Tissue Dual RNA-Seq Allows Fast Discovery of Infection

Home , Bacterial small RNA

Tissue dual RNA-seq allows fast discovery of PNAS PLUS infection-specific functions and riboregulators shaping host–pathogen transcriptomes

Aaron M. Nussa,1, Michael Beckstettea,1, Maria Pimenovaa, Carina Schmühla, Wiebke Opitza, Fabio Pisanoa, Ann Kathrin Herovena, and Petra Derscha,2

aDepartment of Molecular Infection Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany

Edited by Ralph R. Isberg, Howard Hughes Medical Institute/Tufts University School of Medicine, Boston, MA, and approved December 19, 2016 (receivedfor review August 14, 2016) Pathogenic bacteria need to rapidly adjust their virulence and fitness The recent development of “dual RNA-seq” (using probe-in- program to prevent eradication by the host. So far, underlying dependent parallel cDNA sequencing) now offers the possibility to adaptation processes that drive pathogenesis have mostly been simultaneously profile RNA expression in the pathogen and in- studied in vitro, neglecting the true complexity of host-induced fected host cells (10). Taking advantage of this technology, high- stimuli acting on the invading pathogen. In this study, we developed resolution transcriptome profiles have lately been obtained from an unbiased experimental approach that allows simultaneous mon- HeLa cells invaded by Salmonella enterica serovar Typhimurium, itoring of genome-wide infection-linked transcriptional alterations of primary human bronchial epithelial cells infected with Haemophilus the host and colonizing extracellular pathogens. Using this tool for influenza, and macrophages with intracellular Mycobacterium tu- Yersinia pseudotuberculosis- infected lymphatic tissues, we revealed berculosis (11–13). These studies not only revealed novel insights numerous alterations of host transcripts associated with inflamma- into pathogen–host cell cross-talk, but they also illustrated the tory and acute-phase responses, coagulative activities, and transition importance of ncRNAs for fine-tuned intracellular bacterial gene metal ion sequestration, highlighting that the immune response is expression and how it manipulates host cells. dominated by infiltrating neutrophils and elicits a mixed TH17/TH1 ’ As a major drawback, this approach does not address patho- response. In consequence, the pathogen s response is mainly di- gens that live and replicate in extracellular spaces either within rected to prevent phagocytic attacks. Yersinia up-regulates the gene tissues or on the surface of the epithelia that line body cavities and expression dose of the antiphagocytic type III secretion system during the natural course of an infection. Moreover, although cell (T3SS) and induces functions counteracting neutrophil-induced ion culture models are immensely useful for dissecting out pathways deprivation, radical stress, and nutritional restraints. Several conserved bacterial riboregulators were identified that impacted this and the molecular details, they exclude the influence of the host response. The strongest influence on virulence was found for the immune response and cannot substitute for the whole-animal/ loss of the carbon storage regulator (Csr) system, which is shown tissue context. To overcome this limitation, we established a fast, to be essential for the up-regulation of the T3SS on host cell contact. generic tissue dual RNA-seq approach, which allows compre- In summary, our established approach provides a powerful tool for hensive and simultaneous monitoring of infection-linked gene the discovery of infection-specific stimuli, induced host and pathogen expression changes of the infected host and the colonizing responses, and underlying regulatory processes. Significance tissue dual RNA-seq | host–pathogen interaction | host-adapted metabolism | noncoding RNAs | Yersinia Our knowledge of the functions required by extracellular bac-

terial pathogens to grow in host tissues is still limited. Most MICROBIOLOGY ll bacterial pathogens encounter rapidly changing environ- available information refers to studies conducted under labora- Amental conditions and adverse host reactions during the tory growth conditions that mimic host environments but ex- course of infection. Accordingly, a dynamic cascade of events is clude the influence of the host immune system. Tissue dual RNA initiated that triggers a global alteration of gene expression pat- sequencing allows simultaneous transcript profiling of a patho- terns in both interacting organisms to adapt for survival. Moni- gen and its infected host. This sensitive approach led to the toring these infection-linked transcriptome alterations represents identification of host immune responses and virulence-relevant bacterial functions that were not previously reported in the a powerful approach to identify virulence-related factors and context of a Yersinia infection. Application of this tool will allow regulatory processes that drive pathogenesis. Over the past years, transcript profiling of other pathogens to unravel concealed RNA sequencing (RNA-seq) technologies have revolutionized the gene functions that are crucial for survival in different host analyses of transcriptomes by enabling a highly accurate and niches and will improve identification of potential drug targets. sensitive transcriptional profiling on single-nucleotide resolution. Moreover, RNA-seq has permitted the identification of sensory Author contributions: A.M.N., M.B., and P.D. designed research; A.M.N., M.B., M.P., C.S., RNA elements and numerous noncoding RNAs (ncRNAs) in W.O., F.P., A.K.H., and P.D. performed research; A.M.N., M.B., M.P., C.S., W.O., A.K.H., and pathogenic bacteria. A small subset of the few already character- P.D. analyzed data; and A.M.N., M.B., and P.D. wrote the paper. ized riboregulators was found to contribute to pathogenesis (1–3). The authors declare no conflict of interest. Most global gene expression studies of pathogens relied on This article is a PNAS Direct Submission. laboratory growth conditions, which mimic certain host environ- Data deposition: The raw high-throughput read data have been deposited as FASTQ files – in the European Nucleotide Archive (ENA; accession no. PRJEB14242). Split and processed ments (4 6). Only a few RNA-seq studies have used cell culture FASTQ files as well as corresponding BAM files have been deposited in the ENA (accession systems or fluids of infected animals to profile pathogen gene no. PRJEB17683). expression in vivo, but they have commonly neglected the host 1A.M.N. and M.B. contributed equally to this work. – response (7 9). Consequently, our knowledge about the temporal 2To whom correspondence should be addressed. Email: [email protected]. events and gene expression changes in both the pathogen and the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. host that drive pathogenesis is still very limited. 1073/pnas.1613405114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1613405114 PNAS | Published online January 13, 2017 | E791–E800 Downloaded by guest on September 26, 2021 pathogen. In contrast to traditional microarray-based studies, this approach is species-independent (i.e., it does not require the time- consuming design of pathogen- and host-specific custom chips) and can concurrently capture the full-transcript repertoire of the pathogen and the host. We successfully applied this method to investigate the global gene expression pattern of the enteropathogenic bacterium Yersinia pseudotuberculosis growing extracellularly in the gut-associated lymphoid follicles (Peyer’s patches) of infected mice. The obtained high-resolution transcriptomes confirmed known host–pathogen interactions and revealed insights into infection-specific stimuli that induce host responses and provoke bacterial gene expression driving pathogenesis. Collec- tively, our findings show that tissue dual RNA-seq is a fast and powerful tool that can be applied to a broad range of bacteria and host tissues/organs to discover virulence-related functions and defense reactions of the host. Results and Discussion Tissue Dual RNA-Seq of Y. pseudotuberculosis-Infected Peyer’sPatches. We established tissue dual RNA-seq using Y. pseudotuberculosis ’ proliferating in the gut-associated lymphoid tissue (Peyer s patches) Fig. 1. Tissue dual RNA-seq workflow and global reports. (A) For tissue dual of infected mice. The Peyer’s patch is the primary replication site of RNA-seq, female BALB/c mice were orally infected with 2 × 108 cfu Y. pseu- enteropathogenic yersiniae after transcytosis of the intestinal epi- dotuberculosis IP32953 (infected) or 1× PBS (uninfected). For in vitro RNA-seq, thelium. Inside the Peyer’s patches, the bacteria replicate extracel- IP32953wasgrowninLBtoexponentialorstationaryphaseat25°Cor37°C. lularly to high numbers (107–108 cfu/g tissue) (14). A striking feature Total RNA was isolated from Peyer’s patches of mice or bacterial cultures of the pathology of an enteric Yersinia infection is inflammation of processed for preparation of strand-specific barcoded cDNA libraries and se- the intestinal tissue. The murine infection model, which mimics all quenced (SI Appendix). cDNA reads were separated in silico by mapping to the aspects of the human disease, has been proven extremely valuable mm10 and the IP32953 genomes. TAP, tobacco acid pyrophosphatase. (B) Circos plot visualizing reads per kilobase transcript length per million mapped for the characterization of host responses (e.g., cytokine signaling – reads-normalized expression values of in vitro and in vivo RNA-seq data pathways) involved in infection control (15 17). The well-defined for the IP32953 chromosome (NC_006155.1) and pYV virulence plasmid host response associated with the colonization of the Peyer’s (NC_006153.2). E25, exponential phase 25 °C; E37, exponential phase 37 °C; in patches (mainly obtained with Yersinia enterocolitica) will allow us to vivo, infected Peyer’s patches; S25, stationary phase 25 °C; S37, stationary prove the validity of our approach and helps to reveal different and phase 37 °C. (C) Heat map of Euclidian sample distances of rlog-transformed hidden pathogen-triggered host reactions. read counts for triplicate mouse RNA-seq data from RNA pools of uninfected In our tissue dual RNA-seq approach, we systematically cata- and infected Peyer’s patches. (D) Principal component (PC) analysis of mean loged (i) the transcriptome of Y. pseudotuberculosis strain IP32953 centered and scaled rlog-transformed read count values of bacterial in vitro grown in murine Peyer’s patches or under different laboratory andinvivoRNA-seqdatafortheY. pseudotuberculosis IP32953 core genome and the pYV virulence plasmid. growth conditions in vitro and (ii) the host transcriptome of uninfected and infected mice (Fig. 1A). To simultaneously capture pathogen and host transcripts, high-quality total RNA was isolated distinct, and profiles of the three biological triplicates clustered ’ from Peyer s patches 3 d postinfection (SI Appendix, Fig. S1A). together (Fig. 1 C and D). Normalized read counts for all detected Mouse rRNA, which represents the major RNA species within in mouse (SI Appendix,Fig.S2A)orYersinia (SI Appendix,Fig.S2B) vivo RNA pools, was depleted to increase coverage of informative genes were plotted for the three biological replicates, and the mouse and bacterial transcripts (Fig. 1A). Commercially available 6 Pearson correlation coefficient (r) is indicated. All samples closely RNA spike-in mixes covering a 10 -fold concentration range were correlated with their respective replicates from the same biological added to RNA pools from infected and uninfected tissues to de- sample group (r > 0.9). For Yersinia, 1,312 transcriptional start termine the dynamic range within a sample and test for accurate sites of mRNAs (mTSSs) were mapped (Dataset S2), and 129 fold change responses between different biological treatments trans-encoded small ncRNAs, 45 antisense RNAs, and 23 (Fig. 1A and SI Appendix,Fig.S1B and C) (18). Subsequent 3′-UTR–derived RNAs (Dataset S2) were identified. The 5′-UTR strand-specific Illumina-based deep sequencing (SI Appendix) generated ∼201 million cDNA reads from biological triplicate size distribution of mRNAs, the transcriptional start site (TSS) cDNA libraries of infected Peyer’s patches (Dataset S1). About nucleotide use, and the core promoter structure are highly similar 148 million reads mapped to the mm10 genome, whereas more to Y. pseudotuberculosis YPIII (SI Appendix,Fig.S3) (5) and than 8.5 million cDNA reads mapped to the Y. pseudotuberculosis consistent with reports in Escherichia coli and Salmonella (21, 22). ’ genome. From cDNA libraries of uninfected Peyer spatches, Yersinia ∼114 million reads were generated, of which 87 million mapped to -Induced Host Responses. The host gene expression profile the Mus musculus mm10 genome (Dataset S1). Between ∼230,000 was calculated by the differential expression analysis package and 1.6 million uniquely mapped reads for the pathogen (grown in DESeq2 (23), and genes showing statistically significant changes vivo and in vitro) and between 19 and 68 million mapped reads for in the expression level by at least fourfold were considered. Of the host were obtained from cDNA libraries, providing sufficient 19,993 profiled host transcripts, 1,336 were significantly altered ’ coverage for robust RNA profiling (Fig. 1B and Dataset S1) (19, in abundance within infected Peyer s patches (Fig. 2A and 20). By using stringent filter criteria, we observed linearity between Dataset S3). Although 448 transcripts were more abundant as a the read density and RNA input over the entire detection range of response to the bacterial infection, 888 were less abundant (re- “ ” “ ” around 12 log2 units concentration (SI Appendix,Fig.S1B). This ferred to as infection induced or repressed genes). As a span was sufficient to reliably quantify and compare transcript consequence of the Yersinia infection, significant alterations in abundance across a wide dynamic range (19, 20). Highly accurate the host transcriptome were evident, with many of the highly fold change estimates are visualized by the linear fit (SI Appendix, induced genes being associated with (i) inflammatory responses, Fig. S1C). Host and pathogen global gene expression profiles were (ii) acute-phase responses, (iii) coagulative activities, and (iv)

E792 | www.pnas.org/cgi/doi/10.1073/pnas.1613405114 Nuss et al. Downloaded by guest on September 26, 2021 transition metal ion sequestration, which are among the top CXCL3, CXCL5, and CXCL10). They were previously shown PNAS PLUS enriched pathways (Fig. 2B and Dataset S3). Detected changes in to be strongly up-regulated in Peyer’s patches infected with Y. the abundance of host transcripts reflect expression changes but enterocolitica and/or lymph nodes infected with Yersinia pestis and also, variations in the cell composition of the infected tissues (e.g., contribute to the pathology of Yersiniosis and plague (16, 17, 27– because of infiltrating immune cells). DESeq2 estimated fold 29). However, many up-regulated chemokine/lymphokine recep- change responses of selected host transcripts were confirmed by tors and immune cell signaling molecules have not yet been de- quantitative RT-PCR (qRT-PCR) (SI Appendix,Fig.S4A). scribed in the context of a Yersinia infection (Dataset S3). Of those Inflammatory responses. Because inflammation is a hallmark of listed, IL-22 and IL-23a are known to enhance innate and adaptive Yersinia infection, it was expected that various transcripts for immune responses (30, 31). Others (e.g., IL-19 and IL-27) quench host immunomodulatory proteins would be strongly enriched in the inflammatory effects, similar to IL-10, IL-11, the IL-1 receptor infected Peyer’s patches (Fig. 2B and Dataset S3). The pattern of antagonist IL-1Ra, and the decoy receptor IL-1R2, which se- up-regulated inflammatory cytokines/chemokine receptors and quester and inhibit IL-1 (32, 33). Overall, high induction of TH17- strong enrichment of neutrophil-specific transcripts (e.g., stefin type cytokines (IL-17, IL-6, IL-1, and IL-23) and enrichment of A/cystatin A, prokineticin 2, Schlafen 4, Fpr1, and Clec4e) in- the TH1-type cytokine IFN-γ suggest that Y. pseudotuberculosis volved in neutrophil mobilization, neutrophil-mediated phago- triggers adaptive immunity toward a mixed TH17/TH1response. cytosis, and neutrophil extracellular traps formation (24–26) Acute-phase response. Another specific cluster of enriched tran- highlight that the host immune response is dominated by in- scripts is part of the acute-phase response—a complex systemic filtrating neutrophils (Dataset S3). This finding is consistent with reaction activated by trauma, infection, and inflammation with previous observations that the number of neutrophils within the goal of clearing potential pathogens, reestablishing homeo- murine Peyer’s patches increases ∼100-fold (14). Among the stasis, and promoting healing (34). This cluster encompasses highest infection-induced immunomodulatory genes were those mainly the serum amyloids A2, A3, A4, and P (Fig. 2B and of the major proinflammatory cytokines (IL-1α, IL-1β, IL-6, IL- Dataset S3), which contribute to intestinal immunity. Serum 17F, and IFN-γ) and the chemokines (CCL2, CXCL1, CXCL2, amyloids are up-regulated by proinflammatory signals (e.g., IL-1, MICROBIOLOGY

Fig. 2. The host’s transcriptional response to Y. pseudotuberculosis.(A) Volcano plot obtained from DESeq2 analysis of uninfected and infected Peyer’s patches RNA pools. (B) Heat map of the top 50 enriched and depleted host transcripts. Color-coding is based on rlog-transformed read count values. (C) Infection-mediated activation of extrinsic tissue factor-dependent coagulation cascade genes. On IP32953 colonization, extrinsic coagulation cascade transcripts are highly enriched in infected Peyer’s patches. Fibrin clotting is typically induced by bacterial antigens, such as LPS, to trap the invading bacteria and prevent further dissemination. In addition, stimulation of the extrinsic coagulation pathway is triggered by the neutrophil-specific collagenase MMP8, which activates the tissue factor via cleavage of TFPI.

Nuss et al. PNAS | Published online January 13, 2017 | E793 Downloaded by guest on September 26, 2021 TNF-α, and IL-6), which are increased during Yersinia infection. (55). Moreover, transcripts for the heterodimeric S100A8/S100A9 They participate in the systemic modulation of innate and adaptive complex were among the top 10 enriched messengers (83- to 190- immune responses (e.g., TH17 responses and migration/activation fold) (Fig. 2B and Dataset S3). The S100A8/S100A9 complex, also of monocytes, neutrophils, and T cells) (35–38). Moreover, they have known as calprotectin, sequesters the essential trace elements zinc antimicrobial activity and induce expression of matrix metal- and manganese (56, 57) and was found to be released from intestinal loproteases, chitinase-like proteins, and collagenases that are epithelial cells and infiltrating neutrophils during acute inflammation pivotal for tissue remodeling after injury (39). Transcripts of the (55, 58, 59). metalloproteases MMP3 and MMP8 and the chitinase-like proteins In addition, expression of the immunoresponsive gene 1 (Irg1) and CHIL1 and CHIL3 were especially significantly enriched in Yersinia- the histidine decarboxylase gene (Hdc) was highly induced in re- infected Peyer’s patches (Fig. 2 and Dataset S3). A recent study sponse to a Y. pseudotuberculosis infection (Fig. 2B and Dataset S3). further showed that serum amyloid A binds and transports retinoids, HDC is exclusively responsible for production of the biogenic amine which are induced in response to the infection (Dataset S3). Reti- histamine. Histamine signaling via the histamine receptor 2 has been noids promote maturation of immune cells, elicit TH17 responses, previously reported to be important for controlling Y. enterocolitica and facilitate the regeneration of damaged epithelial barriers (40, 41). colonization in the Peyer’s patches, possibly through altering of cy- Coagulative activities. Transcripts related to thecoagulationcascade tokine production and disruption of the TH1–TH2balance(60).The were among the most enriched host messengers, encoding the co- Irg1 gene encodes an enzyme synthesizing the metabolite itaconate agulation factors VII, X, XIIIa1, and FII; the α-, β-, and γ-subunits from the TCA cycle intermediate cis-aconitate. Its production is of the acute-phase reactant fibrinogen (Fga, Fgb, and Fgg); and specifically induced and highly expressed by macrophages on LPS plasminogen (Plg) (Fig. 2 B and C and Dataset S3). In particular, activation, and it is a potent inhibitor of isocitrate lyase, a key enzyme factor VII, factor X, and fibrinogen transcripts were highly of the glyoxylate shunt, which is required for assimilation of fatty enriched, suggesting that a Yersinia infection triggers activation of acids (61). Notably, Y. pestis and Y. pseudotuberculosis encode itaco- the tissue factor-dependent extrinsic coagulation cascade (Fig. 2C). nate-degrading enzymes within the ripCBA operon (62), indicating Enhanced fibrin deposition was shown to physically trap invading that these bacteria evolved detoxification pathways to prevent itaco- pathogens and limit their spread (42). Activation of the extrinsic nate-mediated growth inhibition. However, itaconate degradation coagulation pathway by Y. pseudotuberculosis is further supported by and metabolism seem less relevant for Y. pseudotuberculosis during the vast accumulation of neutrophil-specific Mmp8 transcripts (Fig. Peyer’s patch colonization, because ripCBA transcripts (YPTB1926- 2andDataset S3). MMP8 is stored in specific granules and released 1924) were barely detectable by RNA-seq (Dataset S4). by neutrophils at sites of acute inflammation (43, 44). It contributes Other than the identified up-regulated functions, 888 host to activation of the extrinsic coagulation pathway because of its transcripts were strongly decreased during infection. A large capacity to cleave the tissue factor pathway inhibitor (TFPI), which number of down-regulated transcripts encode metabolic enzymes. was shown to obtain antibacterial activity on proteolytic processing Among them are several monooxygenases, in particular cyto- (45, 46). Similarly, many other coagulation factors, including factor chrome P450 enzymes, solute carriers, and UDP-glucuronosyl- X and prothrombin, contain a catalytic domain at their C terminus transferase systems, which convert lipophilic molecules into with antimicrobial activity after proteolytic processing (47). To date, excretable compounds (Dataset S3). The infection-induced de- it is unknown whether coagulation plays a protective role against a crease of these transcripts may simply reflect an overall shutdown Y. pseudotuberculosis infection. However, both plasminogen and of pivotal cellular functions in response to the infection. A down- fibrinogen have been identified as critical determinants of the in- regulation of monooxygenases was also observed in Y. enter- flammatory response and survival after Y. pestis infection. Y. pestis ocolitica-infected Peyer’s patches, which led to the assumption that infections normally lead to formation of widespread foci containing they may reduce oxidative stress and minimize detrimental effects large numbers of scattered bacteria with low numbers of infiltrated (63). Among the depleted transcripts are several that relate to inflammatory cells and reduced fibrin deposition because of the transport and hydrolytic genes of dietary mono-/disaccharides (e.g., presence of the plasminogen activator gene pla (48–50). This glucose transporters: Slc5a4a, Slc5a11, Slc5a4b, Slc5a1, lactase, pathology is contrary to infections caused by Y. pseudotuberculosis, trehalase, and sucrose isomaltase) (Fig. 2B and Dataset S3), in- which lead to foci of densely packed bacterial microcolonies with dicating Yersinia-mediated malnutrition. Based on the fact that many surrounding inflammatory cells, primarily neutrophils (51, 52). Formation of fibrin-rich matrices at the early stage of microbial unabsorbed carbohydrates increase the flow of water and electro- invasion seems to support recruitment and attachment of phago- lytes into the lumen (64), it is possible that a decrease of these cytizing cells as well as T cell-mediated responses that may help to counteract a Yersinia infection (48–50). Impediment of the inflammatory response, neutrophil recruitment, and reduced survival of fibrin(ogen)-deficient mice after a Y. pestis infection undermine the role of fibrin(ogen) as a critical amplifier of the inflammatory response and crucial determinant of the host defense against pathogenic yersiniae (48, 53). Metal ion sequestration. Among the strongest induced host factors in the infected lymphatic tissue are metal ion sequestration systems (Fig. 2 A and B and Dataset S3). Vertebrates sequester metal ions from invading pathogens as a first line of defense against bacterial infections, an antimicrobial strategy termed “nutritional immunity” (54). Genes encoding the heme and iron-loaded siderophore sequestration systems haptoglobin and lipocalin 2 Fig. 3. Temperature as a major stimulus for virulence plasmid expression in (LCN2) are >100-fold induced on infection; transcripts of lacto- vivo. (A) Principal component (PC) analysis of reads per kilobase transcript transferrin and hemopexin were 7- and 14-fold enriched (Fig. 2 A length per million mapped reads-normalized expression values of in vitro and in vivo RNA-seq data for the IP32953 pYV virulence plasmid B and and Dataset S3). LCN2 is expressed and secreted in a Toll-like (NC_006153.2). (B) Percentage of pYV cDNA read counts obtained from in receptor 4-dependent manner in response to TH17 cell-specific vitro and in vivo RNA-seq data relative to the read counts obtained for the proinflammatory cytokines, such as IL-17 and IL-22 (55), which bacterial chromosome. Unpaired t test (n = 3). E25, exponential phase 25 °C; are also up-regulated during the infection (Dataset S3). LCN2 E37, exponential phase 37 °C; in vivo, infected Peyer’s patches; S25, sta- stimulates the secretion of neutrophil-attracting CXC chemokines tionary phase 25 °C; S37, stationary phase 37 °C. **P ≤ 0.01; ***P ≤ 0.001.

E794 | www.pnas.org/cgi/doi/10.1073/pnas.1613405114 Nuss et al. Downloaded by guest on September 26, 2021 PNAS PLUS

Fig. 4. Bacterial global gene expression analysis uncovers infection-specific metabolic adaptations. (A) Four-way Venn diagram illustrating Yersinia transcripts, which are significantly altered (log2fc ≥ 1; log2fc ≤−1; adjusted P value ≤ 0.05) during Peyer’s patch colonization (in vivo); 283 transcripts were altered in abundance in vivo compared with all four tested in vitro conditions, including those that were enriched under one condition and depleted under another condition. From those 283 transcripts, 116 were consistently enriched, and 62 were consistently depleted in vivo. E25, exponential phase 25 °C; E37, exponential phase 37 °C; in vivo, infected Peyer’s patches; S25, stationary phase 25 °C; S37, stationary phase 37 °C. (B) Heat map of selected bacterial transcripts related to metabolic functions that are strictly enriched (red) or depleted (blue) during infection. Values represent the log2 fold change between indicated conditions. (C) Central carbon metabolism of Y. pseudotuberculosis. Significant changes of the transcriptomic pattern between the in vitro- and in vivo-grown MICROBIOLOGY bacteria are indicated. Enriched transcripts during infection are given in red, and decreased transcripts are indicated in blue.

enzyme activities contributes to the intestinal disorders (diarrhea) tosis and other immune responses, and they induce apoptosis of associated with a Yersinia infection. Additional analysis of Yersinia- the infected host cell (65, 66). mediated alteration of individual transport functions in the brush Expression of virulence factors. Similar mRNA profiles of the pYV- border and association with the intestinal disorders is required to encoded virulence genes at 37 °C suggest that the thermal upshift confirm this hypothesis. on host entry is the primary signal triggering pYV gene expression. The overall ysc/yop mRNA level is further increased during colo- The Host-Responsive mRNA Repertoire of Yersinia. To identify in- nization of Peyer’s patches (Fig. 3B and Dataset S4), showing that fection-specific gene expression alterations, which consequently additional temperature-independent signals enhance pYV gene lead to the precise adjustment of virulence and fitness programs of expression (e.g., via host cell contact) (67). Notably, the levels of the the pathogen, we simultaneously profiled the entire transcriptional copB mRNA, encoding a negative regulator of the pYV replicase landscape of Y. pseudotuberculosis during growth under laboratory gene repA (68), were significantly decreased during infection conditions (in vitro) and Peyer’s patch colonization (in vivo) (Fig. (Dataset S4 and SI Appendix,Fig.S5B and C). The ratio of repA 1A). The bacterial in vivo expression profile of chromosomal transcripts to its antisense RNA copA was strongly increased com- genes was very distinct from all in vitro profiles (Fig. 1D), whereas pared with bacteria grown at 37 °C. This transcriptional profile in- profiles of the virulence plasmid pYV from in vivo- and in vitro- dicated that the replication of pYV is increased during Peyer’s grown bacteria at 37 °C were more alike (Fig. 3 and SI Appendix, patch colonization and supported a recent observation showing that Fig. S5 A and B). The Yersinia virulence plasmid pYV encodes the Yersinia up-regulates the pYV copy number during infection (69). adhesin YadA, the Ysc type III secretion system (T3SS) and the To explicitly unravel other transcriptional responses that allow effectors called Yops, which are translocated by the T3SS mainly the bacteria to resist host defenses and replicate within extra- into neutrophils and macrophages on cell contact. The Yops cellular lymphatic tissue compartments, we screened for tran- modulate eukaryotic signaling pathways to counteract phagocy- scripts that differed significantly in their abundance between

Nuss et al. PNAS | Published online January 13, 2017 | E795 Downloaded by guest on September 26, 2021 in vitro and infection conditions (Fig. 4A, Datasets S4 and S5, trates that loss of a single-ion substitution system is insufficient to and SI Appendix, Fig. S6). During Peyer’s patch colonization, reduce pathogenesis. Most likely, it is compensated by the up- 97 mRNAs were consistently enriched, whereas 60 mRNAs were regulation of other alternative metal ion-independent metabolic consistently depleted. Similar to host gene expression profiles, functions and scavenging systems (Datasets S4 and S5). DESeq2-estimated fold change responses for selected bacterial Host-adapted metabolism. Another prerequisite for the survival of messenger and small ncRNAs were confirmed by qRT-PCR (SI many bacterial pathogens is the adaptation of their metabolism to Appendix, Fig. S4 B and C). the nutrients available within the host tissue. We found that Most of the differentially regulated transcripts encode stress yersiniae colonizing the Peyer’s patches use carbohydrates as their responses and host-adapted metabolic functions but not classical primary carbon source. Particularly, transcripts encoding the glu- virulence factors (e.g., ail). Evidently, their expression is mainly cose, mannose (ptsGI/crr and manXYZ), fructose (fruBKA), and triggered by temperature and not triggered by infection-specific glucuronate (uxaABC) uptake systems as well as the upper part of stimuli (Datasets S4 and S5). glycolysis (gapA, gmpA, eno,andpykF) were highly abundant or Host-induced bacterial stress responses. The bacterial in vivo strongly enriched during infection (Fig. 4B, Datasets S4 and S5, transcriptome revealed that Yersinia is exposed to severe stresses and SI Appendix,Fig.S8A). In agreement, an fruBKA deletion during Peyer’s patch colonization. The overall transcript abun- mutant was significantly impaired, and a pykF mutant was com- dance of important stress resistance genes (e.g., groEL/ES, dnaK, pletely outcompeted in Peyer’s patch colonization (SI Appendix, grpE, rpoS,andcspA/B/C/D) was high (Dataset S4). Moreover, Fig. S8B) (75). A strong down-regulation of TCA cycle compo- several genes involved in detoxification of nitric oxide and other nents (acnB, fumA, sdhCDAB, icdA,andmdh), fatty acid oxidation reactive nitrogen species (e.g., nitric oxide dioxygenase: hmp; enzymes (fadBA, fadJ,andfadL), and cytochrome o ubiquinol glutaredoxin: nrdH) were strongly up-regulated. This upregulation oxidase (cyoABCDE), the terminal complex of the respiratory counteracts induction of nitric oxide synthase 2 in the infected chain, coupled with a high and/or strongly enhanced in vivo ex- tissue (Dataset S3). In contrast, classical oxidative stress-related pression of fermentation genes (adhE, focA-pflB,andldhA)sug- transcripts were not enriched (Datasets S4 and S5). This expres- gest that pyruvate originating from glycolysis is reduced to ethanol, sion profile suggests that Y. pseudotuberculosis is not exposed to formate, and lactate (Fig. 4 B and C and Datasets S4 and S5). reactive oxygen species produced by neutrophils and other innate Parallel use of different fermentation pathways and expression immune cells. However, a recent study showed that the produc- of rescuing alternative enzymes (YPTB0382, YPTB1517, and tion of multiple Yersinia catalases, superoxide dismutases, and YPTB3387) seem to account for the fact that inactivation of the katA sodB sodC trxA thioredoxin ( , , ,and ) is controlled on the highest induced adhE gene was insufficient to affect the outcome posttranscriptional level by thermoresponsive RNA structures of the infection (SI Appendix, Fig. S8B). (RNA thermometers) (70). They act as translational repressor Overall, the Y. pseudotuberculosis metabolism in Peyer’spatches elements at lower temperature but not at 37 °C, and they are thus resembles that of Y. pestis during rat bubo colonization (76–78), not detectable by conventional RNA-seq. indicating that the different lymphatic tissues constitute a similar The most strongly enriched bacterial transcripts in response to nutritional environment, which requires comparable metabolic host defense strategies are those implicated in metal ion acquisition adaptations. Likewise to Y. pseudotuberculosis, Y. pestis was found (Fig. 4B and Datasets S4 and S5). The great majority encodes uptake systems for iron (e.g., heme uptake system: hmuPRSTUV; to strongly up-regulate homologous metal ion acquisition and ferric/ferrous transporters: yfeABCDE; siderophore systems: fecCD, nitrosative stress systems within bubos (76, 77). Similarly, simple alcABC, fyuA-ybtUTE-irp1/2-ybtA-ybtPQXS, fcuA, rhbC, sfuB, tonB, carbohydrates constitute the main carbon source that is metabolized and exbBD). Some also or specifically sequester zinc (znuA-znuCB to pyruvate and then, redirected to anaerobic energy production and ybtPQXS)ormanganese(mntH and yfeABCDE) (71), most of pathways because of the down-regulation of the TCA cycle. How- which are well-known members of the Fur and Zur regulon. The ever, in contrast to Y. pseudotuberculosis, fructose and mannose are strong increase of multiple redundant systems suggests that espe- not important carbon sources for Y. pestis (76, 78). Moreover, cially the availability of iron and zinc is very limited within the in- Y. pestis seems to metabolize its energy sources mainly by anaerobic fected tissue because of the induction of metal ion sequestration respiration (e.g., by the dimethyl sulfoxide reductase DmsABC) and intracellular retention systems by the host (Fig. 2 A and B and (76). The dmsABC transcript was not detected at a significant level Dataset S3)(72). in our study, indicating that these genes are not required for ’ Another strategy to overcome metal ion scarcity in the bacteria Y. pseudotuberculosis during Peyer s patch colonization. is the substitution of ion-containing proteins against nonion-containing homologs. The most striking example is the rpmE2J tran- Growth in Lymphatic Tissues Alters the Abundance of Conserved script encoding the ribosomal subunits RpmE2 and RpmJ, one of Metabolic ncRNAs. Previous in vitro studies identified numerous two alternative L31 and L36 proteins, which showed the highest ncRNAs in Y. pseudotuberculosis (5, 6). Many of them respond to induction (>300-fold) during infection (Datasets S4 and S5). Both temperature, but whether they contribute to virulence remained proteins lack zinc-binding sites in contrast to their paralogs largely unknown. Our in vivo transcriptome analysis revealed encoded by YPTB0102 (RpmE) and YPTB3677 (RpmJ). Recent that the transcript of the general RNA chaperone Hfq as well as studies in E. coli and Bacillus subtilis suggested that the zinc- that of the global regulator Crp, which controls large subsets of containing RpmE protein is replaced by the highly overexpressed ncRNAs in Y. pseudotuberculosis (5, 6), changed significantly nonzinc-containing paralog to mobilize zinc from ribosomes under during infection compared with in vitro growth at 37 °C (Dataset zinc-limiting conditions (73, 74). A highly conserved Zur box was S5). This result indicated that growth in lymphatic tissues alters identified in the Y. pseudotuberculosis rpmE2 regulatory upstream the abundance of regulatory RNAs. In fact, of 197 identified region overlapping the rpmE2 TSS (mRNA mTSS_0385) (Dataset ncRNAs (Dataset S2), 9 trans-encoded ncRNAs (Fig. 5A) were S2 and SI Appendix, Fig. S7A). We could show Zur-dependent at least twofold enriched in vivo compared with all in vitro expression of the rpmE2J operon (SI Appendix,Fig.S7B). This conditions. Some Yersinia-specific in vivo-induced trans-encoded finding implies that a switch to RpmE2J-containing ribosomes RNAs were identified [Ysr189(NU), Ysr206(NU), and Ysr310(NU)], contributes to the survival of the pathogen in low-zinc in vivo but their overall expression level was rather low. In contrast, several environments. However, despite the strong induction during in- of the conserved ncRNAs (SgrS, RyhB1, and RyhB2), which were fection, mouse survival and tissue colonization efficiency of the not or were marginally expressed in vitro, were strongly up-regu- bacteria were not affected by a loss of both alternative ribosomal lated (20- to 250-fold) and highly abundant during infection (Fig. 5 proteins (SI Appendix, Fig. S7 C and D). This observation illus- A and B).

E796 | www.pnas.org/cgi/doi/10.1073/pnas.1613405114 Nuss et al. Downloaded by guest on September 26, 2021 finding strongly suggests that they contribute to the down- PNAS PLUS regulation of the TCA cycle and respiration of Y. pseudotuberculosis during infection. SgrS RNA homologs are found in several γ-proteobacteria, including E. coli and Salmonella (83). SgrS production in these bacteria is induced during accumulation of phosphosugars in the cytoplasm, a harmful growth-restricting condition referred to as glucose-phosphate stress (84, 85). The regulatory function of SgrS involves translational repression and enhanced degradation of mRNAs encoding sugar transporters (ptsG, fruBKA, and manXYZ), which are highly abundant in Yersinia during Peyer’s patch colonization (83, 86, 87). A deletion of sgrS restricted growth of Y. pseudotuberculosis at 37 °C in the presence of the glucose-phosphate stress-inducing agent methyl α-D-glucoside (αMG) (Fig. 5C). This result showed that SgrS fulfills a similar function in Y. pseudotuberculosis, and its up-regulation in vivo supports our data identifying glucose and fructose as dominant carbon sources during Peyer’s patch colonization. Another abundant in vivo–up-regulated ncRNA, GlmY, is involved in cell wall synthesis. Together with its sibling ncRNA GlmZ, they regulate expression of the glucosamine-6-phosphate synthase GlmS. Transcription of glmY is activated from a σ54- dependent promoter and controlled by the GlrR/GlrK two- component system, which plays a role in virulence (88, 89). High abundance and strong in vivo induction of the important metabolic ncRNAs SgrS, RyhB1/RyhB2, and GlmY prompted us to test their role for virulence. The lack of GlmY, SgrS, or RyhB1/2 did

Fig. 5. Highly conserved bacterial ncRNAs contribute to pathogenesis. (A)Heat map of in vivo-enriched (red) and -depleted (blue) trans-encoded bacterial ncRNAs. Values represent the log2 fold change of indicated conditions (adjusted P value ≤ 0.05). (B) Read coverage of the RNA-seq analysis of the sgrS, ryhB1, ryhB2,andglmY loci is illustrated in the Integrated Genome Browser (99). The data were normalized according to the number of uniquely mapped reads. E25, exponential phase 25 °C; E37, exponential phase 37 °C; in vivo, in- MICROBIOLOGY fected Peyer’s patches; S25, stationary phase 25 °C; S37, stationary phase 37 °C. (C) Y. pseudotuberculosis WT strain IP32953 and the isogenic ΔsgrS strain were grown in LB at 37 °C in the presence or absence of 1% αMG, a glucose-phosphate stress-inducing agent. (D) Two independent groups of BALB/c mice (2 × n = 5 per group) were orally infected with an equal mixture of 107 cfu IP32953 (WT) and the isogenic mutant YPIP56 (ΔsgrS/ryhB1/ryhB2/glmY). Data are graphed as competitive index values for tissue samples from one mouse in Peyer’s patches (PPs), mesenteric lymph nodes (MLNs), and organs after 3 days. A competitive index score of one denotes no difference in virulence compared with the WT. The statistical significance between the WT and the mutant was determined by a Wilcoxon signed rank test. **P ≤ 0.01; ***P ≤ 0.001. Fig. 6. CsrA is crucial for virulence and mediates expression of T3SS. (A)Mice were orally infected with 2 × 108 cfu Y. pseudotuberculosis WT strain YPIII or the isogenic ΔcsrA strain. Survival of mice was monitored for 12 d in two in- × ≤ The sibling RNAs RyhB1/RyhB2 are both activated after iron dependent experiments with 2 10 mice per group for each genotype. ***P 0.001; log rank (Mantel–Cox) test. (B) BALB/c mice were orally infected with scarcity by inactivation of the ferric uptake regulator Fur (79). 107 cfu Y. pseudotuberculosis YPIII WT or the isogenic ΔcsrA strain. Mice were Both ncRNAs are functionally redundant, but RyhB1 seems killed 3 d postinfection, and the numbers of bacteria in homogenized Peyer’s slightly more sensitive to alterations of degradosome factors (79, patches (PPs), mesenteric lymph nodes (MLNs),liver,andspleen was determined. 80). They both regulate iron homeostasis by up-regulation of Data from two independent experiments (2 × 5 mice per group) are presented. iron-scavenging systems and repression of nonessential iron- Statistical significances between theWTandmutantsweredeterminedbya containing proteins to liberate iron (79, 81). Notably, in E. coli Mann–Whitney test. The detection limit is indicated by the dotted line. *P ≤ ≤ ≤ and Salmonella, the RyhB RNAs were found to repress expres- 0.05; **P 0.01; ***P 0.001. (C) TCA-precipitated supernatants or (D)Western blots of whole-cell extracts of Y. pseudotuberculosis YPIII WT and the isogenic + sion of iron-containing TCA cycle enzymes, such as succinate ΔcsrA strain with and without the complementing csrA plasmid pAKH56 + dehydrogenase and fumarate reductase (sdhCDAB and frdABCD), grown at 37 °C in the absence of Ca2 are shown. Yops, LcrF, and YadA iden- fumerase (fumA), and aconitase (acnA and acnB), as well as tified by (C) Coomassie blue staining or (D) Western blotting are indicated. The the respiratory-chain components (nuo and fdo)(81,82).This nucleoid-associated protein H-NS was used as loading control.

Nuss et al. PNAS | Published online January 13, 2017 | E797 Downloaded by guest on September 26, 2021 + not have an effect or had only a very mild effect on host colonization influence of CsrA on Yop secretion using Ca2 limitation as a + (SI Appendix,Fig.S8C). This finding illustrates that the network of substitute for cell contact. Ca2 depletion stimulated secretion of cellular functions controlled by the ncRNAs is very robust, because Yops (Fig. 6C)bytheY. pseudotuberculosis WT strain YPIII but their individual loss can be easily compensated. In contrast, a dele- not by the isogenic csrA mutant. Moreover, levels of the T3SS/Yop tion of all four ncRNA genes significantly impaired colonization of regulator LcrF and the adhesin YadA were not increased or only + the Peyer’s patches and mesenteric lymph nodes (Fig. 5D), showing slightly increased in the absence of CsrA on Ca2 depletion (Fig. that pathogenicity is reduced when their functions are eliminated. 6D). The WT phenotype could be restored by csrA expression in Within the entire ncRNA pool, only a set of five trans-encoded trans, indicating that Yop/YadA expression indeed requires CsrA, RNAs was depleted in in vivo compared with in vitro growth (Fig. which promotes induction of LcrF synthesis on host cell contact. 5A and Dataset S4). The most abundant ncRNAs are carbon storage This observation provides evidence that CsrA not only affects the regulator B (CsrB) and CsrC, of which CsrB showed the strongest bacterial metabolic fitness but also, is crucial for type III secretion decrease under infection conditions (Fig. 5A and Dataset S4). De- and pathogenesis during colonization of the Peyer’s patches. crease of CsrB and CsrC levels is likely induced by the down-regulation of Crp and up-regulation of CsrD (Datasets S4 and S5). Crp is Conclusions a known activator of csrB transcription, and CsrD is involved in the One key feature of this work is the ability to simultaneously cap- RNase E-mediated decay of CsrB and CsrC (90, 91). CsrB, CsrC, ture the RNA expression profile of the extracellular pathogen and the RNA-binding protein CsrA form the conserved Csr system, Yersinia and its mammalian host during infection. The established in which both Csr RNAs bind multiple CsrA molecules and prevent tissue dual RNA-seq approach generated a comprehensive, high- CsrA interaction with other target mRNAs. The CsrA regulon of resolution transcriptomic dataset that (i) enabled us to identify Enterobacteriaceae is very complex and includes colonization factors, immune reactions directed against the pathogen and opposed infection-linked metabolic processes, and physiological traits (92, responses of Yersinia at one time point of the infection that drive 93), indicating that an increase of active CsrA is important under pathogenesis with high statistical confidence and (ii)allowedusto infection conditions. We tested a Y. pseudotuberculosis YPIII csrA discover infection-specific stimuli and bacterial riboregulators mutant in the oral mouse model and found that mice infected with a shaping host–pathogen interactions. Future studies including csrA mutant did not develop any disease symptoms and remained earlier and later stages of the infection will provide valuable in- alive 14 d postinfection (Fig. 6A); 10- to 100-fold fewer bacteria were formation about the dynamics of gene expression changes in the recovered from the Peyer’s patches and mesenteric lymph nodes at pathogen and its host during the course of an infection. The day 3 postinfection, and no bacteria could be isolated from the liver bacterial load within infected tissues is the most limiting factor for and spleen (Fig. 6B), showing that CsrA is crucial for virulence. tissue dual RNA-seq, and transcripts with very low abundance might have been missed. However, ongoing improvements in Important Genes for Peyer’s Patch Colonization Are Part of the CsrA sampling, library preparation, and high-throughput sequencing Regulon. A comparison of genes that were differentially expressed technologies will very likely allow a more sensitive detection and during infection with the previously defined CsrA regulon (75) analysis of expression changes in less colonized host tissues in the revealed that CsrA governs numerous metabolic genes implicated near future. Moreover, single-cell RNA-seq (94, 95) will enable us in the adaptation of Yersinia to the lymphatic environment. We to elucidate the transcriptional response of individual host cell detected a strongly reduced transcript abundance of pyruvate– types or different bacterial species (e.g., other enteric pathogens TCA cycle genes (acs, sdhCDAB, icdA, fumA,andacnB)and and intestinal microbiota) and dissect heterogeneity of bacterial fatty acid oxidation genes (fadAB, fadJ,andfadL) in vivo, which and host cell subpopulations during the infection. are repressed by CsrA, and a high abundance of the adhE Our data on Y. pseudotuberculosis revealed many similarities mRNA, which is activated by CsrA (Datasets S4 and S5) (75). but also marked differences in the host response to infection with This finding suggests that adjustment of the CsrABC levels its close relatives Y. enterocolitica and Y. pestis (29, 63). Specifi- during infection plays a crucial role for the adaptation of the cally, integration of the strong enrichment of neutrophil-specific biological fitness during host tissue colonization. transcripts for antimicrobial defense and immune cell dynamics The strong attenuation of a csrA mutant also indicated that during infection illustrates that the immune response directed essential virulence functions (e.g., T3SS/yop genes) are abolished against Y. pseudotuberculosis is dominated by the massive re- in the absence of CsrA. To test this hypothesis, we investigated the cruitment and activation of neutrophils and steers adaptive

Fig. 7. Infection-specific changes of the Yersinia–host transcriptomes. Schematic overview of the complex transcript-based adaptations of Y. pseudotuberculosis and the host during colonization of Peyer’s patches. The most strongly affected inflammatory and acute-phase responses, metabolic and virulence adaptations, and identified differentially regulated bacterial riboregulators with impact on pathogenesis are illustrated. DC, dendritic cell; NK, natural killer cell; NO, nitric oxide; PMN, polymorphonuclear leukocyte.

E798 | www.pnas.org/cgi/doi/10.1073/pnas.1613405114 Nuss et al. Downloaded by guest on September 26, 2021 PNAS PLUS immunity primarily to TH1 and TH17 responses (Fig. 7). This dotuberculosis relies on the combined action of several highly response coincides with an induction of the extrinsic coagulation expressed, conserved ncRNAs, with abundance that is adjusted cascade, shown to form fibrin-rich matrices that encase the in response to ion/nutrient availability under infection conditions bacteria to hamper dissemination and enhance bacterial clear- and/or by the global ncRNA regulators Hfq and Crp. In partic- ance (42, 51). Consequently, the most important defense ular, adjustment of the activity of the global RNA regulator CsrA mechanism of the bacteria is to adjust to the stressful conditions via the ncRNAs CsrB and CsrC is crucial for virulence. Other and counteract neutrophil attack within fibrin clots. than its vital role in the regulation of carbon metabolism (75) Accordingly, the pathogen’s transcriptional response is charac- and invasin gene expression (98), we show that CsrA is essential terized by a marked up-regulation of the antiphagocytic T3SS/Yop for Yop synthesis and secretion to defend against attacks of machinery in response to temperature and an increase of the vir- surrounding phagocytic cells during later stages of the infection. ulence plasmid copy number, which is likely triggered by host cell In summary, these findings argue for the usefulness of the contact (69, 96). This defense strategy is supported by the induction established tissue dual RNA-seq approach to derive meaningful of radical detoxifying genes, such as hmp (Fig. 7). conclusions related to pathogen biology and host response that af- In addition to the immune defense mechanisms, in vivo growth fect the outcome of an infection. The integration of obtained RNA of Yersinia is also accompanied by a strong up-regulation of Fe/Zn/ profile information with data of immune cell migration and bacte- Mn sequestration/uptake systems and a readjustment of metabolic rial pathogenesis offers insights into the complex host–pathogen functions that allows the pathogen to overcome host-imposed ion interaction network and developing disease. It further creates and nutrient constraints (Fig. 7). The plethora of up-regulated possibilities to identify critical processes that govern pathogen redundant ion acquisition systems reflects the high importance of colonization and increase the potential to identify targets for this process to prevent pertinent host measures to restrict bacterial antimicrobial therapies. growth. It also illustrates the difficulties that interfere with this process by target-directed antimicrobial therapies. Materials and Methods Our data further suggest that Yersinia assesses and uses a number Details on methodologies are provided in SI Appendix and include bacterial of diverse nutrients, especially simple sugars, which likely originate growth conditions, DNA cloning procedures and mutant constructions, from dying cells in the infected tissue (97) and remain available be- analysis of reporter gene expression, Yop synthesis and secretion assays, cause of the overall reduction of host cell sugar carriers (Fig. 7). They animal experiments, RNA isolation, qRT-PCR, cDNA library preparation, read are metabolized to pyruvate and converted by anaerobic fermenta- mapping, bioinformatics, and statistics. The custom annotation file including tion into a variable mixture of short-chain fatty acids. This metabolic identified ncRNAs is given in Dataset S6. The animal protocols were diversity relieves Yersinia dependence on a particular energy source approved by the Niedersächsisches Landesamt für Verbraucherschutz und and improves survival when individual nutrients are scarce or fluc- Lebensmittelsicherheit: permission no. 33.9.42502-04-12/1010. tuating during the infection process. It further explains the remark- ’ ACKNOWLEDGMENTS. We thank M. Fenner for discussions and W. Weigel able robustness of the pathogen s metabolism against internal for critical reading of the manuscript. We also thank R. Steinmann, M. Kroll, perturbations but pinpoints limited possibilities for antimicrobial M. Schoof, W. Heine, O. Goldmann, and J. Reinkensmeier for tools and targets (e.g., the phosphoenolpyruvate/pyruvate-acetyl-CoA node). experimental and bioinformatics support and K. Paduch and T. Krause for Furthermore, our data provide an overall view of the ncRNA technical assistance. This work was supported from German Research Foun- dation Grants DE616/4 (to A.M.N.), DE616/6 (to A.M.N.), and SPP1617–Young expression profile within lymphatic tissue, which offers a unique Investigator Start Up Funding (to A.M.N.). M.P. and W.O. received fellow- resource for the identification of important riboregulators during ships from the Helmholtz Centre for Infection Research Graduate School. infection. We show that a successful host infection by Y. pseu- P.D. is supported by the German Center of Infection Research.

1. Papenfort K, Vogel J (2014) Small RNA functions in carbon metabolism and virulence 16. Dube PH, Handley SA, Lewis J, Miller VL (2004) Protective role of interleukin-6 during of enteric pathogens. Front Cell Infect Microbiol 4:91. Yersinia enterocolitica infection is mediated through the modulation of in- 2. Oliva G, Sahr T, Buchrieser C (2015) Small RNAs, 5′ UTR elements and RNA-binding flammatory cytokines. Infect Immun 72(6):3561–3570. proteins in intracellular bacteria: Impact on metabolism and virulence. FEMS 17. Dube PH, Revell PA, Chaplin DD, Lorenz RG, Miller VL (2001) A role for IL-1 alpha in

Microbiol Rev 39(3):331–349. inducing pathologic inflammation during bacterial infection. Proc Natl Acad Sci USA MICROBIOLOGY 3. Heroven AK, Nuss AM, Dersch P (2016) RNA-based mechanisms of virulence control in 98(19):10880–10885. Enterobacteriaceae. RNA Biol 2016:1–17. 18. Locati MD, et al. (2015) Improving small RNA-seq by using a synthetic spike-in set for 4. Kröger C, et al. (2013) An infection-relevant transcriptomic compendium for Salmo- size-range quality control together with a set for data normalization. Nucleic Acids nella enterica Serovar Typhimurium. Cell Host Microbe 14(6):683–695. Res 43(14):e89. 5. Nuss AM, et al. (2015) Transcriptomic profiling of Yersinia pseudotuberculosis reveals 19. Haas BJ, Chin M, Nusbaum C, Birren BW, Livny J (2012) How deep is deep enough for reprogramming of the Crp regulon by temperature and uncovers Crp as a master RNA-Seq profiling of bacterial transcriptomes? BMC Genomics 13:734. regulator of small RNAs. PLoS Genet 11(3):e1005087. 20. Ching T, Huang S, Garmire LX (2014) Power analysis and sample size estimation for 6. Koo JT, Alleyne TM, Schiano CA, Jafari N, Lathem WW (2011) Global discovery of small RNA-Seq differential expression. RNA 20(11):1684–1696. RNAs in Yersinia pseudotuberculosis identifies Yersinia-specific small, noncoding 21. Kim D, et al. (2012) Comparative analysis of regulatory elements between Escherichia RNAs required for virulence. Proc Natl Acad Sci USA 108(37):E709–E717. coli and Klebsiella pneumoniae by genome-wide transcription start site profiling. 7. Srikumar S, et al. (2015) RNA-seq brings new insights to the intra-macrophage tran- PLoS Genet 8(8):e1002867. scriptome of Salmonella Typhimurium. PLoS Pathog 11(11):e1005262. 22. Kröger C, et al. (2012) The transcriptional landscape and small RNAs of Salmonella 8. Mandlik A, et al. (2011) RNA-Seq-based monitoring of infection-linked changes in enterica serovar Typhimurium. Proc Natl Acad Sci USA 109(20):E1277–E1286. Vibrio cholerae gene expression. Cell Host Microbe 10(2):165–174. 23. Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dis- 9. Wu Z, et al. (2014) The Streptococcus suis transcriptional landscape reveals adaptation persion for RNA-seq data with DESeq2. Genome Biol 15(12):550. mechanisms in pig blood and cerebrospinal fluid. RNA 20(6):882–898. 24. Liu M, et al. (2012) Formylpeptide receptors are critical for rapid neutrophil mobili- 10. Westermann AJ, Gorski SA, Vogel J (2012) Dual RNA-seq of pathogen and host. Nat zation in host defense against Listeria monocytogenes. Sci Rep 2:786. Rev Microbiol 10(9):618–630. 25. Sharma A, Steichen AL, Jondle CN, Mishra BB, Sharma J (2014) Protective role of 11. Westermann AJ, et al. (2016) Dual RNA-seq unveils noncoding RNA functions in host- Mincle in bacterial pneumonia by regulation of neutrophil mediated phagocytosis pathogen interactions. Nature 529(7587):496–501. and extracellular trap formation. J Infect Dis 209(11):1837–1846. 12. Baddal B, et al. (2015) Dual RNA-seq of nontypeable haemophilus influenzae and host 26. Kang MJ, et al. (2015) Role of chitinase 3-like-1 in interleukin-18-induced pulmonary cell transcriptomes reveals novel insights into host-pathogen cross talk. MBio 6(6): type 1, type 2, and type 17 inflammation; alveolar destruction; and airway fibrosis in e01765–e15. the murine lung. Am J Respir Cell Mol Biol 53(6):863–871. 13. Rienksma RA, et al. (2015) Comprehensive insights into transcriptional adaptation of in- 27. Autenrieth IB, Kempf V, Sprinz T, Preger S, Schnell A (1996) Defense mechanisms in tracellular mycobacteria by microbe-enriched dual RNA sequencing. BMC Genomics 16:34. Peyer’s patches and mesenteric lymph nodes against Yersinia enterocolitica involve 14. Pisano F, et al. (2014) Influence of PhoP and intra-species variations on virulence of integrins and cytokines. Infect Immun 64(4):1357–1368. Yersinia pseudotuberculosis during the natural oral infection route. PLoS One 9(7): 28. Matteoli G, et al. (2008) Role of IFN-gamma and IL-6 in a protective immune response e103541. to Yersinia enterocolitica in mice. BMC Microbiol 8:153. 15. Carter PB (1975) Animal model of human disease. Yersinia enteritis. Animal model: 29. Comer JE, et al. (2010) Transcriptomic and innate immune responses to Yersinia pestis Oral Yersinia enterocolitica infection of mice. Am J Pathol 81(3):703–706. in the lymph node during bubonic plague. Infect Immun 78(12):5086–5098.

Nuss et al. PNAS | Published online January 13, 2017 | E799 Downloaded by guest on September 26, 2021 30. Nikoopour E, Bellemore SM, Singh B (2015) IL-22, cell regeneration and autoimmu- 66. Durand EA, Maldonado-Arocho FJ, Castillo C, Walsh RL, Mecsas J (2010) The presence nity. Cytokine 74(1):35–42. of professional phagocytes dictates the number of host cells targeted for Yop 31. Wang Y, et al. (2015) Jagged-1 signaling suppresses the IL-6 and TGF-β treatment- translocation during infection. Cell Microbiol 12(8):1064–1082. induced Th17 cell differentiation via the reduction of RORγt/IL-17A/IL-17F/IL-23a/IL- 67. Pettersson J, et al. (1996) Modulation of virulence factor expression by pathogen 12rb1. Sci Rep 5:8234. target cell contact. Science 273(5279):1231–1233. 32. Mascanfroni ID, et al. (2013) IL-27 acts on DCs to suppress the T cell response and 68. Pinto UM, Pappas KM, Winans SC (2012) The ABCs of plasmid replication and seg- autoimmunity by inducing expression of the immunoregulatory molecule CD39. Nat regation. Nat Rev Microbiol 10(11):755–765. Immunol 14(10):1054–1063. 69. Wang H, et al. (2016) Increased plasmid copy-number is essential for Yersinia T3SS 33. Dinarello CA (2009) Immunological and inflammatory functions of the interleukin-1 function and virulence. Science 353(6298):492–495. family. Annu Rev Immunol 27:519–550. 70. Righetti F, et al. (2016) Temperature-responsive in vitro RNA structurome of Yersinia 34. Cray C, Zaias J, Altman NH (2009) Acute phase response in animals: A review. Comp pseudotuberculosis. Proc Natl Acad Sci USA 113(26):7237–7242. Med 59(6):517–526. 71. Perry RD, Bobrov AG, Fetherston JD (2015) The role of transition metal transporters 35. Kanther M, et al. (2014) Commensal microbiota stimulate systemic neutrophil mi- for iron, zinc, manganese, and copper in the pathogenesis of Yersinia pestis. gration through induction of serum amyloid A. Cell Microbiol 16(7):1053–1067. Metallomics 7(6):965–978. 36. Björkman L, et al. (2008) Serum amyloid A mediates human neutrophil production of 72. Soares MP, Weiss G (2015) The Iron age of host-microbe interactions. EMBO Rep reactive oxygen species through a receptor independent of formyl peptide receptor 16(11):1482–1500. like-1. J Leukoc Biol 83(2):245–253. 73. Gabriel SE, Helmann JD (2009) Contributions of Zur-controlled ribosomal proteins to 37. Ather JL, et al. (2011) Serum amyloid A activates the NLRP3 inflammasome and growth under zinc starvation conditions. J Bacteriol 191(19):6116–6122. promotes Th17 allergic asthma in mice. J Immunol 187(1):64–73. 74. Akanuma G, Nanamiya H, Natori Y, Nomura N, Kawamura F (2006) Liberation of zinc- 38. Ivanov II, et al. (2009) Induction of intestinal Th17 cells by segmented filamentous containing L31 (RpmE) from ribosomes by its paralogous gene product, YtiA, in Ba- – bacteria. Cell 139(3):485–498. cillus subtilis. J Bacteriol 188(7):2715 2720. 39. Vallon R, et al. (2001) Serum amyloid A (apoSAA) expression is up-regulated in 75. Bücker R, Heroven AK, Becker J, Dersch P, Wittmann C (2014) The pyruvate-tri- rheumatoid arthritis and induces transcription of matrix metalloproteinases. carboxylic acid cycle node: A focal point of virulence control in the enteric pathogen – J Immunol 166(4):2801–2807. Yersinia pseudotuberculosis. J Biol Chem 289(43):30114 30132. 40. Hall JA, Grainger JR, Spencer SP, Belkaid Y (2011) The role of retinoic acid in tolerance 76. Pradel E, et al. (2014) New insights into how Yersinia pestis adapts to its mammalian and immunity. Immunity 35(1):13–22. host during bubonic plague. PLoS Pathog 10(3):e1004029. 41. Derebe MG, et al. (2014) Serum amyloid A is a retinol binding protein that transports 77. Sebbane F, et al. (2006) Adaptive response of Yersinia pestis to extracellular effectors retinol during bacterial infection. eLife 3:e03206. of innate immunity during bubonic plague. Proc Natl Acad Sci USA 103(31): – 42. van der Poll T, Herwald H (2014) The coagulation system and its function in early 11766 11771. immune defense. Thromb Haemost 112(4):640–648. 78. Palace SG, Proulx MK, Lu S, Baker RE, Goguen JD (2014) Genome-wide mutant fitness 43. Weiss SJ, Peppin G, Ortiz X, Ragsdale C, Test ST (1985) Oxidative autoactivation of profiling identifies nutritional requirements for optimal growth of Yersinia pestis in deep tissue. MBio 5(4):e01385-14. latent collagenase by human neutrophils. Science 227(4688):747–749. 44. Hasty KA, Hibbs MS, Kang AH, Mainardi CL (1986) Secreted forms of human neu- 79. Deng Z, et al. (2012) Two sRNA RyhB homologs from Yersinia pestis biovar microtus expressed invivohave differential Hfq-dependent stability. Res Microbiol 163(6-7):413–418. trophil collagenase. J Biol Chem 261(12):5645–5650. 80. Deng Z, et al. (2014) Rapid degradation of Hfq-free RyhB in Yersinia pestis by PNPase 45. Cunningham AC, Hasty KA, Enghild JJ, Mast AE (2002) Structural and functional independent of putative ribonucleolytic complexes. BioMed Res Int 2014:798918. characterization of tissue factor pathway inhibitor following degradation by matrix 81. Massé E, Gottesman S (2002) A small RNA regulates the expression of genes involved metalloproteinase-8. Biochem J 367(Pt 2):451–458. in iron metabolism in Escherichia coli. Proc Natl Acad Sci USA 99(7):4620–4625. 46. Papareddy P, et al. (2012) Tissue factor pathway inhibitor 2 is found in skin and its 82. Kim JN, Kwon YM (2013) Identification of target transcripts regulated by small RNA C-terminal region encodes for antibacterial activity. PLoS One 7(12):e52772. RyhB homologs in Salmonella: RyhB-2 regulates motility phenotype. Microbiol Res 47. Kasetty G, et al. (2011) The C-terminal sequence of several human serine proteases 168(10):621–629. encodes host defense functions. J Innate Immun 3(5):471–482. 83. Horler RS, Vanderpool CK (2009) Homologs of the small RNA SgrS are broadly dis- 48. Degen JL, Bugge TH, Goguen JD (2007) Fibrin and fibrinolysis in infection and host tributed in enteric bacteria but have diverged in size and sequence. Nucleic Acids Res defense. J Thromb Haemost 5(Suppl 1):24–31. 37(16):5465–5476. 49. Luo D, et al. (2013) Fibrin facilitates both innate and T cell-mediated defense against 84. Vanderpool CK, Gottesman S (2004) Involvement of a novel transcriptional activator Yersinia pestis. J Immunol 190(8):4149–4161. and small RNA in post-transcriptional regulation of the glucose phosphoenolpyruvate 50. Sodeinde OA, et al. (1992) A surface protease and the invasive character of plague. phosphotransferase system. Mol Microbiol 54(4):1076–1089. Science 258(5084):1004–1007. 85. Wadler CS, Vanderpool CK (2007) A dual function for a bacterial small RNA: SgrS 51. Guinet F, et al. (2015) Dissociation of tissue destruction and bacterial expansion performs base pairing-dependent regulation and encodes a functional polypeptide. during bubonic plague. PLoS Pathog 11(10):e1005222. Proc Natl Acad Sci USA 104(51):20454–20459. 52. Davis KM, Mohammadi S, Isberg RR (2015) Community behavior and spatial regula- 86. Wadler CS, Vanderpool CK (2009) Characterization of homologs of the small RNA tion within a bacterial microcolony in deep tissue sites serves to protect against host SgrS reveals diversity in function. Nucleic Acids Res 37(16):5477–5485. attack. Cell Host Microbe 17(1):21–31. 87. Rice JB, Vanderpool CK (2011) The small RNA SgrS controls sugar-phosphate accu- 53. Korhonen TK (2015) Fibrinolytic and procoagulant activities of Yersinia pestis and mulation by regulating multiple PTS genes. Nucleic Acids Res 39(9):3806–3819. Salmonella enterica. J Thromb Haemost 13(Suppl 1):S115–S120. 88. Göpel Y, et al. (2011) Common and divergent features in transcriptional control of the 54. Diaz-Ochoa VE, Jellbauer S, Klaus S, Raffatellu M (2014) Transition metal ions at the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae. Nucleic Acids Res crossroads of mucosal immunity and microbial pathogenesis. Front Cell Infect Microbiol 4:2. 39(4):1294–1309. 55. Raffatellu M, et al. (2009) Lipocalin-2 resistance confers an advantage to Salmonella 89. Flamez C, Ricard I, Arafah S, Simonet M, Marceau M (2008) Phenotypic analysis of enterica serotype Typhimurium for growth and survival in the inflamed intestine. Cell Yersinia pseudotuberculosis 32777 response regulator mutants: New insights into two- – Host Microbe 5(5):476 486. component system regulon plasticity in bacteria. Int J Med Microbiol 298(3-4):193–207. 56. Kehl-Fie TE, et al. (2011) Nutrient metal sequestration by calprotectin inhibits bac- 90. Heroven AK, et al. (2012) Crp induces switching of the CsrB and CsrC RNAs in Yersinia terial superoxide defense, enhancing neutrophil killing of Staphylococcus aureus. Cell pseudotuberculosis and links nutritional status to virulence. Front Cell Infect – Host Microbe 10(2):158 164. Microbiol 2:158. 57. Damo SM, et al. (2013) Molecular basis for manganese sequestration by calprotectin 91. Suzuki K, Babitzke P, Kushner SR, Romeo T (2006) Identification of a novel regulatory and roles in the innate immune response to invading bacterial pathogens. Proc Natl protein (CsrD) that targets the global regulatory RNAs CsrB and CsrC for degradation – Acad Sci USA 110(10):3841 3846. by RNase E. Genes Dev 20(18):2605–2617. 58. Steinbakk M, et al. (1990) Antimicrobial actions of calcium binding leucocyte L1 92. Heroven AK, Böhme K, Dersch P (2012) The Csr/Rsm system of Yersinia and related path- protein, calprotectin. Lancet 336(8718):763–765. ogens: A post-transcriptional strategy for managing virulence. RNA Biol 9(4):379–391. 59. Flo TH, et al. (2004) Lipocalin 2 mediates an innate immune response to bacterial 93. Vakulskas CA, Potts AH, Babitzke P, Ahmer BM, Romeo T (2015) Regulation of bac- infection by sequestrating iron. Nature 432(7019):917–921. terial virulence by Csr (Rsm) systems. Microbiol Mol Biol Rev 79(2):193–224. 60. Handley SA, Dube PH, Miller VL (2006) Histamine signaling through the H(2) receptor 94. Avraham R, et al. (2015) Pathogen cell-to-cell variability drives heterogeneity in host in the Peyer’s patch is important for controlling Yersinia enterocolitica infection. Proc immune responses. Cell 162(6):1309–1321. Natl Acad Sci USA 103(24):9268–9273. 95. Saliba AE, et al. (2016) Single-cell RNA-seq ties macrophage polarization to growth 61. Michelucci A, et al. (2013) Immune-responsive gene 1 protein links metabolism to rate of intracellular Salmonella. Nat Microbiol 2:16206. immunity by catalyzing itaconic acid production. Proc Natl Acad Sci USA 110(19): 96. Böhme K, et al. (2012) Concerted actions of a thermo-labile regulator and a unique 7820–7825. intergenic RNA thermosensor control Yersinia virulence. PLoS Pathog 8(2):e1002518. 62. Sasikaran J, Ziemski M, Zadora PK, Fleig A, Berg IA (2014) Bacterial itaconate deg- 97. Monack DM, Mecsas J, Bouley D, Falkow S (1998) Yersinia-induced apoptosis in vivo radation promotes pathogenicity. Nat Chem Biol 10(5):371–377. aids in the establishment of a systemic infection of mice. J Exp Med 188(11): 63. Handley SA, Miller VL (2007) General and specific host responses to bacterial infection 2127–2137. in Peyer’s patches: A role for stromelysin-1 (matrix metalloproteinase-3) during Sal- 98. Heroven AK, Böhme K, Rohde M, Dersch P (2008) A Csr-type regulatory system, in- monella enterica infection. Mol Microbiol 64(1):94–110. cluding small non-coding RNAs, regulates the global virulence regulator RovA of 64. Gericke B, Amiri M, Naim HY (2016) The multiple roles of sucrase-isomaltase in the Yersinia pseudotuberculosis through RovM. Mol Microbiol 68(5):1179–1195. intestinal physiology. Mol Cell Pediatr 3(1):2. 99. Nicol JW, Helt GA, Blanchard SG, Jr, Raja A, Loraine AE (2009) The Integrated Genome 65. Cornelis GR, et al. (1998) The virulence plasmid of Yersinia, an antihost genome. Browser: Free software for distribution and exploration of genome-scale datasets. Microbiol Mol Biol Rev 62(4):1315–1352. Bioinformatics 25(20):2730–2731.

E800 | www.pnas.org/cgi/doi/10.1073/pnas.1613405114 Nuss et al. Downloaded by guest on September 26, 2021