Petra C. F. Oyston

The complete genome sequence of Francisella tularensis, the causative agent of tularemia.

Francisella tularensis is one of the most infectious human pathogens known. In the past, both the former Soviet Union and the US had programs to develop weapons containing the bacterium. We report the complete genome sequence of a highly virulent isolate of F. tularensis (1,892,819 bp). The sequence uncovers previously uncharacterized genes encoding type IV pili, a surface polysaccharide and iron-acquisition systems. Several virulence-associated genes were located in a putative pathogenicity island, which was duplicated in the genome. More than 10% of the putative coding sequences contained insertion-deletion or substitution mutations and seemed to be deteriorating. The genome is rich in IS elements, including IS630 Tc-1 mariner family transposons, which are not expected in a prokaryote. We used a computational method for predicting metabolic pathways and found an unexpectedly high proportion of disrupted pathways, explaining the fastidious nutritional requirements of the bacterium. The loss of biosynthetic pathways indicates that F. tularensis is an obligate host-dependent bacterium in its natural life cycle. Our results have implications for our understanding of how highly virulent human pathogens evolve and will expedite strategies to combat them.

Vaccines Against Francisella Tularensis

Abstract:  Francisella tularensis is one of the most pathogenic pathogens known, especially when disseminated as a small particle aerosol. Because of this, it was developed into a biological warfare agent by several states during the 20th century. Nowadays, concerns remain about the potential of this pathogen to cause widespread disease, tularemia, in the hands of terrorists. This has resurrected interest in methods to combat it. This article reviews the current status of vaccine development efforts against tularemia. To date most of our understanding of tularemia vaccine efficacy has been derived from the clinical and experimental use of a pragmatically attenuated live vaccine strain of F. tularensis subspecies holarctica. However, this vaccine which has been in existence for more than 50 years is still beset by regulatory issues that continue to hamper its licensure. These issues and possible solutions are highlighted, along with more modern molecular approaches to vaccine development against this highly virulent pathogen.

Direct repeat-mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis.

Francisella tularensis, the causative agent of tularaemia, is a highly infectious and virulent intracellular pathogen. There are two main human pathogenic subspecies, Francisella tularensis ssp. tularensis (type A), and Francisella tularensis ssp. holarctica (type B). So far, knowledge regarding key virulence determinants is limited but it is clear that intracellular survival and multiplication is one major virulence strategy of Francisella. In addition, genome sequencing has revealed the presence of genes encoding type IV pili (Tfp). One genomic region encoding three proteins with signatures typical for type IV pilins contained two 120 bp direct repeats. Here we establish that repeat‐mediated loss of one of the putative pilin genes in a type B strain results in severe virulence attenuation in mice infected by subcutaneous route. Complementation of the mutant by introduction of the pilin gene in cis resulted in complete restoration of virulence. The level of attenuation was similar to that of the live vaccine strain and this strain was also found to lack the pilin gene as result of a similar deletion event mediated by the direct repeats. Presence of the pilin had no major effect on the ability to interact, survive and multiply inside macrophage‐like cell lines. Importantly, the pilin‐negative strain was impaired in its ability to spread from the initial site of infection to the spleen. Our findings indicate that this putative pilin is critical for Francisella infections that occur via peripheral routes.

Galleria mellonella as an alternative infection model for Yersinia pseudotuberculosis

We report that larvae of the wax moth (Galleria mellonella) are susceptible to infection with the human enteropathogen Yersinia pseudotuberculosis at 37 degrees C. Confocal microscopy demonstrated that in the initial stages of infection the bacteria were taken up into haemocytes. To evaluate the utility of this model for screening Y. pseudotuberculosis mutants we constructed and tested a superoxide dismutase C (sodC) mutant. This mutant showed increased susceptibility to superoxide, a key mechanism of killing in insect haemocytes and mammalian phagocytes. It showed reduced virulence in the murine yersiniosis infection model and in contrast to the wild-type strain IP32953 was unable to kill G. mellonella. The complemented mutant regained all phenotypic properties associated with SodC, confirming the important role of this metalloenzyme in two Y. pseudotuberculosis infection models.

Production of a non-toxic site-directed mutant of Clostridium perfringens e-toxin which induces protective immunity in mice

A panel of ten site-directed mutants of Clostridium perfringens e-toxin was generated. All of the mutated proteins expressed in Escherichia coli were recognized in immunoblots by a neutralizing mAb raised against wild-type native e-toxin. The cytotoxicity of the site-directed mutated toxins was assayed in vitro against MDCK cells. One mutation resulting in loss of activity in the assay was identified. This non-toxic protein was derived by substituting a proline for the histidine at residue 106 of the toxin. Immunization of mice with the non-toxic mutated e-toxin resulted in the induction of a specific antibody response and immunized mice were protected against 1000 LD50 doses of wild-type recombinant e-toxin.

Differential ability of novel attenuated targeted deletion mutants of Francisella tularensis subspecies tularensis strain SCHU S4 to protect mice against aerosol challenge with virulent bacteria: effects of host background and route of immunization

Francisella tularensis subspecies tularensis is a highly virulent facultative intracellular pathogen of humans and a potential biological weapon. A live vaccine strain, F. tularensis LVS, was developed more than 50 years ago by pragmatic attenuation of a strain of the less virulent holarctica subspecies. LVS was demonstrated to be highly effective in human volunteers who were exposed to intradermal challenge with fully virulent subsp. tularensis, but was less effective against aerosol exposure. LVS faces regulatory hurdles that to date have prevented its licensure for general use. Therefore, a better defined and more effective vaccine is being sought. To this end we have created gene deletion mutants in the virulent subsp. tularensis strain and tested them for their ability to elicit a protective immune response against systemic or aerosol challenge with the highly virulent wild-type subsp. tularensis strain, SCHU S4. Both oral and intradermal (ID) primary vaccination routes were assessed in BALB/c and C3H/HeN mice as was oral boosting. One SCHU S4 mutant missing the heat shock gene, clpB, was significantly more attenuated than LVS whereas a double deletion mutant missing genes FTT0918 and capB was as attenuated as LVS. In general mice immunized with SCHU S4DeltaclpB were significantly better protected against aerosol challenge than mice immunized with LVS. A single ID immunization of BALB/c mice with SCHU S4DeltaclpB was at least as effective as any other regimen examined. Mice immunized with SCHU S4Delta0918DeltacapB were generally protected to a similar degree as mice immunized with LVS. A preliminary examination of immune responses to vaccination with LVS, SCHU S4DeltaclpB, or SCHU S4Delta0918DeltacapB provided no obvious correlate to their relative efficacies.

Q fever: the neglected biothreat agent.

Coxiella burnetii is the causative agent of Q fever, a disease with a spectrum of presentations from the mild to fatal, including chronic sequelae. Since its discovery in 1935, it has been shown to infect a wide range of hosts, including humans. A recent outbreak in Europe reminds us that this is still a significant pathogen of concern, very transmissible and with a very low infectious dose. For these reasons it has also featured regularly on various threat lists, as it may be considered by the unscrupulous for use as a bioweapon. As an intracellular pathogen, it has remained an enigmatic organism due to the inability to culture it on laboratory media. As a result, interactions with the host have been difficult to elucidate and we still have a very limited understanding of the molecular mechanisms of virulence. However, two recent developments will open up our understanding of C. burnetii: the first axenic growth medium capable of supporting cell-free growth, and the production of the first isogenic mutant. We are approaching an exciting time for expanding our knowledge of this organism in the next few years.

Potential of the β-glucans to enhance innate resistance to biological agents

The use of numerous mushroom species in traditional medicine has been widely documented, with their observed immunomodulatory effects now attributed, in part, to bioactive components called β-glucans. The β-glucans are of particular interest since they are naturally occurring polymers of glucose, are orally active when taken as food supplements and have a long track record of safe use. Due to their immunomodulatory properties, purified β-glucans have been used clinically as part of a combination therapy for a variety of cancers and their potential anti-infective properties have received attention. This review relates the structure of β-glucans to their function, with a particular focus on their documented immunomodulatory effects and the mechanisms by which they affect inter- and intracellular function, resulting in potential antimicrobial benefits. Overall, the benefits of dietary supplementation with β-glucans in order to enhance innate resistance to biological agents are evaluated.

Preliminary analysis and annotation of the partial genome sequence of Francisella tularensis strain Schu 4.

Preliminary analysis and annotation of the partial genome sequence of Francisella tularensis strain Schu 4.

O-Linked Glycosylation of the PilA Pilin Protein of Francisella tularensis: Identification of the Endogenous Protein-Targeting Oligosaccharyltransferase and Characterization of the Native Oligosaccharide

Findings from a number of studies suggest that the PilA pilin proteins may play an important role in the pathogenesis of disease caused by species within the genus Francisella. As such, a thorough understanding of PilA structure and chemistry is warranted. Here, we definitively identified the PglA protein-targeting oligosaccharyltransferase by virtue of its necessity for PilA glycosylation in Francisella tularensis and its sufficiency for PilA glycosylation in Escherichia coli. In addition, we used mass spectrometry to examine PilA affinity purified from Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica and demonstrated that the protein undergoes multisite, O-linked glycosylation with a pentasaccharide of the structure HexNac-Hex-Hex-HexNac-HexNac. Further analyses revealed microheterogeneity related to forms of the pentasaccharide carrying unusual moieties linked to the distal sugar via a phosphate bridge. Type A and type B strains of Francisella subspecies thus express an O-linked protein glycosylation system utilizing core biosynthetic and assembly pathways conserved in other members of the proteobacteria. As PglA appears to be highly conserved in Francisella species, O-linked protein glycosylation may be a feature common to members of this genus.