Streptococcus pyogenes genes that promote pharyngitis in primates

Abstract

Streptococcus pyogenes (group A streptococcus; GAS) causes 600 million cases of pharyngitis annually worldwide. There is no licensed human GAS vaccine despite a century of research. Although the human oropharynx is the primary site of GAS infection, the pathogenic genes and molecular processes used to colonize, cause disease, and persist in the upper respiratory tract are poorly understood. Using dense transposon mutant libraries made with serotype M1 and M28 GAS strains and transposon-directed insertion sequencing, we performed genome-wide screens in the nonhuman primate (NHP) oropharynx. We identified many potentially novel GAS fitness genes, including a common set of 115 genes that contribute to fitness in both genetically distinct GAS strains during experimental NHP pharyngitis. Targeted deletion of 4 identified fitness genes/operons confirmed that our newly identified targets are critical for GAS virulence during experimental pharyngitis. Our screens discovered many surface-exposed or secreted proteins - substrates for vaccine research - that potentially contribute to GAS pharyngitis, including lipoprotein HitA. Pooled human immune globulin reacted with purified HitA, suggesting that humans produce antibodies against this lipoprotein. Our findings provide new information about GAS fitness in the upper respiratory tract that may assist in translational research, including developing novel vaccines.

Keywords: Bacterial infections; Infectious disease.

Figure 1. TraDIS analysis of GAS genes contributing to primate URT infection.

(A) Comparison of M1 and M28 GAS genes contributing to fitness in the NHP URT. (B) Comparison of GAS genes required in the NHP URT and those important for NHP necrotizing fasciitis (NF). Representative fitness genes assigned to each category are shown. Genes selected for further validation and investigation are highlighted in red.

Figure 2. Validation of the contribution of Spy0014, hitABC, irr-ihk, and shp to GAS fitness in the primate URT.

(A) Schematic showing the gene neighborhood of the studied loci, with the deleted regions of each isogenic mutant strain noted. Also shown are transposon insertion sites of each gene determined by TraDIS. Red vertical spikes are forward reads; blue vertical spikes are reverse reads. Read orientations indicate the direction of the transposon insertion. (B) Growth of the isogenic gene deletion mutant strains in rich medium THY. (C and D) Significantly decreased ability of the isogenic mutant strains to colonize the NHP URT, compared with WT parental strain MGAS2221. P values for CFU recovery were determined across the 14-day experiment by 2-way ANOVA with correction for multiple comparisons by the method of Benjamini, Krieger, and Yekutiel. Data are presented as the mean ± standard error of the mean of 4 experiments.

Figure 3. Growth of GAS strains in human body fluids.

Growth of the WT parental strain MGAS2221 and the isogenic gene deletion mutant strains in human saliva (A), human serum (B), and human whole blood (C). *P < 0.05 determined by 1-way ANOVA. Data are presented as the mean ± standard deviation of the mean of 4 experiments.

Figure 4. Spy0014 contributes to maturation of GAS cysteine protease SpeB.

(A) SpeB protease activity of the isogenic Spy0014 deletion mutant strain and the complemented mutant strain (C-Spy0014). (B) Western immunoblot showing the extent of SpeB maturation in the WT parental strain, the Spy0014 deletion strain, and the complemented mutant strain. SpeB-Z, immature zymogen. SpeB-M, mature protease. (C) Contribution of amino acid transporters to GAS SpeB protease activity. *P < 0.05 determined by 1-way ANOVA. Data are presented as the mean ± standard deviation of the mean of 3 experiments.

Figure 5. Irr-Ihk two-component system protects against killing by adherent human PMNs ex vivo.

Killing of WT and irr-ihk–knockout GAS strains by human PMNs in suspension (A) or adherent cells (B). *P ≤ 0.02 using a paired 2-tailed t test (Prism 7 for Windows, GraphPad Software, Inc); ns, not significant. Data are presented as the mean ± standard error of the mean of 4 (A) or 6 (B) separate experiments as indicated.

Figure 6. Distribution of hitABC transporter genes in streptococcal species and immunoreactivity of recombinant HitA.

(A) Distribution of the hitABC transporter genes and the adjacent irr-ihk two-component regulatory genes in other streptococcal species (Streptococcus dysgalactiae subspecies equisimilis [SDSE] strain GGS124, Streptococcus sanguinis strain SK36, Streptococcus cristatus strain NCTC12479, and Streptococcus gordonii strain NCTC7868). Percentages refer to amino acid identities compared with proteins made by serotype M1 GAS reference strain MGAS2221. (B) SDS-PAGE gel showing the purified recombinant GAS lipoproteins HitA and FhuD. BSA was added as a control. (C) Immunoreactivity of purified HitA and FhuD with pooled human immune globulin.