1.
Åhlund, Monika K, et al.
(författare)
Directed screen of Francisella novicida virulence determinants using Drosophila melanogaster
2010
Ingår i: Infection and Immunity. - : ASM International. - 0019-9567 .- 1098-5522. ; 78:7, s. 3118-3128
Tidskriftsartikel (refereegranskat) abstract
Francisella tularensis is a highly virulent, facultative intracellular human pathogen whose virulence mechanisms are not well understood. Occasional outbreaks of tularemia and the potential use of F. tularensis as a bioterrorist agent warrant better knowledge about the pathogenicity of this bacterium. Thus far, genome-wide in vivo screens for virulence factors have been performed in mice, all however restricted by the necessity to apply competition-based, negative-selection assays. We wanted to individually evaluate putative virulence determinants suggested by such assays and performed directed screening of 249 F. novicida transposon insertion mutants by using survival of infected fruit flies as a measure of bacterial virulence. Some 20% of the genes tested were required for normal virulence in flies; most of these had not previously been investigated in detail in vitro or in vivo. We further characterized their involvement in bacterial proliferation and pathogenicity in flies and in mouse macrophages. Hierarchical cluster analysis of mutant phenotypes indicated a functional linkage between clustered genes. One cluster grouped all but four genes of the Francisella pathogenicity island and other loci required for intracellular survival. We also identified genes involved in adaptation to oxidative stress and genes which might induce host energy wasting. Several genes related to type IV pilus formation demonstrated hypervirulent mutant phenotypes. Collectively, the data demonstrate that the bacteria in part use similar virulence mechanisms in mammals as in Drosophila melanogaster but that a considerable proportion of the virulence factors active in mammals are dispensable for pathogenicity in the insect model.
2.
Widerström, Micael, 1958-
(författare)
Molecular epidemiology of coagulase-negative staphylococci in hospitals and in the community
2010
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Background Coagulase-negative staphylococci (CoNS) and in particular Staphylococcus epidermidis have emerged as major pathogens primarily causing nosocomial infections in patients with indwelling medical devices. These infections are often caused by multidrug-resistant strains of S. epidermidis (MDRSE). Other clinical entities due to CoNS are lower urinary tract infections (UTI) in women and native valve endocarditis. The purpose of this work was to investigate the frequency of antibiotic resistance and the molecular epidemiology of both hospital and community-associated isolates of S. epidermidis in order to examine if certain clones are related to MDRSE infections. Furthermore, we aimed to explore if specific clones of S. saprophyticus are associated with UTI in women. Methods A total of 359 hospital-associated methicillin-resistant isolates of CoNS obtained from 11 hospitals in northern Europe and 223 community-associated staphylococcal isolates were examined. Furthermore, 126 isolates of S. saprophyticus isolated from women with uncomplicated UTI from five different locations in northern Europe were analyzed. Pulsed-field gel electrophoresis (PFGE) was used for genotyping. Additionally, some of the S. epidermidis isolates were analyzed with multilocus sequence typing (MLST). Antibiotic susceptibility was determined for all isolates by the disc diffusion test. Results 293 of the 359 (82%) hospital-associated and 124 of the 223 (56%) community-associated isolates belonged to the species S. epidermidis. Among the hospital-associated S. epidermidis isolates, two dominating PFGE types (type A and B) were distinguished, comprising 78 (27%) and 51 (17%) isolates, respectively. Type A, which was detected in a Norwegian and eight Swedish hospitals, corresponded with a novel sequence type (ST215). Type B was discovered in a German, a Danish and seven Swedish hospitals and corresponded with ST2. In contrast, community-associated isolates of S. epidermidis were genetically extremely diverse with no predominating genotype, and showed a low rate of antibiotic resistance; only two (1.6%) methicillin-resistant strains were detected. Among 126 analyzed isolates of S. saprophyticus, 47 different PFGE profiles were identified. Several clusters of genetically highly related isolates were detected among isolates obtained from different locations and periods of time. Conclusion We have demonstrated the occurrence, persistence and potential dissemination of two multidrug-resistant S. epidermidis (MDRSE) genotypes, including a novel sequence type (ST215), within hospitals in northern Europe. Community-associated isolates of S. epidermidis showed a low rate of methicillin-resistance and were genetically heterogeneous. These results indicate that MDRSE by large are confined to the hospital setting in our region. Moreover, although the S. saprophyticus population was quite heterogeneous, indistinguishable isolates of S. saprophyticus causing lower UTI in women were identified in different countries 11 years apart, indicating the persistence and geographical spread of some clones of S. saprophyticus.
3.
Rydén, Patrik, 1969-, et al.
(författare)
Outbreaks of tularemia in a boreal forest region depends on mosquito prevalence
2012
Ingår i: Journal of Infectious Diseases. - : Oxford University Press (OUP). - 0022-1899 .- 1537-6613. ; 205:2, s. 297-304
Tidskriftsartikel (refereegranskat) abstract
Background. We aimed to evaluate the potential association of mosquito prevalence in a boreal forest area with transmission of the bacterial disease tularemia to humans, and model the annual variation of disease using local weather data.Methods. A prediction model for mosquito abundance was built using weather and mosquito catch data. Then a negative binomial regression model based on the predicted mosquito abundance and local weather data was built to predict annual numbers of humans contracting tularemia in Dalarna County, Sweden.Results. Three hundred seventy humans were diagnosed with tularemia between 1981 and 2007, 94% of them during 7 summer outbreaks. Disease transmission was concentrated along rivers in the area. The predicted mosquito abundance was correlated (0.41, P < .05) with the annual number of human cases. The predicted mosquito peaks consistently preceded the median onset time of human tularemia (temporal correlation, 0.76; P < .05). Our final predictive model included 5 environmental variables and identified 6 of the 7 outbreaks.Conclusions. This work suggests that a high prevalence of mosquitoes in late summer is a prerequisite for outbreaks of tularemia in a tularemia-endemic boreal forest area of Sweden and that environmental variables can be used as risk indicators.
4.
Waldenström, Jonas, et al.
(författare)
Migrating birds and tickborne encephalitis virus
2007
Ingår i: Emerging Infectious Diseases. - : Centers for Disease Control and Prevention (CDC). - 1080-6040 .- 1080-6059. ; 13:8, s. 1215-8
Tidskriftsartikel (refereegranskat) abstract
During spring and autumn 2001, we screened 13,260 migrating birds at Ottenby Bird Observatory, Sweden, and found 3.4% were infested with ticks. Four birds, each a different passerine species, carried tickborne encephalitis virus (TBEV)-infected ticks (Ixodes ricinus). Migrating birds may play a role in the geographic dispersal of TBEV-infected ticks.
5.
Alam, Athar, et al.
(författare)
The Role of ClpB in Bacterial Stress Responses and Virulence
2021
Ingår i: Frontiers in Molecular Biosciences. - : Frontiers Media S.A.. - 2296-889X. ; 8
Forskningsöversikt (refereegranskat) abstract
Bacterial survival within a mammalian host is contingent upon sensing environmental perturbations and initiating an appropriate counter-response. To achieve this, sophisticated molecular machineries are used, where bacterial chaperone systems play key roles. The chaperones are a prerequisite for bacterial survival during normal physiological conditions as well as under stressful situations, e.g., infection or inflammation. Specific stress factors include, but are not limited to, high temperature, osmolarity, pH, reactive oxidative species, or bactericidal molecules. ClpB, a member of class 1 AAA+ proteins, is a key chaperone that via its disaggregase activity plays a crucial role for bacterial survival under various forms of stress, in particular heat shock. Recently, it has been reported that ClpB also regulates secretion of bacterial effector molecules related to type VI secretion systems. In this review, the roles of ClpB in stress responses and the mechanisms by which it promotes survival of pathogenic bacteria are discussed.
6.
Elfving, Karin, et al.
(författare)
Dissemination of Spotted Fever Rickettsia Agents in Europe by Migrating Birds
2010
Ingår i: PLOS ONE. - : Public Library of Science. - 1932-6203. ; 5:1
Tidskriftsartikel (refereegranskat) abstract
Migratory birds are known to play a role as long-distance vectors for many microorganisms. To investigate whether this is true of rickettsial agents as well, we characterized tick infestation and gathered ticks from 13,260 migratory passerine birds in Sweden. A total of 1127 Ixodes spp. ticks were removed from these birds and the extracted DNA from 957 of them was available for analyses. The DNA was assayed for detection of Rickettsia spp. using real-time PCR, followed by DNA sequencing for species identification. Rickettsia spp. organisms were detected in 108 (11.3%) of the ticks. Rickettsia helvetica, a spotted fever rickettsia associated with human infections, was predominant among the PCR-positive samples. In 9 (0.8%) of the ticks, the partial sequences of 17kDa and ompB genes showed the greatest similarity to Rickettsia monacensis, an etiologic agent of Mediterranean spotted fever-like illness, previously described in southern Europe as well as to the Rickettsia sp. IrITA3 strain. For 15 (1.4%) of the ticks, the 17kDa, ompB, gltA and ompA genes showed the greatest similarity to Rickettsia sp. strain Davousti, Rickettsia japonica and Rickettsia heilongjiangensis, all closely phylogenetically related, the former previously found in Amblyomma tholloni ticks in Africa and previously not detected in Ixodes spp. ticks. The infestation prevalence of ticks infected with rickettsial organisms was four times higher among ground foraging birds than among other bird species, but the two groups were equally competent in transmitting Rickettsia species. The birds did not seem to serve as reservoir hosts for Rickettsia spp., but in one case it seems likely that the bird was rickettsiemic and that the ticks had acquired the bacteria from the blood of the bird. In conclusion, migratory passerine birds host epidemiologically important vector ticks and Rickettsia species and contribute to the geographic distribution of spotted fever rickettsial agents and their diseases.
7.
Lindgren, Marie, 1981-
(författare)
Characterization of the attenuated Francisella tularensis strain FSC043 : with special focus on the gene pdpC
2013
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Francisella tularensis is a highly infective, intracellular bacterium. It is capable of infecting a wide range of mammals and causes the disease tularemia in humans. As a result of its high infectivity there have been a lot of efforts made to create a generally available vaccine against this pathogen. One potential vaccine candidate is the FSC043 strain, a spontaneous mutant that has acquired mutations making it attenuated for replication both in vitro and in the experimental mouse model. However, it was noted that it afforded protection against challenge with a highly virulent F. tularensis strain.The aim of this thesis has been to delineate the mechanisms of its attenuation to better understand F. tularensis pathogenesis and to obtain a better knowledge about the prerequisites of protective immunity against this potent pathogen. Microarray and whole-genome sequencing revealed four mutations in the attenuated FSC043 strain that were not present in the virulent SCHU S4 isolate. One of these mutations has been described earlier as it results in a fusion protein also found in other attenuated strains. Among the other differences, two mutations were identical nonsense mutations in a duplicated gene region known as the Francisella pathogenicity island (FPI). The affected gene, pdpC, is coding for PdpC (pathogenicity determinant protein C). We found that these mutations resulted in a truncated form of PdpC, and also that the downstream gene was severely downregulated due to these mutations.Further, our studies revealed that the intracellular phenotype of the FSC043 strain differed from other tested strains in that a small portion of the intracellular bacteria were able to escape the phagosome and multiply within the host, while the majority of intracellular bacteria stayed confined to the phagosome. We wanted to study the specific function of pdpC and therefore deleted both copies of it in the virulent SCHU S4strain as well as the Live Vaccine Strain, an empirically attenuated strain often used as a model for the virulent strains of F. tularensis. The resulting mutants showed an attenuated phenotype; no intracellular growth in murine cells, and no virulence in mice. When studying the intracellular localization of the LVS Δpdpc mutant, we found that it was uniformly located adjacent to phagosomal membrane-like structures but that the membrane was markedly disrupted. Further, this mutant induced an MOI-dependent cytotoxicity, measured by LDH release, and also the release of IL-1β, an inflammatory cytokine not induced by phagosomally contained mutants. Studies on markers for host cell death revealed that the LVS ΔpdpC mutant induced mitochondrial instability, phosphatidylserine (PS) presentation, and TUNEL-specific DNA fragmentation in infected cells, rather similar to the wild-type strain, despite its lack of replication.This study reveals that the pdpC gene is an important gene required for F. tularensis virulence. We also show that non-replicating intracellular bacteria can induce host cell death, hypothesizing that release of bacterial components in the host cell cytosol is required for this induction. The FSC043 mutant showed a unique phenotype where a small subset of bacteria was able to escape the phagosome and replicate in the host cell. This was also seen in the pdpC deletion mutant of SCHU S4, but not with the LVS ΔpdpC. However, regardless of genetic background, the ΔpdpC mutant had an effect on phagosomal escape; either by affecting the phagosomal membranes in a unique way or by allowing phagosomal escape of a small proportion of the bacteria.
8.
9.
Mohammadi, Nasibeh, 1981-
(författare)
Determining the role of guanylate-binding proteins for host defense against Francisella tularensis
2022
Doktorsavhandling (övrigt vetenskapligt/konstnärligt) abstract
Francisella tularensis is a highly virulent, intracellular bacterium and the causative agent of the human disease tularemia. This is a zoonotic, often vector-borne disease. Due to its intracellular nature, F. tularensis can infect many cell types, but of special relevance is its ability to infect monocytic cells and avoid their otherwise potent antimicrobial effects. Monocytic cells can; however, control infection after activation with IFN-γ, but the molecular mechanisms behind this control are not well understood. Recently, guanylate-binding proteins (GBPs) have been identified as crucial for the control of intracellular F. tularensis and many other bacteria, viruses, and parasites. They represent a vast family of interferon-inducible proteins, but it is incompletely understood how their ubiquitous abilities to control diverse types of infections are executed. The overall aim of the thesis was to obtain a better understanding of how GBPs execute the control of infection caused by Francisella and how the bacterium counteracts the bactericidal effects of the GBPs and of other immune mediators. To this end, the responses of bone marrow-derived murine macrophages (BMDM) to Francisella was one model investigated and the other employed a co-culture system whereby BMDM were infected and to the cultures immune cells from vaccinated mice were added. To comprehensively understand the host-pathogen interaction, a variety of Francisella strains were utilized; the highly virulent SCHU S4 strain, the human live vaccine strain (LVS), and the widely used surrogate for F. tularensis, the low virulent F. novicida. All strains have similar capability of intracellular multiplication in BMDM, however, activation of the microbicidal ability of BMDM with IFN-γ, significant control of infection was observed for the LVS and F. novicida strains, whereas there was no control of the SCHU S4 infection. The control of the former strains was GBP-dependent, despite that no differences in GBP transcription or translation were observed in the infected cell cultures. Patterns of 18 cytokines very clearly discriminated the different types of infections and high levels were generally observed in F. novicida-infected cultures and very low levels in SCHU S4-infected cultures. Co-infection with F. novicida and SCHU S4 led to significant control of both strains and in these cultures, a majority of cytokines showed intermediate or high levels. A critical component in the immune recognition of Francisella is AIM2, which is a core constituent of a special form of inflammasome, a cytoplasmic multimeric complex. We determined that AIM2-deficient BMDM, despite the central role of AIM2 for immune recognition of F. novicida and LVS, still controlled infection with either of the two strains after activation with IFN-γ. Again, no control of the virulent strain SCHU S4 was observed. The co-culture system revealed further complexity beyond that of the BMDM model. Utilizing splenocytes obtained from immunized C57BL/6 mice as effectors in cultures with BMDM infected with either of the three Francisella strains, we observed that regardless of strain, significant control of replication occurred with wild-type macrophages and immune splenocytes, even for the highly virulent SCHU S4 strain, but not in cultures with immune splenocytes and GBP-deficient macrophages. Supernatants from the cultures demonstrated very distinct patterns for each of the three infections. Thus, the co-culture assay identified, as for the BMDM model, a crucial role of GBPs for the control of intracellular replication of Francisella, however, in contrast to the BMDM model, the co-culture conferred significant control of SCHU S4 infection.Collectively, our studies demonstrate a very important role of GBPs for the IFN-γ-dependent control of Francisella infection, with the notable exception of the highly virulent strain SCHU S4. A GBP-mediated control of SCHU S4 was; however, observed in the co-culture system, thereby identifying additional bactericidal mechanisms, besides those that are IFN-γ-dependent. We also demonstrate that the inflammatory potential of Francisella strains is correlated to their virulence, most notable is the almost complete lack of inflammatory response during infection with the highly virulent SCHU S4 strain, but this anti-inflammatory capacity was counteracted by the strong pro-inflammatory property of F. novicida during co-infection.
10.
Tancred, Linda, 1974-, et al.
(författare)
Administration of a donor of nitric oxide inhibits mglA expression of intracellular Francisella tularensis and counteracts phagosomal escape and subversion of TNF-α secretion
2011
Ingår i: Journal of Medical Microbiology. - : Microbiology Society. - 0022-2615 .- 1473-5644. ; 30:11, s. 1570-1583
Tidskriftsartikel (refereegranskat) abstract
Francisella tularensis is a highly virulent intracellular bacterium capable of rapid multiplication in phagocytic cells. Previous studies have revealed that activation of F. tularensis-infected macrophages leads to control of infection and reactive nitrogen and oxygen species make important contributions to the bacterial killing. We investigated the effects of adding S-nitroso-acetyl-penicillamine (SNAP), which generates nitric oxide or 3-morpholinosydnonimine hydrochloride (SIN-1), which indirectly leads to formation of peroxynitrite, to J774 murine macrophage-like cell cultures infected with F. tularensis LVS. Addition of SNAP led to significantly increased colocalization between LAMP-1 and bacteria, indicating containment of F. tularensis in the phagosome within 2 h, although no killing occurred within 4 h. A specific inhibitory effect on bacterial transcription was observed since the gene encoding the global regulator MglA was inhibited 50- to 100-fold. F. tularensis-infected J774 cells are incapable of secreting TNF-alpha in response to Escherichia coli LPS but addition of SNAP almost completely reversed the suppression. Similarly, infection with an MglA mutant did not inhibit LPS-induced TNF-alpha secretion of J774 cells. Strong staining for nitrotyrosine was observed in SNAP-treated bacteria and mass spectrometry identified nitration of two ribosomal 50S proteins, a CBS domain pair protein, and bacterioferritin. The results demonstrate that addition of SNAP initially did not affect the viability of intracellular F. tularensis LVS but led to containment of the bacteria in the phagosome. Moreover, the treatment resulted in nitration of several F. tularensis proteins.
