Examining the Role of Specific Virulence Mechanisms During Pseudomonas Aeruginosa Infection in a Zebrafish Model of Cystic Fibrosis

Cystic fibrosis (CF) is the most common lethal hereditary disease. CF is caused by recessive mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene and is associated with multi-­-organ defects resulting from improper ion transport across epithelial membranes. Chronic lung infection by the environmentally ubiquitous opportunistic human pathogen Pseudomonas aeruginosa and the subsequent hyperinflammation that occurs as the host immune system combats the bacterium cause substantial morbidity and mortality in CF. Despite numerous studies that have sought to elucidate the role of CFTR in the innate immune response, the links between CFTR, innate immunity, and P. aeruginosa infection remain unclear. The present work highlights the zebrafish as a powerful model organism for human infectious disease. Zebrafish embryos with reduced expression of the cftr gene (Cftr morphants) exhibited reduced respiratory burst response and directed neutrophil migration, supporting a connection between cftr and the innate immune response. Cftr morphants were also found to display a iv significant iron deficiency (ID) compared to control embryos, a symptom commonly diagnosed in CF patients. Cftr morphants were infected with P. aeruginosa or other bacterial species that are commonly associated with infections in CF patients, including Burkholderia cenocepacia, Haemophilus influenzae, and Staphylococcus aureus. Intriguingly, the bacterial burden of P. aeruginosa was found to be significantly higher in zebrafish Cftr morphants than in controls, a phenomenon that was not observed with any of the other bacterial species examined. The bacterial burden in Cftr morphants infected with a P. aeruginosa LasR mutant, a quorum sensing (QS)-­-deficient strain, was comparable to that in control fish indicating that the regulation of virulence factors through QS is required for enhancement of infection in the absence of Cftr. Cftr morphants were then challenged with P. aeruginosa mutants defective in the expression of QS regulated virulence factors. A mutant defective in the production of Exotoxin A (ETA) resulted in similar bacterial clearance in both the Cftr morphant and control embryos. The reduction in directed neutrophil migration to a P. aeruginosa infection was also restored when zebrafish embryos were challenged with the ETA mutant. Taken together, these data point towards a possible explanation for the specificity between P. aeruginosa and CFTR. The zebrafish system provides a multitude of advantages for studying the unique pathophysiology resulting from defective expression of CFTR, investigating the pathogenesis of P. aeruginosa and elucidating the role that the innate system plays in the host response to acute bacterial infections commonly associated with cystic fibrosis

Interleukins 7 and 15 Maintain Human T Cell Proliferative Capacity through STAT5 Signaling

T lymphocytes require signals from self-peptides and cytokines, most notably interleukins 7 and 15 (IL-7, IL-15), for survival. While mouse T cells die rapidly if IL-7 or IL-15 is withdrawn, human T cells can survive prolonged withdrawal of IL-7 and IL-15. Here we show that IL-7 and IL-15 are required to maintain human T cell proliferative capacity through the STAT5 signaling pathway. T cells from humanized mice proliferate better if stimulated in the presence of human IL-7 or IL-15 or if T cells are exposed to human IL-7 or IL-15 in mice. Freshly isolated T cells from human peripheral blood lose proliferative capacity if cultured for 24 hours in the absence of IL-7 or IL-15. We further show that phosphorylation of STAT5 correlates with proliferation and inhibition of STAT5 reduces proliferation. These results reveal a novel role of IL-7 and IL-15 in maintaining human T cell function, provide an explanation for T cell dysfunction in humanized mice, and have significant implications for in vitro studies with human T cells

Dendritic cell nediated inhibition of lentiviral infection

Lentiviral entry to quiescent lymphocytes represents a 'time bomb' waiting for cellular activation to spread infection. In order to undergo immune activation T cells interact with dendritic cells presenting peptide:MHC complexes 'sampling' them to look for agonist peptides and receiving survival signals from self peptides. This makes the dendritic cell:T cell interaction an ideal checkpoint to contain lentiviral infection of quiescent lypmhocytes

Zebrafish: A See-Through Host and a Fluorescent Toolbox to Probe Host–Pathogen Interaction

In many ways, the zebrafish represents a hybrid between mouse and invertebrate infection models. Powerful forwardgenetic tools that have made invertebrates justifiably famous are not only relatively accessible in the zebrafish, but have been exploited to yield new insights into human infectious diseases, including leprosy and tuberculosis [1]. Transgenic technologies have enabled detailed, non-invasive in vivo visualization of macrophages and neutrophils in pitched battle with bacteria and fungi [2,3]. Reverse genetics with morpholinos, vivo-morpholinos, and zinc-finger nucleases (but unfortunately not homologous recombination, which for the moment remains out of reach in this organism) enable examination of the roles of specific genes during infection. Flexible genetic systems such as Gal4-UAS and Cre-Lox permit tissue-specific transformation and ablation ([3]; Figure 1)

Septins restrict inflammation and protect zebrafish larvae from Shigella infection

Shigella flexneri, a Gram-negative enteroinvasive pathogen, causes inflammatory destruction of the human intestinal epithelium. Infection by S. flexneri has been well-studied in vitro and is a paradigm for bacterial interactions with the host immune system. Recent work has revealed that components of the cytoskeleton have important functions in innate immunity and inflammation control. Septins, highly conserved cytoskeletal proteins, have emerged as key players in innate immunity to bacterial infection, yet septin function in vivo is poorly understood. Here, we use S. flexneri infection of zebrafish (Danio rerio) larvae to study in vivo the role of septins in inflammation and infection control. We found that depletion of Sept15 or Sept7b, zebrafish orthologs of human SEPT7, significantly increased host susceptibility to bacterial infection. Live-cell imaging of Sept15-depleted larvae revealed increasing bacterial burdens and a failure of neutrophils to control infection. Strikingly, Sept15-depleted larvae present significantly increased activity of Caspase-1 and more cell death upon S. flexneri infection. Dampening of the inflammatory response with anakinra, an antagonist of interleukin-1 receptor (IL-1R), counteracts Sept15 deficiency in vivo by protecting zebrafish from hyper-inflammation and S. flexneri infection. These findings highlight a new role for septins in host defence against bacterial infection, and suggest that septin dysfunction may be an underlying factor in cases of hyper-inflammation

The Diverse Cellular and Animal Models to Decipher the Physiopathological Traits of Mycobacterium abscessus Infection

Mycobacterium abscessus represents an important respiratory pathogen among the rapidly-growing non-tuberculous mycobacteria. Infections caused by M. abscessus are increasingly found in cystic fibrosis (CF) patients and are often refractory to antibiotic therapy. The underlying immunopathological mechanisms of pathogenesis remain largely unknown. A major reason for the poor advances in M. abscessus research has been a lack of adequate models to study the acute and chronic stages of the disease leading to delayed progress of evaluation of therapeutic efficacy of potentially active antibiotics. However, the recent development of cellular models led to new insights in the interplay between M. abscessus with host macrophages as well as with amoebae, proposed to represent the environmental host and reservoir for non-tuberculous mycobacteria. The zebrafish embryo has also appeared as a useful alternative to more traditional models as it recapitulates the vertebrate immune system and, due to its optical transparency, allows a spatio-temporal visualization of the infection process in a living animal. More sophisticated immunocompromised mice have also been exploited recently to dissect the immune and inflammatory responses to M. abscessus. Herein, we will discuss the limitations, advantages and potential offered by these various models to study the pathophysiology of M. abscessus infection and to assess the preclinical efficacy of compounds active against this emerging human pathogen

Senior Thesis Proposal

Senior Thesis Proposa

Landscape Genetics of the Lesser Antillean Whistling Frog, Eleutherodactylus johnstonei, on Montserrat

With the advent of quick, efficient, and accurate genetic sequencing, ecologists and evolutionary biologists have moved to use genetic methods to better model, represent, and further understand the methods by which life diversifies and speciates. Demonstrating these methods using spatial analysis has resulted in a field dedicated to the integration of spatial mathematics, landscape ecology, and population genetics. This thesis explains the fundamental concepts and ideas of the field of landscape genetics, which aims to combine the practices of landscape ecology and population genetics to allow for an analysis of both over space and time. This sort of analysis allows for the viewing of complex spatial relations that were otherwise unachievable. To apply and practice these concepts, 32 samples of the Lesser Antillean Whistling Frog, Eleutherodactylus johnstonei, were gathered from six major locations on the island of Montserrat, to investigate variation via the Cytochrome-b region over physical space

Interleukins 7 and 15 Maintain Human T Cell Proliferative Capacity through STAT5 Signaling.

T lymphocytes require signals from self-peptides and cytokines, most notably interleukins 7 and 15 (IL-7, IL-15), for survival. While mouse T cells die rapidly if IL-7 or IL-15 is withdrawn, human T cells can survive prolonged withdrawal of IL-7 and IL-15. Here we show that IL-7 and IL-15 are required to maintain human T cell proliferative capacity through the STAT5 signaling pathway. T cells from humanized mice proliferate better if stimulated in the presence of human IL-7 or IL-15 or if T cells are exposed to human IL-7 or IL-15 in mice. Freshly isolated T cells from human peripheral blood lose proliferative capacity if cultured for 24 hours in the absence of IL-7 or IL-15. We further show that phosphorylation of STAT5 correlates with proliferation and inhibition of STAT5 reduces proliferation. These results reveal a novel role of IL-7 and IL-15 in maintaining human T cell function, provide an explanation for T cell dysfunction in humanized mice, and have significant implications for in vitro studies with human T cells