Mariette Barbier

Lyme disease is a tick-borne disease caused by spirochetes of the Borrelia burgdorferi sensu lato complex. Murine models of B. burgdorferi infection allows for the assessment of bacterial dissemination and presence through culture of infected tissue and dark-field microscopic examination after a period of outgrowth. Although valuable, dark-field microscopy can be subjective, complex, and time-consuming. We propose that the limitations of current culture-screening methodologies could be overcome using a fluorescent B. burgdorferi stain for murine challenge studies. To test this, we generated A3-68 Δbbe02 pBBE22G-flgBpGFP, a strain of B. burgdorferi that expresses the fluorescent protein GFP encoded on the plasmid pBBE22G. We then validated that the A3-68 Δbbe02 pBBE22G-flgBpGFP strain ( Bb- GFP) expressed detectable GFP in vitro and that it grew and had a similar infection profile in mice compared to its parental strain, A3-68 Δbbe02 . Next, we compared the accuracy and sensitivity of three different methods to detect B. burgdorferi from infected tissues: flow cytometry, dark-field microscopy, and PCR. In a Bb- GFP murine challenge model, screening of tissue cultures with flow cytometry exhibited sample positivity similar to that of traditional methods, with increased objectivity, speed, and efficiency. Overall, the Bb -GFP fluorescent strain and associated methodology described here provide additional resources for the study of B. burgdorferi pathogenesis. IMPORTANCE: Studying Borrelia burgdorferi pathogenesis can be challenging due to reliance on labor-intensive techniques such as dark-field microscopy to determine the infection status of animals. In this study, we demonstrated that flow cytometry could be used as an alternative approach, allowing for fast, reliable, and comparable results to those of dark-field microscopy in the context of mouse infection models.

Pseudomonas aeruginosa (Pa) is an important opportunistic pathogen that has many virulence factors expressed in a coordinated manner to cause infection. The PilY1 protein is a component of the type IV pili (T4P) but has additional signaling functions outside of the pilus. A major function of PilY1 is to prevent the AlgZ/R two-component system from functioning. However, the complete effects of PilY1 signaling through the control of the AlgZ/R or other regulatory systems are not well understood. In this study, we determine new genes controlled by the AlgZ/R system that are impacted by PilY1. We discovered that PilY1 impacts cAMP by preventing the AlgZ/R system from activating the major adenylate cyclase, cyaB . PilY1 also prevents the AlgZ/R system from activating itself and a putative c-di-GMP phosphodiesterase, PA4781 . Furthermore, PilY1 functions in different P. aeruginosa strains, including mucoid strains. Surprisingly, bacterial survival in the lung, liver, and blood did not require PilY1. Overall, these findings suggest that PilY1 is critical for preventing AlgZ/R activity to prevent improper second messenger production. This work identifies new AlgZ/R targets and implicates the AlgZ/R system in the control of cAMP in P. aeruginosa . This work identifies a possible way to use PilY1 to prevent the expression of the major adenylate cyclase using PilY1 which could be used to impact P. aeruginosa virulence. IMPORTANCE: Understanding PilY1 signaling is important for elucidating how P. aeruginosa adapts to different environments. A major function for PilY1 is to interfere with the AlgZ/R two-component system. Here, we use transcriptomics to determine genes PilY1 affects through the AlgZ/R system. We identified new AlgZ/R targets and established a mechanism for impacting cAMP levels. Our findings further our understanding of PilY1 and the AlgZ/R system and suggest a possible way that PilY1 might be used to prevent cAMP from increasing in P. aeruginosa .

Multidrug-resistant Pseudomonas aeruginosa is a critical pathogen that demands new antibiotics. During P. aeruginosa infection, the extracellular hemophore hasAp and its outer membrane receptor hasR are the most dramatically upregulated genes. The P. aeruginosa ΔhasR strain exhibits significantly reduced growth and virulence. We previously described a gallium salophen complex GaSal that demonstrated antipseudomonal potential by targeting HasAp. Here, we report the development of a water-soluble derivative, GaSal-2, which tightly binds to HasAp, blocks transcriptional activation of the bacterial cell surface signaling cascade, inhibits P. aeruginosa growth, and effectively disrupts P. aeruginosa established biofilms. Moreover, GaSal-2 is not toxic to human lung fibroblasts and hepatocytes. It has shown promising antipseudomonal effects in a murine lung infection model.

Pseudomonas aeruginosa (Pa) is an important opportunistic pathogen that has many virulence factors expressed in a coordinated manner to cause infection. External appendages, such as the type IV pili (T4P), can be used for signal transduction changing gene expression using a mechanism termed mechanosensing. An important role for the minor pilin, PilY1, is to signal that the bacterium has attached to a surface such as a host cell. This mechanosensing is important for triggering changes in bacterial gene expression allowing colonization of host tissues. In this study, we identify new PilY1-controlled genes and demonstrate the importance of PilY1 in controlling second messengers, including cAMP. Genetic analysis showed that PilY1 is important in controlling cAMP levels by regulating the expression of the main adenylate cyclase, cyaB . PilY1 also controls the expression of PA4781 a putative cyclic di-GMP phosphodiesterase. PilY1 also controls the expression of the algZ/R operon by preventing autoregulation of this system. Furthermore, the minor pilin PilY1 functions in different P. aeruginosa strains, including mucoid strains, demonstrating conservation of function. Surprisingly, bacterial survival in the lung, liver, and blood did not require PilY1. Overall, these findings suggest that PilY1 is critical for regulating second messengers by preventing the AlgZ/R system from functioning. Our work provides insight into the mechanisms that P. aeruginosa uses to modulate second messengers which are important in biofilm formation and virulence. Importance. PilY1, a component of type IV pili, is important for signaling environmental cues into the bacterial cell. However, the signaling pathway PilY1 controls is poorly understood. We determined that PilY1 is involved in the regulation of cAMP. Transcriptomic analysis identified new PilY1-controlled genes. We also demonstrate the functionality of PilY1 in other strains, including mucoid strains commonly found in chronic infections. We elucidate the importance of PilY1 controlling cAMP by repressing the AlgZ/R system and preventing cyaB expression. Our data help to explain how regulatory systems interact in P. aeruginosa providing insight that may be useful for therapeutic development.

The incidence of infections attributed to antimicrobial-resistant (AMR) pathogens has increased exponentially over the recent decades reaching 1.27 million deaths worldwide in 2019. Without intervention, these infections are predicted to cause up to 10 million deaths a year and incur costs of up to 100 trillion US dollars globally by 2050. The emergence of AMR bacteria such as the ESKAPEE pathogens, and in particular Pseudomonas aeruginosa and species from the genus Burkholderia , underscores an urgent need for new therapeutic strategies. Monoclonal antibody (mAb) therapy offers a promising alternative to treat and prevent bacterial infections. In this study, we used peptides from highly conserved areas of the bacterial flagellin to generate monoclonal antibodies capable of broad binding to flagellated Gram-negative bacteria. We generated a broadly reactive IgG2bĸ mAb (WVDC-2109) that recognizes P. aeruginosa, Burkholderia sp., and other Gram-negative pathogens of interest. Characterization of the therapeutic potential of this antibody was determined using P. aeruginosa as model. In vitro characterization of WVDC-2109 demonstrated complement-mediated bactericidal activity and enhanced opsonophagocytosis of P. aeruginosa . Prophylactic administration of WVDC-2109 markedly improved survival and outcome in a lethal sepsis model and a sub-lethal murine pneumonia model of P. aeruginosa infection, reducing bacterial burden and inflammation. These findings suggest that WVDC-2109 and similar FliC-targeting antibodies could be valuable in preventing or treating diseases caused by P. aeruginosa as well as other life-threatening diseases of concern. IMPORTANCE: Antimicrobial resistance (AMR) costs hundreds of thousands of lives and billions of dollars annually. To protect the population against these infections, it is imperative to develop new medical countermeasures targeting AMR pathogens like P. aeruginosa and Burkholderia sp. The administration of broadly reactive monoclonal antibodies can represent an alternative to treat and prevent infections caused by multi-drug-resistant bacteria. Unlike vaccines, antibodies can provide protection regardless of the immune status of the infected host. In this study, we generated an antibody capable of recognizing flagellin from P. aeruginosa and B. pseudomallei along with other Gram-negative pathogens of concern. Our findings demonstrate that the administration of the monoclonal antibody WVDC-2109 enhances survival rates and outcomes in different murine models of P. aeruginosa infection. These results carry significant implications in the field given that there are no available vaccines for P. aeruginosa .

Bordetella pertussis, the bacterium responsible for whooping cough, remains a significant public health challenge despite the existing licensed pertussis vaccines. Current acellular pertussis vaccines, though having favorable reactogenicity and efficacy profiles, involve complex and costly production processes. In addition, acellular vaccines have functional challenges such as short-lasting duration of immunity and limited antigen coverage. Filamentous hemagglutinin (FHA) is an adhesin of B. pertussis that is included in all multivalent pertussis vaccine formulations. Antibodies to FHA have been shown to prevent bacterial attachment to respiratory epithelial cells, and T cell responses to FHA facilitate cell-mediated immunity. In this study, FHA’s mature C-terminal domain (MCD) was evaluated as a novel vaccine antigen. MCD was conjugated to virus-like particles via SpyTag-SpyCatcher technology. Prime-boost vaccine studies were performed in mice to characterize immunogenicity and protection against the intranasal B. pertussis challenge. MCD-SpyVLP was more immunogenic than SpyTag-MCD antigen alone, and in Tohama I strain challenge studies, improved protection against challenge was observed in the lungs at day 3 and in the trachea and nasal wash at day 7 post-challenge. Furthermore, a B. pertussis strain encoding genetically inactivated pertussis toxin was used to evaluate MCD-SpyVLP vaccine immunity. Mice vaccinated with MCD-SpyVLP had significantly lower respiratory bacterial burden at both days 3 and 7 post-challenge compared to mock-vaccinated animals. Overall, these data support the use of SpyTag-SpyCatcher VLPs as a platform for use in vaccine development against B. pertussis and other pathogens.

Bordetella pertussis is a Gram-negative bacterium that is the causative agent of the respiratory disease known as pertussis. Since the switch to the acellular vaccines of DTaP and Tap, pertussis cases in the US have risen and cyclically fallen. We have observed that mRNA pertussis vaccines are immunogenic and protective in mice. Here, we further evaluated the pertussis toxoid mRNA antigen and refined the formulation based on optimal pertussis toxin neutralization in vivo . We next evaluated the mRNA pertussis vaccine in Sprague-Dawley rats using an aerosol B. pertussis challenge model paired with whole-body plethysmography to monitor coughing and respiratory function. Female Sprague-Dawley rats were primed and boosted with either commercially available vaccines (DTaP or wP-DTP), an mRNA-DTP vaccine, or mock-vaccinated. The mRNA-DTP vaccine was immunogenic in rats and induced antigen-specific IgG antibodies comparable to DTaP. Rats were then aerosol challenged with a streptomycin-resistant emerging clinical isolate D420Sm1. Bacterial burden was assessed at days 1 and 9 post-challenge, and the mRNA vaccine reduced burden equal to both DTaP and wP-DTP. Whole-body plethysmography revealed that mRNA-DTP vaccinated rats were well protected against coughing which was comparable to the non-challenged group. These data suggest that an mRNA-DTP vaccine is immunogenic in rats and provides protection against aerosolized B. pertussis challenge in Sprague-Dawley rats.

Acellular multivalent vaccines for pertussis (DTaP and Tdap) prevent symptomatic disease and infant mortality, but immunity to Bordetella pertussis infection wanes significantly over time resulting in cyclic epidemics of pertussis. The messenger RNA (mRNA) vaccine platform provides an opportunity to address complex bacterial infections with an adaptable approach providing Th1-biased responses. In this study, immunogenicity and challenge models were used to evaluate the mRNA platform with multivalent vaccine formulations targeting both B. pertussis antigens and diphtheria and tetanus toxoids. Immunization with mRNA formulations were immunogenetic, induced antigen specific antibodies, as well as Th1 T cell responses. Upon challenge with either historical or contemporary B. pertussis strains, 6 and 10 valent mRNA DTP vaccine provided protection equal to that of 1/20th human doses of either DTaP or whole cell pertussis vaccines. mRNA DTP immunized mice were also protected from pertussis toxin challenge as measured by prevention of lymphocytosis and leukocytosis. Collectively these pre-clinical mouse studies illustrate the potential of the mRNA platform for multivalent bacterial pathogen vaccines.