Identification of potential virulence factors of Cronobacter sakazakii isolates by comparative proteomic analysis

• The virulence differences of C. sakazakii isolates were determined by animal experiment and histological analysis.
• Proteomic profiles of virulent and attenuated isolates were firstly developed by 2-DE.
• Potential virulence factors such as Dps, OmpA, LuxS were identified by MAIDI/TOF/TOF coupled with MS.
• Expression abundance of Dps, OmpA, and LuxS genes in G362 were increased using qPCR.

Cronobacter is a group of important foodborne pathogens associated with neonatal meningitis, septicemia, and necrotizing enterocolitis. Among Cronobacter species, Cronobacter sakazakii is the most common species in terms of isolation frequency. However, the molecular basis involved in virulence differences among C. sakazakii isolates is still unknown. In this study, based on the determination of virulence differences of C. sakazakii G362 (virulent isolate) and L3101 (attenuated isolate) through intraperitoneal injection, histopathologic analysis (small intestine, kidney, and liver) further confirmed virulence differences. Thereafter, the potential virulence factors were determined using two-dimensional electrophoresis (2-DE) coupled with MALDI/TOP/TOF mass spectrometry. Among a total of 36 protein spots showing differential expression (fold change > 1.2), we identified 31 different proteins, of which the expression abundance of 22 was increased in G362. These up-regulated proteins in G362 mainly contained DNA starvation/stationary phase protection protein Dps, OmpA, LuxS, ATP-dependent Clp protease ClpC, and ABC transporter substrate-binding proteins, which might be involved in virulence of C. sakazakii. This is the first report to determine the potential virulence factors of C. sakazakii isolates at the proteomic levels.