Martino, R. A. | bioRxiv Science

bioRxiv2025

Genetic dissection of cyclic di-GMP signaling in Pseudomonas aeruginosa via diguanylate cyclase disruption

The second messenger bis-(3'[->]5')-cyclic dimeric guanosine monophosphate (c-di-GMP) governs adaptive responses in the opportunistic pathogen Pseudomonas aeruginosa, including biofilm formation and the transition from acute to chronic infections. Understanding the intricate c-di-GMP signaling network remains challenging due to the overlapping activities of numerous diguanylate cyclases (DGCs). In this study, we employed a CRISPR-based multiplex genome-editing tool to disrupt all 32 GGDEF domain-containing proteins (GCPs) implicated in c-di-GMP signaling in P. aeruginosa UCBPP-PA14. Phenotypic and physiological analyses revealed that the resulting mutant was unable to form biofilms and had attenuated virulence. Residual c-di-GMP levels were still detected despite the extensive GCP disruption, underscoring the robustness of this regulatory network. Taken together, these findings provide insights into the complex c-di-GMP metabolism and showcase the importance of functional overlapping in bacterial signaling. Moreover, our design overcomes the native redundancy in c-di-GMP synthesis, providing a framework to dissect individual DGC functions and paving the way for targeted strategies to address bacterial adaptation and pathogenesis.

microbiology↗

bioRxiv2021

Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis. Author summarySARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. This manuscript demonstrates an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity-matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by pre-emergent members, ACE2-dependent CoVs that are poised for zoonosis.

genetics↗