scholarly journals Proteomic characterization of the Mycobacterium marinum-containing vacuole in Dictyostelium discoideum

2019 ◽  
Author(s):  
Aurélie Guého ◽  
Cristina Bosmani ◽  
Thierry Soldati
Keyword(s):  
Dictyostelium Discoideum ◽  
Model System ◽  
Mycobacterium Marinum ◽  
Close Relative ◽  
Phagosome Maturation ◽  
Fish Pathogen ◽  
Free Living ◽  
Free Living Amoeba ◽  
Professional Phagocyte

ABSTRACTMycobacterium tuberculosis, the causative agent of tuberculosis, is able to manipulate the phagosome compartment where it resides in order to establish a permissive replicative compartment called the Mycobacterium-containing vacuole (MCV). Mycobacterium marinum, a fish pathogen and a close relative of the tuberculosis group, is also able to infect the free-living amoeba and professional phagocyte Dictyostelium discoideum and to manipulate its phagosome maturation. By using this host/pathogen model system, we have established an innovative procedure to isolate MCVs. This procedure allowed us to isolate M. marinum-MCV at 1, 3 and 6 hours post infection to study the early M. marinum-MCV proteome. By using isobaric labelling and mass spectrometry, we quantitatively compared the proteomic composition of those MCVs isolated at different stages of the early infection phase to understand how M. marinum impacts on this compartment to divert it from the normal phagosomal pathway. Furthermore, we also compare the manipulated compartment M. marinum-MCV to non- or less manipulated compartments containing different mycobacteria strains: the non-pathogenic M. smegmatis, the avirulent M. marinum-L1D or the attenuated M. marinum-RD1.

scholarly journals Zn2+ intoxication of Mycobacterium marinum during Dictyostelium discoideum infection is counteracted by induction of the pathogen Zn2+ exporter CtpC

2019 ◽  
Author(s):  
Louise H. Lefrançois ◽  
Vera Kalinina ◽  
Elena Cardenal-Muñoz ◽  
Nabil Hanna ◽  
Hendrik Koliwer-Brandl ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Metal Accumulation ◽  
Chemical Warfare ◽  
Mycobacterium Marinum ◽  
Close Relative ◽  
First Time ◽  
Intracellular Proliferation ◽  
Role Of Zn ◽  
Professional Phagocyte

ABSTRACTMacrophages use diverse strategies to kill or restrict intracellular pathogens. Some of these strategies involve the deprivation of bacteria from (micro)nutrients such as transition metals, and the bacteria intoxication through metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of the human pathogen M. tuberculosis, and its hosts. Here we use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum infection induces the accumulation of Zn2+ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn2+ efflux pumps ZntA and ZntB. In cells lacking the ZntA detoxifying transporter there is further attenuation of M. marinum growth, possibly due to a compensatory efflux of Zn2+ into the MCV. This efflux is presumably carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, M. marinum growth is also impaired in zntB KO cells, where MCVs accumulate less Zn2+. We also demonstrate that M. marinum senses toxic levels of Zn2+ and responds by upregulating its Zn2+ exporter CtpC, which supports bacteria survival under these restrictive conditions. Attenuation of M. marinum intracellular proliferation in zntA and zntB KO cells is accentuated in the absence of CtpC, confirming that mycobacteria face noxious levels of Zn2+. Altogether, we show for the first time that M. marinum infection induces a deleterious Zn2+ elevation in D. discoideum, which is counteracted by the bacteria with the induction of its Zn2+ exporter CtpC.

scholarly journals Disruption of vacuolin microdomains in the host Dictyostelium discoideum increases resistance to Mycobacterium marinum-induced membrane damage and infection

2021 ◽  
Author(s):  
Cristina Bosmani ◽  
Angélique Perret ◽  
Florence Leuba ◽  
Aurélie Guého ◽  
Nabil Hanna ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Membrane Damage ◽  
Mycobacterium Marinum ◽  
Vesicular Trafficking ◽  
Membrane Microdomains ◽  
Membrane Association ◽  
Phagosome Maturation ◽  
Closely Related Species ◽  
Important Host ◽  
And Function

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manipulates the host phagosome maturation pathway to replicate intracellularly. Mycobacterium marinum, a closely-related species, and Dictyostelium discoideum, a social amoeba and alternative phagocytic host, have been used as models to study host-pathogen interactions occurring during mycobacterial infections. Vacuolins, functional homologues of the mammalian flotillins, organize membrane microdomains and play a role in vesicular trafficking. Various pathogens have been reported to manipulate their membrane association and function. During infection of D. discoideum with M. marinum, Vacuolin C was specifically and highly induced and all three vacuolin isoforms were enriched at the mycobacteria-containing-vacuole (MCV). In addition, absence of vacuolins reduced escape from the MCV and conferred resistance to M. marinum infection. Moreover, ESAT-6, the membrane-disrupting virulence factor of M. marinum, was less associated with membranes when vacuolins were absent. Together, these results suggest that vacuolins are important host factors that are manipulated by mycobacteria to inflict membrane damage and escape from their compartment.

Anreicherung und Charakterisierung eines bakteriolytischen Amöbenenzyms / Enrichment and Characterization of a Bacteriolytic Amoebic Enzyme

1970 ◽  
Vol 25 (9) ◽  
pp. 1002-1010 ◽  
Author(s):  
D. Berger ◽  
W. Mannheim
Keyword(s):  
Molecular Weight ◽  
Mode Of Action ◽  
Lytic Activity ◽  
Gram Negative Bacteria ◽  
Free Living ◽  
Ph Optimum ◽  
Gram Negative ◽  
Special Mode ◽  
Free Living Amoeba

Extracts of cultures of a small free-living amoeba, which feeds mainly on gram-negative bacteria, are capable of lysing heat-killed gram-negative bacteria. The lytic activity was enriched 36-fold by means of precipitation with (NH4)2SO4 and of chromatography on Sephadex G-75, followed by ultrafiltration. The activity is less resistant to heat than is lysozyme, and is optimal at 45°C. The pH-optimum is at 6.5. The molecular weight corresponds to that of lysozyme from chicken eggwhite; the isoelecric point is at 10.0. The effect of the amoebic activity on cells of A. metalcaligenes and M. lysodeikticus is different from that of eggwhite lysozyme on the same cells. We conclude that the lytic activity of the amoeba is due to a lysozyme with a special mode of action.

Characterization of a strain-specific inhibitor of cell division from Amoeba cytoplasm

1980 ◽  
Vol 44 (1) ◽  
pp. 273-283
Author(s):  
C.M. Ireland ◽  
S.E. Hawkins
Keyword(s):  
Inhibitory Activity ◽  
Volume Concentration ◽  
Specific Inhibitor ◽  
Void Volume ◽  
Supernatant Fraction ◽  
Main Peak ◽  
Free Living ◽  
Free Living Amoeba ◽  
Microsomal Pellet

The microinjection of cytoplasm taken from one strain of large free-living amoeba into another strain is followed by an incompatibility phenomenon, the inhibition of division amongst the recipient cells. The post-microsomal supernatant fraction from Amoeba discoides (T1D13) injected into A. proteus (T1P) inhibited division in 90% of the injected cells. Further centrifugation of this fraction yielded a pellet which when resuspended and injected, inhibited division in over 95% (and sometimes 100%) of the cells. No inhibitory activity remained in the supernatant after the removal of this pellet. Treatment with 10 micrograms/ml trypsin destroyed the activity of this pellet, while 25 micrograms/ml ribonuclease reduced the inhibitory activity by approximately 40%. Passage of the resuspended post-microsomal pellet through Sephadex G-200 gave one main peak of material which eluted in the void volume. Concentration of this material by either dialysis or lyophilization followed by microinjection into A. proteus showed that this void volume peak contained the inhibitory material, although the most active preparations did not give more than 66% inhibition of division. After elution from Sephadex, the void volume material was analysed by electrophoresis under non-denaturing and denaturing conditions, and by isoelectric focusing. One problem was the loss of inhibitory activity after keeping the pellet at 4 degrees C for 4–5 days, which made further analysis by microinjection difficult. Preliminary experiments using a post-microsomal pellet prepared from Dawson's A. proteus (DP) which inhibited division in A. proteus (T1P) gave a similar profile after Sephadex chromatography and gel electrophoresis.

scholarly journals Zn2+ Intoxication of Mycobacterium marinum during Dictyostelium discoideum Infection Is Counteracted by Induction of the Pathogen Zn2+ Exporter CtpC

mBio ◽  
2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nabil Hanna ◽  
Hendrik Koliwer-Brandl ◽  
Louise H. Lefrançois ◽  
Vera Kalinina ◽  
Elena Cardenal-Muñoz ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Dictyostelium Discoideum ◽  
Bacterial Infections ◽  
Ectopic Expression ◽  
Mycobacterial Infection ◽  
Mycobacterium Marinum ◽  
Close Relative ◽  
Efflux Transporter ◽  
Content Type ◽  
High Concentrations

ABSTRACT Macrophages use diverse strategies to restrict intracellular pathogens, including either depriving the bacteria of (micro)nutrients such as transition metals or intoxicating them via metal accumulation. Little is known about the chemical warfare between Mycobacterium marinum, a close relative of Mycobacterium tuberculosis (Mtb), and its hosts. We use the professional phagocyte Dictyostelium discoideum to investigate the role of Zn2+ during M. marinum infection. We show that M. marinum senses toxic levels of Zn2+ and responds by upregulating one of its isoforms of the Zn2+ efflux transporter CtpC. Deletion of ctpC (MMAR_1271) leads to growth inhibition in broth supplemented with Zn2+ as well as reduced intracellular growth. Both phenotypes were fully rescued by constitutive ectopic expression of the Mtb CtpC orthologue demonstrating that MMAR_1271 is the functional CtpC Zn2+ efflux transporter in M. marinum. Infection leads to the accumulation of Zn2+ inside the Mycobacterium-containing vacuole (MCV), achieved by the induction and recruitment of the D. discoideum Zn2+ efflux pumps ZntA and ZntB. In cells lacking ZntA, there is further attenuation of M. marinum growth, presumably due to a compensatory efflux of Zn2+ into the MCV, carried out by ZntB, the main Zn2+ transporter in endosomes and phagosomes. Counterintuitively, bacterial growth is also impaired in zntB KO cells, in which MCVs appear to accumulate less Zn2+ than in wild-type cells, suggesting restriction by other Zn2+-mediated mechanisms. Absence of CtpC further epistatically attenuates the intracellular proliferation of M. marinum in zntA and zntB KO cells, confirming that mycobacteria face noxious levels of Zn2+. IMPORTANCE Microelements are essential for the function of the innate immune system. A deficiency in zinc or copper results in an increased susceptibility to bacterial infections. Zn2+ serves as an important catalytic and structural cofactor for a variety of enzymes including transcription factors and enzymes involved in cell signaling. But Zn2+ is toxic at high concentrations and represents a cell-autonomous immunity strategy that ensures killing of intracellular bacteria in a process called zinc poisoning. The cytosolic and lumenal Zn2+ concentrations result from the balance of import into the cytosol via ZIP influx transporters and efflux via ZnT transporters. Here, we show that Zn2+ poisoning is involved in restricting Mycobacterium marinum infections. Our study extends observations during Mycobacterium tuberculosis infection and explores for the first time how the interplay of ZnT transporters affects mycobacterial infection by impacting Zn2+ homeostasis.

scholarly journals Bio-characterization of Some Free Living Amoebae in the Surface Water of the City of Yaounde: Relationship to Physico-chemical Parameters of the Medium

2018 ◽  
Vol 6 (1) ◽  
pp. 26
Author(s):  
Kapso Tchouankep Mireille ◽  
Ajeagah Gideon Aghaindum ◽  
Nkeng George Elambo ◽  
Ngassam Pierre
Keyword(s):  
Surface Water ◽  
Suspended Solids ◽  
River System ◽  
Organic Pollution ◽  
Chemical Pollution ◽  
Free Living ◽  
Physico Chemical ◽  
Free Living Amoeba ◽  
High Pollution

A study was carried in Yaounde to evaluate the level of organic pollution in the surface water and to determine the distribution of pathogenic free living amoeba in the aquatic ecosystem. Six points were selected on the Olezoa and the Abiergue streams of Yaounde for our study. Physico-chemical pollution was carried out by standard methods of assessment. The parameters measured were suspended solids, electric conductivity, pH, TDS, Oxydability, BOD5, COD, humidity, Dissolved Oxygen. The free living amoeba were identified by direct observation on microscope and observation after culture on Non Nutritive Agar (NNA)and Page’s Agar Saline Solution (PAS) which are all enriched with Escherichia coli. The data obtained reveal a high pollution of these urban streams of the Mfoundi River system and presents a high densities of telluric amoebae such as Acanthamoeba (21 Cell./L), Balamuthia (11 Cell./L), Naegleria (6 Cell./L), Vanella (17 Cell./L) and Vermamoeba (5 Cell./L). The sizes vary between 12 to 94 μm for the different forms isolated and identified in our study. The presence of amoeba are significantly correlated with hydrometry, temperature, nitrates suspended solids, COD and BOD5 (p <0.001). These organisms in water show contamination of the groundwater by wastewater during the study period.

Balamuthia mandrillaris: A Rare Free-Living Amoeba

2015 ◽  
Vol 2 (1) ◽  
Author(s):  
Jessica Chan ◽  
Mirella Mircescu ◽  
Pratik Shah ◽  
Andrew Liguori ◽  
Aaron Shmookler
Keyword(s):  
Free Living ◽  
Balamuthia Mandrillaris ◽  
Free Living Amoeba
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