Additional file 1 of GroEL protein of the Leptospira spp. interacts with host proteins and induces cytokines secretion on macrophages
Creators: name: Ho, Joana Dias
name: Luiz Eduardo Massao Takara
name: Denize Monaris
name: Gonçalves, Aline Patrícia
name: Souza-Filho, Antonio Francisco
name: Gisele Oliveira De Souza
name: Heinemann, Marcos Bryan
name: Ho, Paulo Lee
name: Abreu, Patrícia Antonia Estima
Publisher: name: figshare
Year: 2021
Subjects: subject: Biochemistry
subject: Microbiology
subject: FOS: Biological sciences
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Biotechnology
subject: 59999 Environmental Sciences not elsewhere classified
subject: FOS: Earth and related environmental sciences
subject: FOS: Earth and related environmental sciences
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Cancer
subject: 110309 Infectious Diseases
subject: FOS: Health sciences
subject: FOS: Health sciences
subject: Plant Biology
subject: 60506 Virology

description: Additional file 1: Figure S1. Multiple sequence alignment performed using Clustal Omega program. a) Chaperonins group I: alignment of amino acid sequences of GroEL from human mitochondria (PDB: 4PJ1_A), L.interrogans (AAS69936.1), Escherichia coli (EFS2935563.1), and Thermus thermophilus (WP_011174077.1). The initial sequence of mitocrondial GroEL, residues 1–26 (MGSHHHHHHHHGSDYDIPTTENLYFQ), was excluded from the figure for better representation. b) Chaperonins group II: Alignment of amino acid sequences of GroEL from human TCP1 (T-complex protein 1) (NP_110379.2), Drosophila TCP1 (AAA28927.1), Escherichia coli (EFS2935563.1), L.interrogans (AAS69936.1), and Thermus thermophilus (WP_011174077.1). The equatorial domain (subdomains E1 and E2) is indicated in blue; intermediate domain (subdomains I1 and I2) in red and apical domain in green. The highly conserved motifs were underlined. (*) asterisk indicates identical residues (fully conserved) in all sequences; (:) colon shows conservation between residues with similar properties (strong conservation), and (.) period represents conservation between residues with weakly similar properties. Protein accession sequences are available in http://www.ncbi.nlm.nih.gov/protein/ . Figure S2. Multiple sequence alignment performed using Clustal Omega program of amino acid sequences of GroEL from L. interrogans Copenhageni strain Fiocruz L1–130 and the most conserved sequences from each leptospiral taxid in the NCBI databases, as described in Table S1. Strains highlighted in yellow are saprophytic. The equatorial domain (subdomains E1 and E2) is indicated in blue box, intermediate domain (subdomains I1 and I2) in red box and apical domain in green box. The conserved motifs were underlined. (*) asterisk indicates identical residues (fully conserved) in all sequences; (:) colon shows conservation between residues with similar proprieties (strong conservation), and (.) period and gray shaded amino acids represent conservation between residues with weakly similar properties. Yellow shaded amino acids are divergent residues. Figure S3. a) Analysis of purified proteins. b) Proteinase K susceptibility of recombinant proteins, which were incubated with two different concentrations of proteinase K (1 and 100 μg/mL). Protein bands were visualized by Coomassie blue staining on 12% SDS-PAGE. M: molecular weight protein markers. Figure S4. Full-length Western blots of Fig. 2. Subcellular localization of the GroEL protein. L. interrogans serovar Copenhageni strain Fiocruz L1–130 was cultivated at 29 °C and in conditions that mimics the host environment, with shift temperatures from 29 to 37 °C for 5 h and in osmolarity of 300 mOsm. a) Whole-cell lysates (W) and cell culture supernatant fractions (S) were analyzed by immunoblotting using anti-GroEL and anti-LipL31 (positive control of cytoplasmatic protein) antisera. b) Whole cell (W), Triton X-114 fractions (A and D), and culture supernatant fraction (S) were analyzed by immunoblotting with anti-GroEL and anti-LipL31. c) Proteinase K accessibility assay. Intact leptospires were incubated with different concentrations of proteinase K and processed for immunoblot analyses using antibodies against GroEL, LipL31 or LigA (positive control of outer membrane protein). Protein ladder used was the color prestained protein Standard, broad range (Biolabs). Figure S5. Full-length Western Blot Overlay of Fig. 4. Collagen type I, collagen type IV, laminin, plasminogen, plasma fibronectin, fibrinogen, and BSA (negative control) were subjected to 4–20% gradient gel (SDS PAGE) under reducing conditions, transferred to nitrocellulose membranes and stained with Ponceau S (right). After incubation of membrane with recombinant proteins, the binding was detected with polyclonal antibodies recognizing Lp25, LigBC and GroEL. Chains of host proteins are indicated by γ (400 kDa), β (270 kDa), α2 (130 kDa), α1 (115 kDa) of collagen type I; α1 (140 kDa), α2 (160 kDa) of collagen type IV; C1 (400 kDa) and C2 (200 kDa) of laminin; α (220 kDa) and β (212 kDa) of plasma fibronectin; α (63.5 kDa), β (56 kDa), γ (47 kDa) of fibrinogen; plasminogen and BSA with 70 kDa and 66 kDa, respectively. Lp25 and LigBC were used as negative and positive binding controls, respectively. Protein ladder used was the prestained full-range rainbow RPN800E (Amersham). Table S1. Name, accession taxid numbers, and sequence identity of GroEL.