Reproducible and quantitative model of infection of Dermacentor variabilis with the live vaccine strain of Francisella tularensis.

Pathogen life cycles in mammalian hosts have been studied extensively, but studies with arthropod vectors represent considerable challenges. In part this is due to the difficulty of delivering a reproducible dose of bacteria to follow arthropod-associated replication. We have established reproducible techniques to introduce known numbers of Francisella tularensis strain LVS from mice into Dermacentor variabilis nymphs. Using this model infection system, we performed dose-response infection experiments and followed bacterial replication through the molt to adults and at later time points. During development to adults, bacteria replicate to high numbers and can be found associated with the gut tissues, salivary glands, and hemolymph of adult ticks. Further, we can transmit a mutant of LVS (LVS ΔpurMCD) that cannot replicate in macrophages in vitro or in mice to nymphs. Our data show that the LVS ΔpurMCD mutant cannot be transstadially transmitted from nymphs to adult ticks. We then show that a plasmid-complemented strain of this mutant is recoverable in adult ticks and necessary for bacterial replication during the molt. In a mixed-infection assay (ΔpurMCD mutant versus ΔpurMCD complement), 98% of the recovered bacteria retained the plasmid marker. These data suggest that the ΔpurMCD mutation cannot be rescued by the presence a complemented strain in a mixed infection. Importantly, our infection model provides a platform to test specific mutants for their replication in ticks, perform competition studies, and use other genetic techniques to identify F. tularensis genes that are expressed or required in this unique environment.

Ubiquitin and ubiquitin-modified proteins activate the Pseudomonas aeruginosa T3SS cytotoxin, ExoU.

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 possesses 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.

Induced conformational changes in the activation of the Pseudomonas aeruginosa type III toxin, ExoU.

ExoU is a 74-kDa, water-soluble toxin injected directly into mammalian cells through the type III secretion system of the opportunistic pathogen, Pseudomonas aeruginosa. Previous studies have shown that ExoU is a Ca(2+)-independent phospholipase that requires a eukaryotic protein cofactor. One protein capable of activating ExoU and serving as a required cofactor was identified by biochemical and proteomic methods as superoxide dismutase (SOD1). In these studies, we carried out site-directed spin-labeling electron paramagnetic resonance spectroscopy to examine the effects of SOD1 and substrate liposomes on the structure and dynamics of ExoU. Local conformational changes within the catalytic site were observed in the presence of substrate liposomes, and were enhanced by the addition of SOD1 in a concentration-dependent manner. Conformational changes in the C-terminal domain of ExoU were observed upon addition of cofactor, even in the absence of liposomes. Double electron-electron resonance experiments indicated that ExoU samples multiple conformations in the resting state. In contrast, addition of SOD1 induced ExoU to adopt a single, well-defined conformation. These studies provide, to our knowledge, the first direct evidence for cofactor- and membrane-induced conformational changes in the mechanism of activation of ExoU.

Quantification of the neutralization of cytokine biological activity by antibody: the ten-fold reduction bioassay of interleukin-6 as growth factor.

The measurement of neutralizing antibodies (NAbs) to biological therapeutic agents is important clinically as well as for the preclinical evaluation of product immunogenicity. To determine whether the theoretical concepts and experimental data from studies of the nature of antibody neutralization of interferons (IFNs) can apply to unrelated protein effector molecules, neutralization experiments were undertaken with interleukin-6 (IL-6), a proinflammatory, highly pleiotropic cytokine. By following IL-6 induction of hybridoma cell growth, we demonstrated that anti-IL-6 monoclonal and polyclonal NAbs can be measured with a bioassay design structured to reduce 10 Laboratory Units (LU)/mL to 1 LU/mL. Results are reported in Ten-fold Reduction Units (TRU)/mL, as recommended for the standardization of IFN NAb unitage. The bioassay was shown to be sensitive, reproducible, and robust in measuring IL-6 potency and NAb titer, as well as for evaluating dose-response curve slope differences. This bioassay design should be applicable to any cytokine, growth factor, protein hormone, or similar effector molecules for which an adequately sensitive cellular response can be quantified.

Identification of Francisella tularensis Himar1-based transposon mutants defective for replication in macrophages.

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.

In vivo Himar1-based transposon mutagenesis of Francisella tularensis.

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.

Use of the Galleria mellonella caterpillar as a model host to study the role of the type III secretion system in Pseudomonas aeruginosa pathogenesis.

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 Delta exoU Delta exoY, Delta exoT Delta exoY, and Delta exoT Delta 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 Delta exoT Delta exoU Delta exoY triple mutant and a Delta 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.

Construction and characterization of a highly efficient Francisella shuttle plasmid.

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 x 10(7) CFU mug of DNA(-1) in F. tularensis LVS, Francisella novicida U112, and E. coli DH5alpha. 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.