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

The role of the Type IV pili system in the virulence of Francisella tularensis

Francisella tularensis is a Gram-negative intracellular pathogen causing the zoonotic disease tularemia. F. tularensis can be found almost all over the world and has been recovered from several animal species, even though the natural reservoir of the bacterium and parts of its life cycle are still unknown. Humans usually get infected after handling infected animals or from bites of blood-feeding arthropod vectors. There are four subspecies of F. tularensis: the highly virulent tularensis (Type A) that causes a very aggressive form of the disease, with mortality as high as 60% if untreated, the moderately virulent holarctica (Type B) and mediasiatica, and the essentially avirulent subspecies F. novicida. So far, our knowledge of the molecular mechanisms that would explain these differences in virulence among the subspecies is poor. However, recent developments of genetic tools and access to genomic sequences have laid the ground for progress in this research field. Analysis of genome sequences have identified several regions that differ between F. tularensis subspecies. One of these regions, RD19, encodes proteins postulated to be involved in assembly of type IV pili (Tfp), organelles that have been implicated in processes like twitching motility, biofilm formation and cell-to-cell communication in pathogenic bacteria. While there have been reports of pili-like structures on the surface of F. tularensis, these have not been linked to the Tfp encoding gene clusters until now. Herein, I present evidence that the Francisella pilin, PilA, can complement pilin-like characteristics and promote assembly of fibers in a heterologous system in Neisseria gonorrhoeae. pilA was demonstrated to be required for full virulence of both type A and type B strains in mice when infected via peripheral routes. A second region, RD18, encoding a protein unique to F. tularensis and without any known function, was verified to be essential for virulence in a type A strain. Interestingly, the non-licensed live vaccine strain, LVS (Type B), lacks both RD18 and RD19 (pilA) due to deletion events mediated by flanking direct repeats. The loss of RD18 and RD19 is responsible for the attenuation of LVS, since re-introducing them in cis could restore the virulence to a level similar to a virulent type B strain. Significantly, these deletion events are irreversible, preventing LVS to revert to a more virulent form. Therefore, this important finding could facilitate the licensing of LVS as a vaccine against tularemia

Sjuksköterskans upplevelse och föreställningar av mötet med våldsutsatta kvinnor

Bakgrund: Kvinnor som utsatts för våld i nära relarion kommer mer frekvent i kontakt med sjukvården än andra kvinnor. Flera kvinnor som söker vård för sina skador identifieras aldrig som offer för våld i nära relation. Detta beror på att flera sjuksköterskor saknar utbildning i att identifiera att våld förekommer. Det finns en osäkerhet hos sjuksköterskan i mötet med våldsutsatta kvinnor som gör att mötet med vården av kvinnan påverkas. Syfte: Syftet var att belysa sjuksköterskans upplevelse och föreställningar av mötet med våldsutsatta kvinnor inom hälso-och sjukvården. Metod: En systematisk litteraturstudie där sex kvalitativa artiklar och två kvantitativa artiklar har granskats, analyserats och kategoriserats. Resultat: Det fanns en tydlig emotionell påverkan hos sjuksköterskor under och efter mötet med våldsutsatta kvinnor. En påverkan som ibland var så stor att det påverkade deras privatliv. Det visade sig även finnas ett stort utbildningsbehov att förbättra vården för våldsutsatta kvinnor och få sjuksköterskor att känna sig tryggare i rollen som vårdare för dessa kvinnor. Slutsats: Det visade sig finnas ett ökat utbildningsbehov för sjuksköterskor i hur de ska bemöta och vårda kvinnor som utsatts för våld i nära relationer. Det fanns även ett behov av att bearbeta de känslor sjuksköterskan upplevde i mötet med kvinnorna.

Iron Content Differs between Francisella tularensis Subspecies tularensis and Subspecies holarctica Strains and Correlates to Their Susceptibility to H2O2-Induced Killing▿ †

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is classified as a category A select agent and a facultative intracellular bacterium. Why F. tularensis subsp. tularensis causes a more severe form of tularemia than F. tularensis subsp. holarctica does is not known. In this study, we have identified prominent phenotypic differences between the subspecies, since we found that F. tularensis subsp. tularensis strains contained less iron than F. tularensis subsp. holarctica strains. Moreover, strain SCHU S4 of F. tularensis subsp. tularensis was less susceptible than FSC200 and the live vaccine strain (LVS) of F. tularensis subsp. holarctica to H2O2-induced killing. The activity of the H2O2-degrading enzyme catalase was similar between the strains, whereas the iron content affected their susceptibility to H2O2, since iron starvation rendered F. tularensis subsp. holarctica strains more resistant to H2O2. Complementing LVS with fupA, which encodes an important virulence factor that regulates iron uptake, reduced its iron content and increased the resistance to H2O2-mediated killing. By real-time PCR, it was demonstrated that FSC200 and LVS expressed higher levels of gene transcripts related to iron uptake and storage than SCHU S4 did, and this likely explained their high iron content. Together, the results suggest that F. tularensis subsp. tularensis strains have restricted iron uptake and storage, which is beneficial for their resistance to H2O2-induced killing. This may be an important factor for the higher virulence of this subspecies of F. tularensis, as reactive oxygen species, such as H2O2, are important bactericidal components during tularemia

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

Upstream land use with microbial downstream consequences : iron and humic substances link to Legionella spp

Intensified land use can disturb water quality, potentially increasing the abundance of bacterial pathogens, threatening public access to clean water. This threat involves both direct contamination of faecal bacteria as well as indirect factors, such as disturbed water chemistry and microbiota, which can lead to contamination. While direct contamination has been well described, the impact of indirect factors is less explored, despite the potential of severe downstream consequences on water supply. To assess direct and indirect downstream effects of buildings, farms, pastures and fields on potential water sources, we studied five Swedish lakes and their inflows. We analysed a total of 160 samples in a gradient of anthropogenic activity spanning four time points, including faecal and water-quality indicators. Through species distribution modelling, Random Forest and network analysis using 16S rRNA amplicon sequencing data, our findings highlight that land use indirectly impacts lakes via inflows. Land use impacted approximately one third of inflow microbiota taxa, in turn impacting â\u88Œ20â\u80\u9350 % of lake taxa. Indirect effects via inflows were also suggested by causal links between e.g. water colour and lake bacterial taxa, where this influenced the abundance of several freshwater bacteria, such as Polynucleobacter and Limnohabitans. However, it was not possible to identify direct effects on the lakes based on analysis of physiochemical- or microbial parameters. To avoid potential downstream consequences on water supply, it is thus important to consider possible indirect effects from upstream land use and inflows, even when no direct effects can be observed on lakes. Legionella (a genus containing bacterial pathogens) illustrated potential consequences, since the genus was particularly abundant in inflows and was shown to increase by the presence of pastures, fields, and farms. The approach presented here could be used to assess the suitability of lakes as alternative raw water sources or help to mitigate contaminations in important water catchments. Continued broad investigations of stressors on the microbial network can identify indirect effects, avoid enrichment of pathogens, and help secure water accessibility

Patient-controlled admissions to inpatient care : A twelve-month naturalistic study of patients with schizophrenia spectrum diagnoses and the effects on admissions to and days in inpatient care

Background: Patients with schizophrenia spectrum diagnoses have a poor prognostic outlook and rates of recovery. Inpatient care is common, but the decision to initiate such care is not solely up to the patient but also influenced by the healthcare providers. Recent ideas about shared decision-making however challenges this idea. Patient-Controlled Admissions (PCA) refers to a care model where a patient signs a contract that allows the decision for admission into inpatient care to be transferred onto the patient. Methods: In Region Stockholm's public healthcare PCA was introduced to patients with schizophrenia spectrum diagnoses deemed to have the greatest care needs. Outcomes of a 12-month naturalistic within-group follow-up was analyzed using Wilcoxon signed-rank test. Results: In total, 56 patients fulfilled the study's inclusion criteria, with between 20 to 42 patients having complete data and being able to analyze statistically, depending on the variable. Number of admissions, inpatient days, number of involuntary admissions, and involuntary admission days decreased, but only significantly so for inpatient days, p < .01 (a mean reduction of 11.5 days). Neither self-rated well-being, as assessed using the EQ5D-3L, or a clinician-administered rating of overall health status, the Clinical Global Impression Scale, demonstrated a significant change. Conclusions: The use of PCA points towards a trend in decreased hospitalization for patients with schizophrenia spectrum diagnoses, although this needs to be explored further in larger samples and over a longer follow-up