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New protein fold revealed by a 1.65 A resolution crystal structure of Francisella tularensis pathogenicity island protein IglC.

ABSTRACT: Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes the fulminating disease tularemia and is considered to be a potential bioweapon. F. tularensis pathogenicity island proteins play a key role in modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm of macrophages. The 23 kDa pathogenicity island protein IglC is essential for the survival and proliferation of F. tularensis in macrophages. Seeking to gain some insight into its function, we determined the crystal structure of IglC at 1.65 A resolution. IglC adopts a beta-sandwich conformation that exhibits no similarity with any known protein structure.

SUBMITTER: Sun P

PROVIDER: S-EPMC2211698 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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New protein fold revealed by a 1.65 A resolution crystal structure of Francisella tularensis pathogenicity island protein IglC.

Sun Ping P Austin Brian P BP Schubot Florian D FD Waugh David S DS

Protein science : a publication of the Protein Society 20070928 11

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes the fulminating disease tularemia and is considered to be a potential bioweapon. F. tularensis pathogenicity island proteins play a key role in modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm of macrophages. The 23 kDa pathogenicity island protein IglC is essential for the survival and proliferation of F. tularensis in macrophages. Seeking to gain some insight into it ...[more]

PMID: 17905833

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Project description:During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides' guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue l-serine hydroxamate. Serine hydroxamate competitively inhibits tRNAser aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC, which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorly understood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials.

Project description:A safer and more effective vaccine than the unlicensed Francisella tularensis Live Vaccine Strain (LVS) is needed to protect against the biowarfare agent F. tularensis. Previously, we developed an LVS ?capB mutant that is significantly safer than LVS and provides potent protective immunity against F. tularensis respiratory challenge when administered intranasally but limited protection when administered intradermally unless as part of a prime-boost vaccination strategy. To improve the immunogenicity and efficacy of LVS ?capB, we developed recombinant LVS ?capB (rLVS ?capB) strains overexpressing various F. tularensis Francisella Pathogenicity Island (FPI) proteins - IglA, IglB and IglC, and a fusion protein (IglABC) comprising immunodominant epitopes of IglA, IglB, and IglC downstream of different Francisella promoters, including the bacterioferritin (bfr) promoter. We show that rLVS ?capB/bfr-iglA, iglB, iglC, and iglABC express more IglA, IglB, IglC or IglABC than parental LVS ?capB in broth and in human macrophages, and stably express FPI proteins in macrophages and mice absent antibiotic selection. In response to IglC and heat-inactivated LVS, spleen cells from mice immunized intradermally with rLVS ?capB/bfr-iglC or bfr-iglABC secrete greater amounts of interferon-gamma and/or interleukin-17 than those from mice immunized with LVS ?capB, comparable to those from LVS-immunized mice. Mice immunized with rLVS ?capB/bfr-iglA, iglB, iglC or iglABC produce serum antibodies at levels similar to LVS-immunized mice. Mice immunized intradermally with rLVS ?capB/bfr-iglABC and challenged intranasally with virulent F. tularensis Schu S4 survive longer than sham- and LVS ?capB-immunized mice. Mice immunized intranasally with rLVS ?capB/bfr-iglABC - but not with LVS - just before or after respiratory challenge with F. tularensis Schu S4 are partially protected; protection is correlated with induction of a strong innate immune response. Thus, rLVS ?capB/bfr-iglABC shows improved immunogenicity and protective efficacy compared with parental LVS ?capB and, in contrast to LVS, has partial efficacy as immediate pre- and post-exposure prophylaxis.

Dataset Information

New protein fold revealed by a 1.65 A resolution crystal structure of Francisella tularensis pathogenicity island protein IglC.

Publications

New protein fold revealed by a 1.65 A resolution crystal structure of Francisella tularensis pathogenicity island protein IglC.

Similar Datasets

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