RESUMO
Bordetella pertussis, the causative agent of whooping cough, secretes and releases adenylate cyclase toxin (ACT), which is a protein bacterial toxin that targets host cells and disarms immune defenses. ACT binds filamentous haemagglutinin (FHA), a surface-displayed adhesin, and until now, the consequences of this interaction were unknown. A B. bronchiseptica mutant lacking ACT produced more biofilm than the parental strain; leading Irie et al. to propose the ACT-FHA interaction could be responsible for biofilm inhibition. Here we characterize the physical interaction of ACT with FHA and provide evidence linking that interaction to inhibition of biofilm in vitro. Exogenous ACT inhibits biofilm formation in a concentration-dependent manner and the N-terminal catalytic domain of ACT (AC domain) is necessary and sufficient for this inhibitory effect. AC Domain interacts with the C-terminal segment of FHA with â¼650 nM affinity. ACT does not inhibit biofilm formation by Bordetella lacking the mature C-terminal domain (MCD), suggesting the direct interaction between AC domain and the MCD is required for the inhibitory effect. Additionally, AC domain disrupts preformed biofilm on abiotic surfaces. The demonstrated inhibition of biofilm formation by a host-directed protein bacterial toxin represents a novel regulatory mechanism and identifies an unprecedented role for ACT.
Assuntos
Toxina Adenilato Ciclase/metabolismo , Adesinas Bacterianas/metabolismo , Biofilmes/crescimento & desenvolvimento , Bordetella bronchiseptica/metabolismo , Bordetella pertussis/fisiologia , Fatores de Virulência de Bordetella/metabolismo , Toxina Adenilato Ciclase/genética , Adesinas Bacterianas/genética , Bordetella bronchiseptica/genética , Bordetella pertussis/genética , Bordetella pertussis/metabolismo , Hemaglutininas/metabolismo , Fatores de Virulência de Bordetella/genéticaRESUMO
As the COVID-19 pandemic transitions into endemicity, seasonal boosters are a plausible reality across the globe. We hypothesize that intranasal vaccines can provide better protection against asymptomatic infections and more transmissible variants of SARS-CoV-2. To formulate a protective intranasal vaccine, we utilized a VLP-based platform. Hepatitis B surface antigen-based virus like particles (VLP) linked with receptor binding domain (RBD) antigen were paired with the TLR4-based agonist adjuvant, BECC 470. K18-hACE2 mice were primed and boosted at four-week intervals with either VLP-RBD-BECC or mRNA-1273. Both VLP-RBD-BECC and mRNA-1273 vaccination resulted in production of RBD-specific IgA antibodies in serum. RBD-specific IgA was also detected in the nasal wash and lung supernatants and were highest in VLP-RBD-BECC vaccinated mice. Interestingly, VLP-RBD-BECC vaccinated mice showed slightly lower levels of pre-challenge IgG responses, decreased RBD-ACE2 binding inhibition, and lower neutralizing activity in vitro than mRNA-1273 vaccinated mice. Both VLP-RBD-BECC and mRNA-1273 vaccinated mice were protected against challenge with a lethal dose of Delta variant SARS-CoV-2. Both vaccines limited viral replication and viral RNA burden in the lungs of mice. CXCL10 is a biomarker of severe SARS-CoV-2 infection and we observed both vaccines limited expression of serum and lung CXCL10. Strikingly, VLP-RBD-BECC when administered intranasally, limited lung inflammation at early timepoints that mRNA-1273 vaccination did not. VLP-RBD-BECC immunization elicited antibodies that do recognize SARS-CoV-2 Omicron variant. However, VLP-RBD-BECC immunized mice were protected from Omicron challenge with low viral burden. Conversely, mRNA-1273 immunized mice had low to no detectable virus in the lungs at day 2. Together, these data suggest that VLP-based vaccines paired with BECC adjuvant can be used to induce protective mucosal and systemic responses against SARS-CoV-2.
Assuntos
COVID-19 , SARS-CoV-2 , Animais , Humanos , Camundongos , Vacina de mRNA-1273 contra 2019-nCoV , Pandemias , COVID-19/prevenção & controle , Adjuvantes Imunológicos , Imunoglobulina A , Anticorpos Antivirais , Anticorpos Neutralizantes , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
Progress in understanding long COVID and developing effective therapeutics is hampered in part by the lack of suitable animal models. Here we used ACE2-transgenic mice recovered from Omicron (BA.1) infection to test for pulmonary and behavioral post-acute sequelae. Through in-depth phenotyping by CyTOF, we demonstrate that naïve mice experiencing a first Omicron infection exhibit profound immune perturbations in the lung after resolving acute infection. This is not observed if mice were first vaccinated with spike-encoding mRNA. The protective effects of vaccination against post-acute sequelae were associated with a highly polyfunctional SARS-CoV-2-specific T cell response that was recalled upon BA.1 breakthrough infection but not seen with BA.1 infection alone. Without vaccination, the chemokine receptor CXCR4 was uniquely upregulated on multiple pulmonary immune subsets in the BA.1 convalescent mice, a process previously connected to severe COVID-19. Taking advantage of recent developments in machine learning and computer vision, we demonstrate that BA.1 convalescent mice exhibited spontaneous behavioral changes, emotional alterations, and cognitive-related deficits in context habituation. Collectively, our data identify immunological and behavioral post-acute sequelae after Omicron infection and uncover a protective effect of vaccination against post-acute pulmonary immune perturbations.
RESUMO
Whole cell vaccines are complex mixtures of antigens, immunogens, and sometimes adjuvants that can trigger potent and protective immune responses. In some instances, such as whole cell Bordetella pertussis vaccination, the immune response to vaccination extends beyond the pathogen the vaccine was intended for and contributes to protection against other clinically significant pathogens. In this study, we describe how B. pertussis whole cell vaccination protects mice against acute pneumonia caused by Pseudomonas aeruginosa. Using ELISA and western blot, we identified that B. pertussis whole cell vaccination induces production of antibodies that bind to lab-adapted and clinical strains of P. aeruginosa, regardless of immunization route or adjuvant used. The cross-reactive antigens were identified using immunoprecipitation, mass spectrometry, and subsequent immunoblotting. We determined that B. pertussis GroEL and OmpA present in the B. pertussis whole cell vaccine led to production of antibodies against P. aeruginosa GroEL and OprF, respectively. Finally, we showed that recombinant B. pertussis OmpA was sufficient to induce protection against P. aeruginosa acute murine pneumonia. This study highlights the potential for use of B. pertussis OmpA as a vaccine antigen for prevention of P. aeruginosa infection, and the potential of broadly protective antigens for vaccine development.
RESUMO
Pseudomonas aeruginosa is a Gram negative, opportunistic pathogen that uses the overproduction of alginate, a surface polysaccharide, to form biofilms in vivo. Overproduction of alginate, also known as mucoidy, affords the bacterium protection from the host's defenses and facilitates the establishment of chronic lung infections in individuals with cystic fibrosis. Expression of the alginate biosynthetic operon is primarily controlled by the alternative sigma factor AlgU (AlgT/σ(22) ). In a nonmucoid strain, AlgU is sequestered by the transmembrane antisigma factor MucA to the cytoplasmic membrane. AlgU can be released from MucA via regulated intramembrane proteolysis by proteases AlgW and MucP causing the conversion to mucoidy. Pseudomonas aeruginosa strain PAO579, a derivative of the nonmucoid strain PAO1, is mucoid due to an unidentified mutation (muc-23). Using whole genome sequencing, we identified 16 nonsynonymous and 15 synonymous single nucleotide polymorphisms (SNP). We then identified three tandem single point mutations in the pilA gene (PA4525), as the cause of mucoidy in PAO579. These tandem mutations generate a premature stop codon resulting in a truncated version of PilA (PilA(108) ), with a C-terminal motif of phenylalanine-threonine-phenylalanine (FTF). Inactivation of pilA(108) confirmed it was required for mucoidy. Additionally, algW and algU were also required for mucoidy of PAO579. Western blot analysis indicated that MucA was less stable in PAO579 than nonmucoid PAO1 or PAO381. The mucoid phenotype and high PalgU and PalgD promoter activities of PAO579 require pilA(108) , algW, algU, and rpoN encoding the alternative sigma factor σ(54) . We also observed that RpoN regulates expression of algW and pilA in PAO579. Together, these results suggest that truncation in type IV pilin in P. aeruginosa strain PAO579 can induce mucoidy through an AlgW/AlgU-dependent pathway.