scholarly journals The Brucella abortus Phosphoglycerate Kinase Mutant Is Highly Attenuated and Induces Protection Superior to That of Vaccine Strain 19 in Immunocompromised and Immunocompetent Mice

2010 ◽  
Vol 78 (5) ◽  
pp. 2283-2291 ◽  
Author(s):  
Cyntia G. M. C. Trant ◽  
Thais L. S. Lacerda ◽  
Natalia B. Carvalho ◽  
Vasco Azevedo ◽  
Gracia M. S. Rosinha ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Brucella Abortus ◽  
Deletion Mutant ◽  
Bacterial Pathogen ◽  
Phosphoglycerate Kinase ◽  
Wild Type ◽  
Gene Encoding ◽  
Commercial Strain ◽  
Immunocompetent Mice

ABSTRACT Brucella abortus is a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. The mechanism of virulence of Brucella spp. is not yet fully understood. Therefore, it is crucial to identify new molecules that can function as virulence factors to better understand the host-pathogen interplay. Herein, we identified the gene encoding the phosphoglycerate kinase (PGK) of B. abortus strain 2308. To test the role of PGK in Brucella pathogenesis, a pgk deletion mutant was constructed. Replacement of the wild-type pgk by recombination was demonstrated by Southern and Western blot analyses. The B. abortus Δpgk mutant strain exhibited extreme attenuation in bone marrow-derived macrophages and in vivo in BALB/c, C57BL/6, 129/Sv, and interferon regulatory factor-1 knockout (IRF-1 KO) mice. Additionally, at 24 h postinfection the Δpgk mutant was not found within the same endoplasmic reticulum-derived compartment as the wild-type bacteria, but, instead, over 60% of Brucella-containing vacuoles (BCVs) retained the late endosomal/lysosomal marker LAMP1. Furthermore, the B. abortus Δpgk deletion mutant was used as a live vaccine. Challenge experiments revealed that the Δpgk mutant strain induced protective immunity in 129/Sv or IRF-1 KO mice that was superior to the protection conferred by commercial strain 19 or RB51. Finally, the results shown here demonstrated that Brucella PGK is critical for full bacterial virulence and that a Δpgk mutant may serve as a potential vaccine candidate in future studies.

scholarly journals Impact of the Listeria monocytogenes Protein InlC on Infection in Mice

2013 ◽  
Vol 81 (4) ◽  
pp. 1334-1340 ◽  
Author(s):  
Nelly Leung ◽  
Antonella Gianfelice ◽  
Scott D. Gray-Owen ◽  
Keith Ireton
Keyword(s):  
Listeria Monocytogenes ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Bacterial Pathogen ◽  
Adaptor Protein ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type

ABSTRACTThe bacterial pathogenListeria monocytogenescauses serious food-borne illnesses in pregnant women and the immunocompromised.L. monocytogenespromotes its internalization into host epithelial cells and then uses an F-actin-dependent motility process to spread from infected cells to surrounding healthy cells. In cultured enterocytes, efficient spread ofL. monocytogenesrequires the secreted bacterial protein InlC. InlC promotes dissemination by physically interacting with and antagonizing the function of the human adaptor protein Tuba. Here we examine the role of InlC and its interaction with host Tuba during infection in mice. The study took advantage of a single-amino-acid substitution (K173A) in InlC that impairs binding to human Tuba but does not affect InlC-mediated inhibition of the NF-κB pathway. Mice were inoculated intravenously with the wild-typeL. monocytogenesstrain EGD, an isogenic strain deleted for theinlCgene (ΔinlC), or a strain expressing K173A mutant InlC (inlC.K173A). The 50% lethal doses (LD50) for the ΔinlCorinlC.K173Amutant strain were approximately 4- or 6-fold greater than that for the wild-type strain, indicating a role forinlCin virulence. Compared to the wild-type strain, theinlC.K173Amutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the twoinlCmutant strains produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-to-cell spreadin vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.

scholarly journals N-3-Hydroxy Dodecanoyl-DL-homoserine Lactone (OH-dDHL) Triggers Apoptosis of Bone Marrow-Derived Macrophages through the ER- and Mitochondria-Mediated Pathways

2021 ◽  
Vol 22 (14) ◽  
pp. 7565
Author(s):  
Kyungho Woo ◽  
Dong Ho Kim ◽  
Man Hwan Oh ◽  
Ho Sung Park ◽  
Chul Hee Choi
Keyword(s):  
Bone Marrow ◽  
Quorum Sensing ◽  
Deletion Mutant ◽  
Transmembrane Protein ◽  
Cell Communication ◽  
Homoserine Lactone ◽  
Host Responses ◽  
Wild Type ◽  
Mutant Strains

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

scholarly journals Deletion of znuA Virulence Factor Attenuates Brucella abortus and Confers Protection against Wild-Type Challenge

2006 ◽  
Vol 74 (7) ◽  
pp. 3874-3879 ◽  
Author(s):  
Xinghong Yang ◽  
Todd Becker ◽  
Nancy Walters ◽  
David W. Pascual
Keyword(s):  
Brucella Abortus ◽  
Deletion Mutant ◽  
Pasteurella Multocida ◽  
Brucella Melitensis ◽  
Normal Growth ◽  
Wild Type ◽  
Minimal Growth ◽  
M Genome ◽  
S19 Vaccine ◽  
Knockout Mutation

ABSTRACT znuA is known to be an important factor for survival and normal growth under low Zn2+ concentrations for Escherichia coli, Haemophilus spp., Neisseria gonorrhoeae, and Pasteurella multocida. We hypothesized that the znuA gene present in Brucella melitensis 16 M would be similar to znuA in B. abortus and questioned whether it may also be an important factor for growth and virulence of Brucella abortus. Using the B. melitensis 16 M genome sequence, primers were designed to construct a B. abortus deletion mutant. A znuA knockout mutation in B. abortus 2308 (ΔznuA) was constructed and found to be lethal in low-Zn2+ medium. When used to infect macrophages, ΔznuA B. abortus showed minimal growth. Further study with ΔznuA B. abortus showed that its virulence in BALB/c mice was attenuated, and most of the bacteria were cleared from the spleen within 8 weeks. Protection studies confirmed the ΔznuA mutant as a potential live vaccine, since protection against wild-type B. abortus 2308 challenge was as effective as that obtained with the RB51 or S19 vaccine strain.

scholarly journals Endothelial cell-specific reduction of heparan sulfate suppresses glioma growth in mice

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Takamasa Kinoshita ◽  
Hiroyuki Tomita ◽  
Hideshi Okada ◽  
Ayumi Niwa ◽  
Fuminori Hyodo ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Heparan Sulfate ◽  
Vascular Endothelium ◽  
Wild Type ◽  
Brain Capillaries ◽  
Gene Encoding ◽  
Glioma Growth ◽  
Underlying Mechanisms ◽  
The Brain

Abstract Purpose Heparan sulfate (HS) is one of the factors that has been suggested to be associated with angiogenesis and invasion of glioblastoma (GBM), an aggressive and fast-growing brain tumor. However, it remains unclear how HS of endothelial cells is involved in angiogenesis in glioblastoma and its prognosis. Thus, we investigated the effect of endothelial cell HS on GBM development. Methods We generated endothelial cell-specific knockout of Ext1, a gene encoding a glycosyltransferase and essential for HS synthesis, and murine GL261 glioblastoma cells were orthotopically transplanted. Two weeks after transplantation, we examined the tumor progression and underlying mechanisms. Results The endothelial cell-specific Ext1 knockout (Ext1CKO) mice exhibited reduced HS expression specifically in the vascular endothelium of the brain capillaries compared with the control wild-type (WT) mice. GBM growth was significantly suppressed in Ext1CKO mice compared with that in WT mice. After GBM transplantation, the survival rate was significantly higher in Ext1CKO mice than in WT mice. We investigated how the effect of fibroblast growth factor 2 (FGF2), which is known as an angiogenesis-promoting factor, differs between Ext1CKO and WT mice by using an in vivo Matrigel assay and demonstrated that endothelial cell-specific HS reduction attenuated the effect of FGF2 on angiogenesis. Conclusions HS reduction in the vascular endothelium of the brain suppressed GBM growth and neovascularization in mice.

scholarly journals Overproduction of SecA Suppresses the Export Defect Caused by a Mutation in the Gene Encoding the Escherichia coli Export Chaperone SecB

1999 ◽  
Vol 181 (10) ◽  
pp. 3010-3017 ◽  
Author(s):  
Heather A. Cook ◽  
Carol A. Kumamoto
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane Proteins ◽  
Protein A ◽  
In Vivo Studies ◽  
Wild Type ◽  
Gene Encoding ◽  
Precursor Proteins ◽  
Additional Support ◽  
Insight Into

ABSTRACT SecB is a cytosolic protein required for rapid and efficient export of particular periplasmic and outer membrane proteins inEscherichia coli. SecB promotes export by stabilizing newly synthesized precursor proteins in a nonnative conformation and by targeting the precursors to the inner membrane. Biochemical studies suggest that SecB facilitates precursor targeting by binding to the SecA protein, a component of the membrane-embedded translocation apparatus. To gain more insight into the functional interaction of SecB and SecA, in vivo, mutations in the secA locus that compensate for the export defect caused by the secBmissense mutation secBL75Q were isolated. Two suppressors were isolated, both of which led to the overproduction of wild-type SecA protein. In vivo studies demonstrated that the SecBL75Q mutant protein releases precursor proteins at a lower rate than does wild-type SecB. Increasing the level of SecA protein in the cell was found to reverse this slow-release defect, indicating that overproduction of SecA stimulates the turnover of SecBL75Q-precursor complexes. These findings lend additional support to the proposed pathway for precursor targeting in which SecB promotes targeting to the translocation apparatus by binding to the SecA protein.

scholarly journals phgABC, a Three-Gene Operon Required for Growth of Streptococcus pneumoniae in Hyperosmotic Medium and In Vivo

2004 ◽  
Vol 72 (8) ◽  
pp. 4579-4588 ◽  
Author(s):  
Jeremy S. Brown ◽  
Sarah M. Gilliland ◽  
Shilpa Basavanna ◽  
David W. Holden
Keyword(s):  
Streptococcus Pneumoniae ◽  
Mutant Strain ◽  
Compatible Solutes ◽  
Wild Type ◽  
Gram Positive Bacteria ◽  
Mutant Strains ◽  
Increased Sensitivity ◽  
Related Proteins ◽  
And Oxidative Stress

ABSTRACT To cause disease, bacterial pathogens need to be able to adapt to the physiological conditions found within the host, including an osmolality of approximately 290 mosmol kg−1. While investigating Streptococcus pneumoniae genes contained within pneumococcal pathogenicity island 1, we identified a three-gene operon of unknown function termed phgABC. PhgC has a domain with similarity to diacylglycerol kinases of eukaryotes and is the first described member of a family of related proteins found in many gram-positive bacteria. phgA and phgC mutant strains were constructed by insertional duplication mutagenesis and found to have impaired growth under conditions of high osmotic and oxidative stress. The compatible solutes proline and glycine betaine improved growth of the wild-type and the phgA mutant strains in hyperosmolar medium, and when analyzed by electron microscopy, the cellular morphology of the phgA mutant strain was unaffected by osmotic stress. The phgA and phgC mutant strains were reduced in virulence in models of both systemic and pulmonary infection. As the virulence of the phgA mutant strain was not restored in gp91phox−/− mice and the phgA and phgC mutant strains had reduced growth in both blood and serum, the reduced virulence of these strains is unlikely to be due to increased sensitivity to the respiratory burst of phagocytes but is, instead, due to impaired growth at physiological osmolality.

Missense mutations in the gene encoding prothrombin corresponding to Arg596 cause antithrombin resistance and thrombomodulin resistance

2016 ◽  
Vol 116 (12) ◽  
pp. 1022-1031 ◽  
Author(s):  
Yuki Takagi ◽  
Moe Murata ◽  
Toshihiro Kozuka ◽  
Yukiko Nakata ◽  
Ryo Hasebe ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Residual Activity ◽  
Base Substitution ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dependent Manner ◽  
Missense Mutations ◽  
Wild Type ◽  
Gene Encoding ◽  
Single Base

SummaryAntithrombin (AT) and thrombomodulin (TM) play important roles in the process of natural anticoagulation in vivo. Recently, we reported that the prothrombin Yukuhashi mutation (p.Arg596Leu) was associated with AT and TM resistance-related thrombophilia. To assess the AT and TM resistances associated with other missense mutations by single base substitution in the Arg596 codon, we generated recombinant variants (596Gln, 596Trp, 596Gly, and 596Pro) and investigated the effects on AT and TM anticoagulant functions. All variants except 596Pro were secreted in amounts comparable to that of the wild-type but exhibited variable procoagulant activities. After a 30-minute inactivation by AT, the relative residual activity of wild-type thrombin decreased to 15 ± 4.0%, in contrast to values of all variants were maintained at above 80%. The thrombin–AT complex formation, as determined by enzyme-linked immunosorbent assay, was reduced with all tested variants in the presence and absence of heparin. In the presence of soluble TM (sTM), the relative fibrinogen clotting activity of wild-type thrombin decreased to 16 ± 0.12%, whereas that of tested variants was 37%–56%. In a surface plasmon resonance assay, missense Arg596 mutations reduced thrombin–TM affinity to an extent similar to the reduction of fibrinogen clotting inhibition. In the presence of sTM or cultured endothelial-like cells, APC generation was enhanced differently by variant thrombins in a thrombin–TM affinity- dependent manner. These data indicate that prothrombin Arg596 missense mutations lead to AT and TM resistance in the variant thrombins and suggest that prothrombin Arg596 is important for AT- and TM- mediated anticoagulation.

scholarly journals Mutation in the relA Gene of Vibrio cholerae Affects In Vitro and In Vivo Expression of Virulence Factors

2003 ◽  
Vol 185 (16) ◽  
pp. 4672-4682 ◽  
Author(s):  
Shruti Haralalka ◽  
Suvobroto Nandi ◽  
Rupak K. Bhadra
Keyword(s):  
Amino Acid ◽  
Vibrio Cholerae ◽  
Mutant Strain ◽  
Virulence Factors ◽  
Antibiotic Production ◽  
Wild Type ◽  
Altered Expression ◽  
Transcript Levels

ABSTRACT The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA VCH) was cloned and sequenced. The relA VCH gene encodes a 738-amino-acid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A ΔrelA::kan allele was generated by replacing ∼31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.

Peptides’ involvement in Pseudomonas putida biofilm formation

2020 ◽  
Author(s):  
Riho Teras ◽  
Hanna Ainelo ◽  
Marge Puhm
Keyword(s):  
Pseudomonas Putida ◽  
Mutant Strain ◽  
Type Strain ◽  
Deletion Mutant ◽  
Wild Type Strain ◽  
Positive Impact ◽  
Proteinase K ◽  
Wild Type ◽  
Positive Effect ◽  
Positively Charged

<p>Pseudomonas putida rapidly forms a biofilm, after which its biomass usually disperses to half its initial amount. We have observed different biofilm dynamics of P. putida in a complex medium LB and a minimal medium M9+glc+CAA and inquired about the importance of extracellular factors for the formation of P. putida biofilm.</p> <p>The proteinaceous component of LB increases the biomass of P. putida biofilm. Supplementation of M9 with tryptone but not CAA increased the biofilm biomass. Proteinase K treatment of LB medium reduced the biomass of P. putida biofilm. At the same time, growth rate or maximum OD of planktic bacteria in used media did not correlate with biofilm biomass of the same media. Thus, peptides appeared to have a positive effect on the biofilm as an extracellular factor and not as a source of C and N.</p> <p>We replaced tryptone in M9 medium with positively charged poly-L-lysine (MW. 1000-5000 Da), negatively charged poly-L-glutaminic acid (MW. 1500-5500 Da) or neutral poly-LD-alanine (MW. 3000-7000). Poly-lysine and poly-glutamic acid had a slight positive effect on the biomass of P. putida wild type strain PSm biofilm and poly-alanine did not affect the biofilm.</p> <p>We have previously shown that overexpression of fis in P. putida strain F15 increases biofilm biomass by increasing the lapA expression, the main adhesin gene of biofilm. Using media similar to that used for the wild-type strain for strain F15, we ascertained that only poly-lysine out of these three polypeptides restored the positive effect of fis-overexpression on the biofilm biomass. At the same time, the positive impact of fis-overexpression was absent in lapA deletion mutant strain, but not in lapF deletion mutant strain.</p> <p>In conclusion, the formation of P. putida biofilm depends on polypeptides in the environment. The enhancing effect of positively charged polypeptides appears to be evident in the presence of LapA, a key factor for P. putida biofilm.</p>

Development of Megakaryocyte-Specific Lentiviral Vectors for Gene Therapy of Platelet Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 5143-5143
Author(s):  
Liesbeth De Waele ◽  
Kathleen Freson ◽  
Chantal Thys ◽  
Christel Van Geet ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Phosphoglycerate Kinase ◽  
Lentiviral Vectors ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Platelet Disorders

Abstract The prevalence of congenital platelet disorders has not been established but for some life-threatening bleeding disorders the current therapies are not adequate, justifying the development of alternative strategies as gene therapy. In the case of platelet dysfunction and thrombocytopenia as described for GATA1 deficiency, potentially lethal internal bleedings can occur. The objective of the study is to develop improved lentiviral vectors for megakaryocyte(MK)-specific long term gene expression by ex vivo transduction of hematopoietic stem cells (HSC) to ultimately use for congenital thrombopathies as GATA1 deficiency. Self-inactivating lentiviral vectors were constructed expressing GFP driven by the murine (m) or human (h) GPIIb promoter. These promoters contain multiple Ets and GATA binding sites directing MK-specificity. To evaluate the cell lineage-specificity and transgene expression potential of the vectors, murine Sca1+ and human CD34+ HSC were transduced in vitro with Lenti-hGPIIb-GFP and Lenti-mGPIIb-GFP vectors. After transduction the HSC were induced to differentiate in vitro along the MK and non-MK lineages. The mGPIIb and hGPIIb promoters drove GFP expression at overall higher levels (20% in murine cells and 25% in human cells) than the ubiquitous CMV (cytomegalovirus) or PGK (phosphoglycerate kinase) promoters, and this exclusively in the MK lineage. Interestingly, in both human and murine HSC the hGPIIb promoter with an extra RUNX and GATA binding site, was more potent in the MK lineage compared to the mGPIIb promoter. Since FLI1 and GATA1 are the main transcription factors regulating GPIIb expression, we tested the Lenti-hGPIIb-GFP construct in GATA1 deficient HSC and obtained comparable transduction efficiencies as for wild-type HSC. To assess the MK-specificity of the lentiviral vectors in vivo, we transplanted irradiated wild-type C57Bl/6 mice with Sca1+ HSC transduced with the Lenti-hGPIIb-GFP constructs. Six months after transplantation we could detect 6% GFP positive platelets without a GFP signal in other cell lineages. Conclusion: In vitro and in vivo MK-specific transgene expression driven by the hGPIIb and mGPIIb promoters could be obtained after ex vivo genetic engineering of HSC by improved lentiviral vectors. Studies are ongoing to study whether this approach can induce phenotypic correction of GATA1 deficient mice by transplantation of ex vivo Lenti-hGPIIb-GATA1 transduced HSC.

Sign in / Sign up

Export Citation Format

Share Document