Loss of rpoE Encoding the δ-Factor of RNA Polymerase Impacts Pathophysiology of the Streptococcus pyogenes M1T1 Strain 5448

Abstract

Streptococcus pyogenes, also known as the Group A Streptococcus (GAS), is a Gram-positive bacterial pathogen of major clinical significance. Despite remaining relatively susceptible to conventional antimicrobial therapeutics, GAS still causes millions of infections and hundreds of thousands of deaths each year worldwide. Thus, a need for prophylactic and therapeutic interventions for GAS is in great demand. In this study, we investigated the importance of the gene encoding the delta (δ) subunit of the GAS RNA polymerase, rpoE, for its impact on virulence during skin and soft-tissue infection. A defined 5448 mutant with an insertionally-inactivated rpoE gene was defective for survival in whole human blood and was attenuated for both disseminated lethality and lesion size upon mono-culture infection in mouse soft tissue. Furthermore, the mutant had reduced competitive fitness when co-infected with wild type (WT) 5448 in the mouse model. We were unable to attribute this attenuation to any observable growth defect, although colony size and the ability to grow at higher temperatures were both affected when grown with nutrient-rich THY media. RNA-seq of GAS grown in THY to late log phase found that mutation of rpoE significantly impacted (>2-fold) the expression of 429 total genes (205 upregulated, 224 downregulated), including multiple virulence and “housekeeping” genes. The arc operon encoding the arginine deiminase (ADI) pathway was the most upregulated in the rpoE mutant and this could be confirmed phenotypically. Taken together, these findings demonstrate that the delta (δ) subunit of RNA polymerase is vital in GAS gene expression and virulence.

Keywords: GAS; RNA-seq; Streptococcus pyogenes; arcA; rpoE; virulence.

Figure 1

Mutation of rpoE in 5448 results in attenuation and loss of fitness in a murine soft-tissue infection model. (A) Disseminated systemic lethality was monitored following subcutaneous infection with wild type (WT, n = 5) 5448 or the rpoE mutant (rpoE, n = 9) at the CFU doses indicated. The p value indicates statistical significance in lethality between the rpoE mutant and WT. (B) A separate experiment was done to measure ulcerative lesion size following subcutaneous infection after 48 h post-inoculation (pi). (C) Competition assays of a Spy_1849 mutant, a covS mutant (covS) and a rpoE mutant each co-infected with an equivalent inoculum of WT 5448 in the mouse model. The control data for Spy_1849 and covS were published [28] whereas the rpoE mutant was assayed in the same experiment but not previously published. CFU was harvested 48 h p.i. and quantified by plate dilution. Asterisk indicates statistical significance (p ≤ 0.05) compared to WT (B) and each strain (C) using an unpaired t-test.

Figure 2

Mutation of rpoE in M1T1 5448 reduces survival in human blood. A Lancefield bactericidal assay in non-immune whole human blood was performed using WT 5448 (WT) and a rpoE mutant (rpoE). Asterisk indicates statistical significance (p ≤ 0.05) by comparison to the WT as determined by an unpaired t-test.

Figure 3

The rpoE mutant alters S. pyogenes colony morphology without impacting growth. (A,B) Serial dilutions were performed from overnight cultures of the wild type (WT), rpoE mutant (rpoE), or the complemented rpoE mutant strain (rpoE^C), and dilutions spotted on tryptic soy agar with 5% sheep’s blood (A) or Todd Hewitt Yeast agar (B) and incubated at 37 °C with 5% CO₂ overnight. (C) Cultures were grown to exponential growth and then turbidity was measured over time at an optical density of 600 nm (OD₆₀₀) after tube inversion to assess aggregation. (D,E) Bacteria were grown from a starting OD₆₀₀ = 0.05 either in nutrient-rich THY broth (D) or nutrient-limiting modified RPMI (E) for 24 h and OD₆₀₀ was recorded over time. Asterisk indicates statistical significance (p ≤ 0.05) of the rpoE mutant by comparison to the WT as determined by an unpaired t-test. NS = not significant.

Figure 4

Mutation of rpoE limits the growth potential for GAS at increasing temperatures. Overnight cultures of WT 5448 (WT), rpoE mutant (rpoE), or the complemented strain (rpoE^C) were diluted to a starting OD₆₀₀ = 0.05 in fresh THY media; 120 µL of culture was then aliquoted into sterile PCR tubes and incubated in a thermocycler at increasing temperature gradient for 24 h and growth measured by absorbance (OD₆₀₀). This experiment was performed in six biological replicates. Asterisks indicate statistical significance (p ≤ 0.05) at indicated growth temperatures of the rpoE mutant by comparison to the WT as determined by an unpaired t-test. NS = not significant.

Figure 5

RNA-seq analysis of the rpoE mutant compared to WT 5448 in THY medium. Transcript levels were measured by RNA-seq for the rpoE mutant relative to the WT 5448 and ontology enrichments were analyzed. Genes that were upregulated (A) and those that were downregulated (B) were plotted based on biological purpose (BP) and in order of confidence based on their predicted BP using goseq software, with those groups most overrepresented being shown in red. (C) Volcano plot of RNA-seq DESeq2 analysis used to determine genes that were differentially expressed (log2) and p-value (log10). All genes greater than two-fold differential expression in the rpoE mutant vs. the WT are shown outside of the vertical black bars. Red symbols in the volcano plot represent genes that show a differential expression with a p-value < 0.05 whereas blue indicates those above that level.

Figure 6

Mutation of rpoE enhances the rate of the deacidification of the extracellular media. Wild type (WT), the rpoE mutant (rpoE), its complemented variant (rpoE^C), or an arcA mutant (arcA) cultures were standardized to a starting OD₆₀₀ = 0.05 and were grown in THY supplemented with phenol red (18 µg mL⁻¹) and supplemental arginine (30 mM). Time points were established based on preliminary studies. Color changes (top) and absorbance changes (bottom) in media pH over time are shown above. The absorbance of spent media was measured at 560 nm (Abs₅₆₀) at the indicated time points. Each experiment is representative of three biological replicates.