Yan Ye

Date of Award

January 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Min Wu

Abstract

Klebsiella pneumoniae (Kp) is a Gram-negative bacterium that can cause serious infections in humans. Autophagy-related gene 7 (Atg7) has been involved in certain bacterial infections; however, the role of Atg7 in macrophage-mediated immunity against Kp infection has not been elucidated. To gain a better understanding of Kp-host interaction which may provide insight into the design of novel and effective therapeutics for this infection, we set out to investigate the potential immune role for autophagy in Kp infection in vitro and in vivo. We found that Atg7 was significantly induced in murine alveolar macrophages (MH-S) upon Kp infection, indicating that Atg7 participated in host defense in this infection. Knocking down Atg7 with siRNA increased bacterial burdens in MH-S cells. Using cell biology assays and whole animal imaging analysis, we found that compared to WT mice, atg7 knockout (KO) mice exhibited increased susceptibility to Kp infection, with decreased survival rates, decreased bacterial clearance, and intensified lung injury. Moreover, Kp infection induced excessive proinflammatory cytokines and superoxide in the lung of atg7 KO mice. Similarly, Atg7 silencing in MH-S cells markedly increased expression levels of proinflammatory cytokines. These findings indicate that Atg7 offers critical resistance to Kp infection by modulating both systemic and local production of proinflammatory cytokines.

We further investigated the molecular mechanism by which Atg7 regulates Kp-induced inflammatory responses. We found that Atg7 expression and p-IκBα level were increased in a time-dependent pattern in MH-S upon Kp infection. We also revealed an interaction between Atg7 and p-IκBα, which was decreased upon Kp infection, whereas the interaction between ubiquitin (Ub) and phosphorylated-IκBα (p-IκBα) was increased in MH-S. We further demonstrated that knocking down Atg7 with siRNA increased p-IκBα ubiquitylation and promoted NF-κB translocation into the nucleus, and increased proinflammatory cytokine production (TNF-α). Moreover, overexpression of Ub in MH-S increased Kp infection-induced proinflammatory cytokines (TNF-α) and silencing ubiquitinase decreased cytokine production. In addition, infection of cells with lentivirus-shUb particles decreased binding of p-IκBα to Ub and inhibited expression of TNF-α in the primary alveolar macrophage (AM) cells and lung tissue of atg7 KO mice upon Kp infection. Thus, loss of Atg7 switched binding of p-IκBα from Atg7 to Ub, resulting in an increased ubiquitylation of p-IκBα, which facilitated NF-κB nuclear translocation and intensified inflammatory responses against Kp. Our current findings reveal a regulatory role of Atg7 in ubiquitylation of p-IκBα. Collectively, research of this dissertation provides new insight into the molecular detail of host-pathogen interaction for Kp, which may be beneficial for design of novel therapeutics to control Kp infection.

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