Bordetella pertussis targets the basolateral membrane of polarized respiratory epithelial cells, gets internalized, and survives in intracellular locations.

The airway epithelial barrier is a continuous highly organized cell layer that separates the exterior from the underlying mucosal tissue, preventing pathogen invasion. Several respiratory pathogens have evolved mechanisms to compromise this barrier, invade and even reside alive within the epithelium. Bordetella pertussis is a persistent pathogen that infects the human airway epithelium, causing whooping cough. Previous studies have shown that B. pertussis survives inside phagocytic and nonphagocytic cells, suggesting that there might be an intracellular stage involved in the bacterial infectious process and/or in the pathogen persistence inside the host. In this study we found evidence that B. pertussis is able to survive inside respiratory epithelial cells. According to our results, this pathogen preferentially attaches near or on top of the tight junctions in polarized human bronchial epithelial cells and disrupts these structures in an adenylate cyclase-dependent manner, exposing their basolateral membrane. We further found that the bacterial internalization is significantly higher in cells exposing this membrane compared with cells only exposing the apical membrane. Once internalized, B. pertussis mainly remains in nondegradative phagosomes with access to nutrients. Taken together, these results point at the respiratory epithelial cells as a potential niche of persistence.

Bordetella pertussis outer membrane vesicles as virulence factor vehicles that influence bacterial interaction with macrophages.

Gram-negative pathogenic bacteria constitutively shed outer membrane vesicles (OMVs) which play a significant role in the host-pathogen interaction, eventually determining the outcome of the infection. We previously found that Bordetella pertussis, the etiological agent of whooping cough, survives the innate interaction with human macrophages remaining alive inside these immune cells. Adenylate cyclase (CyaA), one of the main toxins of this pathogen, was found involved in the modulation of the macrophage defense response, eventually promoting bacterial survival within the cells. We here investigated whether B. pertussis OMVs, loaded with most of the bacterial toxins and CyaA among them, modulate the macrophage response to the bacterial infection. We observed that the pre-incubation of macrophages with OMVs led to a decreased macrophage defense response to the encounter with the bacteria, in a CyaA dependent way. Our results suggest that CyaA delivered by B. pertussis OMVs dampens macrophages protective function by decreasing phagocytosis and the bactericidal capability of these host cells. By increasing the chances of bacterial survival to the innate encounter with the macrophages, B. pertussis OMVs might play a relevant role in the course of infection, promoting bacterial persistence within the host and eventually, shaping the whole infection process.

Hfq modulates global protein pattern and stress response in Bordetella pertussis.

B. pertussis is the etiological agent of whooping cough, a highly contagious respiratory disease which remains uncontrolled worldwide. Understanding how this pathogen responds to the environmental changes and adapts to different niches found inside the host might contribute to gain insight into bacterial pathogenesis. Comparative analyses of previous transcriptomic and proteomic data suggested that post-transcriptional regulatory mechanisms modulate B. pertussis virulence in response to iron availability. Iron scarcity represents one of the major stresses faced by bacterial pathogens inside the host. In this study, we used gel-free nanoLC-MS/MS-based proteomics to investigate whether Hfq, a highly conserved post-transcriptional regulatory protein, is involved in B. pertussis adaptation to low iron environment. To this end, we compared the protein profiles of wild type B. pertussis and its isogenic hfq deletion mutant strain under iron-replete and iron-depleted conditions. Almost of 33% of the proteins identified under iron starvation was found to be Hfq-dependent. Among them, proteins involved in oxidative stress tolerance and virulence factors that play a key role in the early steps of host colonization and bacterial persistence inside the host cells. Altogether these results suggest that Hfq shapes the infective phenotype of B. pertussis. SIGNIFICANCE: In the last years, it became evident that post-transcriptional regulation of gene expression in ba cteria plays a central role in host-pathogen interactions. Hfq is a bacterial protein that regulates gene expression at post-transcriptional level found pivotal in the establishment of successful infections. In this study, we investigated the role of Hfq in Bordetella pertussis response to iron starvation, one of the main stresses imposed by the host. The data demonstrate that Hfq regulates the abundance of a significant number of B. pertussis proteins in response to iron starvation. Among them, virulence factors and proteins involved in oxidative stress tolerance, key players in host colonization and intracellular bacterial survival. Altogether, our results suggest a relevant role of Hfq in B. pertussis adaptation to the different niches found inside the host eventually granting bacterial pathogenesis.