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

Reference standards for follicular density in ovarian cortex from birth to sexual maturity

Research question: Are age-normalized reference values for human ovarian cortical follicular density adequate for tissue quality control in fertility preservation?Design: Published quantitative data on the number of follicles in samples without known ovarian pathology were converted into cortical densities to create reference values. Next, a sample cohort of 126 girls (age 1-24 years, mean +/- SD 11 +/- 6) with cancer, severe haematological disease or Turner syndrome were used to calculate Z-scores for cortical follicular density based on the reference values.Results: No difference was observed between Z-scores in samples from untreated patients (0.3 +/- 3.5, n = 30) and patients treated with (0.5 +/- 2.9, n = 48) and without (0.1 +/- 1.3, n = 6) alkylating chemotherapy. Z-scores were not correlated with increasing cumulative exposure to cytostatics. Nevertheless, Z-scores in young treated patients (0-2 years -2.1 +/- 3.1, n = 10, P = 0.04) were significantly lower than Z-scores in older treated patients (11-19 years, 2 +/- 1.9, n = 15). Samples from patients with Turner syndrome differed significantly from samples from untreated patients (-5.2 +/- 5.1, n = 24, P = 0.003), and a Z-score of -1.7 was identified as a cut-off showing good diagnostic value for identification of patients with Turner syndrome with reduced ovarian reserve. When this cut-off was applied to other patients, analysis showed that those with indications for reduced ovarian reserve (n = 15) were significantly younger (5.9 +/- 4.2 versus 10.7 +/- 5.9 years, P = 0.004) and, when untreated, more often had non-malignant haematologic diseases compared with those with normal ovarian reserve (n = 24, 100% versus 19%, P = 0.009).Conclusion: Z-scores allow the estimation of genetic- and treatment-related effects on follicular density in cortical tissue from young patients stored for fertility preservation. Understanding the quality of cryopreserved tissue facilitates its use during patient counselling. More research is needed regarding the cytostatic effects found in this study.Peer reviewe