Advances in genetic manipulation of obligate intracellular bacterial pathogens - PubMed (original) (raw)

Advances in genetic manipulation of obligate intracellular bacterial pathogens

Paul A Beare et al. Front Microbiol. 2011.

Abstract

Infections by obligate intracellular bacterial pathogens result in significant morbidity and mortality worldwide. These bacteria include Chlamydia spp., which causes millions of cases of sexually transmitted disease and blinding trachoma annually, and members of the α-proteobacterial genera Anaplasma, Ehrlichia, Orientia, and Rickettsia, agents of serious human illnesses including epidemic typhus. Coxiella burnetii, the agent of human Q fever, has also been considered a prototypical obligate intracellular bacterium, but recent host cell-free (axenic) growth has rescued it from obligatism. The historic genetic intractability of obligate intracellular bacteria has severely limited molecular dissection of their unique lifestyles and virulence factors involved in pathogenesis. Host cell restricted growth is a significant barrier to genetic transformation that can make simple procedures for free-living bacteria, such as cloning, exceedingly difficult. Low transformation efficiency requiring long-term culture in host cells to expand small transformant populations is another obstacle. Despite numerous technical limitations, the last decade has witnessed significant gains in genetic manipulation of obligate intracellular bacteria including allelic exchange. Continued development of genetic tools should soon enable routine mutation and complementation strategies for virulence factor discovery and stimulate renewed interest in these refractory pathogens. In this review, we discuss the technical challenges associated with genetic transformation of obligate intracellular bacteria and highlight advances made with individual genera.

Keywords: allelic exchange; antibiotic selection; complementation; electroporation; genetic transformation; shuttle vector; transposon mutagenesis; virulence factor.

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Figures

Figure 1

Figure 1

A Himar1 insertion in R. rickettsii sca2 eliminates actin-based motility. The right micrograph shows wild-type R. rickettsii (arrows) with typical filamentous actin tails (arrowhead). The left micrograph depicts a small plaque-forming mutant of R. rickettsii with a Himar1 insertion in sca2, which encodes an autotransporter protein (Kleba et al., 2010). The mutant rickettsia (arrow) lack actin tails. Rickettsia were stained by immunofluorescence using a specific monoclonal antibody and filamentous actin was stained with Alex Fluor 598 phalloidin. Bar, 3 μm. (Micrographs courtesy of Ted Hackstadt, Rocky Mountain Laboratories).

Figure 2

Figure 2

Characterization C. burnetii Nine Mile (phase II) MC1 and B2c Himar1 transformants. (A) Schematic of the Himar1 chromosomal integration site in C. burnetii MC1. The Himar1 transposon is flanked by inverted terminal repeat (ITR) elements and inserted into an intergenic region between CBU0316 and CBU0317. Flow cytometry (B) and confocal fluorescence microscopy (C) of live Vero cells infected with MC1 or B2c for 5 days. Both assays revealed considerably more mCherry fluorescence from MC1-containing vacuoles, where expression is driven by 311P, then from B2c-containing vacuoles, where expression is driven by 1169P (Beare et al., 2009). The green trace in flow cytometry histograms shows autofluorescence of Vero cells infected with wild-type C. burnetii for 5 days. Bars, 5 μm.

Figure 3

Figure 3

A C. burnetii BlaM translocation assay shuttle vector. (A) Map of the reporter plasmid pJB-CAT-BlaM which contains an RSF1010 ori that functions in both E. coli and C. burnetii. Genes encoding suspected secreted proteins are cloned downstream and in-frame with blaM using the unique SalI site. (B) BlaM translocation assay showing cytosolic delivery of a BlaM-CBUA0015 fusion protein. THP-1 cells were infected with C. burnetii Nine Mile (phase II) containing pJB-CAT-BlaM::CBUA0015 for 48 h, then incubated for 1 h with CCF4/AM. Cleavage of CCF4/AM by cytosolic BlaM results in blue fluorescent cells and indicates secretion of the fusion protein. Bar, 30 μm.

Figure 4

Figure 4

Coxiella burnetii Tn7 transformation system. (A) Maps of two-plasmid C. burnetii miniTn7 transposon system. The suicide plasmid pTNS2_::1169P-tnsABCD_ encodes the tnsABCD operon under control of 1169P. The suicide plasmid pMiniTn7T-CAT::GFP encodes CAT and GFP genes driven independently by 1169P and 311P, respectively. The CAT/GFP cassette is flanked by Tn7L and Tn7R elements. (B) Schematic of the glmS regions in C. burnetii Nine Mile (phase II) and C. burnetii Nine Mile (phase II)/Tn7-CAT-GFP. (C) Fluorescence microscopy of Vero cells infected for 5 days with C. burnetii Nine Mile (phase II)/Tn7-CAT-GFP (green). Cells were fixed with 4% paraformaldehyde, then immunostained for the lysosomal protein CD63 (red). Bar, 5 μm.

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