Author

Kaoru Geddes

Date

January 2007

Document Type

Dissertation

Degree Name

Ph.D.

Department

Dept. of Molecular Microbiology and Immunology

Institution

Oregon Health & Science University

Abstract

Salmonella is an important human pathogen that causes thousands of infections every year due to ingestion of contaminated food. The need to develop new strategies to control Salmonella spread and combat disease are of paramount interest because of the rise in the frequency of antibiotic resistant Salmonella strains. A key to developing new therapeutic strategies is a better understanding of the pathogenesis of Salmonellosis. Although animal and cell culture models have provided much insight into Salmonella infection, many aspects of Salmonellosis warrant further analysis. This dissertation describes the investigation of one of Salmonella's critical virulence determinants, its type III secretion systems (T3S). T3S are syringe like structures that allow pathogenic bacteria to directly inject virulence factors, called effector proteins, into the cytosol of host cells. Salmonella has two T3Ss, encoded in Salmonella pathogenicithy island 1 (SPI-1) and Salmonella pathogenicity island 2 (SPI-2). The SPI-1 encoded T3S (T3S-1) mediates cell invasion and inflammation during the intestinal phase of Salmonella infection, while the SPI-2 encoded T3S (T3S-2) is essential for intracellular survival within phagocytes during the systemic phase of disease. Although, recent research has provided much insight into the molecular mechanisms by which T3S secreted proteins promote Salmonella infection, the T3S effectors mediating several T3S dependent phenotypes have yet to identified. Therefore a strategy was devised to identify secreted effectors, using a transposon that creates random fusions to the calmodulin dependent adenylate cyclase (CyaA') from Bordetella pertussis. This strategy was used to identify three new T3S effectors, SteA, SteB and SteC. Analysis of these proteins revealed that only SteA is required for virulence in mice. Further characterization of SteA demonstrated that it is targeted to the Golgi netword in infected cells. Analysis of the effector proteins that were isolated in the screen for new effectors revealed that they have different secretion patterns. Some effectors are secreted only via T3S-1. some are secreted only by T3S-2, and some are secreted by both T3S-1 and T3S-2. While the current paradigm is that T3S-1 is only involved in the intestinal phase of disease and T3S-2 is only involved in the systemic phase of the disease, some evidence suggests that this may not be the case. Therefore, the analysis of the in vivo secretion patterns of effectors during acute mouse infection was undertaken. Using Salmonella strains that express B-lactamase fusions to several different effectors, spleen cells targeted by T3S could be cleavage of a fluorescent substrate. Only effectors secreted via T3S-2 were secreted into spleen cells, supporting the existing views that T3S-1 is not involved during the systemic phase of infection. FACS analysis of the targeted cells determined secretion was detected in T-cells, B-cells, monocytes and nuetrophils, but not in mature macrophages. Most of the secretion and viable bacteria were actually found in neutrophils, indicating that neutrophils are an important niche for bacterial growth. This result challenges the current view that macrophages are the primary site of intracellular growth.

Identifier

doi:10.6083/M4KP8058

School

School of Medicine

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.