Site-Specific Mutations of GalR Affect Galactose Metabolism in Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae (the pneumococcus) is a formidable human pathogen that is capable of asymptomatically colonising the nasopharynx. Progression from colonisation to invasive disease involves adaptation to distinct host niches, which vary markedly in the availability of key nutrients such as sugars. We have previously reported that cell-cell signalling via the autoinducer-2 (AI-2)/LuxS quorum sensing system boosts the capacity of S. pneumoniae to utilise galactose as a carbon source by upregulation of the Leloir pathway. This resulted in increased capsular polysaccharide production and a hypervirulent phenotype. We hypothesised that this effect was mediated by phosphorylation of GalR, the transcriptional activator of the Leloir pathway. GalR is known to possess three putative phosphorylation sites: S317, T319 and T323. In the present study, derivatives of S. pneumoniae D39 with putative phosphorylation-blocking alanine substitution mutations at each of these GalR sites (singly or in combination) were constructed. Growth assays and transcriptional analyses revealed complex phenotypes for these GalR mutants, with impacts on the regulation of both the Leloir and tagatose 6-phosphate pathways. The alanine substitution mutations significantly reduced the capacity of pneumococci to colonize the nasopharynx, middle ear and lungs in a murine intranasal challenge model. IMPORTANCE Pneumococcal survival in the host and capacity to transition from a commensal to a pathogenic lifestyle is closely linked to its ability to utilise specific nutrients in distinct niches. Galactose is a major carbon source for pneumococci in the upper respiratory tract. We have shown that both the Leloir and tagatose-6-phosphate pathways are necessary for pneumococcal growth in galactose, and demonstrate GalR-mediated interplay between the two pathways. Moreover, the three putative phosphorylation sites in the transcriptional regulator GalR play a critical role in galactose metabolism and are important for pneumococcal colonization of the nasopharynx, middle ear and lungs.

SARS‐CoV‐2 triggered oxidative stress and abnormal energy metabolism in gut microbiota

“…Galactose utilization could result in hypervirulent phenotype of Streptococcus pneumoniae . 74 Dysregulation of the ABC transporter pathway implies that patients might be under toxic stress after exposure to SARS‐CoV‐2. 34 Besides, the accessory genomes of considerable pathogenic bacteria cover ABC transporters that contribute to antimicrobial resistance by multidrug efflux, 75 further explaining antibiotic resistance pathway enrichment.…”

“…Highly active expression of E. coli and upregulation of genes related to biofilm formation supported their association with COVID‐19 severity. Galactose utilization could result in hypervirulent phenotype of Streptococcus pneumoniae 74 . Dysregulation of the ABC transporter pathway implies that patients might be under toxic stress after exposure to SARS‐CoV‐2 34 .…”

Streptococcus pneumoniae Strains Isolated From a Single Pediatric Patient Display Distinct Phenotypes

“…The S. pneumoniae strains used in this study are serotype 15C clinical isolates 60B (SN69534) and 60CSF (SN69531) (isolated from blood and CSF, respectively), D39 (serotype 2 ST595), and Rx1 (unencapsulated). Cells were routinely grown on Columbia agar supplemented with 5% (vol/vol) horse blood (BA), with or without gentamicin (40 µg/mL), at 37°C in 5% CO 2 overnight ( Mclean et al., 2020 ). Growth assays were performed with pneumococci grown in a chemically defined medium (CDM) ( Kloosterman et al., 2006 ) comprising SILAC RPMI 1640 Flex Media, no glucose, no phenol red (Sigma), supplemented with amino acids ( Table 1A ), vitamins ( Table 1B ), uracil (0.01 mg/mL), adenine (0.01 mg/mL), choline chloride (0.005 mg/mL) and catalase (10 U/mL), and either 0.5% glucose, galactose, lactose, raffinose or melibiose.…”

Gene Expression Regulation in Airway Pathogens: Importance for Otitis Media

“…Due to a low availability of glucose in the airways, Spn in the nasopharynx and ME relies on the galactose and mannose derived from the airway mucosal glycan lining, as a main carbon source for energy metabolism and virulence (Paixao et al, 2015a;Paixao et al, 2015b). Several recent studies have unveiled the impact of carbohydrate utilization and metabolic processes in Spn virulence, persistence, and infection at nasopharynx, which involves a series of regulators (galK, galR, hyl, ugl, lacD, nanA, eng, rafK, estA, and auto-inducer AI-2) (Afzal et al, 2015;Mclean et al, 2020;Minhas et al, 2021). Sensing of the host carbohydrates for environmental adaptation is mainly carried out by pneumococcal short hydrophobic peptide (SHP) and phosphatase regulator (Phr) that interact with RRNPP superfamily of transcription regulator, regulator gene of glycosyltrasferase (Rgg) and transcription factor regulated by Phr peptide (Tpr), respectively.…”