title: Beta-lactamase can function as a reporter of bacterial protein export during Mycobacterium tuberculosis infection of host cells.
abstract: Mycobacterium tuberculosis is an intracellular pathogen that is able to avoid destruction by host immune defences. Exported proteins of M. tuberculosis, which include proteins localized to the bacterial surface or secreted into the extracellular environment, are ideally situated to interact with host factors. As a result, these proteins are attractive candidates for virulence factors, drug targets and vaccine components. Here we describe a beta-lactamase reporter system capable of identifying exported proteins of M. tuberculosis during growth in host cells. Because beta-lactams target bacterial cell-wall synthesis, beta-lactamases must be exported beyond the cytoplasm to protect against these drugs. When used in protein fusions, beta-lactamase can report on the subcellular location of another protein as measured by protection from beta-lactam antibiotics. Here we demonstrate that a truncated TEM-1 beta-lactamase lacking a signal sequence for export ('BlaTEM-1) can be used in this manner directly in a mutant strain of M. tuberculosis lacking the major beta-lactamase, BlaC. The 'BlaTEM-1 reporter conferred beta-lactam resistance when fused to both Sec and Tat export signal sequences. We further demonstrate that beta-lactamase fusion proteins report on protein export while M. tuberculosis is growing in THP-1 macrophage-like cells. This genetic system should facilitate the study of proteins exclusively exported in the host environment by intracellular M. tuberculosis.
authors: Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7290, USA.
authors: Jessica R
authors: JR
authors: McCann
authors: Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7290, USA.
authors: Justin A
authors: JA
authors: McDonough
authors: Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
authors: Martin S
authors: MS
authors: Pavelka
authors: Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7290, USA.
authors: Miriam
authors: M
authors: Braunstein
mesh_terms: D019677:Artificial Gene Fusion / Q000379:methods*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D017930:Genes, Reporter; D006801:Humans; D008264:Macrophages / Q000382:microbiology; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000235:genetics; D021381:Protein Transport / Q000235:genetics / Q000502:physiology*; D018440:beta-Lactam Resistance; D001618:beta-Lactamases / Q000032:analysis* / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D021382:Protein Sorting Signals; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1