ABSTRACT
Secretory leukocyte protease inhibitor (SLPI) is an important respiratory tract host defense protein, which is proteolytically inactivated by excessive neutrophil elastase (NE) during chronic Pseudomonas infection in the cystic fibrosis (CF) lung. We generated two putative NE-resistant variants of SLPI by site-directed mutagenesis, SLPI-A16G and SLPI-S15G-A16G, with a view to improving SLPI's proteolytic stability. Both variants showed enhanced resistance to degradation in the presence of excess NE as well as CF patient sputum compared with SLPI-wild type (SLPI-WT). The ability of both variants to bind bacterial lipopolysaccharides and interact with nuclear factor-κB DNA binding sites was also preserved. Finally, we demonstrate increased anti-inflammatory activity of the SLPI-A16G protein compared with SLPI-WT in a murine model of pulmonary Pseudomonas infection. This study demonstrates the increased stability of these SLPI variants compared with SLPI-WT and their therapeutic potential as a putative anti-inflammatory treatment for CF lung disease.
Subject(s)
Cystic Fibrosis/immunology , Leukocyte Elastase/metabolism , Lung/immunology , Pseudomonas Infections/immunology , Pseudomonas aeruginosa/immunology , Secretory Leukocyte Peptidase Inhibitor/metabolism , Animals , Cells, Cultured , Chronic Disease , Cystic Fibrosis/complications , Disease Models, Animal , Humans , Immunity, Innate , Lung/microbiology , Mice , Mice, Inbred C57BL , Mutagenesis, Site-Directed , Mutation/genetics , Neutrophil Infiltration , Proteolysis , Pseudomonas Infections/complications , Secretory Leukocyte Peptidase Inhibitor/geneticsABSTRACT
During intraerythrocytic development, Plasmodium falciparum exports proteins that interact with the host cell plasma membrane and subplasma membrane-associated spectrin network. Parasite-exported proteins modify mechanical properties of host RBCs, resulting in altered cell circulation. In this work, optical tweezers experiments of cell mechanical properties at normal physiological and febrile temperatures are coupled, for the first time, with targeted gene disruption techniques to measure the effect of a single parasite-exported protein on host RBC deformability. We investigate Pf155/Ring-infected erythrocyte surface antigen (RESA), a parasite protein transported to the host spectrin network, on deformability of ring-stage parasite-harboring human RBCs. Using a set of parental, gene-disrupted, and revertant isogenic clones, we found that RESA plays a major role in reducing deformability of host cells at the early ring stage of parasite development, but not at more advanced stage. We also show that the effect of RESA on deformability is more pronounced at febrile temperature, which ring-stage parasite-harboring RBCs can be exposed to during a malaria attack, than at normal body temperature.
