Adaptation of group A Streptococcus to human amniotic fluid

Abstract

Background: For more than 100 years, group A Streptococcus has been identified as a cause of severe and, in many cases, fatal infections of the female urogenital tract. Due to advances in hospital hygiene and the advent of antibiotics, this type of infection has been virtually eradicated. However, within the last three decades there has been an increase in severe intra- and post-partum infections attributed to GAS.

Methodology: We hypothesized that GAS alters its transcriptome to survive in human amniotic fluid (AF) and cause disease. To identify genes that were up or down regulated in response to growth in AF, GAS was grown in human AF or standard laboratory media (THY) and samples for expression microarray analysis were collected during mid-logarithmic, late-logarithmic, and stationary growth phases. Microarray analysis was performed using a custom Affymetrix chip and normalized hybridization values derived from three biological replicates were collected at each growth point. Ratios of AF/THY above a 2-fold change and P-value <0.05 were considered significant.

Principal findings: The majority of changes in the GAS transcriptome involved down regulation of multiple adhesins and virulence factors and activation of the stress response. We observed significant changes in genes involved in the arginine deiminase pathway and in the nucleotide de novo synthesis pathway.

Conclusions/significance: Our work provides new insight into how pathogenic bacteria respond to their environment to establish infection and cause disease.

Figure 1. Characterization of growth of GAS in AF.

A. Individual specimens of AF support growth of GAS at various levels after 24 h of incubation; initial inoculum ∼10⁴ CFU/ml. B. Growth densities of GAS (CFU/ml) in THY and AF.

Figure 2. Number of genes differentially expressed in response to AF in ML, LL and S phases.

Upward arrows represent the number of transcripts with increased expression in AF in ML, LL and S phases; the downward arrows represent the number of transcripts with increased expression in THY (down regulated in AF) in ML, LL and S phases. Intersections of the Venn diagram indicate number of differentially expressed genes during more than one growth phase.

Figure 3. Dynamics of gene expression of GAS genes in ML, LL, and S phase in response to AF.

Each dot represents a single transcript and its coordinates are the average level of expression in THY (x-axis) and AF (y-axis). The dotted lines denote 2-fold change in transcription. Transcripts below the lower dotted line are more highly expressed in THY, transcripts above the upper dotted line are more highly expressed in AF. Thick black lines denote 10-fold differences in transcript level between the studied conditions.

Figure 4. Changes in transcription of genes encoding predicted enzymes in the purine and pyrimidine biosynthetic pathway in GAS and GBS in response to AF.

Both GAS and GBS exhibit similar changes in the expression of genes encoding enzymes involved in nucleotide biosynthesis. Changes in transcription of GBS genes from , changes in transcription of GAS genes, this work. Light green: genes down regulated 2 to 5 fold upon contact with AF; dark green: above 5 fold. Light orange: genes up regulated 2 to 5 fold upon contact with AF; dark orange: above 5 fold. R5P, ribose 5-phosphate; PRPP, phosphoribosylpyrophosphate; PRA, 5-phospho-b-D-ribosylamine; GAR, 5′-phosphoribosylglycinamide; FGAR, 5′-phosphoribosyl-N-formylglycinamide; FGAM, 5′-phosphoribosyl-N-formylglycinamidine; AIR, 5′-phosphoribosyl-5-aminoimidazole; NCAIR, 5′-phosphoribosyl-5-carboxyaminoimidazole; CAIR, 5′-phosphoribosyl-5-aminoimidazole-4-carboxylate; SAICAR, 5′-phosphoribosyl-4-(N-succino-carboxamide)-5-aminoimidazole; AICAR, 5′-phosphoribosyl-4-carboximide-5-aminoimidazole ribonucleotide; FAICAR, 5′-phosphoribosyl-4-carboximide-5-formaminoimidazole; IMP, inosine 5′-monophosphate; XMP, xanthosine monophosphate; GMP, guanosine monophosphate; GTP, guanosine triphosphate; sAMP, adenylosuccinate; AMP, adenosine monophosphate; ATP, adenosine triphosphate.

Figure 5. Addition of arginine, ornithine, purines and pirymidines does not increase growth of GAS in AF.

Cell densities of GAS cultures grown in pooled AF for 24 hours with addition of amino acids (arginine, ornithine) and nucleotides (xanthine, uracil, and adenine) at the concentration that supports growth of GAS in minimal CDM .