Concentration and time-dependent amyloidogenic characteristics of intrinsically disordered N-terminal region of Saccharomyces cerevisiae Stm1.

Saccharomyces cerevisiae Stm1 protein is a ribosomal association factor, which plays an important role in preserving ribosomes in a nutrition-deprived environment. It is also shown to take part in apoptosis-like cell death. Stm1 N-terminal region (Stm1_N1-113) is shown to recognize purine motif DNA triplex and G-quadruplex. Circular dichroism (CD) spectra of Stm1_N1-113 (enriched in positively-charged Lysine and Arginine; negatively-charged Aspartate; polar-uncharged Threonine, Asparagine, Proline and Serine; hydrophobic Alanine, Valine, and Glycine) collected after 0 and 24 h indicate that the protein assumes beta-sheet conformation at the higher concentrations in contrast to intrinsically disordered conformation seen for its monomeric form found in the crystal structure. Thioflavin-T kinetics experiments indicate that the lag phase is influenced by the salt concentration. Atomic force microscopy (AFM) images collected for a variety of Stm1_N1-113 concentrations (in the range of 1-400 µM) in the presence of 150 mM NaCl at 0, 24, and 48 h indicate a threshold concentration requirement to observe the time-dependent amyloid formation. This is prominent seen at the physiological salt concentration of 150 mM NaCl with the fibrillation observed for 400 µM concentration at 48 h, whereas oligomerization or proto-fibrillation is seen for the other concentrations. Such concentration-dependent fibrillation of Stm1_N1-113 explains that amyloid fibrils formed during the overexpression of Stm1_N1-113 may act as a molecular device to trigger apoptosis-like cell death.

Structural diversity among Acinetobacter baumannii K-antigens and its implication in the in silico serotyping.

Acinetobacter baumannii is an emerging opportunistic pathogen. It exhibits multi-, extreme-, and pan-drug resistance against several classes of antibiotics. Capsular polysaccharide (CPS or K-antigen) is one of the major virulence factors which aids A. baumannii in evading the host immune system. K-antigens of A. baumannii exploit the Wzx/Wzy-dependent pathway that involves 13 different proteins for its assembly and transport onto the outer membrane. A total of 64 (out of 237 K-locus(KL) types) known K-antigen sugar repeating structures are discussed here and are classified into seven groups based on their initial sugars, QuiNAc4NAc, GalNAc, GlcNAc, Gal, QuiNAc/FucNAc, FucNAc, and GlcNAc along with Leg5Ac7Ac/Leg5Ac7R. Thus, the corresponding seven initializing glycosyltransferases (ItrA1, ItrA2, ItrA3, ItrA4, ItrB1, ItrB3, and ItrA3 along with ItrB2) exhibit serotype specificity. The modeled 3D-structural repository of the 64 K-antigens can be accessed at https://project.iith.ac.in/ABSD/k_antigen.html. The topology of K-antigens further reveals the presence of 2-6 and 0-4 sugar monomers in the main and side chains, respectively. The presence of negatively (predominant) or neutrally charged K-antigens is observed in A. baumannii. Such diversity in the K-antigen sugar composition provides the K-typing specificity (viz., 18-69% in terms of reliability) for Wza, Wzb, Wzc, Wzx, and Wzy proteins involved in the Wzx/Wzy-dependent pathway. Interestingly, the degree of uniqueness of these proteins among different K-types is estimated to be 76.79%, considering the 237 reference sequences. This article summarizes the A. baumannii K-antigen structural diversity and creation of a K-antigen digital repository and provides a systematic analysis of the K-antigen assembly and transportation marker proteins.

SSP: An In Silico Tool for Salmonella Species Serotyping Using the Sequences of O-Antigen Biosynthesis Proteins and H-Antigen Filament Proteins.

Over 2500 Salmonella species (alternatively, serovars) encompassing different combinations of O-, H1- and H2-antigens are present in nature and cause millions of deaths worldwide every year. Since conventional serotyping is time-consuming, a user-friendly Salmonellaspecies serotyping (SSP) web tool (https://project.iith.ac.in/SSP/) is developed here to predict the serotypes using Salmonella protein(s) or whole proteome sequences. Prior to SSP implementation, a detailed analysis of protein sequences involved in O-antigen biosynthesis and H-antigen formation is carried out to assess their serotype specificity. Intriguingly, the results indicate that the initializing transferases WbaP, WecA and GNE can efficiently distinguish the O-antigens, which have Gal, GlcNAc and GalNAc as initial sugars respectively. Rigorous analysis shows that Wzx and Wzy are sufficient to distinguish the O-types. Exceptionally, some situations warrant additional proteins. Thus, 150 additional transferases, RfbE for O2, O9 and O9,46 types, Orf17.4 for O3,10 and O1,3,19 types, WecB, WbbE and WbbF for O54 and, Wzm and Wzt for O67 are utilized in serotyping. An in-depth analysis of 302 reference datasets representing 56 H1- and 20 H2-types leads to the identification and utilization of 61 unique sequence patterns of FliC and FljB in H-typing. A test dataset of 2136 whole proteome sequences covering 740 Salmonella serovars, including 13 new species are successfully predicted with 99.72% accuracy. Prior to this, all the O-, H1- and H2-antigens are predicted accurately when tested independently. Indeed, SSP also identifies wrongly annotated Salmonella species; hence, it can easily identify new species that emerge with any combination of O-, H1- and H2-antigens. Thus, SSP can act as a valuable tool in the surveillance of Salmonella species.