Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 ؋ 10 7 CFU g of DNA ؊1in F. tularensis LVS, Francisella novicida U112, and E. coli DH5␣. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.

Nonvertebrate model hosts represent valuable tools for the study of host-pathogen interactions because they facilitate the identification of bacterial virulence factors and allow the discovery of novel components involved in host innate immune responses. In this report, we determined that the greater wax moth caterpillar Galleria mellonella is a convenient nonmammalian model host for study of the role of the type III secretion system (TTSS) in Pseudomonas aeruginosa pathogenesis. Based on the observation that a mutation in the TTSS pscD gene of P. aeruginosa strain PA14 resulted in a highly attenuated virulence phenotype in G. mellonella, we examined the roles of the four known effector proteins of P. aeruginosa (ExoS, ExoT, ExoU, and ExoY) in wax moth killing. We determined that in P. aeruginosa strain PA14, only ExoT and ExoU play a significant role in G. mellonella killing. Strain PA14 lacks the coding sequence for the ExoS effector protein and does not seem to express ExoY. Moreover, using ⌬exoU ⌬exoY, ⌬exoT ⌬exoY, and ⌬exoT ⌬exoU double mutants, we determined that individual translocation of either ExoT or ExoU is sufficient to obtain nearly wild-type levels of G. mellonella killing. On the other hand, data obtained with a ⌬exoT ⌬exoU ⌬exoY triple mutant and a ⌬pscD mutant suggested that additional, as-yet-unidentified P. aeruginosa components of type III secretion are involved in virulence in G. mellonella. A high level of correlation between the results obtained in the G. mellonella model and the results of cytopathology assays performed with a mammalian tissue culture system validated the use of G. mellonella for the study of the P. aeruginosa TTSS.The human opportunistic bacterial pathogen Pseudomonas aeruginosa has proven to be a particularly versatile pathogen that is capable of causing diseases in plants, nematodes, and insects as well as in mice and humans (13,31,36,47,51). One particular P. aeruginosa strain, PA14, originally isolated from a human burn wound patient, has been used to demonstrate that P. aeruginosa virulence-related genes important for mouse pathogenesis can be identified by screening for less virulent mutants in plants or nematodes (36, 48, 52). In general, the use of nonvertebrate model hosts has facilitated the identification of bacterial virulence factors in a number of human bacterial pathogens in addition to P. aeruginosa and has led to the identification of new components involved in host innate immune responses (1,9,14,24,35,36,38,41,48,52).In gram-negative plant and animal pathogens, a highly conserved feature of pathogenesis is the so-called type III secretion system (TTSS) required for the translocation of effector proteins (virulence factors) directly into the cytosol of target eukaryotic cells (22,29,34). In mammals, the main targets of the translocated effector proteins include the host cytoskeleton and innate immune response pathways of macrophages and epithelial cells. For example, in Yersinia spp. and P. aeruginosa, TTSS effector proteins alter the normal actin cyto...

Francisella tularensis, the etiologic agent of tularemia in humans, is a potential biological threat due to its low infectious dose and multiple routes of entry. F. tularensis replicates within several cell types, eventually causing cell death by inducing apoptosis. In this study, a modified Himar1 transposon (HimarFT) was used to mutagenize F. tularensis LVS. Approximately 7,000 Km r clones were screened using J774A.1 macrophages for reduction in cytopathogenicity based on retention of the cell monolayer. A total of 441 candidates with significant host cell retention compared to the parent were identified following screening in a high-throughput format. Retesting at a defined multiplicity of infection followed by in vitro growth analyses resulted in identification of approximately 70 candidates representing 26 unique loci involved in macrophage replication and/or cytotoxicity. Mutants carrying insertions in seven hypothetical genes were screened in a mouse model of infection, and all strains tested appeared to be attenuated, which validated the initial in vitro results obtained with cultured macrophages. Complementation and reverse transcription-PCR experiments suggested that the expression of genes adjacent to the HimarFT insertion may be affected depending on the orientation of the constitutive groEL promoter region used to ensure transcription of the selective marker in the transposon. A hypothetical gene, FTL_0706, postulated to be important for lipopolysaccharide biosynthesis, was confirmed to be a gene involved in O-antigen expression in F. tularensis LVS and Schu S4. These and other studies demonstrate that therapeutic targets, vaccine candidates, or virulence-related genes may be discovered utilizing classical genetic approaches in Francisella.Francisella tularensis is a gram-negative intracellular pathogen and the etiologic agent of human tularemia. The CDC has classified F. tularensis as a category A select agent due to its highly infectious nature and ease of dissemination. Four subspecies of F. tularensis have been recognized, including (i) the virulent type A F. tularensis subsp. tularensis, (ii) the less virulent type B F. tularensis subsp. holarctica, (iii) F. tularensis subsp. mediasiatica, and (iv) F. tularensis subsp. novicida. The F. tularensis LVS (live vaccine strain) is derived from F. tularensis subsp. holarctica and is used as a model system to identify Francisella virulence factors since it is attenuated in humans but virulent in mice (8, 21). The limited genetic variation (2 to 4%) between the subspecies of Francisella suggests that there is potential overlap among genes related to pathogenesis (7,54,59). In fact, F. tularensis LVS and Schu S4 vary in genomic sequence by less than 1% (59). Regardless of the high sequence similarity at the genomic level, genome rearrangement and variation at the functional or regulatory level among the subspecies clearly result in phenotypes that impact virulence and pathogenesis (12,15,29,54,73,74).The life cycle of F. tularensis inside the macrophage ...

Summary Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that possesses a type III secretion system (T3SS) critical for evading innate immunity and establishing acute infections in compromised patients. Our research has focused on the structure-activity relationships of ExoU, the most toxic and destructive type III effector produced by P. aeruginosa. ExoU posseses phospholipase activity, which is detectable in vitro only when a eukaryotic cofactor is provided with membrane substrates. We report here that a subpopulation of ubiquitylated yeast SOD1 and other ubiquitylated mammalian proteins activate ExoU. Phospholipase activity was detected using purified ubiquitin of various chain lengths and linkage types; however, free monoubiquitin is sufficient in a genetically engineered dual expression system. The use of ubiquitin by a bacterial enzyme as an activator is unprecedented and represents a new aspect in the manipulation of the eukaryotic ubiquitin system to facilitate bacterial replication and dissemination.

Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host.