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Background: The zoonotic and highly infectious pathogen Francisella tularensis is the etiological agent of tularemia. Tularemia in humans is mainly caused by F. tularensis subspecies tularensis and holarctica, but Francisella species like F. novicida, F. philomiragia, F. hispaniensis and others are known to cause tularemia-like infections in immunocompromised humans. In addition to these Francisella species, further genera of the family Francisellaceae have been described, such as Allofrancisella, Parafrancisella and Pseudofrancisella, but less is known about the distribution and putative virulence of these genera. The methods currently available were not made for a fast and easy detection of all these strains and genera of Francisellaceae. Results: We developed a multiplex quantitative real-time PCR assay that can accurately detect all genera of Francisellaceae, including Francisella, Francisella-like endosymbionts, Allofrancisella, Parafrancisella and Pseudofrancisella. In addition, we developed a qPCR assay to differentiate the major clades (B.4, B.6 and B.12 [B.71 and B.72]) of F. tularensis ssp. holarctica strains. Both primer sets were shown to work on isolated DNA out of human and tick samples. Conclusion: Since the developed qPCRs are able to detect all genera of Francisellaceae tested, an easy and fast identification of opportunistic Francisella strains causing tularemia-like symptoms in humans or animals is possible now. The application of these qPCR assays will thus improve the capability for clinical diagnostics and molecular typing during epidemiological investigations. [ABSTRACT FROM AUTHOR]

Introduction: Tularemia is mainly caused by Francisella tularensis (Ft) subsp. tularensis (Ftt) and Ft subsp. holarctica (Ftt) in humans and in more than 200 animal species including rabbits and hares. Human clinical manifestations depend on the route of infection and range from flu-like symptoms to severe pneumonia with a mortality rate up to 60% without treatment. So far, only 2D cell culture and animal models are used to study Francisella virulence, but the gained results are transferable to human infections only to a certain extent. Method: In this study, we firstly established an ex vivo human lung tissue infection model using different Francisella strains: Ftt Life Vaccine Strain (LVS), Ftt LVS ΔiglC, Ftt human clinical isolate A-660 and a German environmental Francisella species strain W12-1067 (F-W12). Human lung tissue was used to determine the colony forming units and to detect infected cell types by using spectral immunofluorescence and electron microscopy. Chemokine and cytokine levels were measured in culture supernatants. Results: Only LVS and A-660 were able to grow within the human lung explants, whereas LVS ΔiglC and F-W12 did not replicate. Using human lung tissue, we observed a greater increase of bacterial load per explant for patient isolate A-660 compared to LVS, whereas a similar replication of both strains was observed in cell culture models with human macrophages. Alveolar macrophages were mainly infected in human lung tissue, but Ftt was also sporadically detected within white blood cells. Although Ftt replicated within lung tissue, an overall low induction of pro-inflammatory cytokines and chemokines was observed. A-660-infected lung explants secreted slightly less of IL-1β, MCP-1, IP-10 and IL-6 compared to Ftt LVS-infected explants, suggesting a more repressed immune response for patient isolate A-660. When LVS and A-660 were used for simultaneous co-infections, only the ex vivo model reflected the less virulent phenotype of LVS, as it was outcompeted by A-660. Conclusion: We successfully implemented an ex vivo infection model using human lung tissue for Francisella. The model delivers considerable advantages and is able to discriminate virulent Francisella from less-or non-virulent strains and can be used to investigate the role of specific virulence factors. [ABSTRACT FROM AUTHOR]

Biofilms are a matrix-associated lifestyle of microbial communities, often enabling survivability and persistence of such bacteria. The objective of this study was to investigate the survival of the wild-type strain A-271 of Francisella tularensis subsp. holarctica (Fth) in a natural aquatic ex vivo biofilm. To that purpose, we allowed Fth A-271 to produce its own biofilm on solid surfaces but also to colonize naturally formed biofilms from aquatic habitats, which were infected with Francisella in the laboratory. The survival rates of the bacteria in biofilms were compared to those of planktonic bacteria as a function of the employed culture condition. It could be shown by light- and electron microscopy that Fth is able to form a complex, matrix-associated biofilm. The biofilm form of Francisella showed longer cultivability on agar plates in natural water when compared to planktonic (free-living) bacteria. Be it as a part of the existing ex vivo biofilm or free-floating above as planktonic bacteria, more than 80% of Francisella were not only able to survive under these conditions for 28 days, but even managed to establish microcolonies and areas with their own exclusive biofilm architecture within the ex vivo biofilm. Here, we can demonstrate for the first time that a Francisella tularensis wild-type strain (Type B) is able to successfully colonize an aquatic multi-species ex vivo biofilm. It is worthwhile to speculate that Fth might become more persistent in the environment when it forms its own biofilm or integrates in an existing one. Multi-species biofilms have been shown to be more resistant against stress compared to single-species biofilms. This may have an important impact on the long-term survival of Francisella in aquatic habitats and infection cycles in nature. [ABSTRACT FROM AUTHOR]

Francisella tularensis is a Gram-negative bacterium that causes a potentially fatal disease called tularemia. The highly infectious agent can spread via arthropod vectors, including ticks and via rodents such as rabbits or beavers. The facultative intracellular pathogen typically invades human macrophages at the onset of human infection. For intracellular replication of the bacteria, the usage of amino acids from the host cells and gluconeogenesis seem to be detrimental, but other carbon sources including glucose and glycerol are also utilized. Here, we compared the growth phase-dependent degradation of glucose, glycerol, and alanine in F. tularensis subsp. holarctica isolated from an infected beaver with the respective metabolism in the less virulent strains F. novicida strain U112 and Francisella sp. strain W12-1067. To this aim, we performed 13C-labeling experiments with the bacteria growing in medium T supplemented with either [U-13C6]glucose, [U-13C3]glycerol, or [2,3-13C2]alanine during different growth phases. After cell harvest, mechanical disruption, and hydrolysis of cellular fractions, we determined the 13C-profiles in various metabolites by mass spectrometry. The detected 13C-patterns elucidated the metabolic fate of the supplied carbon nutrients and revealed minor, but significant differences indicative of various metabolic phenotypes of the Francisella strains under study. Glucose served as the main substrate for all strains under the experimental conditions. The sugar was degraded via the Embden-Meyerhof-Parnas pathway as the major catabolic route during growth. At lower rates, exogenous glycerol and alanine were used as co-substrates, particularly in the less pathogenic strains during the later growth phases. Our data support the hypothesis that, among other factors, the capability to adapt substrate usages efficiently and metabolic fluxes could determine the virulence of Francisella strains. [ABSTRACT FROM AUTHOR]

Abstract Article Retracted

Abstract Background Legionella pneumophila is an important causative agent of severe pneumonia in humans. Human alveolar epithelium and macrophages are effective barriers for inhaled microorganisms and actively participate in the initiation of innate host defense. The beta defensin-3 (hBD-3), an antimicrobial peptide is an important component of the innate immune response of the human lung. Therefore we hypothesize that hBD-3 might be important for immune defense towards L. pneumophila. Methods We investigated the effects of L. pneumophila and different TLR agonists on pulmonary cells in regard to hBD-3 expression by ELISA. Furthermore, siRNA-mediated inhibition of TLRs as well as chemical inhibition of potential downstream signaling molecules was used for functional analysis. Results L. pneumophila induced release of hBD-3 in pulmonary epithelium and alveolar macrophages. A similar response was observed when epithelial cells were treated with different TLR agonists. Inhibition of TLR2, TLR5, and TLR9 expression led to a decreased hBD-3 expression. Furthermore expression of hBD-3 was mediated through a JNK dependent activation of AP-1 (c-Jun) but appeared to be independent of NF-κB. Additionally, we demonstrate that hBD-3 elicited a strong antimicrobial effect on L. pneumophila replication. Conclusions Taken together, human pulmonary cells produce hBD-3 upon L. pneumophila infection via a TLR-JNK-AP-1-dependent pathway which may contribute to an efficient innate immune defense.

Abstract Background Legionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells. Results Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter. Conclusions Taken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease.

Abstract Background Legionella pneumophila is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of L. pneumophila on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines. Results Infection of L. pneumophila strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with L. pneumophila lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient Legionella. Activation of the IL-8 promoter by L. pneumophila infection occurred through the action of nuclear factor-κB (NF-κB). Transfection of dominant negative mutants of NF-κB-inducing kinase, IκB kinase and IκB inhibited L. pneumophila-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed L. pneumophila-induced IL-8 mRNA due to deactivation of NF-κB. Conclusion Collectively, these results suggest that L. pneumophila induces activation of NF-κB through an intracellular signaling pathway that involves NF-κB-inducing kinase and IκB kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in L. pneumophila-induced IL-8 expression, presumably contributing to immune response in L. pneumophila. The presence of flagellin and a type IV secretion system are critical for Legionella to induce IL-8 expression in lung epithelial cells.

Francisella tularensis is an intracellular pleomorphic bacterium and the causative agent of tularemia, a zoonotic disease with a wide host range. Among the F. tularensis subspecies, especially F. tularensis subsp. holarctica is of clinical relevance for European countries. The study presented herein focuses namely on genetic diversity and spatial segregation of F. tularensis subsp. holarctica in Germany, as still limited information is available. The investigation is based on the analysis of 34 F. tularensis subsp. holarctica isolates and one draft genome from an outbreak strain. The isolates were cultured from sample material being that of primarily human patients (n = 25) and free-living animals (n = 9). For six of 25 human isolates, epidemiological links between disease onset and tick bites could be established, confirming the importance of arthropod linked transmission of tularemia in Germany. The strains were assigned to three of four major F. tularensis subsp. holarctica clades: B.4, B.6, and B.12. Thereby, B.6 and B.12 clade members were predominantly found; only one human isolate was assigned to clade B.4. Also, it turned out that eight isolates which caused pneumonia in patients clustered into the B.6 clade. Altogether, eight different final subclades were assigned to clade B.6 (biovar I, erythromycin sensitive) and six to B.12 (biovar II, erythromycin resistant) in addition to one new final B.12 subclade. Moreover, for 13 human and 3 animal isolates, final subclade subdivisions were not assigned (B.12 subdivisions B.33 and B.34, and B.6 subdivision B.45) because official nomenclatures are not available yet. This gives credit to the genetic variability of F. tularensis subsp. holarctica strains in Germany. The results clearly point out that the given genetic diversity in Germany seems to be comparably high to that found in other European countries including Scandinavian regions. A spatial segregation of B.6 and B.12 strains was found and statistically confirmed, and B.12 clade members were predominantly found in eastern parts and B.6 members more in western to southern parts of Germany. The portion of B.12 clade members in northeastern parts of Germany was 78.5% and in southwestern parts 1.9%. [ABSTRACT FROM AUTHOR]

We recently identified and described a putative prophage on the genomic island FhaGI-1 located within the genome of Francisella hispaniensis AS02-814 (F. tularensis subsp. novicida-like 3523). In this study, we constructed two variants of a Francisella phage integration vector, called pFIV1-Val and pFIV2-Val (Francisella Integration Vector-tRNAVal-specific), using the attL/R-sites and the site-specific integrase (FN3523_1033) of FhaGI-1, a chloramphenicol resistance cassette and a sacB gene for counter selection of transformants against the vector backbone. We inserted the respective sites and genes into vector pUC57-Kana to allow for propagation in Escherichia coli. The constructs generated a circular episomal form in E. coli which could be used to transform Francisella spp. where FIV-Val stably integrated site specifically into the tRNAVal gene of the genome, whereas pUC57-Kana is lost due to counter selection. Functionality of the new vector was demonstrated by the successfully complementation of a Francisella mutant strain. The vectors were stable in vitro and during host-cell infection without selective pressure. Thus, the vectors can be applied as a further genetic tool in Francisella research, expanding the present genetic tools by an integrative element. This new element is suitable to perform long-term experiments with different Francisella species. [ABSTRACT FROM AUTHOR]

Tularemia, also known as "rabbit fever," is a zoonosis caused by the facultative intracellular, gram-negative bacterium Francisella tularensis. Infection occurs through contact with infected animals (often hares), arthropod vectors (such as ticks or deer flies), inhalation of contaminated dust or through contaminated food and water. In this review, we would like to provide an overview of the current epidemiological situation in Germany using published studies and case reports, an analysis of recent surveillance data and our own experience from the laboratory diagnostics, and investigation of cases. While in Germany tularemia is a rarely reported disease, there is evidence of recent re-emergence. We also describe some peculiarities that were observed in Germany, such as a broad genetic diversity, and a recently discovered new genus of Francisella and protracted or severe clinical courses of infectionswith the subspecies holarctica. Because tularemia is a zoonosis, we also touch upon the situation in the animal reservoir and one-health aspects of this disease. Apparently, many pieces of the puzzle need to be found and put into place before the complex interaction between wildlife, the environment and humans are fully understood. Funding for investigations into rare diseases is scarce. Therefore, combining efforts in several countries in the framework of international projects may be necessary to advance further our understanding of this serious but also scientifically interesting disease. [ABSTRACT FROM AUTHOR]

The Legionella genus comprises more than 60 species. In particular, Legionella pneumophila is known to cause severe illnesses in humans. Legionellaceae are ubiquitous inhabitants of aquatic environments. Some Legionellaceae aremotile and their motility is important to move around in habitats. Motility can be considered as a potential virulence factor as already shown for various human pathogens. The genes of the flagellar system, regulator and structural genes, are structured in hierarchical levels described as the flagellar regulon. Their expression is modulated by various environmental factors. For L. pneumophila it was shown that the expression of genes of the flagellar regulon is modulated by the actual growth phase and temperature. Especially, flagellated Legionella are known to express genes during the transmissive phase of growth that are involved in the expression of virulence traits. It has been demonstrated that the alternative sigma-28 factor is part of the link between virulence expression and motility. In the following review, the structure of the flagellar regulon of L. pneumophila is discussed and compared to other flagellar systems of different Legionella species. Recently, it has been described that Legionella micdadei and Legionella fallonii contain a second putative partial flagellar system. Hence, the report will focus on flagellated and non-flagellated Legionella strains, phylogenetic relationships, the role and function of the alternative sigma factor (FliA) and its anti-sigma-28 factor (FlgM). [ABSTRACT FROM AUTHOR]

Francisella tularensis is an intracellular pathogen for many animals causing the infectious disease, tularemia. Whereas F tularensis subsp. holarctica is highly pathogenic for humans, F novicida is almost avirulent for humans, but virulent for mice. In order to compare metabolic fluxes between these strains, we performed 13C-labeling experiments with F tularensis subsp. holarctica wild type (beaver isolate), F tularensis subsp. holarctica strain LVS, or F novicida strain U112 in complex media containing either [U-13C6]glucose, [1,2-13C2]glucose, [U-13C3]serine, or [U-13C3]glycerol. GC/MS-based isotopolog profiling of amino acids, polysaccharide-derived glucose, free fructose, amino sugars derived from the cell wall, fatty acids, 3-hydroxybutyrate, lactate, succinate and malate revealed uptake and metabolic usage of all tracers under the experimental conditions with glucose being the major carbon source for all strains under study. The labeling patterns of the F tularensis subsp. holarctica wild type were highly similar to those of the LVS strain, but showed remarkable differences to the labeling profiles of the metabolites from the F novicida strain. Glucose was directly used for polysaccharide and cell wall biosynthesis with higher rates in F tularensis subsp. holarctica or metabolized, with higher rates in F novicida, via glycolysis and the non-oxidative pentose phosphate pathway (PPP). Catabolic turnover of glucose via gluconeogenesis was also observed. In all strains, Ala was mainly synthesized from pyruvate, although no pathway from pyruvate to Ala is annotated in the genomes of F tularensis and F novicida. Glycerol efficiently served as a gluconeogenetic substrate in F novicida, but only less in the F tularensis subsp. holarctica strains. In any of the studied strains, serine did not serve as a major substrate and was not significantly used for gluconeogenesis under the experimental conditions. Rather, it was only utilized, at low rates, in downstream metabolic processes, e.g., via acetyl-CoA in the citrate cycle and for fatty acid biosynthesis, especially in the F. tularensis subsp. holarctica strains. In summary, the data reflect differential metabolite fluxes in F. tularensis subsp. holarctica and F. novicida suggesting that the different utilization of substrates could be related to host specificity and virulence of Francisella. [ABSTRACT FROM AUTHOR]

The genus Legionella belongs to Gram-negative bacteria found ubiquitously in aquatic habitats, where it grows in natural biofilms and replicates intracellularly in various protozoa (amoebae, ciliates). L. pneumophila is known as the causative agent of Legionnaires' disease, since it is also able to replicate in human alveolar macrophages, finally leading to inflammation of the lung and pneumonia. To withstand the degradation by its host cells, a Legionella-containing vacuole (LCV) is established for intracellular replication, and numerous effector proteins are secreted into the host cytosol using a type four B secretion system (T4BSS). During intracellular replication, Legionella has a biphasic developmental cycle that alternates between a replicative and a transmissive form. New knowledge about the host-adapted and life stage-dependent metabolism of intracellular L. pneumophila revealed a bipartite metabolic network with life stage-specific usages of amino acids (e.g. serine), carbohydrates (e.g. glucose) and glycerol as major substrates. These metabolic features are associated with the differentiation of the intracellular bacteria, and thus have an important impact on the virulence of L. pneumophila. [ABSTRACT FROM AUTHOR]

Background Francisella isolates from patients suffering from tularemia in Germany are generally strains of the species F. tularensis subsp. holarctica. To our knowledge, no other Francisella species are known for Germany. Recently, a new Francisella species could be isolated from a water reservoir of a cooling tower in Germany. Results We identified a Francisella sp. (isolate W12-1067) whose 16S rDNA is 99% identical to the respective nucleotide sequence of the recently published strain F. guangzhouensis. The overall sequence identity of the fopA, gyrA, rpoA, groEL, sdhA and dnaK genes is only 89%, indicating that strain W12-1067 is not identical to F. guangzhouensis. W12-1067 was isolated from a water reservoir of a cooling tower of a hospital in Germany. The growth optimum of the isolate is approximately 30°C, it can grow in the presence of 4-5% NaCl (halotolerant) and is able to grow without additional cysteine within the medium. The strain was able to replicate within a mouse-derived macrophage-like cell line. The whole genome of the strain was sequenced (∼1.7 mbp, 32.2% G + C content) and the draft genome was annotated. Various virulence genes common to the genus Francisella are present, but the Francisella pathogenicity island (FPI) is missing. However, another putative type-VI secretion system is present within the genome of strain W12-1067. Conclusions Isolate W12-1067 is closely related to the recently described F. guangzhouensis species and it replicates within eukaryotic host cells. Since W12-1067 exhibits a putative new type-VI secretion system and F. tularensis subsp. holarctica was found not to be the sole species in Germany, the new isolate is an interesting species to be analyzed in more detail. Further research is needed to investigate the epidemiology, ecology and pathogenicity of Francisella species present in Germany. [ABSTRACT FROM AUTHOR]

Background: The lipopolysaccharide (LPS) is the major immuno-dominant antigen of all Legionella species including L. pneumophila. Its diversity is the basis for the classification of L. pneumophila into serogroups and monoclonal subgroups and is thought to be involved in strain specific virulence. The understanding of the genetic basis of the LPS-antigen is incomplete. Thus, we analyzed the genetic locus involved in LPS-biosynthesis of L. pneumophila serogroup 1 (Sg1) strains with the focus on strain specific gene composition. Results: The LPS-biosynthesis loci of 14 L. pneumophila Sg1 strains comprise two distinct regions: A 15 kb region containing LPS-biosynthesis genes that can be found in all L. pneumophila strains and a Sg1-specific 18 kb region. The 15 kb region is highly conserved among Sg1 strains as reflected by high homologies of single ORFs and by a consistent ORF arrangement. In contrast, the Sg1 specific 18 kb region is variable and partially disrupted by phage related genes. We propose that the region spanning from ORF 6 to ORF 11 of the Sg1-specific region is likely involved in late LPS-modification. Due to the high variability of this small region and various combinations of single ORFs within this region a strain specific LPS-structure could be synthesized including modifications of legionaminic acid derivates. Conclusions: Our data clearly demonstrate that the gene structure of the LPS-biosynthesis locus of L. pneumophila Sg1 strains show significant interstrain variability. These data can be used for further functional analysis of the LPS synthesis to understand pathogenesis and reactivity with monoclonal antibodies. Moreover, variable but strain specific regions can serve as basis for the development of novel genotyping assays. [ABSTRACT FROM AUTHOR]

Background: Legionella pneumophila is the causative agent of human Legionnaire's disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells. Results: Wild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of |κBα, |κB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter. Conclusions: Taken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaire's disease. [ABSTRACT FROM AUTHOR]

Background: Legionella pneumophila is an important causative agent of severe pneumonia in humans. Human alveolar epithelium and macrophages are effective barriers for inhaled microorganisms and actively participate in the initiation of innate host defense. The beta defensin-3 (hBD-3), an antimicrobial peptide is an important component of the innate immune response of the human lung. Therefore we hypothesize that hBD-3 might be important for immune defense towards L. pneumophila. Methods: We investigated the effects of L. pneumophila and different TLR agonists on pulmonary cells in regard to hBD-3 expression by ELISA. Furthermore, siRNA-mediated inhibition of TLRs as well as chemical inhibition of potential downstream signaling molecules was used for functional analysis. Results: L. pneumophila induced release of hBD-3 in pulmonary epithelium and alveolar macrophages. A similar response was observed when epithelial cells were treated with different TLR agonists. Inhibition of TLR2, TLR5, and TLR9 expression led to a decreased hBD-3 expression. Furthermore expression of hBD-3 was mediated through a JNK dependent activation of AP-1 (c-Jun) but appeared to be independent of NF-κ?B. Additionally, we demonstrate that hBD-3 elicited a strong antimicrobial effect on L. pneumophila replication. Conclusions: Taken together, human pulmonary cells produce hBD-3 upon L. pneumophila infection via a TLR-JNK-AP- 1-dependent pathway which may contribute to an efficient innate immune defense. [ABSTRACT FROM AUTHOR]

Background: Legionella pneumophila is a facultative intracellular bacterium, capable of replicating within the phagosomes of macrophages and monocytes, but little is known about its interaction with human lung epithelial cells. We investigated the effect of L. pneumophila on the expression of interleukin-8 (IL-8) in human A549 alveolar and NCI-H292 tracheal epithelial cell lines. Results: Infection of L. pneumophila strain, but not heat-killed strain, resulted in upregulation of IL-8. IL-8 mRNA expression was induced immediately after the infection and its signal became gradually stronger until 24 h after infection. On the other hand, IL-8 expression in A549 cells infected with L. pneumophila lacking a functional type IV secretion system was transient. The IL-8 expression was slightly induced at 16 h and increased at 24 h after infection with flagellin-deficient Legionella. Activation of the IL-8 promoter by L. pneumophila infection occurred through the action of nuclear factor-κB (NF-κB). Transfection of dominant negative mutants of NF-κB-inducing kinase, IκB kinase and IκB inhibited L. pneumophila-mediated activation of IL-8 promoter. Treatment with hsp90 inhibitor suppressed L. pneumophila-induced IL-8 mRNA due to deactivation of NF-κB. Conclusion: Collectively, these results suggest that L. pneumophila induces activation of NF-κB through an intracellular signaling pathway that involves NF-κB-inducing kinase and IκB kinase, leading to IL-8 gene transcription, and that hsp90 acts as a crucial regulator in L. pneumophila-induced IL-8 expression, presumably contributing to immune response in L. pneumophila. The presence of flagellin and a type IV secretion system are critical for Legionella to induce IL-8 expression in lung epithelial cells. [ABSTRACT FROM AUTHOR]

Adaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii. The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner-Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro, may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA (σ28) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival. [ABSTRACT FROM AUTHOR]

The haploid social soil amoebaDictyostelium discoideumhas been established as a host model for several pathogens includingPseudomonas aeruginosa,Cryptococcus neoformans,Mycobacteriumspp. andLegionella pneumophila. The research areas presently pursued include (i) the use ofDictyosteliumwild-type cells as screening system for virulence of extracellular and intracellular pathogens and their corresponding mutants, (ii) the use ofDictyosteliummutant cells to identify genetic host determinants of susceptibility and resistance to infection and (iii) the use of reporter systems inDictyosteliumcells which allow the dissection of the complex host-pathogen cross-talk. The body of information presented in this review demonstrates that the availability of host cell markers, the knowledge of cell signalling pathways, the completion of the genome sequencing project and the tractability for genetic studies qualifiesDictyosteliumfor the study of fundamental cellular processes of pathogenesis. [ABSTRACT FROM AUTHOR]

The gene encoding a major surface protein (MspTL) of Treponema lecithinolyticum, a periodontopathogen, was cloned and sequenced. The mspTL gene has a 1770-bp open reading frame (ORF) encoding a protein of 590 amino acids with a predicted molecular mass of 65 kDa which had a typical prokaryotic signal sequence (19 amino acids). MspTL showed a high level of homology with major sheath protein (MspA) of Treponema maltophilum, phylogenetically the closest relative of T. lecithinolyticum. Southern blot analysis indicated that the mspTL gene exists in a single copy and Northern blot analysis showed that the mspTL transcript is monocistronic. Another ORF located downstream of mspTL was in the same orientation and encoded a putative protein, in which the first N-terminal 291 amino acids were identified. The homologous region of this protein is also a part on the T. maltophilum mspA locus. [Copyright &y& Elsevier]

Reports on the cloning of a Treponema maltophilum flagellar gene cluster containing two flagellin subunit genes and the fliD gene. Aetiologic role of oral treponemes in the mixed bacterial infection periodontitis; Characterization of Treponema maltophilum flagellar gene regulation at the molecular level; Distribution and expression of flagellin genes in various treponeme species.