RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence

Wu, Kejing; Lin, Xingyu; Lu, Yujie; Dong, Rui; Jiang, Hongnian; Svensson, Sarah L.; Zheng, Jiajia; Shen, Ning; Camilli, Andrew; Chao, Yanjie

doi:10.1038/s41467-024-51213-z

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Published: 13 August 2024

RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence

Nature Communications volume 15, Article number: 6946 (2024) Cite this article

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Abstract

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

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Introduction

The Gram-negative Klebsiella pneumoniae is an emerging bacterial pathogen in the era of global antibiotic crisis¹. As a member of the ESKAPE pathogens, antibiotic-resistant K. pneumoniae is a leading cause of nosocomial infections and is difficult to treat by frontline drugs including carbapenems^2,3,4. Whereas most classical K. pneumoniae strains affect immuno-deficient populations, a distinct pathotype of hypervirulent K. pneumoniae (HvKP) emerged in Asia in the 1980s that mainly infects young and healthy people, causing community-acquired pneumonia, liver abscess, endophthalmitis and meningitis^5,6,7. More concerningly, HvKP can evolve to carbapenem-resistant (CR) strains via mobilizing plasmids, giving birth to CR-HvKP superbugs that are responsible for bursting endemics in China that pose urgent threats to global public health and biosecurity^8,9,10,11.

The virulence of HvKP is strongly linked to overproduction of capsular polysaccharides anchored to outer membrane¹². With a limited number of recognized virulence traits such as lipopolysaccharide, fimbriae and siderophores, HvKP has been best characterized by its hypermucoviscosity (HMV) phenotype¹³. HvKP can form elastic strings longer than 5 mm when lifting their colonies from agar plates, a visible feature often used in clinical diagnosis¹¹. HMV is largely attributed to copious capsule production and is required for invasive infections and pathogenesis¹². The emergence of HMV has been associated with lateral acquisition of new capsule regulators^14,15, such as the rmpADC genes that promote capsule biosynthesis, transmembrane export, and chain extension^16,17,18. Besides the capsule biosynthesis gene cluster, dozens of other genes have been found by global genetic screens to promote or suppress HMV (HMV-related genes), belonging to various pathways including central metabolism, iron homeostasis, RNA degradation, lipid transport, as well as global transcription factors such as CRP and OmpR^{19,20,21,22,23}. Mounting evidence supports that HMV is tightly controlled at multiple layers by a complex regulatory network in K. pneumoniae.

Post-transcriptional control of HMV at the RNA level has been implicated in K. pneumoniae, but with little mechanistic understanding. For example, the major RNA chaperone Hfq has been shown to regulate capsule production and pathogenesis^24,25. Hfq, as a conserved RNA-binding protein found in most bacteria, functions to stabilize small regulatory RNAs (sRNAs) and facilitate their base-pairing with target mRNAs (Fig. 1a)^26,27,28. Hfq and its associated sRNAs regulate hundreds of mRNAs up to a quarter of the transcriptome in models such as E. coli and Salmonella, forming large post-transcriptional regulatory networks mediated by direct RNA-RNA interactions^{29,30,31,32,33}. These RNA regulatory networks have been shown to control cell behaviors, stress responses, and metabolic fluxes in a wide range of bacterial pathogens including S. enterica, Pseudomonas aeruginosa, Vibrio cholerae, and Clostridioides difficile^{34,35,36,37,38,39,40}. However, the nature and physiological function of this Hfq-mediated RNA network in the emerging hypervirulent pathotype of K. pneumoniae was little understood.

Fig. 1: iRIL-seq charts global RNA-RNA interactome in hypervirulent *K. pneumoniae.*

Here, we systematically profiled the RNA-RNA interactome of HvKP using an in vivo RNA proximity-ligation approach (iRIL-seq³⁴, Fig. 1a,) to decipher the RNA-mediated regulatory network. We discovered a plethora of Hfq-associated sRNA regulators of HMV and their interacting target mRNAs, establishing an intracellular RNA-RNA interactome map of HvKP and providing molecular insights into the role of post-transcriptional control in HMV. Our study revealed that the conserved enterobacterial sRNA ArcZ and its key targets are crucial regulators of HMV and Klebsiella pathogenesis in mouse models. ArcZ is transcriptionally activated by the master regulator of catabolite repression CRP, establishing a new link between central metabolism and HMV in K. pneumoniae.

Results

Hfq and Hfq-associated sRNAs regulate hypermucoviscosity in K. pneumoniae

Mucoid HvKP resists sedimentation by low-speed centrifugation (1000 g), and thus the OD₆₀₀ of the supernatant from 1 OD of bacteria has been established as a quantitative readout for HMV^18,19. Using this method, we first examined whether the major RNA chaperone Hfq and potentially sRNAs, might regulate HMV. Indeed, we observed a significant increase in supernatant OD values for a Δhfq mutant at multiple growth stages (Fig. 1b, Supplementary Fig. 1), compared to the parental strain ATCC 43816 (serotype O1:K2), a well-known model HvKP strain used as wild-type throughout this study. As a negative control, the i-wza acapsular mutant, which carries a loss-of-function insertion in the capsule biosynthesis gene wza¹⁹, was not mucoid and was pelleted completely by low-speed centrifugation. This result suggests that Hfq and Hfq-associated sRNAs may suppress HMV at the post-transcriptional level in HvKP.

iRIL-seq profiling of the RNA-RNA interactome in K. pneumoniae

To map the Hfq-mediated RNA regulatory network in HvKP, we harnessed the iRIL-seq approach recently established in our lab³⁴, which enables rapid profiling of intracellular RNA-RNA interactomes in live bacteria. Transformation of the pBAD-t4rnl1 plasmid (pYC582) into Klebsiella allowed us to express T4 RNA ligase 1 for proximity-ligation of RNAs in vivo, followed by co-immunoprecipitation (coIP) to enrich the Hfq-bound RNA transcripts (singleton reads) and ligated RNA fragments (chimeras: sRNA-mRNA, sRNA-sRNA, etc) (Fig. 1a). The in vivo ligation followed by Hfq-FLAG-coIP (Hfq-coIP) was carried out in triplicate in cells at early stationary phase of growth in LB (OD₆₀₀ of 3.0), with a monoclonal anti-FLAG antibody for IP and mouse IgG as a negative control (Fig. 1c). The resulting RNA samples were purified and subjected to Illumina paired-end sequencing (Supplementary Data 1).

Bioinformatic analysis of the sequencing reads confirmed a strong enrichment (>10-fold) of Hfq-associated sRNAs (singleton) in the Hfq-coIP samples over the IgG controls (Supplementary Fig. 2). 83 Hfq-associated sRNAs were detected in K. pneumoniae, including 43 sRNAs conserved in the Enterobacteriaceae family (including E. coli and S. enterica), as well as 40 novel sRNA candidates in K. pneumoniae (Supplementary Data 2). Hfq also directly bound to a large number of mRNAs. ~650 mRNAs (singleton) were enriched in our dataset >3-fold (Supplementary Data 3), indicating a large Hfq regulon exists in HvKP.

Ligation-derived chimeric fragments containing Hfq-associated sRNAs were also enriched by Hfq-coIP, occupying ~50% of all chimeric reads (Supplementary Fig. 2). We selected the significant chimeras (S-chimeras), defined as those with at least 10 reads and p_adj < 0.05 using the Fisher’s exact test as previously reported^31,34, for subsequent analyses. The vast majority of S-chimeras (>90%) contained an sRNA at the 3’ half of chimera (RNA2), whereas the 5’ half (RNA1) comprised mostly mRNAs and sRNA fragments, which we consider binding partners of the sRNA (Fig. 1d). Under the condition analyzed here, eight sRNAs represented 90% of all singleton reads, whereas the ArcZ sRNA was highly enriched and ranked as the most abundant sRNA in S-chimeras (Fig. 1e).

Our analysis of the S-chimeras identified a total of ~2800 RNA-RNA interactions involving 76 sRNAs and 1264 target mRNAs, illustrating a large and complex RNA-RNA interaction network in HvKP (Fig. 1f). To better understand these interactions, we systematically analyzed the S-chimeras for sequence motifs. As expected, this analysis revealed a uridine stretch in the RNA2 portion at 3’ ends, consistent with rho-independent terminators found in most sRNAs (Fig. 1g). The RNA1 portion likely contains target mRNAs that basepair with the sRNA portion in RNA2³⁴. Indeed, our analyses revealed many sequence motifs in RNA1 that show complementarity to the cognate sRNA in RNA2 (Fig. 1 h, i). In total, we identified significant sequence motifs for 15 sRNAs in S-chimeras (Supplementary Fig. 3), including several conserved enterobacterial primary (FnrS, MgrR, CyaR) and processed sRNAs (ArcZ, CpxQ, RprA), as well as a new Klebsiella sRNA candidate (KPnc0140). Complementary sequence motifs were discovered in over 90% of mRNA genes in RNA1 with the MAST p < 0.001, bolstering the high reliability of target identification using iRIL-seq analysis in vivo.

Members of the Klebsiella genus have relatively large genomes (>5 Mb) with a core genome similar to that of closely-related E. coli and S. enterica. iRIL-seq analysis in HvKP recapitulated many conserved RNA-RNA interactions previously reported in E. coli and Salmonella (Supplementary Data 4). For example, among 871 interactions involving ArcZ, we captured its orthologous target mRNAs including tpx, sdaC, and a sponge sRNA CyaR^32,41,42. These sRNA-sRNA sponge interactions can titrate one or the other and inhibit their binding to target mRNAs⁴¹. In summary, the iRIL-seq analysis has established a comprehensive map of the RNA-RNA interactome in HvKP.

Multiple sRNAs regulate Klebsiella hypermucoviscosity

To identify potential sRNA regulators of HMV hinted at by our Δhfq phenotype (Fig. 1b), we first inspected sRNA-mediated interactions with the ~20 genes in the capsule biosynthesis cluster¹⁸. This revealed 21 sRNA candidates that may regulate HMV via interaction with up to 14 genes in the cluster (Fig. 2a). Outside the capsule locus, ~100 genes contributing to the HMV phenotype have been previously identified (Supplementary Data 5)^17,18,19,21. Comparing the significant sRNA interactions against these HMV-related genes revealed another 27 sRNA candidates that may regulate HMV via interaction with 49 HMV-related genes (Fig. 2b). By combing both lists, our analysis revealed a large RNA regulatory network of HMV which involves a panel of 31 sRNAs and 64 mRNAs, providing a valuable resource for the study of post-transcriptional regulation of HMV in K. pneumoniae (Supplementary Data 6).

**Fig. 2: The regulatory network of hypermucoviscosity includes many sRNAs.**

To experimentally validate these potential regulators of HMV, we cloned 23 sRNAs from the above list into the pZE12 vector and examined the impact of their overexpression on HMV in WT Klebsiella. While the pZE12 system is widely used to overexpress sRNAs in various bacteria⁴³, we found that it failed to overexpress ArcZ in Klebsiella (Supplementary Fig. 4). This is likely due to transcriptional repression of the P_lacO promoter by ~20 endogenous LacI-like repressors in the Klebsiella genome (Supplementary Fig. 5), and was overcome by a supplementation of the culture with 1 mM IPTG (Supplementary Fig. 4a). Upon IPTG induction, most of the tested sRNAs showed a strong impact on HMV (Fig. 2c), corroborating the robustness of iRIL-seq analysis³⁴. More than half (12/23) of the sRNAs tested reduced HMV vs. overexpression of a scrambled RNA in the control plasmid pJV300, whereas six sRNAs enhanced HMV to various degrees. ArcZ showed the strongest repression (~6-fold) of HMV in the presence of IPTG. Interestingly, HMV was also reduced by leaky expression of ArcZ in the absence of IPTG (Supplementary Fig. 4b), suggesting that HMV is highly sensitive to ArcZ levels.

Clinical K. pneumoniae strains are known for their extraordinary genetic diversity involving numerous sequence types and plasmid constituents¹. To determine whether ArcZ repression of HMV is a conserved regulation or specific to our HvKP strain, we overexpressed ArcZ in a set of different HvKP clinical isolates (Supplementary Data 7). The results in Fig. 2d show that ArcZ induction significantly reduced HMV in all tested HvKP strains. These strains include another widely-used model strain NTUH-K2044⁴⁴, and a recent CR-HvKP clinical isolate (RJ9299) that is responsible for a local hospital outbreak in Shanghai, China¹¹. These data strongly support that the regulation of HMV by ArcZ is a conserved mechanism among HvKP strains with diverse genetic backgrounds.

ArcZ is a conserved repressor of hypermucoviscosity

ArcZ was initially identified in Salmonella in the intergenic region between arcB and elbB (a.k.a. yhbL), and obtained its current name due to its partial overlap with arcB⁴². In E. coli and Salmonella, ArcZ is transcribed as a 129-nt long precursor sRNA (Pre-ArcZ) and further processed into the mature ArcZ sRNA^45,46. In K. pneumoniae, the arcZ gene is located in the same intergenic region (Fig. 3a), and shares a conserved seed sequence with the other enterobacterial homologs (Fig. 3b). The primary sequence of Pre-ArcZ is identical in multiple Klebsiella type strains. Chromosomal deletion of arcZ led to significantly increases in HMV (Fig. 3c) and in the capsule amount (Fig. 3d). These increases in ΔarcZ were successfully complemented by introducing a single-copy of arcZ gene back in the chromosome at another locus. Therefore, the endogenous ArcZ sRNA represses the capsular HMV in Klebsiella, and likely acts in trans independent of its neighboring arcB gene (Supplementary Fig. 6).

**Fig. 3: The ArcZ sRNA is a conserved regulator of multiple HMV-related genes.**

According to our iRIL-seq data, ArcZ may directly interact with many mRNAs including several encoded in the capsule cluster or related to HMV (Fig. 3e, Supplementary Fig. 7). The expression levels of many capsule mRNAs were reduced when ArcZ was overexpressed (Supplementary Fig. 8). To understand the molecular mechanism how ArcZ regulates capsule and HMV, we selected several of these relevant genes and constructed translational GFP reporter fusions using the two-plasmid system⁴³. Of eleven different target genes, ten (91%) were significantly repressed upon ArcZ overexpression (p < 0.05, Student’s t test, Fig. 3f). The strongest suppression was observed for mlaA and fbp, two HMV-related genes located outside of the capsule cluster. MlaA is an outer-membrane lipoprotein that functions in the retrograde phospholipid trafficking pathway^47,48. The fbp gene encodes fructose-1,6-bisphosphatase of the gluconeogenesis pathway⁴⁹. Despite their involvement in different pathways, deletion of mlaA and fbp caused a significant reduction in HMV (Fig. 3g, h), mirroring the effect of ArcZ overexpression in Klebsiella (Fig. 2c, d).

To examine the regulation of MlaA and Fbp proteins in Klebsiella, we inserted chromosomal 3xFLAG tags at their respective C-termini and analyzed their levels by Western blotting. A clear upregulation (~1.7-fold) was observed for the MlaA-3xFLAG protein when arcZ was deleted (Fig. 3i), with little change for Fbp-3xFLAG (Fig. 3j). Overexpression of ArcZ strongly reduced both levels of MlaA and Fbp proteins (Fig. 3k, l). This inhibition was abolished by introducing 2- or 5-nt mutations into the conserved seed sequence of ArcZ (M2 and M5, Fig. 3b), suggesting a direct regulation involving the seed region of ArcZ.

ArcZ represses mlaA and fbp by direct basepairing interactions

ArcZ regulates the expression of mlaA and fbp likely via base-pairing with the mRNAs. Analysis of the iRIL-seq data revealed a large number of ArcZ chimeras mapped to the 5’UTRs of both mlaA and fbp mRNAs (Fig. 4 a, b), indicating that ArcZ basepairs at their 5’UTRs. Indeed, basepairing interactions were predicted, with both IntaRNA and RNAhybrid tools^50,51, between the conserved seed of ArcZ and the translation initiation region of both mRNAs (Fig. 4c, d). Using translational GFP reporters, we confirmed regulation of both targets at the post-transcriptional level. GFP fluorescence analysis showed that the target regulation requires the conserved seed region of ArcZ (Fig. 4e, f), consistent with Western blot results (Supplementary Fig. 9). Finally, introducing compensatory basepairing mutations in the target mRNAs (Fig. 4c, d), restored regulation by the ArcZ-M2 sRNA (Fig. 4e, f). These results show that ArcZ inhibits the translation of mlaA and fbp via direct RNA-RNA basepairing interactions.

**Fig. 4: ArcZ represses *mlaA* and *fbp* via direct basepairing.**

ArcZ and MlaA are crucial for Klebsiella virulence

Given that HMV is a major virulence factor for Klebsiella, we next analyzed the role of ArcZ in virulence regulation using a murine infection model⁵². Compared to the WT strain, mice infected with the ΔarcZ mutant showed a trend of higher CFUs recovered from lung and spleen 48 h post-infection (Supplementary Fig. 10), without reaching statistical significance. Using a more sensitive competition assay with WT and ΔarcZ mixed together, we observed that ΔarcZ showed a significant competitive advantage over WT in mice (Fig. 5a), supporting that ArcZ is a repressor of Klebsiella virulence. In line with this observation, ArcZ expressed from its own promoter on multicopy plasmid (pZE12-P_own-arcZ) led to a significant reduction of Klebsiella load in lungs (Supplementary Fig. 10). Constitutive overexpression of ArcZ from a stronger 16 S rRNA promoter drastically reduced bacterial burden in mice by several orders of magnitude (Fig. 5b–d, Supplementary Fig. 11a). By stark contrast, no effect on bacterial load was observed when the ArcZ-M5 mutant was overexpressed, supporting that ArcZ is a potent and specific inhibitor of HvKP pathogenesis.

The reduced virulence may be due to the diminished expression of ArcZ target genes. Our initial competition experiments revealed that ΔmlaA has reduced fitness in mice. The mlaA mutant was significantly outcompeted in lungs, whereas it did not show any fitness defect in LB in vitro (Supplementary Fig. 11b). When infecting mice with either WT or ΔmlaA in separation, we confirmed that ΔmlaA is severely attenuated (Fig. 5g–i), generating ~1000-fold fewer CFU recovered from lung, spleen, and liver 48 h p.i. compared to WT (p < 0.01, non-parametric Mann-Whitney U test). Next, we complemented the ΔmlaA mutant by introducing a single copy of mlaA gene back to the chromosome at another locus. This complementation strain not only restored the expression of MlaA protein (Fig. 5e), and also successfully rescued HMV and pathogenesis similar to the WT levels (Fig. 5f–i). Collectively, these data show that the ArcZ sRNA and its target mlaA are crucial regulators of HMV and Klebsiella virulence in mice.

MlaA, together with the MlaBCDEF proteins, constitute a phospholipid retrograde transport machinery that maintains lipid asymmetry of the outer-membrane^47,53,54. MlaA removes misplaced phospholipids in the outer leaflet of outer-membrane, and transports them back to the inner membrane via periplasmic MlaC and the MlaBDEF membrane channel. To better understand the mechanism how MlaA contributes to HMV, we constructed a series of MlaA variants based on the recently solved Cryo-EM structures^47,55,56, generating a number of single-copy chromosomal mlaA mutant strains (Fig. 5j). Subsequent analysis revealed that the HMV regulation by MlaA is dependent on several active sites, including residues E163/D166/D169 that are required for phospholipid binding⁴⁷, and residues I212-R222 required for interaction with MlaC⁵⁶. These data indicate that phospholipid retrograde transportation is required for HMV, rather than some unknown activity of the MlaA protein in Klebsiella. The MlaA* mutant, which induces a “short-circuit” of phospholipid flux in the outer-membrane^47,54, also led to reduced HMV levels, indicating the homeostasis of phospholipid is crucial for HMV. Altogether, our genetic characterizations suggest a molecular model in which MlaA removes free phospholipids in the outer leaflet of outer membrane, provides sufficient space to permit the retention of capsular polysaccharide chains to the cell surface, thus promoting the HMV phenotype.

Transcriptional activation of ArcZ by CRP

To better understand the regulatory circuit of ArcZ, we sought to investigate how the sRNA is activated in K. pneumoniae. Previous studies in E. coli and Salmonella have shown that ArcZ is repressed by the ArcAB two-component system under low-oxygen conditions^42,57, but it is unknown which upstream signals activate ArcZ expression in K. pneumoniae. Interestingly, multiple sequence alignment of the ArcZ promoter region from K. pneumoniae and other enterobacteria revealed a conserved binding site potentially recognized by CRP, the master regulator of catabolite repression (Fig. 6a). In line with this observation, induction of catabolite repression by glucose suppressed ArcZ expression levels, suggesting that CRP activates ArcZ transcription (Supplementary Fig. 12). We next constructed a Δcrp mutant and observed a strong reduction in ArcZ levels. This reduction in Δcrp was fully restored to WT levels by the crp gene provided in trans (Fig. 6b). Furthermore, the arcZ gene with its own promoter (pZE12-P_own-arcZ) drove higher levels of Pre-ArcZ only in strains with crp but not in the Δcrp mutant (Fig. 6c). Truncating the potential CRP-binding region from −41 bp upstream (trunc, Fig. 6a; without disturbing the −35 and −10 boxes), or mutating the conserved TCACA motif, both reduced the levels of Pre-ArcZ (Fig. 6c). Although a molecular interaction remains to be proven, these results strongly argue that CRP directly activates the ArcZ promoter. CRP itself acts to repress HMV in K. pneumoniae. Deletion of crp significantly enhanced the mucoid phenotype, which was fully complemented by the crp gene in trans (Fig. 6d). Therefore, we have identified a novel regulatory circuit of HMV, in which the master regulator of catabolite repression CRP activates the ArcZ sRNA, both acting to suppress HMV at the transcriptional and post-transcription level, respectively (Supplementary Fig. 13, Fig. 7).

Fig. 6: CRP activates the transcription of ArcZ in *K. pneumoniae.*

Fig. 7: Regulatory circuit of ArcZ-mediated HMV regulation in *K. pneumoniae.*

Discussion

Post-transcriptional control at the RNA level is crucial for bacterial pathogens to coordinate virulence and fitness under changing conditions during host infection. In this study, we generated a comprehensive RNA-RNA interactome map in the hypervirulent pathotype of K. pneumoniae, revealing the post-transcriptional regulatory network of virulence control in a superbug of global concern. We discovered that HMV in HvKP is regulated by the major RNA chaperone Hfq and a large number of Hfq-associated sRNAs. Our study further demonstrated the molecular mechanism how ArcZ, as an Hfq-dependent sRNA, regulates HMV and virulence via direct sRNA-mRNA interactions (Fig. 7), adding support to the evolving theory that HMV is critically regulated in K. pneumoniae during the process of host infection^58,59,60. Our successful identification of key HMV regulators and regulatory circuit in this study may illustrate a new paradigm of using RNA-RNA interactome to guide functional RNA screens with mechanistic precision to understand important traits in different organisms.

Our systems-wide profiling of the RNA interactome in K. pneumoniae was achieved by using the iRIL-seq approach, which was recently developed and established in Salmonella in our lab³⁴. Taking advantage of expressing T4 RNA ligase in vivo³⁹ and Hfq-coIP⁶¹, iRIL-seq enables proximity-ligation between interacting RNAs in live bacteria and enriches the ligated RNA fragments bound to Hfq. This in vivo approach is highly streamlined compared to crosslinking-dependent RIL-seq^62,63 and CLASH³², facilitating rapid RNA-interactome analyses in various bacteria and eliminating laborious in vitro ligation and digestion steps⁶⁴. iRIL-seq has been proven to effectively capture sRNA-target interactions at the genome-wide scale in Salmonella with high accuracy³⁴. Here the RNA-interactome data obtained in HvKP further support the utility of iRIL-seq. Consensus sequence motifs complementary to sRNAs were identified in over 90% of target mRNA genes (Supplementary Fig. 3). Given its simple setup and streamlined workflow, our HvKP dataset further showcases iRIL-seq is a powerful generic approach that can be easily applied to many different organisms to chart functional RNA regulatory networks.

Our study has discovered a large RNA regulatory network involving many HMV-related genes in the HvKP RNA interactome, while numerous known sRNA-mRNA and sRNA-sRNA interactions from E. coli and Salmonella were also recapitulated. The sRNA regulators of HMV include many core-genome encoded sRNAs conserved in enterobacteria exemplified by ArcZ, and also new sRNA candidates in Klebsiella (Fig. 2c, Supplementary Data 2). Several core sRNA regulators have been previously characterized in model organisms. CpxQ and MicF function in the envelope stress pathway to target membrane proteins^43,65; MgrR in the PhoPQ pathway regulates LPS modification and magnesium transport^66,67; and RprA is an activator of RpoS involved in general stress responses⁶⁸. These sRNAs with diverse functions all strongly inhibit HMV in K. pneumoniae, suggesting that sRNAs from several distinct pathways might converge to modulate Klebsiella virulence during infection. In a recent study, overexpression of the OmrB sRNA was shown to reduce HMV in another HvKP strain⁶³. Besides these core sRNAs, Klebsiella sRNA candidates such as KPnc0122, KPnc0450, and KPnc0107 also repressed HMV, providing future opportunities for molecular and functional characterizations.

Our study discovered that the enterobacterial core sRNA ArcZ is integrated into a new regulatory circuit and is endowed with a new function in HvKP (Fig. 7). In E. coli and Salmonella, the promoter of ArcZ has been shown to be repressed by the ArcAB two-component system^42,57. Our results suggest that ArcZ, instead of being repressed by ArcAB (Supplementary Fig. 12b), is activated by the catabolite repression protein CRP. This indicates that ArcZ might have evolved with new regulatory functions under the control of CRP. Functional enrichment analysis for the ArcZ targets revealed several significant pathways related to carbohydrate and polysaccharide metabolism (Supplementary Fig. 7a), consistent with the observed repressive roles on HMV (Fig. 2c, d) and capsule production (Fig. 3d, Supplementary Fig. 4c). Total RNA-seq analysis also revealed significant down-regulation of capsule genes upon ArcZ overexpression (Supplementary Fig. 8), although direct base-pairings remain to be verified. Our results suggest that even conserved sRNAs may have evolved distinct functions in different organisms. Likewise, a recent study showed that the homologs of ArcZ in Photorhabdus/Xenorhabdus regulate metabolite production and symbiosis with nematodes^46,69.

CRP controls one of the largest regulons to modulate bacterial carbon metabolism and energy expenditure. Consistent with previous reports^20,70, our study suggests that CRP is a negative regulator of HMV. CRP was shown to repress the transcription of capsule gene clusters via direct binding to the promoters. In the meantime, CRP activates the ArcZ sRNA, which in turn binds and represses many other HMV-related mRNAs. ArcZ may function as an extension of CRP to repress more HMV genes at the post-transcriptional level (Fig. 7). We note that the CRP pathway may be more complicated than this simple model. Besides ArcZ, CRP regulates two other sRNAs, Spf and CyaR, according to studies in E. coli and Salmonella^71,72. Both sRNAs positively regulated HMV, in contrast to the repressive roles of ArcZ and Hfq. Thus, sRNAs within the CRP pathway both negatively and positively regulate HMV, indicating the existence of complicated regulatory loops fine-tuning HMV at the post-transcriptional level⁷³.

We show for the first time that the phospholipid trafficking pathway is crucial for Klebsiella virulence and that MlaA is an essential virulence factor in HvKP (Fig. 5g–i). As a key target of ArcZ, mlaA encodes the crucial outer membrane component of the Mla phospholipid retrograde transportation system that maintains lipid asymmetry and membrane integrity^47,48,53. Deletion of mlaA, without showing any growth or fitness defect in LB broth (Supplementary Fig. 11b), resulted in a strong reduction of HMV and a severe attenuation of virulence in the murine model of infection (Fig. 5). Transposon disruption of mlaBCDEF genes also led to decreased HMV¹⁹, supporting the involvement of the entire Mla system and its molecular function. Our further mutational analysis of MlaA revealed that phospholipid transport activity is crucial for capsule retention and HMV, providing a new mechanism underlying the HMV phenotype. Because the ΔmlaA mutant still retains a low level of HMV higher than the acapsular control (Fig. 5f), its strong attenuation in virulence makes ΔmlaA an attractive candidate to develop live attenuated vaccine. MlaA might also represent an interesting drug target located on the bacterial surface for the future development of therapeutics against HvKP. On the same note, ArcZ may also be exploited as a potential anti-infection drug in the form of antisense RNA therapeutics^74,75. Our data demonstrated that ArcZ is effective to reduce HMV in a variety of clinical HvKP strains including CR-HvKP isolates, which urgently demand new treatment options in clinics.

Methods

All the experiments comply with the ethical regulations at the Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences. The animal experiments were conducted following institutional ethics requirements under the animal facility user permit (No. IPS-26-CYJ), and the infection experiments were performed following the animal protocol (A2022053) approved by the institutional animal care and ethics committee. The collection of clinical isolates was approved by the medical science research ethic committee of Peking University Third Hospital (M3022382).

Bacterial strains and growth conditions

Hypervirulent Klebsiella pneumoniae ATCC 43816 is used as wild-type. Information on the clinical isolates is available in Supplementary Methods and Supplementary Data 7. Mutant strains generated in this study are described in Supplementary Data 8. Unless otherwise stated, bacteria were grown in LB medium (10 g/L tryptone, 5 g/L yeast extract, and 5 g/L NaCl) with 220 rpm aeration or on LB agar at 37 °C. Where appropriate, antibiotics were used at the following concentrations: 20 µg/ml chloramphenicol, 50 µg/ml kanamycin, 50 µg/ml spectinomycin, 100 µg/ml hygromycin B, and 50 µg/ml apramycin sulfate. 1 mM IPTG was used for the induction of sRNA expression from pZE12 plasmids.

Deletion strains and chromosomally 3xFLAG epitope tag strains were constructed using the λ Red system^76,77. To construct the ∆arcZ mutant, wild-type Klebsiella cells carrying the temperature-sensitive pACBSR-Hyg helper plasmid were transformed with 1 µg dsDNA fragments, which were amplified using YCO-1438/-YCO-1439 and pFLP-hyg as template. Desired recombinants were confirmed by resistance to apramycin, and by colony PCR with YCO-1038/-YCO-1039 and Sanger sequencing. To introduce 3xFLAG epitope tags, DNA fragments were amplified by PCR with primer pairs YCO-1447/1448 and pSUB11 as template, and electroporated into competent cells expressing λ Red recombinase from pACBSR-Hyg. Correct transformants were selected on kanamycin plates and confirmed by colony PCR.

Chromosomally complemented strains were constructed using the λ Red system and from pACBSR-Hyg^76,77. DNA fragments were amplified from pXG10 vectors containing resistance maker and P_own-arcZ or P_own-mlaA fragments by primer pairs YCO-2321/2322, and electroporated into mutant strains lacking either arcZ or mlaA. Point mutations in MlaA were first introduced into the pXG10-P_own-mlaA vector by site-directed mutagenesis using primers described in Supplementary Data 8. Mutated mlaA fragments were amplified using oligos YCO-2321/2322, and electroporated into ΔmlaA competent cells expressing λ Red recombinase. The DNA fragments were inserted into the putA locus on chromosome by homologous recombination as previously described⁷⁸. Correct transformants were selected on chloramphenicol plates, and confirmed by colony PCR and Sanger sequencing.

Plasmids construction

The oligonucleotides and plasmids used in this study are listed in Supplementary Data 9 and 10, respectively. To express a plasmid-borne t4rnl1 gene from the tightly controlled, L-arabinose-inducible pBAD promoter, pBAD-Hyg was amplified by PCR with oligonucleotides YCO-0453/−0454, and the t4rnl1 gene was amplified from pKH13-t4rnl1 using oligonucleotides YCO-0905/−0906. Vector and insert were ligated following restriction digestion with XbaI. Overexpression plasmids for sRNAs were constructed using pZE12-luc as the scaffold plasmid⁴³. Here, an XbaI-digested PCR product obtained by amplification of pZE12-luc with primers YCO-0557/−0558 was ligated to sRNAs amplified from genomic DNA, and similarly digested with XbaI. Construction of GFP translational fusion plasmids were carried out principally as described⁴³, using the pXG10-SF or pXG30-SF. The pXG10-SF vector was used for monocistronic genes, the pXG30 vector for operons. Briefly, the pXG10-SF backbone was amplified with YCO-1437/−1398, and pXG30-SF backbone was amplified with YCO-1806/−1398 or YCO-1940/−1398. The 5’ regions of target genes, including regions captured in the chimeric fragments, were PCR amplified, digested with NsiI/PstI and NheI, and cloned into pXG10-SF or pXG30-SF backbones digested with the same restriction enzymes. All the plasmids and insertions were verified by colony PCR and Sanger sequencing.

HMV quantification

K. pneumoniae strains were grown overnight in 3 ml LB medium at 37 °C. Next, 1 ml of culture was centrifuged for 5 min at 1000 × g (room temperature), and the OD₆₀₀ of the upper 200 μl supernatant was determined. Final readings were normalized to the OD₆₀₀ in the input culture before centrifugation. At least three biological replicates were prepared for each strain.

Capsule purification and quantification

K. pneumoniae was grown overnight in 1 mL LB medium with 220 rpm at 37 °C. Cultures were added with 100 µl the capsule extraction buffer (500 mM citric acid pH 2.0, 1% Zwittergent 3-10), incubated at 50 °C for 20 min. Cells were pelleted, and the supernatant was mixed with an equal volume of isopropanol, followed by precipitation for 30 min at 4 °C. The capsule was centrifuged for 10 min at maximum speed and resuspended in 150 µl PBS. Protein and membrane contaminants were removed by adding 100 µl buffer P2 (TIANGEN) and 125 µl buffer P3 (TIANGEN). The clarified supernatant was precipitated with an equal volume of isopropanol for 30 min at 4 °C. The capsule was centrifuged at maximum speed for 30 min at 4 °C and washed twice with 75% ethanol. The purified capsule was resuspended in nuclease-free water and confirmed on SDS-PAGE by alcian blue staining. The uronic acid assay was used to quantify the amounts of capsular polysaccharides. 12.5 mM borax (Sangon) in H₂SO₄ was added to samples and boiled before chilling on ice. Followed by addition of 0.15% 3-hydroxydiphenol (Sigma-Aldrich), the mixture was analyzed by the absorbance at 520 nm. Uronic acid content was determined from a standard curve of glucuronic acid (Sangon) and normalized as micrograms per 1 OD of bacterial culture.

Hfq-coIP experiments

The Hfq-coIP was performed using our previously published protocol⁶¹. K. pneumoniae hfq::3xFLAG strain containing the plasmid pYC582 was grown in LB medium with 220 rpm at 37 °C. T4 RNA ligase 1 was induced for 30 min by adding 0.2% L-arabinose when the culture reached OD₆₀₀ of 3.0. Bacterial samples (~100 OD) were collected by centrifugation at 4000 g for 20 min at 4 °C. The bacterial pellets were resuspended in 500 µl ice-cold lysis buffer (20 mM Tris pH 8.0, 150 mM KCl, 1 mM of MgCl₂, 1 mM DTT, 0.05% Tween-20), and broke with 500 µl glass beads using the Cryolys Evolution instrument for 15 min at 4 °C. Lysates were collected by centrifugation at 16,000 g for 30 min at 4 °C, and divided into new tubes equally. Cleared lysates were respectively incubated with anti-FLAG antibody (Sigma) or IgG isolate control (Invitrogen) conjugated on the protein-G magnetic beads (Thermo-Fisher) for 1 h at 4 °C with rotation. The beads were collected by the magnet, and washed five times with 500 µl lysis buffer and resuspended in 50 µl lysis buffer. The Hfq-bound RNA was extracted by RNA clean & concentrator columns (Sangon, Shanghai). The purified RNA was resuspended in nuclease-free water.

RNA extraction

Total RNA was extracted using TRIzol reagent. Briefly, 4 OD of cells were collected with addition of 0.2 vol/vol of STOP solution (95% ethanol, 5% phenol), centrifuged for 5 min, 16,000 g at 4 °C and the supernatant was discarded. For cell lysis, pellets were resuspended with 500 µl lysozyme (A610308, BBI) at 0.5 mg/ml. Cleared lysates were mixed with 1 ml TRIzol and incubated for 10 min at room temperature with shaking. Subsequently, 400 µl chloroform was added, mixed by inversion, and incubated for 5 min at room temperature to allow phase separation. After the samples were centrifuged for 15 min, 16,000 g at 4 °C, the upper phase was transferred to a new tube and 700 µl isopropanol was added and incubated overnight at −20 °C. Finally, samples were centrifuged, washed with cold 75% v/v ethanol and air-dried for 15 min. Precipitated RNA was resuspended in 30 µl nuclease-free water and stored at −80 °C.

Northern blotting

Ten micrograms of total RNA were denatured at 98 °C for 2 min in RNA loading buffer (98% formamide, 1 mM EDTA, 0.1% xylene cyanol, 0.1% Bromphenol blue) and separated on 7 M urea/6% polyacrylamide gels in 1 × TBE buffer. RNAs were transferred onto Hybond-N+ membranes (GE Healthcare) by electroblotting (50 V) for 1 h at 4 °C and fixed to the membrane by UV crosslinking (120 mJ). Detection was performed using the Roche DIG system. Each primary probe was designed to contain an overhang sequence that is complementary the digoxigenin-labeled secondary probe YCO-0550. For instance, mature ArcZ was detected using the specific complementary probe YCO-1745, while CyaR was detected using primary probe YCO-1784. Both primary probe-hybridized sRNAs were detected with secondary probe YCO-0550. Membranes were prehybridized in DIG Easy Hyb (#11796895001, Roche) for 30 min. DNA probes were hybridized overnight at 42 °C. Membranes were washed with 5× SSC/0.1% SDS, 1× SSC/0.1% SDS, and 0.5× SSC/0.1% SDS buffers for 15 min each. Following one wash with maleic acid wash buffer (0.1 M maleic acid, 0.15 M NaCl, 0.3% V/V Tween-20, pH 7.5) for 15 min at 37 °C and blocked with blocking solution (#11096176001, Roche) for 1 h at 37 °C, membrane was hybridized with 75 mU/ml Anti-Digoxigenin-AP (#11093274910, Roche) in blocking solution for 60 min at 37 °C. Wash membrane twice (2 × 15 min) with maleic acid wash buffer and equilibrate membrane 3 min in Detection Buffer (0.1 M Tris–HCl, 0.1 M NaCl, pH 9.5). Signals were visualized by CDP-star (#11759051001, Roche) on a ChemScope imaging station and quantified using ImageJ Software.

Western blotting

Bacterial cells were collected by centrifugation for 5 min at 16,000 g at 4 °C, and pellets were resuspended in protein loading buffer. After heating for 5 min at 95 °C, 0.05 OD₆₀₀ equivalents of samples were separated on 12% SDS-PAGE gel and transferred to PVDF membranes (polyvinylidene fluoride, #10600023, Amersham). Membranes were blocked for 1 h with 5% (w/v) milk powder in TBS-T (Tris-buffered saline-Tween-20) and incubated overnight with primary antibody (monoclonal anti-FLAG, 1:10,000, Sigma #F1804-5MG; anti-GroEL, 1:10,000, Sigma; or anti-GFP, 1:10,000, Roche #11814460001 in 3% bovine serum albumin (BSA)/TBS-T) at 4 °C. Following three washes for 10 min in 1× TBST buffer, membranes were incubated with secondary anti-mouse or anti-rabbit HRP (horseradish peroxidase)-linked antibodies (#D110087-0100 or #D110058-0100, BBI; 1:10000) diluted in 1× TBST buffer containing 1% skim milk. After another three washes for 10 min in 1× TBST buffer, chemiluminescent signals were developed using ECL Prime reagents (#C500044-0100, Sangon) visualized on a ChemScope imaging station and quantified using ImageJ software.

iRIL-seq cDNA library construction

iRIL-seq libraries were constructed using the RNAtag-seq method as described previously^34,64. Briefly, fragmented RNA was treated with DNase I and FastAP. RNA then was ligated to the 3’ barcoded adapter, and depleting ribosomal RNA by Ribo-off rRNA depletion Kit (#N407-01, Vazyme). First strand cDNA was synthesized by HiScript II 1st Strand cDNA Synthesis Kit (#R211-01, Vazyme), and RNA was degraded after reverse transcription by 1 M NaOH and 0.5 M acetic acid. The cDNA was ligated to a second adapter. Samples were purified with 2.5x Agencourt AMPure XP beads and 1.5x isopropanol. Libraries were amplified by PCR with Illumina P5 and P7 primers using Q5 High-Fidelity DNA Polymerase (#M0491L, NEB), and purified with 1.5x AMPure XP beads. The libraries were sequenced by 150 bp paired-end sequencing with an Illumina NovaSeq instrument.

iRIL-seq data computational analysis

Data analysis was performed using the method reported by Melamed et al. ^31,64. with minor modifications. Briefly, clean reads were generated from raw sequencing reads by removing adapter sequences and low-quality sequences using FASTXToolkit (Version 0.0.13), and reads less than 25 nt were discarded. The first 25 nucleotides of each remaining read were mapped to the genome of K. pneumoniae ATCC 43816 (GenBank: NZ_CP009208.1), sorting the fragments into “single” and “chimeric” fragments. Fragments that mapped within a distance of 1000 nt were considered single, whereas fragments that mapped to two different loci were considered chimeric. More details can be found in Supplementary Methods.

Total RNA-seq analysis

RNA-seq libraries were constructed by Novogene Group, Beijing, China. The cDNA libraries were sequenced on Illumina NovaSeq X plus. We used cutadapt 2.10 and fastx toolkit 0.0.14 to clip the adapter sequences and quality filtering. Reads unambiguously aligned to unique position in the reference genome (GenBank: NZ_CP009208.1) were preserved to calculate reads number, CPM (counts per million), and RPKM (reads per kilobase and per million) for each gene. Differential expression was tested using DESeq2, generating p-value and FDR based on the model of negative binomial generalized linear model.

Fluorescence analysis

Single colonies of Klebsiella strains harboring superfolder GFP (sfGFP) translational fusions and sRNA expression plasmids were inoculated into LB medium and grown overnight at 37 °C. The following day, 100 µl of overnight cultures were centrifuged and the pellets were resuspended in 1 ml of PBS. OD₆₀₀ and fluorescence (excitation at 476 nm and emission at 510 nm, using emission cutoff filter of 495 nm) was measured using the Agilent BioTek Synergy H1 plate reader. Fluorescence measurements were normalized to the OD₆₀₀ of cultures. Background fluorescence from control samples not expressing fluorescent proteins was subtracted from experimental samples. The level of regulation was calculated as the ratio between the fluorescence level of a strain carrying the sRNA over-expressing plasmid and a strain carrying the control plasmid. Three biological replicates were prepared for each strain.

Animal infection experiments

Six-to-eight weeks old female Balb/C mice were used and housed at 22 °C with a 12-h light/dark cycle and 45% humidity. Chow and water were provided ad libitum. Mice were randomly assigned to experimental groups, with at least five mice per group. Prior to infection, mice were anaesthetized via intraperitoneal injection of tribromoethanol (0.2 ml/10 g). Mice were intranasally infected with ~10³ CFUs (in 25 µl PBS) per bacterial strain, or a total of 10³ CFUs in competition assays. Mice were weighted daily until the end of experiment. Approximately 48 h post-infection, mice were euthanized and organs including lung, liver, and spleen were processed for CFU analysis. Serial dilution of CFUs were plated on LB agar plates with appropriate antibiotics and incubated overnight at 37 °C.

Statistical analysis

Experiments were repeated at least three times. At least five mice per group were used for animal infection assays. Mean values and standard deviations were shown in figures. Statistical significance (P values) was analyzed using the GraphPad software (version 9) with 95% confidence interval.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The sequencing data have been deposited in the GEO database under accession numbers GSE243246 and GSE260738. Raw data, materials, and regents are available upon request. Source data are provided with this paper.

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Acknowledgements

We are grateful to Drs. Joan Mecsas, Francesca Short, Hong-Yu Ou, Min Li, and Chao Yang for providing Klebsiella strains, Dr. Yunn-Hwen Gan for discussion. This study was financially supported by the National Key R&D Program of China (2022YFE0111800 and 2022YFC2303200 to Y.C.), the Chinese Academy of Sciences (XDB0570000 and 176002GJHZ2022022MI to Y.C.), the Natural Science Foundation of China (32270064 to Y.C., 32200031 to K.W.), Shanghai Municipal Science and Technology Commission (21ZR1471300 and 2019SHZDZX02 to Y.C.), and Shanghai Super Postdoc Fellowship (R.D).

Author information

These authors contributed equally: Kejing Wu, Xingyu Lin, Yujie Lu.

Authors and Affiliations

Microbial RNA Systems Biology Unit, Center for Microbes, Development and Health (CMDH), Shanghai Institute of Immunity and Infection, Chinese Academy of Sciences, Shanghai, China
Kejing Wu, Xingyu Lin, Yujie Lu, Rui Dong, Hongnian Jiang, Sarah L. Svensson & Yanjie Chao
University of Chinese Academy of Sciences, Beijing, China
Xingyu Lin, Yujie Lu, Hongnian Jiang & Yanjie Chao
Center of Infectious Disease, Peking University Third Hospital, Beijing, China
Jiajia Zheng & Ning Shen
Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, MA, USA
Andrew Camilli
Key Laboratory of RNA Innovation, Science and Engineering (RISE), Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, China
Yanjie Chao

Authors

Kejing Wu
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Xingyu Lin
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Yujie Lu
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Rui Dong
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Hongnian Jiang
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Sarah L. Svensson
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Jiajia Zheng
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Ning Shen
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Andrew Camilli
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Yanjie Chao
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Contributions

Y.C. designed the study; K.W. and X.L. performed iRIL-seq analysis; K.W. characterized ArcZ and mechanisms of target regulation; X.L. performed capsule quantifications, characterized regulation by CRP and molecular function of MlaA; Y.L. analyzed clinical isolates and performed mice infection experiments with help from R.D. and H.J.; J.Z., N.S. contributed materials and regents; K.W. processed RNA-seq data; K.W., X.L., S.L.S., Y.C. analyzed results; S.L.S., A.C., Y.C. revised manuscript; Y.C. wrote the manuscript, supervised the project and secured funding.

Corresponding author

Correspondence to Yanjie Chao.

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The authors declare no competing interests.

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Nature Communications thanks Jorgen Johansson, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Wu, K., Lin, X., Lu, Y. et al. RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence. Nat Commun 15, 6946 (2024). https://doi.org/10.1038/s41467-024-51213-z

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Received: 26 April 2024
Accepted: 01 August 2024
Published: 13 August 2024
DOI: https://doi.org/10.1038/s41467-024-51213-z

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