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.
Highlights
In order to distinguish whether a phenotype results from a hostpathogen interaction or an intrinsic bacterial defect, we devised a method to functionally complement and identify hostpathogen interactions in trans
We quantified the amount of beads that bound to both GFP and CellTrace Far Red labeled F. tularensis by flow cytometry and found that 97.3+/20.7% of the beads bound to both GFP and CellTrace Far Red labeled bacteria (Figure 1 D, E, F)
We developed and optimized a method to reliably deliver two distinct F. tularensis bacteria into the same host cell
Summary
In order to distinguish whether a phenotype results from a hostpathogen interaction or an intrinsic bacterial defect, we devised a method to functionally complement and identify hostpathogen interactions in trans. Since cells are consistently infected with both strains of bacteria via the same phagosome, the wildtype bacteria functionally complement the host-pathogen interactions that the neighboring mutant strain fails to initiate. A mutant deficient for phagosomal escape that co-infects a host cell with wild-type bacteria will escape the phagosome because the wild-type bacteria secrete the effectors required for phagosomal escape. Bacterial mutants that exhibit a phenotype caused by intrinsic deficiencies such as defective metabolite production or acquisition will not be functionally complemented by this method since intrinsic defects cannot be trans-complemented by neighboring bacteria. To demonstrate the efficacy of this protocol, we functionally complemented cytokine suppression, phagosomal escape, and intracellular survival in F. tularensis subsp. PdpC contributes to phagosomal escape, intracellular survival and cytokine suppression [5,13]. We used DpdpC as a model of a mutant that contributes to a host-pathogen interaction
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