Leishmania chagasi: Homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model

Yao, Chaoqun; Chen, Yani; Sudan, Bayan; Donelson, John E.; Wilson, Mary E.

doi:10.1016/j.exppara.2007.06.012

Cited by 49 publications

(80 citation statements)

References 29 publications

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“…Consistent with our earlier report, 87.4% Ϯ 0.1% of the cells were viable after exposure to 50% fresh human serum for 30 min at 37°C (Fig. 1A) (18). Cells treated with 10 mM M␤CD were nearly the same as controls, exhibiting 85.3% Ϯ 4.9% viability in 50% serum.…”

Section: Resultssupporting

confidence: 80%

“…One plausible explanation is that M␤CD-treated cells bind to alternate macrophage receptors via ligands exposed by sterol depletion or via the enhanced opsonic C3, such as CR1 or mannose receptor (55). A parallel observation is made in the case of L. infantum chagasi logarithmic-phase promastigotes, which contain only one-fourteenth the level of steady-state MSPs of stationary-phase promastigotes (13) but contain only one-third of the MSPs of metacyclic promastigotes (18), yet their phagocytosis by MDMs is more efficient than that of metacyclic promastigotes (38).…”

Section: Discussionmentioning

confidence: 69%

“…L. infantum chagasi metacyclic promastigotes were isolated from late-stationary-phase promastigote cultures using discontinuous Ficoll gradients as previously described (17,18) and were left untreated (control) or partially depleted of membrane sterols by 60 min of incubation in increasing concentrations of M␤CD (10 to 50 mM). Subsequent measure of viability with propidium iodide and flow cytometry suggested that control promastigotes were highly resistant to complement-mediated lysis.…”

Section: Resultsmentioning

confidence: 99%

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Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

et al. 2013

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The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (M␤CD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. M␤CD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, M␤CD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of M␤CD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that M␤CD promotes the release of proteins into the extracellular medium, including both MSP and MSPlike protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins. Leishmania protozoa shuttle between a mammalian host as intracellular amastigotes and a sand fly vector as flagellated promastigotes. The sand fly acquires amastigote-laden cells during a blood meal. In the sand fly midgut, procyclic promastigotes derived from transformation of amastigotes undergo multiplication and development via intermediate stages, eventually yielding metacyclic promastigotes (1). The infectious metacyclic promastigote is inoculated into a pool of blood in the dermis of a mammalian host formed during a sand fly bite. Parasites are phagocytized by host macrophages, where they transform to amastigotes and multiply in parasitophorous vacuoles. Amastigotes spread to new macrophages at local or disseminated sites, perpetuating the infection and ultimately resulting in asymptomatic infections or symptomatic leishmaniasis (2). Various forms of leishmaniasis are endemic in 88 countries on four continents, leading to approximately two million new cases and 59,000 deaths annually (3).Both host and parasite factors contribute to the success of infection. On one hand, mammalian host environmental risk factors and genetic background influence the clinical manifestations of infection (4). On the other hand, parasite virulence determinants required for disease development include, but are not limited to, the major surface protease (MSP) (also called GP63 or leishmanolysin) and lipophosphoglycan (LPG) (5-8). These two molecules play both overl...

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Section: Resultssupporting

confidence: 80%

Section: Discussionmentioning

confidence: 69%

Section: Resultsmentioning

confidence: 99%

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Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

et al. 2013

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“…Metacyclic promastigotes were isolated from 5-to 6-day cultures of HP and LP parasites using a Ficoll gradient as described previously (32). After gradient centrifugation, the metacyclics were isolated from the 10% Ficoll layer and the procyclic promastigotes from the pellet.…”

Section: Methodsmentioning

confidence: 99%

3′-Nucleotidase/Nuclease Activity Allows Leishmania Parasites To Escape Killing by Neutrophil Extracellular Traps

et al. 2014

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Leishmaniasis is a widespread neglected tropical disease caused by parasites of the Leishmania genus. These parasites express the enzyme 3=-nucleotidase/nuclease (3=NT/NU), which has been described to be involved in parasite nutrition and infection. Bacteria that express nucleases escape the toxic effects of neutrophil extracellular traps (NETs). Hence, we investigated the role of 3=NT/NU in Leishmania survival of NET-mediated killing. Promastigotes of Leishmania infantum were cultured in high-phosphate (HP) or low-phosphate (LP) medium to modulate nuclease activity. We compared the survival of the two different groups of Leishmania during interaction with human neutrophils, assessing the role of neutrophil extracellular traps. As previously reported, we detected higher nuclease activity in parasites cultured in LP medium. Both LP and HP promastigotes were capable of inducing the release of neutrophil extracellular traps from human neutrophils in a dose-and time-dependent manner. LP parasites had 2.4 times more survival than HP promastigotes. NET disruption was prevented by the treatment of the parasites with ammonium tetrathiomolybdate (TTM), a 3=NT/NU inhibitor. Inhibition of 3=NT/NU by 3=-AMP, 5=-GMP, or TTM decreased promastigote survival upon interaction with neutrophils. Our results show that Leishmania infantum induces NET release and that promastigotes can escape NET-mediated killing by 3=-nucleotidase/nuclease activity, thus ascribing a new function to this enzyme. N eutrophils are short-lived cells and the most abundant leukocytes in the blood circulation; they constitute one of the first lines of defense against invading microorganisms (1). These granulocytes can kill microorganisms by phagocytosis, degranulation, and neutrophil extracellular traps (NETs). NETs are weblike structures composed of chromatin, granules, and cytoplasmic proteins that are extruded when neutrophils undergo NETosis, a unique cell death mechanism (2-5). However, recent work challenges NETosis as a cell death mechanism because live neutrophils were detected after NET extrusion in in vivo studies (6). NETs function by killing and containing pathogens, thereby preventing the pathogen's dissemination through the organism. In addition, some studies have indicated that NETs play a role in autoimmune diseases (7-10).A diverse group of stimuli has been described as activating NETosis (5, 11). Among the parasites, Leishmania promastigotes were demonstrated to activate release of NETs (12, 13). Leishmania amazonensis promastigotes interact intimately with NETs and are killed by web-associated histones (12). However, although promastigotes of Leishmania donovani trigger NET release, these parasites escape the toxicity of NETs (13). Groups of microorganisms have evolved different mechanisms of escaping the toxic effects of NETs. Streptococcus pneumoniae, group A Streptococcus, Streptococcus agalactiae, Staphylococcus aureus, and Vibrio cholerae express endonucleases that efficiently degrade DNA filaments from NETs, allowing these bacteria...

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“…Parasites were grown as promastigotes at 26°C in liquid hemoflagellate-modified minimal essential medium (4). Parasite subcultures were grown to stationary phase, and metacyclic promastigotes were enriched on a density gradient as described previously (49).…”

Section: Methodsmentioning

confidence: 99%

Leukocytes Infiltrate the Skin and Draining Lymph Nodes in Response to the ProtozoanLeishmania infantum chagasi

et al. 2011

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The vector-borne protozoan Leishmania infantum chagasi causes minimal inflammation after inoculation into skin but disseminates to cause fatal visceral leishmaniasis. To define the inflammatory response at the parasite inoculation site, we introduced metacyclic L. infantum chagasi promastigotes intradermally into BALB/c mouse ears and studied inflammatory cells over 7 days. Ly6G؉ neutrophils rapidly infiltrated the dermis, peaking after 6 to 24 h. Macrophages and NK cells next infiltrated the dermis, and NK followed by B cells expanded in draining lymph nodes. Parasite-containing phagocytes were tracked with fluorescent mCherry-labeled L. infantum chagasi. Ly6G؉ neutrophils contained the greatest proportion of intracellular parasites 6 to 24 h after inoculation, whereas dermal macrophages harbored the majority of intracellular parasites after 2 to 7 days. These observations were validated microscopically. Low doses of antibody transiently depleted mice of neutrophils, leaving other cells intact. Combined results of in vivo imaging, flow cytometry, and quantitative PCR showed that neutrophil depletion slowed the clearance of extracellular (luciferase-positive) promastigotes during the first 24 h after inoculation yet decreased the numbers of leukocytes containing intracellular (mCherry-positive) parasites. From 3 days onward, total L. infantum chagasi-containing dermal leukocytes and total L. infantum chagasi parasites in draining lymph nodes were similar in both groups. Nonetheless, a second wave of L. infantum chagasi-containing neutrophils occurred 7 days after parasite inoculation into neutrophildepleted mice, corresponding to the time of neutrophil recovery. Thus, neutrophils were recruited to the dermis even late after inoculation, and L. infantum chagasi trafficked through neutrophils in both neutrophil-depleted and control mice, albeit with different kinetics. Recruitment of neutrophils and transient parasite residence in neutrophils may play a role in nonulcerative forms of leishmaniasis.Parasites belonging to the genus Leishmania cause a spectrum of human diseases, the most deadly of which is visceral leishmaniasis. Leishmania infantum chagasi is one of the two most common etiologic agents of visceral leishmaniasis in humans. During natural infection, a bolus of metacyclic promastigotes is delivered into a hemorrhagic dermal lesion formed by a feeding female phlebotomine sand fly (5). Parasites quickly encounter soluble and cellular microbicidal immune elements. Rather than succumb, many are taken up by phagocytic host cells, where they transform to intracellular amastigotes, a form that can multiply and survive in phagolysosomes (9, 48).Although the majority of host cells harboring Leishmania sp. amastigotes are macrophages, intracellular amastigotes have been observed in other mammalian cell types as well, including dendritic cells (DCs), fibroblasts, and neutrophils (6,21,28,33). Recent studies suggest that neutrophils can promote the early establishment of intradermal infection with Leishmania major...

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Leishmania chagasi: Homogenous metacyclic promastigotes isolated by buoyant density are highly virulent in a mouse model

Cited by 49 publications

References 29 publications

Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

3′-Nucleotidase/Nuclease Activity Allows Leishmania Parasites To Escape Killing by Neutrophil Extracellular Traps

Leukocytes Infiltrate the Skin and Draining Lymph Nodes in Response to the ProtozoanLeishmania infantum chagasi

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