Identifying genetic determinants of Streptococcus pyogenes-host interactions in a murine intact skin infection model

Abstract

Streptococcus pyogenes is an obligate human pathobiont associated with many disease states. Here, we present a model of S. pyogenes infection using intact murine epithelium. We were able to perform RNA sequencing to evaluate genetic changes undertaken by both the bacterium and host at 5 and 24 h post-infection. Analysis of these genomic data demonstrate that S. pyogenes undergoes genetic adaptation to successfully infect the murine epithelium, including changes to metabolism and activation of the Rgg2/Rgg3 quorum-sensing (QS) system. Subsequent experiments demonstrate that an intact Rgg2/Rgg3 QS cascade is necessary to establish a stable superficial skin infection. QS cascade activation results in increased murine morbidity and bacterial burden on the skin. This phenotype is associated with gross changes to the murine skin and with evidence of inflammation. These experiments offer a method to investigate S. pyogenes-epithelial interactions and demonstrate that a well-studied QS pathway is critical to a persistent infection.

Keywords: CP: Microbiology; NZ131; RNA-seq; Rgg2/Rgg3; Streptococcus pyogenes; animal models; host response; quorum sensing; skin infection; transcriptome.

Figure 1.. Experimental outline

(A) All experiments begin with depilation on day −1 and inoculation on day 0. The upper limb demonstrates the approach used for RNA-seq experiments. The lower limb demonstrates the approach used for longitudinal experiments. (B) CFU per mouse at 5 and 24 h, at time of RNA-seq sample collection. Each group represents 5 mice, pooled for RNA-seq to generate 3 biological replicates. Black bar represents mean of all 15 mice for a given time point.

Figure 2.. S. pyogenes RNA-seq analysis

(A) PCA of NZ131 grown in THY broth (black) and after 5 h (gold) and 24 h (pink) of murine skin infection. (B and C) Volcano plots demonstrating the distribution of differentially regulated genes at 5 and 24 h post-skin infection. Genes that are part of the Rgg2/Rgg3 QS pathway are labeled with locus names. (D) Venn diagram demonstrating differentially regulated genes at 5 versus 24 h post-inoculation onto murine skin. (E and F) Heatmap of the top differentially expressed S. pyogenes genes, which are normalized counts transformed with Z score method. Each column represents a biologically unique sample, with input controls on the left and 5- or 24-h samples on the right. (G) KEGG pathway analysis of NZ131 gene expression at 5 and 24 h post-inoculation versus input control of the top 45 regulated pathways based on log₂ fold change (FC) >|2|.

Figure 3.. Rgg2/Rgg3 QS pathway is activated during murine skin infection

(A) Schematic demonstrating the Rgg2/Rgg3 QS pathway and associated operons. Activation of the pathway leads to de-repression of the promoters P_shp2 and P_shp3 by displacement of Rgg3 (red) by Rgg2 (green) upon Shp peptide binding. Pathway activation leads to significant upregulation of both of the Shp peptides (dark blue), perpetuating a positive feedback loop. There is also upregulation of associated downstream genes (light blue). Of note, rgg2 was not differentially regulated in this dataset, though rgg3 was found to be repressed 5-fold in mice as compared to broth culture. Mutations used in longitudinal infection experiments are highlighted below the operon. (B and C) Representative IVIS images at 1 and 24 h post-inoculation. Mice were inoculated with NZ131 with integrated P_shp3-luxABCDE transcriptional reporter and monitored longitudinally via IVIS in vivo imaging. (D) Rgg2/3 QS pathway is significantly induced during murine skin infection. Each data point represents the average radiance of 5 mice as technical replicates. 3 individual bacterial preparations were used to generate 3 biological replicates. Error bars represent standard deviation of 3 biological replicates. Radiance reported as [p/s/cm²/sr]. *p < 0.05.

Figure 4.. Mouse transcriptional response at 5 or 24 h post-S. pyogenes NZ131 skin infection

(A) PCA of mouse skin infection. (B) Differential gene expression with significant genes (red, log₂FC < −2 and FDR < 0.05) associated with 5- or 24-h infection (blue, log₂FC > 2 and FDR < 0.05). (C) Heatmap of the top differentially expressed mouse genes, which are normalized counts transformed with Z score method. (D) Gene set enrichment analysis (GSEA) was performed on the FC of 5- and 24-h skin infections. Blue indicates gene sets enriched at 5-h skin infections, while red indicates gene sets enriched at 24-h infections. Bold text indicates significance.

Figure 5.. Rgg2/Rgg3 QS increases bacterial burden and murine morbidity

(A) Mouse weight relative to initial weight. Mice were weighed at indicated time points, with the percentage of weight loss calculated versus initial. For clarity, statistical comparisons to the PBS control group are omitted from the figure. Dashed line indicated 75% threshold for morbidity. (B) CFU per mouse at indicated time points. Dashed line indicates minimum threshold of detection. PBS control mice are excluded for clarity from (B), as no bacterial burden was detected throughout the experiment. 5 mice were included in the PBS control group. (C) Representative photographs of murine skin infection across time. All mice infected with S. pyogenes NZ131 and isogenic mutants demonstrated superficial erythema on day 1 of the experiment. Over time, mice infected with NZ131 capable of active QS demonstrated ongoing skin breakdown, with serous discharge apparent. In comparison, mice infected with NZ131 incapable of QS initially demonstrated limited skin breakdown (day 5) but with marked resolution by day 10. Additional photographs from these experiments are included in Figure S1. All photos have been cropped for clarity. 15 mice were included in each of the experimental groups. For both figures, errors bars indicate mean with standard error of measurement. *p < 0.05, **p < 0.005, ***p < 0.0005, ****p < 0.0001.

Figure 6.. Representative microscopic images from skin biopsies taken on day 10 of longitudinal infection experiments

Samples were fixed in paraformaldehyde prior to sectioning and staining as indicated. Mice infected with NZ131 capable of QS activation demonstrate ulceration of the epidermis and inflammatory infiltrate with both neutrophil (MPO staining) and macrophage (F4:80) staining. Samples from mice infected with QS-null NZ131 show mild acanthosis of the epidermis but limited inflammatory infiltrate and an intake epithelium. Large images taken at 4x magnification, scale bars: 200 μm. Boxes indicate source of inset images. Inset images taken at 40× magnification, scale bars: 50 μm. Images were adjusted in ImageJ with “automatic” window and level correction for clarity.