Emelie Salomonsson

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.

Reintroduction of Two Deleted Virulence Loci Restores Full Virulence to the Live Vaccine Strain of Francisella tularensis

ABSTRACT A disadvantage of several old vaccines is that the genetic events resulting in the attenuation are often largely unknown and reversion to virulence cannot be excluded. In the 1950s, a live vaccine strain, LVS, was developed from a type B strain of Francisella tularensis, the causative agent of tularemia. LVS, which is highly attenuated for humans but still virulent for mice by some infection routes, has been extensively studied and found to protect staff from laboratory-acquired tularemia. The efforts to improve biopreparedness have identified a demand for a vaccine against tularemia. Recently the rapid progress in genomics of different Francisella strains has led to identification of several regions of differences (RDs). Two genes carried within RDs, pilA, encoding a putative type IV pilin, and FTT0918, encoding an outer membrane protein, have been linked to virulence. Interestingly, LVS has lost these two genes via direct repeat-mediated deletions. Here we show that reintroduction of the two deleted regions restores virulence of LVS in a mouse infection model to a level indistinguishable from that of virulent type B strains. The identification of the two attenuating deletion events could facilitate the licensing of LVS for use in humans.

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.

Characterization of protein glycosylation in Francisella tularensis subsp. holarctica; identification of a novel glycosylated lipoprotein required for virulence

FTH_0069 is a previously uncharacterized strongly immunoreactive protein that has been proposed to be a novel virulence factor in Francisella tularensis. Here, the glycan structure modifying two C-terminal peptides of FTH_0069 was identified utilizing high resolution, high mass accuracy mass spectrometry, combined with in-source CID tandem MS experiments. The glycan observed at m/z 1156 was determined to be a hexasaccharide, consisting of two hexoses, three N-acetylhexosamines, and an unknown monosaccharide containing a phosphate group. The monosaccharide sequence of the glycan is tentatively proposed as X-P-HexNAc-HexNAc-Hex-Hex-HexNAc, where X denotes the unknown monosaccharide. The glycan is identical to that of DsbA glycoprotein, as well as to one of the multiple glycan structures modifying the type IV pilin PilA, suggesting a common biosynthetic pathway for the protein modification. Here, we demonstrate that the glycosylation of FTH_0069, DsbA, and PilA was affected in an isogenic mutant with a disrupted wbtDEF gene cluster encoding O-antigen synthesis and in a mutant with a deleted pglA gene encoding pilin oligosaccharyltransferase PglA. Based on our findings, we propose that PglA is involved in both pilin and general F. tularensis protein glycosylation, and we further suggest an inter-relationship between the O-antigen and the glycan synthesis in the early steps in their biosynthetic pathways.

Iron Content Differs between Francisella tularensis Subspecies tularensis and Subspecies holarctica Strains and Correlates to Their Susceptibility to H 2 O 2 -Induced Killing †

ABSTRACT Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is classified as a category A select agent and a facultative intracellular bacterium. Why F. tularensis subsp. tularensis causes a more severe form of tularemia than F. tularensis subsp. holarctica does is not known. In this study, we have identified prominent phenotypic differences between the subspecies, since we found that F. tularensis subsp. tularensis strains contained less iron than F. tularensis subsp. holarctica strains. Moreover, strain SCHU S4 of F. tularensis subsp. tularensis was less susceptible than FSC200 and the live vaccine strain (LVS) of F. tularensis subsp. holarctica to H2O2-induced killing. The activity of the H2O2-degrading enzyme catalase was similar between the strains, whereas the iron content affected their susceptibility to H2O2, since iron starvation rendered F. tularensis subsp. holarctica strains more resistant to H2O2. Complementing LVS with fupA, which encodes an important virulence factor that regulates iron uptake, reduced its iron content and increased the resistance to H2O2-mediated killing. By real-time PCR, it was demonstrated that FSC200 and LVS expressed higher levels of gene transcripts related to iron uptake and storage than SCHU S4 did, and this likely explained their high iron content. Together, the results suggest that F. tularensis subsp. tularensis strains have restricted iron uptake and storage, which is beneficial for their resistance to H2O2-induced killing. This may be an important factor for the higher virulence of this subspecies of F. tularensis, as reactive oxygen species, such as H2O2, are important bactericidal components during tularemia.

Type IV Pili in Francisella – A Virulence Trait in an Intracellular Pathogen

Francisella tularensis is a highly virulent intracellular human pathogen that is capable of rapid proliferation in the infected host. Mutants affected in intracellular survival and growth are highly attenuated which highlights the importance of the intracellular phase of the infection. Genomic analysis has revealed that Francisella encodes all genes required for expression of functional type IV pili (Tfp), and in this focused review we summarize recent findings regarding this system in the pathogenesis of tularemia. Tfp are dynamic adhesive structures that have been identified as major virulence determinants in several human pathogens, but it is not obvious what role these structures could have in an intracellular pathogen like Francisella. In the human pathogenic strains, genes required for secretion and assembly of Tfp and one pilin, PilA, have shown to be required for full virulence. Importantly, specific genetic differences have been identified between the different Francisella subspecies where in the most pathogenic type A variants all genes are intact while several Tfp genes are pseudogenes in the less pathogenic type B strains. This suggests that there has been a selection for expression of Tfp with different properties in the different subspecies. There is also a possibility that the genetic differences reflect adaptation to different environmental niches of the subspecies and plays a role in transmission of tularemia. This is also in line with recent findings where Tfp pilins are found to be glycosylated which could reflect a role for Tfp in the environment to promote survival and transmission. We are still far from understanding the role of Tfp in virulence and transmission of tularemia, but with the genomic information and genetic tools available we are in a good position to address these issues in the future.

Functional analyses of pilin-like proteins from Francisella tularensis: complementation of type IV pilus phenotypes in Neisseria gonorrhoeae.

Accumulating evidence from a number of studies strongly suggests that proteins orthologous to those involved in type IV pili (Tfp) assembly and function are required for Francisella pathogenicity. However, the molecular mechanisms by which the components exert their influence on virulence remain poorly understood. Owing to the conservation and promiscuity of Tfp biogenesis machineries, expression of Tfp pilins in heterologous species has been used successfully to analyse organelle structure-function relationships. In this study we expressed a number of Francisella pilin genes in the Tfp-expressing pathogen Neisseria gonorrhoeae lacking its endogenous pilin subunit. Two gene products, the orthologous PilA proteins from Francisella tularensis subspecies tularensis and novicida, were capable of restoring the expression of Tfp-like appendages that were shown to be dependent upon the neisserial Tfp biogenesis machinery for surface localization. Expression of Francisella PilA pilins also partially restored competence for natural transformation in N. gonorrhoeae. This phenotype was not complemented by expression of the PulG and XcpT proteins, which are equivalent components of the related type II protein secretion system. Taken together, these findings provide compelling, although indirect, evidence of the potential for Francisella PilA proteins to express functional Tfp.

The type IV pilin, PilA, is required for full virulence of Francisella tularensis subspecies tularensis

BackgroundAll four Francisella tularensis subspecies possess gene clusters with potential to express type IV pili (Tfp). These clusters include putative pilin genes, as well as pilB, pilC and pilQ, required for secretion and assembly of Tfp. A hallmark of Tfp is the ability to retract the pilus upon surface contact, a property mediated by the ATPase PilT. Interestingly, out of the two major human pathogenic subspecies only the highly virulent type A strains have a functional pilT gene.ResultsIn a previous study, we were able to show that one pilin gene, pilA, was essential for virulence of a type B strain in a mouse infection model. In this work we have examined the role of several Tfp genes in the virulence of the pathogenic type A strain SCHU S4. pilA, pilC, pilQ, and pilT were mutated by in-frame deletion mutagenesis. Interestingly, when mice were infected with a mixture of each mutant strain and the wild-type strain, the pilA, pilC and pilQ mutants were out-competed, while the pilT mutant was equally competitive as the wild-type.ConclusionsThis suggests that expression and surface localisation of PilA contribute to virulence in the highly virulent type A strain, while PilT was dispensable for virulence in the mouse infection model.

Efficacy and safety profile of the novel antimicrobial peptide PXL150 in a mouse model of infected burn wounds

The urgent need to develop novel antimicrobial therapies has stimulated interest in antimicrobial peptides as therapeutic candidates for the treatment of infectious diseases. The aim of this study was to evaluate the anti-infectious effect of the synthetic antimicrobial peptide PXL150, formulated in hydroxypropyl cellulose (HPC) gel, on Pseudomonas aeruginosa in vitro and in an in vivo mouse model of infected burn wounds as well as to assess the in vivo safety profile of PXL150 in rats and rabbits. Minimal microbicidal concentration analysis showed prominent efficacy of PXL150 against P. aeruginosa in vitro, which was further enhanced in formulating the peptide in HPC gel. Application of 1.25, 2.5, 5, 10 and 20mg/g PXL150 in HPC gel twice daily for four consecutive days significantly reduced bacterial counts in the burn wounds compared with non-treated or placebo-treated controls. Continuous bioluminescence measurements of the bacteria revealed a pronounced anti-infective effect already at the first day post infection by PXL150 in concentrations of ≥2.5mg/g. In the non-clinical safety studies, PXL150 showed a favourable safety profile following repeated administration systemically and locally in rats and rabbits, respectively. In conclusion, these data support that PXL150 has the potential to be an effective and safe drug candidate for the treatment of infected burn wounds. The findings encourage the progression of PXL150 as a novel topical treatment of microbial infections.

Chapter 108 – Francisella

Francisella tularensis, the causative agent of tularaemia, is a zoonotic intracellular pathogen that can be found in a very large number of species ranging from large mammals and vertebrates to invertebrates, arthropods and amoebas. Disease in humans often occurs in parallel with tularaemia in wild animals. Human infection can occur through aerosolization, direct contact with infected animals via arthropod vectors like ticks, mosquitos and biting flies. F. tularensis subspecies tularensis (type A) is one of the most infectious bacteria known and inhalation of as few as ten organisms can be sufficient to establish an infection in humans that if left untreated could be fatal. The success of F. tularensis as a pathogen lies in its unique ability to adapt a lifestyle as an intracellular pathogen and to very timely subvert host defences both at extracellular and intracellular levels. Key events in the infection process are the ability to rapidly escape the phagosome after uptake by phagocytic cells and the ability to replicate to high levels inside the host cells without evoking a host inflammatory response.