Novel Genes Required for the Fitness of Streptococcus pyogenes in Human Saliva

Abstract

Streptococcus pyogenes (group A streptococcus [GAS]) causes 600 million cases of pharyngitis each year. Despite this considerable disease burden, the molecular mechanisms used by GAS to infect, cause clinical pharyngitis, and persist in the human oropharynx are poorly understood. Saliva is ubiquitous in the human oropharynx and is the first material GAS encounters in the upper respiratory tract. Thus, a fuller understanding of how GAS survives and proliferates in saliva may provide valuable insights into the molecular mechanisms at work in the human oropharynx. We generated a highly saturated transposon insertion mutant library in serotype M1 strain MGAS2221, a strain genetically representative of a pandemic clone that arose in the 1980s and spread globally. The transposon mutant library was exposed to human saliva to screen for GAS genes required for wild-type fitness in this clinically relevant fluid. Using transposon-directed insertion site sequencing (TraDIS), we identified 92 genes required for GAS fitness in saliva. The more prevalent categories represented were genes involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. Using six isogenic mutant strains, we confirmed that each of the mutants was significantly impaired for growth or persistence in human saliva ex vivo. Mutants with an inactivated Spy0644 (sptA) or Spy0646 (sptC) gene had especially severe persistence defects. This study is the first to use of TraDIS to study bacterial fitness in human saliva. The new information we obtained will be valuable for future translational maneuvers designed to prevent or treat human GAS infections. IMPORTANCE The human bacterial pathogen Streptococcus pyogenes (group A streptococcus [GAS]) causes more than 600 million cases of pharyngitis annually worldwide, 15 million of which occur in the United States. The human oropharynx is the primary anatomic site for GAS colonization and infection, and saliva is the first material encountered. Using a genome-wide transposon mutant screen, we identified 92 GAS genes required for wild-type fitness in human saliva. Many of the identified genes are involved in carbohydrate transport/metabolism, amino acid transport/metabolism, and inorganic ion transport/metabolism. The new information is potentially valuable for developing novel GAS therapeutics and vaccine research.

Keywords: Streptococcus pyogenes; TraDIS; fitness; human pathogen; saliva; transposon mutagenesis.

FIG 1

Near-random distribution (A) and the high density of transposon insertions (B) of the M1 GAS input mutant library. (A) Insertion index (number of insertion sites divided by gene length; y axis) of each gene (x axis) in the M1 GAS reference genome. (B) A representative section of the transposon insertion map. As expected, the essential gene ftsY has no insertion because it is not represented in the library. Red vertical spikes are forward reads; blue vertical spikes are reverse reads. Read orientations indicate the direction of the transposon insertion.

FIG 2

TraDIS analyses of GAS fitness genes during exposure to human saliva ex vivo. (A to C) Genome-scale summary of the changes in mutant abundance (y axis) for each of the genes (x axis) in the output mutant pools recovered after 12 h, 24 h, and 48 h of incubation in human saliva ex vivo. Gene mutations (insertions) conferring significantly decreased (blue circles) or increased (green circles) fitness are highlighted. Insertion mutations that lacked a significantly altered fitness phenotype (gray circles) are also indicated. (D) Summary of GAS genes identified to be important for fitness in saliva after the indicated period of saliva incubation.

FIG 3

(A) Venn diagram showing the number of GAS genes identified to be important for fitness in saliva after the indicated period of incubation. (B) Functional categorization of the 92 identified GAS saliva fitness genes. Note that in panel A the circle sizes are not proportional to the numbers of genes identified, for the sake of improving the visual presentation and clarity.

FIG 4

Validation of the findings from the TraDIS saliva screen. (A to E) The saliva persistence phenotype was determined for each of six GAS isogenic mutant strains. Highlighted genes (yellow) are the putative saliva fitness genes identified by TraDIS. P values were determined by a repeated-measures 2-way ANOVA. (F) The growth phenotype in rich medium (THY) was also determined for each of the six GAS isogenic mutant strains.

FIG 5

Homologous regions encoding genes for Spy0644 to Spy0646 in GAS and other bacteria. Yellow arrows represent genes for Spy0644, Spy0645, Spy0646, and their homologs. Percentages denote amino acid identities in comparison to serotype M1 GAS strain MGAS2221.