Intracellular Pathogen Francisella Tularensis Research Articles

A Novel Approach to Vaccine Development: Concomitant Pathogen Inactivation and Host Immune Stimulation by Peroxynitrite.

The design of efficient vaccines for long-term protective immunity against pathogens represents an objective of utmost public health priority. In general, live attenuated vaccines are considered to be more effective than inactivated pathogens, yet potentially more reactogenic. Accordingly, inactivation protocols which do not compromise the pathogen’s ability to elicit protective immunity are highly beneficial. One of the sentinel mechanisms of the host innate immune system relies on the production of reactive nitrogen intermediates (RNI), which efficiently inactivate pathogens. Peroxynitrite (PN) is a prevalent RNI, assembled spontaneously upon the interaction of nitric oxide (NO) with superoxide. PN exerts its bactericidal effect by via the efficient oxidation of a broad range of biological molecules. Furthermore, the interaction of PN with proteins results in structural/chemical modifications, such as the oxidation of tryptophan, tyrosine, and cysteine residues, as well as the formation of carbonyl, dityrosine, and nitrotyrosine (NT). In addition to their role in innate immunity, these PN-mediated modifications of pathogen components may also augment the antigenicity of pathogen peptides and proteins, hence contributing to specific humoral responses. In the study reported here, a novel approach for vaccine development, consisting of pathogen inactivation by PN, combined with increased immunity of NT-containing peptides, is implemented as a proof-of-concept for vaccination against the intracellular pathogen Francisella tularensis (F. tularensis). In vivo experiments in a murine model of tularemia confirm that PN-inactivated F. tularensis formulations may rapidly stimulate innate and adaptive immune cells, conferring efficient protection against a lethal challenge, superior to that elicited by bacteria inactivated by the widely used formalin treatment.

A Ribosomal Protein Homolog Governs Gene Expression and Virulence in a Bacterial Pathogen.

ABSTRACTThe molecular machine necessary for protein synthesis, the ribosome, is generally considered constitutively functioning and lacking any inherent regulatory capacity. Yet ribosomes are commonly heterogeneous in composition and the impact of ribosome heterogeneity on translation is not well understood. Here, we determined that changes in ribosome protein composition govern gene expression in the intracellular bacterial pathogen Francisella tularensis. F. tularensis encodes three distinct homologs for bS21, a ribosomal protein involved in translation initiation, and analysis of purified F. tularensis ribosomes revealed they are heterogeneous with respect to bS21. The loss of one homolog, bS21-2, resulted in significant changes to the cellular proteome unlinked to changes in the transcriptome. Among the reduced proteins were components of the type VI secretion system (T6SS), an essential virulence factor encoded by the Francisella Pathogenicity Island. Furthermore, loss of bS21-2 led to an intramacrophage growth defect. Although multiple bS21 homologs complemented the loss of bS21-2 with respect to T6SS protein abundance, bS21-2 was uniquely necessary for robust intramacrophage growth, suggesting bS21-2 modulates additional virulence gene(s) distinct from the T6SS. Our results indicate that ribosome composition in F. tularensis, either directly or indirectly, posttranscriptionally modulates gene expression and virulence. Our findings are consistent with a model in which bS21 homologs function as posttranscriptional regulators, allowing preferential translation of specific subsets of mRNAs, likely at the stage of translation initiation. This work also raises the possibility that bS21 in other organisms may function similarly and that ribosome heterogeneity may permit many bacteria to posttranscriptionally regulate gene expression.IMPORTANCE While bacterial ribosomes are commonly heterogeneous in composition (e.g., incorporating different homologs for a ribosomal protein), how heterogeneity impacts translation is unclear. We found that the intracellular human pathogen Francisella tularensis has heterogeneous ribosomes, incorporating one of three homologs for ribosomal protein bS21. Furthermore, one bS21 homolog posttranscriptionally governs the expression of the F. tularensis type VI secretion system, an essential virulence factor. This bS21 homolog is also uniquely important for robust intracellular growth. Our data support a model in which bS21 heterogeneity leads to modulation of translation, providing another source of posttranscriptional gene regulation. Regulation of translation by bS21, or other sources of ribosomal heterogeneity, may be a conserved mechanism to control gene expression across the bacterial phylogeny.

Neutrophil Survival Signaling During Francisella tularensis Infection.

Neutrophils are the most abundant and shortest-lived leukocytes in humans and tight regulation of neutrophil turnover via constitutive apoptosis is essential for control of infection and resolution of inflammation. Accordingly, aberrant neutrophil turnover is hallmark of many disease states. We have shown in previous work that the intracellular bacterial pathogen Francisella tularensis markedly prolongs human neutrophil lifespan. This is achieved, in part, by changes in neutrophil gene expression. Still unknown is the contribution of major neutrophil pro-survival signaling cascades to this process. The objective of this study was to interrogate the contributions of ERK and p38 MAP kinase, Class I phosphoinositide 3-kinases (PI3K), AKT, and NF-κB to neutrophil survival in our system. We demonstrate that both ERK2 and p38α were activated in F. tularensis-infected neutrophils, but only p38α MAPK was required for delayed apoptosis and the rate of cell death in the absence of infection was unchanged. Apoptosis of both infected and uninfected neutrophils was markedly accelerated by the pan-PI3K inhibitor LY2094002, but AKT phosphorylation was not induced, and neutrophil death was not enhanced by AKT inhibitors. In addition, isoform specific and selective inhibitors revealed a unique role for PI3Kα in neutrophil survival after infection, whereas only simultaneous inhibition of PI3Kα and PI3kδ accelerated death of the uninfected controls. Finally, we show that inhibition of NF-κB triggered rapid death of neutrophil after infection. Thus, we defined roles for p38α, PI3Kα and NF-κB delayed apoptosis of F. tularensis-infected cells and advanced understanding of Class IA PI3K isoform activity in human neutrophil survival.

The intracellular pathogen Francisella tularensis escapes from adaptive immunity by metabolic adaptation.

Intracellular pathogens lose many metabolic genes during their evolution from free-living bacteria, but the pathogenic consequences of their altered metabolic programs on host immunity are poorly understood. Here, we show that a pathogenic strain of Francisella tularensis subsp. tularensis (FT) has five amino acid substitutions in RibD, a converting enzyme of the riboflavin synthetic pathway responsible for generating metabolites recognized by mucosal-associated invariant T (MAIT) cells. Metabolites from a free-living strain, F. tularensis subsp. novicida (FN), activated MAIT cells in a T-cell receptor (TCR)-dependent manner, whereas introduction of FT-type ribD to the free-living strain was sufficient to attenuate this activation in both human and mouse MAIT cells. Intranasal infection in mice showed that the ribD FT-expressing FN strain induced impaired Th1-type MAIT cell expansion and resulted in reduced bacterial clearance and worsened survival compared with the wild-type free-living strain FN. These results demonstrate that F. tularensis can acquire immune evasion capacity by alteration of metabolic programs during evolution.

The Sensor Kinase QseC Regulates the Unlinked PmrA Response Regulator and Downstream Gene Expression in Francisella.

The facultative intracellular bacterial pathogen Francisella tularensis is the causative agent of tularemia in humans and animals. Gram-negative bacteria utilize two-component regulatory systems (TCS) to sense and respond to their changing environment. No classical, tandemly arranged sensor kinase and response regulator TCS genes exist in the human virulent Francisella tularensis subsp. tularensis, but orphaned members are present. PmrA is an orphan response regulator responsible for intramacrophage growth and virulence; however, the regulation of PmrA activity is not understood. We and others have shown that PmrA represses the expression of priM, described to encode an antivirulence determinant. By screening a mutant library for increased priM promoter activity, we identified the sensor kinase homolog QseC as an upstream regulator of priM expression, and this regulation is in part dependent upon the aspartate phosphorylation site of PmrA (D51). Several examined environmental signals, including epinephrine, which is reported to activate QseC in other bacteria, do not affect priM expression in a manner dependent on PmrA. Intramacrophage survival assays also question the finding that PriM is an antivirulence factor. Thus, these data suggest that the PmrA-regulated gene priM is modulated by the QseC-PmrA (QseB) TCS in FrancisellaIMPORTANCE The disease tularemia is caused by the highly infectious Gram-negative pathogen Francisella tularensis This bacterium encodes few regulatory factors (e.g., two-component systems [TCS]). PmrA, required for intramacrophage survival and virulence in the mouse model, is encoded by an orphan TCS response regulator gene. It is unclear how PmrA is responsive to environmental signals to regulate loci, including the PmrA-repressed gene priM We identify an orphan sensor kinase (QseC) that is required for priM repression and further explore both environmental signals that might regulate the QseC-PmrA TCS and the function of PriM.

Tn-Seq reveals hidden complexity in the utilization of host-derived glutathionein Francisellatularensis.

Host-derived glutathione (GSH) is an essential source of cysteine for the intracellular pathogen Francisella tularensis. In a comprehensive transposon insertion sequencing screen, we identified several F. tularensis genes that play central and previously unappreciated roles in the utilization of GSH during the growth of the bacterium in macrophages. We show that one of these, a gene we named dptA, encodes a proton-dependent oligopeptide transporter that enables growth of the organism on the dipeptide Cys-Gly, a key breakdown product of GSH generated by the enzyme γ-glutamyltranspeptidase (GGT). Although GGT was thought to be the principal enzyme involved in GSH breakdown in F. tularensis, our screen identified a second enzyme, referred to as ChaC, that is also involved in the utilization of exogenous GSH. However, unlike GGT and DptA, we show that the importance of ChaC in supporting intramacrophage growth extends beyond cysteine acquisition. Taken together, our findings provide a compendium of F. tularensis genes required for intracellular growth and identify new players in the metabolism of GSH that could be attractive targets for therapeutic intervention.

Formation of the Francisella tularensis Biofilm is Affected by Cell Surface Glycosylation, Growth Medium, and a Glucan Exopolysaccharide

Biofilms are matrix-associated communities that enable bacteria to colonise environments unsuitable for free-living bacteria. The facultative intracellular pathogen Francisella tularensis can persist in water, amoebae, and arthropods, as well as within mammalian macrophages. F. tularensis Types A and B form poor biofilms, but F. tularensis mutants lacking lipopolysaccharide O-antigen, O-antigen capsule, and capsule-like complex formed up to 15-fold more biofilm than fully glycosylated cells. The Type B live vaccine strain was also 50% less capable of initiating surface attachment than mutants deficient in O-antigen and capsule-like complex. However, the growth medium of all strains tested also influenced the formation of biofilm, which contained a novel exopolysaccharide consisting of an amylose-like glucan. In addition, the surface polysaccharide composition of the bacterium affected the protein:DNA:polysaccharide composition of the biofilm matrix. In contrast, F. novicida attached to surfaces more efficiently and made a more robust biofilm than Type A or B strains, but loss of O-antigen or capsule-like complex did not significantly affect F. novicida biofilm formation. These results indicated that suppression of surface polysaccharides may promote biofilm formation by F. tularensis Types A and B. Whether biofilm formation enhances survival of F. tularensis in aquatic or other environmental niches has yet to be determined.

Innate Immune Recognition: Implications for the Interaction of Francisella tularensis with the Host Immune System.

The intracellular bacterial pathogen Francisella tularensis causes serious infectious disease in humans and animals. Moreover, F. tularensis, a highly infectious pathogen, poses a major concern for the public as a bacterium classified under Category A of bioterrorism agents. Unfortunately, research has so far failed to develop effective vaccines, due in part to the fact that the pathogenesis of intracellular bacteria is not fully understood and in part to gaps in our understanding of innate immune recognition processes leading to the induction of adaptive immune response. Recent evidence supports the concept that immune response to external stimuli in the form of bacteria is guided by the primary interaction of the bacterium with the host cell. Based on data from different Francisella models, we present here the basic paradigms of the emerging innate immune recognition concept. According to this concept, the type of cell and its receptor(s) that initially interact with the target constitute the first signaling window; the signals produced in the course of primary interaction of the target with a reacting cell act in a paracrine manner; and the innate immune recognition process as a whole consists in a series of signaling windows modulating adaptive immune response. Finally, the host, in the strict sense, is the interacting cell.

Secreted Effectors Encoded within and outside of the Francisella Pathogenicity Island Promote Intramacrophage Growth.

The intracellular bacterial pathogen Francisella tularensis causes tularemia, a zoonosis that can be fatal. The type VI secretion system (T6SS) encoded by the Francisella pathogenicity island (FPI) is critical for the virulence of this organism. Existing studies suggest that the complete repertoire of T6SS effectors delivered to host cells is encoded by the FPI. Using a proteome-wide approach, we discovered that the FPI-encoded T6SS exports at least three effectors encoded outside of the island. These proteins share features with virulence determinants of other pathogens, and we provide evidence that they can contribute to intramacrophage growth. The remaining proteins that we identified are encoded within the FPI. Two of these FPI-encoded proteins constitute effectors, whereas the others form a unique complex required for core function of the T6SS apparatus. The discovery of secreted effectors mediating interactions between Francisella and its host significantly advances our understanding of the pathogenesis of this organism.

Early Interactions of Murine Macrophages with Francisella tularensis Map to Mouse Chromosome 19.

ABSTRACTDifferences among individuals in susceptibility to infectious diseases can be modulated by host genetics. Much of the research in this field has aimed to identify loci within the host genome that are associated with these differences. In mice, A/J (AJ) and C57BL/6J (B6) mice show differential susceptibilities to various pathogens, including the intracellular pathogen Francisella tularensis. Because macrophages are the main initial target during F. tularensis infection, we explored early interactions of macrophages from these two mouse strains with F. tularensis as well as the genetic factors underlying these interactions. Our results indicate that bacterial interactions with bone marrow-derived macrophages (BMDMs) during early stages of infection are different in the AJ and B6 strains. During these early stages, bacteria are more numerous in B6 than in AJ macrophages and display differences in trafficking and early transcriptional response within these macrophages. To determine the genetic basis for these differences, we infected BMDMs isolated from recombinant inbred (RI) mice derived from reciprocal crosses between AJ and B6, and we followed early bacterial counts within these macrophages. Quantitative trait locus (QTL) analysis revealed a locus on chromosome 19 that is associated with early differences in bacterial counts in AJ versus B6 macrophages. QTL analysis of published data that measured the differential susceptibilities of the same RI mice to an in vivo challenge with F. tularensis confirmed the F. tularensis susceptibility QTL on chromosome 19. Overall, our results show that early interactions of macrophages with F. tularensis are dependent on the macrophage genetic background.

Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis.

The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an anti-bacterial target. In the intracellular pathogen Francisella tularensis, the product of the gene FTT1564 has been identified as a polyphosphate kinase from the polyphosphate kinase 2 (PPK2) family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella. Herein, we report the biochemical and structural characterization of F. tularensis polyphosphate kinase (FtPPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC-based activity assay, the substrate specificity of FtPPK2 was found to include purine but not pyrimidine nts. The activity was also measured using (31)P-NMR. FtPPK2 has been crystallized and the structure determined to 2.23 Å (1 Å=0.1nm) resolution. The structure consists of a six-stranded parallel β-sheet surrounded by 12 α-helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ΔFTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors.

Identification of disulfide bond isomerase substrates reveals bacterial virulence factors.

Bacterial pathogens are exposed to toxic molecules inside the host and require efficient systems to form and maintain correct disulfide bonds for protein stability and function. The intracellular pathogen Francisella tularensis encodes a disulfide bond formation protein ortholog, DsbA, which previously was reported to be required for infection of macrophages and mice. However, the molecular mechanisms by which F. tularensis DsbA contributes to virulence are unknown. Here, we demonstrate that F. tularensis DsbA is a bifunctional protein that oxidizes and, more importantly, isomerizes complex disulfide connectivity in substrates. A single amino acid in the conserved cis-proline loop of the DsbA thioredoxin domain was shown to modulate both isomerase activity and F. tularensis virulence. Trapping experiments in F. tularensis identified over 50 F. tularensis DsbA substrates, including outer membrane proteins, virulence factors, and many hypothetical proteins. Six of these hypothetical proteins were randomly selected and deleted, revealing two novel proteins, FTL_1548 and FTL_1709, which are required for F. tularensis virulence. We propose that the extreme virulence of F. tularensis is partially due to the bifunctional nature of DsbA, that many of the newly identified substrates are required for virulence, and that the development of future DsbA inhibitors could have broad anti-bacterial implications.

Uncovering the components of the Francisella tularensis virulence stealth strategy

Over the last decade, studies on the virulence of the highly pathogenic intracellular bacterial pathogen Francisella tularensis have increased dramatically. The organism produces an inert LPS, a capsule, escapes the phagosome to grow in the cytosol (FPI genes mediate phagosomal escape) of a variety of host cell types that include epithelial, endothelial, dendritic, macrophage, and neutrophil. This review focuses on the work that has identified and characterized individual virulence factors of this organism and we hope to highlight how these factors collectively function to produce the pathogenic strategy of this pathogen. In addition, several recent studies have been published characterizing F. tularensis mutants that induce host immune responses not observed in wild type F. tularensis strains that can induce protection against challenge with virulent F. tularensis. As more detailed studies with attenuated strains are performed, it will be possible to see how host models develop acquired immunity to Francisella. Collectively, detailed insights into the mechanisms of virulence of this pathogen are emerging that will allow the design of anti-infective strategies.

Protective Immunity against Lethal F. tularensis holarctica LVS Provided by Vaccination with Selected Novel CD8+ T Cell Epitopes

Recently we described an unbiased bacterial whole-genome immunoinformatic analysis aimed at selection of potential CTL epitopes located in “hotspots” of predicted MHC-I binders. Applying this approach to the proteome of the facultative intra-cellular pathogen Francisella tularensis resulted in identification of 170 novel CTL epitopes, several of which were shown to elicit highly robust T cell responses. Here we demonstrate that by DNA immunization using a short DNA fragment expressing six of the most prominent identified CTL epitopes a potent and specific CD8+ T cell responses is being induced, to all encoded epitopes, a response not observed in control mice immunized with the DNA vector alone Moreover, this CTL-specific mediated immune response prevented disease development, allowed for a rapid clearance of the bacterial infection and provided complete protection against lethal challenge (10LD50) with F. tularensis holarctica Live Vaccine Strain (LVS) (a total to 30 of 30 immunized mice survived the challenge while all control DNA vector immunized mice succumbed). Furthermore, and in accordance with these results, CD8 deficient mice could not be protected from lethal challenge after immunization with the CTL-polyepitope. Vaccination with the DNA poly-epitope construct could even protect mice (8/10) against the more demanding pulmonary lethal challenge of LVS. Our approach provides a proof-of-principle for selecting and generating a multi-epitpoe CD8 T cell-stimulating vaccine against a model intracellular bacterium.

The ability of CpG oligonucleotides to protect mice against Francisella tularensis live vaccine strain but not fully virulent F. tularensis subspecies holarctica is reflected in cell-based assays

CpG DNA is a potent activator of the innate immune system. Here the protective effects of CpG DNA are assessed against the facultative intracellular pathogen Francisella tularensis. Dosing of mice with CpG DNA provided protection against disease caused by F. tularensis subsp. holarctica live vaccine strain (LVS) but did not protect against the fully virulent F. tularensis subsp holarctica strain HN63. Similarly, in vitro studies in J774A murine macrophage-like cells demonstrated that stimulation with CpG DNA enables control of intracellular replication of LVS but not HN63. These data confirm findings that CpG DNA may have limited efficacy in providing protection against fully virulent strains of F. tularensis and also suggest that in vitro assays may be useful for the evaluation of novel treatments for virulent F. tularensis.

B cells participate on the early stages of immune response against intracellular bacterial pathogen Francisella tularensis

Event Abstract Back to Event B cells participate on the early stages of immune response against intracellular bacterial pathogen Francisella tularensis Zuzana Krocova1*, Klara Kubelkova1, Lenka Plzakova1, Lenka Zarybnicka1, Zuzana Sinkorova1 and Ales Macela1 1 Faculty of Military Health Sciences, University of Defense, Inst Molec Pathol, Czechia Francisella tularensis, as an intracellular bacterial pathogen, adhere, interact, and enter the range of phagocytic and non-phagocytic cells. Recently, we have demonstrated, using in vitro model, that both, vaccine strain F. tularensis LVS and virulent F. tularensis strain FSC200 are able to adhere and even to enter the human (Ramos) and mouse (A20) B cells where survive in non-replicative state. The entrance of F. tularensis into B cells required active participation of bacterium and engagement of B cell receptor. The in vivo analysis of the cellular response during early stages of F. tularensis FSC200 i.p. infection on murine model demonstrated that the cells responding early are the B1b cells and macrophages expressing CD11c marker. In parallel the monocyte-derived CD123+ F4/80+ dendritic cells started to express CD11b and disappeared from the site of infection. During this stage of infection F. tularensis induces increased expression of MHC class II molecules on B1a and B1b cells in peritoneum and activates genes for IL-12, TNF alpha, TGF beta, IFN-gamma, and iNOS at splenic B cells. Twenty-four hours after infection murine splenic B cells produce the specific anti-F. tularensis antibodies. Moreover, the co-cultivation of A20 B cells with the F. tularensis virulent strain FSC200 rendered them to be potent antigen presenting cells presenting F. tularensis antigens. Thus, considering the protective efficacy of circulating antibodies, production of cytokines, and potent antigen-presenting function of B cells, B cell-mediated, as well as T cell-mediated immunity plays an equivalent role in control of F. tularensis infection in mice. Acknowledgements Acknowledgement: The authors are supported by the grant from Grant Agency of Czech Republic No. P302/11/1631 References Crystal L. Jones, B. A. Napier, T. R. Sampson,a, A. C. Llewellyn,, M. R. Schroeder, and D. S. Weissb,c: Subversion of Host Recognition and Defense Systems by Francisella spp. Microbiol Mol Biol Rev. 2012 June; 76(2): 383–404. Krocova Z, Härtlova A, Souckova D, Zivna L, Kroca M, Rudolf E, Macela A, Stulik J. Interaction of B cells with intracellular pathogen Francisella tularensis. Microb Pathog. 2008 Aug;45(2):79-85. Kubelkova K, Krocova Z, Balonova L, Pejchal J, Stulik J, Macela A.: Specific antibodies protect gamma-irradiated mice against Francisella tularensis infection. Microb Pathog. 2012 Nov-Dec;53(5-6):259-68. doi: 10.1016/j.micpath.2012.07.006. Epub 2012 Jul 25. Yang Y, Ghosn EE, Cole LE, Obukhanych TV, Sadate-Ngatchou P, Vogel SN, Herzenberg LA, Herzenberg LA.: Antigen-specific antibody responses in B-1a and their relationship to natural immunity. Proc Natl Acad Sci U S A. 2012 Apr 3;109(14):5382-7. Keywords: B cells, Francisella tularensis, innate immunity, Antigen Presentation, Cytokines Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Krocova Z, Kubelkova K, Plzakova L, Zarybnicka L, Sinkorova Z and Macela A (2013). B cells participate on the early stages of immune response against intracellular bacterial pathogen Francisella tularensis. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00657 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Zuzana Krocova, Faculty of Military Health Sciences, University of Defense, Inst Molec Pathol, Hradec Kralove, 500 01, Czechia, zuzana.krocova@unob.cz Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Zuzana Krocova Klara Kubelkova Lenka Plzakova Lenka Zarybnicka Zuzana Sinkorova Ales Macela Google Zuzana Krocova Klara Kubelkova Lenka Plzakova Lenka Zarybnicka Zuzana Sinkorova Ales Macela Google Scholar Zuzana Krocova Klara Kubelkova Lenka Plzakova Lenka Zarybnicka Zuzana Sinkorova Ales Macela PubMed Zuzana Krocova Klara Kubelkova Lenka Plzakova Lenka Zarybnicka Zuzana Sinkorova Ales Macela Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A mathematical model of CR3/TLR2 crosstalk in the context of Francisella tularensis infection.

Complement Receptor 3 (CR3) and Toll-like Receptor 2 (TLR2) are pattern recognition receptors expressed on the surface of human macrophages. Although these receptors are essential components for recognition by the innate immune system, pathogen coordinated crosstalk between them can suppress the production of protective cytokines and promote infection. Recognition of the virulent Schu S4 strain of the intracellular pathogen Francisella tularensis by host macrophages involves CR3/TLR2 crosstalk. Although experimental data provide evidence that Lyn kinase and PI3K are essential components of the CR3 pathway that influences TLR2 activity, additional responsible upstream signaling components remain unknown. In this paper we construct a mathematical model of CR3 and TLR2 signaling in response to F. tularensis. After demonstrating that the model is consistent with experimental results we perform numerical simulations to evaluate the contributions that Akt and Ras-GAP make to ERK inhibition. The model confirms that phagocytosis-associated changes in the composition of the cell membrane can inhibit ERK activity and predicts that Akt and Ras-GAP synergize to inhibit ERK.

Infection of Mice with Francisella as an Immunological Model

This unit describes the utility of various mouse models of infection for studying pathogenesis and adaptive immune responses to the facultative intracellular bacteria pathogen Francisella tularensis. By judicious use of different combinations of mouse and bacterial strains, as well as different routes of infection, murine tularemia models may be used to explore a complete picture of F. tularensis infection and immunity. Moreover, studies using Francisella, particularly the Live Vaccine Strain (LVS), serve as a convenient and tractable model system that appears to be representative of mammalian host responses to intracellular pathogens in general.

Immunity to Francisella

In recent years, studies on the intracellular pathogen Francisella tularensis have greatly intensified, generating a wealth of new information on the interaction of this organism with the immune system. Here we review the basic elements of the innate and adaptive immune responses that contribute to protective immunity against Francisella species, with special emphasis on new data that has emerged in the last 5 years. Most studies have utilized the mouse model of infection, although there has been an expansion of work on human cells and other new animal models. In mice, basic immune parameters that operate in defense against other intracellular pathogen infections, such as interferon gamma, TNF-α, and reactive nitrogen intermediates, are central for control of Francisella infection. However, new important immune mediators have been revealed, including IL-17A, Toll-like receptor 2, and the inflammasome. Further, a variety of cell types in addition to macrophages are now recognized to support Francisella growth, including epithelial cells and dendritic cells. CD4+ and CD8+ T cells are clearly important for control of primary infection and vaccine-induced protection, but new T cell subpopulations and the mechanisms employed by T cells are only beginning to be defined. A significant role for B cells and specific antibodies has been established, although their contribution varies greatly between bacterial strains of lower and higher virulence. Overall, recent data profile a pathogen that is adept at subverting host immune responses, but susceptible to many elements of the immune system's antimicrobial arsenal.

Cutting Edge: Mutation of Francisella tularensis mviN Leads to Increased Macrophage Absent in Melanoma 2 Inflammasome Activation and a Loss of Virulence

The mechanisms by which the intracellular pathogen Francisella tularensis evades innate immunity are not well defined. We have identified a gene with homology to Escherichia coli mviN, a putative lipid II flippase, which F. tularensis uses to evade activation of innate immune pathways. Infection of mice with a F. tularensis mviN mutant resulted in improved survival and decreased bacterial burdens compared to infection with wild-type F. tularensis. The mviN mutant also induced increased absent in melanoma 2 inflammasome-dependent IL-1beta secretion and cytotoxicity in macrophages. The compromised in vivo virulence of the mviN mutant depended upon inflammasome activation, as caspase 1- and apoptosis-associated speck-like protein containing a caspase recruitment domain-deficient mice did not exhibit preferential survival following infection. This study demonstrates that mviN limits F. tularensis-induced absent in melanoma 2 inflammasome activation, which is critical for its virulence in vivo.