• Chagas disease
• Trypanosoma cruzi
• metacyclic trypomastigote
• metacyclogenesis
• protozoan parasite

• Humans
• Chagas Disease
• Trypanosoma cruzi

• grant 88881.711954/2022-01 Coordenação de Aperfeicoamento de Pessoal de Nível Superior
• INCT-MCTI/CNPq/CAPES/FAPs 16/2014 National Council for Scientific and Technological Development
• 00193-00000833/2021-57 Foundation for Research Support of the Federal District
• 00193-00000229/2021-21 Foundation for Research Support of the Federal District
• 00193-00000825/2021-19 Foundation for Research Support of the Federal District

• Evasion mechanism
• Host immune system
• Intracellular pathogens
• Lysosome
• Phagolysosome

• Lysosomes/microbiology
• Phagocytosis
• Phagosomes/metabolism
• Phagosomes/microbiology
• Endocytosis
• Immune Evasion

• Chagas disease
• Trypanosoma cruzi
• guide
• reservoirs
• triatomine

• R35 GM124918 NIGMS NIH HHS

The Toxoplasma gondii lytic cycle is a repetition of host cell invasion, replication, egress, and re-invasion into the next host cell. While the molecular players involved in egress have been studied in greater detail in recent years, the signals and pathways for triggering egress from the host cell have not been fully elucidated. A perforin-like protein, PLP1, has been shown to be necessary for permeabilizing the parasitophorous vacuole (PV) membrane or exit from the host cell. In vitro studies indicated that PLP1 is most active in acidic conditions, and indirect evidence using superecliptic pHluorin indicated that the PV pH drops prior to parasite egress. Using ratiometric pHluorin, a GFP variant that responds to changes in pH with changes in its bimodal excitation spectrum peaks, allowed us to directly measure the pH in the PV prior to and during egress by live-imaging microscopy. A statistically significant change was observed in PV pH during ionomycin or zaprinast induced egress in both wild-type RH and Δplp1 vacuoles compared to DMSO-treated vacuoles. Interestingly, if parasites are chemically paralyzed, a pH drop is still observed in RH but not in Δplp1 tachyzoites. This indicates that the pH drop is dependent on the presence of PLP1 or motility. Efforts to determine transporters, exchangers, or pumps that could contribute to the drop in PV pH identified two formate-nitrite transporters (FNTs). Auxin induced conditional knockdown and knockouts of FNT1 and FNT2 reduced the levels of lactate and pyruvate released by the parasites and lead to an abatement of vacuolar acidification. While additional transporters and molecules are undoubtedly involved, we provide evidence of a definitive reduction in vacuolar pH associated with induced and natural egress and characterize two transporters that contribute to the acidification.

Toxoplasma gondii is a single celled intracellular parasite that infects many different animals, and it is thought to infect up to one third of the human population. This parasite must rupture out of its replicative compartment and the host cell to spread from one cell to another. Previous studies indicated that a decrease in pH occurs within the replicative compartment near the time of parasite exit from host cells, an event termed egress. However, it remained unknown whether the decrease in pH is directly tied to egress and, if so, what is responsible for the decrease in pH. Here we used a fluorescent reporter protein to directly measure pH within the replicative compartment during parasite egress. We found that pH decreases immediately prior to parasite egress and that this decrease is linked to parasite disruption of membranes. We also identified a family of transporters that release acidic products from parasite use of glucose for energy as contributing to the decrease in pH during egress. Our findings provide new insight that connects parasite glucose metabolism to acidification of its replicative compartment during egress from infected cells.

• Animals
• Hydrogen-Ion Concentration
• Parasites/metabolism
• Perforin/metabolism
• Protozoan Proteins/metabolism
• Toxoplasma/metabolism
• Vacuoles/metabolism

• R01 AI046675 NIAID NIH HHS
• National Institute of Allergy and Infectious Diseases

• Trypanosoma cruzi
• adhesion
• internalization
• recognition

T. cruzi has a complex life cycle involving four developmental stages namely, epimastigotes, metacyclic trypomastigotes, amastigotes and bloodstream trypomastigotes. Although trypomastigotes are the infective forms, extracellular amastigotes have also shown the ability to invade host cells. Both stages can invade a broad spectrum of host tissues, in fact, almost any nucleated cell can be the target of infection. To add complexity, the parasite presents high genetic variability with differential characteristics such as infectivity. In this review, we address the several strategies T. cruzi has developed to subvert the host cell signaling machinery in order to gain access to the host cell cytoplasm. Special attention is made to the numerous parasite/host protein interactions and to the set of signaling cascades activated during the formation of a parasite-containing vesicle, the parasitophorous vacuole, from which the parasite escapes to the cytosol, where differentiation and replication take place.

• autophagic pathway
• exocytic pathway
• host signaling
• host/parasite interaction
• internalization
• invasion
• lysosome-mediated invasion

• Foodborne pathogen
• Phagocytosis
• Phagosomal escape
• Vacuolar escape

• Leishmania major
• Trypanosoma cruzi
• chagas disease
• leishmaniasis
• molecular modelling
• serodiagnosis

• Animals
• Antibodies, Protozoan
• Antigens, Protozoan/chemistry
• Antigens, Protozoan/genetics
• Chagas Disease/diagnosis
• Chagas Disease/immunology
• Cross Reactions
• Epitopes, B-Lymphocyte
• GTP Phosphohydrolases
• Humans
• Leishmania infantum/genetics
• Leishmania infantum/metabolism
• Leishmania major
• Leishmaniasis/immunology
• Leishmaniasis, Cutaneous/immunology
• Leishmaniasis, Cutaneous/parasitology
• Molecular Dynamics Simulation
• Phylogeny
• Protozoan Proteins/chemistry
• Protozoan Proteins/genetics
• Serologic Tests
• Trypanosoma cruzi/genetics
• Trypanosoma cruzi/metabolism

• 120356933228 Ministerio de Ciencia y Tecnología
• RD16/0027/0008 Instituto de Salud Carlos III
• RTI2018-094434-B-I00 Federación Española de Enfermedades Raras

Host cell exit is a critical step in the life-cycle of intracellular pathogens, intimately linked to barrier penetration, tissue dissemination, inflammation, and pathogen transmission. Like cell invasion and intracellular survival, host cell exit represents a well-regulated program that has evolved during host-pathogen co-evolution and that relies on the dynamic and intricate interplay between multiple host and microbial factors. Three distinct pathways of host cell exit have been identified that are employed by three different taxa of intracellular pathogens, bacteria, fungi and protozoa, namely (i) the initiation of programmed cell death, (ii) the active breaching of host cellderived membranes, and (iii) the induced membrane-dependent exit without host cell lysis. Strikingly, an increasing number of studies show that the majority of intracellular pathogens utilize more than one of these strategies, dependent on life-cycle stage, environmental factors and/or host cell type. This review summarizes the diverse exit strategies of intracellular-living bacterial, fungal and protozoan pathogens and discusses the convergently evolved commonalities as well as system-specific variations thereof. Key microbial molecules involved in host cell exit are highlighted and discussed as potential targets for future interventional approaches.

• compartment
• egress
• exit
• host cell
• membrane lysis.
• pathogen
• programmed cell death
• vacuolar escape

• Australia
• Host-parasite interactions
• Marsupials
• Trypanosoma copemani
• Trypanosoma cruzi

• Adenosine Triphosphate/metabolism
• Ammonium Compounds/metabolism
• Gene Deletion
• Genetic Complementation Test
• Glutamate-Ammonia Ligase/metabolism
• Glutamic Acid/metabolism
• Host-Pathogen Interactions
• Saccharomyces cerevisiae/enzymology
• Saccharomyces cerevisiae/genetics
• Trypanosoma cruzi/enzymology
• Trypanosoma cruzi/growth & development
• Vacuoles/parasitology
• Virulence Factors/metabolism

• Fundação de Amparo à Pesquisa do Estado de São Paulo
• Conselho Nacional de Desenvolvimento Científico e Tecnológico

• Discrete Typing Units
• Trypanosoma cruzi virulence
• inactive trans-sialidase
• pathogenesis
• virulence factors

• Animals
• Chagas Disease/parasitology
• Chagas Disease/pathology
• Chlorocebus aethiops
• Glycoproteins/genetics
• Glycoproteins/metabolism
• Humans
• Male
• Mice
• Mice, Inbred BALB C
• Mice, Inbred C3H
• Models, Animal
• Neuraminidase/genetics
• Neuraminidase/metabolism
• Primary Cell Culture
• Rats
• Rats, Sprague-Dawley
• Transfection
• Trypanosoma cruzi/genetics
• Trypanosoma cruzi/pathogenicity
• Vero Cells
• Virulence/genetics
• Virulence Factors/genetics
• Virulence Factors/metabolism

• R01 AI104531 NIAID NIH HHS

• Leishmania sp.
• Trypanosoma brucei sp.
• Trypanosoma cruzi
• Trypanosomatidae family
• immunosuppression
• parasite–host interactions

Chagas disease is a serious illness caused by the protozoan parasite Trypanosoma cruzi. Nearly 30% of chronically infected people develop cardiac, digestive, or mixed alterations, suggesting a broad range of host-parasite interactions that finally impact upon chronic disease outcome. The ability of T. cruzi to persist and cause pathology seems to depend on diverse factors like T. cruzi strains, the infective load and the route of infection, presence of virulence factors, the parasite capacity to avoid protective immune response, the strength and type of host defense mechanisms and the genetic background of the host. The host-parasite interaction is subject to a constant neuro-endocrine regulation that is thought to influence the adaptive immune system, and as the infection proceeds it can lead to a broad range of outcomes, ranging from pathogen elimination to its continued persistence in the host. In this context, T. cruzi evasion strategies and host defense mechanisms can be envisioned as two sides of the same coin, influencing parasite persistence and different outcomes observed in Chagas disease. Understanding how T. cruzi evade host's innate and adaptive immune response will provide important clues to better dissect mechanisms underlying the pathophysiology of Chagas disease.

• Chagas disease
• evasion strategies
• immunoendocrine
• persistence
• thymus
• virulence factors

Intracellular colonization and persistent infection by the kinetoplastid protozoan parasite, Trypanosoma cruzi, underlie the pathogenesis of human Chagas disease. To obtain global insights into the T. cruzi infective process, transcriptome dynamics were simultaneously captured in the parasite and host cells in an infection time course of human fibroblasts. Extensive remodeling of the T. cruzi transcriptome was observed during the early establishment of intracellular infection, coincident with a major developmental transition in the parasite. Contrasting this early response, few additional changes in steady state mRNA levels were detected once mature T. cruzi amastigotes were formed. Our findings suggest that transcriptome remodeling is required to establish a modified template to guide developmental transitions in the parasite, whereas homeostatic functions are regulated independently of transcriptomic changes, similar to that reported in related trypanosomatids. Despite complex mechanisms for regulation of phenotypic expression in T. cruzi, transcriptomic signatures derived from distinct developmental stages mirror known or projected characteristics of T. cruzi biology. Focusing on energy metabolism, we were able to validate predictions forecast in the mRNA expression profiles. We demonstrate measurable differences in the bioenergetic properties of the different mammalian-infective stages of T. cruzi and present additional findings that underscore the importance of mitochondrial electron transport in T. cruzi amastigote growth and survival. Consequences of T. cruzi colonization for the host include dynamic expression of immune response genes and cell cycle regulators with upregulation of host cholesterol and lipid synthesis pathways, which may serve to fuel intracellular T. cruzi growth. Thus, in addition to the biological inferences gained from gene ontology and functional enrichment analysis of differentially expressed genes in parasite and host, our comprehensive, high resolution transcriptomic dataset provides a substantially more detailed interpretation of T. cruzi infection biology and offers a basis for future drug and vaccine discovery efforts.

In-depth knowledge of the functional processes governing host colonization and transmission of pathogenic microorganisms is essential for the advancement of effective intervention strategies. This study focuses on Trypanosoma cruzi, the vector-borne protozoan parasite responsible for human Chagas disease and the leading cause of infectious myocarditis worldwide. To gain global insights into the biology of this parasite and its interaction with mammalian host cells, we have exploited a deep-sequencing approach to generate comprehensive, high-resolution transcriptomic maps for mammalian-infective stages of T. cruzi with the simultaneous interrogation of the human host cell transcriptome across an infection time course. We demonstrate that the establishment of intracellular T. cruzi infection in mammalian host cells is accompanied by extensive remodeling of the parasite and host cell transcriptomes. Despite the lack of transcriptional control mechanisms in trypanosomatids, our analyses identified functionally-enriched processes within sets of developmentally-regulated transcripts in T. cruzi that align with known or predicted biological features of the parasite. The novel insights into the biology of intracellular T. cruzi infection and the regulation of amastigote development gained in this study establish a unique foundation for functional network analyses that will be instrumental in providing functional links between parasite dependencies and host functional pathways that have the potential to be exploited for intervention.

• Chagas disease
• T. cruzi
• host cell invasion
• host cell signaling
• host parasite interaction
• secreted proteins
• secretome
• virulence factor

• Chagas disease
• T. cruzi acute infection
• T. cruzi immune evasion
• immune response
• immunomodulation

• Decellularized human lung
• Decellularized rat liver
• Mass spectrometry
• Matrigel
• Matrisome
• Proteomics

The coordinated exit of intracellular pathogens from host cells is a process critical to the success and spread of an infection. While phospholipases have been shown to play important roles in bacteria host cell egress and virulence, their role in the release of intracellular eukaryotic parasites is largely unknown. We examined a malaria parasite protein with phospholipase activity and found it to be involved in hepatocyte egress. In hepatocytes, Plasmodium parasites are surrounded by a parasitophorous vacuole membrane (PVM), which must be disrupted before parasites are released into the blood. However, on a molecular basis, little is known about how the PVM is ruptured. We show that Plasmodium berghei phospholipase, PbPL, localizes to the PVM in infected hepatocytes. We provide evidence that parasites lacking PbPL undergo completely normal liver stage development until merozoites are produced but have a defect in egress from host hepatocytes. To investigate this further, we established a live-cell imaging-based assay, which enabled us to study the temporal dynamics of PVM rupture on a quantitative basis. Using this assay we could show that PbPL-deficient parasites exhibit impaired PVM rupture, resulting in delayed parasite egress. A wild-type phenotype could be re-established by gene complementation, demonstrating the specificity of the PbPL deletion phenotype. In conclusion, we have identified for the first time a Plasmodium phospholipase that is important for PVM rupture and in turn for parasite exit from the infected hepatocyte and therefore established a key role of a parasite phospholipase in egress.

Leaving their host cell is a crucial process for intracellular pathogens, allowing successful infection of other cells and thereby spreading of infection. Plasmodium parasites infect hepatocytes and red blood cells, and inside these cells they are contained within a vacuole like many other intracellular pathogens. Before parasites can infect other cells, the surrounding parasitophorous vacuole membrane (PVM) needs to be ruptured. However, little is known about this process on a molecular level and Plasmodium proteins mediating lysis of the PVM during parasite egress have not so far been identified. In this study, we characterize a Plasmodium phospholipase and show that it localizes to the PVM of parasites within hepatocytes. We demonstrate that parasites lacking this protein have a defect in rupture of the PVM and thereby in host cell egress. In conclusion, our study shows for the first time that a phospholipase plays a role in PVM disruption of an intracellular eukaryotic parasite.

• Animals
• Disease Models, Animal
• Fluorescent Antibody Technique
• Hepatocytes/enzymology
• Hepatocytes/microbiology
• Malaria/enzymology
• Mice
• Phospholipases/metabolism
• Plasmodium berghei/enzymology
• Plasmodium berghei/pathogenicity
• Protozoan Proteins/metabolism
• Reverse Transcriptase Polymerase Chain Reaction
• Transcriptome
• Vacuoles/enzymology
• Vacuoles/microbiology

The literature has identified complex aspects of intracellular host-parasite relationships, which require systematic, nonreductionist approaches and spatial/temporal information. Increasing and integrating temporal and spatial dimensions in host cell imaging have contributed to elucidating several conceptual gaps in the biology of intracellular parasites. To access and investigate complex and emergent dynamic events, it is mandatory to follow them in the context of living cells and organs, constructing scientific images with integrated high quality spatiotemporal data. This review discusses examples of how advances in microscopy have challenged established conceptual models of the intracellular life cycles of Leishmania spp. and Trypanosoma cruzi protozoan parasites.

• Animals
• Biocatalysis
• Crystallography, X-Ray
• Glycoproteins/chemistry
• Glycoproteins/metabolism
• Models, Molecular
• Neuraminidase/chemistry
• Neuraminidase/metabolism
• Protein Conformation
• Substrate Specificity
• Trypanosoma cruzi/enzymology

To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms.

Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%.

This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization.

Trypanosoma cruzi (T. cruzi), the etiological agent of Chagas disease, affects nearly 18 million people in Latin America and 90 million are at risk of infection. The parasite presents two stages of medical importance in the host, the amastigote, intracellular replicating form, and the extracellular trypomastigote, the infective form. Thus infection by T. cruzi induces a complex immune response that involves effectors and regulatory mechanisms. That is why control of the infection requires a strong humoral and cellular immune response; hence, the outcome of host-parasite interaction in the early stages of infection is extremely important. A critical event during this period of the infection is innate immune response, in which the macrophage’s role is vital. Thus, after being phagocytized, the parasite is able to develop intracellularly; however, during later periods, these cells induce its elimination by means of toxic metabolites. In turn, as the infection progresses, adaptive immune response mechanisms are triggered through the TH1 and TH2 responses. Finally, T. cruzi, like other protozoa such as Leishmania and Toxoplasma, have numerous evasive mechanisms to the immune response that make it possible to spread around the host. In our Laboratory we have developed a vaccination model in mice with Trypanosoma rangeli, nonpathogenic to humans, which modulates the immune response to infection by T. cruzi, thus protecting them. Vaccinated animals showed an important innate response (modulation of NO and other metabolites, cytokines, activation of macrophages), a strong adaptive cellular response and significant increase in specific antibodies. The modulation caused early elimination of the parasites, low parasitaemia, the absence of histological lesions and high survival rates. Even though progress has been made in the knowledge of some of these mechanisms, new studies must be conducted which could target further prophylactic and therapeutic trials against T. cruzi infection.

• Trypanosoma cruzi
• Chagas disease
• Innate and adaptive immune response

In the present short review, we analyze past experiments that addressed the interactions of intracellular pathogenic protozoa (Trypanosoma cruzi, Toxoplasma gondii, and Plasmodium) with host cells and the initial use of the term active penetration to indicate that a protozoan “crossed the host cell membrane, penetrating into the cytoplasm.” However, the subsequent use of transmission electron microscopy showed that, for all of the protozoans and cell types examined, endocytosis, classically defined as involving the formation of a membrane-bound vacuole, took place during the interaction process. As a consequence, the recently penetrated parasites are always within a vacuole, designated the parasitophorous vacuole (PV).

• Chagas disease
• Trypanosoma cruzi
• active penetration
• endocytosis
• phagocytosis

• Trypanosoma cruzi
• apoptosis
• cardiomyocyte
• cell junction
• cell recognition
• cytoskeleton
• endocytosis
• extracellular matrix

Trypanosoma cruzi takes advantage of a sphingomyelinase-dependent plasma membrane repair pathway to gain access to host cells.

Upon host cell contact, the protozoan parasite Trypanosoma cruzi triggers cytosolic Ca²⁺ transients that induce exocytosis of lysosomes, a process required for cell invasion. However, the exact mechanism by which lysosomal exocytosis mediates T. cruzi internalization remains unclear. We show that host cell entry by T. cruzi mimics a process of plasma membrane injury and repair that involves Ca²⁺-dependent exocytosis of lysosomes, delivery of acid sphingomyelinase (ASM) to the outer leaflet of the plasma membrane, and a rapid form of endocytosis that internalizes membrane lesions. Host cells incubated with T. cruzi trypomastigotes are transiently wounded, show increased levels of endocytosis, and become more susceptible to infection when injured with pore-forming toxins. Inhibition or depletion of lysosomal ASM, which blocks plasma membrane repair, markedly reduces the susceptibility of host cells to T. cruzi invasion. Notably, extracellular addition of sphingomyelinase stimulates host cell endocytosis, enhances T. cruzi invasion, and restores normal invasion levels in ASM-depleted cells. Ceramide, the product of sphingomyelin hydrolysis, is detected in newly formed parasitophorous vacuoles containing trypomastigotes but not in the few parasite-containing vacuoles formed in ASM-depleted cells. Thus, T. cruzi subverts the ASM-dependent ceramide-enriched endosomes that function in plasma membrane repair to infect host cells.

• Animals
• Cell Line
• Cells, Cultured
• Chlorocebus aethiops
• Fluorescent Antibody Technique
• Humans
• Leishmania infantum/physiology
• Leishmaniasis, Visceral/complications
• Macrophages/parasitology
• Male
• Mice
• Mice, Inbred BALB C
• Microscopy, Confocal
• Spleen/cytology
• Spleen/parasitology
• Toxoplasma/physiology
• Toxoplasmosis/complications
• Vero Cells

• t. cruzi
• lysosomes
• lamp
• cell invasion

• Animals
• Cells, Cultured
• Fibroblasts/parasitology
• Host-Pathogen Interactions
• Lysosomal-Associated Membrane Protein 2/genetics
• Lysosomal-Associated Membrane Protein 2/metabolism
• Lysosomal Membrane Proteins/deficiency
• Lysosomal Membrane Proteins/metabolism
• Mice
• Mice, Knockout
• Trypanosoma cruzi/pathogenicity

• R37 AI034867 NIAID NIH HHS