Quinazoline-Based Antivirulence Compounds Selectively Target Salmonella PhoP/PhoQ Signal Transduction System

The rapid emergence of multidrug resistance among bacterial pathogens has become a significant challenge to human health in our century. Therefore, development of next-generation antibacterial compounds is an urgent need. Two-component signal transduction systems (TCS) are stimulus-response coupling devices that allow bacteria to sense and elaborate adaptive responses to changing environmental conditions, including the challenges that pathogenic bacteria face inside the host. The differential presence of TCS, present in bacteria but absent in the animal kingdom, makes them attractive targets in the search for new antibacterial compounds. In Salmonella enterica, the PhoP/PhoQ two-component system controls the expression of crucial phenotypes that define the ability of the pathogen to establish infection in the host. We now report the screening of 686 compounds from a GlaxoSmithKline published kinase inhibitor set in a high-throughput whole-cell assay that targets Salmonella enterica serovar Typhimurium PhoP/PhoQ. We identified a series of quinazoline compounds that showed selective and potent downregulation of PhoP/PhoQ-activated genes and define structural attributes required for their efficacy. We demonstrate that their bioactivity is due to repression of the PhoQ sensor autokinase activity mediated by interaction with its catalytic domain, acting as competitive inhibitors of ATP binding. While noncytotoxic, the hit molecules exhibit antivirulence effect by blockage of S. Typhimurium intramacrophage replication. Together, these features make these quinazoline compounds stand out as exciting leads to develop a therapeutic intervention to fight salmonellosis.Fil: Carabajal, María Ayelén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Asquith, Christopher R. M.. University of North Carolina; Estados UnidosFil: Laitinen, Tuomo. University of North Carolina; Estados UnidosFil: Tizzard, Graham J.. University of North Carolina; Estados UnidosFil: Yim, Lucía. Universidad de la República; UruguayFil: Rial, Analía. Universidad de la República; UruguayFil: Chabalgoity, José A.. Universidad de la República; UruguayFil: Zuercher, William J.. University of North Carolina; Estados UnidosFil: Garcia Vescovi, Eleonora. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Ackonc-AWA : A multi-species animal welfare assessment protocol for wild animals under human care to overcome the use of generic welfare checklists

Maintaining a high level of animal welfare is essential in zoos, sanctuaries and aquaria for ethical, legislative and functional reasons. Therefore, it is necessary to have welfare assessment protocols that can be incorporated into daily management programs. Currently, there are different approaches to assessing animal welfare in zoos. Those that can be applied to multiple species consist of checklists or qualitative assessments, with limitations, especially regarding the lack of guidance in the selection and interpretation of indicators. Validated protocols also exist, but they are for very few wild species. This study aimed to develop, test in the field, and describe an animal welfare assessment protocol for wild animals under human care, that can be applied to multiple species, intended to overcome the use of generic welfare checklists and offer an alternative to challenging and time consuming species-specific tools. The development process consisted of the elaboration of a protocol, substantiated by published literature on zoo animal welfare and multidisciplinary focus group work, and its on-field feasibility test. This was performed on 14 species of different taxa housed in an Argentinian zoo. The protocol was structured in two forms: an initial form to serve as scan using various animal-based (ABM), resource-based (RBM), and management-based measurements (MBM), and a follow-up form using exclusively ABM. The protocol also included a user's manual with information about preliminary preparation, equipment required, steps from arrival until completion, and details on how to assess each indicator. The scoring method consisted in rating each indicator on a 3-point scale. 23 ABM, 19 RBM, and three MBM were tested and selected to integrate Ackonc-AWA, a multidimensional protocol covering the five animal welfare domains and applicable to multiple species. This protocol was entirely developed in Spanish and can be applied noninvasively and at a low cost, which constitute features of high relevance for Latin America. Further applications of the described welfare assessment tool in other species and different institutional contexts will reinforce the validation of the proposed measurements and allow the systematic and routine evaluation of animal welfare in zoos

Sublingual flagellin protects against acute pneumococcal pneumonia in a TLR5-dependent and NLRC4-independent fashion.

International audienceAIM: To evaluate efficacy of sublingual flagellin to treat acute pneumonia.MATERIALS & METHODS: Mice were treated sublingually with flagellin and challenged intranasally with a lethal dose of pneumococcus. Flagellins lacking TLR5 or NLRC4 activation domains were used to assess their contribution to protection.RESULTS: Sublingual flagellin protected mice in a TLR5-dependent, NLRC4-independent fashion. Neutrophils were required for protection. Flagellin-stimulated lung epithelial cells recapitulated the lung's transcriptional profile suggesting they could be targeted by flagellin in vivo.CONCLUSION: Ligation of TLR5, a pathogen recognition receptor not naturally engaged by pneumococcus, protects mice from invasive pneumonia when administered via sublingual route. This can be a highly cost-effective alternative therapy against pneumonia

Mucosal Administration of Flagellin Protects Mice from Streptococcus pneumoniae Lung Infection▿

Streptococcus pneumoniae is a major cause of pneumonia in infants and the elderly. Innate defenses are essential to the control of pneumococcal infections, and deficient responses can trigger disease in susceptible individuals. Here we showed that flagellin can locally activate innate immunity and thereby increase the resistance to acute pneumonia. Flagellin mucosal treatment improved S. pneumoniae clearance in the lungs and promoted increased survival of infection. In addition, lung architecture was fully restored after the treatment of infected mice, indicating that flagellin allows the reestablishment of steady-state conditions. Using a flagellin mutant that is unable to signal through Toll-like receptor 5 (TLR5), we established that TLR5 signaling is essential for protection. In the respiratory tract, flagellin induced neutrophil infiltration into airways and upregulated the expression of genes coding for interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), CXCL1, CXCL2, and CCL20. Using depleting antibodies, we demonstrated that neutrophils are major effectors of protection. Further, we found that B- and T-cell-deficient SCID mice clear S. pneumoniae challenge to the same extent as immunocompetent animals, suggesting that these cell populations are not required for flagellin-induced protection. In conclusion, this study emphasizes that mucosal stimulation of innate immunity by a TLR not naturally engaged by S. pneumoniae can increase the potential to cure pneumococcal pneumonia

Table_1_Ackonc-AWA: A multi-species animal welfare assessment protocol for wild animals under human care to overcome the use of generic welfare checklists.xlsx

IntroductionMaintaining a high level of animal welfare is essential in zoos, sanctuaries and aquaria for ethical, legislative and functional reasons. Therefore, it is necessary to have welfare assessment protocols that can be incorporated into daily management programs. Currently, there are different approaches to assessing animal welfare in zoos. Those that can be applied to multiple species consist of checklists or qualitative assessments, with limitations, especially regarding the lack of guidance in the selection and interpretation of indicators. Validated protocols also exist, but they are for very few wild species. This study aimed to develop, test in the field, and describe an animal welfare assessment protocol for wild animals under human care, that can be applied to multiple species, intended to overcome the use of generic welfare checklists and offer an alternative to challenging and time consuming species-specific tools.MethodsThe development process consisted of the elaboration of a protocol, substantiated by published literature on zoo animal welfare and multidisciplinary focus group work, and its on-field feasibility test. This was performed on 14 species of different taxa housed in an Argentinian zoo. The protocol was structured in two forms: an initial form to serve as scan using various animal-based (ABM), resource-based (RBM), and management-based measurements (MBM), and a follow-up form using exclusively ABM. The protocol also included a user's manual with information about preliminary preparation, equipment required, steps from arrival until completion, and details on how to assess each indicator. The scoring method consisted in rating each indicator on a 3-point scale.Results23 ABM, 19 RBM, and three MBM were tested and selected to integrate Ackonc-AWA, a multidimensional protocol covering the five animal welfare domains and applicable to multiple species.DiscussionThis protocol was entirely developed in Spanish and can be applied noninvasively and at a low cost, which constitute features of high relevance for Latin America. Further applications of the described welfare assessment tool in other species and different institutional contexts will reinforce the validation of the proposed measurements and allow the systematic and routine evaluation of animal welfare in zoos.</p