A PTS EII mutant library in Group A Streptococcus identifies a promiscuous man-family PTS transporter influencing SLS-mediated hemolysis

Abstract

The Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive human pathogen that must adapt to unique host environments in order to survive. Links between sugar metabolism and virulence have been demonstrated in GAS, where mutants in the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exhibited Streptolysin S (SLS)-mediated hemolysis during exponential growth. This early onset hemolysis correlated with an increased lesion size and severity in a murine soft tissue infection model when compared with parental M1T1 MGAS5005. To identify the PTS components responsible for this phenotype, we insertionally inactivated the 14 annotated PTS EIIC-encoding genes in the GAS MGAS5005 genome and subjected this library to metabolic and hemolysis assays to functionally characterize each EIIC. It was found that a few EIIs had a very limited influence on PTS sugar metabolism, whereas others were fairly promiscuous. The mannose-specific EII locus, encoded by manLMN, was expressed as a mannose-inducible operon that exhibited the most influence on PTS sugar metabolism, including mannose. Importantly, components of the mannose-specific EII also acted to prevent the early onset of SLS-mediated hemolysis. Interestingly, these roles were not identical in two different M1T1 GAS strains, highlighting the possible versatility of the PTS to adapt to strain-specific needs.

Figure 1. Predicted PTS EII loci present in the GAS M1T1 MGAS5005 annotated genome

Genes encoding EII subunits are indicated in grey and hypothetical proteins are indicated in black. Sugar specificities are provided based on annotation. EIIC genes encoding sugar transporters targeted for mutagenesis are highlighted in red. When known, gene names are listed below Spy number. The two annotated cellobiose EII loci are labeled as 1 and 2 based on their order in the genome.

Figure 2. Growth profiles of the PTS EII mutant library in MGAS5005

(A) Normalized growth analyses were performed as fully described in Experimental Procedures. Growth in a particular PTS sugar of the mutant above WT (2 fold, green), below WT (2 fold, yellow), well below WT (>2 fold, red), or similar to WT (Grey) are indicated. (B) Total yield (ΔOD) was calculated as described in Experimental Procedures. ΔOD of mutants in a particular PTS sugar that were above WT (green, p-value <0.05) or below WT (red, p-value <0.05) are indicated. Only PTS sugars facilitating growth of MGAS5005 are shown. All results represent at least three independent biological replicates.

Figure 3. Carbohydrate utilization profile of the PTS EIIC mutant library in MGAS5005

Utilization of select carbon sources as determined using the API®50CH system as described in Experimental Procedures. Only carbon sources that are utilized by at least one of the strains tested are shown. White boxes indicate utilization (+), grey boxes indicate partial utilization (+/−), and black boxes indicate no utilization (−). Readings for each strain are given for 24h (left box) and 48h (right box).

Figure 4. Influence of each EII on the metabolism of PTS carbohydrates

Heat map of the relative influence of each EII on growth in a particular PTS sugar (Influence Score) was calculated as described in Experimental Procedures. Green indicates a negative influence of an EII for the metabolism of a PTS sugar, red indicates a positive influence of an EII for the metabolism of a PTS sugar, and white indicates no influence. Relative influence is reflected by intensity of color.

Figure 5. Hemolysis profile of the EII mutant library in MGAS5005

Hemolytic activity of culture lysates against red blood cells (RBCs) was assayed as described in Experimental Procedures. Data from three biological replicates is shown comparing occurrence of hemolysis (defined as OD541 > 0.2) on the X-axis and the relative level of hemolytic activity observed on the Y-axis. Hemolysis at an OD600 above 1 was considered normal based on WT MGAS5005 (black bars to right of dotted line). Occurrence of hemolysis was noted as early if it occurred before OD600 of 1 (red bars left of dotted line).

Figure 6. Transcriptional architecture of the mannose PTS EII operon

(A) Graphical representation of the mannose-specific EII genetic locus. Primers for RT-PCR are also shown (arrows) (B) RT-PCR was performed on RNA isolated from MGAS5005 grown to late-logarithmic phase in THY using primers in gene pairs as indicated below. Bands indicate genes are transcribed together. (C) Polarity of each mannose EII subunit was assessed using qPCR. Primers are located in the middle of each gene. (D) Induction of the mannose operon when GAS is grown in mannose was determined. RNA was isolated in MGAS5005 grown in glucose and mannose, and relative transcript levels were compared using qPCR. Each bar is the average of 3 independent biological replicates, depicted with the standard error. Dotted lines represent 2 fold difference in expression.

Figure 7. Metabolic profile of the mannose EII subunit mutants

(A) Growth curve analyses were performed as fully described in Experimental Procedures for manLMN mutants in 5448 (right) or MGAS5005 (left). Growth in a particular PTS sugar of the mutant above WT (green), below WT (yellow), significantly below WT (red), or similar to WT (white) are indicated. (B) Change in growth (ΔOD) of manLMN mutants was calculated as described in Experimental Procedures. ΔOD of mutants in a particular PTS sugar that were significantly above WT (green, p-value <0.05) or below WT (red, p-value <0.05) are indicated. Only PTS sugars facilitating growth of MGAS5005 are shown. All results represent at least three independent biological replicates. (C) Utilization of select carbon sources by manLMN mutants as determined using the API®50CH system and described in Experimental Procedures. Only carbon sources that are utilized by at least one of the strains tested are shown. White boxes indicate utilization (+), grey boxes indicate partial utilization (+/−), and black boxes indicate no utilization (−). Readings for each strain are given for 24h (left box) and 48h (right box). (D) Heat map of the relative influence of manLMN mutants on growth in a particular PTS sugar (Influence Score) was calculated as described in Experimental Procedures. Green indicates a negative influence of an EII for the metabolism of a PTS sugar, red indicates a positive influence of an EII for the metabolism of a PTS sugar, and white indicates no influence. Relative influence is reflected by intensity of color.

Figure 8. Hemolytic activity of the mannose EII subunit mutants

Hemolytic activity of culture lysates against red blood cells (RBCs) for manLMN mutants in 5448 and MGAS5005 was assayed as described in Experimental Procedures. Data from three biological replicates is shown comparing occurrence of hemolysis (defined as OD₅₄₁ > 0.2) on the X-axis and the relative level of hemolytic activity observed on the Y-axis. Hemolysis at an OD₆₀₀ above 1 was considered normal based on WT 5448 or MGAS5005 (black bars to right of dotted line). Occurrence of hemolysis was noted as early if it occurred before OD₆₀₀ of 1 (red bars left of dotted line).

Figure 9. Contribution to virulence of the mannose EII subunits mutants

The mannose EIIC subunit mutant was assayed using a murine subcutaneous infection model. Lesion size and survival of mice were monitored for 7 days. Each strain was tested in at least 10 mice. (A) Depicted representative images of lesions taken at 2 and 3 days post infection. (B) Lesion size and severity was measured using ImageJ. Each data point represents a single mouse lesion measured at each time point. (C) Survival was monitored for 7 days. Significance was determined as described in Experimental Procedures.