Project description:The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Although some Francisella determinants of intracellular growth have been identified, much remains to be understood about the pathogenesis of this organism. In particular, how Francisella responds to its intracellular environment could provide clues about its intracellular biology and reveal pathogenic determinants based on their intracellular expression profiles. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages. Phagocytosed bacteria rapidly responded to their intracellular environment and progressively altered their transcriptional profile over time. Differential gene expression profiles were revealed that correlated with specific intracellular locations of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of a subset of transport and metabolic genes characterized the cytosolic replication stages. Expression of the Francisella Pathogenicity Island (FPI) genes, the functions of which are associated with intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci putatively encoding hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated during the intracellular cycle. In-frame deletion of FTT0383, the Schu S4 ortholog of fevR, of FTT0369c or FTT1676 abolished the ability of Schu S4 to either survive or proliferate intracellularly, demonstrating that bacterial factors of intracellular pathogenesis can be identified based on their intracellular expression profile. In conclusion, establishing the intracellular transcriptome of Francisella has revealed important aspects of its intracellular biology and identified novel virulence determinants of this pathogen. Keywords: Time series

Project description:Francisella tularensis is one of just three bacterial species designated as a Category A select agent by the Centre for Disease Control (CDC), a category indicating agents most likely to be employed as a biological weapon. F. tularensis can be divided into four different subspecies, and it is well known that the type, severity and duration of the disease can differ substantially depending on what subspecies is responsible for the infection. Of the four subspecies, subsp. tularensis (Type A) and subsp. holartica (Type B) are of primary clinical significance, and account for nearly all recorded incidences of the disease in humans. Though Type A is considered to be much more virulent than Type B, recent reports have shown that Type A can be further subdivided into two genetically distinct populations, termed A.I and A.II, which differ with respect to geographical location, disease outcome and source of recovered isolates. Of these two subpopulations, clinical data suggests that Type A.I strains are much more virulent than Type A.II, and Type A.II strains appear to have a disease outcome similar to infections with Type B. During natural infections, host mononuclear phagocytes appear to be the primary target of all F. tularensis subsp. Despite the differences in disease outcome between different subspecies, the mechanisms involved in phagosomal escape, the modulation of phagosomal biogenesis, phagosomal disruption and bacterial egress appears to be indistinguishable between subspecies, at least at a physiological level. However, recent reports suggest that at the molecular level there appears to be significant differences between subspecies during infection, and this may account for the dramatically different disease outcome associated with different subspecies. Our long-term goal is to identify unique molecular markers associated with the more virulent Type A.I strains with the potential for use as vaccine or therapeutic targets, and to provide a greater insight into the molecular differences associated with F. tularensis infection. Our overall objective of this application, which is the next step in the pursuit of that goal, is to obtain a full transcriptional profile of Type A.I, Type A.II and Type B strains following infection of human monocyte derived macrophage (hMDM) using a novel full genome quantitative real time PCR array (qRT-PCR). The central hypothesis of the application is that Type A.I, Type A.II and Type B strains activate different transcriptional networks during the course of infection of macrophage, and that these differences are at least partially responsible for the observed differences in disease outcome between the subspecies. This hypothesis has been formulated on the basis of preliminary data obtained at our laboratory that shows significant differences in the global transcriptional profiles of Type A and Type B strains exposed to environmental stimuli that mimic the intracellular environment (see Preliminary studies).The rationale for the proposed research is that by investigating differences in the transcriptional profiles of the three subspecies, we can identify unique pathogen-associated molecules or networks that can be targeted for future therapeutic research. Complementary to our supportive preliminary data, we are particularly well prepared to undertake the proposed research, with the recent development of a full genome qRT-PCR array that will allow accurate and comprehensive detection of transcriptional changes from pictogram quantities of starting RNA. This is a pilot study

Project description:The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen, whose virulence requires proliferation inside host cells, including macrophages. Although some Francisella determinants of intracellular growth have been identified, much remains to be understood about the pathogenesis of this organism. In particular, how Francisella responds to its intracellular environment could provide clues about its intracellular biology and reveal pathogenic determinants based on their intracellular expression profiles. Here we have performed a global transcriptional profiling of the highly virulent F. tularensis subsp. tularensis Schu S4 strain during its intracellular cycle within primary murine macrophages. Phagocytosed bacteria rapidly responded to their intracellular environment and progressively altered their transcriptional profile over time. Differential gene expression profiles were revealed that correlated with specific intracellular locations of the bacteria. Upregulation of general and oxidative stress response genes was a hallmark of the early phagosomal and late endosomal stages, while induction of a subset of transport and metabolic genes characterized the cytosolic replication stages. Expression of the Francisella Pathogenicity Island (FPI) genes, the functions of which are associated with intracellular proliferation, increased during the intracellular cycle. Similarly, 27 chromosomal loci putatively encoding hypothetical, secreted, outer membrane proteins or transcriptional regulators were identified as upregulated during the intracellular cycle. In-frame deletion of FTT0383, the Schu S4 ortholog of fevR, of FTT0369c or FTT1676 abolished the ability of Schu S4 to either survive or proliferate intracellularly, demonstrating that bacterial factors of intracellular pathogenesis can be identified based on their intracellular expression profile. In conclusion, establishing the intracellular transcriptome of Francisella has revealed important aspects of its intracellular biology and identified novel virulence determinants of this pathogen. Keywords: Time series Intracellular cycle within primary murine macrophages using the time series of zero, one, two, four, eight, twelve, sixteen and twenty-four hours

Dataset Information

Francisella tularensis

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets