Title : Targeting vitamins B1, B3 and B5 metabolism reduces streptococcus pneumoniae virulence in a murine influenza co-infection model
Abstract:
Co-infection studies indicate that primary influenza infection promotes pneumococcal virulence by causing capillary leakage of nutrients into the lower respiratory tract. This study investigates whether limiting the availability or uptake of auxotrophic B-vitamins can mitigate this nutrient-driven advantage and serve as a potential antimicrobial strategy. Specifically, a two-pronged in vivo approach was studied that combines inhibition of pneumococcal vitamin transporters with short-term dietary restriction of the corresponding host B-vitamins. The well-characterised serotype 4 strain, D39, was used both in vitro, and in a murine co-infection model over an 8-day infection period. A chemically defined minimal media supporting robust growth of S. pneumoniae D39 was first formulated de novo. Then, auxotrophic requirements for individual B-vitamins were determined using growth curve assays in which single vitamins were omitted, and vitamins B1, B3 and B5 were identified as auxotrophic requirements for pneumococcus. Putative vitamin transporter genes were selected and deleted, with mutants confirmed by PCR. Dose-response growth assays across a range of vitamin concentrations were then performed to estimate half-maximal effective concentration (EC50). SPD2025, SPD0625to27 (gene cluster of SPD0625, SPD0626, SPD0627), panT and niaX were genes implicated as putative transporters, as deletion, confirmed by PCR, led to increased EC50 for its respective vitamin in these assays. Based on EC50 results, B1 and B5 mutants were prioritised for in vivo testing. C57BL/6J mice (n = 10 per group) were infected intranasally with 30 PFU of influenza A virus, followed 7 days later by intratracheal infection with ~104 CFU of pneumococci; any specific diets were administered from days 0 to 7. Lungs and blood were collected 24h after bacterial infection on day 8, and CFU counts were enumerated on day 9. Bacterial burdens were log???transformed after substituting zero counts with the limit of detection minus one, and groups were compared using unpaired, two?tailed parametric t?tests assuming Gaussian distribution. B1 and B5 transporter deletion mutants exhibited varying levels of reduced virulence in vivo, and short-term depletion of vitamins B1, B3 and B5 from the host diet similarly attenuated wild-type pneumococcal virulence. In summary, genetic disruption of vitamin B1, B3, or B5 transporters increased the requirement for exogenous vitamin supplementation in vitro and significantly attenuated pneumococcal virulence in vivo. Furthermore, dietary depletion of vitamins B1, B3, and B5 in the host also reduced wild?type pneumococcal virulence in vivo. Ultimately, these findings highlight pneumococcal vitamin transporters and short-term vitamin dietary modulation as complementary strategies that could inform the development of novel therapeutic and prophylactic approaches against pneumococcal infection.
