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Effects of the putative transcriptional regulator IclR on Francisella tularensis pathogenesis.

ABSTRACT: Francisella tularensis is a highly virulent Gram-negative bacterium and is the etiological agent of the disease tularemia. IclR, a presumed transcriptional regulator, is required for full virulence of the animal pathogen, F. tularensis subspecies novicida U112 (53). In this study, we investigated the contribution of IclR to the intracellular growth, virulence, and gene regulation of human pathogenic F. tularensis subspecies. Deletion of iclR from the live vaccine strain (LVS) and SchuS4 strain of F. tularensis subsp. holarctica and F. tularensis subsp. tularensis, respectively, did not affect their abilities to replicate within macrophages or epithelial cells. In contrast to F. tularensis subsp. novicida iclR mutants, LVS and SchuS4 ΔiclR strains were as virulent as their wild-type parental strains in intranasal inoculation mouse models of tularemia. Furthermore, wild-type LVS and LVSΔiclR were equally cytotoxic and induced equivalent levels of interleukin-1β expression by infected bone marrow-derived macrophages. Microarray analysis revealed that the relative expression of a limited number of genes differed significantly between LVS wild-type and ΔiclR strains. Interestingly, many of the identified genes were disrupted in LVS and SchuS4 but not in their corresponding F. tularensis subsp. novicida U112 homologs. Thus, despite the impact of iclR deletion on gene expression, and in contrast to the effects of iclR deletion on F. tularensis subsp. novicida virulence, IclR does not contribute significantly to the virulence or pathogenesis of F. tularensis LVS or SchuS4.

SUBMITTER: Mortensen BL

PROVIDER: S-EPMC2981306 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:After infecting a mammalian host, the facultative intracellular bacterium, Francisella tularensis, encounters an elevated environmental temperature. We hypothesized that this temperature change may regulate genes essential for infection.Microarray analysis of F. tularensis LVS shifted from 26 degrees C (environmental) to 37 degrees C (mammalian) showed approximately 11% of this bacterium's genes were differentially-regulated. Importantly, 40% of the protein-coding genes that were induced at 37 degrees C have been previously implicated in virulence or intracellular growth of Francisella in other studies, associating the bacterial response to this temperature shift with pathogenesis. Forty-four percent of the genes induced at 37 degrees C encode proteins of unknown function, suggesting novel Francisella virulence traits are regulated by mammalian temperature. To explore this possibility, we generated two mutants of loci induced at 37 degrees C [FTL_1581 and FTL_1664 (deoB)]. The FTL_1581 mutant was attenuated in a chicken embryo infection model, which was likely attributable to a defect in survival within macrophages. FTL_1581 encodes a novel hypothetical protein that we suggest naming temperature-induced, virulence-associated locus A, tivA. Interestingly, the deoB mutant showed diminished entry into mammalian cells compared to wild-type LVS, including primary human macrophages and dendritic cells, the macrophage-like RAW 264.7 line, and non-phagocytic HEK-293 cells. This is the first study identifying a Francisella gene that contributes to uptake into both phagocytic and non-phagocytic host cells.Our results provide new insight into mechanisms of Francisella virulence regulation and pathogenesis. F. tularensis LVS undergoes considerable gene expression changes in response to mammalian body temperature. This temperature shift is important for the regulation of genes that are critical for the pathogenesis of Francisella. Importantly, the compilation of temperature-regulated genes also defines a rich collection of novel candidate virulence determinants, including tivA (FTL_1581). An analysis of tivA and deoB (FTL_1664) revealed that these genes contribute to intracellular survival and entry into mammalian cells, respectively.

Project description:Francisella tularensis is composed of a number of subspecies with varied geographic distribution, host ranges, and virulence. In view of these marked differences, comparative functional genomics may elucidate some of the molecular mechanism(s) behind these differences. In this study a shared probe microarray was designed that could be used to compare the transcriptomes of Francisella tularensis subsp. tularensis Schu S4 (Ftt), Francisella tularensis subsp. holarctica OR960246 (Fth), Francisella tularensis subsp. holarctica LVS (LVS), and Francisella novicida U112 (Fn). To gain insight into expression differences that may be related to the differences in virulence of these subspecies, transcriptomes were measured from each strain grown in vitro under identical conditions, utilizing a shared probe microarray. The human avirulent Fn strain exhibited high levels of transcription of genes involved in general metabolism, which are pseudogenes in the human virulent Ftt and Fth strains, consistent with the process of genome decay in the virulent strains. Genes encoding an efflux system (emrA2 cluster of genes), siderophore (fsl operon), acid phosphatase, LPS synthesis, polyamine synthesis, and citrulline ureidase were all highly expressed in Ftt when compared to Fn, suggesting that some of these may contribute to the relative high virulence of Ftt. Genes expressed at a higher level in Ftt when compared to the relatively less virulent Fth included genes encoding isochorismatases, cholylglycine hydrolase, polyamine synthesis, citrulline ureidase, Type IV pilus subunit, and the Francisella Pathogenicity Island protein PdpD. Fth and LVS had very few expression differences, consistent with the derivation of LVS from Fth. This study demonstrated that a shared probe microarray designed to detect transcripts in multiple species/subspecies of Francisella enabled comparative transcriptional analyses that may highlight critical differences that underlie the relative pathogenesis of these strains for humans. This strategy could be extended to other closely-related bacterial species for inter-strain and inter-species analyses.

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Effects of the putative transcriptional regulator IclR on Francisella tularensis pathogenesis.

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