Nuclear trafficking of bacterial effector proteins.

Bacterial pathogens can subvert host responses by producing effector proteins that directly target the nucleus of eukaryotic cells in animals and plants. Nuclear-targeting proteins are categorised as either: "nucleomodulins," which have epigenetic-modulating activities; or "cyclomodulins," which specifically interfere with the host cell cycle. Bacteria can deliver these effector proteins to eukaryotic cells via a range of strategies. Despite an increasing number of reports describing the effects of bacterial effector proteins on nuclear processes in host cells, the intracellular pathways used by these proteins to traffic to the nucleus have yet to be fully elucidated. This review will describe current knowledge about how nucleomodulins and cyclomodulins enter eukaryotic cells, exploit endocytic pathways and translocate to the nucleus. We will also discuss the secretion of nuclear-targeting proteins or their release in bacterial membrane vesicles and the trafficking pathways employed by each of these forms. Besides their importance for bacterial pathogenesis, some nuclear-targeting proteins have been implicated in the development of chronic diseases and even cancer. A greater understanding of nuclear-targeting proteins and their actions will provide new insights into the pathogenesis of infectious diseases, as well as contribute to advances in the development of novel therapies against bacterial infections and possibly cancer.

Morphometric and morphokinetic differences in the sperm- and oocyte-originated pronuclei of male and female human zygotes: a time-lapse study.

PURPOSE: To study the morphometric and morphokinetic profiles of pronuclei (PN) between male and female human zygotes. METHOD(S): This retrospective cohort study included 94 consecutive autologous single day 5 transfer cycles leading to a singleton live birth. All oocytes were placed in the EmbryoScope + incubator post-sperm injection with all annotations performed retrospectively by one embryologist (L-SO). Timing parameters included 2nd polar body extrusion (tPB2), sperm-originated PN (tSPNa) or oocyte-originated PN (tOPNa) appearance, and PN fading (tPNF). Morphometrics were evaluated at 8 (stage 1), 4 (stage 2), and 0 h before PNF (stage 3), measuring PN area (um2), PN juxtaposition, and nucleolar precursor bodies (NPB) arrangement. RESULTS: Male zygotes had longer time intervals of tPB2_tSPNa than female zygotes (4.8 ± 0.2 vs 4.2 ± 0.1 h, OR = 1.442, 95% CI 1.009-2.061, p = 0.044). SPN increased in size from stage 1 through 2 to 3 (435.3 ± 7.2, 506.7 ± 8.0, and 556.3 ± 8.9 um2, p = 0.000) and OPN did similarly (399.0 ± 6.1, 464.3 ± 6.7, and 513.8 ± 6.5 um2, p = 0.000), with SPN being significantly larger than OPN at each stage (p < 0.05 respectively). More male than female zygotes reached central PN juxtaposition at stage 1 (76.7% vs 51.0%, p = 0.010), stage 2 (97.7% vs 86.3%, p = 0.048), and stage 3 (97.7% vs 86.3%, p = 0.048). More OPN showed aligned NPBs than in SPN at stage 1 only (44.7% vs 28.7%, p = 0.023). CONCLUSION(S): Embryos with different sexes display different morphokinetic and morphometric features at the zygotic stage. Embryo selection using such parameters may lead to unbalanced sex ratio in resulting offspring.

Fusarium graminearum Infection Strategy in Wheat Involves a Highly Conserved Genetic Program That Controls the Expression of a Core Effectome.

Fusarium graminearum, the main causal agent of Fusarium Head Blight (FHB), is one of the most damaging pathogens in wheat. Because of the complex organization of wheat resistance to FHB, this pathosystem represents a relevant model to elucidate the molecular mechanisms underlying plant susceptibility and to identify their main drivers, the pathogen's effectors. Although the F. graminearum catalog of effectors has been well characterized at the genome scale, in planta studies are needed to confirm their effective accumulation in host tissues and to identify their role during the infection process. Taking advantage of the genetic variability from both species, a RNAseq-based profiling of gene expression was performed during an infection time course using an aggressive F. graminearum strain facing five wheat cultivars of contrasting susceptibility as well as using three strains of contrasting aggressiveness infecting a single susceptible host. Genes coding for secreted proteins and exhibiting significant expression changes along infection progress were selected to identify the effector gene candidates. During its interaction with the five wheat cultivars, 476 effector genes were expressed by the aggressive strain, among which 91% were found in all the infected hosts. Considering three different strains infecting a single susceptible host, 761 effector genes were identified, among which 90% were systematically expressed in the three strains. We revealed a robust F. graminearum core effectome of 357 genes expressed in all the hosts and by all the strains that exhibited conserved expression patterns over time. Several wheat compartments were predicted to be targeted by these putative effectors including apoplast, nucleus, chloroplast and mitochondria. Taken together, our results shed light on a highly conserved parasite strategy. They led to the identification of reliable key fungal genes putatively involved in wheat susceptibility to F. graminearum, and provided valuable information about their putative targets.

mcr-1 Gene Expression Modulates the Inflammatory Response of Human Macrophages to Escherichia coli.

MCR-1 is a plasmid-encoded phosphoethanolamine transferase able to modify the lipid A structure. It confers resistance to colistin and was isolated from human, animal, and environmental strains of Enterobacteriaceae, raising serious global health concerns. In this paper, we used recombinant mcr-1-expressing Escherichia coli to study the impact of MCR-1 products on E. coli-induced activation of inflammatory pathways in activated THP-1 cells, which was used as a model of human macrophages. We found that infection with recombinant mcr-1-expressing E. coli significantly modulated p38-MAPK and Jun N-terminal protein kinase (JNK) activation and pNF-κB nuclear translocation as well as the expression of genes for the relevant proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-12 (IL-12), and IL-1ß compared with mcr-1-negative strains. Caspase-1 activity and IL-1ß secretion were significantly less activated by mcr-1-positive E. coli strains than the mcr-1-negative parental strain. Similar results were obtained with clinical isolates of mcr-1-positive E. coli, suggesting that, in addition to colistin resistance, the expression of mcr-1 allows the escape of early host innate defenses and may promote bacterial survival.

Phytophthora infestans RXLR Effectors Target Parallel Steps in an Immune Signal Transduction Pathway.

The potato (Solanum tuberosum) blight pathogen Phytophthora infestans delivers Arg-X-Leu-Arg (RXLR) effector proteins into host cells to subvert plant immune responses and promote colonization. We show that transient expression and stable transgenic expression of the RXLR effector Pi22926 in Nicotiana benthamiana promotes leaf colonization by P. infestans. Pi22926 suppresses cell death triggered by coexpression of the Cladosporium fulvum avirulence protein Avr4 and the tomato (Solanum lycopersicum) resistance protein Cf4. Pi22926 interacts with a potato mitogen-activated protein kinase kinase kinase, StMAP3Kß2, in the nucleoplasm. Virus-induced gene silencing (VIGS) of the ortholog NbMAP3Kß2 in N. benthamiana enhances P. infestans colonization and attenuates Cf4/Avr4-induced cell death, indicating that this host protein is a positive regulator of immunity. Cell death induced by Cf4/Avr4 is dependent on NbMAP3Kε and NbMAP3Kß2, indicating that these MAP3Ks function in the same signaling pathway. VIGS of NbMAP3Kß2 does not compromise cell death triggered by overexpression of MAP3Kε. Similarly, VIGS of NbMAP3Kε does not attenuate cell death triggered by MAP3Kß2, demonstrating that these MAP3K proteins function in parallel. In agreement, Pi22926 or another RXLR effector, PexRD2, only suppresses cell death triggered by expression of StMAP3Kß2 or StMAP3Kε, respectively. Our data reveal that two P. infestans effectors, PexRD2 and Pi22926, promote P. infestans colonization by targeting MAP3K proteins that act in parallel in the same signal transduction pathway.

Enterospora nucleophila (Microsporidia) in Gilthead Sea Bream (Sparus aurata): Pathological Effects and Cellular Immune Response in Natural Infections.

Enterospora nucleophila is a microsporidian responsible for an emaciative disease in gilthead sea bream (Sparus aurata). Its intranuclear development and the lack of in vitro and in vivo models hinder its research. This study investigated the associated lesions, its detection by quantitative polymerase chain reaction, and the cellular immune response of naturally infected fish. The intensity of infection in the intestine was correlated with stunted growth and reduced body condition. At the beginning of the outbreaks, infection prevalence was highest in intestine and stomach, and in subsequent months, the prevalence decreased in the intestine and increased in hematopoietic organs and stomach. In heavy infections, the intestine had histologic lesions of enterocyte hypercellularity and proliferation of rodlet cells. Infected enterocytes had E. nucleophila spores in the cytoplasm, and a pyknotic nucleus, karyorhexis or karyolysis. Lymphocytes were present at the base of the mucosa, and eosinophilic granule cells were located between the enterocytes. In intestinal submucosa, macrophage aggregates containing spores were surrounded by lymphocytes and granulocytes, with submucosal infiltration of granulocytes. Macrophage aggregates appeared to develop into granulomata with necrotic areas containing parasite remnants. Immunohistochemistry revealed mast cells as the main type of granulocyte involved. Abundant IgM+ and IgT+ cells were identified by in situ hybridization in the submucosa when intracytoplasmic stages were present. This study describes the lesions of E. nucleophila in gilthead sea bream, an important aquaculture species.

Infection and nuclear interaction in mammalian cells by 'Candidatus Berkiella cookevillensis', a novel bacterium isolated from amoebae.

BACKGROUND: 'Candidatus Berkiella cookevillensis' and 'Ca. Berkiella aquae' have previously been described as intranuclear bacteria of amoebae. Both bacteria were isolated from amoebae and were described as appearing within the nuclei of Acanthamoeba polyphaga and ultimately lysing their host cells within 4 days. Both bacteria are Gammaproteobacteria in the order Legionellales with the greatest similarity to Coxiella burnetii. Neither bacterium grows axenically in artificial culture media. In this study, we further characterized 'Ca. B. cookevillensis' by demonstrating association with nuclei of human phagocytic and nonphagocytic cell lines. RESULTS: Transmission electron microscopy (TEM) and confocal microscopy were used to confirm nuclear co-localization of 'Ca. B. cookevillensis' in the amoeba host A. polyphaga with 100% of cells having bacteria co-localized with host nuclei by 48 h. TEM and confocal microscopy demonstrated that the bacterium was also observed to be closely associated with nuclei of human U937 and THP-1 differentiated macrophage cell lines and nonphagocytic HeLa human epithelial-like cells. Immunofluorescent staining revealed that the bacteria-containing vacuole invaginates the nuclear membranes and appears to cross from the cytoplasm into the nucleus as an intact vacuole. CONCLUSION: Results of this study indicate that a novel coccoid bacterium isolated from amoebae can infect human cell lines by associating with the host cell nuclei, either by crossing the nuclear membranes or by deeply invaginating the nuclear membranes. When associated with the nuclei, the bacteria appear to be bound within a vacuole and replicate to high numbers by 48 h. We believe this is the first report of such a process involving bacteria and human cell lines.

Lactoferrin translocates to the nucleus of bovine rectal epithelial cells in the presence of Escherichia coli O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a foodborne pathogen which causes illness in humans. Ruminants are the main reservoirs and EHEC predominantly colonizes the epithelium of the recto-anal junction of cattle. Immunosuppression by EHEC promotes re-infection of cattle. However, bovine lactoferrin (bLF) apparently can overrule the immunosuppression by inducing EHEC-specific IgA responses at the mucosal site. The IgA responses are significantly correlated with reduced EHEC shedding and the absence of colonization at the rectal mucosa following re-infection. Therefore, to examine the interaction between bLF and bovine rectal epithelial cells, we first developed a method to establish a primary cell culture of epithelial cells of the rectum of cattle. Furthermore, we used LC-MS/MS to demonstrate the presence of secreted lactoferrin in bovine milk and the absence of a "delta" isoform which is known to translocate to the nucleus of cells. Nevertheless, lactoferrin derived from bovine milk was internalized by rectal epithelial cells and translocated to the nuclei. Moreover, nuclear translocation of bLF was significantly enhanced when the epithelial cells were inoculated with EHEC, as demonstrated by confocal fluorescence microscopy and confirmed by Raman microscopy and 3D imaging.

Cytosolic activation of cell death and stem rust resistance by cereal MLA-family CC-NLR proteins.

Plants possess intracellular immune receptors designated "nucleotide-binding domain and leucine-rich repeat" (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively.

Detection of the intranuclear microsporidian Enterospora nucleophila in gilthead sea bream by in situ hybridization.

Enterospora nucleophila is an intranuclear microsporidian responsible for emaciative microsporidiosis of gilthead sea bream (GSB). Its minute size and cryptic nature make it easily misdiagnosed. An in situ hybridization (ISH) technique based on antisense oligonucleotide probes specific for the parasite was developed and used in clinically infected GSB in combination with calcofluor white stain (CW) and other histopathological techniques. The ISH method was found to label very conspicuously the cells containing parasite stages, with the signal concentrating in merogonial and sporogonial plasmodia within the infected cell nuclei. Comparison with CW demonstrated limited ISH signal in cells containing mature spores, which was attributed mostly to the scarcity of probe targets present in these stages. Although spores were detected in other organs of the digestive system as well as in the peripheral blood, proliferative stages or parasite reservoirs were not found in this work outside the intestines. The study demonstrated a frequent disassociation between the presence of abundant spores and the intensity of the infections as determined by the parasite activity. The ISH allows confirmatory diagnosis of GSB microsporidiosis and estimation of infection intensity and will be a valuable tool for a more precise determination of parasite dissemination pathways and pathogeny mechanisms.

Intracellular Staphylococcus aureus Control by Virulent Bacteriophages within MAC-T Bovine Mammary Epithelial Cells.

Bacteriophages (phages) are known to effectively kill extracellular multiplying bacteria. The present study demonstrated that phages penetrated bovine mammary epithelial cells and cleared intracellular Staphylococcus aureus in a time-dependent manner. In particular, phage vB_SauM_JS25 reached the nucleus within 3 h postincubation. The phages had an endocytotic efficiency of 12%. This ability to kill intracellular host bacteria suggests the utility of phage-based therapies and may protect patients from recurrent infection and treatment failure.

Intrasperm vertical symbiont transmission.

Symbiotic bacteria are commonly associated with cells and tissues of diverse animals and other organisms, which affect hosts' biology in a variety of ways. Most of these symbionts are present in the cytoplasm of host cells and maternally transmitted through host generations. The paucity of paternal symbiont transmission is likely relevant to the extremely streamlined sperm structure: the head consisting of condensed nucleus and the tail made of microtubule bundles, without the symbiont-harboring cytoplasm that is discarded in the process of spermatogenesis. Here, we report a previously unknown mechanism of paternal symbiont transmission via an intrasperm passage. In the leafhopper Nephotettix cincticeps, a facultative Rickettsia symbiont was found not only in the cytoplasm but also in the nucleus of host cells. In male insects, strikingly, most sperm heads contained multiple intranuclear Rickettsia cells. The Rickettsia infection scarcely affected the host fitness including normal sperm functioning. Mating experiments revealed both maternal and paternal transmission of the Rickettsia symbiont through host generations. When cultured with mosquito and silkworm cell lines, the Rickettsia symbiont was preferentially localized within the insect cell nuclei, indicating that the Rickettsia symbiont itself must have a mechanism for targeting nucleus. The mechanisms underlying the sperm head infection without disturbing sperm functioning are, although currently unknown, of both basic and applied interest.

Helicobacter pylori HP0425 Targets the Nucleus with DNase I-Like Activity.

BACKGROUND AND AIMS: Nuclear targeting of bacterial proteins has a significant impact on host cell pathology. Helicobacter pylori have many nuclear targeting proteins that translocate into the nucleus of host cells. H. pylori HP0425, annotated as hypothetical, has a nuclear localization signal (NLS) sequence, but its function has not been demonstrated. The aim of this experiment was to address the nuclear translocation of HP0425 and determine the effect of HP0425 pathology on host cells. MATERIALS AND METHODS: To investigate the nuclear localization of HP0425, it was expressed in AGS and MKN-1 cells as a GFP fusion protein (pEGFP-HP0425), and its localization was analyzed by confocal microscopy. Recombinant HP0425 (rHP0425) protein was overproduced as a GST fusion protein in Escherichia coli and purified by glutathione-affinity column chromatography. Purified rHP0425 was examined for cytotoxicity and DNase activity. RESULTS: The pEGFP-HP0425 fluorescence was expressed in the nucleus and cytosol fraction of cells, while it was localized in the cytoplasm in the negative control. This protein exhibited DNase activity under various conditions, with the highest DNase activity in the presence of manganese. In addition, the rHP0425 protein efficiently decreased cell viability in a concentration-dependent manner. CONCLUSIONS: These results suggest that HP0425 carrying a nuclear localization signal sequence translocates into the nucleus of host cells and degrades genomic DNA by DNase I-like enzymatic activity, which is a new pathogenic strategy of H. pylori in the host.

Evaluation of Micronuclei, Nuclear Anomalies and the Nuclear/Cytoplasmic Ratio of Exfoliated Cervical Epithelial Cells in Genital Candidiasis.

OBJECTIVE: Candida is the most common cause of fungal infections. The aim of this study was to fill the gaps in the current knowledge on the frequencies of micronuclei and nuclear anomalies, and the nucleus/cytoplasmic ratio in genital candidiasis. STUDY DESIGN: A total of 88 Papanicolaou- stained cervical smears, which comprised Candida spp. (n = 44) and control cases with no infectious agent (n = 44), were studied. In each smear, cells with micronuclei and nuclear anomalies were counted in 1,000 epithelial cells and also nuclear and cellular areas were evaluated using image analysis software at a magnification of ×400. RESULTS: The frequencies of micronucleated and binucleated cells and cells with perinuclear halos, and the nucleus/cytoplasmic ratio of epithelial cells were higher in the Candida-infected group compared with the control group (p < 0.05). CONCLUSIONS: Genital candidiasis is able to induce changes in the size and shape of epithelial cells. The nuclear/cytoplasmic ratio and the frequency of micronuclei may reflect the DNA damage in the cervical epithelium. Micronucleus scoring could be used to screen the genomic damage profile of epithelial cells in candidiasis.

Detecting and characterizing cellular responses to Mycobacterium tuberculosis from histology slides.

Infection with Mycobacterium tuberculosis (M.tb) results in immune cell recruitment to the lungs, forming macrophage-rich regions (granulomas) and lymphocyte-rich regions (lymphocytic cuffs). The objective of this study was to accurately identify and characterize these regions from hematoxylin and eosin (H&E)-stained tissue slides. The two target regions (granulomas and lymphocytic cuffs) can be identified by their morphological characteristics. Their most differentiating characteristic on H&E slides is cell density. We developed a computational framework, called DeHiDe, to detect and classify high cell-density regions in histology slides. DeHiDe employed a novel internuclei geodesic distance calculation and Dulmange Mendelsohn permutation to detect and classify high cell-density regions. Lung tissue slides of mice experimentally infected with M.tb were stained with H&E and digitized. A total of 21 digital slides were used to develop and train the computational framework. The performance of the framework was evaluated using two main outcome measures: correct detection of potential regions, and correct classification of potential regions into granulomas and lymphocytic cuffs. DeHiDe provided a detection accuracy of 99.39% while it correctly classified 90.87% of the detected regions for the images where the expert pathologist produced the same ground truth during the first and second round of annotations. We showed that DeHiDe could detect high cell-density regions in a heterogeneous cell environment with non-convex tissue shapes.

When bacteria target the nucleus: the emerging family of nucleomodulins.

The nucleus, at the heart of the eukaryotic cell, hosts and protects the genetic material, governs gene expression and regulates the whole cell physiology, including cell division. A growing number of studies indicate that various animal and plant pathogenic bacteria can deliver factors to this central organelle to subvert host defences by directly interfering with transcription, chromatin-remodelling, RNA splicing or DNA replication and repair. Such bacterial molecules entering the nucleus, which we propose to term 'nucleomodulins', use diverse strategies to hijack nuclear processes by targeting host DNA or an array of nuclear proteins. In some cases, bacteria can even enter the nucleus. These bacterial 'nuclear attacks' might have permanent genetic or long-term epigenetic effects on the host. Studying nucleomodulins and endonuclear bacteria can thus generate new insights into long-term impacts of infectious diseases and create novel tools for biotechnological applications and for deciphering the regulation of nuclear dynamics.

[Effects of oil-refining microbes (genus Acinetobacter) on cytogenetical structures of human lymphocytes in cell cultures].

The objective of this study was to assess ability of oil-refining bacteria Acinetobacter calcoaceticus and A. valentis to induce karyopathological abnormalities and chromosomal aberrations in human lymphocyte cultures. It was found that the cultures infected with A. calcoaceticus showed significantly high frequencies of cytogenetical effects and chromosomal aberrant cells as compared to the intact cultures and cultures infected with A. valentis. The most of chromosomal aberrations, mainly chromatid aberrations, were located in 1 and 2 chromosomes. Moreover, the aberrations were detected in some specific chromosome areas. Abnormalities of mitotic cell division and nucleus morphology were determined in lymphocyte cultures infected with A. calcoaceticus. There were found significantly high frequencies of cells with micronuclei, nucleus protrusions, anaphase or metaphase chromosome and chromosomal fragments lagging as well as multipolar and C-mitoses. Thus, the oil-refining bacteria A. calcoaceticus in contrast to A. valentis demonstrated strong genotoxic effects in human lymphocyte cultures in vitro.

Formation of simian immunodeficiency virus long terminal repeat circles in resting T cells requires both T cell receptor- and IL-2-dependent activation.

Although immunodeficiency viruses can enter resting CD4+ T lymphocytes, activation of T cells is required for complete viral cDNA synthesis and transport of double-stranded viral DNA to the nucleus. Cross-linking T cell receptors (TCRs) on resting CD4+ T cells induces reverse transcription of full-length simian immunodeficiency virus (SIV) genomes, but TCR engagement alone is insufficient to stimulate SIV DNA to move to the nucleus and form long terminal repeat (LTR) circles. Neither ligation of TCR or CD28 receptors nor interleukin 2 (IL-2) alone induces formation of LTR circles; however, the combination of TCR ligation with either CD28 ligation or IL-2 doses. Anti-IL-2 serum inhibits the formation of LTR circles induced by cross-linking CD3 and CD28, but has no effect on the induction of increased viral reverse transcription. Thus, two signals appear to be required for immunodeficiency viruses to move to the T cell nucleus, one from the TCR to promote reverse transcription of the viral genome, the other through an IL-2-dependent process leading to formation of LTR circles.

Epidemiology of Nucleus-Dwelling Holospora: Infection, Transmission, Adaptation, and Interaction with Paramecium.

The chapter describes the exceptional symbiotic associations formed between the ciliate Paramecium and Holospora, highly infectious bacteria residing in the host nuclei. Holospora and Holospora-like bacteria (Alphaproteobacteria) are characterized by their ability for vertical and horizontal transmission in host populations, a complex biphasic life cycle, and pronounced preference for host species and colonized cell compartment. These bacteria are obligate intracellular parasites; thus, their metabolic repertoire is dramatically reduced. Nevertheless, they perform complex interactions with the host ciliate. We review ongoing efforts to unravel the molecular adaptations of these bacteria to their unusual lifestyle and the host's employment in the symbiosis. Furthermore, we summarize current knowledge on the genetic and genomic background of Paramecium-Holospora symbiosis and provide insights into the ecological and evolutionary consequences of this interaction. The diversity and occurrence of symbioses between ciliates and Holospora-like bacteria in nature is discussed in connection with transmission modes of symbionts, host specificity and compatibility of the partners. We aim to summarize 50 years of research devoted to these symbiotic systems and conclude trying to predict some perspectives for further studies.