The facultative intracellular bacterium Francisella tularensis is a highly virulent and contagious organism, and little is known about its intracellular survival mechanisms. We studied the intracellular localization of the attenuated human vaccine strain, F. tularensis LVS, in adherent mouse peritoneal cells, in mouse macrophagelike cell line J774A.1, and in human macrophage cell line THP-1. Confocal microscopy of infected J774A.1 cells indicated that during the first hour of infection the bacteria colocalized with the late endosomal-lysosomal glycoprotein LAMP-1, but within 3 h this colocalization decreased significantly from approximately 60% to 30%. Transmission electron microscopy revealed that >90% of bacteria were not enclosed by a phagosomal membrane after 2 h of infection, and some bacteria were in vacuoles that were only partially surrounded by a limiting membrane. Similar findings were obtained with all three host cell types. Immunoelectron microscopy performed with an F. tularensis LVS-specific polyclonal rabbit antiserum showed that the antiserum stained a thick, evenly distributed capsule-like material in bacteria grown in broth. In contrast, intracellular F. tularensis LVS cells were only marginally stained with this antiserum. Instead, most of the immunoreactive material was diffusely localized in the phagosomes or was associated with the phagosomal membrane. Our findings indicate that F. tularensis LVS is able to escape from the phagosomes of macrophages via a mechanism that may involve degradation of the phagosomal membrane.
Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.
Survival of microorganisms in aerobiological samples is often assessed by a survival ratio (SR), which is the ratio between the viable or metabolic active number (MA) of microorganisms to the total number (TOT) of microorganisms. A method to determine survival ratios with flow cytometry was developed for Francisella tularensis, the causative agent of tularemia. F. tularensis is a fastidious bacteria that can be transmitted by aerosol and constitute as such a valid model organism for aerobiological studies. The total number of F. tularensis cells was determined by specific targeting with monoclonal antibodies and detected by phycoerytrine (PE) conjugated secondary antibodies. The metabolic active part of the targeted F. tularensis cells was quantified by staining with rhodamine 123 (Rh123). Application of the presented method showed higher precision compared to an earlier developed method for survival ratios, achieved with plate count (VC) and Coulter Counter (CC) measurements. The coefficient of variation between samples for the new method was below 5% for the survival ratio. Comparison of VC yield with MA yield showed consistently higher values for MA. The survival ratios of F. tularensis in samples taken before and after aerosolisation were analysed. SR for F. tularensis determined with the new method decreased approximately 19% whereas SR determined with VC and CC decreased 62% after passage through aerosol state.
The host-pathogen interaction represents a complex and dynamic biological system. The outcome of this interaction is dependent on the microbial pathogen properties to establish infection and the ability of the host to control infection. Although bacterial pathogens have evolved a variety of strategies to subvert host defense functions, several general mechanisms have been shown to be shared among these pathogens. As a result, host effectors that are critical for pathogen entry, survival and replication inside the host cells have become a new paradigm for antimicrobial targeting. This review focuses on the potential utility of a proteomics approach in defining the host-pathogen interaction from the host's perspective.
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