scholarly journals Haemophilus influenzaeMicroarrays: Virulence and Vaccines

2002 ◽  
Vol 3 (4) ◽  
pp. 358-361
Author(s):  
Tahir R. Ali ◽  
J. Simon Kroll ◽  
Paul R. Langford
Keyword(s):  
Genome Sequence ◽  
Regulatory Networks ◽  
Living Organism ◽  
Dna Arrays ◽  
Wild Type ◽  
Free Living ◽  
Mutant Strains ◽  
Genome Array ◽  
Global Strategies

In 1995 the genome sequence of theHaemophilus influenzaeKW20 (Rd) strain was published, the first available for a free-living organism. The genome has been invaluable in global strategies to identify certain virulence-related genes, e.g. those involved in LPS synthesis, and also essential genes, but there is a paucity of wholegenome transcriptome studies. We have now constructed a whole-genome array consisting of genes from Rd, additional genes identified in other strains ofH. influenzaeand controls (from eukaryotic sources and other bacteria). We intend to use this array in studies aimed at understanding the bacterium’s basic metabolism and its response to changing environments; deciphering global regulatory networks (by comparison of wild-type and mutant strains); and identifying genes expressedin vivo. The use ofH. influenzaeDNA arrays combined with proteomic approaches will enhance our understanding of the metabolism and virulence of the organism. Additionally, the genome sequence of a non-typableH. influenzaestrain is in progress. The sequence from this isolate will be invaluable not only in identifying potential novel antibiotic targets and putative vaccine candidates but also in the design of a microarray for genome-typing purposes.

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 Roles of rpoN, fliA,and flgR in Expression of Flagella inCampylobacter jejuni

2001 ◽  
Vol 183 (9) ◽  
pp. 2937-2942 ◽  
Author(s):  
Aparna Jagannathan ◽  
Chrystala Constantinidou ◽  
Charles W. Penn
Keyword(s):  
Campylobacter Jejuni ◽  
Genome Sequence ◽  
Sigma Factor ◽  
Transcriptional Activator ◽  
Alternative Sigma Factor ◽  
Wild Type ◽  
Electron Microscopic ◽  
Global Regulation ◽  
Mutant Strains ◽  
Microscopic Studies

ABSTRACT Three potential regulators of flagellar expression present in the genome sequence of Campylobacter jejuni NCTC 11168, the genes rpoN, flgR, andfliA, which encode the alternative sigma factor ς54, the ς54-associated transcriptional activator FlgR, and the flagellar sigma factor ς28, respectively, were investigated for their role in global regulation of flagellar expression. The three genes were insertionally inactivated inC. jejuni strains NCTC 11168 and NCTC 11828. Electron microscopic studies of the wild-type and mutant strains showed that therpoN and flgR mutants were nonflagellate and that the fliA mutant had truncated flagella. Immunoblotting experiments with the three mutants confirmed the roles of rpoN, flgR, and fliA in the expression of flagellin.

Calreticulin Mediates Anesthetic Sensitivity in Drosophila melanogaster 

2003 ◽  
Vol 99 (4) ◽  
pp. 867-875 ◽  
Author(s):  
Sumiko Gamo ◽  
Junya Tomida ◽  
Katsuyuki Dodo ◽  
Dai Keyakidani ◽  
Hitoshi Matakatsu ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Double Mutant ◽  
Developmental Stages ◽  
Northern Blotting ◽  
General Anesthetics ◽  
Wild Type ◽  
Mutant Strains ◽  
Low Expression

Background Various species, e.g., Caenorhabditis elegans, Drosophila melanogaster, and mice, have been used to explore the mechanisms of action of general anesthetics in vivo. The authors isolated a Drosophila mutant, ethas311, that was hypersensitive to diethylether and characterized the calreticulin (crc) gene as a candidate of altered anesthetic sensitivity. Methods Molecular analysis of crc included cloning and sequencing of the cDNA, Northern blotting, and in situ hybridization to accomplish the function of the gene and its mutation. For anesthetic phenotype assay, the 50% anesthetizing concentrations were determined for ethas311, revertants, and double-mutant strains (wild-type crc transgene plus ethas311). Results Expression of the crc 1.4-kb transcript was lower in the mutant ethas311 than in the wild type at all developmental stages. The highest expression at 19 h after pupation was observed in the brain of the wild type but was still low in the mutant at that stage. The mutant showed resistance to isoflurane as well as hypersensitivity to diethylether, whereas it showed the wild phenotype to halothane. Both mutant phenotypes were restored to the wild type in the revertants and double-mutant strains. Conclusion ethas311 is a mutation of low expression of the Drosophila calreticulin gene. The authors demonstrated that hypersensitivity to diethylether and resistance to isoflurane are associated with low expression of the gene. In Drosophila, calreticulin seems to mediate these anesthetic sensitivities, and it is a possible target for diethylether and isoflurane, although the predicted anesthetic targets based on many studies in vitro and in vivo are the membrane proteins, such as ion channels and receptors.

scholarly journals The Entner-Doudoroff Pathway Has Little Effect on Helicobacter pylori Colonization of Mice

2003 ◽  
Vol 71 (5) ◽  
pp. 2920-2923 ◽  
Author(s):  
Amy E. Wanken ◽  
Tyrrell Conway ◽  
Kathryn A. Eaton
Keyword(s):  
Helicobacter Pylori ◽  
Wild Type ◽  
Mutant Strains ◽  
H Pylori ◽  
Type Strains

ABSTRACT Helicobacter pylori mutants deficient in 6-phosphogluconate dehydratase (6PGD) were constructed. Colonization densities were lower and minimum infectious doses were higher for mutant strains than for wild-type strains. In spite of better colonization, however, wild-type strains did not displace the mutant in cocolonization experiments. Loss of 6PGD diminishes the fitness of H. pylori in vivo, but the pathway is nonessential for colonization.

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.

scholarly journals Modulation of the Effects of Lung Immune Response on Bone Marrow by Oral Antigen Exposure

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
P. Xavier-Elsas ◽  
C. L. C. A. Silva ◽  
L. Pinto ◽  
T. Queto ◽  
B. M. Vieira ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Lymphocytes ◽  
Ex Vivo ◽  
Allergen Challenge ◽  
Allergic Airway Inflammation ◽  
Oral Exposure ◽  
Wild Type ◽  
Homologous Antigen ◽  
Mutant Strains

Allergic airway inflammation is attenuated by oral tolerization (oral exposure to allergen, followed by conventional sensitization and challenge with homologous antigen), which decreases airway allergen challenge-induced eosinophilic infiltration of the lungs and bone marrow eosinophilia. We examined its effects on bone marrow eosinophil and neutrophil production. Mice of wild type (BP-2, BALB/c, and C57BL/6) and mutant strains (lacking iNOS or CD95L) were given ovalbumin (OVA) or water (vehicle) orally and subsequently sensitized and challenged with OVA (OVA/OVA/OVA and H2O/OVA/OVA groups, resp.). Anti-OVA IgG and IgE, bone marrow eosinophil and neutrophil numbers, and eosinophil and neutrophil production ex vivo were evaluated. T lymphocytes from OVA/OVA/OVA or control H2O/OVA/OVA donors were transferred into naïve syngeneic recipients, which were subsequently sensitized/challenged with OVA. Alternatively, T lymphocytes were cocultured with bone marrow eosinophil precursors from histocompatible sensitized/challenged mice. OVA/OVA/OVA mice of the BP-2 and BALB/c strains showed, relative to H2O/OVA/OVA controls, significantly decreased bone marrow eosinophil counts and ex vivo eosinopoiesis/neutropoiesis. Full effectiveness in vivo required sequential oral/subcutaneous/intranasal exposures to the same allergen. Transfer of splenic T lymphocytes from OVA/OVA/OVA donors to naive recipients prevented bone marrow eosinophilia and eosinopoiesis in response to recipient sensitization/challenge and supressed eosinopoiesis upon coculture with syngeneic bone marrow precursors from sensitized/challenged donors.

scholarly journals Natural Selection in the Chicken Host Identifies 3-Deoxy-d-manno- Octulosonic Acid Kinase Residues Essential for Phosphorylation of Pasteurella multocida Lipopolysaccharide

2010 ◽  
Vol 78 (9) ◽  
pp. 3669-3677 ◽  
Author(s):  
Marina Harper ◽  
Andrew D. Cox ◽  
Frank St. Michael ◽  
Mark Ford ◽  
Ian W. Wilkie ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Pasteurella Multocida ◽  
Inner Core ◽  
Single Amino Acid ◽  
Sequencing Analysis ◽  
Wild Type ◽  
Fowl Cholera ◽  
Mutant Strains ◽  
High Doses

ABSTRACT Pasteurella multocida is the causative agent of a number of diseases in animals, including fowl cholera. P. multocida strains simultaneously express two lipopolysaccharide (LPS) glycoforms (glycoforms A and B) that differ only in their inner core structure. Glycoform A contains a single 3-deoxy-d-manno-octulosonic acid (Kdo) residue that is phosphorylated by the Kdo kinase, KdkA, whereas glycoform B contains two unphosphorylated Kdo residues. We have previously shown that P. multocida mutants lacking the heptosyltransferase, HptA, produce full-length glycoform B LPS and a large amount of truncated glycoform A LPS, as they cannot add heptose to the glycoform A inner core. These hptA mutants were attenuated in chickens because the truncated LPS made them vulnerable to host defense mechanisms, including antimicrobial peptides. However, here we show that birds inoculated with high doses of the hptA mutant developed fowl cholera and the P. multocida isolates recovered from diseased birds no longer expressed truncated LPS. Sequencing analysis revealed that the in vivo-derived isolates had mutations in kdkA, thereby suppressing the production of glycoform A LPS. Interestingly, a number of the spontaneous KdkA mutant strains produced KdkA with a single amino acid substitution (A112V, R123P, H168Y, or D193N). LPS structural analysis showed that complementation of a P. multocida kdkA mutant with wild-type kdkA restored expression of glycoform A to wild-type levels, whereas complementation with any of the mutated kdkA genes did not. We conclude that in P. multocida KdkA, the amino acids A112, R123, H168, and D193 are critical for Kdo kinase function and therefore for glycoform A LPS assembly.

Metribuzin-Resistant Mutants of Chlamydomonas reinhardii

1984 ◽  
Vol 39 (5) ◽  
pp. 437-439 ◽  
Author(s):  
N. Pucheu ◽  
W. Oettmeier ◽  
U. Heisterkamp ◽  
K. Masson ◽  
G.F. Wildner
Keyword(s):  
Electron Transport ◽  
Photosynthetic Electron Transport ◽  
Wild Type ◽  
Binding Studies ◽  
Molecular Weight Range ◽  
Chlamydomonas Reinhardii ◽  
Thylakoid Membrane Proteins ◽  
Mutant Strains ◽  
Resistant Mutants

Herbicide resistance in Chlamydomonas reinhardii cells was induced by mutagenesis with 5-fluorodeoxyuridine and ethylmethanesulfonate. Four mutant strains were isolated and analyzed for resistance against DCMU-type or phenolic inhibitors of photosynthetic electron transport. The mutants were different in both the extent and the pattern of their resistance: the R/S value, i.e. the ratio of I50 values of the inhibition of photosynthetic electron transport in isolated resistant and susceptible thylakoids, varied for metribuzin from 10 000 to 36. The mutant MZ-1 was resistant against metribuzin, atrazine and DCMU, whereas the mutant MZ-2 showed resistance mainly against metribuzin and atrazine. The mutant MZ-3 was similar to MZ-1, but showed a lesser extent of resistance against DCMU. The mutant MZ-4 showed resistance against metribuzin, but not against atrazine. These results demonstrate that the resistance against one herbicide of the DCMU-type (metribuzin) must not be accompanied by similar resistance against te other inhibitors. Binding studies with radioactively labeled herbicides, [14C]metribuzin, [14C]atrazine and [3H]DCMU, and isolated thylakoids supported these observations. Phosphorylation of thylakoid membrane proteins was studied with wild-type cells and resistant mutants under in vivo conditions in the light. The 32P-labeled main proteins bands were in the molecular weight range of 10-14 kDa, 26-29 kDa, 32-35 kDa and 46-48 kDa. The pattern and the extent of incorporation of 32P were similar for the mutants and the wild-type cells.

scholarly journals Deletion of the Dynein Heavy-Chain Gene DYN1 Leads to Aberrant Nuclear Positioning and Defective Hyphal Development in Candida albicans

2004 ◽  
Vol 3 (6) ◽  
pp. 1574-1588 ◽  
Author(s):  
R. Martin ◽  
A. Walther ◽  
J. Wendland
Keyword(s):  
Candida Albicans ◽  
Heavy Chain ◽  
Time Lapse ◽  
Yeast Cells ◽  
Chain Gene ◽  
Wild Type ◽  
Heavy Chain Gene ◽  
Mutant Strains ◽  
Mutant Phenotypes

ABSTRACT Cytoplasmic dynein is a microtubule-associated minus-end-directed motor protein. CaDYN1 encodes the single dynein heavy-chain gene of Candida albicans. The open reading frames of both alleles of CaDYN1 were completely deleted via a PCR-based approach. Cadyn1 mutants are viable but grow more slowly than the wild type. In vivo time-lapse microscopy was used to compare growth of wild-type (SC5314) and dyn1 mutant strains during yeast growth and after hyphal induction. During yeast-like growth, Cadyn1 strains formed chains of cells. Chromosomal TUB1-GFP and HHF1-GFP alleles were used both in wild-type and mutant strains to monitor the orientation of mitotic spindles and nuclear positioning in C. albicans. In vivo fluorescence time-lapse analyses with HHF1-GFP over several generations indicated defects in dyn1 cells in the realignment of spindles with the mother-daughter axis of yeast cells compared to that of the wild type. Mitosis in the dyn1 mutant, in contrast to that of wild-type yeast cells, was very frequently completed in the mother cells. Nevertheless, daughter nuclei were faithfully transported into the daughter cells, resulting in only a small number of multinucleate cells. Cadyn1 mutant strains responded to hypha-inducing media containing l-proline or serum with initial germ tube formation. Elongation of the hyphal tubes eventually came to a halt, and these tubes showed a defect in the tipward localization of nuclei. Using a heterozygous DYN1/dyn1 strain in which the remaining copy was controlled by the regulatable MAL2 promoter, we could switch between wild-type and mutant phenotypes depending on the carbon source, indicating that the observed mutant phenotypes were solely due to deletion of DYN1.

scholarly journals Increased Pho Regulon Activation Correlates with Decreased Virulence of an Avian Pathogenic Escherichia coli O78 Strain

2010 ◽  
Vol 78 (12) ◽  
pp. 5324-5331 ◽  
Author(s):  
Nicolas Bertrand ◽  
Sébastien Houle ◽  
Guillaume LeBihan ◽  
Édith Poirier ◽  
Charles M. Dozois ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory System ◽  
Rabbit Serum ◽  
Infection Model ◽  
Pho Regulon ◽  
Wild Type ◽  
Mutant Strains ◽  
Pst System

ABSTRACT Avian pathogenic Escherichia coli (APEC) strains are associated with respiratory infections, septicemia, cellulitis, peritonitis, and other conditions, since colibacillosis manifests in many ways. The Pho regulon is jointly controlled by the two-component regulatory system PhoBR and by the phosphate-specific transport (Pst) system. To determine the specific roles of the PhoBR regulon and the Pst system in the pathogenesis of the APEC O78 strain χ7122, different phoBR and pst mutant strains were tested in vivo in chickens and in vitro for virulence traits. Mutations resulting in constitutive activation of the Pho regulon rendered strains more sensitive than the wild type to hydrogen peroxide and to the bactericidal effects of rabbit serum. In addition, production of type 1 fimbriae was also impaired in these strains. Using a chicken competitive infection model, all PhoB constitutive mutants were outcompeted by the wild-type parent, including strains containing a functional Pst system. Cumulative inactivation of the Pst system and the PhoB regulator resulted in a restoration of virulence. In addition, loss of the PhoB regulator alone did not affect virulence in the chicken infection model. Interestingly, the level of attenuation of the mutant strains correlated directly with the level of activation of the Pho regulon. Overall, results indicate that activation of the Pho regulon rather than phosphate transport by the Pst system plays a major role in the attenuation of the APEC O78 strain χ7122.

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