Systems approach to investigating host-pathogen interactions in infections with the biothreat agent Francisella. Constraints-based model of Francisella tularensis

Abstract

BackgroundFrancisella tularensis is a prototypic example of a pathogen for which few experimental datasets exist, but for which copious high-throughout data are becoming available because of its re-emerging significance as biothreat agent. The virulence of Francisella tularensis depends on its growth capabilities within a defined environmental niche of the host cell.ResultsWe reconstructed the metabolism of Francisella as a stoichiometric matrix. This systems biology approach demonstrated that changes in carbohydrate utilization and amino acid metabolism play a pivotal role in growth, acid resistance, and energy homeostasis during infection with Francisella. We also show how varying the expression of certain metabolic genes in different environments efficiently controls the metabolic capacity of F. tularensis. Selective gene-expression analysis showed modulation of sugar catabolism by switching from oxidative metabolism (TCA cycle) in the initial stages of infection to fatty acid oxidation and gluconeogenesis later on. Computational analysis with constraints derived from experimental data revealed a limited set of metabolic genes that are operational during infection.ConclusionsThis integrated systems approach provides an important tool to understand the pathogenesis of an ill-characterized biothreat agent and to identify potential novel drug targets when rapid target identification is required should such microbes be intentionally released or become epidemic.

Importance of Branched-Chain Amino Acid Utilization in Francisella Intracellular Adaptation

“…The plasmid was introduced into F. tularensis LVS by cryo-transformation. The same twostep allelic exchange procedure was used to create the chromosomal ⌬FTL_1803 mutant (14).…”

“…Bacteria were suspended at a final OD 600 of 0.5 in the same medium containing 50 mg ml Ϫ1 of chloramphenicol. After 15 min of preincubation at 25°C, uptake was started by the addition of either L-[U- 14 C]isoleucine or L-[U-14 C]leucine (Ile and Leu; PerkinElmer) at various concentrations (ranging from 1 to 100 M). The 14 C-radiolabeled amino acids were at a specific activity of 7.4 GBq mmol Ϫ1 .…”

“…novicida U112 by electroporation. A standard two-step allelic exchange procedure was used to create the chromosomal ⌬FTN_1654 mutant (14). The mutant strains were checked for loss of the corresponding wild-type genes by PCR sequencing (GATC Biotech) using specific primers.…”

“…Recent studies from our group (7)(8)(9) and from several others groups (10)(11)(12)(13) have highlighted the importance of nutrient acquisition and metabolic adaptations in Francisella pathogenesis. In particular, amino acids are thought to represent major sources of carbon and energy for F. tularensis (13)(14)(15). However, the mammalian cell cytosol contains variable amounts of amino acids.…”

Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*

“…It has, therefore, developed sophisticated tools recently designated as "nutritional virulence" attributes (6,7). Amino acids have been described as the essential nutrient source that Francisella has evolved to scavenge, by different means, during its intracellular life cycle (8). For examples, activation of the host macro-autophagy degradation machinery (9), and exploitation of host polypeptides such as glutathione (10), allow Francisella to obtain its essential amino acids.…”