Intracellular pathogens are microorganisms able to grow and reproduce in host cells. Some of them cause disease.
In relation to the degree of dependency on the host cell resources, they can be classified in two different groups: facultative and obligate intracellular pathogens. Facultative intracellular pathogens like Listeria monocytogenes can survive both inside or outside cells, while obligate intracellular pathogens like Toxoplasma gondii need host cells in order to reproduce.
Host-pathogen interaction is the term used to describe how infectious agents survive within the host organisms.
In order to understand host-pathogen interactions at a cellular level, we need to overcome conventional microscopy methods limitations such as the impossibility to obtain 3D information or perform long-term live cell imaging avoiding phototoxicity and image quality loss.
Digital, marker-free, non-invasive imaging from Nanolive’s 3D Cell Explorer allows for a time-reduced sample preparation, high quality live-cell imaging at unique spatiotemporal resolution (<200nm; 1img/2sec) without any problem of photobleaching or phototoxicity.
A microscopic view of label-free and living Listeria bacteria
What is Listeria and where does it come from?
Infection from Listeria monocytogenes is a food borne bacterial illness that can be very serious for pregnant women and people with impaired immune systems. Listeria infection can be contracted by eating badly preserved meat and unpasteurized milk products (including soft cheese, ice cream, and yogurt).
Healthy individuals are normally resistant to listeria infection, but the disease can be fatal to unborn babies and very young babies. Immunodepressed people also are at higher risk. Listeria bacteria is very resistant and can survive refrigeration and even freezing. This is why people at risk should just avoid consuming products at risk.
Mouse macrophages infected with Listeria
In this video we observe mouse macrophages that have been infected with Listeria monocytogenes.
At the beginning of the video, the cell at the top of the field of view is going through mitosis. The chromosomes’ condensation is clearly observable in the center of the cell. Listeria bacteria are observable in this same host cell: they are either free to move around in the cytoplasm or inside cytoplasmic vacuoles, replicating. When the host cell divides, we can observe the transmission of these pathogenic bacteria to the daughter cells.
In the second part of the video, we move the attention to the cell below. At the end of its reproductive cell cycle, the Listeria bacterium is released through the host cell’s plasma membrane and is free to infect other cells. Here we clearly see the vacuole localization of Listeria followed by membrane destruction and cell death during its release.
P. falciparum's intra-erythrocytic cycle, an important immune escape strategy in Malaria
Malaria is a preventable and curable disease caused by the genus Plasmodium. In 2017, around 219 million cases were estimated, from which 435000 resulted in death. As many as 87 different countries registered malaria cases, but India and sub-Saharan Africa carried almost 80% of the disease prevalence.
The most severe form of human malaria is caused by the protozoan parasite Plasmodium falciparum. Female Anopheles mosquitoes are vectors in the transmission of the parasite.
In order to avoid misdiagnosis, which results in increased morbidity and mortality rates, the World Health Organization calls for an early, accurate and high-quality diagnosis of the disease. Currently, the diagnose is made using microscopy, rapid diagnostic tests and nucleic acid amplification-based diagnostics.
P. Falciparum infected human red blood cells
Internalization of the parasite in the host red blood cells entails not just avoiding macrophages detection, but also degrading huge amounts of haemoglobin. This loss of haemoglobin is visible under the microscope. During its life cycle, the parasite’s shape does not remain constant. Indeed, its life cycle goes through different stages as shown above.
Read our very detailed blog post about Malaria here: https://nanolive.ch/pfalciparums-intra-erythrocytic-cycle/.
Macrophages - The Big Eaters
Macrophages are present in almost all tissues. They are contributing to various processes in the healthy organism, such as development, wound healing, infection and tissue homeostasis. They can rapidly change their phenotype in response to variations in their environment. Macrophages are known for their classical function as antimicrobial phagocytes but support immune function as well by the presentation of antigens. Their research applications are vast, and in vitro assays are increasingly used in a wide range of research areas, including immunology, bacteriology and parasitology, as well as in biomedical and transplantation studies. Two advantages of macrophages in cell culture are that they are relatively easy to generate and to cultivate.
In these videos – obtained with Nanolive’s 3D Cell Explorer – we present cryopreserved human M1 macrophages from PromoCell in cell culture. The 3D Cell Explorer allows to image these living macrophages in a novel, marker-free fashion. A special note goes to the visualization of membrane ruffling as waves arising at the leading edge of lamellipodia that move centripetally toward the main cell body.
E.coli being engulfed through phagocytosis
Phagocytosis Assay Kits by PromoCell were used to test the viability and cellular functioning of the macrophages (video 2 & 3). E.coli particles, visible as small ellipsoid particles, are trapped by the cells, transported and lysed. This system can be used to provide a robust screening system for activators and/or inhibitors of phagocytosis and Toll-like Receptor (TLR) ligands.
Dictyostelium discoideum, known as the social amoeba, interacts with bacteria in order to predate them and form symbiotic associations, but it can also be harmed or infected by them.
D.discoideum’s pseudopods are cytoplasmatic projections of its cell membrane that help internalize food like bacteria, algae or protozoa from the surrounding water. Digestion enzymes are used to digest the internalized products, which are then absorbed. This step is followed by the disappearance of the food vacuole.
This high temporal time-lapse resolution footage (3D/1.7s) obtained with Nanolive’s 3D Cell Explorer, allows us to observe Dictyostelium amoebae phagocyting Escherichia coli, after an overnight incubation. The time-lapse imaging had a total length of 9 minutes, and images were captured every 4 seconds.
Toxoplasma gondii is an obligate intracellular parasite responsible for toxoplasmosis, a non-severe disease in adults, but that may lead to birth defects if infected during pregnancy. Domestic cats and other felids are its definitive hosts, but it can virtually infect any kind of warm-blooded animal.
During its life cycle it undergoes a sexual and an asexual phase. While the sexual cycle can only be completed within their definitive hosts, the asexual cycle can be carried out in any of their intermediate hosts (warm-blooded animals like humans or birds).
Ingestion of oocysts that have been released into the felid’s intestinal lumen and shed within their faeces is the usual infection route of the parasite’s intermediate host.
Different stages are observed in T. gondii’s life cycle:
- Bradyzoites: contained in tissue cysts, they invade host epithelial cells, where they divide.
- Sporozoites: released from the oocysts, they penetrate and divide in intestinal cells.
- Tachyzoites: evolved from bradyzoites or sporozoites, they kill cells using them to multiply and form more tachyzoites that will spread though the host bloodstream.
- Cysts: containing bradyzoites.
- Oocysts: containing sporozoites.
- Merozoites: evolved from the bradyzoites, they divide within intestinal epithelial cells, where they convert into the sexual stages of the parasite (oocysts).
Under Nanolive’s 3D Cell Explorer, the identification of the parasitophorous vacuoles created during parasitic entry to the cell is possible, as well as a follow-up on the tachyzoites multiplication within the vacuoles. Thanks to the 3D Cell Explorer live cell imaging capabilities, the monitoring of host cell death and burst, followed by tachyzoites release can be observed.
In this footage, we can distinguish T. gondii’s apical ring-shaped complex in tachyzoites from a Toxoplasma gondii infection in mouse fibroblasts (MEF1 T2). This structure plays a key role in host cell invasion processes. The 3D render obtained solely from the sample’s refractive index, an inherent characteristic from it (thus, from a non-stained sample) is compared to a static image obtained from confocal laser-scanning microscopy1.
1 Katris NJ, van Dooren GG, McMillan PJ, Hanssen E, Tilley L, Waller RF (2014) The Apical Complex Provides a Regulated Gateway for Secretion of Invasion Factors in Toxoplasma. PLoS Pathog 10(4): e1004074. https://doi.org/10.1371/journal.ppat.1004074
Scientific Paper: labelfree holotomographic microscopy reveals virus-induced cytopathic effects in live cells
In this work from the Greber group (University of Zurich), the 3D Cell Explorer’s technology is described as the only microscopy solution available to characterize the cytopathic effect induced by virus in live cells in a label-free fashion.
Cytopathic effects (CPEs) are a hallmark of infections. CPEs can be observed by phase contrast or fluorescence light microscopy, albeit at the cost of phototoxicity. We report that digital holo-tomographic microscopy (DHTM) reveals distinct patterns of virus infections in live cells with minimal perturbation. DHTM is label-free, and records the phase shift of low energy light passing through the specimen on a transparent surface. DHTM infers a 3-dimensional (3D) tomogram based on the refractive index (RI). By measuring RI and computing the refractive index gradient (RIG) values DHTM unveils on optical heterogeneity in cells upon virus infection. We find that vaccinia virus (VACV), herpes simplex virus (HSV) and rhinovirus (RV) infections progressively and distinctly increased RIG. VACV, but not HSV and RV infection induced oscillations of cell volume, while all three viruses altered cytoplasmic membrane dynamics, and induced apoptotic features akin to the chemical compound staurosporin, but with virus-specific signatures. In sum, we introduce DHTM for quantitative label-free microscopy in infection research, and uncover virus-type specific changes and CPE in living cells at minimal interference.
Find the publication here: https://msphere.asm.org/content/3/6/e00599-18.
Scientific Paper: Besnoitia besnoiti infection alters both endogenous cholesterol de novo synthesis and exogenous LDL uptake in host endothelial cells
Besnoitia besnoiti is an emerging parasite in Europe, responsible for bovine besnoitiosis, an illness causing a significant economic impact on cattle industry.
Similarly to T. gondii, already featured here, B. besnoiti also needs to scavenge cholesterol from its host cell in order to replicate. However, the mechanism used by B. besnoiti to obtain cholesterol is still controversial.
On this work, Silva and colleagues from the Justus-Liebig University of Giessen study the model of cholesterol salvage by analysing B. besnoiti infection of primary bovine endothelial cells in vivo.
Their results demonstrate that B. besnoiti can obtain cholesterol both via endogenous than exogeneous uptake. This finding reinforces the theory that the successful B. besnoiti infections in primary bovine endothelial host cells rely on both the parasite and the cell type.
The 3D Cell Explorer- fluo was used to confirm the presence of abundant lipid droplet-like structures in B. besnoiti-infected cells. Images were later analysed using STEVE software (Figure 1).
The full publication is available here!
Figure 1. Live cell holographic tomography-based illustration of lipid droplets in non-infected and B. besnoiti- infected BUVEC.
Scientific Paper: Toxoplasma gondii-induced host cellular cell cycle dysregulation is linked to chromosome missegregation and cytokinesis failure in primary endothelial host cells
Toxoplasma gondii is an obligate intracellular parasite that induces lysis in host cells as a result of its fast proliferation. Traditionally, T. gondii’s infection effects on cell cycle have been studied in immortalized cell lines, which may behave differently than primary cells. Moreover, some recent data suggested that T. gondii might cause cell-type-specific reactions, thus the need to analyze the effects of the infection in typical host cell for T. gondii. For that purpose, Velasquez and colleagues studied the influence of T.gondii’s tachyzoites on host cell cycle progression in primary endothelial cells.
Infected primary bovine umbilical vein endothelial cells (BUVEC) were analyzed by immunohistochemistry and physicochemical analyses. They were also observed under different types of microscopy, including the 3D Cell Explorer-fluo from Nanolive, that allowed for nuclei visualization in a live cell experiment.
Nuclei visualization unveiled a significantly higher presence of binucleated or multinucleate cells in the infected sample with a range from two to five nuclei in infected BUVEC cells (27.7%) compared to non-infected BUVEC samples (6.8%). Furthermore, infected BUVEC cells failed in successfully accomplishing cytokinesis. The blockage of cytokinesis seemed to be mediated by the downregulation of the Aurora B kinase expression.
T. gondii’s infection stopped cell cycle in G2-M phase without affecting expression of G2-specific cyclin B1. Mitosis dysfunctions included chromosome segregation errors and supernumerary centrosome formation.
Read the full publication here: https://www.ncbi.nlm.nih.gov/pubmed/31467333.
Figure 1. Vital staining of live binucleate T. gondii-infected BUVEC. Cells infected with T. gondii were stained with the vital stain DRAQ5 (red) for nuclei detection. Figure from .