed genes (DEGs) between uninfected and S. aureus-infected A/J mice, of which 2,756 were found to be up-regulated in response to infection, while 2,784 were downregulated (Supplementary Data 1). In C57BL/6 mice, 3,559 genes were differentially expressed between uninfected and S. aureus-infected mice, of which 1,758 were upregulated and 1,801 were down-regulated in response to infection (Supplementary Data 2). The numbers of unique and overlapping DEGs between S. aureus-infected A/J and C57BL/6 mice are shown in Fig. 2a. Functional classification of the DEGs using KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis, revealed that a large group of host genes with increased expression in response to infection in both A/J and C57BL/6 mice belonged to the groups ‘cytokine-cytokine receptor interaction’ and ‘chemokine signalling pathway’ (Fig. 2b). Particularly, genes encoding inflammatory cytokines such as IL-6, IL-1a, IL-1band TNF-aas well as chemokines involved in the chemoattraction of monocytes/macrophages such as Cxcl1, Cxcl2 and Cxcl3, were upregulated in both A/J and C57BL/6 mice in response to S. aureus infection (Supplementary Data 1 and 2). Also, host genes encoding acute phase proteins such as Saa1, Saa2, haptoglobin and the calcium-binding proteins S100a8 and S100a9 were highly induced in infected A/J and C57BL/6 mice. Although the global analysis of the transcriptional data suggested that a ‘core’ set of inflammation-related genes was highly expressed in both A/J and C57BL/6 mice in response to S. aureus infection, the average fold change of expression in this set of genes was markedly higher in infected A/J than in infected C57BL/6 mice (Fig. 2). This suggested that A/J mice developed a more intense systemic inflammation than C57BL/6 mice in response to S. aureus infection, which is indicative of severe sepsis leading to death. Besides the systemic hyperinflammation, the increased expression of the gene encoding the coagulation activator tissue factor (F3) and of the gene encoding the fibrinolysis inhibitor PAI-1 (Serpine1) in A/J mice (Supplementary Data 1), revealed a net pro-coagulant status that is typical for severe sepsis23. Altered coagulation, coupled with microvascular dysfunction occurring during sepsis, decreases tissue perfusion. This leads to perturbations of oxygen supply, resulting in tissue hypoxia and the activation of the hypoxiainducible factor alpha encoded by Hif1a24,25. The significant induction of Hif1a observed in A/J, but not in C57BL/6 mice, in response to infection (Supplementary Tables 2 and 3), indicated more severe tissue hypoxia in the kidneys of infected A/J mice than in those of C57BL/6 mice. Furthermore, the genes encoding arginase 1 (Arg1) and arginase 2 (Arg2) were expressed to a greater extent in infected A/J than in infected C57BL/6 mice 10 9 85 4 3 7 7100 80 60 40 20 0 0246 Time of infection (days) Percent survival 810 A/J C57BL/6 P < 0.01 * 6 6 A/J C57BL/6 A/J C57BL/6 Resistant C57BL/6 mice 4 × 107 CFU S. aureus 4 × 107 CFU S. aureus 48 h Kidneys Dual RNA-seq Resistant host transcriptome Susceptible host transcriptome Validation with isogenic mutants S. aureus transcriptome in resistant host S. aureus transcriptome in susceptible host Dual RNA-seq Susceptible A/J mice * CFU of S. aureus in kidneys (Log10) CFU of S. aureus in liver (Log10) abc d Figure 1 | A/J and C57BL/6 mice exhibit opposed levels of resistance to S. aureus.Bacterial loads in the kidneys (a) and liver (b) of A/J and C57BL/6 mice at 48 h after intravenous inoculation with 4 107CFU of S. aureus SH1000. Each symbol represents the bacterial counts determined in an individual mouse and the horizontal lines represent the average±s.d. for each mouse strain. One representative experiment out of three independent experiments is shown (n¼6, t-test, *Po0.05). (c) Survival curves of A/J and C57BL/6 mice intravenously infected with 4 107CFU of S. aureus SH1000 (n¼5, log-rank test, Po0.01). (d) Schematic summary of the experimental design for dual RNA-seq analysis. Susceptible A/J mice and resistant C57BL/6 mice were infected intravenously with 4 107CFU of S. aureus SH1000, their kidneys removed at 48 h after bacterial inoculation and subjected to dual RNA-seq analysis to simultaneously determine the gene expression profile of the host and pathogen in the same sample. The genes differentially expressed by S. aureus in A/J and C57BL/6 mice were identified and related to the infection-associated transcriptional response of the corresponding mouse strain. The effect of targeting a virulence factor differentially expressed by S. aureus SH1000 between infection of A/J and C57BL/6 mice was also determined. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14268 ARTICLE NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications 3 (Supplementary Data 1). Arginase can contribute to endothelial cell dysfunction by depleting extra-cellular L-arginine and nitric oxide (NO) bioavailability, leading to endothelial nitric oxide synthase uncoupling and consequently to the production of high levels of harmful reactive oxygen species (ROS)26. Taken together, these findings suggest that the micro-environment in the infected tissue is highly different between A/J and C57BL/6 mice, which could significantly affect the expression of virulence determinants by S. aureus. S. aureus transcriptome during infection of A/J or C57BL/6. In parallel, we analysed the transcriptome of S. aureus during the infection of resistant C57BL/6 and susceptible A/J mice to determine the impact of the different physiological conditions present at the site of infection on the pathogen’s transcriptional response. Hierarchical ordination (Supplementary Fig. 2a) and PCA (Supplementary Fig. 2b) showed high within-group reproducibility, while PERMANOVA demonstrated that the transcriptional response of S. aureus infecting susceptible A/J mice differed significantly from that of S. aureus infecting resistant C57BL/6 mice (Supplementary Table 2). Transcript abundance was determined by normalizing the number of raw reads in each data set for gene length and expressed as transcripts per Kilobases per Million (TPM) (Supplementary Data 3). A total of 85 genes were identified as differentially expressed (probability value Z0.95) by S. aureus between infection of A/J and infection of C57BL/6 mice using NOISeq (Supplementary Data 4). Of those, transcripts of 20 genes were more abundant in S. aureus infecting A/J mice (Fig. 3a, Table 1, Supplementary Table 4), 65 genes exhibited greater expression in S. aureus during infection of C57BL/6 mice (Fig. 3a, Table 2, Supplementary Table 5) and 594 were expressed at a similar level by S. aureus in A/J and C57BL/6 mice (Fig. 3a, Supplementary Data 4). One of the most prominent operons expressed by S. aureus to a greater extent during infection of A/J mice, than during infection of C57BL/6 mice, was the arc operon, which encodes the arginine Increased expression A/J A/J 1,722 1,012 Log2 fold change 2 Toll-like receptor signalling pathway T cell receptor signalling pathway RIG-I-like receptor signalling pathway Natural killer mediated cytotoxicity NOD-like receptor signalling pathway Intestinal immune network for IgA production Hematopoietic cell lineage Cytosolic DNA-sensing pathway Cytokine-cytokine receptor interaction Complement and coagulation cascades Chemokine signalling pathway B cell receptor pathway A/J 0 20 40 60 80 100 Number of genes C57BL/6 A/J C57BL/6 3456 789 C57BL/6 C57BL/6 791 967 1,789 Decreased expression a b Figure 2 | Gene expression analysis of S. aureus-infected kidneys from A/J and C57BL/6 mice. (a) Venn diagram showing the number of DEGs with increased (left) or decreased (right) expression in response to S. aureus infection that are unique or common between A/J and C57BL/6 mice. (b) Heat map of the mean log 2 -fold change of gene expression of the significantly up-regulated genes determined between S. aureus-infected versus uninfected A/J and C57BL/6 mice within the KEGG ‘immune system’ category (left part). The corresponding numbers of significantly up-regulated genes in response to S. aureus infection in A/J (blue bars) and C57BL/6 (red bars) are shown in the right part of the figure. Table 1 | DEGs between S. aureus infecting A/J and C57BL/6 mice with greater transcript abundance during infection of A/J mice. Locus tag Gene symbol Description SAOUHSC_00845 Hypothetical SAOUHSC_02853 Hypothetical SAOUHSC_00371 yflT Hypothetical SAOUHSC_02964 arcR Hypothetical SAOUHSC_01477 Hypothetical SAOUHSC_01969 gvpP Hypothetical SAOUHSC_00101 drm Phosphopentomutase SAOUHSC_01181 xynA Hypothetical SAOUHSC_02967 arcD Arginine/ornithine antiporter SAOUHSC_01191 rpmB 50S ribosomal protein L28 SAOUHSC_00686 Hypothetical SAOUHSC_01803 aapA Hypothetical SAOUHSC_02862 clpL ATP-dependent Clp protease, ATP-binding subunit ClpC SAOUHSC_01403 cspA Cold shock protein SAOUHSC_02850 cidB Hypothetical SAOUHSC_01002 qoxB quinol oxidase AA3 subunit II SAOUHSC_01024 graF Hypothetical SAOUHSC_02702 Hypothetical SAOUHSC_02697 tcyC Amino acid ABC transporter ATP-binding protein SAOUHSC_02665 Hypothetical DEG, differentially expressed gene. The complete data of the differentially expressed genes with higher expression by S. aureus during infection of the susceptible A/J mice are displayed in Supplementary Table 4. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14268 4NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications deiminase (ADI) system (Fig. 3b, Supplementary Data 4). The arc operon comprises the genes coding for arginine deiminase (arcA), ornithine transcarbamylase (arcB), carbamate kinase (arcC), the arginine/ornithine antiporter (arcD) and the transcriptional regulator ArcR (arcR). These enzymes catalyse the conversion of arginine to ornithine, ammonia, and CO 2 , while producing ATP. This not only enables S. aureus to utilize arginine as an energy source under anaerobic conditions27 but also generates ammonia, which confers protection against acidic stress28. The expression of all genes of the ADI pathway was greater in S. aureus during infection of A/J than during infection of C57BL/ 6 mice, even though only the differences in the level of arcR and arcD expression reached statistical significance (false discovery rate (FDR) o0.05, NOISeq analysis), (Fig. 3b, Supplementary Data 4). Other genes, which were differentially expressed by S. aureus during infection of A/J and C57BL/6 mice, were those encoding proteins for amino acid transport and biosynthesis. Thus, transcripts of the genes encoding a hypothetical cysteine permease (tcyC), a hypothetical lysine permease (lysP2), glutamate synthase (gltB, gltD) and homoserine dehydrogenase (dhoM) were more abundant in S. aureus during infection of C57BL/6 mice than during infection of A/J mice (Table 2). Because de novo amino acid synthesis pathways and amino acids transport systems are under control of catabolite repression in the presence of preferred carbon sources29 in S. aureus, the increased expression of the genes encoding these pathways during infection of C57BL/6 mice is consistent with more severe nutrient limitation in the tissue microenvironment of C57BL/6 than in the tissue of A/J mice. This nutrient limitation leads to de-repression of genes that enable S. aureus to use secondary carbon sources such as amino acids. Furthermore, genes encoding exoproteases such as staphopain (sspB2), serine proteases (sspA, splA,splE and splF), staphostatin B (sspC), aureolysin (aur) and a cysteine protease (sspB) were also expressed by S. aureus to a greater extent in C57BL/6 than in A/J mice (Fig. 4a). These proteases can generate peptides in the extracellular environment that can be imported by the bacterium via specialized transport systems and used to retrieve amino acids30. Besides their role in metabolism, extracellular proteases are potent virulence factors that help S. aureus to evade the host immune defenses31,32. Other genes encoding important virulence factors such as the immunodominant staphylococcal antigen B (isaB), the extracellular fibrinogen binding protein Efb (efb), which is involved in inhibition of phagocytosis33, the pore-forming cytolysin alpha-toxin (hla), and the amphipathic a-helical phenol-soluble modulins psma1, psma2 and psma3, which can kill host cells by damaging the plasma membrane34, were also expressed by S. aureus to a higher extent during infection of C57BL/6 mice (Fig. 4b). These findings indicate that S. aureus express greater levels of virulence factors during infection of C57BL/6 than during infection of A/J mice, which is most probably driven by the different growth phase of the bacteria in the two mouse strains. The expression of these virulence factors is controlled by regulatory elements such as two-component regulatory systems (TCRS) and transcriptional regulatory systems in response to environmental cues encountered by the bacterium during infection35. The staphylococcal quorum-sensing system accessory gene regulator (agr) was found highly expressed by S. aureus during infection of both A/J and C57BL/6 mice. The agr system comprises two divergent transcripts, RNAII and RNAIII, which are under the control of two distinct promoters, P2 and P3, respectively36. RNA II encodes the quorum-sensing elements AgrB, AgrD, AgrC and AgrA that represent an autocatalytic sensory transduction system. RNAIII encodes delta-hemolysin (hld)37 and is a major regulator of virulence factors in S. aureus, inducing the transcription of various extracellular proteases and toxins38. Although the genes encoding the agr P2 operon SAO in A/J mice 500 400 300 200 100 0 500 400 300 200 100 0 arcA arcB arcD arcC arcR arcA arcB arcD arcC arcR TPM TPM 500 400 300 200 100 0 TPM 500 400 300 200 100 0 TPM 600 800 1,000 * * 400 200 0 TPM SAO in C57BL/6 mice SAO in A/J 20 65 594 SAO in C57BL/6 a b Figure 3 | Gene expression analysis of S. aureus during infection of A/J and C57BL/6 mice. (a) Venn diagram showing the number of unique and common expressed genes between S. aureus infecting A/J and C57BL/6 mice based on differential gene expression analysis determined with NOISeq. (b) Gene composition and organization of the genes of the ADI operon and their level of expression in S. aureus during infection of A/J (blue bars) or C57BL/6 (red bars) mice. Each bar represents the mean of TPM±s.d. of triplicates. NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14268 ARTICLE NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications 5 (agrBDCA) were expressed by S. aureus to a similar level in both mouse strains, the expression level of RNAIII/hld was greater during infection of C57BL/6 mice (Fig. 4c, Supplementary Data 4). The gene encoding the transcriptional regulator SarR was also upregulated by S. aureus infecting C57BL/6 mice (Fig. 4c, Supplementary Data 4). This could explain the higher level of transcripts encoding proteases and toxins detected in S. aureus during infection of C57BL/6 mice, since both RNAIII (ref. 38) and SarR39 activate their transcription. Antimicrobial peptides (AMPs) are an important part of the host innate immune defense against S. aureus by directly impairing the integrity of the bacterial cell wall40. The bacterial gene encoding phosphatidylglycerol lysyltransferase (mprF), which is part of the cell wall stress stimulon and mediates Table 2 | DEGs between S. aureus infecting A/J and C57BL/6 mice with greater transcript abundance during infection of C57BL/6 mice Locus tag Gene symbol Description SAOUHSC_02260 hld Delta-hemolysin SAOUHSC_00411.1 psma1 Alpha phenol soluble modulin SAOUHSC_02566 sarR Hypothetical SAOUHSC_02971 aur Zinc metalloproteinase aureolysin SAOUHSC_00435 gltB Glutamate synthase large subunit SAOUHSC_01788 thrS Threonyl-tRNA synthetase SAOUHSC_00987 sspB Cysteine protease SAOUHSC_00248 lytM Peptidoglycan hydrolase SAOUHSC_02571 ssaA Secretory antigen SAOUHSC_00427 sle1 Autolysin SAOUHSC_02941 nrdG Hypothetical SAOUHSC_01001 qoxA Quinol oxidase subunit I SAOUHSC_00964 Hypothetical SAOUHSC_00401 Hypothetical SAOUHSC_00717 saeP Hypothetical SAOUHSC_00741 nrdI Ribonucleotide reductase stimulatory protein SAOUHSC_01942 splA Serine protease SplA SAOUHSC_00083 sbnI Hypothetical SAOUHSC_00348 rpsF 30S ribosomal protein S6 SAOUHSC_00436 gltD Glutamate synthase subunit beta SAOUHSC_00051 plc 1-phosphatidylinositol phosphodiesterase SAOUHSC_00411.2 psma2 Alpha phenol soluble modulin SAOUHSC_01121 hla Alpha-hemolysin SAOUHSC_00272 Hypothetical SAOUHSC_00801 secG Preprotein translocase subunit SecG SAOUHSC_01935 splF Serine protease SplF SAOUHSC_02369 rpoE DNA-directed RNA polymerase subunit delta SAOUHSC_00268 Hypothetical SAOUHSC_01110 efb Fibrinogen-binding protein-like protein SAOUHSC_01688 lepA GTP-binding protein LepA SAOUHSC_02855 amiD2 LysM domain-containing protein SAOUHSC_02762 Hypothetical SAOUHSC_02114 dagK Putative lipid kinase SAOUHSC_02372 Hypothetical SAOUHSC_02430 htsA ABC transporter periplasmic binding protein SAOUHSC_01320 dhoM Homoserine dehydrogenase SAOUHSC_00986 sspC Cysteine protease SAOUHSC_00411.3 psma3 Alpha phenol soluble modulin SAOUHSC_02112 Hypothetical SAOUHSC_02972 isaB Immunodominant antigen B SAOUHSC_02885 Hypothetical SAOUHSC_01326 lysP2 Hypothetical SAOUHSC_02127 sspB2 Staphopain thiol proteinase SAOUHSC_01936 splE Serine protease SplE SAOUHSC_00728 ltaS Hypothetical SAOUHSC_00625 mnhA Putative monovalent cation/H þantiporter subunit A SAOUHSC_02763 opp-1F Peptide ABC transporter ATP-binding protein SAOUHSC_00988 sspA Glutamyl endopeptidase SAOUHSC_00711 Hypothetical SAOUHSC_00561 vraX Hypothetical SAOUHSC_02550 FdhD Formate dehydrogenase accessory protein SAOUHSC_00875 ndh2 Hypothetical SAOUHSC_01359 mprF Hypothetical SAOUHSC_01192 vfrA Hypothetical SAOUHSC_02887 isaA Immunodominant antigen A SAOUHSC_02254 groEL chaperonin GroEL SAOUHSC_02485 rpoA DNA-directed RNA polymerase subunit alpha SAOUHSC_01462 gpsB Hypothetical SAOUHSC_00367 tcyP Hypothetical SAOUHSC_01062 Hypothetical SAOUHSC_00893 namA FMN oxidoreductase SAOUHSC_00144 ausA Hypothetical SAOUHSC_00652 fhuA Iron compound ABC transporter ATP-binding protein SAOUHSC_02883 ssaA LysM domain-containing protein SAOUHSC_01431 msrB Methionine sulfoxide reductase B DEG, differentially expressed gene. The complete data of the differentially expressed genes with higher expression by S. aureus during infection of the resistant C57BL/6 mice are displayed in Supplementary Table 5. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14268 6NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications resistance to cationic AMPs, by reducing the negative charge of the membrane surface41, was expressed by S. aureus to a larger extent during infection of resistant C57BL/6 than during infection of A/J mice. Similarly, other genes of the cell wall stress stimulon including vraX, autolysin encoding genes (ssaA, lytM, amiD2 and isaA), genes involved in cell separation (gpsB and sle1), groEL encoding a chaperonin, and the lipoteichoic acid synthase encoding gene ltaS exhibited greater expression levels in S. aureus during infection of C57BL/6 mice than during infection of A/J mice. The higher induction of these genes might reflect an adaptive response of S. aureus to keep cell wall integrity when confronted with high levels of cell wall damaging agents in the tissue of C57BL/6 mice. Notably, genes related to central metabolism, iron acquisition, general and oxidative stress responses were expressed by S. aureus to a similar extent during infection of A/J and C57BL/6 mice (Supplementary Data 4), indicating that their expression was not influenced by the levels of host resistance to infection. The RNA-seq results were validated in a selected set of genes from host and pathogen by quantitative reverse transcription– PCR (RT–PCR; Supplementary Fig. 3) and at the protein level by ELISA (Supplementary Fig. 4). Effect of decreased host resistance on S. aureus transcription. Next, we determined if depressing specific mechanisms of host resistance in C57BL/6 mice affected the transcriptional response of S. aureus during infection. First, we hypothesized that apolipoprotein B (ApoB), the major structural protein of very lowdensity (VLDL) and low-density lipoproteins (LDL), contributes to the resistance of C57BL/6 mice to S. aureus in our infection model, because the gene encoding Apo B (Apob) was expressed to a greater extent in the kidneys of C57BL/6 than in the kidneys of A/J mice at both gene expression (Fig. 5a) and protein level (Fig. 5b), before and after infection (Fig. 5c). Furthermore, ApoB has been shown to contribute to the host defense against S. aureus in experimental models of skin42 and respiratory43 infection by antagonizing the agr quorum sensing system of S. aureus. To validate this hypothesis, A/J and C57BL/6 mice were treated with 4-aminopyrazolopyrimidine (4-APP), a drug that impairs low-density lipoprotein secretion44, and subsequently infected intravenously with S. aureus. Whereas inhibition of ApoB rendered C57BL/6 mice more susceptible to S. aureus, demonstrated by the significantly higher bacterial loads in kidneys at 48 h infection (P-valueo0.05, t-test), (Fig. 5d), it did not affect the level of susceptibility of A/J mice (Fig. 5d). Next, we investigated if the reduced resistance in C57BL/6 mice after attenuation of ApoB secretion impacted the transcriptional response of S. aureus during infection. For this purpose, we compared the expression of a set of virulence-related genes in S. aureus infecting 4-APP-treated C57BL/6 mice with the expression of the same set of genes in S. aureus infecting C57BL/6 mice treated with vehicle alone. The results show that the level of expression of all genes tested hld/RNAIII, sarR,hla, sspA,aur,vraX and gltB was decreased by 4-APP-treatment, while the expression of the hypothetical alanine permease (aapA) was higher in S. aureus infecting 4-APP-treated than in vehicletreated C57BL/6 mice (Table 3). These genes were also found to be differentially expressed between S. aureus infecting C57BL/6 mice and S. aureus infecting C57BL/6 mice deficient in the expression of MyD88, an adaptor molecule that is essential for the signalling of IL-1R/TLR family (Table 3). Since MyD88-deficient mice are more susceptible to S. aureus than wild type C57BL/6 mice45, these observations further demonstrated the remarkable influence of the level of host resistance on the transcriptional response of S. aureus during infection. Target expression affects efficacy of anti-virulence approaches. After having demonstrated the influence of the levels of host resistance on both the quality and quantity of S. aureus transcriptional response, we sought to determine the consequences of this dependence on the effectiveness of anti-virulence strategies. For this purpose, we assessed the effect of neutralizing a virulence factor that differed in expression between S. aureus infecting A/J and C57BL/6 mice on the bacteria fitness during infection. We chose aureolysin, which has been shown to be important for full virulence of S. aureus in experimental infection models46,47 and was expressed to a significantly greater extent by S. aureus during infection of C57BL/6 than during infection of A/J mice in our study (FDRo0.05, NOISeq analysis). C57BL/6 and A/J mice were simultaneously challenged with wild type and an aureolysindeficient strain of S. aureus (wild type, Daur) and the amount of each bacterial strain was determined in the kidneys of infected mice at 48h of infection. The overall amount of S. aureus bacteria counting both wild type and Daur strains was significantly greater (P¼0.0138, t-test) in the kidneys of A/J (1.3 108±4.3 107) than in the kidneys of C57BL/6 mice (1.03 107±5.4 106). Moreover, while a lower amount of Daur than wild type S. aureus was recovered from C57BL/6 mice, the amount of Daur recovered from A/J mice was comparable to that of wild type strain (Fig. 6a). Thus, the Daur had a competitive disadvantage when coadministered with wild type S. aureus in C57BL/6 mice (mean competitive index for Daur/wild type S. aureus of 0.013), while Daur and wild type S. aureus were equally competitive after co-administration in A/J mice (mean competitive index for Daur/wild type S. aureus of 1.595) (Fig. 6b). Taken together, these results indicate that the efficacy of targeting a virulence factor by anti-virulence strategies will strongly depend on its level of expression by the pathogen during infection, which in turn is highly influenced by the intrinsic levels of host resistance to infection. Discussion Anti-virulence strategies based on attenuation of bacterial pathogenesis by the specific inhibition of virulence factors essential for the pathogen’s survival during infection48, have received increasing attention as novel treatment options for infections caused by antibiotic-resistant pathogens48. The concept of anti-virulence therapy is still very much in its infancy and therefore more research is needed to explore its practicability. One important aspect that should be considered carefully when designing anti-virulence strategies is that the expression of virulence traits by the pathogens is not constitutive but rather influenced by the specific environment encountered during infection. Consequently, absent expression of the targeted virulence factors could render anti-virulence strategies completely ineffective. Therefore, it is essential to understand the impact of the wide-ranging, inter-individual variation of the host response on the pathogen’s expression of virulence determinants during infection. Our study supports the idea that the host genetic background affects the transcriptional response of S. aureus during infection. The limited capability of the immune defense mechanisms of susceptible A/J mice to control S. aureus growth led to the development of an intense inflammatory response, apparent by the disproportionate expression of inflammatory cytokines and damage-associated molecular patterns. Transcriptional reprogramming of the eukaryotic cells, possibly resulting from the concomitant accumulation of acidic products and the lowered oxygen tension (hypoxia) in the infected tissue, involved the induction of Hif1a that encodes the central mediator of transcriptional responses to hypoxia HIF-1a49,50. Therefore, the NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14268 ARTICLE NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications 7 major challenge faced by S. aureus in susceptible A/J mice seems to be the adaptation to the adverse conditions imposed by the hyperinflammatory response and hypoxic microenvironments. To survive in the septic A/J mice, S. aureus increased expression of the ADI operon (arcABDCR), which is generally induced under anaerobic conditions27 and is important for energy generation, but also protects S. aureus against acidic stress28. The superior resistance mechanisms of C57BL/6 mice against S. aureus enabled a better control of bacterial multiplication. Therefore, S. aureus faced the main challenge of counteracting the powerful host defense mechanisms of the resistant C57BL/6 mice. The transcription data indicated that S. aureus responded to the adverse environment encountered within C57BL/6 mice by increasing the expression of cytotoxins and extracellular proteases. Cytotoxins such as alpha-hemolysin (hla) and PSMs (psma1-3 and hld) help S. aureus to avoid phagocytic killing by inducing pores in the membrane of host cells, leading to cell death51 and can promote bacterial spreading by disrupting the 2,000 20,000 10,000 8,000 6,000 4,000 2,000 SAO in A/J mice SAO in C57BL/6 mice 0 40,000 1,000 3,000 SarR –+ + SarR * 2,500 2,000 1,500 1,000 500 0 1,000 2,000 3,000 4,000 5,000 6,000 0 800 700 600 * * * * * * * ** * * TPM TPM TPM TPM TPM TPM 500 400 300 200 100 0 sspB2 sspB sspA sspC AIP AgrB AgrD AgrA AgrC P P P3 P2 + RNAIII RNAII agrA agrA agrC aur 600 0 25,000 50,000 75,000 100,000 125,000 150,000 175,000 500 400 300 200 * * 100 0aur agrC agrB agrB agrD agrD hld hld hld AIP O SM I F D CT S Y O SM I F D CT S Y spIA spIE spIF psma1 psma2 psma3 hla ab c Figure 4 | Expression of selected genes by S. aureus during infection of C57BL/6 or A/J mice. (a) Expression levels of genes encoding proteases. (b) Expression levels of genes encoding toxins. (c) Schematic representation and level of expression of genes encoding the global regulator Agr and SarR. Red bars show expression values of the genes expressed by S. aureus in C57BL/6 mice and blue bars show expression values of the genes expressed by S. aureus in A/J mice. Each bar represents the mean TPM±s.d. of triplicates. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14268 8NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications epithelial barrier52. The strong induction of genes encoding key exoproteases, including the metalloproteinase aureolysin (aur), serine proteases (splA,sspA,splE and splF), staphostatin B (sspC) and cysteine protease (sspB), further accentuates the importance of immune evasion for S. aureus survival in C57BL/6 mice. These proteases can cleave and degrade components of the complement system53 and can inhibit neutrophil chemotaxis54. The expression of these virulence determinants is orchestrated through transcriptional and post-transcriptional regulation by regulatory systems. The expression of RNAIII, a major regulator of these factors38, was higher expressed by S. aureus during infection of C57BL/6 mice than in A/J mice, while the expression of the autocatalytic sensory transduction system did not differ between both mouse strains. This apparent discrepancy could, however, be explained by greater transcript abundance of SarR in S. aureus infecting C57BL/6 mice, which mitigates the expression of the agr P2 operon, while having no apparent effect on the expression of RNAIII (ref. 55). Moreover, SarR enhances the expression of genes encoding several extracellular proteases regulated by RNAIII (ref. 39) and, therefore, may act in synergy with RNAIII to boost the expression of virulence factors, required for survival under the strong immune pressure in C57BL/6 mice. The strong immune pressure in the resistant mice could also explain the greater expression of several genes of the cell wall stress stimulon by S. aureus infecting C57BL/6 mice. It has been shown that the magnitude of cell wall stimulon induction strongly 600 Uninfected A/J C57BL/6 A/J C57BL/6 *** 10 8 6 4 2 Vehicle Vehicle 4-APP 4-APP *** ** Fold change in Apob expression (C57BL/6 relative to A/J) Fold change in Apob expression (C57BL/6 relative to A/J) CFU S.aureus in kidneys (Log10) 400 200 0 RNA-Seq RNA-Seq 25 20 15 10 5 0 S. aureus RT-PCR RT-PCR ab cd Figure 5 | Apolipoprotein B (ApoB) contributes to resistance against S. aureus bloodstream infection in C57BL/6 mice. (a) Relative fold change of Apob expression values in the kidneys of uninfected C57BL/6 respect to those in the kidneys of uninfected A/J mice determined by either RNA-Seq (white bars) or qRT–PCR (black bars). Each bar represents the mean relative fold change±s.d. of triplicates. (b) Expression of ApoB in kidneys of A/J (left) and C57BL/ 6 (right) mice determined by immunostaining of kidney tissue using specific antibodies against ApoB. Magnification X40. (c) Relative fold change of Apob expression values in the kidneys of S. aureus-infected C57BL/6 in comparison to those in the kidneys of S. aureus-infected A/J mice at 48 h of infection determined by either RNA-Seq (white bars) or qRT-PCR (black bars). Each bar represents the mean relative fold change±s.d. of triplicates. (d) Bacterial loads in the kidneys of A/J (blue symbols) and C57BL/6 (red symbols) mice treated with 4-Aminopyrazolo[3,4-d]pyrimidine (4-APP) (open symbols) or with vehicle alone (solid symbols) at 48 after intravenous inoculation with 2 107CFU of S. aureus strain SH1000. Each symbol represents the bacterial counts determined in an individual mouse and the horizontal lines represent the average±s.d. for each mouse strain (n¼6, t-test, **Po0.01, ***Po0.001). Table 3 | S. aureus gene expression during infection of 4-APP-treated and MyD88-deficient C57BL/6 mice. Locus tag Gene symbol Description Relative fold change in 4-APP-treated to vehicle-treated C57BL/6 mice (mean (s.e.)) Relative fold change in MyD88deficient to wild type C57BL/6 mice (mean (s.e.)) SAOUHSC_02265 agrA Accessory gene regulator protein A 1.60 (0.22) 1.62 (0.5) SAOUHSC_02566 sarR Accessory regulator R 1.83 (0.22) 1.23 (0.09) SAOUHSC_01121 hla Alpha-hemolysin 3.32 (1.56) 18.06 (0.16) SAOUHSC_02971 aur Zinc metalloproteinase aureolysin 2.31 (0.96) 2.59 (0.75) SAOUHSC_00988 sspA Glutamyl endopeptidase 2.19 (0.01) 2.32 (0.46) SAOUHSC_00561 vraX Protein VraX 3.91 (2.76) 1.11 (0.04) SAOUHSC_00436 gltD Glutamate synthase subunit beta 2.41 (1.11) 6.03 (1.66) SAOUHSC_01803 aapA D-serine/D-alanine/glycine transporter 1.21 (2.32) 1.69 (0.29) RNAIII Regulatory RNA 1.80 (0.53) 2.52 (0.08) Level of expression of a subset of genes by S. aureus during infection of 4-APP-treated compared with vehicle-treated C57BL/6, respectively MyD88-deficient compared with wild-type C57BL/6 mice determined by quantitative reverse transcription–PCR (qRT–PCR). NATURE COMMUNICATIONS | DOI: 10.1038/ncomms14268 ARTICLE NATURE COMMUNICATIONS | 8:14268 | DOI: 10.1038/ncomms14268 | www.nature.com/naturecommunications 9