Single-copy chromosomal integration systems for Francisella tularensis

Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642, USA.
Microbiology (Impact Factor: 2.56). 05/2009; 155(Pt 4):1152-63. DOI: 10.1099/mic.0.022491-0
Source: PubMed


Francisella tularensis is a fastidious Gram-negative bacterium responsible for the zoonotic disease tularemia. Investigation of the biology and molecular pathogenesis of F. tularensis has been limited by the difficulties in manipulating such a highly pathogenic organism and by a lack of genetic tools. However, recent advances have substantially improved the ability of researchers to genetically manipulate this organism. To expand the molecular toolbox we have developed two systems to stably integrate genetic elements in single-copy into the F. tularensis genome. The first system is based upon the ability of transposon Tn7 to insert in both a site- and orientation-specific manner at high frequency into the attTn7 site located downstream of the highly conserved glmS gene. The second system consists of a sacB-based suicide plasmid used for allelic exchange of unmarked elements with the blaB gene, encoding a beta-lactamase, resulting in the replacement of blaB with the element and the loss of ampicillin resistance. To test these new tools we used them to complement a novel d-glutamate auxotroph of F. tularensis LVS, created using an improved sacB-based allelic exchange plasmid. These new systems will be helpful for the genetic manipulation of F. tularensis in studies of tularemia biology, especially where the use of multi-copy plasmids or antibiotic markers may not be suitable.

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Available from: Martin S Pavelka, Nov 21, 2014
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    • "In brief, the two pdpA alleles in F. tularensis LVS were sequentially removed using the approach previously used to remove the one pdpA allele from F. novicida [26]. Genetic complementation was accomplished by introducing a copy of pdpA on a derivative of plasmid pMP831 [66]. F. tularensis LVS ΔiglC and its complement F. tularensis LVS ΔiglC::iglC KmR were kindly provided by Dr. Anders Sjostedt. "
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    ABSTRACT: The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis 'Type B' Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "Primers were used to delete all but the first five amino acid (MNDKY) and the last five codons (KISS stop) while keeping the deletion in frame. After blunt end cloning into pCR BLUNT II (Invitrogen), the SOED pdpC fragment was removed by BamHI-Not I digest and cloned into suicide vector pMP812 [23]. Integrants were selected on kanamycin (10 ug/ml). "
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    ABSTRACT: Francisella tularensis is a highly infectious bacterial pathogen that invades and replicates within numerous host cell types. After uptake, F. tularensis bacteria escape the phagosome, replicate within the cytosol, and suppress cytokine responses. However, the mechanisms employed by F. tularensis to thrive within host cells are mostly unknown. Potential F. tularensis mutants involved in host-pathogen interactions are typically discovered by negative selection screens for intracellular replication or virulence. Mutants that fulfill these criteria fall into two categories: mutants with intrinsic intracellular growth defects and mutants that fail to modify detrimental host cell processes. It is often difficult and time consuming to discriminate between these two possibilities. We devised a method to functionally trans-complement and thus identify mutants that fail to modify the host response. In this assay, host cells are consistently and reproducibly infected with two different F. tularensis strains by physically tethering the bacteria to antibody-coated beads. To examine the efficacy of this protocol, we tested phagosomal escape, cytokine suppression, and intracellular replication for F. tularensis ΔripA and ΔpdpC. ΔripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1β secretion. In the co-infection model, ΔripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ΔripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ΔpdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1β secretion. Wild-type bacteria that entered through the same phagosome as ΔpdpC rescued all of these phenotypes, indicating that ΔpdpC failed to properly manipulate the host. In summary, functional trans-complementation using bead-bound bacteria co-infections is a method to rapidly identify mutants that fail to modify a host response. Francisella tularensis is a facultative intracellular bacterial pathogen and is the causative agent of the disease tularemia. F. tularensis enters host cells through phagocytosis, escapes the phagosome, and replicates in the host cell cytosol while suppressing cytokine secretion [1]-[4]. Although substantial progress has been made in understanding the intracellular life cycle of F. tularensis, the F. tularensis proteins responsible for manipulating many host cell pathways are unknown. Identifying novel host-pathogen effector proteins is difficult because there is no rapid method to reliably distinguish between bacterial proteins that modify host processes and proteins that are involved in bacterial processes that are required for the bacteria to survive or replicate in the intracellular environment. The ability to identify mutants that are deficient for host-pathogen interactions is important because it can aid in prioritizing the investigation of genes of interest and in downstream experimental design. Moreover, certain mutant phenotypes, such as decreased phagosomal escape, hinder investigation of other potential phenotypes. A method to specifically complement these phenotypes would allow for further characterizations of certain F. tularensis mutants. Thus we sought to develop a method to easily identify and functionally complement mutants that are deficient for interactions with the host.
    Full-text · Article · Feb 2014 · PLoS ONE
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    • "200 μM of each was used as a template for PCR with primers p15/p18 and treated with 30 cycles of PCR (94°C for 30 s, 54°C for 30 s and 72°C for 120 s). The gel-purified 1996 bp fragment was digested with BamHI and NotI (New England Biolabs) and cloned into BamHI–NotI digested pMP812 (LoVullo et al., 2009). The plasmid is introduced into F. tularensis LVS by electroporation. "
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    ABSTRACT: In order to develop a successful infectious cycle, intracellular bacterial pathogens must be able to adapt their metabolism to optimally utilize the nutrients available in the cellular compartments and tissues where they reside. Francisella tularensis, the agent of the zoonotic disease tularemia, is a highly infectious bacterium for a large number of animal species. This bacterium replicates in its mammalian hosts mainly in the cytosol of infected macrophages. We report here the identification of a novel amino acid transporter of the major facilitator superfamily of secondary transporters that is required for bacterial intracellular multiplication and systemic dissemination. We show that inactivation of this transporter does not affect phagosomal escape but prevents multiplication in the cytosol of all cell types tested. Remarkably, the intracellular growth defect of the mutant was fully and specifically reversed by addition of asparagine or asparagine-containing dipeptides as well as by simultaneous addition of aspartic acid and ammonium. Importantly, bacterial virulence was also restored in vivo, in the mouse model, by asparagine supplementation. This work unravels thus, for the first time, the importance of asparagine for cytosolic multiplication of Francisella. Amino acid transporters are likely to constitute underappreciated players in bacterial intracellular parasitism.
    Full-text · Article · Oct 2013 · Cellular Microbiology
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