Label-free proteomic analysis of intracellular Bordetella pertussis

LAMBERTI, YANINA; SCHMIDT, FRANK; SURMANN, KRISTIN; CAFIERO, HILARIO; RODRIGUEZ, MARIA EUGENIA

Congreso; 10th International Symposium on Bordetella; 2013

Previous studies indicated that B. pertussis can invade and replicate within human macrophages. In order to investigate factors involved in the intracellular survival, we analyzed the proteome of intracellular B. pertussis. To this end, THP-I differentiated into macrophages were infected with GFP-expressing B. pertussis (100 bacteria per cell) and incubated for 2 and 48 h. Intracellular bacteria were isolated by sucrose gradient and bacterial proteins were identified using nano-HPLC coupled to a mass spectrometer (MS/MS).. We observed stark differences in gene expression profiles of intracellular bacteria as compared to extracellular bacteria. Around 200 proteins showed significant differences in the level of expression (P< 0.05). Adaptive intracellular gene expression involved proteins associated with virulence, stress response, transport, and many genes with unknown functions. Proteins involved in stress response were induced early after infection, including ClpX, GroEL and HsIU. Two proteins involved in iron uptake were found upregulated as early as 2 h post-infection and remained over expressed 48 h post-infection. These proteins are BfrD and BfrE, recently identified as TonB-dependent outer membrane catecholamine receptors involved in iron uptake from transferrin in B. bronchiseptica. Interestingly, we found at least three proteins of the Type III Secretion System (TTSS) up regulated already at 2 h after infection, the proteins BteA, BscC and BopN, suggesting that TTSS becomes operative when bacteria infect human macrophages. These results represent the first characterization of intracellular B. pertussis proteome and demonstrate that this pathogen undergoes an adaptive response that enables the development of an intracellular infection.