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

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

Published onlineJournal ArticleThis is the final version of the article. Available from BioMed Central via the DOI in this record.BACKGROUND: All 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. RESULTS: In 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. CONCLUSIONS: This 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

Validation guidelines for PCR workflows in bioterrorism preparedness, food safety and forensics

The polymerase chain reaction (PCR) is the backbone of contemporary DNA/RNA analysis, ideally enabling detection of one or just a few target molecules. However, when analysing food or forensic samples the analytical procedure is often challenged by low amounts of poor quality template molecules and complex matrices. Applying optimised and validated methods in all steps of the analysis workflow, i.e. sampling, sample treatment, DNA/RNA extraction and PCR (including reverse transcription for RNA analysis), is thus necessary to ensure the reliability of analysis. In this paper, we describe how in-house validation can be performed for the different modules of the diagnostic PCR process, providing practical examples as tools for laboratories in their planning of validation studies. The focus is analysis of heterogeneous samples with interfering matrices, with relevance in food testing, forensic DNA analysis, bioterrorism preparedness and veterinary medicine. Our objective is to enable rational in-house validation for reliable and swift quality assurance when results are urgent, for example in the event of a crisis such as a foodborne outbreak or a crime requiring the analysis of a large number of diverse samples. To that end, we explain the performance characteristics associated with method validation from a PCR and biological sample matrix perspective and suggest which characteristics to investigate depending on the type of method to be validated. Also, we include a modular approach to validation within the PCR workflow, aiming at efficient validation and a flexible use of methods

Long-Term Survival of Virulent Tularemia Pathogens outside a Host in Conditions That Mimic Natural Aquatic Environments

Francisella tularensis, the causative agent of the zoonotic disease tularemia, can cause seasonal outbreaks of acute febrile illness in humans with disease peaks in late summer to autumn. Interestingly, its mechanisms for environmental persistence between outbreaks are poorly understood. One hypothesis is that F. tularensis forms biofilms in aquatic environments. We utilized two fully virulent wild-type strains: FSC200 (Francisella tularensis subsp. holarctica) and Schu S4 (Francisella tularensis subsp. tularensis) and three control strains, the attenuated live vaccine strain (LVS; F. tularensis subsp. holarctica), a Schu S4 DwbtI mutant that is documented to form biofilms, and the low-virulence strain U112 of the closely related species Francisella novicida. Strains were incubated in saline solution (0.9% NaCl) microcosms for 24 weeks at both 4°C and 20°C, whereupon viability and biofilm formation were measured. These temperatures were selected to approximate winter and summer temperatures of fresh water in Scandinavia, respectively. U112 and Schu S4 DwbtI formed biofilms, but F. tularensis strains FSC200 and Schu S4 and the LVS did not. All strains exhibited prolonged viability at 4°C compared to 20°C. U112 and FSC200 displayed remarkable long-term persistence at 4°C, with only 1- and 2-fold log reductions, respectively, of viable cells after 24weeks. Schu S4 exhibited lower survival, yielding no viable cells by week 20. At 24weeks, cells from FSC200, but not from Schu S4, were still fully virulent in mice. Taken together, these results demonstrate biofilm-independent, long-term survival of pathogenic F. tularensis subsp. holarctica in conditions that mimic overwinter survival in aquatic environments