Dataset Information

The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.

ABSTRACT: Francisella tularensis causes the human disease tularemia. F. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. F. tularensis virulence is dependent upon the Francisella pathogenicity island (FPI), a cluster of genes that we show here shares homology with type VI secretion gene clusters in Vibrio cholerae and Pseudomonas aeruginosa. We demonstrate that two FPI proteins, VgrG and IglI, are secreted into the cytosol of infected macrophages. VgrG and IglI are required for F. tularensis phagosomal escape, intramacrophage growth, inflammasome activation and virulence in mice. Interestingly, VgrG secretion does not require the other FPI genes. However, VgrG and other FPI genes, including PdpB (an IcmF homologue), are required for the secretion of IglI into the macrophage cytosol, suggesting that VgrG and other FPI factors are components of a secretion system. This is the first report of F. tularensis FPI virulence proteins required for intramacrophage growth that are translocated into the macrophage.

SUBMITTER: Barker JR

PROVIDER: S-EPMC2814410 | biostudies-literature | 2009 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.

Barker Jeffrey R JR Chong Audrey A Wehrly Tara D TD Yu Jieh-Juen JJ Rodriguez Stephen A SA Liu Jirong J Celli Jean J Arulanandam Bernard P BP Klose Karl E KE

Molecular microbiology 20091201 6

Francisella tularensis causes the human disease tularemia. F. tularensis is able to survive and replicate within macrophages, a trait that has been correlated with its high virulence, but it is unclear the exact mechanism(s) this organism uses to escape killing within this hostile environment. F. tularensis virulence is dependent upon the Francisella pathogenicity island (FPI), a cluster of genes that we show here shares homology with type VI secretion gene clusters in Vibrio cholerae and Pseudo ...[more]

PMID: 20054881

Similar Datasets

Project description:Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of approximately 21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals.

Project description:pool 16 and pool 29 arrays represent the Francisella transposon insertion mutants isolated from the spleens of two separate groups of 5 intraperitoneally infected mice. skin arrays represent the Francisella transposon insertion mutants isolated from the skin of 5 individual, subcutaneously infected mice. input arrays represent Francisella transposon insertion mutants surviving after growth in TSB broth. Detailed Materials and Methods from Weiss et al, PNAS, 2007, In vivo negative selection screen identifies genes required for Francisella virulence: Mice and Francisella infections Female C57BL/6 mice (Jackson) between 7 and 10 wk of age were kept under specific pathogen-free conditions in filter-top cages at Stanford University and provided with sterile water and food ad libitum. Experimental studies were in accordance with IACUC Guidelines. Mice were inoculated with 2x105 cfu subcutaneously in 50 ul sterile PBS or intraperitoneally (IP) in 500 ul. Spleens were harvested 2 days post-infection, homogenized, and dilutions were plated on supplemented MH agar plates containing kanamycin (infections with library) or MH plates with and without the appropriate antibiotic (competition experiments). Plates were grown overnight at 37oC, 5% CO2 and colonies were enumerated. After infections with the library, approximately 10^5 cfu were collected in sterile PBS for isolation of DNA. Competitive Index (CI) values were calculated using the formula CI = (mutant cfu in output/wild-type cfu in output)/(mutant cfu in input/wild-type cfu in input). Microarrays Genomic DNA was purified from bacterial pellets by phenol-chloroform extraction. Each DNA sample was divided in two and digested separately with BfaI or RsaI (NEB). The digested DNA was used as the template for in vitro transcription with the AmpliScribe T7-Flash transcription kit (Epicentre) following the manufacturer's protocol, except that 2 ?g of digested DNA was used, and the reaction was allowed to proceed for 12 to 16 h. Purified RNA was used in a RT reaction using Superscript II(?) (Invitrogen) and random hexamers as primers. cDNA was labeled with amino-allyl dUTP using the Klenow (exo-) enzyme. The ssDNA containing amino-allyl dUTP from the mouse output or the library input pools were labeled with Cy3 and Cy5, respectively, prior to hybridization to our Francisella microarray as described previously. Data analysis Normalized data were downloaded from the Stanford Microarray Database according to the median log2 Cy5/Cy3 (logRAT2N). Filters for feature quality, including a Cy3 net median intensity of 150 and regression correlation of >0.6, were applied. To compare data from separate IP infection experiments, each experimental sample was zero-transformed to the input/input control. Features (spots) missing values for 40% of the arrays were removed from the data set. The data sets were analyzed with the SAM program, using the two-class analysis option to identify features that consistently deviated from the input and samples across all of the arrays with a false discovery rate of 1%.

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.

Dataset Information

The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.

Publications

The Francisella tularensis pathogenicity island encodes a secretion system that is required for phagosome escape and virulence.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets