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The Utility of Zebrafish to Study Bacterial Pathogenesis
thesis
posted on 2017-02-13, 04:43 authored by Md. Saruar BhuiyanZebrafish
(Danio rerio) are now established as an excellent model to study infections and
their pathogenesis. Zebrafish have many advantages over other vertebrates in
studying host immune responses to infectious diseases. Most importantly, the
ease of manipulating host genes to generate transgenic fish lines, and the
optical transparency of zebrafish that facilitates imaging of real-time
interactions between host and pathogen with excellent resolution. In my PhD
project, I optimized and applied a zebrafish infection model to study multiple
virulence features of two major nosocomial pathogens, these being Acinetobacter
baumannii and Staphylococcus aureus.
In the first part of my PhD, I used zebrafish as a substitute host to study A. baumannii infections and the genes required for its virulence. Bacterial mutants involving genes responsible for quorum-sensing and global virulence regulation (gacSA) were studied in zebrafish, and results corroborated with those observed in a murine model of infection. Neutrophil and macrophage migration responses were also studied to find the primary phagocyte responsible for initial killing of bacteria. These experiments led to an important finding related to bacterial-driven neutrophil chemoattraction. When zebrafish were infected with the A. baumannii gacS mutant, it induced prompt neutrophil swarming and dwelling at the site of infection, which was not observed with wild-type bacteria. To understand the mechanism of this neutrophil swarming, I then tested a number of mutants with deletion in genes controlled by the two-component global virulence regulator (gacS). These experiments led me to find that a mutation in the phenylacetic acid metabolic pathway gene (paaA) was responsible for neutrophil dwelling at a localised site of infection. Investigation of metabolites in culture supernatant revealed that phenylactic acid was the chemoattractant for neutrophils, and this facilitated the survival of fish by clearing the infection more effectively.
The second aim was focused on another problematic hospital-acquired pathogen; Staphylococcus aureus. More specifically, to understand the impact of resistance to a last-line antibiotic, known as daptomycin, on host-pathogen interactions. Daptomycin resistance in S. aureus is associated with changes in the bacterial membrane lipid composition. My work assessed how these changes in membrane lipid composition contributed to host immune evasion and persistent infection. Studying the lipid moieties present in the membrane of daptomycin-resistant clinical strains showed that there was an increase in cardiolipin content and a reduction in phosphatidylglycerol. Clinically relevant point mutations were generated in the S. aureus cardiolipin synthase (cls2) gene, which allowed a detailed assessment of the impact of bacterial membrane phospholipid changes on antibiotic resistance and interaction with the immune system. By using neutrophil migration assays in zebrafish, I showed that less neutrophils migrated to the site of infection caused by the point mutants compared to wild-type infection. To investigate the role of individual lipids in neutrophil migration, I used purified cardiolipin and phosphatidylglycerol isolated from wild-type bacteria and showed that phosphatidylglycerol was a neutrophil chemoattractant, whereas cardiolipin had no effect. These results indicated that gaining more cardiolipin and reducing phosphatidylglycerol, as was being observed in our clinical daptomycin-resistant strains, caused less neutrophil migration to the site of infection promoting bacterial survival and persistence.
Overall, I used zebrafish to study the pathogenesis of two common hospital acquired organisms. The work has supported the importance of zebrafish as an infection model to study bacterial pathogenesis and has contributed novel findings related to drug resistance and bacterial driven neutrophil chemoattraction.
In the first part of my PhD, I used zebrafish as a substitute host to study A. baumannii infections and the genes required for its virulence. Bacterial mutants involving genes responsible for quorum-sensing and global virulence regulation (gacSA) were studied in zebrafish, and results corroborated with those observed in a murine model of infection. Neutrophil and macrophage migration responses were also studied to find the primary phagocyte responsible for initial killing of bacteria. These experiments led to an important finding related to bacterial-driven neutrophil chemoattraction. When zebrafish were infected with the A. baumannii gacS mutant, it induced prompt neutrophil swarming and dwelling at the site of infection, which was not observed with wild-type bacteria. To understand the mechanism of this neutrophil swarming, I then tested a number of mutants with deletion in genes controlled by the two-component global virulence regulator (gacS). These experiments led me to find that a mutation in the phenylacetic acid metabolic pathway gene (paaA) was responsible for neutrophil dwelling at a localised site of infection. Investigation of metabolites in culture supernatant revealed that phenylactic acid was the chemoattractant for neutrophils, and this facilitated the survival of fish by clearing the infection more effectively.
The second aim was focused on another problematic hospital-acquired pathogen; Staphylococcus aureus. More specifically, to understand the impact of resistance to a last-line antibiotic, known as daptomycin, on host-pathogen interactions. Daptomycin resistance in S. aureus is associated with changes in the bacterial membrane lipid composition. My work assessed how these changes in membrane lipid composition contributed to host immune evasion and persistent infection. Studying the lipid moieties present in the membrane of daptomycin-resistant clinical strains showed that there was an increase in cardiolipin content and a reduction in phosphatidylglycerol. Clinically relevant point mutations were generated in the S. aureus cardiolipin synthase (cls2) gene, which allowed a detailed assessment of the impact of bacterial membrane phospholipid changes on antibiotic resistance and interaction with the immune system. By using neutrophil migration assays in zebrafish, I showed that less neutrophils migrated to the site of infection caused by the point mutants compared to wild-type infection. To investigate the role of individual lipids in neutrophil migration, I used purified cardiolipin and phosphatidylglycerol isolated from wild-type bacteria and showed that phosphatidylglycerol was a neutrophil chemoattractant, whereas cardiolipin had no effect. These results indicated that gaining more cardiolipin and reducing phosphatidylglycerol, as was being observed in our clinical daptomycin-resistant strains, caused less neutrophil migration to the site of infection promoting bacterial survival and persistence.
Overall, I used zebrafish to study the pathogenesis of two common hospital acquired organisms. The work has supported the importance of zebrafish as an infection model to study bacterial pathogenesis and has contributed novel findings related to drug resistance and bacterial driven neutrophil chemoattraction.
