The adhesive PitA pilus protein from the early dental plaque colonizerStreptococcus oralis: expression, purification, crystallization and X-ray diffraction analysis

Abstract

PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size (∼100 kDa), flexibility and complicated folding of PitA, obtaining diffraction-quality crystals has been a challenge. However, by the use of limited proteolysis with α-chymotrypsin, the diffraction quality of the PitA crystals was considerably enhanced to 2.16 Å resolution. These crystals belonged to space group P1, with unit-cell parameters a = 61.48, b = 70.87, c = 82.46 Å, α = 80.08, β = 87.02, γ = 87.70°. The anomalous signal from the terbium derivative of α-chymotrypsin-treated PitA crystals prepared with terbium crystallophore (Tb-Xo4) was sufficient to obtain an interpretable electron-density map via terbium SAD phasing.

Digital imaging and qPCR analysis and comparison of short-term plaque removal effects of toothbrushing

“…As one of the earliest colonized bacteria, oral Streptococcus has stronger adhesion than the later colonized bacteria, so it may be more difficult to remove during oral cleaning. The proportion of Lactobacillus in the whole oral flora is very small, usually about 0.01%-1% (13). Yang Ran et al found that the detection rates of Actinomyces odontolyticus and Actinomyces naeslundii in a caries-free group were significantly higher than those in a caries-sensitive group, and speculated that Actinomyces odontolyticus and Actinomyces naeslundii may be cariesbeneficial bacteria (14).…”

LrpCBA pilus proteins of gut-dwelling Ligilactobacillus ruminis: crystallization and X-ray diffraction analysis

“…Characteristically, the formation of these covalent bonds requires triad-sequestered Lys and Asn/Asp residues and an autocatalytic glutamate (or aspartate) in a hydrophobic environment. Among the three pilin types, the adhesive tip subunits are the largest, consisting of an N-terminal receptor-binding region that is often a globular domain and a C-terminal stalk-like region of CnaA/ CnaB domains (Izoré et al, 2010;Kant et al, 2020;Linke-Winnebeck et al, 2014;Pointon et al, 2010;Kumari Yadav & Krishnan, 2020). Backbone subunits typically consist of two to four CnaA/CnaB domains (Krishnan, 2015).…”

Tracking Crystallophore Nucleating Properties: Setting Up a Database for Statistical Analysis

“…18,19 Finally, Tb-Xo4 was exploited to determine new protein structures including multi-protein complexes [20][21][22] and can be used as a routine nucleating and phasing tool by the community. [23][24][25][26][27] To understand the origins of the crystallophore nucleating and phasing properties with the aim to further improve them, we analyzed the binding of Tb-Xo4 at the surface of proteins by coupling crystal structure analysis, obtained on 4 different proteins, with evaluation of the interaction energies determined by density functional theory calculations. 28 This detailed analysis of the interaction sites on the proteins surfaces clearly demonstrated the great versatility of Tb-Xo4 binding through various supramolecular interactions via anionic, cationic or hydrophobic amino-acid residues.…”

“…The story so far of the Xo4 system started with its terbium variant Tb-Xo4. Its unique properties were first highlighted with a set of eight proteins, and its efficiency was confirmed in solving issues often dealt by crystallographers in their quest to assess the protein structure. , Finally, Tb-Xo4 was exploited to determine new protein structures including multiprotein complexes − and can be used as a routine nucleating and phasing tool by the community. − …”