Acanthamoeba castellanii trophozoites escape killing by neutrophil extracellular traps using their 3'-nucleotidase/nuclease activity.

Acanthamoeba castellanii is a free-living amoeba that acts as an opportunistic pathogen for humans and is the pathogenic agent of Acanthamoeba keratitis (AK). A. castellanii may present as proliferative and infective trophozoites or as resistant cysts during their life cycle. The immune response against AK is still poorly explored; however, it is well established that macrophages and neutrophils play essential roles in controlling corneal infection during the disease outcome. The release of NETs is one of the innate immune strategies to prevent parasite infection, especially when neutrophils interact with microorganisms that are too large to be phagocytosed, which is the case for amoeba species. The present work demonstrated that A. castellanii trophozoites can trigger NET formation upon in vitro interaction with neutrophils. Using DNase as a control, we observed increased parasite survival after coinciding with neutrophils, which may be correlated with NET degradation. Indeed, A. castellanii trophozoites degrade the NET DNA scaffold. Molecular analysis confirmed the occurrence of a 3'-nucleotidase/nuclease (3'-NT/NU) in the A. castellanii genome. We also demonstrated that trophozoites exhibit significantly higher 3'-NT/NU activity than cysts, which cannot trigger NET release. Considering that previous studies indicated the pathological role of 3'-NT-/NU in parasite infection, we suggest that this enzyme may act as the mechanism of escape of A. castellanii trophozoites from NETs.

A detailed kinetic model of glycolysis in Plasmodium falciparum-infected red blood cells for antimalarial drug target identification.

Upon infection by the malaria parasite Plasmodium falciparum, the glycolytic rate of a red blood cell increases up to 100-fold, possibly contributing to lactic acidosis and hypoglycemia in patients with severe malaria. This dramatic increase in glucose uptake and metabolism was correctly predicted by a newly constructed detailed enzyme kinetic model of glucose metabolism in the trophozoite-infected red blood cell. Subsequently, we expanded the model to simulate an infected red blood cell culture, including the different asexual blood-stage forms of the malaria parasite. The model simulations were in good agreement with experimental data, for which the measured parasitic volume was an important parameter. Upon further analysis of the model, we identified glucose transport as a drug target that would specifically affect infected red blood cells, which was confirmed experimentally with inhibitor titrations. This model can be a first step in constructing a whole-body model for glucose metabolism in malaria patients to evaluate the contribution of the parasite's metabolism to the disease state.

Curcumin effect on Acanthamoeba triangularis encystation under nutrient starvation.

Background: Curcumin is an active compound derived from turmeric, Curcuma longa, and is known for its benefits to human health. The amoebicidal activity of curcumin against Acanthamoeba triangularis was recently discovered. However, a physiological change of intracellular pathways related to A. triangularis encystation mechanism, including autophagy in the surviving amoeba after curcumin treatment, has never been reported. This study aims to investigate the effect of curcumin on the survival of A. triangularis under nutrient starvation and nutrient-rich condition, as well as to evaluate the A. triangularis encystation and a physiological change of Acanthamoeba autophagy at the mRNA level. Methods: In this study, A. triangularis amoebas were treated with a sublethal dose of curcumin under nutrient starvation and nutrient-rich condition and the surviving amoebas was investigated. Cysts formation and vacuolization were examined by microscopy and transcriptional expression of autophagy-related genes and other encystation-related genes were evaluated by real-time PCR. Results: A. triangularis cysts were formed under nutrient starvation. However, in the presence of the autophagy inhibitor, 3-methyladenine (3-MA), the percentage of cysts was significantly reduced. Interestingly, in the presence of curcumin, most of the parasites remained in the trophozoite stage in both the starvation and nutrient-rich condition. In vacuolization analysis, the percentage of amoebas with enlarged vacuole was increased upon starvation. However, the percentage was significantly declined in the presence of curcumin and 3-MA. Molecular analysis of A. triangularis autophagy-related (ATG) genes showed that the mRNA expression of the ATG genes, ATG3, ATG8b, ATG12, ATG16, under the starvation with curcumin was at a basal level along the treatment. The results were similar to those of the curcumin-treated amoebas under a nutrient-rich condition, except AcATG16 which increased later. On the other hand, mRNA expression of encystation-related genes, cellulose synthase and serine proteinase, remained unchanged during the first 18 h, but significantly increased at 24 h post treatment. Conclusion: Curcumin inhibits cyst formation in surviving trophozoites, which may result from its effect on mRNA expression of key Acanthamoeba ATG-related genes. However, further investigation into the mechanism of curcumin in A. triangularis trophozoites arrest and its association with autophagy or other encystation-related pathways is needed to support the future use of curcumin.

Anaerobic ATP synthesis pathways and inorganic phosphate transport and their possible roles in encystment in Acanthamoeba castellanii.

Acanthamoeba castellanii is the etiological agent of amoebic keratitis and is present in the environment in trophozoite or cyst forms. Both forms can infect the vertebrate host and colonize different tissues. The high resistance of cysts to standard drugs used in clinics contributes to the lack of effective treatments. Therefore, in this context, studies have emerged to understand cyst physiology and metabolism. Phosphate transporters are proteins responsible for the uptake of extracellular inorganic phosphate and transport to the cytosol. This work aims to verify the relationship between Pi transport and energetic metabolism in cysts of A. castellanii. The phosphate uptake ratio was higher in cysts compared with trophozoites. Recently, three sequences related to phosphate transporters have been identified in the A. castellanii genome (AcPHS1, AcPHS2, and AcPHS3); the messenger RNA expression levels of which differ depending on the amoeba life form. Pi uptake in cysts displayed peak activity at alkaline pH, whereas Pi transport in trophozoites was not affected in the same pH ranges. Cysts harbor a low-affinity Pi transport system (K0,5 and Vmax values of 1.76 ± 0.26 mM and 104.6 ± 6.3 nmol Pi × h-1 × 106 cells) compared to the trophozoite phosphate transport system. Pi transport seems important for anaerobic adenosine triphosphate synthesis in cysts, which initially occurs through the glycolytic pathway and subsequently through the pyruvate ferredoxin oxidoreductase pathway. Altogether, these results suggest that contrary to that previously postulated, cysts are active metabolic forms, and, as noted in trophozoites, phosphate uptake is important for energetic metabolism.

An update on cell division of Giardia duodenalis trophozoites.

Giardia duodenalis is a flagellated protozoan that is responsible for many cases of diarrheal disease worldwide and is characterized by its great divergence from the model organisms commonly used in studies of basic cellular processes. The life cycle of Giardia involves an infectious cyst form and a proliferative and mobile trophozoite form. Each Giardia trophozoite has two nuclei and a complex microtubule cytoskeleton that consists of eight flagellar axonemes, basal bodies, the adhesive disc, the funis and the median body. Since the success of Giardia infecting other organisms depends on its ability to divide and proliferate efficiently, Giardia must coordinate its cell division to ensure the duplication and partitioning of both nuclei and the multiple cytoskeletal structures. The purpose of this review is to summarize current knowledge about cell division and its regulation in this protist.

Modified DMEM xenic culture medium for propagation, isolation and maintenance of Balantioides coli.

Balantioides coli is a known ciliated zoonotic protozoan that mainly causes diarrhea in humans and pigs. An efficient and reliable culture system for this parasite remains unavailable until now. In this study, a modified Dulbecco's modified eagle medium (DMEM) with pH 7.0-7.5, containing 5 mg/mL starch and 20% new calf serum, was optimized for propagation of B. coli at 28°C-32°C. At the growth-peaking stage, the average trophozoite density was up to 12,970 trophozoites per milliliter. A reproducible protocol for isolation and maintenance of this parasite was also developed based on the modified DMEM culture medium. Moreover, cloning results of B. colipopulations showed that 250 trophozoites in 3 mL modified DMEM medium were the minimal number of trophozoites that propagated to the growth-peaking stage, and finally obtained the individual population. However, less than 250 trophozoites failed to continuously grow in the modified DMEM culture medium under the optimal conditions for growth of B. coli. These data showed that the modified DMEM culture medium is an ideal and efficient medium for propagation and maintenance of B. coli in vitro and will help studies on its biology, genome, transcriptome, proteome, and drug screening.

Laurinterol from Laurencia johnstonii eliminates Naegleria fowleri triggering PCD by inhibition of ATPases.

Primary amoebic encephalitis (PAM) is a lethal disease caused by the opportunistic pathogen, Naegleria fowleri. This amoebic species is able to live freely in warm aquatic habitats and to infect children and young adults when they perform risk activities in these water bodies such as swimming or splashing. Besides the need to increase awareness of PAM which will allow an early diagnosis, the development of fully effective therapeutic agents is needed. Current treatment options are amphotericin B and miltefosine which are not fully effective and also present toxicity issues. In this study, the in vitro activity of various sesquiterpenes isolated from the red alga Laurencia johnstonii were tested against the trophozoite stage of a strain of Naegleria fowleri. Moreover, the induced effects (apoptotic cell death) of the most active compound, laurinterol (1), was evaluated by measuring DNA condensation, damages at the mitochondrial level, cell membrane disruption and production of reactive oxygen species (ROS). The obtained results demonstrated that laurinterol was able to eliminate the amoebae at concentrations of 13.42 ± 2.57 µM and also to induced programmed cell death (PCD) in the treated amoebae. Moreover, since ATP levels were highly affected and laurinterol has been previously reported as an inhibitor of the Na+/K+-ATPase sodium-potassium ion pump, comparison with known inhibitors of ATPases were carried out. Our results points out that laurinterol was able to inhibit ENA ATPase pump at concentrations 100 times lower than furosemide.

Giardia lamblia: Laboratory Maintenance, Lifecycle Induction, and Infection of Murine Models.

Giardia lamblia is a protozoan parasite that is found ubiquitously throughout the world and is a major contributor to diarrheal disease. Giardia exhibits a biphasic lifestyle existing as either a dormant cyst or a vegetative trophozoite. Infections are typically initiated through the consumption of cyst-contaminated water or food. Giardia was first axenized in the 1970s and can be readily maintained in a laboratory setting. Additionally, Giardia is one of the few protozoans that can be induced to complete its complete lifecycle using laboratory methods. In this article, we outline protocols to maintain Giardia and induce passage through its lifecycle. We also provide protocols for infecting and quantifying parasites in an animal infection model. © 2020 Wiley Periodicals LLC. Basic Protocol 1: In vitro maintenance and growth of Giardia trophozoites Basic Protocol 2: In vitro encystation of Giardia cysts Basic Protocol 3: In vivo infections using Giardia trophozoites.

The malaria parasite Plasmodium falciparum in red blood cells selectively takes up serum proteins that affect host pathogenicity.

BACKGROUND: The malaria parasite Plasmodium falciparum is a protozoan that develops in red blood cells (RBCs) and requires various host factors. For its development in RBCs, nutrients not only from the RBC cytosol but also from the extracellular milieu must be acquired. Although the utilization of host nutrients by P. falciparum has been extensively analysed, only a few studies have reported its utilization of host serum proteins. Hence, the aim of the current study was to comprehensively identify host serum proteins taken up by P. falciparum parasites and to elucidate their role in pathogenesis. METHODS: Plasmodium falciparum was cultured with human serum in vitro. Uptake of serum proteins by parasites was comprehensively determined via shotgun liquid chromatography-mass spectrometry/mass spectrometry and western blotting. The calcium ion concentration in serum was also evaluated, and coagulation activity of the parasite lysate was assessed. RESULTS: Three proteins, vitamin K-dependent protein S, prothrombin, and vitronectin, were selectively internalized under sufficient Ca2+ levels in the culture medium. The uptake of these proteins was initiated before DNA replication, and increased during the trophozoite and schizont stages, irrespective of the assembly/disassembly of actin filaments. Coagulation assay revealed that prothrombin was activated and thereby induced blood coagulation. CONCLUSIONS: Serum proteins were taken up by parasites under culture conditions with sufficient Ca2+ levels. This uptake phenomenon was associated with their pathogenicity.

Phenotypic masquerade: Polymorphism in the life cycle of the centrohelid heliozoan Raphidiophrys heterophryoidea (Haptista: Centroplasthelida).

The life cycle of the centrohelid heliozoan Raphidiophrys heterophryoidea Zlatogursky, 2012 was studied with light and electron microscopy in clonal cultures from the type locality. The alternation of two types of trophozoites, having contrastingly different morphology, was observed. Type 1 trophozoites morphology matched the original description. Type 2 trophozoites tended to form colonies usually of 6-8 individuals, connected with cytoplasmic bridges and their cell size was noticeably bigger, namely 43-45 µm compared to 24.5 µm on average in type 1 trophozoites. Some colonies were forming stalks composed of three or four axopodia covered with scales. Spicules were lacking completely, while plate-scales differed from those of type 1 trophozoites: they had oblong-elliptical shape, larger (5.9-14.1 × 2.4-5.8 µm) size, non-branching septa always reaching scale centre, solid upper plate. The conspecificity of the two trophozoite types was confirmed with the comparison of SSU rDNA gene sequence data. Both types of trophozoites were capable of encystment and excysted individuals always were type 1 trophozoites. A new type of cyst-scales (cup-scales) was described. Transitions between cysts and the two trophozoites types were documented. The diagnosis of R. heterophryoidea was improved accordingly. The possible functions, driving forces, and taxonomic consequences of the polymorphism were discussed.

In vitro effects of environmental isolates of Acanthamoeba T4 and T5 over human erythrocytes and platelets.

Free-living amoebae of the genus Acanthamoeba have been associated with keratitis and encephalitis. Some factors related to their pathogenic potential have been described, including the release of hydrolytic enzymes, and the adhesion and phagocytosis processes. However, other factors such as their effect over the hemodynamics and microcirculation elements have not been fully investigated. This work determines the in vitro activity of potentially pathogenic environmental isolates of Acanthamoeba genotype T4 and T5 over erythrocytes and platelets. The hemolytic activity (dependent and independent of contact), as well as the production of ADP of ten environmental isolates of Acanthamoeba obtained from dental units, combined emergency showers, dust, and hospital water, were measured. Tests were carried out over erythrocytes in suspension and blood agar plates, incubated at 4 °C, room temperature and 37 °C. Erythrophagocytosis and platelet aggregation assays were also performed. Live trophozoites of all of the isolates tested showed a hemolytic activity that was temperature-dependent. Over erythrocytes in suspension, variable hemolysis percentages were obtained: a maximum of 41% and a minimum of 15%. Regarding hemolysis over agar plates, two patterns of hemolysis were observed: double and simple halos. Conditioned medium and crude extracts of trophozoites did not show hemolytic activity. Erythrophagocytosis by Acanthamoeba was also observed; however, no production of ADP was determined by the employed methodology.

Vorinostat, a possible alternative to metronidazole for the treatment of amebiasis caused by Entamoeba histolytica.

AbbreviationsSAHAsuberoylanilide hydroxamic acidEhHDACHistone Deacetylase from Entamoeba histolyticaRgRadius of gyrationRMSDroot-mean-square deviationRMSFroot-mean-square fluctuationMDSmolecular dynamics simulationVMDVisual Molecular DynamicsNAMDNanoscale Molecular DynamicsPBCperiodic boundary conditionsPMEParticle Mesh Ewald3Dthree-dimensionalCαalpha carbonFDAFood and Drug AdministrationnsnanosecondsGPU CUDAGraphics Processing Unit Compute Unified Device ArchitectureCommunicated by Ramaswamy H. Sarma.

Experimental keratitis in rats caused by Acanthamoeba griffini: A kinetic histopathological study.

The aim of this study was to evaluate the inflammation process that resulted from the inoculation of Wistar Rats with Acanthamoeba griffini, a virulent T3 Acanthamoeba genotype that produces keratitis. Haematoxylin and eosin, periodic acid stain, immunohistochemistry and morphometry were used to analyse tissues from rats of an Acanthamoeba keratitis (AK) model. Two weeks after inoculating the rats with A griffini trophozoites, the thickness of the stroma had diminished, followed by an increase in thickness at 4 weeks. At the latter time, an abundance of inflammatory infiltrate cells was observed, some found to express IL-1ß, IL-10 and/or caspase 3. Intercellular adhesion molecule-1 was expressed in corneal blood vessels amid the abundant vascularization characteristic of the development of AK. Through an immunohistochemical technique, trophozoites were detected at 2 and 4 weeks post-inoculation. By 8 weeks, there were a low number of trophozoites and cysts and the corneas of infected rats were similar in thickness to those of the controls. Thus, the rats were capable of healing experimental AK in the present rat model. Diverse immunological mechanisms regulated the inflammatory process in acute AK induced by A griffini in a murine model.

EFFECT OF LACTOBACILLUS POSTBIOTICS ON ENTAMOEBA HISTOLYTICA TROPHOZOITES.

BACKGROUND: Amebiasis is an infectious disease caused by Entamoeba histolytica. It represents one of the three worldwide leading causes of death by parasites and a public health problem due to its frequency, morbidity, mortality, and easy dispersion. OBJECTIVE: The study was aimed to evaluate the in vitro effect of Lactobacillus spp. postbiotics on E. histolytica trophozoites (HM1-IMSS strain) and to determine morphometric changes in trophozoite membrane by atomic force microscopy (AFM). METHODS: Bioassays on trophozoites were conducted with lyophilized postbiotics at 0.1, 0.3, and 0.5 mg/mL concentrations, and trophozoite samples were obtained for AFM analysis. RESULTS: Results indicated postbiotic inhibitory activity; the highest percentage inhibition was 89.63% at 0.5 mg/mL. Trophozoites nanomechanical analysis showed 28.32% increase in ruggedness and 56% decrease in size with treatments compared to the control. CONCLUSION: Our study showed that the synergy of Lactobacillus postbiotics inhibited E. histolytica HM1-IMSS in vitro growth under axenic conditions, inducing morphometric alterations in trophozoites' cell membrane. These results would allow designing strategies or treatments aimed at E. histolytica control in the future.

Effect of lactobacillus postbiotics on entamoeba histolytica trophozoites

ABSTRACT Background Amebiasis is an infectious disease caused by Entamoeba histolytica. It represents one of the three worldwide leading causes of death by parasites and a public health problem due to its frequency, morbidity, mortality, and easy dispersion. Objective The study was aimed to evaluate the in vitro effect of Lactobacillus spp. postbiotics on E. histolytica trophozoites (HM1-IMSS strain) and to determine morphometric changes in trophozoite membrane by atomic force microscopy (AFM). Methods Bioassays on trophozoites were conducted with lyophilized postbiotics at 0.1, 0.3, and 0.5 mg/mL concentrations, and trophozoite samples were obtained for AFM analysis Results Results indicated postbiotic inhibitory activity; the highest percentage inhibition was 89.63% at 0.5 mg/mL. Trophozoites nanomechanical analysis showed 28.32% increase in ruggedness and 56% decrease in size with treatments compared to the control. Conclusion Our study showed that the synergy of Lactobacillus postbiotics inhibited E. histolytica HM1-IMSS in vitro growth under axenic conditions, inducing morphometric alterations in trophozoites’ cell membrane. These results would allow designing strategies or treatments aimed at E. histolytica control in the future.

Cytopathic Change and Inflammatory Response of Human Corneal Epithelial Cells Induced by Acanthamoeba castellanii Trophozoites and Cysts.

Acanthamoeba castellanii has ubiquitous distribution and causes primary acanthamoebic keratitis (AK). AK is a common disease in contact lens wearers and results in permanent visual impairment or blindness. In this study, we observed the cytopathic effect, in vitro cytotoxicity, and secretion pattern of cytokines in human corneal epithelial cells (HCECs) induced by A. castellanii trophozoites and/or cysts. Morphological observation revealed that panked dendritic HCECs co-cultured with amoeba cysts had changed into round shape and gradually died. Such changes were more severe in co-culture with cyst than those of co-cultivation with trophozoites. In vitro cytotoxicity assay revealed the highest cytotoxicity to HCECs in the co-culture system with amoeba cysts. A. castellanii induced the expression of IL-1α, IL-6, IL-8, and CXCL1 in HCECs. Secreted levels of IL-1α, IL-6, and IL-8 in HCECs co-cultured with both trophozoites and cysts were increased at an early incubation time (3 and 6 hr). These results suggested that cytopathic changes and pro-inflammatory cytokines release of HCECs in response to A. castellanii, especially amoebic cysts, are an important mechanism for AK development.

Interactions of Pseudomonas aeruginosa with Acanthamoeba polyphaga Observed by Imaging Flow Cytometry.

Pseudomonas aeruginosa is a Gram-negative bacterium that is abundant in the environment and water systems, with strains that cause serious infections, especially in patients with compromised immune systems. In times of stress or as part of its natural life cycle, P. aeruginosa can adopt a viable but not culturable (VBNC) state, which renders it undetectable by current conventional food and water testing methods and makes it highly resistant to antibiotic treatment. Specific conditions can resuscitate these coccoid VBNC P. aeruginosa cells, which returns them to their active, virulent rod-shaped form. Underreporting the VBNC cells of P. aeruginosa by standard culture-based methods in water distribution systems may therefore pose serious risks to public health. As such, being able to accurately detect and quantify the presence of VBNC P. aeruginosa, especially in a hospital setting, is of critical importance. Herein, we describe a method to analyze VBNC P. aeruginosa using imaging flow cytometry. With this technique, we can accurately distinguish between active and VBNC forms. We also show here that association of VBNC P. aeruginosa with Acanthamoeba polyphaga results in resuscitation of P. aeruginosa to an active form within 2 h. Our approach could provide an alternative, reliable detection method of VBNC P. aeruginosa when coupled with species-specific staining. Most importantly, our experiments demonstrate that the coculture with amoebae can lead to a resuscitation of P. aeruginosa of culturable morphology after only 2 h, indicating that VBNC P. aeruginosa could potentially resuscitate in piped water (healthcare) environments colonized with amoebae. © 2019 International Society for Advancement of Cytometry.

Swarming and Aggregation in the Parasitic Diplomonad Flagellate Spironucleus vortens.

Pathogenicity, evolutionary history, and unusual cell organization of diplomonads are well known, particularly for Giardia and Spironucleus; however, behavior of these aerotolerant anaerobes is largely unknown. Addressing this deficit, we studied behavior of the piscine diplomonad Spironucleus vortens (ATCC 50386) in in vitro culture. Spironucleus vortens trophozoites from Angelfish, Pterophyllum scalare, were maintained axenically in modified liver digest, yeast extract, and iron (LYI) medium, at 22 °C in the dark, and subcultured weekly. Cultures were monitored every 1-2 d, by removing an aliquot, and loading cells into a hemocytometer chamber, or onto a regular microscope slide. We observed three distinct swimming behaviors: (i) spontaneous formation of swarms, reaching 200 µm in diameter, persisting for up to several min in situ, (ii) directional movement of the swarm, via collective motility, and (iii) independent swimming of trophozoites to form a band (aggregation), presumably at the location of optimal environmental conditions. These behaviors have not previously been reported in Spironucleus. The observation that flagellate motility can change, from individual self-propulsion to complex collective swarming motility, prompts us to advocate S. vortens as a new model for study of group behavioral dynamics, complementing emerging studies of collective swimming in flagellated bacteria.

Influence of Micropatterned Grill Lines on Entamoeba histolytica Trophozoites Morphology and Migration.

Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a-better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified.

Interactions of free-living amoebae with rice bacterial pathogens Xanthomonas oryzae pathovars oryzae and oryzicola.

BACKGROUND: Free-living amoebae (FLA) are voracious feeders, consuming bacteria and other microbes during colonization of the phytobiome. FLA are also known to secrete bacteriocidal or bacteriostatic compounds into their growth environment. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we explore the impacts of co-cultivation of five FLA species, including Acanthamoeba castellanii, A. lenticulata, A. polyphaga, Dictyostelium discoideum and Vermamoeba vermiformis, on survival of two devastating bacterial pathogens of rice, Xanthomonas oryzae pathovars (pv.) oryzae and oryzicola. In co-cultivation assays, the five FLA species were either bacteriostatic or bactericidal to X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Despite these effects, bacteria were rarely detected inside amoebal cells. Furthermore, amoebae did not disrupt X. oryzae biofilms. The bactericidal effects persisted when bacteria were added to a cell-free supernatant from amoebal cultures, suggesting some amoebae produce an extracellular bactericidal compound. CONCLUSIONS/SIGNIFICANCE: This work establishes novel, basal dynamics between important plant pathogenic bacteria and diverse amoebae, and lays the framework for future mechanistic studies.