Conclusions The results obtained in this study show that adhesion and invasion are not necessarily coupled processes. Adhesion rates are not strictly correlated with pili formation and in summary the pili repertoire of the investigated LEE011 cell line strains is highly variable. As shown by genome comparisons [23] it is check details necessary to investigate various isolates on a molecular level to understand and to predict the colonization process of different C. diphtheriae strains.

Methods Bacterial strains and growth Strains used in this study are listed in Table 1. C. diphtheriae strains were grown in Heart Infusion (HI) broth or on Columbia agar with sheep blood (Oxoid, Wesel, Germany) at 37°C. S. Typhimurium and Escherichia coli DH5αMCR were grown in Luria Broth (LB) [25] at 37°C. If appropriate, kanamycin was added (30 μg ml-1 for E. coli; 50 μg ml-1 for C. diphtheriae).

Table 1 Bacterial strains and eukaryotic cell lines used in this study Strains Description Reference C. diphtheriae     DSM43988 non-toxigenic, isolated from throat culture DSMZ, Braunschweig, Germany DSM43989 tox +, unknown source DSMZ, Braunschweig, Germany DSM44123 non-toxigenic isolate, type-strain, unknown source DSMZ, Braunschweig, Germany ISS3319 C. diphtheriae var. mitis, non-toxigenic, isolated from patients affected by pharyngitis/tonsilitis [1] ISS4060 selleck kinase inhibitor C. diphtheriae var. gravis, non-toxigenic, isolated from patients affected by pharyngitis/tonsilitis [1] ISS4746 C. diphtheriae var. gravis, non-toxigenic, isolated from patients affected by pharyngitis/tonsilitis [21] ISS4749 C. diphtheriae

var. gravis, non-toxigenic, isolated from patients affected by pharyngitis/tonsilitis [21] NCTC13129 C. diphtheriae var. gravis, non-toxigenic, isolated from pharyngeal membrane, patient with clinical diphtheria [2] E. coli     DH5αMCR endA1 supE44 thi-1 λ- recA1 gyrA96 relA1 deoR Δ(lacZYA-argF) U196 ϕ80ΔlacZ ΔM15mcrA Δ(mmr hsdRMSmcrBC) [9] Salmonella enterica serovar Typhimurium ( S . Typhimurium)     NCTC12023 wild type identical to ATCC14028 NCTC, Colindale, UK Cell lines     Detroit562 human hypopharyngeal carcinoma cells [20] Transformation of competent C. diphtheriae 3-oxoacyl-(acyl-carrier-protein) reductase For preparation of electrocompetent cells, 10 ml of an overnight culture of C. diphtheriae were inoculated in 200 ml of Brain Heart Infusion (BHI) containing 2% glycine and 15% sucrose, at 37°C in an orbital shaker until an OD600 nm of 0.5 was reached. After storing the cells on ice for 15 min, bacteria were harvested by centrifugation (4,000 × g, 4°C), washed thrice with 15% glycerol, and resuspended in 1 ml of 15% glycerol. 100 μl aliquots of the competent cells were frozen in liquid nitrogen and stored at -80°C. For transformation the aliquots were thawed on ice. Plasmid DNA used for transformation was extracted from E. coli strain DH5αMCR, which is unable to methylate DNA. One microgram of plasmid DNA was used to transform C. diphtheriae cells using a GenePulser II apparatus (Bio-Rad, Munich, Germany) and 200 Ω, 2.5 kV, 25 μF.