Crystal Structure of Glyceraldehyde-3-Phosphate Dehydrogenase from the Gram-Positive Bacterial Pathogen A. vaginae, an Immunoevasive Factor that Interacts with the Human C5a Anaphylatoxin.

Sara Gómez,Cecilia Melchor-Tafur,Ana V Marin,Virginia Franco-Hidalgo,M Cristina Vega,Javier Querol-García,José R Regueiro,Francisco J Fernández,Daniel Fullà,Jordi Juanhuix,Santiago Rodríguez De Córdoba,Sebastián Albertí

doi:10.3389/fmicb.2017.00541

Abstract

The Gram-positive anaerobic human pathogenic bacterium Atopobium vaginae causes most diagnosed cases of bacterial vaginosis as well as opportunistic infections in immunocompromised patients. In addition to its well-established role in carbohydrate metabolism, D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Streptococcus pyogenes and S. pneumoniae have been reported to act as extracellular virulence factors during streptococcal infections. Here, we report the crystal structure of GAPDH from A. vaginae (AvGAPDH) at 2.19 Å resolution. The refined model has a crystallographic Rfree of 22.6%. AvGAPDH is a homotetramer wherein each subunit is bound to a nicotinamide adenine dinucleotide (NAD+) molecule. The AvGAPDH enzyme fulfills essential glycolytic as well as moonlight (non-glycolytic) functions, both of which might be targets of chemotherapeutic intervention. We report that AvGAPDH interacts in vitro with the human C5a anaphylatoxin and inhibits C5a-specific granulocyte chemotaxis, thereby suggesting the participation of AvGAPDH in complement-targeted immunoevasion in a context of infection. The availability of high-quality structures of AvGAPDH and other homologous virulence factors from Gram-positive pathogens is critical for drug discovery programs. In this study, sequence and structural differences between AvGAPDH and related bacterial and eukaryotic GAPDH enzymes are reported in an effort to understand how to subvert the immunoevasive properties of GAPDH and evaluate the potential of AvGAPDH as a druggable target.

Highlights

Atopobium vaginae is a Gram-positive anaerobic bacterium of the Coriobacteriaceae family that is held responsible for about 80% of diagnosed cases of bacterial vaginosis (BV) in humans, a condition that can lead to obstetric complications and gynecological disorders (Polatti, 2012; Datcu, 2014; Xiao et al, 2016)
The quaternary structure shows four subunits designated O, P, Q, and R with three non-equivalent intersubunit interfaces related by three mutually perpendicular dyad axes (P, Q, and R; nomenclature consistent with Protein Data Bank (PDB) code 1CER; Tanner et al, 1996) and one NAD+ molecule bound per active site, which is clearly defined in electron density (Buehner et al, 1974; Roitel et al, 2003)
Anaphylatoxin In addition to the essential glycolytic role of AvGAPDH, we investigated whether AvGAPDH could function as a virulence factor by sequestering the human C5a anaphylatoxin, a complement factor 5 (C5)-derived proteolytic activation fragment which diffuses out of the area wherein C5 is activated to chemoattract neutrophils and macrophages (Marder et al, 1985; Guo and Ward, 2005)

Summary

Introduction

Atopobium vaginae is a Gram-positive anaerobic bacterium of the Coriobacteriaceae family that is held responsible for about 80% of diagnosed cases of bacterial vaginosis (BV) in humans, a condition that can lead to obstetric complications and gynecological disorders (Polatti, 2012; Datcu, 2014; Xiao et al, 2016). Phylogenetically diverse bacteria have been shown to encode virulence factors that interfere with the proper function of the human complement system, one of the first barriers of defense against bacterial infections from the innate immune system. One such virulence factor is the glycolytic enzyme D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, E.C. 1.2.1.12). There are two different Pi sites depending on the precise location within the active site of the inorganic phosphate moiety, which are termed the “classical” and “new” Pi sites (Yun et al, 2000; Mukherjee et al, 2010)

Methods

Results

Conclusion