Kdo Hydrolase Is Required for Francisella tularensis Virulence and Evasion of TLR2-Mediated Innate Immunity

ABSTRACT The highly virulent Francisella tularensis subsp. tularensis has been classified as a category A bioterrorism agent. A live vaccine strain (LVS) has been developed but remains unlicensed in the United States because of an incomplete understanding of its attenuation. Lipopolysaccharide (LPS) modification is a common strategy employed by bacterial pathogens to avoid innate immunity. A novel modification enzyme has recently been identified in F. tularensis and Helicobacter pylori. This enzyme, a two-component Kdo (3-deoxy-d-manno-octulosonic acid) hydrolase, catalyzes the removal of a side chain Kdo sugar from LPS precursors. The biological significance of this modification has not yet been studied. To address the role of the two-component Kdo hydrolase KdhAB in F. tularensis pathogenesis, a ΔkdhAB deletion mutant was constructed from the LVS strain. In intranasal infection of mice, the ΔkdhAB mutant strain had a 50% lethal dose (LD50) 2 log10 units higher than that of the parental LVS strain. The levels of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) in bronchoalveolar lavage fluid were significantly higher (2-fold) in mice infected with the ΔkdhAB mutant than in mice infected with LVS. In vitro stimulation of bone marrow-derived macrophages with the ΔkdhAB mutant induced higher levels of TNF-α and IL-1β in a TLR2-dependent manner. In addition, TLR2−/− mice were more susceptible than wild-type mice to ΔkdhAB bacterial infection. Finally, immunization of mice with ΔkdhAB bacteria elicited a high level of protection against the highly virulent F. tularensis subsp. tularensis strain Schu S4. These findings suggest an important role for the Francisella Kdo hydrolase system in virulence and offer a novel mutant as a candidate vaccine. IMPORTANCE The first line of defense against a bacterial pathogen is innate immunity, which slows the progress of infection and allows time for adaptive immunity to develop. Some bacterial pathogens, such as Francisella tularensis, suppress the early innate immune response, killing the host before adaptive immunity can mature. To avoid an innate immune response, F. tularensis enzymatically modifies its lipopolysaccharide (LPS). A novel LPS modification—Kdo (3-deoxy-d-manno-octulosonic acid) saccharide removal—has recently been reported in F. tularensis. We found that the ∆kdhAB mutant was significantly attenuated in mice. Additionally, the mutant strain induced an early innate immune response in mice both in vitro and in vivo. Immunization of mice with this mutant provided protection against the highly virulent F. tularensis strain Schu S4. Thus, our study has identified a novel LPS modification important for microbial virulence. A mutant lacking this modification may be used as a live attenuated vaccine against tularemia.

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Ursodeoxycholic Acid (UDCA) Mitigates the Host Inflammatory Response during Clostridioides difficile Infection by Altering Gut Bile Acids

Infection and Immunity ◽

10.1128/iai.00045-20 ◽

2020 ◽

Vol 88 (6) ◽

Author(s):

Jenessa A. Winston ◽

Alissa J. Rivera ◽

Jingwei Cai ◽

Rajani Thanissery ◽

Stephanie A. Montgomery ◽

...

Keyword(s):

Immune Response ◽

Bile Acid ◽

Inflammatory Response ◽

Innate Immune Response ◽

Innate Immune ◽

Colonization Resistance ◽

Content Type ◽

Clostridioides Difficile

ABSTRACT Clostridioides difficile infection (CDI) is associated with increasing morbidity and mortality posing an urgent threat to public health. Recurrence of CDI after successful treatment with antibiotics is high, thus necessitating discovery of novel therapeutics against this enteric pathogen. Administration of the secondary bile acid ursodeoxycholic acid (UDCA; ursodiol) inhibits the life cycles of various strains of C. difficile in vitro, suggesting that the FDA-approved formulation of UDCA, known as ursodiol, may be able to restore colonization resistance against C. difficile in vivo. However, the mechanism(s) by which ursodiol is able to restore colonization resistance against C. difficile remains unknown. Here, we confirmed that ursodiol inhibits C. difficile R20291 spore germination and outgrowth, growth, and toxin activity in a dose-dependent manner in vitro. In a murine model of CDI, exogenous administration of ursodiol resulted in significant alterations in the bile acid metabolome with little to no changes in gut microbial community structure. Ursodiol pretreatment resulted in attenuation of CDI pathogenesis early in the course of disease, which coincided with alterations in the cecal and colonic inflammatory transcriptome, bile acid-activated receptors nuclear farnesoid X receptor (FXR) and transmembrane G-protein-coupled membrane receptor 5 (TGR5), which are able to modulate the innate immune response through signaling pathways such as NF-κB. Although ursodiol pretreatment did not result in a consistent decrease in the C. difficile life cycle in vivo, it was able to attenuate an overly robust inflammatory response that is detrimental to the host during CDI. Ursodiol remains a viable nonantibiotic treatment and/or prevention strategy against CDI. Likewise, modulation of the host innate immune response via bile acid-activated receptors FXR and TGR5 represents a new potential treatment strategy for patients with CDI.

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A newly established in vitro culture using transgenic Drosophila reveals functional coupling between the phospholipase A2-generated fatty acid cascade and lipopolysaccharide-dependent activation of the immune deficiency (imd) pathway in insect immunity

Biochemical Journal ◽

10.1042/bj20021603 ◽

2003 ◽

Vol 371 (1) ◽

pp. 205-210 ◽

Cited By ~ 69

Author(s):

Masashi YAJIMA ◽

Masatoshi TAKADA ◽

Nahoko TAKAHASHI ◽

Haruhisa KIKUCHI ◽

Shunji NATORI ◽

...

Keyword(s):

Immune Deficiency ◽

Immune Response ◽

Innate Immunity ◽

Arachidonic Acid ◽

Fatty Acid ◽

Innate Immune Response ◽

Innate Immune ◽

Insect Immunity ◽

Imd Pathway

Innate immunity is the first line of defence against infectious micro-organisms, and the basic mechanisms of pathogen recognition and response activation are evolutionarily conserved. In mammals, the innate immune response in combination with antigen-specific recognition is required for the activation of adaptive immunity. Therefore, innate immunity is a pharmaceutical target for the development of immune regulators. Here, for the purpose of pharmaceutical screening, we established an in vitro culture based on the innate immune response of Drosophila. The in vitro system is capable of measuring lipopolysaccharide (LPS)-dependent activation of the immune deficiency (imd) pathway, which is similar to the tumour necrosis factor signalling pathway in mammals. Screening revealed that well-known inhibitors of phospholipase A2 (PLA2), dexamethasone (Dex) and p-bromophenacyl bromide (BPB) inhibit LPS-dependent activation of the imd pathway. The inhibitory effects of Dex and BPB were suppressed by the addition of an excess of three (arachidonic acid, eicosapentaenoic acid and γ-linolenic acid) of the fatty acids so far tested. Arachidonic acid, however, did not activate the imd pathway when used as the sole agonist. These findings indicate that PLA2 participates in LPS-dependent activation of the imd pathway via the generation of arachidonic acid and other mediators, but requires additional signalling from LPS stimulation. Moreover, PLA2 was activated in response to bacterial infection in Sarcophaga. These results suggest a functional link between the PLA2-generated fatty acid cascade and the LPS-stimulated imd pathway in insect immunity.

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Effects of Dietary Maltol on Innate Immunity, Gut Health, and Growth Performance of Broiler Chickens Challenged With Eimeria maxima

Frontiers in Veterinary Science ◽

10.3389/fvets.2021.667425 ◽

2021 ◽

Vol 8 ◽

Author(s):

Inkyung Park ◽

Doyun Goo ◽

Hyoyoun Nam ◽

Samiru S. Wickramasuriya ◽

Kichoon Lee ◽

...

Keyword(s):

Immune Response ◽

Innate Immunity ◽

Growth Performance ◽

Innate Immune Response ◽

Broiler Chickens ◽

Muscle Cells ◽

Innate Immune ◽

Gut Health ◽

Eimeria Maxima

Two studies were conducted to evaluate the effects of maltol as a postbiotic on innate immunity, gut health, and enteric infection. In the first study, an in vitro culture system was used to evaluate the effects of maltol on the innate immune response of chicken macrophage cells (CMC), gut integrity of chicken intestinal epithelial cells (IEC), anti-parasitic activity against Eimeria maxima, and differentiation of quail muscle cells (QMC) and primary chicken embryonic muscle cells (PMC). All cells seeded in the 24-well plates were treated with maltol at concentrations of 0.1, 1.0, and 10.0 μg. CMC and IEC were stimulated by lipopolysaccharide to induce an innate immune response, and QMC and PMC were treated with 0.5 and 2% fetal bovine serum, respectively. After 18 h of incubation, pro-inflammatory cytokines, tight junction proteins (TJPs), and muscle cell growth markers were measured. In the second study, the dietary effect of maltol was evaluated on disease parameters in broiler chickens infected with E. maxima. Eighty male 1-day-old broiler chickens were allocated into the following four treatment groups: (1) Control group without infection, (2) Basal diet with E. maxima, (3) High maltol (HI; 10.0 mg /kg feed) with E. maxima, and (4) Low maltol (LO; 1.0 mg/kg feed) with E. maxima. Body weights (BW) were measured on days 0, 7, 14, 20, and 22. All chickens except the CON group were orally infected with 104E. maxima per chicken on day 14. Jejunum samples were collected for gut lesion scoring, and the gene expression of cytokines and TJPs. Data was analyzed using PROC MIXED in SAS. In vitro, maltol not only increased TJPs in IEC and cytokines in the LPS-stimulated CMC but also showed direct cytotoxicity against sporozoites of E. maxima. In vivo, the HI group improved the BW, reduced the gut lesion scores and fecal oocyst shedding, and decreased jejunal TNFSF15 and IL-1β expression in E. maxima-infected chickens. In conclusion, these results demonstrate the beneficial effects of dietary maltol in the enhancement of growth performance, gut health, and coccidiosis resistance and the applicability of maltol as a postbiotic for the replacement of antibiotic growth promoters in commercial poultry production.

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DIFFERENCES IN THE EXPRESSION OF INNATE IMMUNE RESPONSE-MODULATING GENES IN BLOOD MONOCYTES BETWEEN SUBCLINICALLY PORCINE CIRCOVIRUS TYPE 2 (PCV2)-INFECTED AND PCV2-FREE PIGS PRIOR TO AND AFTER LIPOPOLYSACCHARIDE STIMULATIONIN VITRO

Taiwan Veterinary Journal ◽

10.1142/s168264851450005x ◽

2014 ◽

Vol 40 (01) ◽

pp. 37-48

Author(s):

Yi-Chieh Tsai ◽

Chian-Ren Jeng ◽

Chih-Cheng Chang ◽

Shih-Hsuan Hsiao ◽

Hui-Wen Chang ◽

...

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Secondary Infection ◽

Porcine Circovirus Type ◽

Innate Immune ◽

Porcine Circovirus ◽

Blood Monocytes ◽

Expression Levels ◽

Tnf Α

The objective of the present study was to characterize and compare the differences in gene expression associated with innate immune response of blood monocytes (Mos) between healthy subclinically PCV2-infected and PCV2-free pigs prior to and after lipopolysaccharide (LPS) stimulation in vitro by relative quantitative real-time PCR (q-rt-PCR). Genes coding for 24 innate molecules, including toll-like receptors (TLRs), interferon-regulatory factors (IRFs), nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) and pro-inflammatory cytokines were evaluated. When compared with PCV2-free pigs, Mos from subclinically PCV2-infected pigs showed significantly lower mRNA expression levels in TLR-9, IRF-3, IRF-6, IRF-7, IL-6, IL-12p35, IL-12p40 and IFN-α under no further stimulation. Following LPS stimulation in vitro, a broad and/or obvious reduction in TLRs, IRFs, IL-12 and IFN-α along with increase in IL-1α, IL-6, IL-8, IL-10 and/or TNF-α were seen in both PCV2-free and subclinically PCV2-infected pigs; when compared with PCV2-free pigs, the subclinically PCV2-infected pigs had significantly higher expression levels in TLR-10 and IRF-1 but lower expression levels in IRF-6, IL-1α and IL-12p40. On the contrary, the expression level of NF-κB was consistently higher in subclinically PCV2-infected pigs than in PCV2-free pigs with or without LPS stimulation. The changes seen in the present study suggest that the subclinically PCV2-infected pigs may look healthy clinically, but their innate immunity has become dis-regulated or is in an improper status. The adverse condition may become even worse when exposed to certain bacterial products such as endotoxin. Such alterations in the innate immune system may make the subclinically PCV2-infected pigs more vulnerable to the secondary infection and subsequent PCV2-associated disease development.

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A Role for NFAT in Innate Immunity: Neutrophil Effector Functions in Patients after Allogeneic Hematopoietic Stem Cell Transplantation Under Cyclosporine Α Treatment

Blood ◽

10.1182/blood.v124.21.2495.2495 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2495-2495

Author(s):

Katharina Plein ◽

Daniel Teschner ◽

Christian Michel ◽

Eva-Maria Wagner ◽

Pamela Stein ◽

...

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Inflammatory Mediators ◽

Ex Vivo ◽

Innate Immune ◽

Hematopoietic Stem ◽

Effector Functions ◽

Healthy Donors ◽

Tnf Α

Abstract Background and Aims: Patients after allogeneic hematopoietic stem cell transplantation (HSCT) suffer from immunodeficiency, in part due to long-term immunosuppressive medication e.g. by calcineurin inhibitors like cyclosporine A (CsA). Additionally, these patients have an increased risk for opportunistic fungal infections like invasive aspergillosis (IA). The nuclear factor of activated T cells (NFAT) is known as an important transcription factor in signaling-pathways downstream of calcineurin in the adaptive immune systems, e.g. in T cells, but also plays an important role in innate immune response as indicated by recent data in rodent models. These studies showed a relevant impact of NFAT-/calcineurin inhibition on the innate immune response by polymorphonuclear neutrophils (PMN) in a candida sepsis model. To clarify whether this is also relevant in humans, we investigated the role of NFAT signaling pathways in PMN activation and effector functions in healthy volunteer donors and patients under immunosuppressive treatment with CsA after HSCT. Methods: Firstly, we performed in vitro experiments using PMN from healthy donors analyzing their effector functions in absence or presence of CsA in titrated doses according to therapeutic levels. In detail, we examined phagocytosis, activation, generation of reactive oxygen species (ROS) and release of inflammatory mediators like IL-8 and TNF-α. After activation with lipopolysaccharide (LPS) and zymosan, phagocytosis and activation-induced shedding of CD62L were measured by flow cytometry using polychromatic microspheres and matching surface markers (CD11b, CD62L, CD66b). In addition, generation of ROS was analyzed by dichlorofluorescein assay (DCF), whereas activation-induced release of inflammatory mediators was measured by enzyme-linked immunosorbent assay (ELISA) and intracellular flow cytometry. In addition, blood samples of patients after HSCT under continuous CsA medication (n=6) and healthy volunteer donors (n=6) were analyzed ex vivo at two different time points after allogeneic HSCT (day 25-35 and 125-135) concerning their PMN effector functions in the same manner as described above. Results: Analysis of healthy donors PMN in vitro showed that CsA had no significant influence on expression of activation markers and shedding of CD62L. Moreover, no substantial influence of CsA on generation of ROS was detected compared to untreated controls (5245 RFU +/- 354 (CsA) vs. 5763 +/- 520 (control) after stimulation with LPS, mean +/- SEM). Furthermore, activation-induced synthesis of IL-8 was not reduced in presence of CsA (519pg/ml +/- 81 vs. 463 +/- 131 (control) after stimulation with LPS). In contrast, CsA rather enhanced phagocytosis after stimulation with LPS (83.5% +/- 1.7 vs. 71.0 +/- 1.5 (control)). Regarding the ex vivo analysis of HSCT patient and healthy donor blood samples, production of ROS was not affected under CsA therapy (35.1% +/- 9.4 vs. 31.1 +/- 6.6 (control) after stimulation with zymosan). Furthermore, CsA medication showed a stimulating effect on PMN phagocytosis which is in line with our in vitro data (58.4% +/- 7.1 vs. 44.3 +/- 2.3 (control) after stimulation with LPS). Interestingly, in several patients an increased production of IL-8 and TNF-α was detectable after stimulation with zymosan (IL-8: 9.1% +/- 3.6 vs. 1.5 +/- 0.3 (control); TNF-α: 16.6% +/- 6.2 vs. 1.0 +/- 0.4 (control)). Conclusions: In contrast to previous results by others in murine model systems, we found an increased phagocytic activity in vitro and ex vivo in human PMN upon NFAT/calcineurin inhibition, whereas other effector mechanisms were unaffected. In addition, HSCT patients under CsA treatment displayed enhanced inflammatory mediators production in PMN. It is currently unclear whether these findings are clinically relevant for the innate immune response after HSCT, e. g. in terms of anti-fungal immunity. Further studies are needed to address whether these enhanced PMN functions in the presence of CsA contribute to a dysregulated innate immune response in humans. Alternatively, our discrepant results may be due to differences in PMN functionality and CsA responsiveness in mice and man. However, while CsA strongly suppresses adaptive immune responses, i.e. T-cell responses in graft-versus-host disease (GvHD), our results so far suggest that CsA does not affect innate immune effector functions by humans PMN in a comparable way. Disclosures Radsak: Celgene: Research Funding.

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High content screening and computational prediction reveal viral genes that suppress innate immune response

10.1101/2021.12.14.472572 ◽

2021 ◽

Author(s):

Tai L Ng ◽

Erika J Olson ◽

Tae Yeon Yoo ◽

H. Sloane Weiss ◽

Yukiye Koide ◽

...

Keyword(s):

Immune Response ◽

Innate Immune Response ◽

Nuclear Transport ◽

Computational Prediction ◽

Viral Proteins ◽

Innate Immune ◽

Type I ◽

Viral Genes ◽

Genes Encoding

Suppression of the host innate immune response is a critical aspect of viral replication. Upon infection, viruses may introduce one or more proteins that inhibit key immune pathways, such as the type I interferon pathway. However, the ability to predict and evaluate viral protein bioactivity on targeted pathways remains challenging and is typically done on a single virus/gene basis. Here, we present a medium-throughput high-content cell-based assay to reveal the immunosuppressive effects of viral proteins. To test the predictive power of our approach, we developed a library of 800 genes encoding known, predicted, and uncharacterized human viral genes. We find that previously known immune suppressors from numerous viral families such as Picornaviridae and Flaviviridae recorded positive responses. These include a number of viral proteases for which we further confirmed that innate immune suppression depends on protease activity. A class of predicted inhibitors encoded by Rhabdoviridae viruses was demonstrated to block nuclear transport, and several previously uncharacterized proteins from uncultivated viruses were shown to inhibit nuclear transport of the transcription factors NF-kB and IRF3. We propose that this pathway-based assay, together with early sequencing, gene synthesis, and viral infection studies, could partly serve as the basis for rapid in vitro characterization of novel viral proteins.

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The Hepatitis C Virus-Induced Membranous Web in Liver Tissue

Cells ◽

10.3390/cells7110191 ◽

2018 ◽

Vol 7 (11) ◽

pp. 191

Author(s):

Emmanuelle Blanchard ◽

Philippe Roingeard

Keyword(s):

Immune Response ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Innate Immune Response ◽

Liver Tissue ◽

Hepatoma Cell ◽

Membrane Vesicles ◽

Innate Immune ◽

Host Cell Membrane

Host cell membrane rearrangements induced by the hepatitis C virus (HCV) have been exclusively studied in vitro. These studies have shown that HCV induces double-membrane vesicles (DMVs), which probably serve to separate replication sites from the cytoplasmic sensors of the innate immune response. We report for the first time the observation of HCV-induced membrane rearrangements in liver biopsy specimens from patients chronically infected with HCV. Unlike observations performed in vitro, the membranous web detected in liver tissue seems essentially made of clusters of single-membrane vesicles derived from the endoplasmic reticulum and close to lipid droplets. This suggests that the DMVs could be a hallmark of laboratory-adapted HCV strains, possibly due to their ability to achieve a high level of replication. Alternatively, the concealment of viral RNA in DMVs may be part of innate immune response mechanisms particularly developed in hepatoma cell lines cultured in vitro. In any case, this constitutes the first report showing the differences in the membranous web established by HCV in vitro and in vivo.

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An In Vitro Assessment of Immunostimulatory Responses to Ten Model Innate Immune Response Modulating Impurities (IIRMIs) and Peptide Drug Product, Teriparatide

Molecules ◽

10.3390/molecules26247461 ◽

2021 ◽

Vol 26 (24) ◽

pp. 7461

Author(s):

Claire K. Holley ◽

Edward Cedrone ◽

Duncan Donohue ◽

Barry W. Neun ◽

Daniela Verthelyi ◽

...

Keyword(s):

Immune Response ◽

Immune Responses ◽

Innate Immune Response ◽

Innate Immune ◽

Cytokine Secretion ◽

Poly I:C ◽

In Vitro Assays ◽

Innate Immune Responses ◽

Vitro Assay

Understanding, predicting, and minimizing the immunogenicity of peptide-based therapeutics are of paramount importance for ensuring the safety and efficacy of these products. The so-called anti-drug antibodies (ADA) may have various clinical consequences, including but not limited to the alteration in the product’s distribution, biological activity, and clearance profiles. The immunogenicity of biotherapeutics can be influenced by immunostimulation triggered by the presence of innate immune response modulating impurities (IIRMIs) inadvertently introduced during the manufacturing process. Herein, we evaluate the applicability of several in vitro assays (i.e., complement activation, leukocyte proliferation, and cytokine secretion) for the screening of innate immune responses induced by ten common IIRMIs (Bacillus subtilis flagellin, FSL-1, zymosan, ODN2006, poly(I:C) HMW, poly(I:C) LMW, CLO75, MDP, ODN2216, and Escherichia coli O111:B4 LPS), and a model biotherapeutic Forteo™ (teriparatide). Our study identifies cytokine secretion from healthy human donor peripheral blood mononuclear cells (PBMC) as a sensitive method for the in vitro monitoring of innate immune responses to individual IIRMIs and teriparatide (TP). We identify signature cytokines, evaluate both broad and narrow multiplex cytokine panels, and discuss how the assay logistics influence the performance of this in vitro assay.

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RNA sensors as a mechanism of innate immune evasion among SARS-CoV2, HIV and Nipah viruses

Current Protein and Peptide Science ◽

10.2174/1389203722666210322142725 ◽

2021 ◽

Vol 22 ◽

Author(s):

Dalia Cicily Kattiparambil Dixon ◽

Chameli Ratan ◽

Bhagyalakshmi Nair ◽

Sabitha Mangalath ◽

Rachy Abraham ◽

...

Keyword(s):

Immune Response ◽

Innate Immunity ◽

Innate Immune Response ◽

Vaccine Development ◽

Innate Immune ◽

Interferon Response ◽

Host Immune System ◽

Type I ◽

Adaptive Responses ◽

Rna Sensors

: Innate immunity is the first line of defence elicited by the host immune system to fight against invading pathogens such as viruses and bacteria. From this elementary immune response, the more complex antigen-specific adaptive responses are recruited to provide a long-lasting memory against the pathogens. Innate immunity gets activated when the host cell utilizes a diverse set of receptors known as pattern recognition receptors (PRR) to recognize the viruses that have penetrated the host and respond with cellular processes like complement system, phagocytosis, cytokine release and inflammation and destruction of NK cells. Viral RNA or DNA or viral intermediate products are recognized by receptors like toll-like receptors(TLRs), nucleotide oligomerization domain(NOD)-like receptors (NLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) thereby, inducing type I interferon response (IFN) and other proinflammatory cytokines in infected cells or other immune cells. But certain viruses can evade the host innate immune response to replicate efficiently, triggering the spread of the viral infection. The present review describes the similarity in the mechanism chosen by viruses from different families -HIV, SARS-CoV2 and Nipah viruses to evade the innate immune response and how efficiently they establish the infection in the host. The review also addresses the stages of developments of various vaccines against these viral diseases and the challenges encountered by the researchers during vaccine development.

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Global Analysis of Genes Essential forFrancisella tularensisSchu S4 GrowthIn Vitroand for Fitness during Competitive Infection of Fischer 344 Rats

Journal of Bacteriology ◽

10.1128/jb.00630-18 ◽

2019 ◽

Vol 201 (7) ◽

Cited By ~ 8

Author(s):

Philip M. Ireland ◽

Helen L. Bullifent ◽

Nicola J. Senior ◽

Stephanie J. Southern ◽

Zheng Rong Yang ◽

...

Keyword(s):

Francisella Tularensis ◽

Insertion Site ◽

Therapeutic Drug ◽

Content Type ◽

Fischer 344 ◽

A Genome ◽

Fischer 344 Rat ◽

Schu S4

ABSTRACTThe highly virulent intracellular pathogenFrancisella tularensisis a Gram-negative bacterium that has a wide host range, including humans, and is the causative agent of tularemia. To identify new therapeutic drug targets and vaccine candidates and investigate the genetic basis ofFrancisellavirulence in the Fischer 344 rat, we have constructed anF. tularensisSchu S4 transposon library. This library consists of more than 300,000 unique transposon mutants and represents a transposon insertion for every 6 bp of the genome. A transposon-directed insertion site sequencing (TraDIS) approach was used to identify 453 genes essential for growthin vitro. Many of these essential genes were mapped to key metabolic pathways, including glycolysis/gluconeogenesis, peptidoglycan synthesis, fatty acid biosynthesis, and the tricarboxylic acid (TCA) cycle. Additionally, 163 genes were identified as required for fitness during colonization of the Fischer 344 rat spleen. Thisin vivoselection screen was validated through the generation of marked deletion mutants that were individually assessed within a competitive index study against the wild-typeF. tularensisSchu S4 strain.IMPORTANCEThe intracellular bacterial pathogenFrancisella tularensiscauses a disease in humans characterized by the rapid onset of nonspecific symptoms such as swollen lymph glands, fever, and headaches.F. tularensisis one of the most infectious bacteria known and following pulmonary exposure can have a mortality rate exceeding 50% if left untreated. The low infectious dose of this organism and concerns surrounding its potential as a biological weapon have heightened the need for effective and safe therapies. To expand the repertoire of targets for therapeutic development, we initiated a genome-wide analysis. This study has identified genes that are important forF. tularensisunderin vitroandin vivoconditions, providing candidates that can be evaluated for vaccine or antibacterial development.

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