Molecular characterization of group A Streptococcus maltodextrin catabolism and its role in pharyngitis

Abstract

We previously demonstrated that the cell-surface lipoprotein MalE contributes to GAS maltose/maltodextrin utilization, but MalE inactivation does not completely abrogate GAS catabolism of maltose or maltotriose. Using a genome-wide approach, we identified the GAS phosphotransferase system (PTS) responsible for non-MalE maltose/maltotriose transport. This PTS is encoded by an open reading frame (M5005_spy1692) previously annotated as ptsG based on homology with the glucose PTS in Bacillus subtilis. Genetic inactivation of M5005_spy1692 significantly reduced transport rates of radiolabelled maltose and maltotriose, but not glucose, leading us to propose its reannotation as malT for maltose transporter. The DeltamalT, DeltamalE and DeltamalE:malT strains were significantly attenuated in their growth in human saliva and in their ability to catabolize alpha-glucans digested by purified human salivary alpha-amylase. Compared with wild-type, the three isogenic mutant strains were significantly impaired in their ability to colonize the mouse oropharynx. Finally, we discovered that the transcript levels of maltodextrin utilization genes are regulated by competitive binding of the maltose repressor MalR and catabolite control protein A. These data provide novel insights into regulation of the GAS maltodextrin genes and their role in GAS host-pathogen interaction, thereby increasing the understanding of links between nutrient acquisition and virulence in common human pathogens.

Fig. 1

Transcript level analysis of known and putative carbohydrate transport systems in GAS. Strain MGAS5005 was grown to mid-exponential phase in either standard laboratory media (THY, white bars), a chemically defined medium (CDM) containing either 0.5% (wt/vol) glucose (grey bars) or 0.5% maltose (textured bars) or human saliva (black bars). TaqMan real-time QRT-PCR was performed using probe and primers listed in Table S2. The transcript levels of target genes indicated by their M5005_spy gene numbers on the X-axis are shown relative to those of proS, a gene expressed constitutively throughout the GAS cell cycle and whose transcript levels are similar whether grown in THY or saliva (Shelburne et al., 2005a; Virtaneva et al., 2003). (A) Genes whose transcript levels were elevated in human saliva compared to other media; (B) Genes whose transcript levels were low in all media; (C) Genes whose transcript levels were high in all media; (D) Genes whose transcript levels were high in maltose-medium and human saliva. The two genes with the final pattern included malE, which was previously demonstrated to participate in maltodextrin catabolism, and M5005_spy1692 (Shelburne et al., 2007a). Transcript levels are presented as the mean ± standard deviation of four independent experiments done on two separate occasions.

Fig. 2

Growth of indicated GAS strains under various conditions. OD₆₀₀ readings were taken at indicated times for growth in nutrient-rich medium (THY) and chemically defined medium (CDM) with 0.5% (wt/vol) of indicated carbohydrate. Growth in human saliva was monitored using CFU as previously described (Shelburne et al., 2005b). Growth media were: (A) THY; (B) glucose-medium; (C) maltose-medium; (D) maltotriose-medium; (E) human saliva; (F) starch-medium with purified human salivary α-amylase at 200 U/mL. Data graphed are mean values ± standard deviation for five independent experiments done on two separate occasions.

Fig. 3

Colonization and colony forming unit (CFU) recovery rates among mice infected with GAS. Adult outbred CD-1 mice (25 per group) were inoculated with 1 × 10⁷ CFU of indicated GAS strains. Mice oropharynx were swabbed daily onto BSA. Plates were incubated for 24 hrs, and β-hemolytic colonies were counted and tested for GAS carbohydrate antigen using latex agglutination. (A) % of mice with GAS isolated from oropharynx by day; (B) average number of GAS CFU per mouse isolated by day. Data graphed are mean values ± standard deviation. P values refer to repeated measures analysis of indicated strains compared to parental wild-type strain MGAS5005.

Fig. 4

Presence of malT and malE transcripts in vivo during pharyngitis in humans. malT and malE transcript levels for 6 patients with GAS pharyngitis were determined by TaqMan real-time PCR. X-axis label indicate the M serotype of the infecting GAS strains. Column height indicates the median level of the indicated gene compared to the endogenous GAS control gene proS (43). Transcript levels are presented as the mean ± standard deviation of four independent experiments done on two separate occasions with samples analyzed in triplicate.

Fig. 5

Influence of MalR and CcpA on maltodextrin utilization genes. Strain MGAS5005 and its isogenic ΔmalR or ΔccpA derivatives were grown to indicated growth phases in standard laboratory media (THY), a maltotriose-medium, or human saliva. TaqMan real-time QRT-PCR was performed using probe and primers listed in Table S2. The transcript levels of target genes were normalized to those of proS, a gene expressed constitutively throughout the GAS cell cycle and whose transcript levels are similar during growth in THY or saliva (Shelburne et al., 2005a; Virtaneva et al., 2003). (A) malT and M5005_spy1691 transcript levels are expressed as log₂ of the fold difference in the ΔmalR versus wild-type strain (ΔΔC_T method), therefore positive values indicates higher transcript levels in the ΔmalR strain. (B) Gene transcript levels in the ΔmalR strain relative to proS (ΔC_T method). (C) and (D) Log₂ fold difference of maltodextrin utilization genes for ΔmalR (C) and ΔccpA (D) strains compared to wild-type during growth in THY. (E) and (F) Log₂ fold difference of maltodextrin utilization genes for ΔmalR (E) and ΔccpA (F) strains compared to wild-type during growth in maltotriose-medium. Data are presented as the mean ± standard deviation of quadruplicate measurements done on two separate occasions with samples analyzed in triplicate.

Fig. 6

Binding of GAS CcpA and MalR to a representative maltodextrin utilization gene cre site. (A) Purified GAS CcpA was titrated into a binding buffer containing 1 nM fluorescein-labeled pulA cre (open circle) or 1 nM fluorescein labelled ftsX promoter DNA (negative control, closed triangle) in the presence of 50µM HPr-Ser46-P, a co-effector necessary for high affinity CcpA binding to cre DNA. Millipolarization units (mP) are plotted against the CcpA concentration (nM). (B) Comparison of CcpA pulA-cre biding in the presence (open circle) and absence (closed triangle) of 50 µM HPr-Ser46-P. (C) Purified GAS MalR was titrated as for CcpA except that Hpr-Ser46-P was not included in the binding buffer. (D) Comparison of MalR pulA-cre binding in the presence (closed triangle) and absence (open circle) of 10 mM maltotriose. The hyperbolic nature of the CcpA-HPr-Ser46-P and MalR interaction with the pulA cre indicates specific binding to this dideoxynucleotide.

Fig. 7

Schematic for potential contribution of GAS maltodextrin utilization to pharyngitis including regulatory mechanisms. Host-ingested polysaccharides are broken down by salivary α-amylase to maltodextrins that are mainly transported by MalE with some transport of maltose and maltotriose by MalT. The entry of maltodextrins leads to release of MalR from the promoter regions of the maltodextrin utilization genes with subsequent binding by CcpA. CcpA binding induces transcription of maltodextrin utilization genes allowing for further transport and subsequent catabolism of maltodextrins. The transported maltodextrins eventually enter into glycolytic pathways providing energy needed for proliferation and subsequent pharyngitis.