CRYSTAL STRUCTURE RESOLUTION OF STREPTOCOCCUS PNEUMONIAE NEWLY IDENTIFIED ADHESIN, PHOSPHOENOL PYRUVATE PHOSPHOTRANSFERASE (PTSA)

Tatyana Kushnir ^1,2,3 Anat Shahar ⁵ Raz Zarivach ^4,5 Marilou Shagan ^1,2,3 Ron Dagan ^1,2,3 Yaffa Mizrachi Nebenzahl ^1,2,3

¹Shraga Segal Department of Microbiology, Immunology and Genetics, Ben-Gurion University of the Negev, Beer-Sheva, Israel
²Pediatric Infectious Disease Unit, Soroka University Medical Center, Beer-Sheva, Israel
³The Faculty of Health Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
⁴Department of Biosciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel
⁵National Institute for Biotechnology in the Negev, Ben-Gurion University of the Negev, Beer-Sheva, Israel

Streptococcus pneumoniae is a commensal pathogen, commonly carried asymptomatically in the nasopharynx. However, otitis media, bacteremia, pneumonia and meningitis are initiated by S. pneumoniae colonization of the nasopharynx through adhesion followed by spread to and invasion of the respective tissues. S. pneumoniae adhesion to the host cells is mediated by adhesins, surface attached proteins that interact with their corresponding host target receptors. One such adhesin is Phosphoenol-pyruvate Phospho-transferase A (PtsA). Primarily PtsA functions as the first enzyme (EI) of the PTS system, translocating the phosphoryl group of the phosphoenol pyruvate (PEP) to the next in line Histidine phosphocarrier protein (HPr).

Our previous studies revealed cell-wall localization of PtsA and its host target receptors, residing on the epithelial and neuronal cells. Additionally, host target receptors` derived peptides inhibited pneumococcal adhesion in concentrations within the micromolar range, in line with their Microscale Thermophoresis (MST) determined affinity to the rPtsA. Sequence alignment of the target receptors derived peptides with HPr revealed two sites of homology, implicating their putative binding sites on PtsA.

To characterize the interactions of these peptides with PtsA, its crystal structure was resolved for the first time. PtsA was crystallized in optimized crystallizing conditions, restricting conformational flexibility inherent to phosphotransferase enzymes. The attained favorable conformation of PtsA structure was resolved by Molecular Replacement method relying on the existing structure of E. coli E1 of the PTS system, which shares 65% of homology with PtsA sequence.

The limited protection of the currently existing vaccines increasing antibiotic-resistance of S. pneumoniae points to the importance of new therapeutic approaches. The target receptor-derived peptides may be thus considered as candidates for potential effective adjunct therapies in the future.