PI: Anders Sjöstedt, professor
Department for Clinical Microbiology, Umeå University

Diagnosis of tularemia tradionally relies on cultivation, PCR and serology, but there is still a need to improve the diagnostic possibilities, e.g., with regard to speed and prognostic markers. Moreover, some patients present with uncharacteristic symptoms and are therefore difficult to diagnose. The relatively high number of samples handled by the laboratory means that the laboratory staff may be exposed to the bacterium during routine work and since it is highly contagious, being at risk to contract tularemia. Therefore, improved assays that allow very rapid diagnosis are of high priority. We will now develop methods based on the characterization of the host response or secreted bacterial factors as rapid diagnostic tools that also can have prognostic potential. Moreover, we will develop very rapid methods for identification and typing of the bacterium to enhance the laboratory safety.

Rapid development within the various areas designated “omics”; genomics, proteomics, and metabonomics, have led to an enormous expansion of the molecular knowledge regarding the human body. The techniques used allow a precise profiling of the host response for specific infection diagnosis and prognostic information. They can also demonstrate the degree of specific organ involvement and thereby give important information to assist in decisions regarding institution of early interventions and therapeutic regimens against infections. The project will implement several large-scale techniques to develop rapid diagnostic and prognostic assays.

We will collect samples from tularemia during a 2 week period of the infection. The sample types will be serum, saliva, and urine. We plan to first analyze samples from approximately a dozen patients to assess the usefulness of the analytical methods. The presence of soluble markers of inflammation and infection will be determined in serum collected at the indicated time points by a multiplex analysis using existing equipment based on the Luminex (xMAP) technology and also by use of metabonomic analyses. The latter technique will also be used to analyze urine and saliva samples.

The rationale for this work is that such analyses if useful will be a rapid and easy-to-use test for diagnosis of tularemia and will provide a much faster diagnosis that cultivation or serology. Although the PCR-based method gives a relatively rapid diagnosis of cases of ulceroglandular tularemia, the method cannot be used in a considerable number of all cases if there is an absence of a primary ulcer or for patients with other forms of tularemia. Often the latter cases are of the respiratory variant and these cases may have a more severe disease and therefore methods that provide prognostic information will be very valuable.
The project will also analyze the same type of samples from individuals with other actute infectious diseases to characterize the specificity of the assays and also their utility for diagnosis of several infectious diseases.

A type VI secretion system of Francisella tularensis - defining the functions of its components and their contribution to virulence

PI: Anders Sjöstedt, Department of Clinical Microbiology, Umeå University
There are at least 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. We are investigating the contribution of almost 20 proteins encoded by a pathogenicity island of Francisella tularensis that may contribute to a putative T6SS.

Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These genes include iglA and iglB encoding IglA and IglB, homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA necessary for efficient binding to IglB, as well as for IglAB protein stability and intramacrophage growth. In particular, a highly conserved -helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in the yeast Saccharomyces cerevisiae but markedly impaired intramacrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to type VI secretion, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica serovar Typhimurium, and Escherichia coli were also shown to interact in yeast, and the interaction was dependent on preservation of the same helix. Heterologous interactions between nonnative IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity, and the same interaction is conserved in other human pathogens.

The project is aimed to reveal the roles of the individual proteins of this complex. The work is performed by combing state-of-the-art techniques in microbiology, molecular biology, biochemistry and structural analysis. The wrok will be basedon in vitro, cell-infection, and in vivo assays. The relevance of homologous IglAB complexes in other T6SSs will be investigated by targeted mutagenesis. Screening will be performed to identify small molecular inhibitors of the interaction. Our work will help to identify the molecular mechanisms of a poorly understood secretion system essential for virulence of many human pathogens.

Contact:

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