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Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts.

ABSTRACT: Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.

SUBMITTER: Escudero R

PROVIDER: S-EPMC2546735 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts.

Escudero Raquel R Toledo A A Gil Horacio H Kovácsová Katarina K Rodríguez-Vargas Manuela M Jado Isabel I García-Amil Cristina C Lobo Bruno B Bhide Mangesh M Anda Pedro P

Journal of clinical microbiology 20080723 9

Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and se ...[more]

PMID: 18650358

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Project description: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 ?ripA and ?pdpC. ?ripA has an intracellular growth defect that is likely due to an intrinsic defect and fails to suppress IL-1? secretion. In the co-infection model, ?ripA was unable to replicate in the host cell when wild-type bacteria infected the same cell, but cytokine suppression was rescued. Therefore, ?ripA intracellular growth is due to an intrinsic bacterial defect while cytokine secretion results from a failed host-pathogen interaction. Likewise, ?pdpC is deficient for phagosomal escape, intracellular survival and suppression of IL-1? secretion. Wild-type bacteria that entered through the same phagosome as ?pdpC rescued all of these phenotypes, indicating that ?pdpC 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.

Dataset Information

Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts.

Publications

Molecular method for discrimination between Francisella tularensis and Francisella-like endosymbionts.

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