Abstract
Pneumolysin (Ply) of Streptococcus pneumoniae (pneumococcus) at relatively high and low levels facilitates pneumococcal invasion into the lung and brain, respectively; however, the regulatory mechanisms of Ply expression are poorly understood. Here, we find that a small RNA plyT, processed from the 3’UTR of the ply operon, is expressed higher in anaerobically- than in statically-cultured pneumococcus D39. Using bioinformatic, biochemical and genetic approaches, we reveal that PlyT inhibits Ply synthesis and hemolytic activities by pairing with an RBS-embedded intergenic region of the ply operon. The RNA-binding protein SPD_1558 facilitates the pairing. Importantly, PlyT inhibition of Ply synthesis is stronger in anaerobic culture and leads to lower Ply abundance. Deletion of plyT decreases the number of pneumococci in the infected mouse brain and reduces the virulence, demonstrating that PlyT-regulated lower Ply in oxygen-void microenvironments, such as the blood, is important for pneumococcus to cross the blood-brain barrier and invade the brain. PlyT-mediated repression of Ply synthesis at anoxic niches is also verified in pneumococcal serotype 4 and 14 strains; moreover, the ply operon with a 3’UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus.
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Introduction
Streptococcus pneumoniae (pneumococcus) is an opportunistic pathogen that commonly colonizes the human nasopharynx and invades deeper tissues and organs in immunocompromised individuals, causing pneumonia, bacteremia, and meningitis1,2. Pneumococcal infections are estimated to cause up to half a million deaths annually in children under 5 years3. Particularly, pneumococcus is a leading cause of bacterial meningitis, which has a mortality rate of up to 30%, and ~50% of survivors experience severe sequelae, such as cognitive impairment and hearing loss4,5,6.
Pneumococcus relies on many virulence factors to achieve deeper tissue invasion, and pneumolysin (Ply) plays key roles in pneumococcal transmission, colonization and invasion7,8,9. As a member of the cholesterol-dependent cytolysin family of proteins, Ply exerts cytotoxic effects by binding to cholesterol on the cellular membrane, resulting in the perforation and breakage of the host cells, thus promoting pneumococcal invasion10,11,12,13. In addition, Ply induces host inflammatory reactions, causing the shedding and transmission of colonized pneumococcus to new hosts14,15,16. Although higher amounts of Ply protein benefit pneumococcal transmission and lung infection16,17, recently pneumococcus with lower Ply expression was found to be less susceptible to killing by microvascular endothelial cells, allowing easier translocation across the blood‒brain barrier (BBB)18, a crucial step in meningitis pathogenesis4,19. Therefore, maintaining a lower Ply abundance could be essential for pneumococcal brain invasion. However, it is unknown how the pneumococcus regulates ply expression to accomplish the invasion of different tissues.
To achieve deeper tissue invasion, such as the brain, pneumococcus spreads from the nasopharynx to the lung and blood and then transits across the BBB20,21. The aforementioned tissues contain different O2 contents; for example, the nasopharynx, lung and blood have O2 contents of 21, 5, and 0%, respectively22. Therefore, to infect the brain successfully, pneumococcus must finely regulate the Ply expression in response to different O2 levels in various tissues. In a previous experiment, we observed higher Ply contents in statically cultured than anaerobically cultured pneumococcus serotype 2 strain D39; however, the ply transcript abundances were similar (unpublished data). This suggests post-transcriptional regulation of Ply synthesis in response to O2.
Small RNAs are known to be major responders to environmental changes, exerting post-transcriptional regulation by affecting translation efficacy and transcript stability23,24,25,26. Using sRNA sequencing, Acebo et al. identified a 89 nt-sRNA trn0884 in the ply 3’-untranslated region (3’ UTR) of the pneumococcal serotype 4 strain TIGR427. In this study, we conducted bioinformatics, physiological, genetic, and animal experiments, and found that a sRNA plyT is located at the 3’UTR of the ply operon in strain D39 and produced by processing from the ply polycistronic transcript. PlyT inhibits Ply synthesis by base pairing with the ribosomal binding site (RBS)-embedded intergenic region (IGR) of the ply operon under the facilitation of the RNA-binding protein SPD_1558. Remarkably, higher levels of the plyT transcript and its inhibition of Ply synthesis are found in anaerobically- than in statically-grown D39 cells, thus resulting in lower Ply protein levels in anoxic niches, such as the blood. Deletion of plyT retarded the ability of the pneumococcus to cross the BBB and reduced the death rate of the experimental mice, highlighting the importance of PlyT-regulated lower Ply levels in the blood in pneumococcal brain invasion. PlyT-repressed Ply synthesis under anaerobic condition is verified in two other serotype strains; moreover, the ply operon with a 3’UTR-embedded plyT, and the pairing sequences of IGR and plyT are highly conserved among pneumococcal strains, implying PlyT-regulated Ply synthesis might be widely employed by pneumococcus.
Results
The ply 3’ UTR encodes an actively expressed sRNA
Previously, Cappable-seq and deep transcriptome sequencing of strain D39 predicted that ply (SPD_1726) is co-transcribed with three upstream hypothetical protein genes, SPD_1729, SPD_1728, and SPD_172728. TransTermHP predicted a Rho-independent terminator at the 3’-end of the ply transcript, which has a 195 nt 3’ UTR (Fig. 1a). Coincidently, small noncoding RNA sequencing revealed an 89-nt sRNA, named trn0884, at the 3’ UTR of ply in the pneumococcal serotype 4 strain TIGR427. We then used the trn0884 sRNA sequence as a probe to blast the D39 genome and found an 89-nt fragment at the 3’ UTR of the 4-gene ply operon at 98% sequence identity (Fig. 1a), thus named plyT.
a Schematic illustration of the ply operon and sRNA plyT (red stem-loop) situated in the 195 nt 3’ UTR of the ply operon. The DNA sequences of sRNA trn0884 in TIGR4 and its homolog in strain D39 are framed with dashed lines; differential nucleotides between D39 and TIGR4 are shown in red letters, and the italics indicate the terminator sequences. The solid arrow and the stem-loop structure indicate the transcription start site and terminator, respectively. Gene accession numbers and encoded proteins are labeled inside and on top of the bullet symbols, respectively. b Northern blot analysis of the plyT transcripts in the wild-type (WT), plyT deletion (ΔplyT), and ply deletion (Δply) strains. Total RNA was separated on 5% (upper panel) and 10% (lower panel) Urea-PAGE gels, respectively. A plyT probe was used to determine the plyT transcripts in the ply operon-plyT polycistron transcript (left upper panel) and the independent plyT transcripts (left lower panel). The ply probe was used to detect the polycistronic transcript of ply operon (right panels). 5S rRNA was used as the loading control. RNA size marker is shown at the gel left. The experiment was repeated three times; one representative picture is shown. c An aliquot of total RNA from D39 strain was treated with or without 5’ pyrophosphohydrolase (RppH). Then, 5’ and 3’ RACE experiments were performed as described in “Methods”. The left and middle panels show the 5’ RACE-amplified (Up and Down (Dn)) and 3’ RACE-amplified products (black arrow), respectively. The right panel shows the plyT and flanking DNA sequence. Nucleotides shadowed in blue and green specify the 5’-ends of 5’ RACE “Up” and “Dn)” bands, with the major 5’-end nucleotides designated as “5’1” and “5’2”, respectively; nucleotide shadowed in orange shows the 3’ RACE-assayed 3’-end. Nucleotides in blue are the homogeneous sequence of sRNA trn0884. M, DNA molecular marker. d GFP fluorescence measurement of D39 strains harboring transcriptional fusion of respective 300 bp DNA fragment upstream of the two major 5’-end nucleotides (5′1up and 5′2up) of plyT with sf-gfp gene (top panel). ply operon promoter (Pply)-sf-gfp fusion was used as the positive control. The results are expressed as fluorescence intensity/OD600 (lower panel). The experiment was repeated three times; Averages ± SD (n = 3) of one experiment with three independent batches of culture are shown.
Next, a Northern blot was performed to measure the expression of the sRNA plyT in strain D39 and the plyT deletion strain ΔplyT using a DNA fragment complementary to plyT as the probe. An 10% Urea-PAGE gel was used to better separate short RNA transcripts. Three RNA fragments, ~90, 120, and 180 nt in length, the latter of which being the most abundant, were detected in the wild-type but not in the ΔplyT strain (Fig. 1b, lower panel). This finding suggested that the sRNA plyT is actively expressed as multiple transcript isoforms, with 180 nt being the major one.
To further determine the 5’- and 3’-boundaries of the plyT RNA isoforms, 5’ and 3’ rapid amplifications of cDNA ends (RACE) were implemented. One aliquot of the total RNA was treated with 5’ pyrophosphohydrolase (RppH), and another was left untreated to determine the 5’-end status. Consistent with the Northern blot results (Fig. 1b, lower panel), 5’ RACE amplified two identical products in lengths of ~180 and 90 bp (assigned as up and down, respectively), with the 180-bp overrepresented, in RppH-treated and -untreated RNAs (Fig. 1c, left panel). This indicated that sRNA plyT was in the 5’ monophosphorylated form, implying that it is not a primary transcript and could be produced through RNA processing. While 3’ RACE only amplified one product (Fig. 1c, middle panel). DNA sequencing and subsequent BLAST searches of the sequences against the D39 genome revealed that ~60 and 75% clones of 5’ RACE amplified ~90- and 180-bp DNA fragments, respectively, had an adenine nucleotide at their 5’-ends, located at 2- (labeled as 5’1) and 91-nt (labeled as 5’2), respectively, upstream of the 5’-end of the sRNA trn0884 in strain TIGR4. All 3’ RACE amplified products consistently matched a thymine located 2 nt downstream of the 3’ end of the sRNA trn0884 (Fig. 1c, right panel). This result indicates that the plyT isoforms should have a single 3’-end but multiple 5’-ends. Notably, some clones of 5’ RACE-amplified products matched diverse nucleotides adjacent to the major 5’-end (Fig. 1c, right panel). In conclusion, the sRNA plyT is expressed mainly as a 182-nt transcript in pneumococcus D39.
sRNA plyT is co-transcribed with the ply operon and produced by mRNA processing
Given that sRNA plyT has the diverse 5’ ends, we next determined its generation mode. First, the co-transcription of plyT with ply and the three upstream genes (SPD_1729-SPD_1727) was examined. A Northern blot using 5% Urea-PAGE gel to separate long RNA transcripts and the plyT probe revealed a large RNA transcript of ~3100 nt in length in the wild-type but not in the ΔplyT strain (Fig. 1b, upper panel). This length matches the polycistronic transcript of the ply operon comprising SPD_1729, SPD_1728, SPD_1727, SPD_1726, and plyT. A probe targeting the ply gene also detected the large RNA fragments in D39 wild-type but not in the Δply strain. Furthermore, using primers paired with the respective intergenic regions of the ply operon including plyT, RT–PCR only amplified products with the predicted lengths from the cDNA but not from the DNase-treated total RNA (Supplementary Fig. 1a, b). These results indicate that the four genes and plyT are co-transcribed. RT–PCR also detected transcription of the IGR between ply-plyT-SPD_1725; however, the transcription level was ~1.98-fold lower than that of the ply-plyT IGR (Supplementary Fig. 1c, d). In addition, Northern blot revealed only a ~3100 nt transcript of the ply operon (Fig. 1b, upper panel), but not the ~5000 nt transcript including SPD_1725. Thus, we conclude that the ply polycistronic transcription should mostly terminate at the terminator embedded in plyT. This finding is consistent with the 93% terminating efficiency of the ply operon terminator reported by Slager et al28. RT–PCR did not detect IGR transcripts between SPD_1725 and SPD_1724 (Supplementary Fig. 1c).
Given that the 5’ RACE assay-determined plyT transcripts to be 5’-p forms (Fig. 1c), we reasonably predicted that the sRNA plyT transcripts are the processing products of the ply polycistronic transcript. To confirm this prediction, we examined the promoter activity of a 300-bp DNA fragment upstream of the 5’1- and 5’2-ends of plyT by constructing a transcription fusion with the RBS-containing green fluorescence protein sf-gfp gene on the shuttle plasmid pDL278 in strain D39. Compared with that of the ply promoter fusion, almost no GFP fluorescence was detected in the strains harboring the DNA sequences upstream of the 5’1 or 5’2 ends (Fig. 1d), verifying that these DNA sequences have no promoter activity. This finding confirmed that the plyT transcripts are produced from the ply operon by mRNA processing.
sRNA PlyT represses the translation of the ply operon proteins, including Ply
As plyT is processed from the 3’ UTR of the ply operon transcript, its effect on the expression of the operon genes was examined in statically grown wild-type and plyT deletion strains. Notably, deletion of plyT did not affect the growth of strain D39 (Fig. 2a). qRT–PCR did not detect differential transcriptions of SPD_1729, SPD_1728, SPD_1727, or ply genes upon plyT deletion (Fig. 2b); however, using the same amount of cellular proteins and spent cultures, and glycerol-3-phosphate dehydrogenase (GpsA) abundance determination (Supplementary Fig. 2a) as a loading control, Western blot revealed a 1.4- and 1.3-fold higher Ply amount in the cell lysates and spent culture, respectively, of the plyT deletion strain than in the wild-type strain (Fig. 2c). These findings indicate that the sRNA PlyT suppresses translation of Ply protein. To further verify the role of PlyT in the regulation of Ply protein production, the plyT ectopically complementary strain, plyTcom, was constructed by transferring the shuttle plasmid pDL278-Pply-plyT, carrying a transcriptional fusion of ply operon promoter DNA fragment and a 182 nt plyT sequence, into the ΔplyT strain. The expression of plyT in plyTcom was verified by Northern blot (Supplementary Fig. 3), and Western blot revealed lower Ply abundances in the cell lysates and spent cultures of the plyTcom strain than in those of the wild-type strain (Fig. 2c).
a Growth profiles of D39 wild type (WT) and plyT deletion (ΔplyT) strains under static conditions were measured for optical density at 600 nm. b qRT–PCR quantifying the transcript abundances of the ply operon genes in late-exponential statically cultured D39 wild-type (WT) and ΔplyT cells. 16S rRNA gene was used as the biomass reference, and transcript copies of the tested genes per 1000 16S rRNA copies are shown. c Western blot assays Ply abundances in statically cultured D39 wild-type (WT), ΔplyT, and plyT complementary (Com) strains. Cell lysates (CFE) with 10 µg of total proteins and the same volume of spent culture (SP) concentrated through a 10-kD ultrafiltration tube were loaded on a 12% SDS–PAGE gel, and Ply abundances were detected using anti-Ply monoclonal antibody (upper panel). Glycerol-3-phosphate dehydrogenase (GpsA) was detected using mouse-raised antibody as the loading control (middle panel). No GpsA was detected in the SP as it is a cytoplasmic protein. Band intensities were quantified using ImageJ software and are shown as the percentiles of that in the wild-type strain; Relative abundance ratios of Ply to GpsA were calculated by setting that in the wild-type strain at 100% (lower panel). Protein molecular markers are shown on the right of the gel. d Hemolysis assay of D39 wild-type (WT), ΔplyT, and plyT complementary (Com) strains. CFE with 40 µg of total protein and 15 µL of SP were 2-fold serially diluted as indicated on the top, and each was incubated with equal volumes of 2% red blood cells at 37 °C. After 30 min, the non-hemolytic blood cells were collected by centrifugation (upper panel), and the supernatants were removed to measure the optical density at 540 nm (lower panel). e Western blot assays the protein abundances of SPD_1729, SPD_1728, and SPD_1727 in the statically grown D39 wild-type (WT) and ΔplyT strains. SPD_1729 protein was determined using anti-GFP antibody in SPD_1729::GFP fusion strain. SPD_1728 and SPD_1727 proteins were detected using mouse-raised antibodies. GpsA abundances were determined as loading controls (upper panel). Band intensity quantification and calculation of the relative abundance ratios of Ply to GpsA (lower panel) were performed as described in (c). The molecular markers are shown on the right of the gel. All the experiments were repeated three times; for (a, b), Averages ± SD (n = 3) of one experiment with three independent batches of culture are shown. For (c–e), Averages ± SD (n = 3) of three replicate experiments and representative images are shown. Significant difference from the wild-type strain is indicated by the P value or *, P < 0.05, **, P < 0.01, ***, P < 0.001; n.s. no significant difference between the samples (P > 0.05) (b, e, Student’s t test; c, d, One-way ANOVA, Tukey’s test).
As the Ply protein is a hemolysin and can perforate red blood cells12, hemolytic experiments were subsequently performed to verify the regulation of PlyT on Ply protein synthesis. The wild-type, plyT deletion and plyTcom strains were all statically grown in BHI broth, and the late-exponential cultures were used to prepare cell lysates and spent cultures. All the preparations were 2-fold serially diluted and determined for hemolysis activity. Consistent with the results of Western blot (Fig. 2c), significant elevated and decreased hemolytic activities were found in both the cell lysate and spent culture of the plyT deletion and plyTcom strains, respectively, in comparison with that of the wild-type strain (Fig. 2d), further verifying the inhibitory effect of PlyT on Ply protein synthesis.
PlyT-mediated inhibition of the protein synthesis of the other ply operon genes, SPD_1729, SPD_1728, and SPD_1727, was also tested. As shown in Fig. 2e, using the respective antibodies (Supplementary Fig. 2b, c), Western blot revealed 1.3- and 1.4-fold higher abundances of the SPD_1728 and SPD_1727 proteins, respectively, in cell lysates of ΔplyT versus those in the wild-type strain. However, deletion of plyT did not increase the SPD_1729 protein level, which was determined in the SPD_1729::GFP fusion strain using an antibody against GFP (Fig. 2e upper panel), because we failed to overexpress this membrane protein in Escherichia coli. These results suggest that PlyT regulates the protein synthesis of the ply operon genes, mostly likely by interacting with the RNA sequences downstream of SPD_1729.
PlyT represses Ply protein synthesis likely by pairing with the RBS-embedded IGR between the SPD_1729 and SPD_1728 genes
Given that small RNA regulation of translation generally occurs through base pairing with a transcript 5’ UTR29, to identify the regulatory targets of the sRNA PlyT on Ply translation, we searched for the possible plyT base-paired RNA sequences in the ply operon. Using the IntaRNA algorithm30, an RNA sequence embedded with an RBS in the IGR between SPD_1729 and SPD_1728 (IGR1729/1728 and flanking sequence) was predicted to base-pair with plyT at a free energy of –33.28 kJ/mol (Fig. 3a). RNAfold WebServer predicted that the plyT transcript would form a stable secondary structure, and the loop region could base-pair with the IGR1729/1728 and flanking RNA sequence (Fig. 3b). To verify this base pairing, we constructed the translation fusion plasmid pDL278-Pply-IGR-gfp-plyT, in which a DNA fragment consisting of the ply operon promoter, a 168 bp DNA fragment containing 27 bp of the SPD_1729 3’end, a 12 bp intergenic sequence, and a 129 bp 5’-terminal sequence of SPD_1728 (IGR), was fused upstream of the gfp-plyT gene fusion. A translation fusion plasmid without plyT, pDL278-Pply-IGR-gfp, was constructed as a plyT-vacant control (Fig. 3c). The two plasmids were respectively transformed into the plyT deletion strain. After verifying the expression of the cloned plyT by Northern blot (Fig. 3d), we monitored GFP fluorescence during the growth of the ΔplyT strains harboring the two different plasmids. As shown in Fig. 3e, ~1.5-fold lower GFP fluorescence intensity was determined in the strain overexpressing plyT than in the strain without its overexpression. However, after substituting the 168 bp IGR sequence with the 5’ UTR sequence of the ply operon, no difference in GFP fluorescence was detected between the strains with and without plyT overexpression (Supplementary Fig. 4). These experiments suggest that PlyT could repress protein translation by pairing with IGR1729/1728 and flanking RNA.
a The genome context of the ply operon, including sRNA plyT, is shown on top, and IntaRNA software-predicted base pairing between plyT and IGR1729/1728 flanking sequence at a free energy of –33.28 KJ/mol is shown at the bottom. Nucleotides in blue represent the 3’ end fragment of the SPD_1729 gene, ATG in red, and nucleotides in italics indicate the start codon and SD sequence of SPD_1728, respectively. Nucleotides in orange are the mutated (Mut) plyT in the base-pair region. b Prediction of sRNA plyT secondary structure using RNAfold WebServer. The 5’ end (No. 1) of plyT and the two predicted base-paired regions (nucleotides between 49–62 and 68–82) are shown. c The GFP translation fusion plasmid pDL278-Pply-IGR-gfp (IGR-gfp) was constructed by fusing a DNA fragment containing IGR1729/1728 and flanking sequence and 129 bp 5’–terminal sequence of SPD_1728 downstream of the ply operon promoter (P) and upstream of the gfp gene (left). Then, wild-type and paired nucleotides-mutated plyT, as indicated in (a), were each fused downstream of gfp to construct pDL278-Pply-IGR-gfp-plyT (IGR-gfp-plyT) (middle) and pDL278-Pply-IGR-gfp-plyTMut (IGR-gfp-plyTMut) (right). d Northern blot verified the expression of the ectopic plyT in plyT deletion strains harboring one of the three different plasmids shown in (c). RNA size marker is shown at the gel left. e GFP fluorescence measurement of the strains in (d) during the growth. The early-exponential (Early-exp), middle-exponential (Mid-exp), and late-exponential (Late-exp) phase cells were collected and washed twice with PBS. After air exposure in the dark for 30 min, GFP fluorescence intensity and cell biomass (OD600) of cells were measured. The results are expressed as GFP fluorescence intensity/OD600. Experiments were repeated three times in (d, e); one representative picture is shown in (d), and averages ± SD (n = 3) of one experiment with three independent batches of culture are shown in (e). Significance between the samples is shown by the P value (One-way ANOVA, Tukey’s test; P < 0.05).
To further verify that plyT pairs with the IGR1729/1728 and flanking RNA sequence, we constructed a translation fusion of the gfp reporter gene with the plyT mutant, pDL278-Pply-IGR-gfp-plyTMut, in which cytosine and guanine nucleotides in the predicted pairing region with the RBS-containing IGR1729/1728 was substituted by guanine and cytosine, respectively (Fig. 3a, c, d). As shown in Fig. 3e, the GFP fluorescence in the strain harboring pDL278-Pply-IGR-gfp-plyTMut was not significantly different from that in the strain without plyT overexpression, suggesting that PlyT-mediated suppression of Ply protein synthesis occurs through base pairing with the RBS of SPD_1728. As complementary mutation of nucleotides in RBS region of IGR1729/1728 would influence the translation of the SPD_1728 gene, we instead deleted three hypothetical protein-encoding genes, SPD_1729, SPD_1728, and SPD_1727, and named this strain Δ3HP. Western blot revealed an ~1.7-fold elevation of Ply protein contents in the cell lysate and spent culture of the Δ3HP strain versus that in those from the wild-type strain (Supplementary Fig. 5), indicating that deletion of the IGR1729/1728 and flanking RNA sequence paired with plyT increased Ply abundance. This result further confirmed that PlyT represses Ply synthesis through pairing with the IGR1729/1728 and flanking RNA sequence.
plyT binds to the SPD_1728 RBS-embedded IGR sequence in the presence of an RNA-binding protein
To determine whether plyT binds to the RBS of SPD_1728, RNA EMSA (rEMSA) was used to assay RNA hybridization between the 182 nt plyT and a 55 nt IGR1729/1728 and flanking RNA sequence embedded with the RBS of SPD_1728. However, no RNA hybrid products were detected (Supplementary Fig. 6a). To explore whether a chaperone protein is needed, we searched for four pneumococcal RNA-binding proteins (RBPs): SPD_0675 and SPD_1849, which contain a KH domain31, SPD_1777, annotated as a 3’–5’ exoribonuclease32, and SPD_1558, recently identified as an actively expressed RBP32. By examining the deletion mutants of each of the four genes in strain D39, Western blot, and hemolytic experiments revealed that only the deletion of SPD_1558 and SPD_1777 caused elevated Ply synthesis in aerobically cultured pneumococcus (Fig. 4a and b).
a Western blot determines Ply abundances in D39 wild-type (WT) and four RNA binding protein encoding genes, SPD_0675 (Δ0675), SPD_1558 (Δ1558), SPD_1849 (Δ1849) and SPD_1777 (Δ1777) deletion mutants. GpsA abundances were determined as loading controls (upper panel). Band intensities were quantified using ImageJ software and are shown as percentiles of that in the wild-type strain. Relative abundance ratios of Ply to GpsA were calculated by setting that in the wild-type strain as 100% (lower panel). The molecular markers are shown at the gel right. b The cell lysates, with 40 µg of total protein, of strains in (a) were 2-fold serially diluted as indicated on top, and incubated with equal volumes of 2% red blood cells. After a 30-min incubation at 37 °C, non-hemolytic blood cells were collected by centrifugation (upper panel), and the supernatants were measured for optical density at 540 nm (lower panel). For (a, b), Averages ± SD (n = 3) of three independent experiments are shown. Significant difference from the wild-type strain is indicated by *, P < 0.05, **, P < 0.01, ***, P < 0.001 (One-way ANOVA, Tukey’s test). c RNA-EMSA (rEMSA) revealed that the recombinant SPD_1558 promotes the pairing between sRNA plyT and IGR1729/1728 flanking RNA sequence (IGR). Gradient concentrations of SPD_1558 proteins were incubated with 3’-biotin-labeled IGR RNA or plyT, or with the two RNA fragments together. Black and red arrows specify the free 3’-biotin-labeled IGR and 3’-biotin-labeled plyT, respectively; open arrows in blue, green, and purple indicate the binding complexes of SPD_1558-IGR, SPD_1558-plyT, and SPD_1558-IGR-plyT, respectively. d Forty-eight micrograms of total protein from wild-type (WT) and 6XHis tagged SPD_1558 (1558-6His) strain were incubated with Anti-His Magnetic Beads, and RNA immune-precipitation (RIP) experiments were implemented as described in “Methods”. Western blot (upper panel) and Northern blot (lower panel) were used to assess the immune-precipitated 1558-6His protein (upper panel) and sRNA plyT (lower panel) in the input and eluted (RIP) samples using an anti-His-tag antibody and plyT probe, respectively. e Northern blot assayed the plyT transcript abundances in the wild-type (WT) and SPD_1558 deletion (Δ1558) strains using the plyT probe on a 10% Urea–PAGE gel. 5S rRNA (5S) was used as a loading control. RNA size markers are shown at the gel right. All the experiments were repeated three times, and representative images are shown.
rEMSA was performed to determine the RNA chaperone activity of the SPD_1558 and SPD_1777 proteins by observing whether they could facilitate 182 nt plyT pairing with the 55 nt IGR1729/1728 and flanking RNA. The results showed that the heterogeneously expressed SPD_1558 protein not only bound to either the IGR1729/1728 and flanking RNA (Fig. 4c, lanes 3 and 4) or plyT (Fig. 4c, lanes 5 and 6) but also appeared to facilitate the formation of an SPD_1558-plyT-IGR tripartite complex (Fig. 4c, lanes 7 and 8), which migrated significantly slower than the SPD_1558-IGR duplex complex and slightly slower than the SPD_1558-plyT duplex complex. These findings indicate that the SPD_1558 protein behaves like an RNA chaperone. Consistent with the fact that chaperone protein can bind and protect sRNAs from degradation, an RNA immunoprecipitation (RIP) assay coupled with Northern blot experiment revealed that SPD_1558 bound plyT in vivo (Fig. 4d); additionally, reduced cellular plyT expression was found in the SPD_1558 deletion mutant (Fig. 4e).
Although rEMSA also revealed the binding of the overexpressed SPD_1777 protein to the IGR1729/1728 flanking RNA (Supplementary Fig. 6b, lanes 2–5), no promotion of plyT and IGR1729/1728 flanking RNA pairing was found (Supplementary Fig. 6b, lanes 6–9), precluding its role in facilitating plyT-IGR1729/1728 flanking RNA pairing.
PlyT regulation is responsible for the decreased Ply protein level in anaerobic culture
Occasionally, we found higher Ply abundances in statically cultured than anaerobically grown pneumococcus. To confirm this phenomenon in relation to oxygen, we cultured strain D39 statically and anaerobically under N2 gas. As pneumococcus produces plenty of H2O2 in the presence of O233, to exclude the effect of H2O2, 1 KU mL−1 catalase was added to the static culture, while H2O2 was added to the anaerobic culture to a final concentration of 100 µM, as low concentrations of H2O2 (40–100 µM) could function as a signal molecule and induce the antioxidant genes expression in an oral streptococcus34. Western blot revealed a consistent approximate 2-fold increase in Ply content in the static cultures compared with that in the anaerobic cultures, irrespective of the supplementation with catalase or H2O2 (Fig. 5a). These results indicate that pneumococcus regulates Ply production in response to O2, but not H2O2, and produces more Ply protein in aerobic than anaerobic niches. However, the same abundances of the ply transcripts were found in the four types of cultures of pneumococcus D39 (Supplementary Fig. 7), indicating that O2-responsive Ply protein production is likely post-transcriptionally regulated. To determine the linkage between PlyT regulation and O2-responsive Ply production, we compared the Ply contents in the anaerobic cultures of the wild-type, plyT deletion, and plyTcom strains with those in the respective static cultures (Fig. 2c, d). As shown in Fig. 5b, Western blot revealed an ~1.7- and 2.9-fold higher abundance of Ply in cell lysates and spent culture, respectively, of anaerobically grown ΔplyT than in the wild-type and plyTcom strains. Moreover, an ~8-fold higher level of hemolytic activity was found in anaerobically grown ΔplyT than that of the wild-type and plyTcom strains (Fig. 5c). In comparison, plyT deletion only caused an ~1.3-fold increase in sum Ply abundance (Fig. 2c), and an ~2-fold increase in the level of hemolytic activity in static cultures (Fig. 2d). This finding indicates that the sRNA PlyT suppresses Ply synthesis more strongly under anaerobic conditions and is thus responsible for the O2-responsive Ply protein production in pneumococcus.
a Pneumococcus D39 was grown statically (Static) with or without the addition of 1 KU mL−1 catalase (Static+catalase) and anaerobically (Anaerobic) with or without 100 µM H2O2 treatment (Anaerobic+H2O2). Western blot revealed the abundances of Ply (upper panel) and the loading control GpsA protein (middle panel) in 10 mg total protein using the respective antibodies. Band intensities were quantified using ImageJ software; Ply, GpsA contents, and relative abundance ratios of Ply to GpsA (lower panel) are shown as the percentiles of that in the statically grown wild-type strain. b The wild-type (WT), ΔplyT, and plyT complementary (Com) strains were cultured anaerobically, and the late-exponential cells and spent culture were collected. Cell lysates (CFE) with 10 µg of total protein and the same volume of spent culture (SP) concentrated through a 10-kD ultrafiltration tube were used for Western blotting. Protein quantification (upper and middle panel) and relative abundance ratios of Ply to GpsA (lower panel) were expressed as in (a). No GpsA was detected in the SP as it is a cytoplasmic protein. Molecular markers are shown at the gel right. c Cell lysates (CFE) with 40 µg of total protein and 15 µL of spent culture (SP) were 2-fold serially diluted and incubated with equal volumes of 2% red blood cells for 30 min at 37 °C. Residual non-hemolytic blood cells were collected by centrifugation (left panel), and the supernatants were measured for optical density at 540 nm (right panel). d Total RNA was extracted from the early-(Early-exp) and late-exponential (Late-exp) cultures of aerobically (Static) and anaerobically (Anae) grown pneumococcus D39 and Northern blotting was used to assay the plyT transcript using the plyT probe (upper panel). 5S rRNA (5S) was used as a loading control (middle panel). Band intensities were quantified using ImageJ software; plyT, 5S rRNA contents, and relative abundance ratios of plyT to 5S rRNA (lower panel) are shown as the percentiles of that in the statically grown wild-type strain. RNA size marker is shown on the right of the gel image. The experiments were repeated three times in (a–d); Averages ± SD (n = 3) and representative pictures are shown. Significance is indicated by the P value in (a, b, d) and difference from the wild-type strain in (c) indicated by *, P < 0.05, **, P < 0.01, ***, P < 0.001 (One-way ANOVA, Tukey’s test (P < 0.05)). e, f BALB/c mice were infected by 100 µL of PBS containing 5 × 106 CFUs of D39 wild-type, plyT deletion (ΔplyT), or plyT complementary (plyTcom) strains via intraperitoneal injection. PBS and the ply deletion strain (Δply) at the same CFUs were administered to mice and included as controls. The dying mice infected with various pneumococcal strains were anesthetized, blood was collected by retroorbital bleeding, and the mice were euthanized to obtain the brain tissues. All the samples were 10-fold serially diluted and CFUs of pneumococcus in the blood (e) and brain (f) were enumerated. Experiments were repeated two times, and the average ± SD of CFUs in mice (n = 5–10) of one independent experiment is shown. The datum of the Δply strain is statistically different from that of the other strains in (e), and the data of ΔplyT and Δply strains are statistically different from those of the other strains in (f) (*, P < 0.05, **, P < 0.01, ***, P < 0.001; One-way ANOVA, Tukey’s test). g A representative survival curve of BALB/c mice (n = 8-10, female) infected by the various pneumococcal strains from two independent animal experiments. h The survival time of the mice infected with various pneumococcal strains in (g). Data are presented as means ± SEM (n = 8-10). Statistical significance with the wild-type and plyTcom strains is indicated by *, P < 0.05, **, P < 0.01, ***, P < 0.001; for (h), Log-rank Mantel-Cox test; for e, One-way ANOVA, Tukey’s test.
The Northern blot analysis revealed more plyT transcripts in anaerobic cultures than in static cultures in both the early- and late-exponential phases (Fig. 5d). Given that the Ply protein content is important for pneumococcus to transit the BBB18 and that the bacterium must cross through O2-void blood to enter the brain21, PlyT-regulated O2-responsive Ply synthesis is likely important for pneumococcal meningitis.
PlyT-regulated lower Ply production in anoxic niches is important for pneumococcus crossing the murine BBB and enhancing virulence
Given that pneumococci with lower Ply contents usually evade degradation by lysosome of brain microvascular endothelial cells and thus transit across the BBB18, we presumed that PlyT-regulated lower Ply production in anaerobic niches, such as blood, could contribute to the pneumococcal invasion into brain. Eight to ten BALB/c mice (SPF grade, female, 6–8 weeks) were infected with the D39 wild-type, plyT deletion, or plyT complementary strains via intraperitoneal injection of 5 × 106 CFUs of each strain in 100 µL of PBS. PBS- and Δply-infected mice were included as the negative and Ply protein null controls, respectively. Mouse survival was observed for 7 days, and colony forming units (CFUs) of various pneumococcal strains in blood and brain tissue of the dying mice were counted. Compared with that in the mice infected by wild-type and plyT complementary strains, significantly fewer CFUs were detected in the brains of the ΔplyT-infected mice, whereas comparable CFUs were observed in blood (Fig. 5e, f). This indicates that PlyT-regulated lower Ply abundance in anoxic blood benefits the infection of brain tissue by pneumococcus. Although significantly fewer CFUs in blood and brain were observed in Δply-infected mice than in those infected with the wild-type strain (Fig. 5e, f), a higher CFUs ratio of brain to blood was found in Δply- (0.033 ± 0.01) versus wild-type-infected mice (0.025 ± 0.01), consistent with the finding that Ply impedes brain microvascular endothelial cell crossing by pneumococcus18. Accordingly, ΔplyT-infected mice demonstrated significantly higher survival percentages and longer mean survival times than those infected with the wild-type and plyT complementary strains (Fig. 5g, h). These results highlight the importance of sRNA PlyT-regulated suitable content of the Ply protein in pneumococcal brain invasion and virulence.
PlyT regulation of Ply synthesis could be widely employed by pneumococcus
To explore the wide distribution of sRNA PlyT regulation of Ply synthesis in pneumococcus strains, we used the sequences of ply operon genes, IGR1729/1728 and sRNA plyT of strain D39 as probes to blast 158 complete pneumococcal genomes available in the NCBI GenBank Database (updated on 2023/09/05). The analysis revealed the ubiquitous distribution of the ply operon with a plyT embedded 3’ UTR among the 158 genomes (Fig. 6a, Supplementary Data 1), and 100% and 97.25% to 100% sequence identities were found for the IGR1729/1728 and plyT sequences, respectively (Fig. 6a, Supplementary Data 1). Although variations in at most 5 nucleotides were found among 158 pneumococcal plyT orthologues (Supplementary Data 1), 100% identities were found in the plyT sequence pairing with IGR1729/1728 (Fig. 6a). This finding suggests that pneumococcus might widely employ sRNA PlyT-regulated Ply synthesis. Indeed, deletion of plyT resulted in a 3.9-fold increase in Ply protein content in spent culture of anaerobically grown serotype 4 TIGR4 strain, and a 1.5-fold increase in both the cell lysates and spent culture of anaerobically grown serotype 14 34359 strains (Fig. 6b), indicating that PlyT-repressed Ply synthesis in anoxic niches occurs in other pneumococcal serotype strains.
a Scheme of conservation of ply operon organization and the pairing sequences between IGR and sRNA plyT in representative pneumococcal serotypes strains. The middle panel shows the organization of the ply operon of strain D39, and the bullets in the same color indicate the orthologs of the corresponding genes in other pneumococcal strains. The red and gray rectangles represent IGR1729/1728 (IGR) 55 nt in length and 3’UTR embedded plyT, respectively. The numbers inside the IGR red rectangles represent sequence identity (%) with that of the D39 strain. The right panel shows the D39 plyT 182 nt in length, and the nucleotide numbers are labeled above. The black vertical lines indicate the locations of the nucleotides that are different from those in D39 plyT, respectively, at the 5’ end (orange rectangle) and the 3’ end with the terminator signal (blue rectangle). No difference was found in the nucleotide sequences pairing with IGR1729/1728, which are shown as red rectangles. The 16S rRNA consensus phylogenetic tree (left panel) was constructed using MEGA 7.0 software. b Western blot assays Ply abundance in the pneumococcus serotype 4 strain TIGR4 and serotype 14 strain 34359 (WT) and their respective plyT deletion mutant (ΔplyT). The tested strains were statically or anaerobically cultured until the late-exponential phase, and the cells and spent cultures were collected by centrifugation. Cell lysates (CFE) with 10 µg of total protein and the same volume of spent culture (SP) concentrated through a 10-kD ultrafiltration tube were loaded on 12% SDS-PAGE gels. Abundance of Ply (left upper panel) and GpsA (left lower panel), which was used as a loading control, was detected using the respective antibodies. Protein molecular markers are shown on the right of the gel. No GpsA was detected in the SP as it is a cytoplasmic protein. Band intensities were quantified using ImageJ software. Ply and GpsA contents and relative abundance ratios of Ply to GpsA (right panel) are shown as the percentiles of those in the statically or anaerobically grown wild-type strain. The experiments were performed three times; Averages ± SD (n = 3) and representative images are shown. Significance from the wild-type strain is indicated by the P value (Student’s t test, P < 0.05).
Discussion
Pneumolysin Ply, an important virulence factor of the pathobiont S. pneumoniae, plays key roles in pneumococcal transmission, colonization, and invasion via its cytotoxicity and modulation of host inflammatory reactions7,11,12. Although higher Ply protein abundances are believed to promote pneumococcal transmission and lung infection17,35, it was recently reported that lower levels of Ply promote pneumococcus to transit the blood-brain barrier and thus cause meningitis18. Therefore, controlling lower Ply production in the anaerobic blood is important for pneumococcal invasion into brain tissue. As depicted in Fig. 7, this work revealed that sRNA PlyT, processed from the 3’ UTR of the ply operon, suppresses Ply synthesis and hemolytic activity. PlyT regulates Ply translation by pairing with an RBS-embedded intergenic RNA sequence between the first and second genes of the ply operon. The RNA binding protein SPD_1558 facilitates the pairing. Higher levels of the plyT transcript and more significant inhibition of PlyT in Ply synthesis are found in anaerobically cultured pneumococcal cells than in statically grown cultures, resulting in lower Ply contents in anaerobic niches, such as the bloodstream. Deletion of plyT impedes the ability of pneumococcus to cross the BBB in the infected mice and thus reduces its pathogenesis. These findings demonstrate that PlyT-regulated lower Ply production in O2-void microenvironments, such as the bloodstream contributes to pneumococcal brain invasion.
a The sRNA plyT (red stem-loop) in the 3’ UTR of ply operon is produced by an unknown ribonuclease processing and represses Ply protein translation by pairing with the ribosomal binding site-embedded intergenic RNA (red line) between SPD_1729 and SPD_1728 in the ply operon. The RNA binding protein SPD_1558 facilitates the pairing, and thus inhibits translation of the Ply protein. Thus, higher levels of Ply are produced in the plyT deletion strain (ΔplyT) than in the wild-type pneumococcus. b As reported by Surve et al18., pneumococcus with lower Ply abundance enters brain tissue more readily by escaping from the lysosomal degradation in brain microvascular endothelial cells. The ΔplyT strain, which produces more Ply protein than the wild-type strain, displays a decreased ability to cross the blood-brain barrier (BBB) and enter the brain. As sRNA PlyT represses Ply production more significantly in anaerobic niches such as the blood, the sRNA-regulated lowering of Ply production to certain levels is important for the pneumococcus to cross the BBB and enhances its virulence.
To achieve brain invasion, pneumococci must detach from the nasopharynx, enter the lung and bloodstream, and then cross BBB. During this journey inside the host, the pneumococcus must adjust its virulence factor expression to adapt to the new microenvironments and leads to continuous infection36,37,38. Hence, the altered expression of Ply, the primary pneumococcal virulence factor, among different host anatomical sites has been intensively investigated. RNA-sequencing revealed reduced ply transcription in pneumococcus grown in media mimicking blood components or supplemented with human plasma, indicating that pneumococcus alters ply transcription in response to blood components21,39. In addition, qRT-PCR assays revealed elevated ply expression in pneumococcus in the mouse nasopharynx and bloodstream versus that grown in in vitro culture40. Recently, ZomB protein, encoded by a mobile genetic element, was found to regulate the ply expression of S. pneumoniae serotype 23F, and the elevated Ply protein helps the transmission of pneumococcus among hosts41. These findings indicate that elaborate regulation of Ply expression can confer pneumococcus the capability of transmission, colonization, and invasion. The present study revealed an sRNA PlyT-mediated post-transcriptional regulation mechanism controlling Ply expression, highlighting the multifaceted regulatory modes of this pneumococcus virulence factor.
sRNAs are well-known post-transcriptional regulators that affect important physiological behaviors and virulence in bacteria responding to environmental signals42,43. The previously reported sRNAs are frequently located within intergenic regions or 5’ UTRs and are independently transcribed from the inherent promoters44. Recently, some 3’ UTR-derived sRNAs have been reported and regarded as “dark regulatory materials” because previously short bacterial 3’ UTRs were not considered to play regulatory roles44,45,46. However, the ply operon harbors a remarkably long 3’UTR of 195 nt (Fig. 1a), which is within the top 5% of the 3’ UTR lengths (median 46 nt) among the 361 pneumococcal operons (Supplementary Fig. 8). Noticeably, this long 3’UTR is embedded with an sRNA plyT, which is most likely cropped from the polycistronic transcript of the ply operon encoding the virulence factor Ply and pairs with the RBS-embedded IGR of the ply operon to inhibit Ply protein translation (Fig. 4). Thus, the greater abundance of the ply transcript, the greater the PlyT level, and in turn, the greater suppression of Ply protein translation. This fine-tuned post-transcriptional regulatory mechanism provides feedback suppression to leverage the Ply content during pneumococcal infection.
Pneumococcus encounters varying O2 levels in different host organs; such as 21, 5, and 0% O2 in the nasopharynx, lung, and blood, respectively21,22. The Ply contents are believed to be related to the different actions of pneumococcus; for example, higher Ply contents in the O2-sufficient nasopharynx promote transmission of the bacterium to other hosts16, whereas lower Ply levels in the O2-void blood favor its transition across the BBB18. Here, we demonstrated lower Ply production in anaerobically cultured than statically cultured pneumococcus (Fig. 5a), revealing O2-responsive Ply production by pneumococcus. Furthermore, this fine-tuned regulation is controlled by the small RNA PlyT at the post-transcription level, as more pronounced suppression of Ply translation by PlyT was observed in anaerobic environments (Fig. 2c vs 5b), such as blood. Importantly, PlyT-regulated lower Ply production in anoxic niches plays a key role in pneumococcal brain invasion (Fig. 5e–h). Gene organization of the ply operon with 3’ UTR embedded plyT and the plyT and IGR1729/1728 sequences are conserved among pneumococcus strains (Fig. 6a). Moreover, PlyT-mediated inhibition of Ply protein production under anaerobic conditions was also found in pneumococcal serotype 4 and 14 strains (Fig. 6b), suggesting that PlyT-regulated Ply translation can be widely employed by pneumococcus strains. Considering that PlyT inhibits Ply production weaker in D39 under aerobic conditions, we tested PlyT’s role in pneumococcal pathogenesis via intranasal challenge. plyT deletion only slightly reduced the pneumococcal lethality in mice (Supplementary Fig. 9). This finding does not conform with Ply-related lethality; therefore, we hypothesize that elevated Ply expression could promote pneumococcus transmission by inducing nasopharyngeal inflammatory reactions in the host16,41, reducing invasion of the lung.
Notably, higher plyT transcript abundances are found in anaerobically cultured pneumococcus (Fig. 5d), leading to O2-responsive Ply production. However, no O2-related differential transcription of the ply operon genes was found (Supplementary Fig. 7); therefore, we speculated that the higher sRNA plyT levels in anaerobic culture could be attributable to elevated unknown 3’ UTR processing ribonuclease levels or RNA-binding protein SPD_1558, since the two proteins respectively crop sRNA plyT from the ply operon and stabilize it. Additionally, epigenetic modifications of sRNA plyT at different O2 levels might influence its stability.
In conclusion, this study revealed that pneumococcus D39 uses an sRNA derived from the 3’ UTR of the ply operon to feedback-regulate the production of Ply, the major virulence factor, ensuring that the protein levels are appropriate for pneumococcal brain invasion. This novel regulatory strategy could be employed by pneumococcus.
Methods
Experimental strains and culture conditions
Bacterial strains used in this study were listed in Supplementary Table 1. Pneumococcal serotype 2 strain D39, serotype 4 stain TIGR4, serotype 14 strain 34359, and their derivates were grown in brain heart infusion (BHI, Difco, BD) broth or on BHI agar plate supplemented with 3% (v/v) defibrillated sheep blood at 37 °C with 5% CO2. For aerobic growth, pneumococcus was incubated statically as a standing culture; for anaerobic growth, pneumococcus was incubated in 100% N2. Escherichia coli strains were grown in Luria-Bertani (LB) medium at 37 °C under shaken at 200 rpm/min. For the transformants selection, spectinomycin (200 and 50 μg mL−1), kanamycin (250 and 50 μg mL−1), chloramphenicol (5 and 34 μg mL−1), gentamycin (50 and 40 μg mL−1) were used for the pneumococcus and E. coli, respectively, and streptomycin (150 μg mL−1) was used for the pneumococcus.
Construction of mutation strains
All primers used in this study are listed in Supplementary Table 1. sRNA plyT and other gene deletion strains were constructed using PCR ligation methods47. Briefly, the DNA fragments upstream and downstream of plyT and respective genes, ~1000 bp in length, were PCR amplified, respectively, using pairs of primers listed in Supplementary Table 1. Meanwhile, kanamycin or spectinomycin resistance gene cassette was amplified from pALH12448 or pDL27849. After purification, the three PCR products were fused by overlapping PCR and then transformed into S. pneumoniae D39, TIGR4, or 34359 strain as described previously50,51. The correct transformants were selected on BHI blood agar plates supplemented with kanamycin or spectinomycin, and verified by PCR amplification and sequencing.
For construction of plyT ectopically complementary strain, the promoter fragment of ply operon was PCR amplified, purified, and fused with PCR amplified plyT gene fragment by overlapping PCR to obtain Pply-plyT fragment. The purified Pply-plyT fragment was integrated into the shuttle plasmid pDL278 using Gibson assembly cloning kit (NEB, Beverly, MA). The constructed plasmid pDL278-Pply-plyT was transformed into ΔplyT strain, and verified by PCR amplification and sequencing.
Construction of GFP reporter and Histidine-tagged strains
The promoter fragment of ply operon and a 168 bp DNA fragment containing 27 bp of SPD_1729 3’end, 12 bp intergenic sequence, and a 129 bp 5’–terminal sequence of SPD_1728 (IGR) were respectively amplified from D39 genome DNA and fused by overlapping PCR. Meanwhile, DNA fragment of super-folding green fluorescence protein gene (sf-gfp) was amplified from plasmid pYES2-So-aqpA-gfp52, and then the three DNA fragments were fused by overlapping PCR to obtain Pply-IGR-gfp fragment. Additionally, DNA sequence of sRNA plyT was amplified from genome DNA, and fused with Pply-IGR-gfp by overlapping PCR to get Pply-IGR-gfp-plyT. Then DNA fragments Pply-IGR-gfp and Pply-IGR-gfp-plyT were integrated into the shuttle plasmid pDL278 using Gibson assembly cloning kit (NEB, Beverly, MA), then the plasmids pDL278-Pply-IGR-gfp and pDL278-Pply-IGR-gfp-plyT were transformed into D39 ΔplyT strain, and verified by PCR amplification and sequencing. To get the GFP reporters with mutated plyT sequence, wild-type plyT sequence on pDL278-Pply-IGR-gfp-plyT was substituted by the chemically synthesized mutated plyT sequence, in which nucleotides paired with IGR1729-1728 RNA were mutated, using Gibson assembly, and then the correct plasmid pDL278-Pply-IGR-gfp-plyTMut were transformed into D39 ΔplyT strain and verified by PCR amplification and sequencing.
For construction of SPD_1729::GFP fusion strain, a sweet Janus cassette53 was first used to replace SPD_1729 gene in D39S to construct D39SΔSPD_1729. Approximate 1000 bp length DNA sequence upstream and 3’-end 30 nucleotides downstream of SPD_1729 gene was respectively PCR amplified, meanwhile, SPD_1729 gene sequence without the 3’-end 30 nucleotides was fused with gfp gene by overlapping PCR, then the three PCR products were fused by overlapping PCR to get up-SPD_1729::gfp-dn DNA fragment. After purification, this fragment was transformed into D39SΔSPD_1729 strain. Correct SPD_1729::GFP strain was verified by PCR amplification, in which sweet Janus cassette was substituted by SPD_1729::gfp fusion. sRNA plyT was deleted in SPD_1729::GFP strain to obtain ΔplyT SPD_1729::GFP.
For construction of the SPD_1558-3XFlag-6XHistidine tagged strain (SPD_1558-6His), a DNA fragment (SPD_1558-6His) containing SPD_1558 gene and intergenic region between SPD_1558 and the upstream SPD_1559, at a length of ~540 bp, was PCR amplified using a pair of primers with a reverse primer carrying 3XFlag and 6 histidine encoding sequences immediately before the stop codon. As SPD_1558 is situated in a nine-gene operon, the promoter fragment of the operon was PCR amplified and fused with SPD_1558-6His fragment by overlapping PCR. Then, purified Poperon-SPD_1558-6His fragment was integrated into vector pPEPX150 using Gibson assembly. Correct plasmid pPEPX1-Poperon-SPD_1558-6His was linearized using NcoI and then transformed into the SPD_1558 deletion strain. The correct SPD_1558-6His strain was verified by PCR amplification and sequencing.
GFP fluorescence measurement
The GFP reporter cells were collected, twice washed, and resuspended in PBS. After exposing air for 30 min in the dark, 200 µL of cells were added into a 96-well plate, and GFP fluorescence intensities were measured using Synergy H4 hybrid microplate reader (BioTek) at excitation wavelength of 485 nm and emission of 528 nm. Meanwhile, the growth mass (optical density) of GFP reporter cells were measured using visible light spectrophotometer at 600 nm. The results were expressed as GFP fluorescence/OD600. The experiments were repeated for three times with three independent batches of cultures each time.
Northern blot
RNA samples were separated on 5% or 10% Urea-PAGE gel, transferred to positively CHGD nylon membranes (GE Healthcare, Piscataway, NJ) and cross-linked by using GS Gene Linker™ UV Chamber. The membranes were pre-hybridized in buffer (5 × SSC, 5 × Denhardt’s, 50% (v/v) deionized formamide, 0.5% (w/v) SDS and 200 µg mL−1 salmon sperm DNA) for 4 h, then were hybridized with biotin-labeled probes (Supplementary Table 1) at 42 °C for overnight, and detected by Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Waltham, MA).
5’ and 3’ RACE
Total RNA was purified on 10% Urea-PAGE gels using a ZR small-RNA PAGE Recovery Kit (Zymo Research, Irvine, CA, USA), and treated with or without RNA 5’ pyrophosphohydrolase (RppH). Then the 5’ and 3’ ends were ligated with 50 pmol of universal miRNA cloning linker 5′-CAGACUGGAUCCGUCCUC-App-3′ and 5′-AppCUGUAGGCACCAUCAAU-ddC-3′ (synthesized by Integrated DNA, Coralville, IA, USA), respectively, with the addition of 20 U of T4 RNA ligase (Ambion, Austin, TX, USA) in the presence of 40 U of RNasin-Plus. The reactions were incubated at 18 °C for 16 h, and the 5’- and 3’-linker-ligated RNAs were recovered by isopropanol precipitation, and then mixed with 2 pmol of random primer and 100 pmol of 3’ RACE RT primer (5′-ATTGATGGTGCCTACAG-3′; complementary to the universal miRNA cloning linker), respectively. Full-length cDNAs were synthesized by using 200 U SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The 5’ and 3’ end containing cDNA fragments were next amplified using nested PCR using primers listed in Supplementary Table 1. PCR products were recovered from a 2% (w/v) agarose gel, and then TA-cloned into pMD19-T (Takara, Dalian, China), followed by colony PCR and sequencing.
Hemolytic activities analysis
Pneumococcal strains were grown statically or anaerobically until OD600 reached ~0.7, and then the cells and spent cultures were collected. The cells were washed with PBS twice and resuspended in PBS containing 0.1% sodium deoxycholate, and lysed by sonication for 15 min. After centrifugation at 12,000 rpm for 5 min, the cell lysates were taken out and protein concentrations were determined using a Bicinchoninic Acid (BCA) protein assay kit (ThermoFisher Scientific, Waltham, MA, USA). Cell lysates with 40 µg of total protein and 15 µL of spent cultures were 2-fold serially diluted using PBS containing 10 mM Dithiothreitol (DTT) and 0.1% BSA. One-hundred microliter of diluted samples were added into the 96-well plate containing 100 µL 2% sheep red blood cells. The reactions were maintained at 37 °C for 30 min, then the plates were centrifuged at 3000 rpm for 10 min to collect the un-lysed red blood cells. The supernatants were removed to measure the optical density at 540 nm using visible light spectrophotometer. The red blood cells incubated with PBS or PBS containing 0.1% sodium deoxycholate were used as negative controls, and red blood cells incubated with PBS containing 1% triton X-100 were used as positive controls.
Western blot
Pneumococcal cells were lysed in RIPA lysis buffer (Beyotime Biotechnology) containing 1 mM PMSF by sonication and cell lysates were collected by centrifugation. Meanwhile, the spent cultures were concentrated using 10-kD ultrafiltration tube. Same amounts of cell lysates or concentrated spent cultures were separated by SDS-PAGE and hybridized with Anti-Pneumolysin antibody (PLY-4) (ab71810) (Abcam, Cambridge, England) at a 1:5000 dilution, anti-His-tag antibody (Abmart Company, Shanghai, China) at a 1:2000 dilution, Horseradish peroxidase (HRP)-conjugated Anti-GFP monoclonal antibody (Easybio, Beijing, China) at a 1:5000 dilution, or mice raised anti-SPD_1728, anti-SPD_1727, and anti-GpsA (SPD_1918) polyclonal antibody at a 1:1000 or 1:5000 dilution. HRP-conjugated anti-mice antibody (Easybio, Beijing, China) at a 1:2000 dilution was used as secondary antibody. Western blot signals were detected using the Amersham ECL Prime Western blot detection reagent (GE Healthcare). Intensities of target bands were quantified using Image J.
Protein overexpression and purification
A 609, 312, 954 and 1017-bp DNA fragments containing the entire SPD_1728, SPD_1558, SPD_1777, and gpsA (SPD_1918) coding region were PCR amplified with primers listed in Supplementary Table 1 and then was integrated into pET-28a (Novagen, Madison, WI, USA) by Gibson assembly (NEB, Beverly, MA, USA) to produce pET-28a-SPD_1728, -SPD_1558, -SPD_1777 and -gpsA. Verified constructs were transformed into E. coli BL21 (DE3) (Novagen). Positive transformants were grown at 37 °C to an OD600 of 0.6–0.8, and 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma-Aldrich, St Louis, MO, USA) was added to the cultures to induce protein expression and the cultures were further incubated at 22 °C overnight. Then cells were collected by centrifugation, resuspended in binding buffer (20 mM Tris-HCl, 500 mM NaCl, 20 mM imidazole, and 1 mM DTT, pH 7.4), and lysed by sonication for 45 min. The supernatants were filtered and applied to a Ni2+-charged chelating column (GE Healthcare, Piscataway, NJ, USA) previously equilibrated with binding buffer. Proteins were then eluted using elution buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, and 1 mM DTT, pH 7.4). The fractions with desired proteins were pooled and dialyzed against buffer containing 20 mM Tris-HCl, 100 mM NaCl, 1 mM DTT, and 1 mM EDTA three times. Then the proteins were further purified with an anion or cation exchange column and heparin column according to protocols of the commerce’s supply. Purified proteins were then stored in aliquots in 10% glycerol at −80 °C until use.
A 411-bp DNA fragment containing the entire SPD_1727 coding gene was PCR amplified. The purified PCR product was inserted into the plasmid pGEX-4T-1 via Gibson assembly (NEB, Beverly, MA), the produced pGEX-SPD_1727 was transformed into E. coli BL21 (DE3) (Novagen, Madison, WI). Correct transformants were grown at 37 °C to OD600 of 0.4–0.6, 0.1 mM IPTG (Sigma-Aldrich) was added to induce SPD_1727-GST expression at 22 °C for overnight. Then cells were collected by centrifugation and resuspended in binding buffer (phosphate buffer saline (PBS, 10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, and 2.7 mM KCl, pH 7.4)), and then lysed by sonication for 30 min. As SPD_1727-GST was expressed as inclusion body, the cell lysate was centrifuged at 8000 g for 30 min and the precipitates were collected. The inclusion body proteins were washed with washing solution (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl) and resuspended in a solution containing 8 M urea for overnight at 4 °C. Then 8 M urea was diluted with binding buffer (20 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 5 mM imidazole) to 1.5 M, and the purity of proteins was analyzed by electrophoresis on a 12% SDS-PAGE gel. Then purified proteins were stored in aliquots in 10% (v/v) glycerol at 80 °C until use.
In vitro transcription
In vitro transcription assays were performed using the MEGAshortscript T7 Transcriptional Kit (ThermoFisher Scientific) per the manufacturer’s protocol. DNA fragment of sRNA plyT was amplified from the genome DNA using pairs of primers, with the 5’ end primer containing a T7 RNA polymerase promoter sequence (Supplementary Table 1). PCR products were gel purified and used as templates. The reaction mixture (20 µL) contained 2 µL 10 × T7 reaction buffer, 2 µL T7 Enzyme Mix, 75 mM each of ATP, CTP, GTP, and UTP, 400 mM GMP, 40 U RNase inhibitor (Promega, Madison, WI, USA), and 2 µg of template. Transcription reactions were performed at 37 °C for 4 h. Template DNA and unincorporated nucleotides were removed using TURBO DNase (Promega) and G50 spin columns (Amersham), respectively. After denaturation at 70 °C for 10 min, the products were immediately put on ice to open transcript secondary structures. Transcript purity was examined by 10% Urea-PAGE gels that were stained with 3,8-Diamino-5-ethyl-6-phenylphenanthridinium Bromide (EB). Aliquots of sRNA plyT were 3’-biotin-labeled using an RNA 3’ End Biotinylation Kit (Pierce, Rockford, IL, USA), and biotin-labeled and -unlabeled plyT were preserved at −80 °C until use.
RNA electrophoretic mobility shift assays (rEMSA)
Biotin-labeled sRNA plyT was produced by in vitro transcription from a DNA template and 3’-biotin-labeled using an RNA 3’ End Biotinylation Kit (Pierce), whereas 3’-biotin-labeled IGR1729-1728 RNA was synthesized by Genescript (Nanjing, China). 0.5- or 4-nM biotin-labeled RNA and increasing amounts of purified SPD_1558 or SPD_1777 protein (500–2000 nM) were incubated in 20 µL binding mixture, which contained 20 mM Tris-HCl (pH 8.0), 100 mM KCl, 1 mM DTT, 1 mM MgCl2, 100 mM NaCl, 5% glycerol, and 80 U RNase inhibitor (Promega, Madison, WI, USA). The reactions were kept at 37 °C for 30 min and then loading onto a 6% nondenaturing polyacrylamide gel. After electrophoresis at room temperature, the free RNA or protein-RNA complex was transferred onto a nylon membrane and cross-linked by using GS Gene Linker™ UV Chamber (Bio-Rad Laboratories, Hercules, CA), and then detected by Chemiluminescent Nucleic Acid Detection Module Kit (Thermo Fisher Scientific, Waltham, MA).
RNA immunoprecipitation (RIP) assay
D39 wild-type and SPD_1558-6His strains were statically grown in BHI broth and the late-exponential cells were collected and washed with PBS three times. Then, the cells were resuspended in 500 µL RNase-free RIP lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton-100, 1 mM PMSF), and sonicated on ice for 5 min. After centrifugation at 12,000 rpm for 10 min, the cell lysates were pre-incubated with BeyoMag™ Mouse IgG Magnetic Beads (Beyotime, Shanghai, China) at room temperature for 3 h, and then incubated with 50 µL of BeyoMag™ Anti-His Magnetic Beads (Beyotime) at 4 ˚C for 12 h. After being washed 4 times with RNase-free 1X TBS (20 mM Tris-HCl (pH 7.4–7.6), 137 mM NaCl), the immunoprecipitated 6His-tagged SPD_1558 protein and bound RNA were eluted from the beads by boiling in nonreducing SDS sample buffer (50 mM Tris-HC (pH 7.5), 1% SDS, 10% glycerol). Then SPD_1558 protein and sRNA plyT in the elution were detected by Western blot and Northern blot experiments.
Animal experiments
BALB/c mice (SPF grade, female, 6- to 8-week-old) were purchased from Vital River Company (Beijing, China). Animal experiments were approved by the Biomedical Research Ethics Committee of Institute of Microbiology, Chinese Academy of Sciences. The protocol was approved by the Institutional Animal Care and Use Committee. The mice were infected by S. pneumoniae D39 and derivative strains at 5 × 106 CFUs in 100 μL PBS via intraperitoneal injection, PBS-administrated BALB/c mice were included as controls. Survivals (8–10 mice per group) were monitored for 7 days. Blood from the orbit and brain tissues were collected from the dying mice under anaesthetization with 250 μL 20 mg mL−1 Tribromoethanol via intraperitoneal injection, and CFU numbers of the pneumococcus were counted in these samples. In addition, S. pneumoniae D39 and derivative strains at 5 × 107 CFUs in 40 μL PBS were used to infect BALB/c mice (10 mice per group) via intranasal administration under anaesthetization with 250 μL 20 mg mL−1 Tribromoethanol. PBS-administrated BALB/c mice were included as controls. Survivals of mice were monitored for 7 days.
Statistics and reproducibility
One-way ANOVA followed by Tukey’s post hoc test and Student’s t test were performed using PASW Statistics 18 or Excel, respectively. Log-rank Mantel-Cox test was performed using GraphPad Prism 8.0. The level of significance was determined at P < 0.05.
All the experiments were repeated for three times; For growth profiles, GFP fluorescence measurement, and qRT-PCR experiments, three independent batches of culture were included in each experiment. No data were excluded from the analyses.
Data availability
All data that support the findings of this study are available in the article, Supplementary information and Supplementary Data files. The uncropped gel images are provided in Supplementary Figs. 10–15. The source data underlying the graphs in this paper are provided in Supplementary Data 2. All other data are available from the corresponding author upon reasonable request.
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Acknowledgements
This study was supported by the National Natural Science Foundation of China, grant No. 31970035. We thank Associate professor Jinghua Yang in Institute of Microbiology, Chinese Academy of Sciences for bestowal of serotype 14 strain 34359.
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K.S., W.M., and L.Z. contributed to the investigation, methodology, data collection, and formal analysis; Q.H. contributed to the investigation and methodology; F.R. contributed to the methodology and writing-original draft; X.D. and H.T. contributed to the conceptualization, methodology, formal analysis, project administration, and writing-original draft and revisions.
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Shen, K., Miao, W., Zhu, L. et al. A 3’UTR-derived small RNA represses pneumolysin synthesis and facilitates pneumococcal brain invasion. Commun Biol 7, 1130 (2024). https://doi.org/10.1038/s42003-024-06845-8
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DOI: https://doi.org/10.1038/s42003-024-06845-8
