Neuraminidase activity in representatives of the genus Francisella]

In two Francisella species (F. tularensis and F. novicida) neuraminidase activity, heretofore unknown, was detected. The enzyme exhibited specificity with respect to the substrates used in the investigation, neutralizing natural mucins, but not other compounds (glycoproteins and glycoproteins). All F. tularensis strains were found to have enzymatic activity irrespective of their subspecies, but neuraminidase activity was higher in the strains belonging to the American subspecies. Experimentally obtained F. tularensis noncapsular variants possessed higher neuraminidase activity than the capsular parent strains. The conclusion on the possible role of this enzyme in F. tularensis colonization of the host body was made.     

Optimized application of surface-enhanced laser desorption/ionization time-of-flight MS to differentiate Francisella tularensis at the level of subspecies and individual strains

Francisella tularensis, the causative agent of tularaemia, is a potential agent of bioterrorism. The phenotypic discrimination of the closely related F. tularensis subspecies and individual strains with traditional methods is difficult and time consuming, often producing ambiguous results. Surface-enhanced laser desorption/ionization time-of-flight MS (SELDI-TOF MS) was used in this study to discriminate the different species and subspecies of the genus Francisella. We tested 18 Francisella strains including at least one representative of each species/subspecies on four different types of chromatographic chip surfaces. Multivariate analysis (hierarchical clustering and principal component analysis) allowed grouping of the strains according to their designated subspecies. Furthermore, single strains within F. tularensis subspecies could be discriminated.     

Superoxide dismutase activity in representatives of the genus Francisella]

Superoxide dismutase (SOD) activity has been registered in representatives of the genus Francisella. The electrophoretic mobility of the enzyme and a number of its isoforms in F. tularensis are linked with the subdivision of this species into several subspecies. Avirulent and noncapsular variants of F. tularensis are characterized by a higher SOD activity than the initial virulent strains. Attempts to detect catalase activity in F. tularensis have failed.     

Evolution of subspecies of Francisella tularensis

Analysis of unidirectional genomic deletion events and single nucleotide variations suggested that the four subspecies of Francisella tularensis have evolved by vertical descent. The analysis indicated an evolutionary scenario where the highly virulent F. tularensis subsp. tularensis (type A) appeared before the less virulent F. tularensis subsp. holarctica (type B). Compared to their virulent progenitors, attenuated strains of F. tularensis exhibited specific unidirectional gene losses.     

Genetic diversity of Francisella tularensis subspecies holarctica strains isolated in Japan

The recently developed MLVA has high discriminatory power for the typing of individual strains or isolates of Francisella tularensis. In the present study, MLVA was applied to 33 Japanese F. tularensis subspecies holarctica strains to examine the genetic diversity of F. tularensis isolated. Among the seven VNTR loci analyzed, Ft-M2, Ft-M10, and Ft-M20 loci showed high genetic polymorphism in Japanese strains, whereas Ft-M3 was most variable in non-Japanese strains. These results provide novel extended information about the genomic diversity among the strains of F. tularensis ssp. holarctica distributed in Japan and enable determination of whether a given isolate is indigenous to Japan by examining these loci using MLVA.     

Paired-end sequence mapping detects extensive genomic rearrangement and translocation during divergence of Francisella tularensis subsp. tularensis and Francisella tularensis subsp. holarctica populations

Comparative genome hybridization of the Francisella tularensis subsp. tularensis and F. tularensis subsp. holarctica populations have shown that genome content is highly conserved, with relatively few genes in the F. tularensis subsp. tularensis genome being absent in other F. tularensis subspecies. To determine if organization of the genome differs between global populations of F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, we have used paired-end sequence mapping (PESM) to identify regions of the genome where synteny is broken. The PESM approach compares the physical distances between paired-end sequencing reads of a library of a wild-type reference F. tularensis subsp. holarctica strain to the predicted lengths between the reads based on map coordinates of two different F. tularensis genome sequences. A total of 17 different continuous regions were identified in the F. tularensis subsp. holarctica genome (CR(holar)(c)(tica)) which are noncontiguous in the F. tularensis subsp. tularensis genome. Six of the 17 different CR(holarctica) are positioned as adjacent pairs in the F. tularensis subsp. tularensis genome sequence but are translocated in F. tularensis subsp. holarctica, implying that their arrangements are ancestral in F. tularensis subsp. tularensis and derived in F. tularensis subsp. holarctica. PCR analysis of the CR(holarctica) in 88 additional F. tularensis subsp. tularensis and F. tularensis subsp. holarctica isolates showed that the arrangements of the CR(holarctica) are highly conserved, particularly in F. tularensis subsp. holarctica, consistent with the hypothesis that global populations of F. tularensis subsp. holarctica have recently experienced a periodic selection event or they have emerged from a recent clonal expansion. Two unique F. tularensis subsp. tularensis-like strains were also observed which likely are derived from evolutionary intermediates and may represent a new taxonomic unit.     

Population structure of Francisella tularensis

We have sequenced fragments of five metabolic housekeeping genes and two genes encoding outer membrane proteins from 81 isolates of Francisella tularensis, representing all four subspecies. Phylogenetic clustering of gene sequences from F. tularensis subsp. tularensis and F. tularensis subsp. holarctica aligned well with subspecies affiliations. In contrast, F. tularensis subsp. novicida and F. tularensis subsp. mediasiatica were indicated to be phylogenetically incoherent taxa. Incongruent gene trees and mosaic structures of housekeeping genes provided evidence for genetic recombination in F. tularensis.     

Influence of nutrient status and grazing pressure on the fate of Francisella tularensis in lake water

The natural reservoir of Francisella tularensis , the causative agent of tularaemia, is yet to be identified. We investigated the possibility that Francisella persists in natural aquatic ecosystems between outbreaks. It was hypothesized that nutrient-rich environments, with strong protozoan predation, favour the occurrence of the tularaemia bacterium. To investigate the differences in adaptation to aquatic environments of the species and subspecies of Francisella , we screened 23 strains for their ability to survive grazing by the ciliate Tetrahymena pyriformis . All the Francisella strains tested were consumed at a low rate, although significant differences between subspecies were found. The survival and virulence of gfp -labelled F. tularensis ssp. holarctica were then studied in a microcosm experiment using natural lake water, with varying food web complexities and nutrient availabilities. High nutrient conditions in combination with high abundances of nanoflagellates were found to favour F. tularensis ssp. holarctica . The bacterium was observed both free-living and within the cells of a nanoflagellate. Francisella tularensis entered a viable but nonculturable state during the microcosm experiment. When studied over a longer period of time, F. tularensis ssp. holarctica survived in the lake water, but loss of virulence was not prevented by either high nutrient availability or the presence of predators.     

Discrimination of Francisella tularensis subspecies using surface enhanced laser desorption ionization mass spectrometry and multivariate data analysis

Francisella tularensis causes the zoonotic disease tularemia, and is considered a potential bioterrorist agent due to its extremely low infection dose and potential for airborne transmission. Presently, F. tularensis is divided into four subspecies; tularensis, holarctica, mediasiatica and novicida. Phenotypic discrimination of the closely related subspecies with traditional methods is difficult and tedious. Furthermore, the results may be vague and they often need to be complemented with virulence tests in animals. Here, we have used surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to discriminate between the four subspecies of F. tularensis. The method is based on the differential binding of protein subsets to chemically modified surfaces. Bacterial thermolysates were added to anionic, cationic, and copper ion-loaded immobilized metal affinity SELDI chip surfaces. After binding, washing, and SELDI-TOF-MS different protein profiles were obtained. The spectra generated from the different surfaces were then used to characterize each bacterial strain. The results showed that the method was reproducible, with an average intensity variation of 21%, and that the mass precision was good (300-450 ppm). Moreover, in subsequent cluster analysis and principal component analysis (PCA) data for the analyzed Francisella strains grouped according to the recognized subspecies. Partial least squares-discriminant analysis (PLS-DA) of the protein profiles also identified proteins that differed between the strains. Thus, the protein profiling approach based on SELDI-TOF-MS holds great promise for rapid high-resolution phenotypic identification of bacteria.     

Targeted inactivation of francisella tularensis genes by group II introns

Studies of the molecular mechanisms of pathogenesis of Francisella tularensis, the causative agent of tularemia, have been hampered by a lack of genetic techniques for rapid targeted gene disruption in the most virulent subspecies. Here we describe efficient targeted gene disruption in F. tularensis utilizing mobile group II introns (targetrons) specifically optimized for F. tularensis. Utilizing a targetron targeted to blaB, which encodes ampicillin resistance, we showed that the system works at high efficiency in three different subspecies: F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, and "F. tularensis subsp. novicida." A targetron was also utilized to inactivate F. tularensis subsp. holarctica iglC, a gene required for virulence. The iglC gene is located within the Francisella pathogenicity island (FPI), which has been duplicated in the most virulent subspecies. Importantly, the iglC targetron targeted both copies simultaneously, resulting in a strain mutated in both iglC genes in a single step. This system will help illuminate the contributions of specific genes, and especially those within the FPI, to the pathogenesis of this poorly studied organism.     

Comparative proteome analysis of cellular proteins extracted from highly virulent Francisella tularensis ssp. tularensis and less virulent F. tularensis ssp. holarctica and F. tularensis ssp. mediaasiatica

Francisella tularensis is the causative agent of the zoonotic disease tularemia. Four subspecies of this pathogen, namely ssp. tularensis, mediaasiatica, holarctica, and novicida are spread throughout the northern hemisphere. Although there are marked variations in their virulence to mammals, the subspecies are difficult to identify as they are closely genetically related. We carried out the comparative proteome analysis of cellular extracts from isolates representing the highly virulent subspecies tularensis, and the less virulent subspecies mediaasiatica and holarctica in order to identify new diagnostic markers and putative factors of virulence. We identified 27 protein spots that were either specifically present or at significantly higher abundance in ssp. tularensis strains, 22 proteins in ssp. mediaasiatica strains, and 26 proteins in ssp. holarctica strains. Subspecies tularensis-specific proteins might represent putative virulence factors. Of 27 identified tularensis-specific spots 17 represented charge and mass variants of proteins occurring in other subspecies, 7 spots were found to be present at higher abundance, and 3 spots were specifically present in tularensis strains. Amongst them, PilP protein, as a component necessary for the biogenesis of the type IV pilus, virulence and adhesion factor for many human pathogen, was identified. Furthermore, the identification of additional 27 proteins common for ssp. tularensis and mediaasiatica, and 19 proteins shared by ssp. mediaasiatica and holarctica documented apparent closer genetic similarity between ssp. tularensis and mediaasiatica.     

Immunoproteomic analysis of the murine antibody response to successful and failed immunization with live anti-Francisella vaccines

Francisella tularensis subspecies tularensis is one of the most virulent of bacterial pathogens for humans. Protective immunity against the pathogen can be induced in humans and some, but not all, mouse strains by vaccination with live, but not killed, vaccines. In mice, this protection is mediated predominantly by CD4+ and CD8+ T cells. This is thought to be the case too for humans. Nevertheless, it is possible that successful vaccination elicits antigen-specific antibodies that can serve as correlates of protection. To test this hypothesis we examined the repertoire of antibodies induced following successful immunization of BALB/c and CH3/HeN mice versus unsuccessful vaccination of C57BL/6 and DBA\2 mice with F. tularensis Live Vaccine Strain or following unsuccessful vaccination of BALB/c mice with highly related subspecies, F. novicida. The results showed that successful vaccination elicited antibodies to at least six proteins that were not recognized by antisera from vaccinated but unprotected mice.     

Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis

The intracellular bacterium Francisella tularensis is the causative agent of tularemia and poses a serious threat as an agent of bioterrorism. We have developed a highly effective molecular subtyping system from 25 variable-number tandem repeat (VNTR) loci. In our study, multiple-locus VNTR analysis (MLVA) was used to analyze genetic relationships and potential population structure within a global collection of 192 F. tularensis isolates, including representatives from each of the four subspecies. The VNTR loci displayed between 2 and 31 alleles with Nei's diversity values between 0.05 and 0.95. Neighbor-joining cluster analysis of VNTR data revealed 120 genotypes among the 192 F. tularensis isolates, including accurate subspecies identification. F. tularensis subsp. tularensis (type A) isolates showed great diversity at VNTR loci, while F. tularensis subsp. holarctica (type B) isolates showed much lower levels despite a much broader geographical prevalence. The resolution of two distinct clades within F. tularensis subsp. tularensis (designated A.I and A.II) revealed a previously unrecognized genetic division within this highly virulent subspecies. F. tularensis subsp. holarctica appears to have recently spread globally across continents from a single origin, while F. tularensis subsp. tularensis has a long and complex evolutionary history almost exclusively in North America. The sole non-North American type A isolates (Slovakian) were closely related to the SCHU S4 strain. Significant linkage disequilibrium was detected among VNTR loci of F. tularensis consistent with a clonal population structure. Overall, this work greatly augments the study of tularemia ecology and epidemiology, while providing a framework for future forensic analysis of F. tularensis isolates.     

Detection of diverse new Francisella-like bacteria in environmental samples

Following detection of putative Francisella species in aerosol samples from Houston, Texas, we surveyed soil and water samples from the area for the agent of tularemia, Francisella tularensis, and related species. The initial survey used 16S rRNA gene primers to detect Francisella species and related organisms by PCR amplification of DNA extracts from environmental samples. This analysis indicated that sequences related to Francisella were present in one water and seven soil samples. This is the first report of the detection of Francisella-related species in soil samples by DNA-based methods. Cloning and sequencing of PCR products indicated the presence of a wide variety of Francisella-related species. Sequences from two soil samples were 99.9% similar to previously reported sequences from F. tularensis isolates and may represent new subspecies. Additional analyses with primer sets developed for detection and differentiation of F. tularensis subspecies support the finding of very close relatives to known F. tularensis strains in some samples. While the pathogenicity of these organisms is unknown, they have the potential to be detected in F. tularensis-specific assays. Similarly, a potential new subspecies of Francisella philomiragia was identified. The majority of sequences obtained, while more similar to those of Francisella than to any other genus, were phylogenetically distinct from known species and formed several new clades potentially representing new species or genera. The results of this study revise our understanding of the diversity and distribution of Francisella and have implications for tularemia epidemiology and our ability to detect bioterrorist activities.     

Genetic dissection of the Francisella novicida restriction barrier

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10(6)-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.     

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.     

Host-pathogen o-methyltransferase similarity and its specific presence in highly virulent strains of Francisella tularensis suggests molecular mimicry

Whole genome comparative studies of many bacterial pathogens have shown an overall high similarity of gene content (>95%) between phylogenetically distinct subspecies. In highly clonal species that share the bulk of their genomes subtle changes in gene content and small-scale polymorphisms, especially those that may alter gene expression and protein-protein interactions, are more likely to have a significant effect on the pathogen's biology. In order to better understand molecular attributes that may mediate the adaptation of virulence in infectious bacteria, a comparative study was done to further analyze the evolution of a gene encoding an o-methyltransferase that was previously identified as a candidate virulence factor due to its conservation specifically in highly pathogenic Francisella tularensis subsp. tularensis strains. The o-methyltransferase gene is located in the genomic neighborhood of a known pathogenicity island and predicted site of rearrangement. Distinct o-methyltransferase subtypes are present in different Francisella tularensis subspecies. Related protein families were identified in several host species as well as species of pathogenic bacteria that are otherwise very distant phylogenetically from Francisella, including species of Mycobacterium. A conserved sequence motif profile is present in the mammalian host and pathogen protein sequences, and sites of non-synonymous variation conserved in Francisella subspecies specific o-methyltransferases map proximally to the predicted active site of the orthologous human protein structure. Altogether, evidence suggests a role of the F. t. subsp. tularensis protein in a mechanism of molecular mimicry, similar perhaps to Legionella and Coxiella. These findings therefore provide insights into the evolution of niche-restriction and virulence in Francisella, and have broader implications regarding the molecular mechanisms that mediate host-pathogen relationships.     

Species- and genus-specific antigenic epitopes of Francisella tularensis lipopolysaccharides

Lipopolysaccharide (LPS) antigenic epitopes of natural virulent and isogenic avirulent Francisella tularensis strains and other species of the Francisella genus (F. novicida, F. novicida-like, and F. philomiragia) were studied by dot and immunoblotting. Polyclonal rabbit and human sera to virulent F. tularensis strains and monoclonal antibodies to F. tularensis LPS O-side chain were used for detecting species- and genus-specific LPS epitopes. Typical virulent F. tularensis strains produce two types of S-LPS with different antigenic specificity simultaneously. Antigenic determinants of two LPS types were located in LPS O-polysaccharide but not in the core oligosaccharide. The epitopes of the first LPS type were characterized by species specificity for F. tularensis in contrast to determinants of the second LPS type, which had epitopes common with F. novicida. Cross exhaustion of human and rabbit antitularemic sera by F. tularensis and F. novicida LPS showed that F. novicida LPS molecules contained at least two epitopes--highly specific for F. novicida and common with the second type of F. tularensis LPS. The immune response of rabbits and humans to F. tularensis LPS epitopes was different in principle. Sera from rabbits immunized with vaccine and virulent F. tularensis strains contained antibodies "recognizing" antigenic epitopes of two S-LPS forms of the bacterium: type 1 species-specific (in high titers) and type 2 epitopes common with F. novicida LPS (in low titers). In addition to these, sera from patients with tularemia contain immunoglobulins to species-specific epitopes of F. novicida LPS in high titers. Experiments on avirulent mutants showed that in some cases attenuation of F. tularensis can involve loss of species-specific LPS form, while S-LPS with epitopes common with F. novicida LPS will be retained. The difference in specificity of human and rabbit antitularemic antibodies is due to individual features in the host immune system.     

The preventive activity of monoclonal antibodies specific to the lipopolysaccharide of Francisella tularensis]

The preventive activity of five monoclonal antibodies (McAb) in experimental tularemia was evaluated. McAb produced by hybridoma FB11-k (IgG2a), specific to F. tularensis lipopolysaccharide, prevented the death of mice and guinea pigs infected with F. tularensis virulent strain 503 of the holarctic subspecies.