RESUMO
Engineering pathogens is a useful method for discovering new details of microbial pathogenesis and host defense. However, engineering can result in off-target effects. We previously engineered Salmonella enterica serovar Typhimurium to overexpress the secretion signal of the type 3 secretion system effector SspH1 fused with domains of other proteins as cargo. Such engineering had no virulence cost to the bacteria for the first 48 hours post infection in mice. Here, we show that after 48 hours, the engineered bacteria manifest an attenuation that correlates with the quantity of the SspH1 translocation signal expressed. In IFN-γ-deficient mice, this attenuation was weakened. Conversely, the attenuation was accelerated in the context of a pre-existing infection. We speculate that inflammatory signals change aspects of the target cell's physiology, which makes host cells less permissive to S. Typhimurium infection. This increased degree of difficulty requires the bacteria to utilize its T3SS at peak efficiency, which can be disrupted by engineered effectors.
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Salmonella typhimurium , Sistemas de Secreção Tipo III , Animais , Camundongos , Virulência , Sistemas de Secreção Tipo III/genética , Fatores de Virulência/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismoRESUMO
Latent liver stages termed hypnozoites cause relapsing Plasmodium vivax malaria infection and represent a major obstacle in the goal of malaria elimination. Hypnozoites are clinically undetectable, and presently, there are no biomarkers of this persistent parasite reservoir in the human liver. Here, we have identified parasite and human proteins associated with extracellular vesicles (EVs) secreted from in vivo infections exclusively containing hypnozoites. We used P. vivax-infected human liver-chimeric (huHEP) FRG KO mice treated with the schizonticidal experimental drug MMV048 as hypnozoite infection model. Immunofluorescence-based quantification of P. vivax liver forms showed that MMV048 removed schizonts from chimeric mice livers. Proteomic analysis of EVs derived from FRG huHEP mice showed that human EV cargo from infected FRG huHEP mice contain inflammation markers associated with active schizont replication and identified 66 P. vivax proteins. To identify hypnozoite-specific proteins associated with EVs, we mined the proteome data from MMV048-treated mice and performed an analysis involving intragroup and intergroup comparisons across all experimental conditions followed by a peptide compatibility analysis with predicted spectra to warrant robust identification. Only one protein fulfilled this stringent top-down selection, a putative filamin domain-containing protein. This study sets the stage to unveil biological features of human liver infections and identify biomarkers of hypnozoite infection associated with EVs.
Assuntos
Vesículas Extracelulares , Malária Vivax , Parasitos , Humanos , Camundongos , Animais , Malária Vivax/tratamento farmacológico , Malária Vivax/parasitologia , Plasmodium vivax , Proteômica , Proteoma , Filaminas , Fígado , Biomarcadores , Espectrometria de MassasRESUMO
BACKGROUND: Plasmodium 18S rRNA is a biomarker used to monitor blood-stage infections in malaria clinical trials. Plasmodium sporozoites also express this biomarker, and there is conflicting evidence about how long sporozoite-derived 18S rRNA persists in peripheral blood. If present in blood for an extended timeframe, sporozoite-derived 18S rRNA could complicate use as a blood-stage biomarker. METHODS: Blood samples from Plasmodium yoelii infected mice were tested for Plasmodium 18S rRNA and their coding genes (rDNA) using sensitive quantitative reverse transcription PCR and quantitative PCR assays, respectively. Blood and tissues from Plasmodium falciparum sporozoite (PfSPZ)-infected rhesus macaques were similarly tested. RESULTS: In mice, when P. yoelii sporozoite inoculation and blood collection were performed at the same site (tail vein), low level rDNA positivity persisted for 2 days post-infection. Compared to intact parasites with high rRNA-to-rDNA ratios, this low level positivity was accompanied by no increase in rRNA-to-rDNA, indicating detection of residual, non-viable parasite rDNA. When P. yoelii sporozoites were administered via the retro-orbital vein and blood sampled by cardiac puncture, neither P. yoelii 18S rRNA nor rDNA were detected 24 h post-infection. Similarly, there was no P. falciparum 18S rRNA detected in blood of rhesus macaques 3 days after intravenous injection with extremely high doses of PfSPZ. Plasmodium 18S rRNA in the rhesus livers increased by approximately 101-fold from 3 to 6 days post infection, indicating liver-stage proliferation. CONCLUSIONS: Beyond the first few hours after injection, sporozoite-derived Plasmodium 18S rRNA was not detected in peripheral blood. Diagnostics based on 18S rRNA are unlikely to be confounded by sporozoite inocula in human clinical trials.
Assuntos
Plasmodium yoelii/fisiologia , RNA de Protozoário/análise , RNA Ribossômico 18S/análise , Administração Intravenosa , Animais , Feminino , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Esporozoítos/químicaRESUMO
BACKGROUND: Plasmodium gametocytes are sexual stages transmitted to female Anopheles mosquitoes. While Plasmodium parasites can be differentiated microscopically on Giemsa-stained blood smears, molecular methods are increasingly used because of their increased sensitivity. Molecular detection of gametocytes requires methods that discriminate between asexual and sexual stage parasites. Commonly tested gametocyte-specific mRNAs are pfs25 and pfs230 detected by reverse transcription polymerase chain reaction (RT-PCR). However, detection of these unspliced mRNA targets requires preceding DNase treatment of nucleic acids to eliminate co-purified genomic DNA. If gametocyte-specific, spliced mRNAs could be identified, DNase treatment could be eliminated and one-step multiplexed molecular methods utilized. RESULTS: Expression data was used to identify highly-expressed mRNAs in mature gametocytes that were also low in antisense RNA expression in non-gametocyte stages. After testing numerous candidate mRNAs, the spliced female Pf3D7_0630000 mRNA was selected as a Plasmodium falciparum gametocyte-specific biomarker compatible with Plasmodium 18S rRNA RT-PCR. This mRNA was only detected in samples containing mature gametocytes and was absent in those containing only asexual stage parasites or uninfected human blood. PF3D7_0630000 RT-PCR detected gametocytes across a wide range of parasite densities in both spiked and clinical samples and agreed with pfs25 RT-PCR, the gold standard for RT-PCR-based gametocyte detection. PF3D7_0630000 multiplexed with Plasmodium 18S rRNA RT-PCR was more sensitive than other spliced mRNA targets for one-step RT-PCR gametocyte detection. CONCLUSIONS: Because the spliced target does not require DNase treatment, the PF3D7_0630000 assay can be multiplexed with Plasmodium 18S rRNA for direct one-step detection of gametocytes from whole human blood.
Assuntos
Reação em Cadeia da Polimerase Multiplex/métodos , Plasmodium falciparum/genética , RNA Mensageiro/genética , RNA de Protozoário/genética , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
Malaria infection caused by Plasmodium parasites continues to cause enormous morbidity and mortality in areas where it is endemic, and there is no licensed vaccine capable of inducing sterile protection. Hyperimmunization with attenuated whole sporozoites can induce sterile protective immune responses targeting preerythrocytic antigens. Most animal models of hyperimmunization rely on sporozoites dissected from mosquito salivary glands and injected without further purification. In BALB/c mice, repeated small doses of P. yoelii sporozoites progressively expand the population of sporozoite-specific CD8(+) T cells. In this study, large secondary doses of unpurified sporozoites unexpectedly led to contraction of sporozoite-specific CD8(+) T cell responses in sporozoite-primed mice. While sporozoite-primed CD8(+) T cells alternatively can be expanded by secondary exposure to Listeria monocytogenes expressing recombinant Plasmodium antigens, such expansion was potently inhibited by coinjection of large doses of unpurified sporozoites and by uninfected salivary glands alone. Purification of sporozoites away from mosquito salivary gland debris by density gradient centrifugation eliminated salivary gland-associated inhibition. Thus, the inhibitory effect appears to be due to exposure to uninfected mosquito salivary glands rather than sporozoites. To further assess the effect of salivary gland exposure on later sporozoite vaccinations, mice were immunized with uninfected salivary glands from a single mosquito. Compared to naive mice, salivary gland presensitization reduced subsequent liver burdens by 71%. These data show that a component(s) in mosquito salivary glands reduces liver infection, thereby limiting antigen dose and contributing to lower-magnitude T cell responses. These findings suggest that sporozoite immunogenicity studies be performed using purified sporozoites whenever feasible.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Malária/imunologia , Malária/parasitologia , Plasmodium/imunologia , Esporozoítos/imunologia , Animais , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Imunização , Vacinas Antimaláricas/imunologia , CamundongosRESUMO
BACKGROUND: Whole Plasmodium sporozoites serve as both experimental tools and potentially as deployable vaccines in the fight against malaria infection. Live sporozoites infect hepatocytes and induce a diverse repertoire of CD8(+) T cell responses, some of which are capable of killing Plasmodium-infected hepatocytes. Previous studies in Plasmodium yoelii-immunized BALB/c mice showed that some CD8(+) T cell responses expanded with repeated parasite exposure, whereas other responses did not. RESULTS: Here, similar outcomes were observed using known Plasmodium berghei epitopes in C57BL/6 mice. With the exception of the response to PbTRAP, IFNγ-producing T cell responses to most studied antigens, such as PbGAP50, failed to re-expand in mice immunized with two doses of irradiated P. berghei sporozoites. In an effort to boost secondary CD8(+) T cell responses, heterologous cross-species immunizations were performed. Alignment of P. yoelii 17XNL and P. berghei ANKA proteins revealed that >60 % of the amino acids in syntenic orthologous proteins are continuously homologous in fragments ≥8-amino acids long, suggesting that cross-species immunization could potentially trigger responses to a large number of common Class I epitopes. Heterologous immunization resulted in a larger liver burden than homologous immunization. Amongst seven tested antigen-specific responses, only CSP- and TRAP-specific CD8(+) T cell responses were expanded by secondary homologous sporozoite immunization and only those to the L3 ribosomal protein and S20 could be re-expanded by heterologous immunization. In general, heterologous late-arresting, genetically attenuated sporozoites were better at secondarily expanding L3-specific responses than were irradiated sporozoites. GAP50 and several other antigens shared between P. berghei and P. yoelii induced a large number of IFNγ-positive T cells during primary immunization, yet these responses could not be re-expanded by either homologous or heterologous secondary immunization. CONCLUSIONS: These studies highlight how responses to different sporozoite antigens can markedly differ in recall following repeated sporozoite vaccinations. Cross-species immunization broadens the secondary response to sporozoites and may represent a novel strategy for candidate antigen discovery.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Plasmodium berghei/imunologia , Plasmodium yoelii/imunologia , Proteínas de Protozoários/imunologia , Esporozoítos/imunologia , Animais , Linfócitos T CD8-Positivos/parasitologia , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Vacinas Antimaláricas/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Protozoários/metabolismo , Análise de Sequência de DNARESUMO
Eliminating malaria parasites during the asymptomatic but obligate liver stages (LSs) of infection would stop disease and subsequent transmission. Unfortunately, only a single licensed drug that targets all LSs, Primaquine, is available. Targeting host proteins might significantly expand the repertoire of prophylactic drugs against malaria. Here, we demonstrate that both Bcl-2 inhibitors and P53 agonists dramatically reduce LS burden in a mouse malaria model in vitro and in vivo by altering the activity of key hepatocyte factors on which the parasite relies. Bcl-2 inhibitors act primarily by inducing apoptosis in infected hepatocytes, whereas P53 agonists eliminate parasites in an apoptosis-independent fashion. In combination, Bcl-2 inhibitors and P53 agonists act synergistically to delay, and in some cases completely prevent, the onset of blood stage disease. Both families of drugs are highly effective at doses that do not cause substantial hepatocyte cell death in vitro or liver damage in vivo. P53 agonists and Bcl-2 inhibitors were also effective when administered to humanized mice infected with Plasmodium falciparum. Our data demonstrate that host-based prophylaxis could be developed into an effective intervention strategy that eliminates LS parasites before the onset of clinical disease and thus opens a new avenue to prevent malaria.
Assuntos
Antimaláricos/farmacologia , Estágios do Ciclo de Vida/efeitos dos fármacos , Fígado/parasitologia , Malária/parasitologia , Plasmodium/efeitos dos fármacos , Plasmodium/fisiologia , Profilaxia Pós-Exposição , Animais , Antimaláricos/administração & dosagem , Linhagem Celular , Modelos Animais de Doenças , Feminino , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Indóis , Malária/tratamento farmacológico , Malária/metabolismo , Malária/prevenção & controle , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Malária Falciparum/prevenção & controle , Camundongos , Camundongos Transgênicos , Carga Parasitária , Piperazinas/administração & dosagem , Piperazinas/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Pirróis/administração & dosagem , Pirróis/farmacologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismoRESUMO
A patient in Washington State harbored a fish tapeworm most likely acquired from eating raw salmon. Diphyllobothrium nihonkaiense was identified by cox1 sequence analysis. Although this is the first documented human D. nihonkaiense infection in the United States, the parasite may have been present earlier but misidentified as Diphyllobothrium latum.
Assuntos
Difilobotríase/parasitologia , Diphyllobothrium/isolamento & purificação , Animais , Anti-Helmínticos/uso terapêutico , Difilobotríase/tratamento farmacológico , Diphyllobothrium/enzimologia , Diphyllobothrium/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Parasitologia de Alimentos , Humanos , Filogenia , Praziquantel/uso terapêutico , Adulto JovemRESUMO
Inflammasomes are multiprotein complexes that form in response to ligands originating from pathogens as well as alterations of normal cell physiology caused by infection or tissue damage. These structures engage a robust inflammatory immune response that eradicates environmental microbes before they cause disease, and slow the growth of bona fide pathogens. Despite their undeniable utility in immunity, inflammasomes are radically reduced in birds. Perhaps most surprising is that, within all birds, NLRP3 is retained, while its signaling adapter ASC is lost, suggesting that NLRP3 signals via a novel unknown adapter. Crocodilian reptiles and turtles, which share a more recent common ancestor with birds, retain many of the lost inflammasome components, indicating that the deletion of inflammasomes occurred after birds diverged from crocodiles. Some bird lineages have even more extensive inflammasome loss, with songbirds continuing to pare down their inflammasomes until only NLRP3 and CARD8 remain. Remarkably, songbirds have lost caspase-1 but retain the downstream targets of caspase-1: IL-1ß, IL-18, and the YVAD-linker encoding gasdermin A. This suggests that inflammasomes can signal through alternative proteases to activate cytokine maturation and pyroptosis in songbirds. These observations may reveal new contexts of activation that may be relevant to mammalian inflammasomes and may suggest new avenues of research to uncover the enigmatic nature of the poorly understood NLRP3 inflammasome.
Assuntos
Aves , Inflamassomos , Animais , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Evolução MolecularRESUMO
Gasdermins oligomerize to form pores in the cell membrane, causing regulated lytic cell death called pyroptosis. Mammals encode five gasdermins that can trigger pyroptosis: GSDMA, B, C, D, and E. Caspase and granzyme proteases cleave the linker regions of and activate GSDMB, C, D, and E, but no endogenous activation pathways are yet known for GSDMA. Here, we perform a comprehensive evolutionary analysis of the gasdermin family. A gene duplication of GSDMA in the common ancestor of caecilian amphibians, reptiles and birds gave rise to GSDMA-D in mammals. Uniquely in our tree, amphibian, reptile and bird GSDMA group in a separate clade than mammal GSDMA. Remarkably, GSDMA in numerous bird species contain caspase-1 cleavage sites like YVAD or FASD in the linker. We show that GSDMA from birds, amphibians, and reptiles are all cleaved by caspase-1. Thus, GSDMA was originally cleaved by the host-encoded protease caspase-1. In mammals the caspase-1 cleavage site in GSDMA is disrupted; instead, a new protein, GSDMD, is the target of caspase-1. Mammal caspase-1 uses exosite interactions with the GSDMD C-terminal domain to confer the specificity of this interaction, whereas we show that bird caspase-1 uses a stereotypical tetrapeptide sequence to confer specificity for bird GSDMA. Our results reveal an evolutionarily stable association between caspase-1 and the gasdermin family, albeit a shifting one. Caspase-1 repeatedly changes its target gasdermin over evolutionary time at speciation junctures, initially cleaving GSDME in fish, then GSDMA in amphibians/reptiles/birds, and finally GSDMD in mammals.
RESUMO
Inflammasomes are sensors that detect cytosolic microbial molecules or cellular damage, and in response they initiate a form of lytic regulated cell death called pyroptosis. Inflammasomes signal via homotypic protein-protein interactions where CARD or PYD domains are crucial for recruiting downstream partners. Here, we screened these domains from NLR family proteins, and found that the PYD domain of NLRP6 and NLRP12 could activate caspase-1 to induce cleavage of IL-1ß and GSDMD. Inflammasome reconstitution verified that full length NLRP6 and NLRP12 formed inflammasomes in vitro, and NLRP6 was more prone to auto-activation. NLRP6 was highly expressed in intestinal epithelial cells (IEC), but not in immune cells. Molecular phylogeny analysis found that NLRP12 was closely related to NLRP3, but the activation mechanisms are different. NLRP3 was highly expressed in monocytes and macrophages, and was modestly but appreciably expressed in neutrophils. In contrast, NLRP12 was specifically expressed in neutrophils and eosinophils, but was not detectable in macrophages. NLRP12 mutations cause a periodic fever syndrome called NLRP12 autoinflammatory disease. We found that several of these patient mutations caused spontaneous activation of caspase-1 in vitro, which likely causes their autoinflammatory disease. Different cell types have unique cellular physiology and structures which could be perturbed by a pathogen, necessitating expression of distinct inflammasome sensors to monitor for signs of infection.
Assuntos
Proteínas Reguladoras de Apoptose , Inflamassomos , Peptídeos e Proteínas de Sinalização Intracelular , Proteína 3 que Contém Domínio de Pirina da Família NLR , Inflamassomos/metabolismo , Humanos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Animais , Células HEK293RESUMO
Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. Although pyroptosis and apoptosis have distinct signaling pathways, when a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we also now engineer S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. In both engineered strains, apoptosis occurred somewhat slower than pyroptosis. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche, but failed to clear the bacteria in the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. In order to clear an infection, distinct cell types may have specific tasks that they must complete before they die. In some cells, either apoptotic or pyroptotic signaling may initiate the same tasks, whereas in other cell types these modes of cell death may lead to different tasks that may not be identical in defense against infection. We recently suggested that such diverse tasks can be considered as different cellular 'bucket lists' to be accomplished before a cell dies.
RESUMO
Pyroptosis and apoptosis are two forms of regulated cell death that can defend against intracellular infection. When a cell fails to complete pyroptosis, backup pathways will initiate apoptosis. Here, we investigated the utility of apoptosis compared to pyroptosis in defense against an intracellular bacterial infection. We previously engineered Salmonella enterica serovar Typhimurium to persistently express flagellin, and thereby activate NLRC4 during systemic infection in mice. The resulting pyroptosis clears this flagellin-engineered strain. We now show that infection of caspase-1 or gasdermin D deficient macrophages by this flagellin-engineered S. Typhimurium induces apoptosis in vitro. Additionally, we engineered S. Typhimurium to translocate the pro-apoptotic BH3 domain of BID, which also triggers apoptosis in macrophages in vitro. During mouse infection, the apoptotic pathway successfully cleared these engineered S. Typhimurium from the intestinal niche but failed to clear the bacteria from the myeloid niche in the spleen or lymph nodes. In contrast, the pyroptotic pathway was beneficial in defense of both niches. To clear an infection, cells may have specific tasks that they must complete before they die; different modes of cell death could initiate these 'bucket lists' in either convergent or divergent ways.
Although alive and healthy cells are essential for survival, in certain circumstances such as when a cell becomes infected it is beneficial for cells to deliberately die through a process known as regulated cell death. There are several types of regulated cell death, each with distinct pathways and mechanisms. However, if the initial pathway is blocked, cells can use an alternative one, suggesting that they can compensate for one other. Two forms of regulated cell death named pyroptosis and apoptosis can be used by infected cells to limit the spread of pathogens. However, it was not clear if these two forms or additional 'back-up' apoptosis pathways which are induced when pyroptosis fails are equally efficient at clearing infections and how they might vary in different cell types. To address this, Abele et al. investigated cell death in live mice infected with the bacterium Salmonella. Different organs in which the bacterium infects distinct cell types were examined. Experiments showed that pyroptosis could eliminate bacteria from both intestinal cells as well as immune cells found throughout the body, called macrophages. In contrast, apoptosis was only able to clear infection from intestinal cells. The findings can be explained by prior studies showing both apoptosis and pyroptosis lead to the same outcome in intestinal cells dead cells are expelled from the body through a process called extrusion to maintain the barrier function of the intestine. However, in macrophages, the different pathways lead to different outcomes, indicating they are not entirely interchangeable. Overall, the findings of Abele et al. underscore the complexity of cellular responses to infection and the nuanced roles of different cell death pathways. This provides further evidence that cells might have specific tasks they need to complete before death in order to effectively clear an infection. These tasks may differ depending on cell type and the form of regulated cell death, and may not be equally efficient at clearing an infection.
Assuntos
Apoptose , Flagelina , Animais , Camundongos , Morte Celular , Caspase 1/metabolismo , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Piroptose , Inflamassomos/metabolismoRESUMO
Granulomas often form around pathogens that cause chronic infections. Here, we discover a novel granuloma model in mice. Chromobacterium violaceum is an environmental bacterium that stimulates granuloma formation that not only successfully walls off but also clears the infection. The infected lesion can arise from a single bacterium that replicates in the presence of a neutrophil swarm. Bacterial replication ceases when macrophages organize around the infection and form a granuloma. This granuloma response is accomplished independently of adaptive immunity that is typically required to organize granulomas. The C. violaceum -induced granuloma requires at least two separate defense pathways, gasdermin D and iNOS, to maintain the integrity of the granuloma architecture. These innate granulomas successfully eradicate C. violaceum infection. Therefore, this new C. violaceum -induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby eradicate infection by an environmental pathogen.
RESUMO
Granulomas often form around pathogens that cause chronic infections. Here, we discover an innate granuloma model in mice with an environmental bacterium called Chromobacterium violaceum. Granuloma formation not only successfully walls off, but also clears, the infection. The infected lesion can arise from a single bacterium that replicates despite the presence of a neutrophil swarm. Bacterial replication ceases when macrophages organize around the infection and form a granuloma. This granuloma response is accomplished independently of adaptive immunity that is typically required to organize granulomas. The C. violaceum-induced granuloma requires at least two separate defense pathways, gasdermin D and iNOS, to maintain the integrity of the granuloma architecture. This innate granuloma successfully eradicates C. violaceum infection. Therefore, this C. violaceum-induced granuloma model demonstrates that innate immune cells successfully organize a granuloma and thereby resolve infection by an environmental pathogen.
Assuntos
Granuloma , Neutrófilos , Animais , Camundongos , Macrófagos/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Liver stage (LS) Plasmodia mature in 2-2.5 days in rodents compared to 5-6 days in humans. Plasmodium-specific CD8+ T cell expansion differs across these varied timespans. To mimic the kinetics of CD8+ T cells of human Plasmodium infection, a two-dose challenge mouse model that achieved 4-5 days of LS antigen exposure was developed. In this model, mice were inoculated with a non-protective, low dose of late-arresting, genetically attenuated sporozoites to initiate T cell activation and then re-inoculated 2-3 days later with wild-type sporozoites. Vaccines that partially protected against traditional challenge completely protected against two-dose challenge. During the challenge period, CD8+ T cell frequencies increased in the livers of two-dose challenged mice but not in traditionally challenged mice, further suggesting that this model better recapitulates kinetics of CD8+ T cell expansion in humans during the P. falciparum LS. Vaccine development and antigen discovery efforts may be aided by using the two-dose challenge strategy.
RESUMO
Plasmodium vivax is a malaria-causing pathogen that establishes a dormant form in the liver (the hypnozoite), which can activate weeks, months, or years after the primary infection to cause a relapse, characterized by secondary blood-stage infection. These asymptomatic and undetectable latent liver infections present a significant obstacle to the goal of global malaria eradication. We use a human liver-chimeric mouse model (FRG huHep) to study P. vivax hypnozoite latency and activation in an in vivo model system. Functional activation of hypnozoites and formation of secondary schizonts is demonstrated by first eliminating primary liver schizonts using a schizont-specific antimalarial tool compound, and then measuring recurrence of secondary liver schizonts in the tissue and an increase in parasite RNA within the liver. We also reveal that, while primaquine does not immediately eliminate hypnozoites from the liver, it arrests developing schizonts and prevents activation of hypnozoites, consistent with its clinical activity in humans. Our findings demonstrate that the FRG huHep model can be used to study the biology of P. vivax infection and latency and assess the activity of anti-relapse drugs.
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18S rRNA is a biomarker that provides an alternative to thick blood smears in controlled human malaria infection (CHMI) trials. We reviewed data from CHMI trials at non-endemic sites that used blood smears and Plasmodium 18S rRNA/rDNA biomarker nucleic acid tests (NATs) for time to positivity. We validated a multiplex quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for Plasmodium 18S rRNA, prospectively compared blood smears and qRT-PCR for three trials, and modeled treatment effects at different biomarker-defined parasite densities to assess the impact on infection detection, symptom reduction, and measured intervention efficacy. Literature review demonstrated accelerated NAT-based infection detection compared with blood smears (mean acceleration: 3.2-3.6 days). For prospectively tested trials, the validated Plasmodium 18S rRNA qRT-PCR positivity was earlier (7.6 days; 95% CI: 7.1-8.1 days) than blood smears (11.0 days; 95% CI: 10.3-11.8 days) and significantly preceded the onset of grade 2 malaria-related symptoms (12.2 days; 95% CI: 10.6-13.3 days). Discrepant analysis showed that the risk of a blood smear-positive, biomarker-negative result was negligible. Data modeling predicted that treatment triggered by specific biomarker-defined thresholds can differentiate complete, partial, and non-protective outcomes and eliminate many grade 2 and most grade 3 malaria-related symptoms post-CHMI. Plasmodium 18S rRNA is a sensitive and specific biomarker that can justifiably replace blood smears for infection detection in CHMI trials in non-endemic settings. This study led to biomarker qualification through the U.S. Food and Drug Administration for use in CHMI studies at non-endemic sites, which will facilitate biomarker use for the qualified context of use in drug and vaccine trials.
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Malária/diagnóstico , Plasmodium/genética , RNA de Protozoário/genética , RNA Ribossômico 18S/sangue , Biomarcadores/sangue , Humanos , Reação em Cadeia da Polimerase Multiplex , Plasmodium/isolamento & purificação , RNA Ribossômico 18S/genética , Reação em Cadeia da Polimerase em Tempo Real , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The invention of liver-humanized mouse models has made it possible to directly study the preerythrocytic stages of Plasmodium falciparum. In contrast, the current models to directly study blood stage infection in vivo are extremely limited. Humanization of the mouse blood stream is achievable by frequent injections of human red blood cells (hRBCs) and is currently the only system with which to study human malaria blood stage infections in a small animal model. Infections have been primarily achieved by direct injection of P. falciparum-infected RBCs but as such, this modality of infection does not model the natural route of infection by mosquito bite and lacks the transition of parasites from liver stage infection to blood stage infection. Including these life cycle transition points in a small animal model is of relevance for testing therapeutic interventions. To this end, we used FRGN KO mice that were engrafted with human hepatocytes and performed a blood exchange under immune modulation to engraft the animals with more than 50% hRBCs. These mice were infected by mosquito bite with sporozoite stages of a luciferase-expressing P. falciparum parasite, resulting in noninvasively measurable liver stage burden by in vivo bioluminescent imaging (IVIS) at days 5-7 postinfection. Transition to blood stage infection was observed by IVIS from day 8 onward and then blood stage parasitemia increased with a kinetic similar to that observed in controlled human malaria infection. To assess the utility of this model, we tested whether a monoclonal antibody targeting the erythrocyte invasion ligand reticulocyte-binding protein homolog 5 (with known growth inhibitory activity in vitro) was capable of blocking blood stage infection in vivo when parasites emerge from the liver and found it highly effective. Together, these results show that a combined liver-humanized and blood-humanized FRGN mouse model infected with luciferase-expressing P. falciparum will be a useful tool to study P. falciparum preerythrocytic and erythrocytic stages and enables the testing of interventions that target either one or both stages of parasite infection.
Assuntos
Modelos Animais de Doenças , Malária Falciparum , Animais , Anticorpos Monoclonais/farmacologia , Proteínas de Transporte/imunologia , Eritrócitos/parasitologia , Humanos , Hepatopatias/parasitologia , Malária Falciparum/parasitologia , Camundongos Knockout , Parasitemia/parasitologia , Plasmodium falciparum , Proteínas de Protozoários/imunologiaRESUMO
Babesia are increasingly appreciated as a cause of transfusion-transmitted infection. Sensitive methods are needed to screen blood products. We report herein that B. microti 18S rRNA is over 1,000-fold more abundant than its coding genes, making reverse transcription PCR (RT-PCR) much more sensitive than PCR. Babesia 18S rRNA may be useful for screening the blood supply.