scholarly journals Francisella tularensis Schu S4 Lipopolysaccharide Core Sugar and O-Antigen Mutants Are Attenuated in a Mouse Model of Tularemia

2014 ◽  
Vol 82 (4) ◽  
pp. 1523-1539 ◽  
Author(s):  
Jed A. Rasmussen ◽  
Deborah M. B. Post ◽  
Bradford W. Gibson ◽  
Stephen R. Lindemann ◽  
Michael A. Apicella ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Francisella Tularensis ◽  
Lethal Dose ◽  
Protective Effects ◽  
Wild Type ◽  
Content Type ◽  
O Antigen ◽  
Capsule Production ◽  
Mutant Strains ◽  
Schu S4

ABSTRACTThe virulence factors mediatingFrancisellapathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms.Francisella tularensisproduces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using anF. tularensisSchu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236andFTT1238c) revealed similarity to those forwaa(rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to theF. tularensismutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type AF. tularensischallenge.

scholarly journals A Francisella tularensis Locus Required for Spermine Responsiveness Is Necessary for Virulence

2011 ◽  
Vol 79 (9) ◽  
pp. 3665-3676 ◽  
Author(s):  
Brian C. Russo ◽  
Joseph Horzempa ◽  
Dawn M. O'Dee ◽  
Deanna M. Schmitt ◽  
Matthew J. Brown ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Febrile Illness ◽  
Host Cells ◽  
High Morbidity ◽  
Wild Type ◽  
Content Type ◽  
Infectious Dose ◽  
Schu S4

ABSTRACTTularemia is a debilitating febrile illness caused by the category A biodefense agentFrancisella tularensis. This pathogen infects over 250 different hosts, has a low infectious dose, and causes high morbidity and mortality. Our understanding of the mechanisms by whichF. tularensissenses and adapts to host environments is incomplete. Polyamines, including spermine, regulate the interactions ofF. tularensiswith host cells. However, it is not known whether responsiveness to polyamines is necessary for the virulence of the organism. Through transposon mutagenesis ofF. tularensissubsp.holarcticalive vaccine strain (LVS), we identified FTL_0883 as a gene important for spermine responsiveness. In-frame deletion mutants of FTL_0883 and FTT_0615c, the homologue of FTL_0883 inF. tularensissubsp.tularensisSchu S4 (Schu S4), elicited higher levels of cytokines from human and murine macrophages compared to wild-type strains. Although deletion of FTL_0883 attenuated LVS replication within macrophagesin vitro, the Schu S4 mutant with a deletion in FTT_0615c replicated similarly to wild-type Schu S4. Nevertheless, both the LVS and the Schu S4 mutants were significantly attenuatedin vivo. Growth and dissemination of the Schu S4 mutant was severely reduced in the murine model of pneumonic tularemia. This attenuation depended on host responses to elevated levels of proinflammatory cytokines. These data associate responsiveness to polyamines with tularemia pathogenesis and define FTL_0883/FTT_0615c as anF. tularensisgene important for virulence and evasion of the host immune response.

scholarly journals Pathogenicity of Yersinia pestis Synthesis of 1-Dephosphorylated Lipid A

2013 ◽  
Vol 81 (4) ◽  
pp. 1172-1185 ◽  
Author(s):  
Wei Sun ◽  
David A. Six ◽  
C. Michael Reynolds ◽  
Hak Suk Chung ◽  
Christian R. H. Raetz ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Yersinia Pestis ◽  
Francisella Tularensis ◽  
Inflammatory Reaction ◽  
Lipid A ◽  
Lethal Dose ◽  
Wild Type ◽  
Content Type ◽  
Significant Attenuation

ABSTRACTSynthesis ofEscherichia coliLpxL, which transfers a secondary laurate chain to the 2′ position of lipid A, inYersinia pestisproduced bisphosphoryl hexa-acylated lipid A at 37°C, leading to significant attenuation of virulence. Our previous observations also indicated that strain χ10015(pCD1Ap) (ΔlpxP32::PlpxLlpxL) stimulated a strong inflammatory reaction but sickened mice before recovery and retained virulence via intranasal (i.n.) infection. The development of live, attenuatedY. pestisvaccines may be facilitated by detoxification of its lipopolysaccharide (LPS). Heterologous expression of the lipid A 1-phosphatase, LpxE, fromFrancisella tularensisinY. pestisyields predominantly 1-dephosphorylated lipid A, as confirmed by mass spectrometry. Results indicated that expression of LpxE on top of LpxL provided no significant reduction in virulence ofY. pestisin mice when it was administered i.n. but actually reduced the 50% lethal dose (LD50) by 3 orders of magnitude when the strain was administered subcutaneously (s.c.). Additionally, LpxE synthesis in wild-typeY. pestisKIM6+(pCD1Ap) led to slight attenuation by s.c. inoculation but no virulence change by i.n. inoculation in mice. In contrast toSalmonella enterica, expression of LpxE does not attenuate the virulence ofY. pestis.

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

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Nihal A. Okan ◽  
Sabina Chalabaev ◽  
Tae-Hyun Kim ◽  
Avner Fink ◽  
Robin A. Ross ◽  
...  
Keyword(s):  
Immune Response ◽  
Innate Immunity ◽  
Innate Immune Response ◽  
Francisella Tularensis ◽  
Innate Immune ◽  
Content Type ◽  
Tnf Α ◽  
Schu S4 ◽  
Highly Virulent

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.

scholarly journals Prostaglandin E2Receptor Antagonist with Antimicrobial Activity against Methicillin-ResistantStaphylococcus aureus

2017 ◽  
Vol 62 (3) ◽  
Author(s):  
Mélanie A. C. Ikeh ◽  
Paul L. Fidel ◽  
Mairi C. Noverr
Keyword(s):  
Antimicrobial Activity ◽  
Receptor Antagonist ◽  
Enzyme Inhibitors ◽  
Lethal Dose ◽  
Prostaglandin E ◽  
Protective Effects ◽  
Physiological Concentration ◽  
Content Type ◽  
Abdominal Infections

ABSTRACTPolymicrobial intra-abdominal infections (IAI) involvingCandida albicansandStaphylococcus aureusare associated with severe morbidity and mortality (∼80%). Our laboratory discovered that the immunomodulatory eicosanoid prostaglandin E2(PGE2) plays a key role in the lethal inflammatory response during polymicrobial IAI using a mouse model of infection. In studies designed to uncover key PGE2biosynthesis/signaling components involved in the response, selective eicosanoid enzyme inhibitors and receptor antagonists were selected and prescreened for antimicrobial activity againstC. albicansorS. aureus. Unexpectedly, we found that the EP4receptor antagonist L-161,982 had direct growth-inhibitory effects onS. aureusin vitroat the physiological concentration required to block the PGE2interaction with EP4. This antimicrobial activity was observed with methicillin-sensitiveS. aureusand methicillin-resistantS. aureus(MRSA) strains, with the MIC and minimum bactericidal concentration values for planktonic cells being 50 μg/ml and 100 μg/ml, respectively. In addition, L-161,982 inhibitedS. aureusbiofilm formation and had activity against preformed mature biofilms. More importantly, treatment of mice with L-161,982 following intraperitoneal inoculation with a lethal dose of MRSA significantly reduced the bioburden and enhanced survival. Furthermore, L-161,982 protected mice against the synergistic lethality induced by coinfection withC. albicansandS. aureus. The antimicrobial activity of L-161,982 is independent of EP4receptor inhibitory activity; an alternative EP4receptor antagonist exerted no antimicrobial or protective effects. Taken together, these findings demonstrate that L-161,982 has potent antimicrobial activity against MRSA and may represent a significant therapeutic alternative in improving the prognosis of mono- or polymicrobial infections involving MRSA.

scholarly journals Pneumococcal Surface Protein C Contributes to Sepsis Caused by Streptococcus pneumoniae in Mice

2004 ◽  
Vol 72 (5) ◽  
pp. 3077-3080 ◽  
Author(s):  
Francesco Iannelli ◽  
Damiana Chiavolini ◽  
Susanna Ricci ◽  
Marco Rinaldo Oggioni ◽  
Gianni Pozzi
Keyword(s):  
Streptococcus Pneumoniae ◽  
Protein C ◽  
Lethal Dose ◽  
Surface Protein ◽  
Infection Model ◽  
Wild Type ◽  
Mutant Strains ◽  
Type 3

ABSTRACT The role of pneumococcal surface protein C (PspC; also called SpsA, CbpA, and Hic) in sepsis by Streptococcus pneumoniae was investigated in a murine infection model. The pspC gene was deleted in strains D39 (type 2) and A66 (type 3), and the mutants were tested by being injected intravenously into mice. The animals infected with the mutant strains showed a significant increase in survival, with the 50% lethal dose up to 250-fold higher than that for the wild type. Our findings indicate that PspC affords a decisive contribution to sepsis development.

scholarly journals Regulated Delayed Expression of rfaH in an Attenuated Salmonellaenterica Serovar Typhimurium Vaccine Enhances Immunogenicity of Outer Membrane Proteins and a Heterologous Antigen

2009 ◽  
Vol 77 (12) ◽  
pp. 5572-5582 ◽  
Author(s):  
Qingke Kong ◽  
Qing Liu ◽  
Kenneth L. Roland ◽  
Roy Curtiss
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Lethal Dose ◽  
Enteric Bacteria ◽  
Lymphoid Tissues ◽  
Heterologous Antigen ◽  
O Antigen ◽  
Mutant Strains ◽  
Serovar Typhimurium

ABSTRACT RfaH is a transcriptional antiterminator that reduces the polarity of long operons encoding secreted and surface-associated cell components of Salmonella enterica serovar Typhimurium, including O antigen and lipopolysaccharide core sugars. A ΔrfaH mutant strain is attenuated in mice (50% lethal dose [LD50], >108 CFU). To examine the potential for using rfaH in conjunction with other attenuating mutations, we designed a series of strains in which we replaced the native rfaH promoter with the tightly regulated arabinose-dependent araC PBAD promoter so that rfaH expression was dependent on exogenously supplied arabinose provided during in vitro growth. Following colonization of host lymphoid tissues, where arabinose was not available, the PBAD promoter was no longer active and rfaH was not expressed. In the absence of RfaH, O antigen and core sugars were not synthesized. We constructed three mutant strains that expressed different levels of RfaH by altering the ribosome-binding sequence and start codon. One mutation, ΔPrfaH178, was introduced into the attenuated vaccine strain χ9241 (ΔpabA ΔpabB ΔasdA) expressing the pneumococcal surface protein PspA from an Asd+ balanced-lethal plasmid. Mice immunized with this strain and boosted 4 weeks later induced higher levels of serum immunoglobulin G specific for PspA and for outer membrane proteins from other enteric bacteria than either an isogenic ΔrfaH derivative or the isogenic RfaH+ parent. Eight weeks after primary oral immunization, mice were challenged with 200 LD50 of virulent S treptococcus pneumoniae WU2. Immunization with ΔPrfaH178 mutant strains led to increased levels of protection compared to that of the parent χ9241 and of a ΔrfaH derivative of χ9241.

scholarly journals The Type VI Secretion System Modulates Flagellar Gene Expression and Secretion in Citrobacter freundii and Contributes to Adhesion and Cytotoxicity to Host Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2596-2604 ◽  
Author(s):  
Liyun Liu ◽  
Shuai Hao ◽  
Ruiting Lan ◽  
Guangxia Wang ◽  
Di Xiao ◽  
...  
Keyword(s):  
Secretion System ◽  
Host Cells ◽  
Target Cells ◽  
Deletion Mutants ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Vi Secretion System ◽  
Content Type ◽  
Type Vi Secretion ◽  
Mutant Strains

The type VI secretion system (T6SS) as a virulence factor-releasing system contributes to virulence development of various pathogens and is often activated upon contact with target cells.Citrobacter freundiistrain CF74 has a complete T6SS genomic island (GI) that containsclpV,hcp-2, andvgrT6SS genes. We constructedclpV,hcp-2,vgr, and T6SS GI deletion mutants in CF74 and analyzed their effects on the transcriptome overall and, specifically, on the flagellar system at the levels of transcription and translation. Deletion of the T6SS GI affected the transcription of 84 genes, with 15 and 69 genes exhibiting higher and lower levels of transcription, respectively. Members of the cell motility class of downregulated genes of the CF74ΔT6SS mutant were mainly flagellar genes, including effector proteins, chaperones, and regulators. Moreover, the production and secretion of FliC were also decreased inclpV,hcp-2,vgr, or T6SS GI deletion mutants in CF74 and were restored upon complementation. In swimming motility assays, the mutant strains were found to be less motile than the wild type, and motility was restored by complementation. The mutant strains were defective in adhesion to HEp-2 cells and were restored partially upon complementation. Further, the CF74ΔT6SS, CF74ΔclpV, and CF74Δhcp-2mutants induced lower cytotoxicity to HEp-2 cells than the wild type. These results suggested that the T6SS GI in CF74 regulates the flagellar system, enhances motility, is involved in adherence to host cells, and induces cytotoxicity to host cells. Thus, the T6SS plays a wide-ranging role inC. freundii.

scholarly journals Data-Driven Models Reveal Mutant Cell Behaviors Important for Myxobacterial Aggregation

mSystems ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Zhaoyang Zhang ◽  
Christopher R. Cotter ◽  
Zhe Lyu ◽  
Lawrence J. Shimkets ◽  
Oleg A. Igoshin
Keyword(s):  
Cell Tracking ◽  
Mutant Cell ◽  
Myxococcus Xanthus ◽  
Data Driven ◽  
Self Organization ◽  
Cell Behavior ◽  
Significant Heterogeneity ◽  
Wild Type ◽  
Content Type ◽  
Mutant Strains

ABSTRACT Single mutations frequently alter several aspects of cell behavior but rarely reveal whether a particular statistically significant change is biologically significant. To determine which behavioral changes are most important for multicellular self-organization, we devised a new methodology using Myxococcus xanthus as a model system. During development, myxobacteria coordinate their movement to aggregate into spore-filled fruiting bodies. We investigate how aggregation is restored in two mutants, csgA and pilC, that cannot aggregate unless mixed with wild-type (WT) cells. To this end, we use cell tracking to follow the movement of fluorescently labeled cells in combination with data-driven agent-based modeling. The results indicate that just like WT cells, both mutants bias their movement toward aggregates and reduce motility inside aggregates. However, several aspects of mutant behavior remain uncorrected by WT, demonstrating that perfect recreation of WT behavior is unnecessary. In fact, synergies between errant behaviors can make aggregation robust. IMPORTANCE Self-organization into spatial patterns is evident in many multicellular phenomena. Even for the best-studied systems, our ability to dissect the mechanisms driving coordinated cell movement is limited. While genetic approaches can identify mutations perturbing multicellular patterns, the diverse nature of the signaling cues coupled to significant heterogeneity of individual cell behavior impedes our ability to mechanistically connect genes with phenotype. Small differences in the behaviors of mutant strains could be irrelevant or could sometimes lead to large differences in the emergent patterns. Here, we investigate rescue of multicellular aggregation in two mutant strains of Myxococcus xanthus mixed with wild-type cells. The results demonstrate how careful quantification of cell behavior coupled to data-driven modeling can identify specific motility features responsible for cell aggregation and thereby reveal important synergies and compensatory mechanisms. Notably, mutant cells do not need to precisely recreate wild-type behaviors to achieve complete aggregation.

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

2019 ◽  
Vol 201 (7) ◽  
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.

scholarly journals Monophosphoryl Lipid A Enhances Efficacy of a Francisella tularensis LVS-Catanionic Nanoparticle Subunit Vaccine against F. tularensis Schu S4 Challenge by Augmenting both Humoral and Cellular Immunity

2017 ◽  
Vol 24 (3) ◽  
Author(s):  
Katharina Richard ◽  
Barbara J. Mann ◽  
Aiping Qin ◽  
Eileen M. Barry ◽  
Robert K. Ernst ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Subunit Vaccine ◽  
Lipid A ◽  
Ex Vivo ◽  
The United States ◽  
Content Type ◽  
Monophosphoryl Lipid A ◽  
Monophosphoryl Lipid ◽  
Ifn Γ ◽  
Schu S4

ABSTRACT Francisella tularensis, a bacterial biothreat agent, has no approved vaccine in the United States. Previously, we showed that incorporating lysates from partially attenuated F. tularensis LVS or fully virulent F. tularensis Schu S4 strains into catanionic surfactant vesicle (V) nanoparticles (LVS-V and Schu S4-V, respectively) protected fully against F. tularensis LVS intraperitoneal (i.p.) challenge in mice. However, we achieved only partial protection against F. tularensis Schu S4 intranasal (i.n.) challenge, even when employing heterologous prime-boost immunization strategies. We now extend these findings to show that both LVS-V and Schu S4-V immunization (i.p./i.p.) elicited similarly high titers of anti-F. tularensis IgG and that the titers could be further increased by adding monophosphoryl lipid A (MPL), a nontoxic Toll-like receptor 4 (TLR4) adjuvant that is included in several U.S. FDA-approved vaccines. LVS-V+MPL immune sera also detected more F. tularensis antigens than LVS-V immune sera and, after passive transfer to naive mice, significantly delayed the time to death against F. tularensis Schu S4 subcutaneous (s.c.) but not i.n. challenge. Active immunization with LVS-V+MPL (i.p./i.p.) also increased the frequency of gamma interferon (IFN-γ)-secreting activated helper T cells, IFN-γ production, and the ability of splenocytes to control intramacrophage F. tularensis LVS replication ex vivo. Active LVS-V+MPL immunization via heterologous routes (i.p./i.n.) significantly elevated IgA and IgG levels in bronchoalveolar lavage fluid and significantly enhanced protection against i.n. F. tularensis Schu S4 challenge (to ∼60%). These data represent a significant step in the development of a subunit vaccine against the highly virulent type A strains.

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