Functional and Proteomic Analysis of Streptococcus pyogenes Virulence Upon Loss of Its Native Cas9 Nuclease

Abstract

The public health impact of Streptococcus pyogenes (group A Streptococcus, GAS) as a top 10 cause of infection-related mortality in humans contrasts with its benefit to biotechnology as the main natural source of Cas9 nuclease, the key component of the revolutionary CRISPR-Cas9 gene editing platform. Despite widespread knowledge acquired in the last decade on the molecular mechanisms by which GAS Cas9 achieves precise DNA targeting, the functions of Cas9 in the biology and pathogenesis of its native organism remain unknown. In this study, we generated an isogenic serotype M1 GAS mutant deficient in Cas9 protein and compared its behavior and phenotypes to the wild-type parent strain. Absence of Cas9 was linked to reduced GAS epithelial cell adherence, reduced growth in human whole blood ex vivo, and attenuation of virulence in a murine necrotizing skin infection model. Virulence defects of the GAS Δcas9 strain were explored through quantitative proteomic analysis, revealing a significant reduction in the abundance of key GAS virulence determinants. Similarly, deletion of cas9 affected the expression of several known virulence regulatory proteins, indicating that Cas9 impacts the global architecture of GAS gene regulation.

Keywords: CRISPR-Cas; Cas9; Streptococcus pyogenes; bacterial virulence; group A Streptococcus; pathogenesis; proteomics; regulation.

FIGURE 1

Deletion of the cas9 gene does not affect GAS growth and morphology. (A) Schematic of the genomic organization of type II-A CRISPR-Cas loci in GAS 5448 wild type (top) and Δcas9 (bottom) strains. The cas genes are represented in light gray with cas9 highlighted in black. The tracrRNA is shown in dark gray. Substitution of cas9 by the cat gene in the Δcas9 strain is represented in white. The CRISPR array of GAS 5448 is constituted by the leader sequence (dark gray bar), four repeats (black diamonds) and three spacers (squares). (B,C) Quantification of cas9, cas1, cas2, and csn2 mRNA transcripts by RT-qPCR (B) and expression of Cas9 protein by western blot (C) from wild type (WT) and cas9 mutant (Δcas9) GAS strains grown to mid-exponential (Exp) or stationary (Stat) cell growth phases. (D) Cellular growth curves of WT Δcas9 GAS strains grown at 37°C in THB media. (E) Microscopic visualization (top panels) and colonies morphology (bottom panels) of WT and Δcas9 GAS strains grown to stationary growth phase in THB media or on THA agar plates, respectively. Scale bar (10 μm) is indicated. For each experiment, samples were assayed at least in triplicate. Data in (B) and (D) are plotted as the mean ± SEM and are pooled and representative of three independent experiments, respectively. Data in (B) was analyzed by two-way ANOVA multiple comparisons. N.S, not significant (p > 0.05); ^****P < 0.0001.

FIGURE 2

Cas9-deficiency is associated with RAST functional enrichment and differential abundance of GAS virulence determinants and regulators. (A) Comparison of significant differential protein abundance at the RAST subcategory level (Log2 normalized abundance ratio) between wild type (WT) and isogenic cas9 mutant (Δcas9) GAS strains. Significant enrichment in protein abundance in the wild-type (up in WT) or in the cas9-deficien mutant strain (up in Δcas9) is indicated (top panel), with a matching color code of the corresponding RAST subcategory (bottom panel). (B) Statistical representation of the differential abundance of proteins identified in the WT or Δcas9 GAS strains and grouped under “virulence, disease, and defense” (blue bar) and “adhesion” (red bar) RAST subcategories. Color pattern of the normalized protein abundance is indicated (C,D) Comparison of normalized abundance of transcriptional repressors (C) and activators (D) of GAS virulence identified by proteomics in the WT and Δcas9 GAS strains. Proteins annotated with asterisks indicate that were manually added (curated) into the relevant RAST subcategory based on published evidence. All differential proteins listed in (B,D) were significant by t-test (p < 0.05).

FIGURE 3

Lack of Cas9 is impaired with significant changes in key virulence factors and pathogenic functionalities of GAS. (A–F) GAS WT and Δcas9 strains were assessed for (A) Capsule expression by ELISA, (B) Quantification of M protein-anchored to the cell wall by flow cytometry, (C) SpeB protease activity by azocasein assay, (D) Capacity of adhesion to HaCaT human skin keratinocytes, (E) β-hemolysis on blood-agar media, and (F) Efficiency on human red-blood cell lysis. Isogenic GAS mutant strains in capsule (hasA), M protein (Δemm) SpeB (ΔspeB), and SLO (Δslo) were used as negative controls in (A–C,F), respectively. For each experiment, samples were assayed at least in triplicate. Data in (A–D,F) are plotted as the mean ± SEM, pooled from three independent experiments and analyzed by Student’s t test. N.S, non-significant (p > 0.05); ^∗P < 0.05; ^∗∗P < 0.01; ^****P < 0.001.

FIGURE 4

Loss of Cas9 attenuates GAS virulence. (A) Comparison of the ability of GAS WT and Δcas9 strains for growth on human whole blood. Growth index in blood was calculated as the ratio of recovered CFUs after incubation over the initial inoculum. (B,C) Subcutaneous infection of C57BL/6 mice with GAS WT and Δcas9 strains. (B) Representative images (left panel) of lesions triggered by GAS WT (top) or Δcas9 (bottom) strains, and average lesion sizes (right panel). (C) Enumeration of CFUs recovered from excised lesions 48 h post-infection. Data are plotted as the mean ± SEM and are pooled from three (A) and two (B,C) independent experiments and analyzed by Student’s t test. ^∗P < 0.05; ^****P < 0.001.

FIGURE 5

Schematic representation of the network of GAS regulatory proteins and virulence factors affected upon loss of Cas9. Cas9 may directly (gray arrows) activate expression of several GAS virulence determinants (highlighted in pink) through an unknown molecular mechanism (?). Cas9 also upregulates (solid red arrows) some transcriptional activators of virulence (highlighted in teal), further augmenting the expression of numerous virulence factors (black arrows). Conversely, Cas9 negatively controls (dashed red lines) the expression of several transcriptional repressors of virulence (highlighted in orange). Consequently, Cas9 blocks the repressor role (dashed black lines) of these regulators on the expression of key GAS virulence factors. Virulence determinants downstream of transcription factors experimentally confirmed in this study are highlighted in gray. Other virulence factors or functions known from previous studies to be regulated by the highlighted repressors and activators of GAS virulence are depicted with black text only. Carb. Metabolism, carbohydrates metabolism, A.A. metabolism, amino acids metabolism.