Francisella tularensis resistance to bactericidal action of normal human serum

Abstract Lipopolysaccharide and outer membranes from the three virulent encapsulated (Cap⁺) strains of three subspecies of Francisella tularensis and their isogenic avirulent capsule-deficient (Cap⁻) mutants were isolated. It was shown that the Cap⁻ cells and their outer membranes almost completely consumed the available complement of normal human serum whereas Cap⁻ LPS (R-LPS), Cap⁺ cells and their components activated the complement less effectively. Absorption of normal human serum with Cap⁻ strain dramatically reduced the complement consumption for homologous strain and its surface structures. This reduction reflected the loss of bactericidal antibodies. Addition of antibodies to whole cells of F. tularensis completely restored complement activity. The cross-absorbing experiments demonstrated that Cap⁻ cells more effectively deplete bactericidal antibodies than homologous virulent strain. From these results it can be concluded that normal human serum is bactericidal for serum-sensitive Cap⁻ F. tularensis strains through the action of complement initiated by the classical complement pathway and serum resistance of virulent strains is not due to absence of targets for bactericidal antibodies, but is due to their low accessibility because of O-side chains of lipopolysaccharide.     

Outer membranes of Francisella tularensis and their protein composition

Four strains of the species Francisella tularensis were used in the present work: a live vaccine strain 15/10 and three virulent strains (503, Schu, 543) from three different subspecies. The bacterial membranes were prepared by the 0.5% N-laurylsarcosinate (Sarcosyl) treatment. These membranes were identified as the outer membranes by morphological, immunological and biochemical analyses. The outer membrane proteins contained up to 30-35 polypeptides with three dominant fractions having the 63, 48 and 41-43 kD molecular masses. Despite the significant similarity between the membranes protein profiles there were some quantitative and qualitative differences between the three variants of Francisella tularensis in polypeptides compositions and patterns.     

Nucleolin, a shuttle protein promoting infection of human monocytes by Francisella tularensis

Background

Francisella tularensis is a highly virulent facultative intracellular bacterium, disseminating in vivo mainly within host mononuclear phagocytes. After entry into macrophages, F. tularensis initially resides in a phagosomal compartment, whose maturation is then arrested. Bacteria escape rapidly into the cytoplasm, where they replicate freely. We recently demonstrated that nucleolin, an eukaryotic protein able to traffic from the nucleus to the cell surface, acted as a surface receptor for F. tularensis LVS on human monocyte-like THP-1 cells.

Methodology/Principal Findings

Here, we followed the fate of nucleolin once F. tularensis has been endocytosed. We first confirmed by siRNA silencing experiments that expression of nucleolin protein was essential for binding of LVS on human macrophage-type THP-1 cells. We then showed that nucleolin co-localized with intracellular bacteria in the phagosomal compartment. Strikingly, in that compartment, nucleolin also co-localized with LAMP-1, a late endosomal marker. Co-immunoprecipation assays further demonstrated an interaction of nucleolin with LAMP-1. Co-localization of nucleolin with LVS was no longer detectable at 24 h when bacteria were multiplying in the cytoplasm. In contrast, with an iglC mutant of LVS, which remains trapped into the phagosomal compartment, or with inert particles, nucleolin/bacteria co-localization remained almost constant.

Conclusions/Significance

We herein confirm the importance of nucleolin expression for LVS binding and its specificity as nucleolin is not involved in binding of another intracellular pathogen as L. monocytogenes or an inert particle. Association of nucleolin with F. tularensis during infection continues intracellularly after endocytosis of the bacteria. The present work therefore unravels for the first time the presence of nucleolin in the phagosomal compartment of macrophages.     

Francisella tularensis RipA protein topology and identification of functional domains

Francisella tularensis is a Gram-negative coccobacillus and is the etiological agent of the disease tularemia. Expression of the cytoplasmic membrane protein RipA is required for Francisella replication within macrophages and other cell types; however, the function of this protein remains unknown. RipA is conserved among all sequenced Francisella species, and RipA-like proteins are present in a number of individual strains of a wide variety of species scattered throughout the prokaryotic kingdom. Cross-linking studies revealed that RipA forms homoligomers. Using a panel of RipA-green fluorescent protein and RipA-PhoA fusion constructs, we determined that RipA has a unique topology within the cytoplasmic membrane, with the N and C termini in the cytoplasm and periplasm, respectively. RipA has two significant cytoplasmic domains, one composed roughly of amino acids 1 to 50 and the second flanked by the second and third transmembrane domains and comprising amino acids 104 to 152. RipA functional domains were identified by measuring the effects of deletion mutations, amino acid substitution mutations, and spontaneously arising intragenic suppressor mutations on intracellular replication, induction of interleukin-1β (IL-1β) secretion by infected macrophages, and oligomer formation. Results from these experiments demonstrated that each of the cytoplasmic domains and specific amino acids within these domains are required for RipA function.     

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.     

Persistence factors of Francisella tularensis

The study of the persistence potential of 64 F. tularensis strains isolated from different sources was carried out. The wide spread of the antilysozyme, antilactoferrin and anticomplementory activities of F. tularensis were detected. F. tularensis, isolated from ticks and water, were characterized by the highest level of the expression of antilysozyme activity, while anticomplementory and antilactoferrin activities of the infective agents were characteristic of those microorganisms which were isolated from rodents and their excrements.     

Chlorine disinfection of Francisella tularensis

Aims: To determine the range of free available chlorine (FAC) required for disinfection of the live vaccine strain (LVS) and wild‐type strains of Francisella tularensis. Methods and Results: Seven strains of planktonic F. tularensis were exposed to 0·5 mg·l^?1 FAC for two pH values, 7 and 8, at 5 and 25°C. LVS was inactivated 2 to 4 times more quickly than any of the wild‐type F. tularensis strains at pH 8 and 5°C. Conclusions: Free available chlorine residual concentrations routinely maintained in drinking water distribution systems would require up to two hours to reduce all F. tularensis strains by 4 log₁₀. LVS was inactivated most quickly of the tested strains. Significance and Impact of the Study: This work provides contact time (CT) values that are useful for drinking water risk assessment and also suggests that LVS may not be a good surrogate in disinfection studies.     

Noncultivatable forms of Francisella tularensis

Conditions for the appearance of F. tularensis uncultivated forms and for their reversion into the initial state have been studied. As revealed in this study, the combined influence of stress factors (starvation and low temperature) may result in the transition of F. tularensis into the uncultivated state in which it persists in the environment during the period between epidemics. The reversion of F. tularensis uncultivated forms into the initial state has been carried out with the use of sensitive animals. The uncultivated state of F. tularensis should be regarded as the actual form of the existence of the causative agent of tularemia in soil and water ecosystems.     

Phase variations of Francisella tularensis lipopolysaccharide in human infection and immunization

The comparative study of the specificity of antibodies in human sera after tularemia infection and immunization with live tularemia infection was carried out with the use of passive hemagglutination and immunoblotting techniques. The sera of tularemia patients contained two different types of immunoglobulins: strictly specific to the antigenic epitopes of F. tularensis Iipopolysaccharide (LPS) and strictly specific to F. tularensis subsp. novicida LPS. Such phenomenon may be due to phase variations of the antigenic structure of F. tularensis LPS in the body of a slightly susceptible host. The immune sera of vaccinated were found to contain antibodies, strictly specific only to F. tularensis LPS. At the same time in one vaccinee by the presence of pronounced postvaccinal reactions was found sharply defined interaction between serum imunoglobulins and F. tularensis subsp. novicida LPS. As the result, the data on the possibility of the antigenic modification of F. tularensis in tularemia infection in humans were obtained. At the same time antigenic epitopes, characteristic of faintly pathogenic and closely related F. tularensis novicida LPS, appeared in the structure of F. tularensis LPS.     

Glycosylation of DsbA in Francisella tularensis subsp. tularensis

In Francisella tularensis subsp. tularensis, DsbA has been shown to be an essential virulence factor and has been observed to migrate to multiple protein spots on two-dimensional electrophoresis gels. In this work, we show that the protein is modified with a 1,156-Da glycan moiety in O-linkage. The results of mass spectrometry studies suggest that the glycan is a hexasaccharide, comprised of N-acetylhexosamines, hexoses, and an unknown monosaccharide. Disruption of two genes within the FTT0789-FTT0800 putative polysaccharide locus, including a galE homologue (FTT0791) and a putative glycosyltransferase (FTT0798), resulted in loss of glycan modification of DsbA. The F. tularensis subsp. tularensis ΔFTT0798 and ΔFTT0791::Cm mutants remained virulent in the murine model of subcutaneous tularemia. This indicates that glycosylation of DsbA does not play a major role in virulence under these conditions. This is the first report of the detailed characterization of the DsbA glycan and putative role of the FTT0789-FTT0800 gene cluster in glycan biosynthesis.     

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.     

Natural penicillin resistance of Francisella tularensis

Altered viable forms of F. tularensis with spheroplast specific damages of the surface structures were isolated after the culture exposure to lithium chloride (0.5 and 1%). Study of natural penicillin resistance in the spheroplasts and bacterial forms of F. tularensis revealed their difference: the spheroplasts of the strains tested had a lower resistance to beta-lactam antibiotics than the bacterial forms while the activity of spheroplast beta-lactamase did not differ from that of the enzyme of the bacterial form and equalled 224 to 252 U/ml of the cell suspension. Therefore, on the model of the lithium-induced spheroplasts it appeared possible to show that the damages of the surface structures of the cell walls of F. tularensis changed the penicillin resistance level which was indicative of involvement of the F. tularensis cell walls in the phenomenon of the natural resistance to beta-lactams.     

Multilocus VNTR-typing of Francisella tularensis strains

In the analysis of F. tularensis genome with the use of the specially developed program "DNA" a great number of loci containing tandem repeats were found. For analysis, 3 of them were selected and designated as FtA, FtB, FtC. The study of DNA of 40 F. tularensis strains in the polymerase chain reaction with specific primers to these loci a great variability in the number of repeats was established, the presence of 17 alleles being found in locus FtA, 5 alleles in locus FtB and 5 alleles in locus FtC. The strains under study formed 24 variants of genotypes, whose occurrence varied from 0.025 to 0.125. Taking into account the variability of the detected loci and a great number of potential loci VNTR in the genome, further development of this method will facilitate the creation of local and general data bases of the strains, thus ensuring more effective genetic typing of F. tularensis.     

Towards proteome database of Francisella tularensis

The accessibility of the partial genome sequence of Francisella tularensis strain Schu 4 was the starting point for a comprehensive proteome analysis of the intracellular pathogen F. tularensis. The main goal of this study is identification of protein candidates of value for the development of diagnostics, therapeutics and vaccines. In this review, the current status of 2-DE F. tularensis database building, approaches used for identification of biologically important subsets of F. tularensis proteins, and functional and topological assignments of identified proteins using various prediction programs and database homology searches are presented.     

Properties of Aldolase from Francisella tularensis

The aldolase of Francisella tularensis resembles Class II aldolases in its requirement for divalent ions and its inactivation by metal chelating agents. Cysteine and other reducing agents stimulated the activity of the enzyme.     

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.     

Purine and pyrimidine metabolism in Francisella tularensis

The assimilation and mutual transformation of exogenous purine and pyrimidine bases and their nucleosides in the known subspecies of F. tularensis have been studied by means of radio-labeled compounds. The possibility of using the specific features of the metabolism of these compounds in F. tularensis, established in this study, for taxonomy and differential diagnosis has been demonstrated.