scholarly journals Contribution of the Shigella flexneri Sit, Iuc, and Feo Iron Acquisition Systems to Iron Acquisition In Vitro and in Cultured Cells

2003 ◽  
Vol 71 (4) ◽  
pp. 1919-1928 ◽  
Author(s):  
L. J. Runyen-Janecky ◽  
S. A. Reeves ◽  
E. G. Gonzales ◽  
S. M. Payne
Keyword(s):  
Iron Transport ◽  
Cultured Cells ◽  
Iron Acquisition ◽  
Shigella Flexneri ◽  
Luria Broth ◽  
Iron Chelator ◽  
Wild Type ◽  
Cell Monolayers ◽  
Serovar Typhimurium

ABSTRACT Shigella flexneri possesses multiple iron acquisition systems, including proteins involved in the synthesis and uptake of siderophores and the Feo system for ferrous iron utilization. We identified an additional S. flexneri putative iron transport gene, sitA, in a screen for S. flexneri genes that are induced in the eukaryotic intracellular environment. sitA was present in all Shigella species and in most enteroinvasive Escherichia coli strains but not in any other E. coli isolates tested. The sit locus consists of four genes encoding a potential ABC transport system. The deduced amino acid sequence of the S. flexneri sit locus was homologous to the Salmonella enterica serovar Typhimurium Sit and Yersinia pestis Yfe systems, which mediate both manganese and iron transport. The S. flexneri sit promoter was repressed by either iron or manganese, and the iron repression was partially dependent upon Fur. A sitA::cam mutation was constructed in S. flexneri. The sitA mutant showed reduced growth, relative to the wild type, in Luria broth containing an iron chelator but formed wild-type plaques on Henle cell monolayers, indicating that the sitA mutant was able to acquire iron and/or manganese in the host cell. However, mutants defective in two of these iron acquisition systems (sitA iucD, sitA feoB, and feoB iucD) formed slightly smaller plaques on Henle cell monolayers. A strain carrying mutations in sitA, feoB, and iucD did not form plaques on Henle cell monolayers.

A DNA adenine methylase mutant of Shigella flexneri shows no significant attenuation of virulence

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 1073-1078 ◽  
Author(s):  
Yasuko Honma ◽  
Reinaldo E. Fernández ◽  
Anthony T. Maurelli
Keyword(s):  
Shigella Flexneri ◽  
Spontaneous Mutation ◽  
Bacterial Pathogen ◽  
Wild Type ◽  
Vaccine Strategy ◽  
Cell Monolayers ◽  
Dna Adenine Methylase ◽  
Significant Attenuation

Mutants of Salmonella defective in DNA adenine methylase (dam) have been reported to be attenuated for virulence and to provide protective immunity when used as vaccine strains. To determine whether these observations could be extended to Shigella, a dam mutant of Shigella flexneri 2a was characterized and examined for the role of dam in pathogenesis. The Shigella dam mutant showed some unique characteristics; however, it retained virulence in vivo as well as in vitro. The mutant invaded cultured L2 monolayer cells as efficiently as the wild-type parent, but its intracellular growth was suppressed up to 7 h post-invasion. Furthermore, the invading dam mutant formed smaller plaques in cell monolayers compared to the parent strain. However, the mutant produced keratoconjunctivitis in the Sereny test in guinea pigs only slightly more slowly than the wild-type. While the effect of the dam mutation on virulence was modest, the rate of spontaneous mutation in the dam mutant was 1000-fold greater compared with the wild-type. The virulence and high mutability displayed by the dam mutant of Sh. flexneri suggest that a general anti-bacterial pathogen vaccine strategy based on mutations in dam needs to be re-evaluated.

scholarly journals Role and Regulation of the Shigella flexneri Sit and MntH Systems

2006 ◽  
Vol 74 (8) ◽  
pp. 4666-4672 ◽  
Author(s):  
Laura Runyen-Janecky ◽  
Ellyn Dazenski ◽  
Stephanie Hawkins ◽  
Lisa Warner
Keyword(s):  
Hydrogen Peroxide ◽  
Wild Type Strain ◽  
Shigella Flexneri ◽  
Luria Broth ◽  
Divalent Metal ◽  
Wild Type ◽  
Cell Monolayers ◽  
Metal Chelator ◽  
Activated Macrophage ◽  
Genes Encoding

ABSTRACT Shigella flexneri possesses at least two putative high-affinity manganese acquisition systems, SitABCD and MntH. Mutations in the genes encoding the components of both of these systems were constructed in S. flexneri. The sitA mntH mutant showed reduced growth, relative to the wild type, in Luria broth (L broth) containing the divalent metal chelator ethylene diamino-o-dihydroxyphenyl acetic acid, and the addition of either iron or manganese restored growth to the level of the wild-type strain. Although the sitA mntH mutant was not defective in surviving exposure to superoxide generators, it was defective in surviving exposure to hydrogen peroxide. The sitA mntH mutant formed wild-type plaques on Henle cell monolayers but had a reduced ability to survive in activated macrophage lines. Expression of the S. flexneri sit and mntH promoters was higher when Shigella was in Henle cells than when it was in L broth. Expression of both the sit and mntH promoters was repressed by either iron or manganese, and this repression was partially dependent upon Fur and MntR, respectively. The mntH promoter, but not the sit promoter, exhibited OxyR-dependent induction in the presence of hydrogen peroxide.

scholarly journals SseJ Deacylase Activity by Salmonella enterica Serovar Typhimurium Promotes Virulence in Mice

2005 ◽  
Vol 73 (10) ◽  
pp. 6249-6259 ◽  
Author(s):  
Maikke B. Ohlson ◽  
Kerry Fluhr ◽  
Cheryl L. Birmingham ◽  
John H. Brumell ◽  
Samuel I. Miller
Keyword(s):  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Cultured Cells ◽  
Effector Protein ◽  
Null Mutant ◽  
Wild Type ◽  
Active Site Residues ◽  
Serovar Typhimurium ◽  
Phagosomal Membrane

ABSTRACT Salmonella enterica serovar Typhimurium utilizes a type III secretion system (TTSS) encoded on Salmonella pathogenicity island-2 (SPI2) to promote intracellular replication during infection, but little is known about the molecular function of SPI2-translocated effectors and how they contribute to this process. SseJ is a SPI2 TTSS effector protein that is homologous to enzymes called glycerophospholipid-cholesterol acyltransferases and, following translocation, localizes to the Salmonella-containing vacuole and Salmonella-induced filaments. Full virulence requires SseJ, as sseJ null mutants exhibit decreased replication in cultured cells and host tissues. This work demonstrates that SseJ is an enzyme with deacylase activity in vitro and identifies three active-site residues. Catalytic SseJ mutants display wild-type translocation and subcellular localization but fail to complement the virulence defect of an sseJ null mutant. In contrast to the wild type, SseJ catalytic mutants fail to down regulate Salmonella-induced filament formation and fail to restore the sifA null mutant phenotype of loss of phagosomal membrane to sifA sseJ null double mutants, suggesting that wild-type SseJ modifies the vacuolar membrane. This is the first demonstration of an enzymatic activity for a SPI2 effector protein and provides support for the hypothesis that the deacylation of lipids on the Salmonella-containing vacuole membrane is important to bacterial pathogenesis.

scholarly journals Shigella flexneri DegP Facilitates IcsA Surface Expression and Is Required for Efficient Intercellular Spread

2002 ◽  
Vol 70 (11) ◽  
pp. 6355-6364 ◽  
Author(s):  
Georgiana E. Purdy ◽  
Mei Hong ◽  
Shelley M. Payne
Keyword(s):  
Actin Polymerization ◽  
Elevated Temperatures ◽  
Cultured Cells ◽  
Shigella Flexneri ◽  
Surface Expression ◽  
Wild Type ◽  
Cell Monolayers ◽  
E Coli ◽  
Induced Apoptosis ◽  
Intercellular Spread

ABSTRACT A degP mutant of Shigella flexneri was identified in a screen for insertion mutants that invaded cultured cells but did not form wild-type plaques in monolayers. The degP mutant SM1100 invaded Henle cells at wild-type levels and induced apoptosis in macrophages but formed smaller plaques than those formed by wild-type S. flexneri in confluent monolayers of Henle and Caco-2 cells. The proportion of SM1100 bacteria with IcsA localized to the bacterial pole, a process required for actin polymerization into actin “tails,” was reduced compared to results with wild-type bacteria. The reduction in proper IcsA localization may account for the reduced plaque size of the degP mutant. Although DegP is a protease, the protease activity of S. flexneri DegP was not required for IcsA localization or the formation of plaques in Henle cell monolayers. DegP was also required for efficient polar IcsA localization in E. coli expressing icsA. In addition, the growth or survival of SM1100 was compromised compared to that of the wild type at elevated temperatures and in acidic conditions.

Transmembrane prolyl 4-hydroxylase is a fourth prolyl 4-hydroxylase regulating EPO production and erythropoiesis

Blood ◽  
2012 ◽  
Vol 120 (16) ◽  
pp. 3336-3344 ◽  
Author(s):  
Anu Laitala ◽  
Ellinoora Aro ◽  
Gail Walkinshaw ◽  
Joni M. Mäki ◽  
Maarit Rossi ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mrna Level ◽  
Hypoxia Inducible Factor ◽  
Mrna Levels ◽  
Wild Type ◽  
Α Subunit ◽  
Hepcidin Expression ◽  
In The Wild ◽  
Hepcidin Mrna

AbstractAn endoplasmic reticulum transmembrane prolyl 4-hydroxylase (P4H-TM) is able to hydroxylate the α subunit of the hypoxia-inducible factor (HIF) in vitro and in cultured cells, but nothing is known about its roles in mammalian erythropoiesis. We studied such roles here by administering a HIF-P4H inhibitor, FG-4497, to P4h-tm−/− mice. This caused larger increases in serum Epo concentration and kidney but not liver Hif-1α and Hif-2α protein and Epo mRNA levels than in wild-type mice, while the liver Hepcidin mRNA level was lower in the P4h-tm−/− mice than in the wild-type. Similar, but not identical, differences were also seen between FG-4497–treated Hif-p4h-2 hypomorphic (Hif-p4h-2gt/gt) and Hif-p4h-3−/− mice versus wild-type mice. FG-4497 administration increased hemoglobin and hematocrit values similarly in the P4h-tm−/− and wild-type mice, but caused higher increases in both values in the Hif-p4h-2gt/gt mice and in hematocrit value in the Hif-p4h-3−/− mice than in the wild-type. Hif-p4h-2gt/gt/P4h-tm−/− double gene-modified mice nevertheless had increased hemoglobin and hematocrit values without any FG-4497 administration, although no such abnormalities were seen in the Hif-p4h-2gt/gt or P4h-tm−/− mice. Our data thus indicate that P4H-TM plays a role in the regulation of EPO production, hepcidin expression, and erythropoiesis.

scholarly journals Production of the Siderophore 2,3-Dihydroxybenzoic Acid Is Required for Wild-Type Growth of Brucella abortus in the Presence of Erythritol under Low-Iron Conditions In Vitro

2003 ◽  
Vol 71 (5) ◽  
pp. 2927-2832 ◽  
Author(s):  
Bryan H. Bellaire ◽  
Philip H. Elzer ◽  
Cynthia L. Baldwin ◽  
R. Martin Roop
Keyword(s):  
Brucella Abortus ◽  
Reproductive Tract ◽  
Iron Acquisition ◽  
Energy Sources ◽  
Growth Defect ◽  
Wild Type ◽  
Dihydroxybenzoic Acid ◽  
Experimental Findings ◽  
The Relationship

ABSTRACT Production of the siderophore 2,3-dihyroxybenzoic acid (2,3-DHBA) is required for the wild-type virulence of Brucella abortus in cattle. A possible explanation for this requirement was uncovered when it was determined that a B. abortus dhbC mutant (BHB1) defective in 2,3-DHBA production displays marked growth restriction in comparison to its parent strain, B. abortus 2308, when cultured in the presence of erythritol under low-iron conditions. This phenotype is not displayed when these strains are cultured under low-iron conditions in the presence of other readily utilizable carbon and energy sources. The addition of either exogenous 2,3-DHBA or FeCl3 relieves this growth defect, suggesting that the inability of the B. abortus dhbC mutant to display wild-type growth in the presence of erythritol under iron-limiting conditions is due to a defect in iron acquisition. Restoring 2,3-DHBA production to the B. abortus dhbC mutant by genetic complementation abolished the erythritol-specific growth defect exhibited by this strain in low-iron medium, verifying the relationship between 2,3-DHBA production and efficient growth in the presence of erythritol under low-iron conditions. The positive correlation between 2,3-DHBA production and growth in the presence of erythritol was further substantiated by the observation that the addition of erythritol to low-iron cultures of B. abortus 2308 stimulated the production of 2,3-DHBA by increasing the transcription of the dhbCEBA operon. Correspondingly, the level of exogenous iron needed to repress dhbCEBA expression in B. abortus 2308 was also greater when this strain was cultured in the presence of erythritol than that required when it was cultured in the presence of any of the other readily utilizable carbon and energy sources tested. The tissues of the bovine reproductive tract are rich in erythritol during the latter stages of pregnancy, and the ability to metabolize erythritol is thought to be important to the virulence of B. abortus in pregnant ruminants. Consequently, the experimental findings presented here offer a plausible explanation for the attenuation of the B. abortus 2,3-DHBA-deficient mutant BHB1 in pregnant ruminants.

scholarly journals Comparison between Pseudomonas aeruginosa siderophores and desferrioxamine for iron acquisition from ferritin

2010 ◽  
Vol 4 (4) ◽  
pp. 631-635 ◽  
Author(s):  
Somporn Srifuengfung ◽  
Susan Assanasen ◽  
Malulee Tuntawiroon ◽  
Sumonrat Meejanpetch
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Iron Acquisition ◽  
Iron Chelator ◽  
Dialysis Membrane ◽  
In Vitro Experiment ◽  
Iron Mobilization ◽  
Ph Values ◽  
Minimum Medium ◽  
Stimulation Of

Abstract Background: Siderophore is an iron chelator produced by microorganism. Pseudomonas aeruginosa produces two siderophores (pyoverdin and pyochelin). Desferrioxamine is a siderophore used in thalassemia patients to treat an iron overload of vital organs. Objective: Compare the ability of pyoverdin, pyochelin, and desferrioxamine for iron mobilization from ferritin. Materials and Methods: In vitro experiment, the ability of P. aeruginosa siderophores and desferrioxamine for iron mobilization from ferritin was compared by using a dialysis membrane assay at pH values of 7.4 and 6.0. Stimulation of P. aeruginosa PAO1 growth by all siderophores was studied in glucose minimum medium. Results: All three compounds were capable of iron mobilization at both pHs. At pH 6.0, the most effectiveness compound was desferrioxamine (31.6%), followed by pyoverdin (21.5%) and pyochelin (13.7%) compared on weight basis, each at 10 μg/mL. At equimolar concentration, their activities were desferrioxamine (38.5±1.2%), followed by pyoverdin (32.0±4.8%) and pyochelin (26.7±1.9%), respectively. Conclusion: The most effective compound in iron mobilization from ferritin was desferrioxamine, followed by pyoverdin and pyochelin respectively.

scholarly journals The Pseudomonas aeruginosa product pyochelin interferes with Trypanosoma cruzi infection and multiplication in vitro

2020 ◽  
Vol 114 (7) ◽  
pp. 492-498 ◽  
Author(s):  
Gabriele Sass ◽  
Laura C Miller Conrad ◽  
Terrence-Thang H Nguyen ◽  
David A Stevens
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Trypanosoma Cruzi ◽  
Mammalian Cells ◽  
Iron Acquisition ◽  
Vero Cells ◽  
Dependent Manner ◽  
Wild Type ◽  
Current Standard ◽  
Cruzi Infection

Abstract Background Bacteria are sources of numerous molecules used in treatment of infectious diseases. We investigated effects of molecules produced by 26 Pseudomonas aeruginosa strains against infection of mammalian cell cultures with Trypanosoma cruzi, the aetiological agent of Chagas disease. Methods Vero cells were infected with T. cruzi in the presence of wild-type P. aeruginosa supernatants or supernatants of mutants with defects in the production of various virulence, quorum sensing and iron acquisition factors. Quantification of T. cruzi infection (percentage of infected cells) and multiplication (number of amastigotes per infected cell) was performed and cell viability was determined. Results Wild-type P. aeruginosa products negatively affected T. cruzi infection and multiplication in a dose-dependent manner, without evident toxicity for mammalian cells. PvdD/pchE mutation (loss of the P. aeruginosa siderophores pyoverdine and pyochelin) had the greatest impact on anti–T. cruzi activity. Negative effects on T. cruzi infection by pure pyochelin, but not pyoverdine, or other P. aeruginosa exoproducts studied, were quantitatively similar to the effects of benznidazole, the current standard therapy against T. cruzi. Conclusions The P. aeruginosa product pyochelin showed promising activity against T. cruzi and might become a new lead molecule for therapy development.

scholarly journals Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

2001 ◽  
Vol 69 (12) ◽  
pp. 7413-7418 ◽  
Author(s):  
Tahar van der Straaten ◽  
Angela van Diepen ◽  
Kitty Kwappenberg ◽  
Sjaak van Voorden ◽  
Kees Franken ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Function ◽  
Salmonella Enterica ◽  
Host Cells ◽  
Wild Type ◽  
Intracellular Replication ◽  
Oxygen Intermediates ◽  
Serovar Typhimurium

ABSTRACT Upon contact with host cells, the intracellular pathogenSalmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104 to 107bacteria in C3H/HeN and 101 to 104 bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

scholarly journals Mammalian Dynamin-like Protein DLP1 Tubulates Membranes

2001 ◽  
Vol 12 (9) ◽  
pp. 2894-2905 ◽  
Author(s):  
Yisang Yoon ◽  
Kelly R. Pitts ◽  
Mark A. McNiven
Keyword(s):  
Cultured Cells ◽  
Immunogold Labeling ◽  
Wild Type ◽  
Tubule Formation ◽  
Defective Mutant ◽  
Ring Structures ◽  
Membrane Tubules ◽  
Large Gtpases

Dynamins are large GTPases with mechanochemical properties that are known to constrict and tubulate membranes. A recently identified mammalian dynamin-like protein (DLP1) is essential for the proper cellular distribution of mitochondria and the endoplasmic reticulum in cultured cells. In this study, we investigated the ability of DLP1 to remodel membranes similar to conventional dynamin. We found that the expression of a GTPase-defective mutant, DLP1-K38A, in cultured cells led to the formation of large cytoplasmic aggregates. Electron microscopy (EM) of cells expressing DLP1-K38A revealed that these aggregates were comprised of membrane tubules of a consistent diameter. High-magnification EM revealed the presence of many regular striations along individual membrane tubules, and immunogold labeling confirmed the association of DLP1 with these structures. Biochemical experiments with the use of recombinant DLP1 and labeled GTP demonstrated that DLP1-K38A binds but does not hydrolyze or release GTP. Furthermore, the affinity of DLP1-K38A for membrane is increased compared with wild-type DLP1. To test whether DLP1 could tubulate membrane in vitro, recombinant DLP1 was combined with synthetic liposomes and nucleotides. We found that DLP1 protein alone assembled into sedimentable macromolecular structures in the presence of guanosine-5′-O-(3-thio)triphosphate (GTPγS) but not GTP. EM of the GTPγS-treated DLP1 revealed clusters of stacked helical ring structures. When liposomes were included with DLP1, formation of long membrane tubules similar in size to those formed in vivo was observed. Addition of GTPγS greatly enhanced membrane tubule formation, suggesting the GTP-bound form of DLP1 deforms liposomes into tubules as the DLP1-K38A does in vivo. These results provide the first evidence that the dynamin family member, DLP1, is able to tubulate membranes both in living cells and in vitro. Furthermore, these findings also indicate that despite the limited homology to conventional dynamins (35%) these proteins remodel membranes in a similar manner.

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