Protozoan Host Cells Research Articles

Association between Legionella species and humic substances during early summer in the northern Baltic Sea

Climate change is projected to cause alterations in northern coastal systems, including humification and intensified nutrient loads, which can lead to ecosystem imbalances and establishment of new bacterial species. Several potential pathogens, such as different species of Legionella, hide in the environment between infections, some by living inside protozoan host cells. Knowledge about the occurrence of Legionella in natural waters is missing, which disable risk assessments of exposure. We performed a study of the species diversity of Legionella in the northern Baltic Sea (Gulf of Bothnia) during early summer to map their occurrence and to identify possible environmental drivers. We detected Legionella and potential protozoan hosts along gradients of the Gulf of Bothnia. We also for the first time present third generation full-length 16S rRNA amplicon sequencing (Nanopore) to resolve environmental species classification of Legionella, with a method suitable to study all bacteria. Our data show that full length 16S rRNA sequences is sufficient to resolve Legionella while the standard short Illumina sequences did not capture the entire diversity. For accurate species classification of Legionella, harmonization between the Nanopore classification methods is still needed and the bias toward the well-studied Legionella pneumophila need to be resolved. Different Legionella species occurred both in the Bothnian Sea and in the Bothnian Bay and their abundance were linked to humic substances and low salinity. The relative abundance of Legionella was higher in the humic-rich northern waters of the Bothnian Bay. The link between Legionella species and humic substances may be indirect via promotion of the heterotrophic microbial food web, allowing Legionella species and similar bacteria to establish. Humic substances are rich in iron, which has been shown crucial for growth of Legionella species and other pathogens. Considering climate change projections in this regional area, with increased humification and freshwater inflow, this bacterial niche containing potential pathogens might become more widespread in the future Baltic Sea. This study demonstrates the significance of DNA sequencing to monitor public health relevant bacteria like Legionella species in the environment. Including sequencing of bacteria and protozoa in the environmental monitoring programs could be used to identify ecosystem imbalances, which enable appropriate responses to emerging diseases.

Knowledge to Predict Pathogens: Legionella pneumophila Lifecycle Systematic Review Part II Growth within and Egress from a Host Cell.

Legionella pneumophila (L. pneumophila) is a pathogenic bacterium of increasing concern, due to its ability to cause a severe pneumonia, Legionnaires’ Disease (LD), and the challenges in controlling the bacteria within premise plumbing systems. L. pneumophila can thrive within the biofilm of premise plumbing systems, utilizing protozoan hosts for protection from environmental stressors and to increase its growth rate, which increases the bacteria’s infectivity to human host cells. Typical disinfectant techniques have proven to be inadequate in controlling L. pneumophila in the premise plumbing system, exposing users to LD risks. As the bacteria have limited infectivity to human macrophages without replicating within a host protozoan cell, the replication within, and egress from, a protozoan host cell is an integral part of the bacteria’s lifecycle. While there is a great deal of information regarding how L. pneumophila interacts with protozoa, the ability to use this data in a model to attempt to predict a concentration of L. pneumophila in a water system is not known. This systematic review summarizes the information in the literature regarding L. pneumophila’s growth within and egress from the host cell, summarizes the genes which affect these processes, and calculates how oxidative stress can downregulate those genes.

Impact of temperature on Legionella pneumophila, its protozoan host cells, and the microbial diversity of the biofilm community of a pilot cooling tower

Legionella pneumophila is a waterborne bacterium known for causing Legionnaires' Disease, a severe pneumonia. Cooling towers are a major source of outbreaks, since they provide ideal conditions for L. pneumophila growth and produce aerosols. In such systems, L. pneumophila typically grow inside protozoan hosts. Several abiotic factors such as water temperature, pipe material and disinfection regime affect the colonization of cooling towers by L. pneumophila. The local physical and biological factors promoting the growth of L. pneumophila in water systems and its spatial distribution are not well understood. Therefore, we built a lab-scale cooling tower to study the dynamics of L. pneumophila colonization in relationship to the resident microbiota and spatial distribution. The pilot was filled with water from an operating cooling tower harboring low levels of L. pneumophila. It was seeded with Vermamoeba vermiformis, a natural host of L. pneumophila, and then inoculated with L. pneumophila. After 92 days of operation, the pilot was disassembled, the water was collected, and biofilm was extracted from the pipes. The microbiome was studied using 16S rRNA and 18S rRNA genes amplicon sequencing. The communities of the water and of the biofilm were highly dissimilar. The relative abundance of Legionella in water samples reached up to 11% whereas abundance in the biofilm was extremely low (≤0.5%). In contrast, the host cells were mainly present in the biofilm. This suggests that L. pneumophila grows in host cells associated with biofilm and is then released back into the water following host cell lysis. In addition, water temperature shaped the bacterial and eukaryotic community of the biofilm, indicating that different parts of the systems may have different effects on Legionella growth.

Exosomes or microvesicles? Two kinds of extracellular vesicles with different routes to modify protozoan-host cell interaction.

Parasite-host cell interaction can be modulated by a dynamic communication between extracellular vesicles (EVs). They should play key roles in cell-cell communications transferring biomolecules (miRNA, proteins, soluble factors) from one cell to another cell. While many names have been used to denominate EVs, a better comprehension to understand these vesicles is raised when we classify it according to biogenesis: originated from multivesicular bodies, named exosomes, and from plasmatic membranes, denominated microvesicles. Here, we have reviewed EV participation during the protozoan-host cell interaction and reinforced the differences and similarities between exosomes and microvesicles, suggesting different intracellular routes and functions. We also discussed perspectives to study EVs and the role of EVs in diagnosis and chemotherapies of infectious diseases.

Is Trichomonas tenax a Parasite or a Commensal?

Trichomonas tenax is considered a commensal organism found under poor oral hygiene conditions. T. tenax presents morphological similarities with T. vaginalis, and there are doubts concerning whether this protist is a parasite and whether it is a genetic variant of T. vaginalis. This study aimed to investigate the capacity of T. tenax to cause mammalian cell damage and compare its cytotoxicity with that of T. vaginalis. Protozoan-host cell interaction assays were performed with Madin-Darby canine kidney, HeLa, and gum cells and 3D spheroids, which were examined by scanning electron and transmission electron microscopy. Cellular viability experiments were also performed. T. tenax attached and had different forms when interacting with mammalian cells and caused damage with time-dependent host-cell viability. We observed that T. tenax produced plasma membrane projections and phagocytosed portions of the mammalian cells. In addition, T. tenax caused membrane blebbing and apoptotic bodies in HeLa cells, thus inducing cell death. Spheroids were also used in interaction assays with T. tenax and they were damaged by these cells. This study shows that T. tenax fulfills the requisites of a parasite, causing damage to different mammalian cells and behaving similarly to T. vaginalis when in contact with target cells in vitro.

Trypanosoma cruzi-Host Cell Interaction.

Chagas disease was discovered by Carlos Chagas in Brazil in 1909 (1). It is caused by the pathogenic protozoan Trypanosoma cruzi, member of Trypanosomatidae family, Kinetoplastida order. Chagas disease is recognized by the World Health Organization as one of the main neglected tropical diseases, affecting about 8–15 million individuals in 18 countries in Central and South America, where it is endemic, as well as countries in North America and Europe (2). At least 30 million people are at risk. Public health programs have significantly reduced transmission of Chagas disease, however, blood and organ transplant transmission in non-endemic countries and a growing number of food borne (especially fruit juices) infections still require special attention. In addition, an increase in the rate of infection in the Amazon region has become a challenge for the control of Chagas disease (3). Trypanosoma cruzi presents a complex life cycle both in the vertebrate and invertebrate hosts, involving dramatic changes in cell shape (4). Its life cycle involves several developmental stages that are known as amastigotes, epimastigotes, and trypomastigotes. The first two stages are able to divide inside and outside host cells, respectively. The trypomastigote and amastigote stages are also able to infect host cells where they multiply, amplifying the number of parasites, and releasing millions of the infective trypomastigote forms in the intercellular spaces. This thematic issue deals with the ability of T. cruzi to penetrate into host cells. In the first article, de Souza and de Carvalho (5) make a review of the concept of active penetration and suggest that T. cruzi always penetrates the host cell through an endocytic process with the formation of a transient parasitophorous vacuole. The second article by Barrias et al. (6) reviews the various mechanisms of endocytosis, which are used by the protozoan to gain access to the intracellular portion of the host cells. These include processes such as classical phagocytosis, participation of membrane rafts, macropinocytosis, and clathrin-mediated endocytosis. In the third article, Calvet et al. (7) analyze in detail the process of interaction of T. cruzi with cardiomyocytes, an important host cell, because in vivo many of the parasite strains have a tropism for the heart. In the fourth article, Tonelli et al. (8) call the attention to the fact that most probably a large number of molecules are involved in the process of protozoan–host cell interaction and discuss the use of more powerful technologies, such as peptide-based phage display and oligonucleotide-based aptamer techniques. Using these approaches, the results obtained by the group highlight the importance of members of the 85-kDa family on the process of interaction. In the fifth article, Freire-de-Lima et al. (9) point out the relevance of a unique system of sialoglycoproteins and sialyl-binding proteins, which in the case of T. cruzi are represented by trans-sialidases. These proteins are involved in parasite–host cell recognition, infectivity, and survival. The sixth and seventh articles by Nde et al. (10) and Ferreira et al. (11) respectively, analyze the role played by components of the extracellular matrix during the interaction of the trypomastigote and amastigote forms of T. cruzi with the host cells. Infective trypomastigotes up-regulate the expression of laminin-gamma−1 and thrombospondin to facilitate recruitment of parasites to enhance cell infection. The extracellular matrix interactome network seems to be regulated by T. cruzi and its gp 83 ligand. The eighth article by Maeda et al. (12) reviews the cell signaling process that takes place during the interaction of metacyclic trypomastigotes, infective forms found in the invertebrate host, with host cells. Special emphasis is given to intracellular calcium mobilization and the triggering the exocytosis of host cell lysosomes during the interaction process mediated by a surface-expressed parasite glycoprotein of 82 kDa. This process leads to the activation of mammalian target of rapamycin (mTor), phosphatidylinositol 3-kinase, and protein kinase C. The last article, by Scharfstein et al. (13) initially analyses the process by which T. cruzi trypomastigotes elicit an inflammatory edema that stimulates protective type-1 effector cells through the activation of the kallikrein–kinin system, providing a framework to investigate the intertwined proteolytic circuits that couple the anti-parasite immunity to inflammation and fibrosis.

In the case of transmission of <i>Mycobacterium ulcerans</i> in Buruli ulcer disease <i>Acanthamoeba </i>species stand accused

Buruli ulcer disease caused by Mycobacterium ulcerans results in extensive destruction of skin and soft tissue and long-term functional disabilities that ultimately require surgery and rehabilitation. The disease is associated with aquatic and swampy environments with the mycobacterium occurring in biofilms, soil, aquatic insects, fish and wildlife however, the mode of transmission to humans remains an enigma. Current transmission ideas including bites from predatory water bugs and mosquitoes, do not explain satisfactorily the spasmodic disease distribution in human populations. Here we argue that Acanthamoeba species are the natural hosts of M. ulcerans and are mainly responsible for disease transmission because; (i) Acanthamoebae are known natural hosts of several microbial pathogens including M. marinum, M. avium and Legionella pneumophila, (ii) culture of slow-to-grow microbial pathogens hosted in nature by Acanthamoeba spp is enhanced when the media is seeded with the protozoa, (iii) acanthamoebae and M. ulcerans share similar bio-ecological and epidemiological settings, (iv) documented evidence that prior growth of L. pneumophila and M. avium in acanthamoebae influences entry mechanisms, intracellular growth and virulence in human monocytes, (v) Acanthamoeba spp also infect humans and cause diseases via routes of openings including broken skin and sites of trauma similar to M. ulcerans and (vi) M. ulcerans is rather a fastidious intracellular organism as recent analysis of the genome indicate. We argue further that temperature plays a significant role in transmission determining the fate of either the intracellular microbe or the host cells. Also, Acanthamoeba-pathogen association has a long evolutionary history because the same set of bacterial genes and gene products e.g. in L. pneumophila are required for survival in both mammalian and protozoan host cells. We suggest that the involvement of Acanthamoeba in the transmission of M. ulcerans to humans better explains the disease's epidemiology.

Pseudomonas aeruginosa uses type III secretion system to kill biofilm-associated amoebae

Bacteria and protozoa coexist in a wide range of biofilm communities of natural, technical and medical importance. Generally, this interaction is characterized by the extensive grazing activity of protozoa on bacterial prey populations. We hypothesized that the close spatial coexistence in biofilms should allow opportunistic pathogenic bacteria to utilize their eukaryote-targeting arsenal to attack and exploit protozoan host cells. Studying cocultures of the environmental pathogen Pseudomonas aeruginosa and the amoeba Acanthamoeba castellanii, we found that P. aeruginosa rapidly colonized and killed biofilm-associated amoebae by a quorum-sensing independent mechanism. Analysis of the amoeba-induced transcriptome indicated the involvement of the P. aeruginosa type III secretion system (T3SS) in this interaction. A comparison of mutants with specific defects in the T3SS demonstrated the use of the secretion apparatus and the effectors ExoU, ExoS and ExoT in the killing process, of which ExoU had the greatest impact. T3SS-mediated virulence towards A. castellanii was found to be controlled by the global regulators RpoN and RpoS and through modulation of cAMP and alginate biosynthesis. Our findings suggest that conserved virulence pathways and specifically the T3SS play a central role in bacteria-protozoa interactions in biofilms and may be instrumental for the environmental persistence and evolution of opportunistic bacterial pathogens.

Predator or prey? Chlamydophila abortus infections of a free-living amoebae, Acanthamoeba castellani 9GU

Limited evidence exists to suggest that the ability to invade and escape protozoan host cell bactericidal activity extends to members of the Chlamydiaceae, intracellular pathogens of humans and animals and evolutionary descendants of amoeba-resisting Chlamydia-like organisms. PCR and microscopic analyses of Chlamydophila abortus infections of Acanthamoeba castellani revealed uptake of this chlamydial pathogen but, unlike the well-described inhabitant of A. castellani, Parachlamydia acanthamoebae, Cp. abortus did not appear to propagate and is likely digested by its amoebal host. These data raise doubts about the ability of free-living amoebae to serve as hosts and vectors of pathogenic members of the Chlamydiaceae but reveal opportunities, via comparative genomics, to understand virulence mechanisms used by Chlamydia-like organisms to avoid amoebal digestion.

Coevolution between nonhomologous but functionally similar proteins and their conserved partners in the Legionella pathogenesis system

Legionella pneumophila, the causative agent of Legionnaires' disease, and other pathogenic Legionella species multiply inside protozoa and human macrophages by using the intracellular multiplication (Icm)/defect in organelle trafficking (Dot) type-IV secretion system. The IcmQ protein, which possesses pore-forming activity, and IcmR, which regulates the IcmQ activity, are two essential components of this system. Analysis of the region expected to contain these two genes from 29 Legionella species revealed the presence of a conserved icmQ gene and a large hypervariable gene family [functional homologues of icmR (fir) genes], located at the icmR genomic position. Although hypervariable in their sequence, the fir genes from all 29 Legionella species were found, together with their corresponding icmQ genes, to function similarly during infection. In addition, all FIR proteins we examined were found to interact with their corresponding IcmQ proteins. Detailed bioinformatic, biochemical, and genetic analysis of the interaction between the variable FIR proteins and conserved IcmQ proteins revealed that their interaction depends on a variable region located between two conserved domains of IcmQ. This variable region was also found to be critical for IcmQ self-interaction, and the region probably coevolved with the corresponding FIR protein. A FIR-IcmQ pair was also found in Coxiella burnetii, the only known non-Legionella bacterium that contains an Icm/Dot system, indicating the significance of this protein pair for the function of this type-IV secretion system. We hypothesize that this gene variation, which is probably mediated by positive selection, plays an important role in the evolutionary arms race between the protozoan host cell and the pathogen.

Impact of the bacterial type I cytochrome c maturation system on different biological processes

In the alpha-, beta- and gamma-Proteobacteria, the so-called cytochrome c maturation (Ccm) system is known to promote the covalent attachment of the haem to periplasmic apocytochrome c. However, in species of Pseudomonas, Rhizobium, Paracoccus and Legionella, mutations in ccm genes result in phenotypes that cannot be readily explained by the simple loss of a c-type cytochrome. These phenotypes include loss of siderophore production and utilization, reduced abilities to grow in low-iron conditions and in mammalian and protozoan host cells, and alterations in copper sensitivity and manganese oxidation. These various data suggest that Ccm proteins may perform one or more functions in addition to Ccm, which are critical for bacterial physiology and growth. Novel hypotheses that should be explored include the utilization of Ccm-associated haem for processes besides attachment to apocytochrome c, the export of a non-haem compound through the Ccm system, and the negative effects of protoporphyrin IX accumulation.

Characterization of the icmH and icmF genes required for Legionella pneumophila intracellular growth, genes that are present in many bacteria associated with eukaryotic cells.

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process. Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells. Here, we have characterized the L. pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined. The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity. Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A. castellanii. Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements. In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA- and RelA-dependent manner. Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L. pneumophila pathogenesis.

Legionella Effectors That Promote Nonlytic Release from Protozoa

Legionella pneumophila, the bacterial agent of legionnaires' disease, replicates intracellularly within a specialized vacuole of mammalian and protozoan host cells. Little is known about the specialized vacuole except that the Icm/Dot type IV secretion system is essential for its formation and maintenance. The Legionella genome database contains two open reading frames encoding polypeptides (LepA and LepB) with predicted coiled-coil regions and weak homology to SNAREs; these are delivered to host cells by an Icm/Dot-dependent mechanism. Analysis of mutant strains suggests that the Lep proteins may enable the Legionella to commandeer a protozoan exocytic pathway for dissemination of the pathogen.

Identification and subcellular localization of the Legionella pneumophila IcmX protein: a factor essential for establishment of a replicative organelle in eukaryotic host cells.

The gram-negative respiratory pathogen Legionella pneumophila infects and grows within mammalian macrophages and protozoan host cells. Upon uptake into macrophages, L. pneumophila establishes a replicative organelle that avoids fusion with endocytic vesicles. There are 24 dot/icm genes on the L. pneumophila chromosome required for biogenesis of this vacuole. Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been investigating the function of L. pneumophila dot/icm gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis. The icmX gene product falls into this category. To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an L. pneumophila icmX deletion mutant and murine bone marrow-derived macrophages. These data demonstrate that icmX is required for biogenesis of the L. pneumophila replicative organelle. Immunoblot analysis indicates that the icmX gene product is a polypeptide with an estimated molecular mass of 50 kDa. The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal sec-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-type L. pneumophila growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other Legionella species by immunoblot analysis using a monoclonal antibody specific for L. pneumophila IcmX protein. From these data, we conclude that the IcmX protein is an essential component of the dot/icm secretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the Legionellaceae family.

Legionella pneumophila kills human phagocytes but not protozoan host cells by inducing apoptotic cell death

Legionella pneumophila is a facultative intracellular parasite able to replicate within and to kill a variety of eukaryotic cells. One possible killing mechanism is the induction of programmed cell death. Based on electron microscopy and flow cytometry studies using the phosphatidylserine binding protein annexin V, we could demonstrate that L. pneumophila is able to induce apoptosis in human monocytes which was clearly dependent on the multiplicity of infection, the time postinfection and the intracellular location of the bacteria. Furthermore, it became evident that Legionella-induced apoptosis does not require the TNF-α mediated signal-transduction pathway. By studying infection in Acanthamoeba castellanii, we found that L. pneumophila is not able to induce programmed cell death in their natural host cells indicating that different mechanisms are responsible for host cell killing in protozoan and human cells.