Advances in genetic manipulation of obligate intracellular bacterial pathogens

Review Article published: 02 May 2011 doi: 10.3389/fmicb.2011.00097 Advances in genetic manipulation of obligate intracellular bacterial pathogens Paul A. Beare1, Kelsi M. Sandoz2, Anders Omsland1, Daniel D. Rockey2 and Robert A. Heinzen1* Coxiella Pathogenesis Section, Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, USA 2 Department of Biomedical Sciences, Oregon State University, Corvallis, OR, USA 1 Edited by: Rey Carabeo, Imperial College London, UK Reviewed by: Mikhail A. Gavrilin, Ohio State University, USA Andres Vazquez-Torres, University of Colorado Medical School, USA *Correspondence: Robert A. Heinzen, Rocky Mountain Laboratories, Laboratory of Intracellular Parasites, 903 South Fourth Street, Hamilton, MT 59840, USA. e-mail: 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: transposon mutagenesis, electroporation, antibiotic selection, allelic exchange, genetic transformation, virulence factor, shuttle vector, complementation Introduction Obligate intracellular bacterial pathogens are an understudied but significant group of human disease agents. These bacteria are thought to have emerged from ancestral non-pathogens through a pathoadaptive evolutionary process that involves significant genome reduction (Pallen and Wren, 2007). Ongoing gene loss in obligate intracellular bacteria is indicated by the presence of pseudogenes whose functions are often compensated for by the host cell. Some obligate intracellular bacteria, such as Rickettsia prowazekii and Chlamydia trachomatis, appear in the final stages of host cell adaptation, having cleared most pseudogenes from their respective genomes (Andersson and Andersson, 1999). Relative to these bacteria, Coxiella burnetii has a sophisticated genome with central metabolic pathways largely intact, suggesting a more recent adaptation to an obligate intracellular lifestyle (Seshadri et al., 2003). Despite dramatically reduced genomes relative to most freeliving bacterial pathogens, obligate intracellular bacterial pathogens still retain potent pathogenetic potential that can manifest in infections ranging from asymptomatic to fulminating and deadly (Walker, 1989; Maurin and Raoult, 1999). Unfortunately, the historic lack of genetic tools for obligates has severely limited molecular dissection of mechanisms associated with intracellular parasitism and animal pathogenesis. Many genes encoding putative www.frontiersin.org virulence factors have been revealed by pathogen genome sequences (Andersson et al., 1998; Seshadri et al., 2003). However, because methods for site-specific gene inactivation and complementation are lacking, molecular Koch’s postulates (Falkow, 2004) have been impossible to fulfill for these genes. Consequently, gene function and regulation have often been explored using heterologous expression in surrogate hosts (Whitworth et al., 2005; Raghavan et al., 2008; Voth et al., 2009). In this review, we discuss the experimental hurdles associated with developing genetic transformation systems for obligate intracellular bacteria and review the genetic tools that are currently available. Technical considerations in transforming obligate intracellular bacteria A pathogen’s obligate reliance on a eukaryotic host cell for growth complicates several steps in genetic transformation that are easily conducted with free-living bacteria. Nonetheless, by employing tenacity and attention to detail, several investigators have overcome technical hurdles to establish at least rudimentary genetic systems for most pathogenic obligate intracellular bacteria. In this section, we highlight the special experimental considerations associated with genetic transformation systems of these bacteria. May 2011 | Volume 2 | Article 97 | 1 Beare et al. Bacterial purification Before any genetic transformation procedure, obligate intracellular bacteria must be purified to some extent from host cells and concentrated to high density in a viable form. Depending on the degree of purity, the procedure can involve several centrifugation steps that take nearly a full day to complete (Shannon and Heinzen, 2007). For organisms that grow to low density in host cells, such as spotted fever group (SFG) rickettsia, yields can be poor and allow for only a few electroporation experiments (Kleba et al., 2010). To ensure utmost viability, some obligate intracellular bacteria are electroporated immediately after purification (Qin et al., 2004), thereby eliminating the convenience of storing purified bacteria for subsequent transformation experiments. Several low ionic strength electroporation buffers have been used, ranging from distilled water (Binet and Maurelli, 2009) to buffers containing osmoprotectants such as sucrose and glycerol (Beare et al., 2009). Organisms are washed several times in buffers and resuspended at high density (approx. 1010 bacteria per ml) prior to electroporation. A consideration when purifying obligate intracellular bacteria for transformation experiments is that many display developmental forms that may be differentially infective and/or receptive to electroporation. For example, the large reticulate cell (RC) of Anaplasma phagocytophilum may be more amenable to electroporation than the smaller dense-cored cell (DC) with (...truncated)