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Type VI secretion systems of Gram-negative bacteria - defining the functions of T6SS components and their contribution to virulence

PI: Anders Sjöstedt, Professor
Department of Clinical Microbiology
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There are six specialized secretion systems in Gram-negative bacteria. The Type VI secretion systems (T6SSs), identified in almost 100 different bacterial species, are essential for virulence of many important human pathogens such as Vibrio cholera and Pseudomonas aeruginosa. Still, the understanding of T6SSs in general is very incomplete. The overall aim of our work is to delineate the regulation and functions of effector proteins of T6SS, primarily Francisella tularensis, but also Vibrio cholerawill be studied. We will utilize molecular techniques to generate specific mutants as well as tagged T6SS components. Their subsequent localization and secretion during infection will then be detected by a variety of techniques, including direct microinjection of bacteria or T6SS components into host cells. We are also studying the possibility to inhibit the functions of T6SS by means of small molecular inhibitors. Specific aims are described in detail below.

 

Small compound screening as a mean to inhibit the T6SS of V. cholerae and F. tularensis.
We have initiated small compound screenings with the aim to identify inhibitors of T6S. Three different protocols will be used, which potentially will enable us to identify inhibitors that are directed against specific molecular targets, such as QseBC or VipAB, or with a general impact on T6S and where the molecular target(s) initially are not known. Since inhibitors of T6S target a virulence mechanism, they should not be deleterious for bacteria during 'normal' environmental growth conditions. Such anti-virulence strategies are advantageous to avoid development of resistance.

Structural determination of the IglA-IglB and VipA-VipB complexes and identification of their interaction partners.
Since structural information greatly contributes to the understanding of how proteins carry out their biological functions, we plan to solve the structure of the IglA-IglB and VipA-VipB complexes using X-ray crystallography. The work will be done in collaboration with experts on structural analyses in general and crystallography specifically.

Determining the function of the FPI proteins.

The exact molecular mechanisms exerted by the FPI proteins are enigmatic. While our generation of specific FPI mutants has contributed to an increased understanding of their biological roles, it does not allow us to determine whether their functions are effectuated exclusively during the intra-phagosomal phase of the infection or if they also execute important functions in the cytosolic location since many of them never reach the latter compartment. To clarify this, we have implemented a technique where GFP-expressing bacteria are injected directly into the cytoplasm of macrophages, thus circumventing the requirement for phagosomal escape. The morphological changes of microinjected cells and bacterial replication have then been assessed by live cell microscopy. In addition, an array of methods has established to determine the effects on host cell signaling and inflammatory responses in the eukaryotic host cells.

A model for prediction of tularemia outbreaks – relevance of climate change for future outbreaks

Tularemia, a zoonotic disease caused by Francisella tularensis, has been endemic in certain areas of northern Sweden for almost a century and it has emerged in areas of middle Sweden during the past decade. In 2010 some 500 individuals were diagnosed with tularemia. The infection is a local public health threat and incidences in Sweden are as high as 500/100,000. It is very contagious and a common laboratory-acquired infection. The pathogen is also a bioterrorism agent, since aerosols of ssp. tularensis are highly infectious and cause a life-threatening disease. The disease occurs with a seasonal pattern and is especially prevalent in late summer and autumn. The reasons for its geographical distribution and seasonal occurrence remain largely unexplained since epidemiological information is scarce. The most prevalent subspecies is ssp. holarctica (type B). Areas in Sweden and Finland have the highest incidences of the disease in the world and in each there have been around 2,000 cases per 5 years and therefore present unique opportunities to study the transmission and life cycle of the pathogen.


We have a working hypothesis that the predominant spread of F. tularensis in Sweden is through mosquitoes and that they acquire the bacteria in the water. This hypothesis is controversial since it is, to our knowledge, the only bacterial disease proposed to be disseminated mostly through mosquitoes and no experimental models have demonstrated how mosquitoes can acquire bacteria by other means than by blood-feeding. However, we believe that our recent work has provided strong support for the hypothesis and help to explain the critical role of mosquitoes for the life cycle. We have demonstrated that i) diverse F. tularensis-like organisms, including ssp. holarctica, persist in natural waters in the endemic areas in Sweden (Broman et al., 2011); ii) mosquito larvae from the same areas were reared to adults in the laboratory and PCR analysis revealed that 1/3 of the mosquito pools contained F. tularensis, including human-feeding mosquito species, implying that mosquitoes may acquire the bacterium already during their larval stage (Lundström et al., 2011); iii) a model was created using meteorological and hydrological data to predict mosquito prevalence in Dalarna and together with data on tularemia cases and local environmental variables, a predictive model for human tularemia cases was generated. The model showed that peak numbers of mosquitoes preceded tularemia among humans and the final model predicted 6 out of 7 actual outbreaks (Rydén et al., 2012); iv) an extensive investigation of the bacterial diversity during outbreaks has been performed using high-resolution genotyping of F. tularensis. Analysis of isolates from 136 patients revealed a strong spatial association between bacterial subpopulations and presumed location for disease acquisition. Disease clusters were in the vicinity of water-associated recreational areas (Svensson et al., 2009a). Thus, we have obtained extensive evidence to support our original hypothesis that the primary natural reservoir for F. tularensis in endemic areas is water-associated and that mosquitoes play a key role in the transmission cycle, possibly through ingestion of bacteria in their larval stage. Moreover, in view of the specific association between outbreaks and meteorological and hydrological variables, it is very likely that future outbreaks of tularemia will be affected by climate change. To this end, we performed a simulation based on an IPCC climate change scenario. Tularemia outbreaks in the counties of Dalarna, Gävleborg, Norrbotten, Värmland and Örebro were forecasted and durations were predicted to increase by 3.5-6.6 weeks until 2100 (Ryden et al., 2009). Thus, future climate changes will lead to an increased duration of tularemia in high-endemic areas of Sweden.

We have received support from Formas for the period 2013-2015 to continue the work and we now aim to identify parameters that significantly contribute to the outbreaks of tularemia in Sweden and to develop predictive models for these outbreaks and thereafter to; i) validate the models on retrospective data from tularemia endemic areas in Sweden, ii) to prognosticate the future occurrence of tularemia in Sweden and how it will be affected by climate change, and iii) implement the models so they will be operational in real-time and thereby can predict future outbreaks so these can be confined.

Tularemia is a significant health threat in endemic regions, and today there are no practical means to forecast an outbreak. With our Prognostic model at hand, health authorities could be alerted for the upcoming situation, which in turn would expedite preventive measures, diagnosis, and treatment of patients. Further, by defining the major transmission risk factors, useful recommendations on how to reduce the risk for transmission could be given to the public. The increased knowledge on the ecology that will be gained as a result of the project will be important tools in the surveillance of F. tularensis. A better understanding of the environmental prerequisites for spread of F. tularensis in aquatic milieus, and of its transmission routes, will help in the planning of constructed wetlands. The project will bring new insights and allow precise modeling of the effect of climate factors, such as global warming, on the occurrence and spread of tularemia in the future. In addition, it will serve as proof-of-concept and form a basis for the modeling of the spread of other vector-borne diseases.

References
Broman, T., Sjöstedt, A., Johansson, A (2011). Molecular Detection of Francisella tularensis in Natural Waters. Int J Microbiol 2011.
Lundström, J.O., Forsman, M., and Thelaus, J. (2011). Transstadial transmission of Francisella tularensis holarctica in mosquitoes, Sweden. Emerg Inf Dis DOI: 10.3201/eid1705.100426.

Rydén, P., Björk, R., Schäfer, M., Forsman, M., Sjöstedt, A., and Johansson, A. (2012). Swedish outbreaks of tularemia in man are explained by late summer mosquito prevalence. J. Inf. Dis. 205:297-304.

Ryden, P., Sjöstedt, A., and Johansson, A. (2009). Effects of climate change on tularaemia disease activity in Sweden. Glob Health Action 2.
Svensson, K., Larsson, P., Forsman, M., and Johansson, A. (2009a). Landscape epidemiology of tularemia outbreaks in Sweden. Emerg Infect Dis 15, 1937-1947.

Svensson, K., Forsman, M., and Johansson, A. (2009b). A real-time PCR array for hierarchical identification of Francisella isolates. PLoS One 4, e8360.


Contact:
Anders Sjöstedt, Professor
Molecular Infection Medicine Sweden (MIMS)
and
Department for Clinical Microbiology
90182 Umeå
Sweden
phone:+46 90 7851120
Email: This email address is being protected from spambots. You need JavaScript enabled to view it.

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