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.

SummaryFrancisella 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 pilinnegative 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.

We have determined the sequence of the gene cluster encoding the O antigen in Francisella novicida and compared it to the previously reported O-antigen cluster in Francisella tularensis subsp. tularensis. Immunization with purified lipopolysaccharide (LPS) from F. tularensis subsp. tularensis or F. novicida protected against challenge with Francisella tularensis subsp. holarctica and F. novicida, respectively. The LPS from F. tularensis subsp. tularensis did not confer protection against challenge with F. novicida, and the LPS from F. novicida did not confer protection against challenge with F. tularensis subsp. holarctica. Allelic replacement mutants of F. tularensis subsp. tularensis or F. novicida which failed to produce O antigen were attenuated, but exposure to these mutants did not induce a protective immune response. The O antigen of F. tularensis subsp. tularensis appeared to be important for intracellular survival whereas the O antigen of F. novicida appeared to be critical for serum resistance and less important for intracellular survival.

The Mycobacterium bovis antigens MPB70 and MPB83 are homologous cross-reactive proteins. It has been reported previously that MPB83 is glycosylated and exists in two forms with apparent molecular masses of 23kDa and 25kDa, whereas the apparent molecular mass of MPB70 is 22kDa. Using a monoclonal antibody, SB10, which recognizes an epitope common to both MPB70 and MPB83, we compared the expression of these proteins in M. bovis BCG, virulent M. bovis and virulent Mycobacterium tuberculosis by Western blotting of bacterial lysates. The previously described pattern of high and low producing substrains of BCG for MPB70 was also applicable for MPB83. Virulent M. bovis was found to express high levels of MPB70 and MPB83. Immunoblotting experiments using sera from Balb/c mice infected with live M. tuberculosis H37Rv revealed that although the MPB83 homologue of M. tuberculosis, MPT83, is expressed at low levels in M. tuberculosis when grown in vitro, the protein is highly immunogenic during infection with live bacteria. A clone from a mycobacterial shuttle cosmid library of M. tuberculosis H37Rv was isolated which expressed both MPT70 and MPT83. Genetic analysis of this cosmid revealed that MPT70 and MPT83 were encoded by separate genes with the gene encoding MPT83 situated 2.4kb upstream of mpt70. Both genes are transcribed in the same direction. The gene encoding MPT83 was cloned and DNA sequencing revealed an open reading frame of 660bp encoding a protein with a predicted molecular mass of 22kDa. Recombinant MPT83 was expressed in Escherichia coli from the native AUG initiation codon by translational coupling. In E. coli MPT83 was expressed as a 23kDa antigen whereas in the rapid growing mycobacterium Mycobacterium smegmatis the protein was expressed as a 25kDa protein indicating post-translational modification of the protein by M. smegmatis. In recombinant M. smegmatis MPT83 was predominantly cell associated whereas MPT70 was secreted into the culture medium. Amino acid sequence comparison between MPT83 and MPT70 revealed a 61% identity between the proteins, although little homology was apparent at the amino terminus. In MPT83 this region contained a typical lipoprotein signal peptide cleavage motif and a putative signal motif for O glycosylation. Both these motifs were absent from the amino acid sequence of MPT70.

Background: The aim of this study was to characterize the elution of four antibiotics from pharmaceuticalgrade calcium sulfate beads and show that the eluted antibiotics retained efficacy. Methods: Calcium sulfate was combined with gentamicin, tobramycin, vancomycin, or rifampicin (ratio: 20 g of calcium sulfate, to 240 mg, 500 mg, 900 mg, and 600 mg of antibiotic, respectively). Three grams of beads were immersed in 4 mL of sterile phosphate-buffered saline (PBS) at 37°C. At each time point (4, 8, 24 h; 2, 7, 14, 28, 42 d), eluates were removed for analysis by liquid chromatography-mass spectrometry. The antimicrobial efficacy of antibiotics combined with calcium sulfate beads after 42 d was tested by a modified KirbyBauer disc diffusion assay. Results: All samples showed a generally exponential decay in the eluted antibiotic concentration. At the first time point, both gentamicin and tobramycin had eluted to a peak concentration of approximately 10,000 mcg/mL. For rifampicin, the peak concentration occurred at 24 h, whereas for vancomycin, it occurred at 48 h. The eluted concentrations exceeded the minimum inhibitory concentration for common periprosthetic joint infection pathogens for the entire span of the 42 study days. Mass spectrometry confirmed all antibiotics were unchanged when eluted from the calcium sulfate carrier. Antimicrobial efficacy was unaltered after 42 d in combination with calcium sulfate at 37°C. Conclusions: Pharmaceutical-grade calcium sulfate has the potential for targeted local release of tobramycin, gentamicin, vancomycin, and rifampicin over a clinically meaningful time period.