title: Corynebacterium diphtheriae 67-72p hemagglutinin, characterized as the protein DIP0733, contributes to invasion and induction of apoptosis in HEp-2 cells.
abstract: Although Corynebacterium diphtheriae has been classically described as an exclusively extracellular pathogen, there is growing evidence that it may be internalized by epithelial cells. The aim of the present report was to investigate the nature and involvement of the surface-exposed non-fimbrial 67-72 kDa proteins (67-72p), previously characterized as adhesin/hemagglutinin, in C. diphtheriae internalization by HEp-2 cells. Transmission electron microscopy and bacterial internalization inhibition assays indicated the role of 67-72p as invasin for strains of varied sources. Cytoskeletal changes with accumulation of polymerized actin in HEp-2 cells beneath adherent 67-72p-adsorbed microspheres were observed by the Fluorescent actin staining test. Trypan blue staining method and Methylthiazole tetrazolium reduction assay showed a significant decrease in viability of HEp-2 cells treated with 67-72p. Morphological changes in HEp-2 cells observed after treatment with 67-72p included vacuolization, nuclear fragmentation and the formation of apoptotic bodies. Flow cytometry revealed an apoptotic volume decrease in HEp-2 cells treated with 67-72p. Moreover, a double-staining assay using Propidium Iodide/Annexin V gave information about the numbers of vital vs. early apoptotic cells and late apoptotic or secondary necrotic cells. The comparative analysis of MALDI-TOF MS experiments with the probes provided for 67-72p CDC-E8392 with an in silico proteome deduced from the complete genome sequence of C. diphtheriae identified with significant scores 67-72p as the protein DIP0733. In conclusion, DIP0733 (67-72p) may be directly implicated in bacterial invasion and apoptosis of epithelial cells in the early stages of diphtheria and C. diphtheriae invasive infection.
authors.affiliation: Faculdade de Ciências Médicas, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, RJ, Brazil.
authors.forename: Priscila Soares
authors.initials: PS
authors.lastname: Sabbadini
authors.forename: Maria Cristina
authors.initials: MC
authors.lastname: Assis
authors.forename: Eva
authors.initials: E
authors.lastname: Trost
authors.forename: Débora Leandro Rama
authors.initials: DL
authors.lastname: Gomes
authors.forename: Lilian Oliveira
authors.initials: LO
authors.lastname: Moreira
authors.forename: Cíntia Silva
authors.initials: CS
authors.lastname: Dos Santos
authors.forename: Gabriela Andrade
authors.initials: GA
authors.lastname: Pereira
authors.forename: Prescilla Emy
authors.initials: PE
authors.lastname: Nagao
authors.forename: Vasco Ariston de Carvalho
authors.initials: VA
authors.lastname: Azevedo
authors.forename: Raphael
authors.initials: R
authors.lastname: Hirata Júnior
authors.forename: André Luis Souza
authors.initials: AL
authors.lastname: Dos Santos
authors.forename: Andreas
authors.initials: A
authors.lastname: Tauch
authors.forename: Ana Luíza
authors.initials: AL
authors.lastname: Mattos-Guaraldi
mesh_terms: D000199:Actins / Q000378:metabolism; D017209:Apoptosis; D002460:Cell Line; D002470:Cell Survival; D003353:Corynebacterium diphtheriae / Q000235:genetics / Q000472:pathogenicity*; D004705:Endocytosis; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism*; D022781:Hepatocytes / Q000382:microbiology* / Q000502:physiology*; D006801:Humans; D055503:Protein Multimerization; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000199:Actins; D006388:Hemagglutinins; D037521:Virulence Factors