scholarly journals Legionella pneumophila feoAB Promotes Ferrous Iron Uptake and Intracellular Infection

2002 ◽  
Vol 70 (10) ◽  
pp. 5659-5669 ◽  
Author(s):  
Marianne Robey ◽  
Nicholas P. Cianciotto
Keyword(s):  
Yeast Extract ◽  
Legionella Pneumophila ◽  
Ferrous Iron ◽  
Iron Uptake ◽  
Iron Transport ◽  
Growth Defect ◽  
Wild Type ◽  
Extract Agar ◽  
Ferrous Iron Transport ◽  
Yeast Extract Agar

ABSTRACT In order to determine the role of ferrous iron transport in Legionella pathogenesis, we identified and mutated the feoB gene in virulent Legionella pneumophila strain 130b. As it is in Escherichia coli, the L. pneumophila feoB gene was contained within a putative feoAB operon. L. pneumophila feoB insertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss of L. pneumophila FeoB. However, the L. pneumophila feoB mutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition of feoB in trans. In unsupplemented buffered yeast extract broth, the feoB mutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2′-dipyridyl. The feoB mutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of the feoB mutant was noted within iron-depleted Hartmannella vermiformis amoebae and human U937 cell macrophages. The reduced intracellular infectivity of the feoB mutant was complemented by the introduction of a plasmid containing feoAB. The L. pneumophila feoB gene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate that L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.

scholarly journals Secreted Pyomelanin of Legionella pneumophila Promotes Bacterial Iron Uptake and Growth under Iron-Limiting Conditions

2013 ◽  
Vol 81 (11) ◽  
pp. 4182-4191 ◽  
Author(s):  
Huaixin Zheng ◽  
Christa H. Chatfield ◽  
Mark R. Liles ◽  
Nicholas P. Cianciotto
Keyword(s):  
Legionella Pneumophila ◽  
Ferrous Iron ◽  
Iron Uptake ◽  
Iron Transport ◽  
Ferric Iron ◽  
Iron Acquisition ◽  
Ferric Hydroxide ◽  
Supernatant Fraction ◽  
Content Type ◽  
Ferrous Iron Transport

ABSTRACTIron acquisition is critical to the growth and virulence ofLegionella pneumophila. Previously, we found thatL. pneumophilauses both a ferrisiderophore pathway and ferrous iron transport to obtain iron. We now report that two molecules secreted byL. pneumophila, homogentisic acid (HGA) and its polymerized variant (HGA-melanin, a pyomelanin), are able to directly mediate the reduction of various ferric iron salts. Furthermore, HGA, synthetic HGA-melanin, and HGA-melanin derived from bacterial supernatants enhanced the ability ofL. pneumophilaand other species ofLegionellato take up radiolabeled iron. Enhanced iron uptake was not observed with a ferrous iron transport mutant. Thus, HGA and HGA-melanin mediate ferric iron reduction, with the resulting ferrous iron being available to the bacterium for uptake. Upon further testing ofL. pneumophilaculture supernatants, we found that significant amounts of ferric and ferrous iron were associated with secreted HGA-melanin. Importantly, a pyomelanin-containing fraction obtained from a wild-type culture supernatant was able to stimulate the growth of iron-starved legionellae. That the corresponding supernatant fraction obtained from a nonpigmented mutant culture did not stimulate growth demonstrated that HGA-melanin is able to both promote iron uptake and enhance growth under iron-limiting conditions. Indicative of a complementary role in iron acquisition, HGA-melanin levels were inversely related to the levels of siderophore activity. Compatible with a role in the ecology and pathogenesis ofL. pneumophila, HGA and HGA-melanin were effective at reducing and releasing iron from both insoluble ferric hydroxide and the mammalian iron chelates ferritin and transferrin.

scholarly journals Toward a mechanistic understanding of Feo-mediated ferrous iron uptake

Metallomics ◽  
2018 ◽  
Vol 10 (7) ◽  
pp. 887-898 ◽  
Author(s):  
Alexandrea E. Sestok ◽  
Richard O. Linkous ◽  
Aaron T. Smith
Keyword(s):  
Ferrous Iron ◽  
Iron Uptake ◽  
Iron Transport ◽  
Structure And Function ◽  
Ferrous Iron Transport ◽  
Mechanistic Understanding ◽  
And Function

The ferrous iron transport (Feo) system is the predominant mode of bacterial Fe2+import. Advancements in the structure and function of FeoB provide glimpses into the mechanism of Fe2+uptake.

Charcoal-yeast extract agar: primary isolation medium for Legionella pneumophila.

1979 ◽  
Vol 10 (4) ◽  
pp. 437-441 ◽  
Author(s):  
J C Feeley ◽  
R J Gibson ◽  
G W Gorman ◽  
N C Langford ◽  
J K Rasheed ◽  
...  
Keyword(s):  
Yeast Extract ◽  
Legionella Pneumophila ◽  
Isolation Medium ◽  
Primary Isolation ◽  
Extract Agar ◽  
Yeast Extract Agar

The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 167-182 ◽  
Author(s):  
Urs Albers ◽  
Katrin Reus ◽  
Howard A. Shuman ◽  
Hubert Hilbi
Keyword(s):  
Legionella Pneumophila ◽  
Lipid A ◽  
Sequence Similarity ◽  
Acanthamoeba Castellanii ◽  
Growth Defect ◽  
Wild Type ◽  
Intracellular Growth ◽  
Plate Test ◽  
Raw264.7 Macrophages ◽  
Mutant Strains

Legionella pneumophila is a bacterial parasite of freshwater amoebae which also grows in alveolar macrophages and thus causes the potentially fatal pneumonia Legionnaires' disease. Intracellular growth within amoebae and macrophages is mechanistically similar and requires the Icm/Dot type IV secretion system. This paper reports the development of an assay, the amoebae plate test (APT), to analyse growth of L. pneumophila wild-type and icm/dot mutant strains spotted on agar plates in the presence of Acanthamoeba castellanii. In the APT, wild-type L. pneumophila formed robust colonies even at high dilutions, icmT, -R, -P or dotB mutants failed to grow, and icmS or -G mutants were partially growth defective. The icmS or icmG mutant strains were used to screen an L. pneumophila chromosomal library for genes that suppress the growth defect in the presence of the amoebae. An icmS suppressor plasmid was isolated that harboured the icmS and flanking icm genes, indicating that this plasmid complements the intracellular growth defect of the mutant. In contrast, different icmG suppressor plasmids rendered the icmG mutant more cytotoxic for A. castellanii without enhancing intracellular multiplication in amoebae or RAW264.7 macrophages. Deletion of individual genes in the suppressor plasmids inserts identified lcs (Legionella cytotoxic suppressor) -A, -B, -C and -D as being required for enhanced cytotoxicity of an icmG mutant strain. The corresponding proteins show sequence similarity to hydrolases, NlpD-related metalloproteases, lipid A disaccharide synthases and ABC transporters, respectively. Overexpression of LcsC, a putative paralogue of the lipid A disaccharide synthase LpxB, increased cytotoxicity of an icmG mutant but not that of other icm/dot or rpoS mutant strains against A. castellanii. Based on sequence comparison and chromosomal location, lcsB and lcsC probably encode enzymes involved in cell wall maintenance and peptidoglycan metabolism. The APT established here may prove useful to identify other bacterial factors relevant for interactions with amoeba hosts.

Comparison of agar-based methods for the isolation and enumeration of heterotrophic bacteria with the new Multidose IDEXX(tm) SimPlate method

2004 ◽  
Vol 50 (1) ◽  
pp. 277-280
Author(s):  
M. Vulindlu ◽  
A. Charlett ◽  
S. Surman ◽  
J.V. Lee
Keyword(s):  
Yeast Extract ◽  
Heterotrophic Bacteria ◽  
Treatment Plant ◽  
Plant Performance ◽  
Extract Agar ◽  
Quality Of Water ◽  
Heterotrophic Microorganisms ◽  
External Sources ◽  
Conventional Methods ◽  
Yeast Extract Agar

Pour and spread plates are the conventional methods of choice for the isolation and enumeration of heterotrophic microorganisms in treated water supplies. The tests are performed at 22°C and 37°C for 72 h and 48 h respectively. Counts at 22°C are associated with pollution of water systems from external sources, while counts at 37°C are used as an indication of treatment plant performance and the deterioration of the general quality of water. Conventional methods using Yeast Extract Agar for a pour plate and R2A agar for a spread plate were compared with the multidose IDEXXTM SimPlate method for the isolation and enumeration of heterotrophic bacteria in water. SimPlate gave a significantly higher count on average than the conventional methods. The R2A method showed the next highest count, being significantly higher than Yeast Extract Agar. In addition, unlike the pour and spread plate methods, SimPlate was easier to use, reduced labour, and the test results were far easier to read.

Functional properties of transfected human DMT1 iron transporter

2000 ◽  
Vol 279 (6) ◽  
pp. G1265-G1273 ◽  
Author(s):  
Mark T. Worthington ◽  
Lauren Browne ◽  
Emily H. Battle ◽  
Roger Qi Luo
Keyword(s):  
Transition Metals ◽  
Iron Uptake ◽  
Iron Transport ◽  
Potential Role ◽  
Human Gene ◽  
Wild Type ◽  
Ph Optimum ◽  
Transfected Cells ◽  
Proton Current ◽  
Intestinal Enterocytes

Recently, mutation of the DMT1 gene has been discovered to cause ineffective intestinal iron uptake and abnormal body iron metabolism in the anemic Belgrade rat and mkmouse. DMT1 transports first-series transition metals, but only iron turns on an inward proton current. The process of iron transport was studied by transfection of human DMT1 into the COS-7 cell line. Native and epitope-tagged human DMT1 led to increased iron uptake. The human gene with the Belgrade rat mutation was found to have one-fifth of the activity of the wild-type protein. The pH optimum of human DMT1 iron uptake was 6.75, which is equivalent to the pH of the duodenal brush border. The transporter demonstrates uptake without saturation from 0 to 50 μM iron, recapitulating earlier studies of isolated intestinal enterocytes. Diethylpyrocarbonate inhibition of iron uptake in DMT1-transfected cells suggests a functional role for histidine residues. Finally, a model is presented that incorporates the selectivity of the DMT1 transporter for transition metals and a potential role for the inward proton current.

Nannocystis exedens: A Potential Biocompetitive Agent against Aspergillus flavus and Aspergillus parasiticus

2001 ◽  
Vol 64 (7) ◽  
pp. 1030-1034 ◽  
Author(s):  
WILLIE J. TAYLOR ◽  
FRANCES A. DRAUGHON
Keyword(s):  
Aspergillus Flavus ◽  
Yeast Extract ◽  
Biocontrol Agent ◽  
Aspergillus Parasiticus ◽  
Extract Agar ◽  
Potential Biocontrol Agent ◽  
Aflatoxigenic Fungi ◽  
Close Proximity ◽  
Zones Of Inhibition ◽  
Yeast Extract Agar

This study examined the potential for controlling toxigenic Aspergillus flavus and Aspergillus parasiticus by biological means using a myxobacterium commonly found in soil. The ability of Nannocystis exedens to antagonize A. flavus ATCC 16875, A. flavus ATCC 26946, and A. parasiticus NRRL 3145 was discovered. Cultures of aflatoxigenic fungi were grown on 0.3% Trypticase peptone yeast extract agar for 14 days at 28°C. When N. exedens was grown in close proximity with an aflatoxigenic mold, zones of inhibition (10 to 20 mm) developed between the bacterium and mold colony. A flattening of the mold colony on the sides nearest N. exedens and general stunting of growth of the mold colony were also observed. When N. exedens was added to the center of the cross-streak of a mold colony, lysis of the colony by the bacterium was observed after 24 h. Microscopic observations revealed that N. exedens grew on spores, germinating spores, hyphae, and sclerotia of the molds. These results indicate that N. exedens may be a potential biocontrol agent against A. flavus and A. parasiticus.

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