Proteomic and phosphoproteomic analyses to unravel relevant aspects of Bordetella pertussis pathogenesis

Bordetella pertussis is the etiological agent of whooping cough (or pertussis), a highly contagious, though vaccine-preventable, respiratory illness that is especially life-threatening to young children and is experiencing a global resurgence. Pertussis resurgence can be attributed to several factors, including waning immunity after vaccination with current vaccines and the emergence of B. pertussis strains with an adapted genome and virulence factor repertoire. Furthermore, intracellular persistence allows B. pertussis to evade the host's immune system, contributing to infection and transmission. Therefore, understanding the mechanisms underlying B. pertussis virulence regulation during intracellular persistence is essential for developing novel preventive strategies, to avoid intracellular persistence upon infection and subsequently, transmission.

Previously, we used a proteomics approach to investigate B. pertussis" adaptation to human macrophages. Currently, we are monitoring the proteome adaptation of this pathogen, as well as the host adaptation after bacterial infection, in polarized human bronchial epithelial 16HBE14o- cells. To achieve this, we use a combined approach of fluorescence-based cell sorting and mass spectrometry in data independent mode. These data indicate a niche specific adaptation of B. pertussis, e.g. by different abundance changes of proteins involved in bacterial stress response and of some virulence factors upon internalization.

However, the mechanisms of how B. pertussis adapts its proteomic profile to host-provided environmental conditions, remain poorly understood. In bacteria, genetic adaptation is primarily governed by two-component systems (TCS) with the BvgAS system serving as the master regulator of virulence genes in B. pertussis. Besides BvgAS, only two more (PlrRS, RisA/RisK) out of about 20 potential TCS have been functionally characterized in B. pertussis. Recently, we found that BP1092, a histidine kinase putatively belonging to a TCS, is involved in the fine-tuning of virulence factor regulation in B. pertussis. In order to understand the function of until now poorly characterized TCS, we apply phosphoproteomics of B. pertussis under different infection-mimicking conditions. To do so, we cultivate B. pertussis under standard conditions as well as e.g., during iron and oxygen limitation and harvest bacterial samples in the late exponential growth phase. Then, we apply phosphoproteomic protocols adapted to reproducibly detect histidine- and aspartate phosphorylation at TCS components (e.g., Potel et al., Nat Methods, 2018). By doing so, we expect to characterize the activity of yet undescribed TCS.

To summarize, we show that B. pertussis is able to adapt to several niches of the human host by specific proteome adaptations and we provide a method to further improve our understanding of the regulation of adaptation processes relevant during pertussis infection.