BPP0974 is a Bordetella parapertussis adhesin expressed in the avirulent phase, implicated in biofilm formation and intracellular survival.

B. parapertussis is a bacterium that causes whooping cough, a severe respiratory infection disease, that has shown an increased incidence in the population. Upon transmission through aerosol droplets, the initial steps of host colonization critically depend on the bacterial adhesins. We here described BPP0974, a B. parapertussis protein that exhibits the typical domain architecture of the large repetitive RTX adhesin family. BPP0974 was found to be retained in the bacterial membrane and secreted into the culture medium. This protein was found overexpressed in the avirulent phase of B. parapertussis, the phenotype proposed for initial host colonization. Interestingly, BPP0974 was found relevant for the biofilm formation as well as involved in the bacterial attachment to and survival within the respiratory epithelial cells. Taken together, our results suggest a role for BPP0974 in the early host colonization and pathogenesis of B. parapertussis.

Adenylate cyclase toxin of Bordetella parapertussis disrupts the epithelial barrier granting the bacterial access to the intracellular space of epithelial cells.

B. parapertussis is one of the etiological agents of whooping cough. Once inhaled, the bacteria bind to the respiratory epithelium and start the infection. Little is known about this first step of host colonization and the role of the human airway epithelial barrier on B. parapertussis infection. We here investigated the outcome of the interaction of B. parapertussis with a polarized monolayer of respiratory epithelial cells. Our results show that B. parapertussis preferentially attaches to the intercellular boundaries, and causes the disruption of the tight junction integrity through the action of adenylate cyclase toxin (CyaA). We further found evidence indicating that this disruption enables the bacterial access to components of the basolateral membrane of epithelial cells to which B. parapertussis efficiently attaches and gains access to the intracellular location, where it can survive and eventually spread back into the extracellular environment. Altogether, these results suggest that the adenylate cyclase toxin enables B. parapertussis to overcome the epithelial barrier and eventually establish a niche of persistence within the respiratory epithelial cells.

Receptor-binding domain-based SARS-CoV-2 vaccine adjuvanted with cyclic di-adenosine monophosphate enhances humoral and cellular immunity in mice.

Novel adjuvants are highly desired to improve immune responses of SARS-CoV-2 vaccines. This work reports the potential of the stimulator of interferon genes (STING) agonist adjuvant, the cyclic di-adenosine monophosphate (c-di-AMP), in a SARS-CoV-2 vaccine based on the receptor binding domain (RBD). Here, mice immunized with two doses of monomeric RBD adjuvanted with c-di-AMP intramuscularly were found to exhibit stronger immune responses compared to mice vaccinated with RBD adjuvanted with aluminum hydroxide (Al(OH)3 ) or without adjuvant. After two immunizations, consistent enhancements in the magnitude of RBD-specific immunoglobulin G (IgG) antibody response were observed by RBD + c-di-AMP (mean: 15360) compared to RBD + Al(OH)3 (mean: 3280) and RBD alone (n.d.). Analysis of IgG subtypes indicated a predominantly Th1-biased immune response (IgG2c, mean: 14480; IgG2b, mean: 1040, IgG1, mean: 470) in mice vaccinated with RBD + c-di-AMP compared to a Th2-biased response in those vaccinated with RBD + Al(OH)3 (IgG2c, mean: 60; IgG2b: n.d.; IgG1, mean: 16660). In addition, the RBD + c-di-AMP group showed better neutralizing antibody responses as determined by pseudovirus neutralization assay and by plaque reduction neutralization assay with SARS-CoV-2 wild type. Moreover, the RBD + c-di-AMP vaccine promoted interferon-γ secretion of spleen cell cultures after RBD stimulation. Furthermore, evaluation of IgG-antibody titers in aged mice showed that di-AMP was able to improve RBD-immunogenicity at old age after 3 doses (mean: 4000). These data suggest that c-di-AMP improves immune responses of a SARS-CoV-2 vaccine based on RBD, and would be considered a promising option for future COVID-19 vaccines.

Bordetella parapertussis adenylate cyclase toxin promotes the bacterial survival to the encounter with macrophages.

B. parapertussis is a whooping cough etiological agent, whose incidence in the population has increased remarkably. Virulence factors involved in the bacterial infection, however, remain poorly investigated. We here studied the role of adenylate cyclase (CyaA), the main toxin of B. parapertussis, in the outcome of the bacterial interaction with macrophages. Our results showed that B. parapertussis CyaA intoxicates human macrophages, prevents bacterial phagocytosis and precludes phago-lysosomal fusion eventually promoting the bacterial survival to the encounter with these immune cells. Accordingly, we found that B. parapertussis CyaA induces the transcriptional downregulation of host genes encoding for antimicrobial peptides, proteins involved in bacterial intracellular killing, and the pro-inflammatory cytokine TNF-α, while induces the upregulation of the anti-inflammatory cytokine IL-10. Together with previous reports suggesting a protective role of B. parapertussis CyaA against neutrophils bactericidal activity, the results of this study suggest a central role of CyaA in B. parapertussis immune evasion and persistence.

Bordetella pertussis modulates human macrophage defense gene expression.

Bordetella pertussis, the etiological agent of whooping cough, still causes outbreaks. We recently found evidence that B. pertussis can survive and even replicate inside human macrophages, indicating that this host cell might serve as a niche for persistence. In this work, we examined the interaction of B. pertussis with a human monocyte cell line (THP-1) that differentiates into macrophages in culture in order to investigate the host cell response to the infection and the mechanisms that promote that intracellular survival. To that end, we investigated the expression profile of a selected number of genes involved in cellular bactericidal activity and the inflammatory response during the early and late phases of infection. The bactericidal and inflammatory response of infected macrophages was progressively downregulated, while the number of THP-1 cells heavily loaded with live bacteria increased over time postinfection. Two of the main toxins of B. pertussis, pertussis toxin (Ptx) and adenylate cyclase (CyaA), were found to be involved in manipulating the host cell response. Therefore, failure to express either toxin proved detrimental to the development of intracellular infections by those bacteria. Taken together, these results support the relevance of host defense gene manipulation to the outcome of the interaction between B. pertussis and macrophages.