Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein

Abstract

The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg2+-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg2+-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg2+ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.

NaCl promotes antibiotic resistance by reducing redox states in Vibrio alginolyticus

“…The Na(+)-NQR is a unique respiratory enzyme that causes an uncoupling of the proton gradient to electrogenic pumping of sodium across the cell membrane in bacteria, especially in Vibrio species (Hayashi et al, 2001;Li et al, 2012;Deka et al, 2015). It oxidizes NADH and reduces quinone to quinol and generates a redoxdriven transmembrane electrochemical potential.…”

Treponema pallidum, the syphilis spirochete: making a living as a stealth pathogen

“…A contemporary bioinformatics-based analysis of the possible functions of the more than 400 ‘hypothetical’ proteins encoded in the T. pallidum genome 40 may inform future attempts. Recent recognition of the importance of riboflavin uptake and flavin utilization for spirochete metabolism and energy generation may be pertinent as well 41,42 . Therefore, investigators today still rely upon intratesticular inoculation of rabbits to isolate T. pallidum strains from clinical samples and propagate them for experimentation 43 .…”

“…Another lipoprotein, TP0655, was shown to be the SBP for a polyamine transporter (PotABCD (TP0652-0655)) with nanomolar binding affinities for putrescine and spermidine 99 . The X-ray structure of TP0298, also a lipoprotein, revealed it to be the SBP of a riboflavin transporter (RfuABCD (TP0298-0302)) 41 which collaborates with a dual function FAD pyrophosphatase-FMN transferase (Ftp (TP0796)) 42,100 to meet the T. pallidum ’s prodigious requirements for flavin cofactors. A longstanding question has been how T. pallidum , a fatty acid auxotroph, obtains essential long-chain fatty acids (LCFAs).…”

“…To resolve the second dilemma, with the added dividend of boosting energy production, T. pallidum is believed to couple the generation of an electrochemical gradient by a non-canonical flavin-dependent R hodobacter n itrogen f ixation (Rnf) Na + /H + redox pump to ATP synthesis by one or both of its A-type ATP synthases (formerly annotated as V-type ATPases) (Fig. 2) 42,115 . Transfer of electrons from reduced flavodoxin, generated by PFOR, drives the electrogenic pump 115 with NAD + , generated by LDH or NOX-2 depending upon oxygen availability, the ultimate electron acceptor.…”

Chemerin Induced byTreponema pallidumPredicted Membrane Protein Tp0965 Mediates Endothelial Dysfunction via Activating MAPK Signaling Pathway

“…Based on an expanded corpus of molecular immunological observations, research focused on bacteria structure and physiology of bacteria made considerable progress over the past two decades [4-6]. However, investigators found that T. pallidum outer membrane, unlike other Gram-negative bacteria, is fragile and contains limited antigens.…”