Global Discovery of Small Rnas in Yersinia Pseudotuberculosis Identi

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Global discovery of small RNAs in Yersinia PNAS PLUS pseudotuberculosis identiﬁes Yersinia-speciﬁc small, noncoding RNAs required for virulence

Jovanka T. Kooa, Trevis M. Alleyneb,1, Chelsea A. Schianoa, Nadereh Jafarib, and Wyndham W. Lathema,2

aDepartment of Microbiology-Immunology and bCenter for Genetic Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL, 60611

Edited by Susan Gottesman, National Cancer Institute, Bethesda, MD, and approved June 7, 2011 (received for review January 28, 2011) A major class of bacterial small, noncoding RNAs (sRNAs) acts by environment. This regulation includes the adaptation of patho- base-pairing with mRNAs to alter the translation from and/or genic bacteria to the host and the coordination of expression of stability of the transcript. Our laboratory has shown that Hfq, the virulence determinants. sRNAs can regulate the expression of chaperone that mediates the interaction of many sRNAs with their virulence genes directly [e.g., RNAIII regulation of staphylo- targets, is required for the virulence of the enteropathogen Yersi- coccal protein A and the α-toxin gene mRNAs in Staphylococcus nia pseudotuberculosis. This finding suggests that sRNAs play aureus (8, 9)] or control global regulators [e.g., quorum regula- a critical role in the regulation of virulence in this pathogen, but tory sRNAs regulate the hemagglutinin/protease regulator hapR these sRNAs are not known. Using a deep sequencing approach, mRNA in Vibrio cholerae (10)]. The end result is the fine-tuning we identified the global set of sRNAs expressed in vitro by Y. of metabolic requirements of pathogenic bacteria to endure the pseudotuberculosis. Sequencing of RNA libraries from bacteria stress imposed by the host and the synthesis of virulence factors. grown at 26 °C and 37 °C resulted in the identification of 150 un- There are three pathogenic Yersinia species that cause disease annotated sRNAs. The majority of these sRNAs are Yersinia spe- in humans: Y. pestis, Y. pseudotuberculosis and Y. enterocolitica. cific, without orthologs in either Escherichia coli or Salmonella Y. pestis is thought to have evolved from Y. pseudotuberculosis typhimurium. Six sRNAs are Y. pseudotuberculosis specific and ∼1,500–20,000 y ago. Y. enterocolitica is more distantly related, are absent from the genome of the closely related species Yersinia having diverged from a common ancestral Yersinia species 41– pestis. We found that the expression of many sRNAs conserved 186 million y ago (11, 12). Y. pestis is the causative agent of the between Y. pseudotuberculosis and Y. pestis differs in both timing disease plague. The bubonic form of the disease occurs following and dependence on Hfq, suggesting evolutionary changes in post- the transmission of Y. pestis via fleabite and multiplication of transcriptional regulation between these species. Deletion of mul- bacteria in the lymph nodes; pneumonic plague, the most severe Y. pseudotuberculosis tiple sRNAs in leads to attenuation of the form of disease, occurs if the bacteria colonize and multiply in pathogen in a mouse model of yersiniosis, as does the inactivation the lungs (13). Y. pestis can be spread from person to person by Y. pestis Yersinia fi in of a conserved, -speci c sRNA in a mouse infectious respiratory droplets, and without early treatment, model of pneumonic plague. Finally, we determined the regulon pneumonic plague is 100% fatal. controlled by one of these sRNAs, revealing potential virulence In contrast to Y. pestis, the closely related Y. pseudotuberculosis Y. pseudotuberculosis determinants in that are regulated in a post- is a soil- and water-borne enteropathogen that primarily infects transcriptional manner. wild animals and birds. In humans it causes a mild, self-limiting gastrointestinal disease called “yersiniosis” and is transmitted by Ysr29 | RybB | stress | Illumina-Solexa | 2D-DIGE the fecal–oral route. Yersiniosis caused by Y. pseudotuberculosis is characterized by ileitis and mesenteric lymphadenitis, as well istorically it was assumed that only proteins act as regulators as fever and diarrhea (14). Although Y. pseudotuberculosis and Y. Hof gene expression. There has been increasing recognition, pestis are highly genetically related (they share >97% identity in however, that small, noncoding RNAs (sRNAs) serve as major 75% of their chromosomal genes), they differ radically in their components of diverse regulatory circuits in bacteria. sRNAs are pathogenesis and disease etiologies (15). RNA molecules that typically are encoded in intergenic regions, Despite these differences, recent evidence indicates that the are independently transcribed, contain their own promoters and sRNA chaperone Hfq is required for the full virulence of both ρ – -independent terminators, and range from 50 500 nt in length Y. pestis (16) and Y. pseudotuberculosis (17) in mouse models of (1). Many were discovered initially by computational methods infection (s.c. or i.v. and intragastric, respectively) and suggests involving homology searches of closely related bacterial species a role for sRNAs in the regulation of pathogenesis. The arsenal fi and more recently have been identi ed by a variety of experi- of sRNAs expressed by Y. pestis and Y. pseudotuberculosis that mental approaches (i.e., genetic screens, deep sequencing, tiling control gene expression has not been examined experimentally microarrays, coimmunoprecipitation with proteins) (2). Most on a global level, however. Thus, we sought to gain a greater sRNAs control gene expression at the posttranscriptional level understanding of the roles that sRNAs play in manipulating the by base-pairing with target mRNAs, resulting in alterations in mRNA target translation or half-life (3, 4). The predominant – outcome of the sRNA target mRNA interaction is the down- Author contributions: J.T.K. and W.W.L. designed research; J.T.K. and W.W.L. performed research; J.T.K. and C.A.S. contributed new reagents/analytic tools; J.T.K., T.M.A., N.J., and regulation of gene expression [e.g., repression of outer mem- MICROBIOLOGY brane protein synthesis by the sRNAs MicA and RybB (5)], but W.W.L. analyzed data; and J.T.K. and W.W.L. wrote the paper. positive regulation by sRNAs also has been described [e.g., The authors declare no conflict of interest. regulation of the stationary phase Sigma factor RpoS by the This article is a PNAS Direct Submission. sRNAs DsrA and RprA (6)]. In most cases, the RNA chaperone 1Present address: École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzer- protein Hfq is required, presumably to stabilize the sRNA– land. mRNA interaction, because the sRNA contact on the target 2To whom correspondence should be addressed. E-mail: [email protected]. typically is short and imperfect (7). See Author Summary on page 15029. Like protein regulators of gene expression, sRNAs act to in- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tegrate extracellular signals that aid bacteria in adjusting to their 1073/pnas.1101655108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1101655108 PNAS | September 13, 2011 | vol. 108 | no. 37 | E709–E717 Downloaded by guest on October 1, 2021 synthesis of virulence factors in these bacteria. To do so, we used a deep sequencing approach to perform a transcriptome-wide analysis to identify sRNAs encoded by the chromosome and plasmids of Y. pseudotuberculosis strain IP32953. We identified a total of 150 unannotated sRNAs, the majority of which are Yersinia specific. The deletion of multiple sRNAs led to the attenuation of Y. pseudotuberculosis in a mouse model of yersiniosis, suggesting that these sRNAs regulate factors involved in the pathogenesis of this species. In addition, we found that the deletion of one of these virulence-associated, Yersinia-specific sRNAs that is conserved between Y. pseudotuberculosis and Y. pestis resulted in the reduced virulence of the plague pathogen in an intranasal mouse model of infection. These results may have implications for the evolution of mechanisms that regulate virulence in Yersinia. Results Global Identification of sRNAs Expressed by Y. pseudotuberculosis. Our goal was to identify sRNAs expressed by Y. pseudotuberculosis in an unbiased fashion. For this purpose we generated sRNA libraries of Y. pseudotuberculosis grown to early-log, mid- log, late-log, and stationary phase in broth culture at 26 °C and 37 °C and subjected these libraries to deep sequencing based on the Illumina-Solexa platform (18). This analysis resulted in ∼2.4– 17 million 36-nt-long reads that were aligned exactly to the Y. pseudotuberculosis Fig. 1. Experimental identification of annotated sRNAs in Y. pseudotu- chromosome and plasmids. The mapped berculosis.(A) All previously annotated Y. pseudotuberculosis sRNAs were reads were categorized into mRNA, ribosomal RNA (rRNA), identified by Illumina-Solexa sequencing. Percent of total reads for anno- tRNA, tmRNA, and miscellaneous (misc.) RNAs, and the re- tated RNAs identified at 37 °C shows a flux in sRNA levels over the course of mainder of the reads was classified as intergenic RNA sequences. bacterial growth. (B) The expression levels of the sRNA spf (Spot 42) as de- The distribution of mapped reads into categories at the two termined by deep sequencing reads (red bars in A) correlate with the levels temperatures is shown in SI Appendix, Fig. S1. The miscellaneous of the sRNA as measured by Northern blot analysis. RNAs category contains all previously annotated sRNAs in Y. pseudotuberculosis (the annotation of which was based on se- Y. pseudotuberculosis fi quence homology with Escherichia coli and Salmonella typhimu- the plasmid pYptb and identi ed only a rium sRNAs), and our analysis was able simultaneously to single sRNA on the plasmid pYV. confirm and map the changing expression patterns of these Yersinia fi fi sRNAs over time (Fig. 1A). Spot 42 (Spf) is a previously anno- -Speci c sRNAs. The 150 clusters that satis ed the criteria for presumed sRNAs are referred to hereafter as “Ysrs” (for tated sRNA for which the numbers of deep sequencing reads Yersinia correlated well with its expression as determined by Northern small RNAs). The predicted genomic coordinates and blot (Fig. 1B). Additionally, the sensitivity of Illumina-based sizes, orientation with respect to surrounding ORFs, and num- deep sequencing in identifying sRNAs was validated, because bers of deep sequencing reads generated at the different tem- low-abundance sRNAs such as SraG, which was represented by peratures and time points for all the Ysrs are listed in Dataset as few as 191 reads and could not be detected by Northern blot S1. For 32 Ysrs, BlastN analysis revealed orthologous sequences E. coli S. typhimurium using as much as 10 μg of total RNA, are detected by this in the and genomes; these sequences in- method. Notably, the major RNA species in the cell, rRNA, was clude the previously characterized sRNAs MicA (Ysr7), FnrS/ reduced to ∼0.01–0.7% of total reads by rRNA depletion with Stnc520 (Ysr11), RprA (Ysr40), GcvB (Ysr45), RybB (Ysr48), MicrobExpress (Ambion). The proportion of tRNA reads in the MicM (Ysr145), RyhB (Ysr146.1 and Ysr146.2), GlmY (Ysr147), total also is small (0.3–19%) compared with previous global GlmZ (Ysr148), and OmrA/B (Ysr149) (3). Seventy-nine percent sRNA identification efforts (19). The intergenic reads mapped to (118/150) of the Ysrs are specifictoY. pseudotuberculosis and the chromosome of Y. pseudotuberculosis (29.6–43.3% of total Y. pestis in that they do not show sequence conservation with reads) contain the 5′ and 3′ UTRs of transcribed mRNAs, cis- other bacterial species (Fig. 2, highlighted in red). Ninety-two encoded antisense RNAs, and putative trans-encoded sRNAs. Ysrs also are absent from other members of the Yersinia genus To identify these sRNAs specifically, the intergenic reads were such as Y. ruckeri, Y. frederiksenii, and Y. intermedia and also grouped further into clusters at least 50 nt in length (e.g., this from the closely related genus Photorhabdus (Dataset S1, sheet grouping yielded ∼3,200 clusters for the 6-h time-point at 37 °C). 2). We identified six Ysrs that are Y. pseudotuberculosis specific A filtering algorithm was developed to eliminate the majority of (Ysr29, Ysr53, Ysr70, Ysr84, Ysr94, Ysr118), having no homol- the 5′ and 3′ UTRs in the clustered intergenic groups by ana- ogous sequences in the genome of Y. pestis. In addition, 63/144 lyzing the expression level of clusters compared with the sur- Ysrs encoded by both Y. pseudotuberculosis and Y. pestis contain rounding ORFs. The clusters whose expression levels differed single or multiple differences in sequence (i.e., mismatches, from the expression levels of a neighboring ORF by less than deletions, or insertions highlighted in yellow in Fig. 2). threefold were removed from further analysis. The remaining At 26 °C, the majority of identified sRNAs are not expressed intergenic clusters (e.g., 1,313 clusters for the 6-h time point at in earlier stages of growth (based on number of deep sequencing 37 °C) then were assessed using the Integrated Genome Browser reads) and start to accumulate at later time points (SI Appendix, (20). Computational prediction of putative promoter and ρ-in- Fig. S2). The exceptions are Ysr8, Ysr32, and Ysr45/GcvB. Also, dependent terminator sequences, which are characteristic fea- at this temperature, Ysr3 levels peak in late-log phase and then tures of bacterial sRNAs (21), led to the identification of 150 decrease in stationary phase. Although there are more excep- putative unannotated sRNAs in Y. pseudotuberculosis. Analysis tions to this trend at 37 °C than at 26 °C, a large proportion of of the intergenic clusters did not reveal any potential sRNAs on sRNAs accumulate over the growth curve (SI Appendix, Fig. S2).

E710 | www.pnas.org/cgi/doi/10.1073/pnas.1101655108 Koo et al. Downloaded by guest on October 1, 2021 PNAS PLUS

Fig. 2. Yersinia small RNAs (Ysrs). The sequences of sRNAs identiﬁed by deep sequencing in Y. pseudotuberculosis strain IP32953 were compared with the genomes of Y. pestis strain CO92, Y. enterocolitica 8081, E. coli strain K12 (substrain MG1655), and Salmonella enterica serovar Typhimurium strain LT2 by BlastN analysis. Ysrs present in the genome and identical in sequence are shown in green, and Ysrs present in the genome with sequence differences are shown in yellow. For an sRNA to be considered present in a genome, at least 50 nt of its sequence had to contain homology in the genome of interest. Ysrs absent from the genome are shown in red. Detailed information for each Ysr is given in Dataset S1.

The most notable exception at 37 °C is Ysr45/GcvB, the levels of of Ysr11, which exhibits the highest expression level at late-log which are highest at the start of growth and decrease over time. phase and then decreases in stationary phase. This sRNA dis- The most abundant sRNA at 26 °C is Ysr45/GcvB, whereas at played a similar pattern of expression when examined in S. 37 °C, the most sequencing reads were obtained for Ysr7/MicA typhimurium (STnc520) (23). Unlike S. typhimurium,however,the and Ysr149/OmrA/B (SI Appendix, Fig. S2). abundance of Ysr11 is not dependent on Hfq at either temperature. Ysr4 and Ysr48/RybB are the only sRNAs examined whose Verification of sRNA Expression. Northern blot analysis was used to expression levels are Hfq dependent at 37 °C. For all the sRNAs detect newly identified sRNAs in Y. pseudotuberculosis.We tested at both temperatures, only the expression pattern of Ysr45/ performed the analysis for 49 Ysrs and detected 29 of these GcvB at 37 °C by Northern blot appears to be different from that RNAs using biotinylated oligonucleotide probes (Dataset S1). predicted by the numbers of deep sequencing reads. For most of the sRNAs detected, the band sizes correlated with Because all but six sRNAs identified in Y. pseudotuberculosis the lengths predicted by the deep sequencing (i.e., Ysr4, Ysr7, contain an at least partially homologous sequence in the Y. pestis and Ysr11). Some Ysrs, however, yielded bands corresponding to genome, Northern blot analysis was used to examine the expres- larger transcripts (i.e., Ysr1, Ysr2, and Ysr9). For nine of these sion of the seven sRNAs tested above in Y. pestis grown under the Ysrs, we performed simultaneous 5′ and 3′ RACE to map the same conditions (26 °C and 37 °C, wild-type and hfq mutant) (Fig. boundaries of primary transcripts and to distinguish them from 3B). In Y. pestis, at 26 °C the sRNAs examined show mostly steady- the processing of longer transcripts. The genomic coordinates state levels over time and little dependence on Hfq. At 37 °C, and lengths of these sRNAs are listed in Dataset S1, and for however, these Ysrs are expressed differently than in Y. pseudo- eight of the nine Ysrs, the coordinates are identical to or fall tuberculosis: Most show stable levels or accumulation over time within 20 nt of the borders predicted by deep sequencing. with a peak at late-log phase and are almost undetectable in sta- Because many genes in Yersiniae are under thermal regulation tionary phase. Additionally, unlike in Y. pseudotuberculosis,allthe and are expressed at either 26 °C or 37 °C (22), it was of interest to sRNAs tested require Hfq for their stability/expression. examine the expression of Ysrs over the time course of growth at Ysr29 is one of six Y. pseudotuberculosis specific sRNAs. In- both these temperatures. Additionally, because most sRNAs terestingly, this sRNA is unique in that it is specific for the identified in enteric bacteria are dependent on Hfq for their IP32953 strain of Y. pseudotuberculosis and is absent from other functions, we examined whether the absence of hfq from the Y. sequenced Y. pseudotuberculosis isolates. It is encoded in the pseudotuberculosis genome led to differential expression and/or intergenic region between the adenylate cyclase gene cyaA and stability of the Ysr in question. The expression levels of all Ysrs antisense to a portion of the 3′ end of YPTB0186 (encoding tested increase over time and culminate in stationary phase at a putative transposase for IS285). Even though there are multi- 26 °C (Fig. 3A). At this temperature only Ysr4 shows strong ple copies of this transposase in the genomes of Y. pseudotu- dependence on Hfq for its expression or stability. All the Ysrs berculosis and Y. pestis, the Ysr29 locus is the only occurrence of tested at 37 °C are expressed similarly at 26 °C, with the exception this sRNA sequence. The Ysr29 transcript accumulates in sta- MICROBIOLOGY

Fig. 3. Veriﬁcation of Ysr expression. (A) Northern blot analysis was performed to examine the expression of identiﬁed Ysrs. (B) Northern blot analysis of Y. pseudotuberculosis and Y. pestis wild-type and Δhfq strains. Representative blots of at least two replicate reproducible experiments are shown. Black triangles above the blots indicate different time points on the bacterial growth curve at which the cultures were collected for RNA isolation (i.e., early-log, mid-log, late-log, and stationary phase). 5S rRNA is shown as a loading control.

Koo et al. PNAS | September 13, 2011 | vol. 108 | no. 37 | E711 Downloaded by guest on October 1, 2021 tionary phase of growth at both 26 °C and 37 °C, with slightly dicator of their overall health. Mice infected with wild-type elevated levels at the optimal growth temperature for Y. pseu- bacteria displayed significant weight loss by day 6 before succumb- dotuberculosis (Fig. 3A). This sRNA may be classified as Hfq ing to infection, whereas the animals infected with Ysr mutants that dependent, because its levels decrease in the absence of hfq, survived the infection were able to recover after an initial weight particularly at 26 °C. decline and continued to maintain or gain weight (Fig. 4B). Ysr35 is 339 nt long, and the ExPasyTranslate Tool (24) Contribution of Newly Identified Yersinia sRNAs to Virulence. Pre- predicts that a small ORF may be encoded within the sRNA. To vious work from our laboratory has established that Hfq is crit- determine if a peptide is synthesized from the predicted ORF, ical for the pathogenesis of Y. pseudotuberculosis, and the we generated by allelic replacement a version of the ORF with deletion of hfq results in an ∼10,000-fold attenuation of the the influenza HA epitope tag on the predicted C-terminal end. bacteria in a mouse model of yersiniosis (17). This result suggests Under the conditions in which Ysr35 is expressed, we were un- that Hfq, together with sRNAs, regulates essential virulence able to detect a HA-tagged peptide by immunoblot (SI Appendix, determinants in Y. pseudotuberculosis. To test this hypothesis, the Fig. S3B). Hfq-dependent sRNAs Ysr29 and Ysr48/RybB and the Hfq-in- Because Ysr23, Ysr35, and Ysr48/RybB (but not Ysr29) also dependent sRNAs Ysr23 and Ysr35 (SI Appendix, Fig. S3A) were are encoded in the genome of Y. pestis and are transcribed deleted from the Y. pseudotuberculosis chromosome by homol- (Dataset S1 and Fig. 3), we deleted these sRNAs from Y. pestis ogous recombination. Deletions of these Ysrs were confirmed by (SI Appendix, Fig. S3D) and examined their contributions to the PCR (SI Appendix, Fig. S3C). The strains carrying deletions of development of pneumonic plague. Similarly to Y. pseudotuber- Ysr23, Ysr29, Ysr35, and Ysr48/RybB grew comparably to the culosis, the inactivation of both Ysr23 (although affecting the wild-type strain at both 26 °C and 37 °C (SI Appendix, Fig. S3E expression of one of the neighboring ORFs, ybcI)(SI Appendix, and F). In addition, the deletion of Ysr23, Ysr29, Ysr35, and Fig. S4) and Ysr48/RybB does not affect the ability of Y. pestis to Ysr48/RybB did not alter significantly the expression of the cause disease and the subsequent death of mice when delivered surrounding ORFs as measured by quantitative RT-PCR (qRT- via the intranasal route (Fig. 4C). However, much as in Y. PCR) (SI Appendix, Fig. S4). To determine whether these pseudotuberculosis, the deletion of Ysr35 results in the attenua- sRNAs contribute to the virulence of Y. pseudotuberculosis, wild- tion of Y. pestis following intranasal infection, significantly type and Ysr-deleted strains were used to infect BALB/c mice via delaying the time to death of mice as compared with the fully the intragastric route. Although 90% of mice infected with 2.0 × 105 virulent, wild-type strain (Fig. 4C). This observation suggests that cfu of wild-type Y. pseudotuberculosis succumbed by day 8 post- Ysr35 may play a role in virulence that is conserved between the infection, 10% of mice infected with ΔYsr48/RybB and 50% of two Yersinia species. mice infected with ΔYsr29 or with ΔYsr35 survived for the du- ration of the experiment (Fig. 4A). Infection of mice with the Identification of sRNA-Regulated Proteins. Considering the unique- ΔYsr23 strain did not result in a significant difference in survival ness of Ysr29 to the IP32953 strain of Y. pseudotuberculosis compared with the wild-type but showed a trend toward atten- and its contribution to virulence, we performed a proteomic uation. We also measured the weights of the animals as an in- analysis using 2D differential gel electrophoresis (2D-DIGE) to

Fig. 4. Contribution of Ysrs to the virulence of Y. pseudotuberculosis and Y. pestis.(A) Groups of 10 mice were inoculated via oral gavage with Y. pseudotuberculosis wild-type, ΔYsr23, ΔYsr29, ΔYsr35, and ΔYsr48/RybB strains (∼2.0 × 105 cfu). Survival of infected mice was monitored over 21 d. P values were determined by Mantel–Cox survival analysis log-rank test. P = 0.2254 for ΔYsr23 compared with wild-type (not significant); *P = 0.0202 for ΔYsr29 compared with wild-type; ***P = 0.0002 for ΔYsr35 compared with wild-type; P = 0.9154 for ΔYsr48/RybB compared with wild-type (not significant). Data are representative of three independent experiments. (B) Body weight over 21 d of mice infected with Y. pseudotuberculosis. The plot shows median weight, indicated by a solid line; boxes represent the 25th and 75th percentiles, and whiskers represent the range. Significance was calculated by student’s unpaired t-test. (C) Groups of 10 mice were inoculated intranasally with Y. pestis wild-type, ΔYsr23, ΔYsr35, and ΔYsr48/RybB strains (∼1.0 × 104 cfu). Survival of infected mice was monitored over 7 d. P values were determined by Mantel–Cox survival analysis log-rank test. P = 0.9066 for ΔYsr23 compared with wild-type and P = 0.0946 for ΔYsr48/RybB compared with wild-type (both not significant); ***P < 0.0001 for ΔYsr35 compared with wild-type. Data are representative of two independent experiments.

E712 | www.pnas.org/cgi/doi/10.1073/pnas.1101655108 Koo et al. Downloaded by guest on October 1, 2021 determine the regulated targets of this sRNA. Protein profiles transcript levels for the genes in question are equivalent in the PNAS PLUS from whole-cell lysates of wild-type Y. pseudotuberculosis were wild-type and ΔYsr29 strains (Fig. 5B). compared with those of the ΔYsr29 strain grown to stationary Therefore, we examined the effects of Ysr29 at the post- phase at 26 °C, the time point at which this sRNA is most transcriptional level by generating chromosomal in-frame fusions abundant. The comparison of protein profiles between the wild- of the GST, RpsA, OmpA, and GroEL coding regions with the type and ΔYsr29 strains showed 16 spots with 1.5-fold or more HA-epitope tag in both wild-type and ΔYsr29 strains. Levels of difference in fluorescence intensity (Fig. 5A). Thirteen spots fusion proteins were measured by immunoblot analysis using an were analyzed successfully by MALDI-TOF mass spectroscopy anti-HA antibody (Fig. 5C). In accordance with 2D-DIGE, we and correspond to eight proteins: 30S ribosomal protein S1 found that GST is more abundant in the ΔYsr29 strain than in (RpsA), outer membrane protein A (OmpA), the chaperonin the wild-type background, whereas RpsA, OmpA, and GroEL GroEL, glutathione-S-transferase (GST), the molecular chap- are elevated in the wild-type compared with the ΔYsr29 strain, erone DnaK, peroxidase (AhpC), ribosome recycling factor demonstrating posttranscriptional regulation by this sRNA. Be- (RRF), and urease (UreC) (SI Appendix, Table S1). Ysr29 does cause the overexpression of sRNAs can result in more dramatic not appear to regulate the identified proteins at the level effects on regulated target protein levels than sRNA deletions, of transcription, because qRT-PCR analysis shows that the we generated an isopropyl-β-D-thiogalactopyranoside (IPTG)-

Fig. 5. Posttranscriptional regulation of targets by Ysr29. (A) Proteomic comparison of Y. pseudotuberculosis wild-type and ΔYsr29 strains by 2D-DIGE. Wild- type proteins were labeled with Cy3, and ΔYsr29 mutant proteins were labeled with Cy5. The gel image shows 1.5× or greater differences in spot volume ratio > Δ

for 16 marked spots. Blue indicates spots with a Cy3/Cy5 ratio 0 (increased expression in Ysr29 compared with the wild-type); red indicates spots with a Cy3/ MICROBIOLOGY Cy5 ratio <0 (increased expression in wild-type compared with ΔYsr29). Molecular masses in kilodaltons, indicated by the numbers to the right of the image, are approximate. (B) Expression of Ysr29 targets. Cultures of wild-type and ΔYsr29 strains were grown to stationary phase at 26 °C, and transcript levels of targets identiﬁed by 2D-DIGE were examined by qRT-PCR. The fold change in RNA levels is relative to wild-type, which was set to 1. (C) Western blots of whole-cell lysates from wild-type and ΔYsr29 strains expressing chromosomal HA fusions of GroEL, OmpA, RpsA, and GST. (Upper) Anti-HA antibody. (Lower) Anti-RpoA antibody (loading control). (D) The effect of Ysr29 overexpression on GroEL and GST. ΔYsr29 strains with the GroEL-HA and GST-HA fusions were

electroporated with either the pACY177 vector or a Ptac-Ysr29 overexpression construct. (Upper) Northern blots showing overexpression of Ysr29 upon addition of 1 mM IPTG to the growth medium. Note: The band in the pACYC177 samples is nonspeciﬁc and migrates at a slightly higher molecular weight than Ysr29. 5S rRNA is shown as a loading control. (Lower) Immunoblots showing the effect of Ysr29 overexpression on GST-HA, GroEL-HA, and RpoA (loading control). (E) IntaRNA predictions of base-pairing between Ysr29 and the target mRNAs. The software predicts at least hepta-nucleotide pairing of Ysr29 with the 5′ UTRs of all four target mRNAs.

Koo et al. PNAS | September 13, 2011 | vol. 108 | no. 37 | E713 Downloaded by guest on October 1, 2021 inducible Ysr29 plasmid and introduced this construct into the express a DsrA ortholog, for example, raises the question of how ΔYsr29 strains carrying the GST-HA and GroEL-HA fusions. Y. pseudotuberculosis regulates the stationary phase Sigma factor As expected, upon induction of Ysr29 expression with IPTG σS synthesis. One possibility is that some of the Yersinia-specific (Fig. 5D, Upper), the abundance of GroEL-HA was elevated, and sRNAs that we have identified functionally replace the above- the levels of GST-HA were reduced (Fig. 5D, Lower). Because mentioned enteric sRNAs. Alternatively, RpoS in Yersiniae may Ysr29 expression is not fully repressed in the GST-HA strain not require regulation by sRNAs for its synthesis. Another dif- (Ptac-Ysr29, 0-min Northern blot in Fig. 5D), GST-HA protein is ference is that Y. pseudotuberculosis encodes two distinct copies not detected even at the 0-min time point, demonstrating the of the iron level-regulated sRNA RyhB (Ysr146.1 and Ysr146.2). substantial effect of extended Ysr29 overexpression on GST Because the expression levels of these sRNAs in Y. pseudotu- synthesis. Finally, we used a computational approach to predict if berculosis are low in iron-replete conditions, it would be in- Ysr29 could interact with the 5′ UTRs of these target mRNAs. teresting to examine the expression of the two RyhB copies in Y. The IntaRNA alignment tool (25) predicts at least 7-nt-long pseudotuberculosis in the iron-limiting conditions that are known base-pairing interactions of Ysr29 with the 5′ UTRs of all four to induce RyhB in E. coli (26, 35). validated targets (Fig. 5E). We also identified 118 Yersinia-specific sRNAs (78.7%) without a sequence match in available databases. Similarly, an Discussion analysis of S. typhimurium-specific genetic islands by Padalon- Although small regulatory RNAs have become increasingly Brauch et al. (36) led to the identification of 28 candidate recognized as major modulators of gene expression in bacteria sRNAs, 19 of which were verified. These observations suggest (26), only a limited number of studies have sought to identify the that related bacterial species have evolved distinct species-spe- global set of expressed sRNAs, particularly in bacterial patho- cific regulatory networks that rely on sRNAs that are required gens (27–29). Additionally, the expression of only a small num- for the adaptation to a particular niche or, in the case of ber of sRNAs has been verified in Yersiniae relative to other pathogens or symbionts, for the interaction with the host. Ad- enteric bacteria (3). Here, we describe the deep sequencing- ditionally, we identified sRNAs that are conserved between Y. based identification of sRNAs in the human gastrointestinal pseudotuberculosis and Y. pestis, two species of Yersiniae that pathogen Y. pseudotuberculosis. A similar approach has been share 97% identity in 75% of their protein-coding genes (15). used to identify sRNAs and mRNA targets that interact with Hfq Remarkably, a substantial number of these sRNAs (43.75%) in S. typhimurium (23) and in an RNomic analysis of Bur- contain nucleotide mismatches, insertions, or deletions. Al- kholderia cenocepacia (30). Analysis of sRNA libraries generated though these hypotheses are not yet tested, it is interesting to from Y. pseudotuberculosis grown at 26 °C and at 37 °C led to the speculate that these differences in sRNA sequences might alter identification of 150 putative sRNAs as well as confirming the the secondary structure of the molecules or that a single-nucle- expression of 15 previously annotated sRNAs. The ability to otide mismatch between the sRNA and its target mRNA might identify all the “miscellaneous” RNAs in an impartial manner affect the outcome of the interaction. To determine whether the validated the deep sequencing approach for discovery of pre- nucleotide differences present in Ysrs in Y. pseudotuberculosis viously unidentified sRNAs. The number of putative sRNAs is and Y. pestis are localized or are spread across the entire se- much smaller than the 1,478 sRNAs predicted for the closely quence of the sRNA, we performed ClustalW2 alignments of all related Y. pestis by Livny et al. (31) who used a computer algo- Ysrs containing mismatches between the two species (SI Ap- rithm that relies on conservation of intergenic sequences and pendix, Table S2). The differences do not localize to either the 5′ ρ-independent terminators, but is comparable to the number of or the 3′ end of Ysrs but rather are spread throughout the length sRNAs predicted and verified in other enteric bacteria (3). It is of the sRNAs. Thus, although the mismatches in the conserved possible, however, that additional sRNAs may be expressed un- sRNAs could lead to differential regulation of targets by the two der conditions not examined here, such as upon host-cell contact closely related species, it also is possible that these differences or during animal infection. may have little or no effect on sRNA function. Thirty-two identified sRNAs are orthologs of potential sRNAs We also observed that sRNAs conserved between Y. pseudo- encoded in the genomes of E. coli and S. typhimurium. This tuberculosis and Y. pestis demonstrated divergent expression finding is not surprising, because sequence conservation in patterns. Most Ysrs accumulate over time in Y. pseudotubercu- intergenic regions of closely related bacteria was one of the losis, but in Y. pestis the levels of those same Ysrs peak in late-log original criteria used to identify sRNAs (32). The unexpected phase and decrease in stationary phase. This difference suggests observation is that many of the well-characterized sRNAs from that the temporal regulation of Ysr gene expression or stability E. coli and S. typhimurium are not encoded by the genome of may affect the outcomes of Ysrs in modulating target mRNA Y. pseudotuberculosis as determined by BlastN alignment analy- expression. This hypothesis is supported by the work of Bai et al. sis. These sRNAs include MicC, OxyS, ArcZ, and DsrA, among (37), which showed that the deletion of hfq causes a severe others. Although informative, this type of analysis also was un- growth defect at 37 °C in Y. pestis but a less drastic effect in Y. able to identify SgrS in the genome of Y. pseudotuberculosis,an pseudotuberculosis. Thus, our findings, together with those of Bai sRNA that has been described previously in this species (33, 34). et al. (37), further support the hypothesis that these two genet- Interestingly, the filtering algorithm we used to identify sRNAs ically linked species could differentially regulate gene expression from our deep sequencing data also did not reveal SgrS, but via sRNAs to result in divergent disease manifestations. when the parameters were relaxed (i.e., the position of the Most trans-acting sRNAs in bacteria require the RNA chap- cluster with respect to any ORF was moved within 1 kb on the erone Hfq for their functions. Although the exact role of Hfq is same strand), the deep sequencing reads for SgrS were indeed not fully understood, it appears that the chaperone promotes present (Ysr150 in Dataset S1). This observation suggests that base-pairing interactions of the sRNA and the mRNA target by using multiple tools may enhance the comparison of sRNA increasing the rate of sRNA–mRNA association in addition to sequences between species and that adjusting the parameters of protecting the sRNAs from degradation (38, 39). Here we note deep sequencing data analysis could reveal additional sRNAs another difference between Y. pseudotuberculosis and E. coli and encoded in the genome of Y. pseudotuberculosis. Salmonella.InE. coli and Salmonella, MicA and GcvB are Given the limitations discussed above, it is interesting to hy- known to rely on Hfq for proper expression/stability and also for pothesize how Y. pseudotuberculosis may have evolved to regu- the regulation of their mRNA targets (40, 41), but in Y. pseu- late the expression of genes that are conserved between the dotuberculosis, expression levels of Ysr7/MicA, Ysr20, Ysr23, enteric bacteria. The fact that Y. pseudotuberculosis may not and Ysr45/GcvB do not differ between the wild-type and Δhfq

E714 | www.pnas.org/cgi/doi/10.1073/pnas.1101655108 Koo et al. Downloaded by guest on October 1, 2021 strains. Our finding that the majority of sRNAs tested in Y. outer membrane protein synthesis (51)]. In sum, the global PNAS PLUS pseudotuberculosis do not require Hfq for their expression and/or identification of sRNAs in Y. pseudotuberculosis provides an stability suggests the possibility of an alternative sRNA chaper- opportunity to discover mechanisms of virulence gene regulation one. Y. pseudotuberculosis may rely on SmpB, an Sm-like chap- in this pathogenic bacterium, particularly in comparison with erone of tmRNA (42), or an as yet unidentified protein to closely related species such as Y. pestis and in contrast to other stabilize sRNAs and promote interaction with mRNAs. bacteria such as E. coli and Salmonella, the prototype species for Hfq is known to play a role in the virulence of Y. pseudotu- studying sRNAs. berculosis (17), and in other species a majority of sRNAs require Hfq for their function (reviewed in ref. 3). Therefore, we ex- Materials and Methods amined the contribution of several sRNAs, whose stability/ex- RNA Isolation. Overnight cultures of Y. pseudotuberculosis strain IP32953 pression either relies on or does not require Hfq, to the virulence were subcultured in brain heart infusion (BHI) broth supplemented with 2.5 of Y. pseudotuberculosis. The deletion of both Ysr29 and Ysr35 mM CaCl2 to an OD620 of 0.1 and grown to early-log (OD620 0.3, 1.5 h), mid- resulted in increased survival of mutant-infected mice compared log (OD620 0.65, 3.5 h), late-log (OD620 4.3, 6 h), and stationary phase (OD620 with those infected with wild-type bacteria, whereas the deletion 7.6, 11 h) at either 26 °C or 37 °C. Five OD equivalents were collected, and RNA was stabilized with the RNAprotect Bacteria reagent (Qiagen). RNA of Ysr23 delayed the overall time to death of mice but did not enriched for sRNAs was extracted using the RiboPure Bacteria kit (Ambion) impact overall survival. Interestingly, deletion of the Hfq-de- with modifications. Specifically, the provided columns (which are designed pendent sRNA Ysr48/RybB did not result in a significant de- to eliminate small RNAs) were omitted, and instead the RNA was pre- crease in virulence of Y. pseudotuberculosis, suggesting that not all cipitated overnight with isopropanol. Isolated RNA was treated with DNase I Hfq-binding sRNAs play a direct role in virulence. The attenua- (Ambion), and the quality was assessed using the Experion automated tion of individual sRNA-deleted strains is not as dramatic as that electrophoresis system (Bio-Rad). RNA also was isolated as above from Y. of a Δhfq strain (17), suggesting that in the absence of Hfq the pestis strain CO92 Δpgm for experiments comparing the expression of fi dysregulation of multiple sRNA-dependent targets may result in identi ed sRNAs by Northern blot analysis. For Y. pestis RNA isolation, the severe attenuation of Y. pseudotuberculosis. It is possible that overnight cultures diluted to an OD620 of 0.1 were grown to early-log (OD620 0.2, 2.5 h), mid-log (OD620 0.8, 5.5 h), late-log (OD620 1.8, 8.5 h), and sta- a double mutant of Ysr35 and Ysr29 may have a phenotype that tionary phase (OD 4.5, 15 h) at 26 °C and at 37 °C. more closely resembles that of the Δhfq mutant. 620 Yersinia fi Because Ysr35 is a -speci c sRNA that is conserved sRNA Library Preparation and Illumina-Solexa Sequencing. Ribosomal RNA was between Y. pseudotuberculosis and Y. pestis, we deleted this eliminated by the MicrobExpress kit (Ambion), and the integrity of the RNA sRNA from the genome of the plague bacillus and tested its role was reanalyzed by Experion electrophoresis. sRNA libraries were generated in virulence. The Ysr35 deletion is attenuated in a mouse model using the Illumina small RNA v1.5 kit with the following modifications: RNA of pneumonic plague (Fig. 4C), suggesting that the sRNA may was treated with 5′ RNA polyphosphatase (Epicentre) followed by the liga- play a similar role in virulence in Y. pseudotuberculosis and Y. tion of RNA adapter oligonucleotides, cDNA synthesis, and PCR amplification pestis. It will be of interest to determine whether Ysr35 regulates for 12 cycles. The library then was separated on 6% acrylamide gels, and – the same or distinct targets in the two species. In addition, al- fragments between 90 500 nt were excised. cDNA was eluted from the gels and precipitated. Cluster generation was performed according to the man- though we were unable to detect the production of a Ysr35- ufacturer’s protocols (Illumina), and 36-nt single-end reads were generated encoded peptide by immunoblot in vitro, our results do not on a Solexa Genome Analyzer at the Institute for Genomics and Systems discount the possibility that the ORF still may be translated Biology (Argonne National Laboratory, Argonne, IL). The Solexa reads that under other conditions. passed the purity filtering and had a unique alignment were mapped to the Hfq-binding sRNAs in E. coli and S. typhimurium exert a variety Y. pseudotuberculosis IP32953 reference chromosome (NC_006155.1) and of physiological roles mediated by the base-pairing interactions of plasmids (pYV, NC_006154; pYtb, NC_006153). the sRNA to the mRNA target. We sought to determine how Ysr29 may control virulence in Y. pseudotuberculosis by identifying Bioinformatics. Solexa sequencing reads that overlapped the annotated the regulated targets of this unique sRNA. Proteomic analyses of miscellaneous RNA, mRNA, rRNA, tmRNA, and tRNA genes (based on the wild-type and ΔYsr29 strains resulted in identification of eight above GenBank records of Y. pseudotuberculosis IP32953 chromosome and putative targets of Ysr29, although it is not yet known whether plasmids) were extracted and counted. The reads not overlapping these annotations were considered to be intergenic and include 5′ and 3′ UTRs, cis- Ysr29 regulates these factors directly or acts indirectly to alter encoded antisense RNAs, and potential trans-encoded sRNAs. Clusters of at their synthesis. Nevertheless, Ysr29 may act on these targets least 50 bp in length that form a continuous region of coverage were posttranscriptionally, because the absence of the sRNA does not extracted from the intergenic category of reads on each strand of the affect their transcript levels but instead alters the levels of protein. chromosome or plasmids. These clusters were analyzed further by calculat- This finding suggests that 2D-DIGE accompanied by target-fusion ing the expression level in reads per kilobase for each intergenic cluster and (or direct Western blot analysis, if antibodies are available) is an all miscellaneous RNA, mRNA, rRNA, tmRNA, and tRNA genes. To aid in appropriate approach for validating target gene expression, be- eliminating 5′ and 3′ UTRs from the analysis of intergenic clusters, intergenic cause transcriptional profiling by microarray probably would not clusters differentially expressed with respect to nearby ORFs were de- reveal the targets identified by our analysis. termined. The candidate clusters were required to have a difference in expression level above or below a threshold of at least threefold with respect All the Ysr29-regulated targets validated by immunoblot Y. to any ORF within 1 kb on the same strand. Generated cluster sets then were analysis have the potential to be involved in the virulence of analyzed using the Integrated Genome Browser (http://www.bioviz.org/igb/). pseudotuberculosis. For example, GST participates in protecting Predicted sRNAs were inspected for the presence of promoters and ρ- cells against the damage of oxidative stress (43, 44), and Ysr29 independent terminators using the BProm and TermFind/RNAFold programs repression of GST levels may prevent an aberrant response to (Softberry).

this stress. In addition, OmpA is an outer membrane protein MICROBIOLOGY known to play a role in the pathogenesis of E. coli K1 and acts Animal Experiments. All procedures involving animals were carried out in as an adhesin, invasin, and immune evasin (45–47). Interestingly, compliance with protocols approved by the Northwestern University in- in E. coli, the expression of OmpA is posttranscriptionally re- stitutional animal care and use committee. For Y. pseudotuberculosis pressed by MicA (48). It is not known if MicA acts similarly in infections, 8-wk-old female BALB/c mice were purchased from Harlan Lab- Y. pseudotuberculosis oratories and allowed to acclimate in the animal facility for 5–7 d before , but we show here that Ysr29 acts to en- infection. To prepare the inocula, Y. pseudotuberculosis strains were cul- hance OmpA levels. There are other examples of targets being tured overnight in BHI broth at 26 °C, diluted to an OD620 of 0.1 in BHI broth, differently regulated by multiple sRNAs [e.g., RpoS regulation and incubated at 26 °C with shaking (250 rpm) to an OD620 of 0.6. The cells by DsrA, RprA, ArcZ, and OxyS (49, 50)] and also of multiple were harvested by centrifugation, washed once with sterile PBS, and diluted

targets under the control of one sRNA [e.g., RybB regulation of to the appropriate OD620 in PBS. Groups of 10 mice were inoculated intra-

Koo et al. PNAS | September 13, 2011 | vol. 108 | no. 37 | E715 Downloaded by guest on October 1, 2021 gastrically using a 22-gauge feeding needle with ∼2 × 105 cfu of Y. pseu- (GE Healthcare) according to the manufacturer’s instructions. Both samples dotuberculosis and monitored for 21 d. The weights of individual mice were were run in a single polyacrylamide gel but were imaged separately at recorded every third day as an indicator of health status. Input colony- a wavelength of 570 nm for Cy3 and at a wavelength of 670 nm for Cy5. forming units were determined by serial plating onto BHI agar. Animals Protein spots were selected based on >1.5-fold differential expression be- infected with wild-type Y. pseudotuberculosis that demonstrated weight tween the wild-type and ΔYsr29 mutant and were subjected to robotic spot loss at any point during the infection were included in the analysis. For Y. excision, trypsin digestion, and MALDI-TOF. Protein identification was based pestis infections, pathogen-free 6- to 8-wk-old female C57BL/6 mice were on highly accurate masses and MS/MS sequence data from multiple tryptic obtained from the Jackson Laboratory and allowed to acclimate as above. peptides. The 2D-DIGE and subsequent analyses were performed at the Bacteria were grown in BHI broth as described in SI Appendix, SI Materials and Methods, washed once with sterile PBS, and maintained at 37 °C. Groups W. M. Keck Foundation Biotechnology Resource Laboratory (Yale University, of 10 mice were anesthetized lightly with ketamine and xylazine and in- New Haven, CT). oculated by the intranasal route with wild-type, fully virulent Y. pestis or Ysr deletion strains (1 × 104 cfu) diluted in PBS as described previously (52). Mice Additional Methods. Bacterial strains and plasmids (SI Appendix, Table S3), were monitored twice daily for 7 d. All animal infections were repeated at oligonucleotides (SI Appendix, Table S4), and growth conditions and least twice, unless stated otherwise. Statistical analysis was performed using reagents, as well as methods for sRNA and hfq deletions, Northern blot, 5′/3′ the log-rank (Mantel–Cox) test. The weights of infected animals were RACE, qRT-PCR, sRNA target verification, and Ysr29 overexpression are de- compared using the student’s unpaired t test. A P value of ≤0.05 was tailed in SI Appendix, SI Materials and Methods. considered significant. ACKNOWLEDGMENTS. We thank Brian Fritz for guidance and technical sRNA Target Identification. Overnight cultures of wild-type and ΔYsr29 Y. support and Marc Domanus for Illumina-Solexa sequencing. We also thank

pseudotuberculosis were diluted to an OD620 of 0.1 in BHI broth and grown Terence Wu for assistance with 2D-DIGE and mass spectroscopy analysis. We thank Lauren Bellows for outstanding technical assistance and Hank Seifert to stationary phase (OD620 ∼7, 11 h) at 26 °C. Ten OD equivalents of culture were collected, bacteria were centrifuged, washed once in ice-cold PBS, and for helpful discussions. This work was sponsored by the Northwestern Uni- versity Feinberg School of Medicine and the National Institutes of Health/ incubated with lysozyme (10 mg/mL) for 30 min on ice. Cells were sonicated ﬁ National Institute of Allergy and Infectious Diseases Regional Center of Ex- (three 20-s pulses) on ice, and whole-cell lysates were clari ed by centrifu- cellence (RCE) Research Program for Bio-defense and Emerging Infectious ﬁ gation. Protein content of the lysates was quanti ed by the Bradford assay Diseases. The authors acknowledge membership within and support from (Bio-Rad). The wild-type sample was labeled with Cy3 dye, and the ΔYsr29 the Region V ‘Great Lakes’ RCE (National Institutes of Health Award U54- sample was labeled with Cy5 dye using the CyDye DIGE Fluor Labeling Kit AI-057153).

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