Flavin Reductase Contributes to Pneumococcal Virulence by Protecting from Oxidative Stress and Mediating Adhesion and Elicits Protection Against Pneumococcal Challenge

Abstract

Pneumococcal flavin reductase (FlaR) is known to be cell-wall associated and possess age dependent antigenicity in children. This study aimed at characterizing FlaR and elucidating its involvement in pneumococcal physiology and virulence. Bioinformatic analysis of FlaR sequence identified three-conserved cysteine residues, suggesting a transition metal-binding capacity. Recombinant FlaR (rFlaR) bound Fe2+ and exhibited FAD-dependent NADP-reductase activity, which increased in the presence of cysteine or excess Fe2+ and inhibited by divalent-chelating agents. flaR mutant was highly susceptible to H2O2 compared to its wild type (WT) and complemented strains, suggesting a role for FlaR in pneumococcal oxidative stress resistance. Additionally, flaR mutant demonstrated significantly decreased mice mortality following intraperitoneal infection. Interestingly, lack of FlaR did not affect the extent of phagocytosis by primary mouse peritoneal macrophages but reduced adhesion to A549 cells compared to the WT and complemented strains. Noteworthy are the findings that immunization with rFlaR elicited protection in mice against intraperitoneal lethal challenge and anti-FlaR antisera neutralized bacterial virulence. Taken together, FlaR’s roles in pneumococcal physiology and virulence, combined with its lack of significant homology to human proteins, point towards rFlaR as a vaccine candidate.

Streptococcus pneumoniae Cell Wall-Localized Trigger Factor Elicits a Protective Immune Response and Contributes to Bacterial Adhesion to the Host

“…Previously we found TF in the CW fraction of S. pneumoniae 27 , 28 . Notably, we recently showed that a bona fide cytoplasmic protein, malonyl-CoA:ACP transacylase (FabD), which is involved in lipid metabolism could not be found in the CW protein extract 28 , 33 . To further verify that TF is indeed CW-localized, a monoclonal anti-rTF antibody was incubated with a live unencapsulated, serotype 2-derived, R6 strain and flow cytometry analysis was performed.…”

“…The untagged rTF protein was commercially produced by Protein laboratories, Ltd. (Israel), which amplified tig from TIGR4 genome 69 , and cloned, expressed and purified the protein as previously described 28 , 33 . Validation was also performed by the company using MS.…”

“…Microtiter plates (F96 Maxisorp Nunc, ThermoFisher) were coated with 1 µg/ml solution of rHis-TF in bicarbonate buffer pH 9.6. ELISA was performed as previously described 33 . Sera obtained from mice after the second (TF II) and third (TF III) rHis-TF immunizations served as primary antibodies.…”

Practical observations on the use of fluorescent reporter systems in C. difficile

“…Riboflavin is an essential precursor of compounds such as the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) that are cofactors of flavoproteins. Flavoproteins have pleiotropic functions [42] and have been implicated in the oxidative stress response in several anaerobes [43,44]. In C. difficile, flavoproteins are overrepresented in the regulon of the general stress response sigma factor σB [45,46] and have been implicated in spore resistance [47].…”

“…Flavoproteins act as electron transfer proteins and mediate redox reactions, for instance in the metabolism of carbohydrates, oxidative stress response or even in the extracellular environment (Garcia-Angulo 2017). In Streptococcus pneumoniae, the flavin reductase FlaR contributes to the resistance to H2O2 [43]. The expression of genes involved in the synthesis and uptake of flavins is regulated through FMNdependent riboswitches [56,57].…”