Time-resolved RNA-seq analysis to unravel the in vivo competence induction by Streptococcus pneumoniae during pneumonia-derived sepsis

Abstract

Competence development in Streptococcus pneumoniae (pneumococcus) is tightly intertwined with virulence. In addition to genes encoding genetic transformation machinery, the competence regulon also regulates the expression of allolytic factors, bacteriocins, and cytotoxins. Pneumococcal competence system has been extensively interrogated in vitro where the short transient competent state upregulates the expression of three distinct phases of "early," "late," and "delayed" genes. Recently, we have demonstrated that the pneumococcal competent state develops naturally in mouse models of pneumonia-derived sepsis. To unravel the underlying adaptive mechanisms driving the development of the competent state, we conducted a time-resolved transcriptomic analysis guided by the spatiotemporal live in vivo imaging system of competence induction during pneumonia-derived sepsis. Mouse lungs infected by the serotype 2 strain D39 expressing a competent state-specific reporter gene (D39-ssbB-luc) were subjected to RNA sequencing guided by monitoring the competence development at 0, 12, 24, and, at the moribund state, >40 hours post-infection (hpi). Transcriptomic analysis revealed that the competence-specific gene expression patterns in vivo were distinct from those under in vitro conditions. There was significant upregulation of early, late, and some delayed phase competence-specific genes as early as 12 hpi, suggesting that the pneumococcal competence regulon is important for adaptation to the lung environment. Additionally, members of the histidine triad (pht) gene family were sharply upregulated at 12 hpi followed by a steep decline throughout the rest of the infection cycle, suggesting that Pht proteins participate in the early adaptation to the lung environment. Further analysis revealed that Pht proteins execute a metal ion-dependent regulatory role in competence induction.IMPORTANCEThe induction of pneumococcal competence for genetic transformation has been extensively studied in vitro but poorly understood during lung infection. We utilized a combination of live imaging and RNA sequencing to monitor the development of a competent state during acute pneumonia. Upregulation of competence-specific genes was observed as early as 12 hour post-infection, suggesting that the pneumococcal competence regulon plays an important role in adapting pneumococcus to the stressful lung environment. Among others, we report novel finding that the pneumococcal histidine triad (pht) family of genes participates in the adaptation to the lung environment and regulates pneumococcal competence induction.

Keywords: Pht histidine triad proteins; RNA-seq; Streptococcus pneumoniae; competent state; host adaptation; pneumonia-derived sepsis.

Fig 1

RNA-seq captures full repertoire of pneumococcal transcripts within murine lungs. (A) Schematic workflow of time-resolved RNA-seq comprising of T1 = 0 hpi, T2 = 12 hpi, T3 = 24 hpi, and T4 = >40 hpi in CD-1 mouse lungs (n = 5) intranasally infected with 5 × 10⁷ CFU of the D39-ssbB-luc. Before harvesting lungs for total RNA isolation, each cohort was imaged with IVIS SpectrumCT to examine spatiotemporal induction of the competent state. (B) Representative images captured by the IVIS SpectrumCT on the competence induction in mice infected by D39-ssbB-luc. (C) RNA read counts of D39-ssbB-luc across four time points. (D) PCA plot displays unique variance across the pneumococcal data sets demonstrating disease progression-specific clustering of the top 500 most variable pneumococcal genes. (E) Venn diagram displaying the number of genes expressed across the time points, organized into unique expressions and overlapping expressions.

Fig 2

Heatmaps of differentially expressed pneumococcal genes from infected mouse lungs. (A) Each heatmap showcases up to 50 genes above the P_adj cutoff value that have undergone upregulation between the indicated time points based on the fold change. Scale bar (bottom right) indicates the gene expression level based on the log₂ fold-change value between each time points. The superscripts indicate early, late, or delayed competence-specific genes (a: early, b: late, and c: delayed). The asterisk (*) indicates the unannotated locus tags. Blank annotations are shown in Table S3. All of the genes represented in the heatmaps are listed in Tables S5 to S7. (B) Volcano plots showing differentially expressed pneumococcal genes above the cutoff value set by the log₂ fold change (x-axis) and the log₁₀ adjusted P value (y-axis), indicated by color codes where blue and red dots represent downregulated and upregulated genes, respectively.

Fig 3

Functional annotation of differentially expressed pneumococcal genes in mouse lung across infection timespan when compared to initial basal expression. Each point symbol represents the ratio of genes pertaining to the corresponding gene ontology term to total number of upregulated genes. The color of symbols reflects the adjusted significance (P_adj) by the Benjamini-Hochberg method, as illustrated in the scale. The size of the symbol reflects the number of genes represented, as indicated by the count scale. Detailed list of genes represented by the symbols is provided in Table S4.

Fig 4

qRT-PCR validation of the expression of representative competence-specific genes revealed by RNA-seq. The same RNA samples isolated from the D39-ssbB-luc-infected lungs were used for qRT-PCR. Log₂ fold-change values reflecting the differential gene expressions as validated by qRT-PCR. Ten competence-specific genes were analyzed, resulting in 30 plotted samples. A high degree of correlation was observed with the associated P value of <0.0001 (R² = 0.727, Pearson). X-axis: log₂ fold change (RNA-seq). Y-axis: log₂ fold change (qPCR).

Fig 5

Changes in expression of selected genes across timespan in infected lungs shown in linear scale. (A) Seven representative pneumococcal genes encoding 6-phospho-β-glucosidases (bglA, bglA2, celA, and bguA) and 6-phospho-galactosidases (lacG1 and lacG2). RegR regulates lacG1 and LacG2. (B–D) Early, late, and delayed competence-specific signature genes. (E) Competence-regulated genes of the blp locus encoding bacteriocin-inducing peptides and immunity proteins of the BIR. (F) The phtABDE and adcA and adcAII genes involved in zinc acquisition. The numerical values of fold change and corresponding standard error values are provided in Table S8.

Fig 6

Loss of pht genes leads to metal-dependent dysregulated competence phenotypes in vitro. The firefly luc reporter gene was genetically fused to competence-specific ssbB in a hypercompetent pneumococcal strain CP1250 to monitor spontaneous competence induction in pht mutants. (A) In a metal-rich (+Cu²⁺, +Fe²⁺, and +Zn²⁺) C + Y_A (pH = 7.8), ∆phtA, ∆phtB, ∆phtD, and ∆phtE show varying degrees of expedited onset of the competent state as well as higher levels of competence. (B) In C + Y_A (pH = 7.8), ∆phtABDE enters competence state earlier with higher degree of competence. (C) In metal-deficient (−Cu²⁺, −Fe²⁺, and −Zn²⁺) C + Y_B, CP1250-ssbB-luc fails to enter competent state naturally unless provided with 432 µg L⁻¹ of CuSO₄, FeSO₄•7H₂O, ZnSO₄•7H₂O, or any combination of the divalent metals. (D) In C + Y_B, CP1250-ΔphtABDE-ssbB-luc fails to enter competent state naturally even with supplementation of 432 µg L⁻¹ of CuSO₄, FeSO₄•7H₂O, ZnSO₄•7H₂O, or any combinations of the divalent metals. Typical results from one of the three independent experiments are shown. (E) In metal-sufficient C + Y_A, ΔphtABDE mutant derived from the pneumococcal strain D39 displays increased rate of genetic transformation frequency. P = 0.0001 by one-way analysis of variance test. (F) The qRT-PCR validation of the RNA-seq expression of the pht genes from the infected mouse lungs with the P value of 0.0004 (R² = 0.7301, Pearson). X-axis: log₂ fold change (RNA-seq). Y-axis: log₂ fold change (qPCR).

Fig 7

Pht proteins regulate entry into spontaneous competent state during pneumonia-derived sepsis. (A and B) CD-1 mice (7 weeks old, 10/group) were intranasally inoculated with 5 × 10⁷ CFU of D39-ssbB-luc (A) or D39-ΔphtABDE-ssbB-luc (B), and competence induction was monitored for 72 hours with IVIS imaging by detecting the bioluminescent signals. (C and D) Stacked bar graphs were constructed to indicate numbers of mice displaying either non-competent pneumococci (gray), competent pneumococci (red), or euthanized (black) status at each time point. (E) Average radiant efficiency of each group was quantified at each time point to determine the level of competence induction with threshold set to 20%.