Host-glycan metabolism is regulated by a species-conserved two-component system in Streptococcus pneumoniae

Abstract

Pathogens of the Streptococcus genus inhabit many different environmental niches during the course of an infection in a human host and the bacteria must adjust their metabolism according to available nutrients. Despite their lack of the citric-acid cycle, some streptococci proliferate in niches devoid of a readily available carbohydrate source. Instead they rely on carbohydrate scavenging for energy acquisition, which are obtained from the host. Here we discover a two-component system (TCS07) of Streptococcus pneumoniae that responds to glycoconjugated structures on proteins present on the host cells. Using next-generation RNA sequencing we find that the uncharacterized TCS07 regulon encodes proteins important for host-glycan processing and transporters of the released glycans, as well as intracellular carbohydrate catabolizing enzymes. We find that a functional TCS07 allele is required for growth on the glycoconjugated model protein fetuin. Consistently, we see a TCS07-dependent activation of the glycan degradation pathway. Thus, we pinpoint the molecular constituents responsible for sensing host derived glycans and link this to the induction of the proteins necessary for glycan degradation. Furthermore, we connect the TCS07 regulon to virulence in a mouse model, thereby establishing that host-derived glycan-metabolism is important for infection in vivo. Finally, a comparative phylogenomic analysis of strains from the Streptococcus genus reveal that TCS07 and most of its regulon is specifically conserved in species that utilize host-glycans for growth.

Fig 1. Overexpression of RR07 alters transcription of genes involved in host-glycan and carbohydrate metabolism.

The transcriptome of wild-type D39 and the isogenic RR07 overexpressing mutant was compared by RNA-seq. Strains were grown in quadruplicates in C+Y to OD₆₀₀ = 0.5 at which point cells were harvested and RNA extracted. The RNA was prepared for and sequenced as described in materials and methods. Sequence reads were mapped to the genome of D39, normalized and quantified. Log₂ fold changes (Log₂FC), false-discovery rates (FDR) and reads per million (RPM) were calculated for each gene. Genes were considered upregulated if Log₂FC > 2, FDR < 0.05 and RPM > 500 in the RR07 overexpressing strain. Genes were considered downregulated if Log₂FC < -2, FDR < 0.05 and RPM > 50 in the wild type strain. A) Circular plot of D39 genome with log₂FC at y-axis and genomic location at x-axis. Up- or downregulated genes are color coded according to known or predicted functions (grey: genes not considered regulated based on above criteria). B) Log₂FC of up- or downregulated genes sorted according to predicted or known function. 11 genes involved in host-glycan metabolism and 7 in carbohydrate metabolism are all upregulated.

Fig 2. Validation of RNA-seq by RT-qPCR.

Validation was performed by repeating the experimental setup for RNA-seq with inclusion of a tcs07 mutant and performing RT-qPCR on the extracted RNA with primer sets for selected upregulated genes and hk07, nanA and bgaA. mRNA levels were normalized to gyrA and mRNA fold-changes are relative to the wild-type. Performed in biological duplicates with standard deviation as error bars.

Fig 3. Growth experiments in glycan-derived sugars.

Indicated strains were grown to exponential phase (OD₆₀₀ = 0.3–0.4) in C+Y, at which point they were spun down and resuspended to OD₆₀₀ = 0.1 in PBS. 20 μL was added to 180 μL CDM supplemented with 0.5% of the indicated sugar. Standard deviation depicted as colored-shadow. Performed in triplicates at least three independent times.

Fig 4. TCS07 is required for growth in and responding to a model glycan protein, fetuin.

A) Growth experiments in fetuin (glycosylated) and BSA (not glycosylated). Indicated strains were grown to exponential phase (OD₆₀₀ = 0.3–0.4) in C+Y, at which point they were spun down and resuspended to OD₆₀₀ = 0.1 in PBS. 20 μL was added to 180 μL CDM supplemented with 2% fetuin or BSA. Lines depict replicates and colored shadows depict standard deviation. B) Growth in fetuin increase transcription of host-glycan metabolizing genes. D39 wild-type was grown to exponential phase (OD₆₀₀ = 0.3–0.4) in C+Y, at which point the culture was spun down and resuspended to OD₆₀₀ = 0.1 in PBS. 200 μL was added to 1800 μL CDM supplemented with 0.5% glucose or 2% fetuin. Cultures were grown to OD₆₀₀ = 0.2 at which point cells were harvested and RNA extracted. RT-qPCR was performed on the extracted RNA with primer sets for indicated genes. mRNA levels were normalized to gyrA and mRNA fold-changes for each gene are relative to glucose. Faded genes (nanA and bgaA) is not part of the identified regulon of TCS07. Performed in biological duplicates with standard deviation as error bars. C) TCS07 is required for induction of the gh125-gh38-ROK-gh20 operon. Fluorescence microscopy of wild type and ΔTCS07 strains with gh20::mKate2 fusion. The wild type strain with the gh20::mKate fusion was incubated in CDM with 2% fetuin for 24 h (after growth initiated) or in CDM with 0.5% glucose until exponential phase. The wild type strain without the fusion was incubated in CDM with fetuin as a negative control. The wild type and ΔTCS07 with the gh20::mKate2 fusion were incubated in CDM with 2% fetuin for 16 h (before growth initiated). Scale bar is 10 μm.

Fig 5. TCS07 does not affect adherence or invasion.

Wild type and ΔTCS07 were inoculated from plate into DMEM to an OD₆₀₀ = 0.2 and added to a confluent monolayer of A549 cells. Cells were incubated for 2 or 3 h for adherence or invasion assays, respectively. For adherence, non-adherent bacterial cells were washed away with PBS, and adherent cells were spread-plated to determine CFU counts. For invasion, non-invasive bacterial cells were killed by replacing the medium with DMEM supplemented with penicillin G and gentamycin and incubated for 1 h, before washing and plating to determine CFU counts. Adherence assays were performed in triplicates four individual times and invasion assays were performed in triplicates two times; error bars depicting standard deviation.

Fig 6. TCS07 increase virulence in a mouse infection model.

5 weeks old anesthetized female Swiss mice were challenged intranasally with 10⁷ CFU of wild type D39 or ΔTCS07. After 24 or 48 h mice were euthanised, blood and tissues were harvested and CFU was determined by serial dilution and plating. One mouse in the 48 h wild type group died from the infection. Statistical analysis was performed with a linear mixed effect model, (details in materials and methods). Deletion of TCS07 significantly reduced virulence (p-value = 0.02916, χ²(1) = 4.7579), and on average reduced bacterial loads 9.24-fold ± 2.67 (standard deviation).

Fig 7. TCS07 is specifically enriched amongst species that catabolize glycans.

Unrooted phylogenetic tree of important species from the Streptococcus genus. A maximum of 10 strains from each species were used for the phylogenetic tree. Numbers represent percentages of strains with a TCS07 homolog with the total number of strains from each species in parenthesis. All species with evidence for glycan catabolism have TCS07 homologs and all species with evidence against lack homologs. Evidence for or against is based on the literature (Table 2).

Fig 8. Most of the identified regulon clusters with TCS07 in the genome in other species of Streptococci.

The genomic distance from the response regulator (rr07) to each gene of the regulon was determined in strains with a TCS07 homolog. For genes in operons, only the distance to the first gene was used, and each dot represents a gene homolog. The regulon is spread out in the genome of S. pneumoniae, whereas it mostly clusters around the TCS07 in other species. Except for strH, all genes cluster to TCS07 in at least two of the species. SPD_0156, a hypothetical protein, is part of the TCS07 operon in many species and otherwise absent. gh29 is absent in three of the species. In two of them, endoD has replaced it genomic location, while it is absent in the last.

Fig 9. Model of N-glycan metabolism and TCS07 regulation.

N-glycans are sequentially cleaved by extracellular and intracellular glycoside hydrolases, most of which are transcriptionally activated by TCS07 (HK07 and RR07). Genes upregulated by TCS07 and corresponding protein products are colored yellow. First step of degrading complex N-glycan structures is cleavage of sialic acid and galactose by NanA and BgaA, respectively, neither of which are part of the identified TCS07 regulon. All other enzymatic activities related to N-glycan metabolism can be attributed to protein products of TCS07 regulated genes as follows: GlcNAc exposed by NanA and BgaA is cleaved by StrH, and the resulting Man₃GlcNAc₂-core is cleaved between the GlcNAc moieties by EndoD. For high-mannose N-glycans, the Man_6-9GlcNAc₂ is trimmed by GH92 to Man₅GlcNAc₂, which is cleaved by EndoD to Man₅GlcNAc. The released Man₃GlcNAc and Man₅GlcNAc are imported by the NgtS-P1-P2 ABC transporter system. Intracellularly GH38 hydrolyze the 1,3-glycoside bonds, and GH125 hydrolyze the 1,6-glycoside bonds of the Man₃GlcNAc and Man₅GlcNAc resulting in ManGlcNAc. Mannose released by GH38 and GH125 is possibly phosphorylated by ScrK into Mannose-6-phosphate, which is converted by ManA to fructose-6-phosphate. Finally, fructose-6-phosphate enters glycolysis. Genes that are faded are genes that has not been attributed a function in the model. Genes marked with * are genes that were not considered upregulated based on criteria defined in Fig 1 but are part of an operon with genes considered upregulated.