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The capBCA Locus is Required for Intracellular Growth of Francisella tularensis LVS.

ABSTRACT: Francisella tularensis is the causative agent of tularemia and a category A bioterrorism agent. The molecular basis for the extreme virulence of F. tularensis remains unclear. Our recent study found that capBCA, three neighboring genes, are necessary for the infection of F. tularensis live vaccine strain (LVS) in a respiratory infection mouse model. We here show that the capBCA genes are necessary for in vivo growth of F. tularensis LVS in the lungs, spleens, and livers of BALB/c mice. Unmarked deletion of capBCA in type A strain Schu S4 resulted in significant attenuation in virulence although the level of the attenuation in Schu S4 was much less profound than in LVS. We further demonstrated that CapB protein is produced at a low level under the in vitro culture conditions, and capB alone is necessary for in vivo growth of F. tularensis LVS in the lungs of BALB/c mice. Finally, deletional mutations in capB alone or capBCA significantly impaired intracellular growth of F. tularensis LVS in cultured macrophages, thus suggesting that the capBCA genes are necessary for intracellular adaptation of F. tularensis. The requirement of this gene locus in intracellular adaption at least in part explains the significant attenuation of F. tularensis capBCA mutants in virulence.

SUBMITTER: Su J

PROVIDER: S-EPMC3128946 | biostudies-other | 2011

REPOSITORIES: biostudies-other

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The capBCA Locus is Required for Intracellular Growth of Francisella tularensis LVS.

Su Jingliang J Asare Rexford R Yang Jun J Nair Manoj Kumar Mohan MK Mazurkiewicz Joseph E JE Abu-Kwaik Yousef Y Zhang Jing-Ren JR

Frontiers in microbiology 20110425

Francisella tularensis is the causative agent of tularemia and a category A bioterrorism agent. The molecular basis for the extreme virulence of F. tularensis remains unclear. Our recent study found that capBCA, three neighboring genes, are necessary for the infection of F. tularensis live vaccine strain (LVS) in a respiratory infection mouse model. We here show that the capBCA genes are necessary for in vivo growth of F. tularensis LVS in the lungs, spleens, and livers of BALB/c mice. Unmarked ...[more]

PMID: 21747799

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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

The capBCA Locus is Required for Intracellular Growth of Francisella tularensis LVS.

Publications

The capBCA Locus is Required for Intracellular Growth of Francisella tularensis LVS.

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