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Legionella longbeachae and Legionella pneumophila are the most common causative agents of Legionnaires' disease. While the clinical manifestations caused by both species are similar, species-specific differences exist in environmental niches, disease epidemiology, and genomic content. One such difference is the presence of a genomic locus predicted to encode a capsule. Here, we show that L. longbeachae indeed expresses a capsule in post-exponential growth phase as evidenced by electron microscopy analyses, and that capsule expression is abrogated when deleting a capsule transporter gene. Capsule purification and its analysis via HLPC revealed the presence of a highly anionic polysaccharide that is absent in the capsule mutant. The capsule is important for replication and virulence in vivo in a mouse model of infection and in the natural host Acanthamoeba castellanii. It has anti-phagocytic function when encountering innate immune cells such as human macrophages and it is involved in the low cytokine responses in mice and in human monocyte derived macrophages, thus dampening the innate immune response. Thus, the here characterized L. longbeachae capsule is a novel virulence factor, unique among the known Legionella species, which may aid L. longbeachae to survive in its specific niches and which partly confers L. longbeachae its unique infection characteristics., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Schmidt et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Exploration of interspecies interactions between microorganisms can have taxonomic, ecological, evolutionary, or medical applications. To better explore interactions between microorganisms it is important to establish the ideal conditions that ensure survival of all species involved. In this study, we sought to identify the ideal biotic and abiotic factors that would result in high co-culture viability of two interkingdom species, Pseudomonas aeruginosa and Acanthamoeba castellanii, two soil dwelling microbes. There have been limited studies showing long-term interactions between these two organisms as co-culture can result in high mortality for one or both organisms suggesting a predator-predator interaction may exist between them. In this study, we identified biotic and abiotic conditions that resulted in a high viability for both organisms in long-term co-culture, including optimizing temperature, nutrient concentration, choice of bacterial strains, and the initial ratio of interacting partners. These two species represent ideal partners for studying microbial interactions because amoebae act similarly to mammalian immune cells in many respects, and this can allow researchers to study host-pathogen interactions in vitro. Therefore, long-term interaction studies between these microbes might reveal the evolutionary steps that occur in bacteria when subjected to intense predation, like what occurs when pathogens enter the human body. The culture conditions characterized here resulted in high viability for both organisms for at least 14-days in co-culture suggesting that long-term experimental studies between these species can be achieved using these culture conditions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Cecil, Yoder-Himes. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Free-living amoebae of the genus Acanthamoeba are infected by various bacteria in nature, and thus bacteria can protect themselves from adverse environmental conditions. Contrary to this ameba-bacteria relationship whether Acanthamoeba has antibacterial effects on bacteria is the different aspect of the relationship between these microorganisms. In this study, we investigate various Acanthamoeba strains have antibacterial effects on various Staphylococcus strains. Three environmental Acanthamoeba strains, isolated from various aquatic environments in Turkey, and Acanthamoeba castellanii ATCC 50373 standard strains were used in the study. The antistaphylococcal effect of cell-free supernatant (CFS) obtained from these amoebae against 12 different Staphylococcus bacteria was investigated by colony counting method. In addition, the pathogenicity of the tested Acanthamoeba strains was determined using osmotolerance and thermotolerance tests. CFSs obtained from Acanthamoeba were found to have varying degrees of antistaphylococcal effects on various Staphylococcus strains (0%-100%). It was determined that the CFS of the standard Acanthamoeba strain showed 100% inhibitory effect against one clinical methicillin-resistant Staphylococcus aureus strain (M2). Also, CFS of Ugöl strain showed 99.97% inhibitory effect against one clinical methicillin-sensitive Staphylococcus epidermidis strain (L3). It was determined that all Acanthamoeba isolates had no pathogenic potential. According to the results, it has been observed that Acanthamoeba produces antibacterial substance(s) against Staphylococcus bacteria and that the ameba-bacteria relationship may also result in the detriment of the bacteria. Furthermore, the current study indicates that new and natural antimicrobial agents from Acanthamoeba can be used as an alternative to infections caused by Staphylococcus., (© 2024 Wiley‐VCH GmbH.)

Acanthamoeba species are clinically relevant free-living amoebae (FLA) ubiquitously found in soil and water bodies. Metabolically active trophozoites graze on diverse microbes via phagocytosis. However, functional studies on Rab GTPases (Rabs), which are critical for controlling vesicle trafficking and maturation, are scarce for this FLA. This knowledge gap can be partly explained by the limited genetic tools available for Acanthamoeba cell biology. Here, we developed plasmids to generate fusions of A. castellanii strain Neff proteins to the N- or C-termini of mEGFP and mCherry2. Phylogenomic and structural analyses of the 11 Neff Rab7 paralogs found in the RefSeq assembly revealed that eight of them had non-canonical sequences. After correcting the gene annotation for the Rab7A ortholog, we generated a line stably expressing an mEGFP-Rab7A fusion, demonstrating its correct localization to acidified macropinocytic and phagocytic vacuoles using fluorescence microscopy live cell imaging (LCI). Direct labeling of live Stenotrophomonas maltophilia ESTM1D_MKCAZ16_6a (Sm18) cells with pHrodo Red, a pH-sensitive dye, demonstrated that they reside within acidified, Rab7A-positive vacuoles. We constructed new mini-Tn7 delivery plasmids and tagged Sm18 with constitutively expressed mScarlet-I. Co-culture experiments of Neff trophozoites with Sm18::mTn7TC1_Pc_mScarlet-I, coupled with LCI and microplate reader assays, demonstrated that Sm18 underwent multiple replication rounds before reaching the extracellular medium via non-lytic exocytosis. We conclude that S. maltophilia belongs to the class of bacteria that can use amoeba as an intracellular replication niche within a Stenotrophomonas -containing vacuole that interacts extensively with the endocytic pathway.IMPORTANCEDiverse Acanthamoeba lineages (genotypes) are of increasing clinical concern, mainly causing amoebic keratitis and granulomatous amebic encephalitis among other infections. S. maltophilia ranks among the top 10 most prevalent multidrug-resistant opportunistic nosocomial pathogens and is a recurrent member of the microbiome hosted by Acanthamoeba and other free-living amoebae. However, little is known about the molecular strategies deployed by Stenotrophomonas for an intracellular lifestyle in amoebae and other professional phagocytes such as macrophages, which allow the bacterium to evade the immune system and the action of antibiotics. Our plasmids and easy-to-use microtiter plate co-culture assays should facilitate investigations into the cellular microbiology of Acanthamoeba interactions with Stenotrophomonas and other opportunistic pathogens, which may ultimately lead to the discovery of new molecular targets and antimicrobial therapies to combat difficult-to-treat infections caused by these ubiquitous microbes., Competing Interests: The authors declare no conflict of interest.

Protozoan predation is a major cause of bacterial mortality. The first step of predation for phagocytic amoebae is the recognition of their prey. Lipopolysaccharide (LPS) is a major component of Gram-negative bacteria and is only present on the outer leaflet of the outer membrane lipid bilayer. LPS consists of three distinct regions: lipid A, an oligosaccharide core, and O -polysaccharide. Previous research in our lab determined that the oligosaccharide (OS) region of LPS mediates the recognition and internalization of Escherichia coli by Acanthamoeba castellanii. The oligosaccharide region is conceptually divided into the inner core and outer core. The LPS of any given E. coli strain contains only one of five different OS structures: K-12 and R1 to R4. All OSs contain the same inner core sugars but different outer core sugars. Here, we show that the Kdo2 moiety of the inner core is necessary and sufficient for E. coli recognition and internalization by A. castellanii. We also show that the precise composition of the variable outer core OS region modulates the efficiency with which A. castellanii consumes bacteria. The latter finding indicates that outer core OS composition plays a role in bacterial defense against phagocytic predators. IMPORTANCE Rather than being transmitted from host to host, most opportunistic bacterial pathogens reside in the environment for significant amounts of time. Protist predation is a major cause of bacterial mortality. To enhance their survival in the environment, bacteria have evolved various defense strategies such as filamentation, increased motility, biofilm formation, toxin release, and modification of cell wall structure; strategies which also enhance their virulence to humans. This work shows that the major component of the bacterial cell wall, LPS, also known as bacterial endotoxin, is a "dual use" factor, regulating amoeba predation of bacteria in addition to its well-known role as a human virulence factor. Both these functions are governed by the same parts of LPS. Thus, the structure and composition of this "dual use" factor likely evolved as a response to constant voracious protist grazing pressure in the environment, rather than during short-term infections of human and animals.

Evolutionary selection pressures that resulted in microbes found within environmental reservoirs that can cause diseases in animals are unknown. One hypothesis is that predatory organisms select microbes able to counteract animal immune cells. Here, a non-pathogenic yeast, Sporobolomyces primogenomicus, was exposed to predation by Acanthamoeba castellanii. Strains emerged that were resistant to being killed by this amoeba. All these strains had altered morphology, growing as pseudohyphae. The mutation in one strain was identified: CNA1 encodes the calcineurin A subunit that is highly conserved in fungi and where it is essential for their virulence in hosts including mammals, insects, and plants., (© The Author(s) 2023. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology.)

The unicellular amoeba Acanthamoeba castellanii is ubiquitous in aquatic environments, where it preys on bacteria. The organism also hosts bacterial endosymbionts, some of which are parasitic, including human pathogens such as Chlamydia and Legionella spp. Here we report complete, high-quality genome sequences for two extensively studied A. castellanii strains, Neff and C3. Combining long- and short-read data with Hi-C, we generated near chromosome-level assemblies for both strains with 90% of the genome contained in 29 scaffolds for the Neff strain and 31 for the C3 strain. Comparative genomics revealed strain-specific functional enrichment, most notably genes related to signal transduction in the C3 strain and to viral replication in Neff. Furthermore, we characterized the spatial organization of the A. castellanii genome and showed that it is reorganized during infection by Legionella pneumophila Infection-dependent chromatin loops were found to be enriched in genes for signal transduction and phosphorylation processes. In genomic regions where chromatin organization changed during Legionella infection, we found functional enrichment for genes associated with metabolism, organelle assembly, and cytoskeleton organization. Given Legionella infection is known to alter its host's cell cycle, to exploit the host's organelles, and to modulate the host's metabolism in its favor, these changes in chromatin organization may partly be related to mechanisms of host control during Legionella infection., (© 2022 Matthey-Doret et al.; Published by Cold Spring Harbor Laboratory Press.)

Aliarcobacter butzleri (formerly known as Arcobacter butzleri) is an emerging food-borne zoonotic pathogen that establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba. Previously, we described that this bacterium acts as an endocytobiont of A. castellanii, surviving for at least 10 days in absence of bacterial replication. Thus, the aim of this study was to evaluate the ability of A. butzleri to survive as a long-term endosymbiont of A. castellanii for 30 days in two models of symbiotic interaction with A. castellanii: (i) endosymbiotic culture followed by gentamicin protection assay and (ii) transwell co-culture assay. The results allow us to conclude that A. butzleri is capable of surviving as an endosymbiont of A. castellanii for at least 30 days, without multiplying, under controlled laboratory conditions. In addition, in the absence of nutrients and as both microorganisms remain in the same culture, separated by semi-permeable membranes, A. castellanii does not promote the survival of A. butzleri, nor does it multiply. Our findings suggest that the greater survival capacity of A. butzleri is associated with their endosymbiont status inside A. castellanii, pointing out the complexity of this type of symbiotic relationship., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Encystment is a common stress response of most protists, including free-living amoebae. Cyst formation protects the amoebae from eradication and can increase virulence of the bacteria they harbor. Here, we mapped the global molecular changes that occur in the facultatively pathogenic amoeba Acanthamoeba castellanii during the early steps of the poorly understood process of encystment. By performing transcriptomic, proteomic, and phosphoproteomic experiments during encystment, we identified more than 150,000 previously undescribed transcripts and thousands of protein sequences absent from the reference genome. These results provide molecular details to the regulation of expected biological processes, such as cell proliferation shutdown, and reveal new insights such as a rapid phospho-regulation of sites involved in cytoskeleton remodeling and translation regulation. This work constitutes the first time-resolved molecular atlas of an encysting organism and a useful resource for further investigation of amoebae encystment to allow for a better control of pathogenic amoebae., (© 2022. The Author(s).)

Acanthamoeba castellanii ( Ac ) is a species of free-living amoebae (FLAs) that has been widely applied as a model for the study of host-parasite interactions and characterization of environmental symbionts. The sharing of niches between Ac and potential pathogens, such as fungi, favors associations between these organisms. Through predatory behavior, Ac enhances fungal survival, dissemination, and virulence in their intracellular milieu, training these pathogens and granting subsequent success in events of infections to more evolved hosts. In recent studies, our group characterized the amoeboid mannose binding proteins (MBPs) as one of the main fungal recognition pathways. Similarly, mannose-binding lectins play a key role in activating antifungal responses by immune cells. Even in the face of similarities, the distinct impacts and degrees of affinity of fungal recognition for mannose receptors in amoeboid and animal hosts are poorly understood. In this work, we have identified high-affinity ligands for mannosylated fungal cell wall residues expressed on the surface of amoebas and macrophages and determined the relative importance of these pathways in the antifungal responses comparing both phagocytic models. Mannose-purified surface proteins (MPPs) from both phagocytes showed binding to isolated mannose/mannans and mannosylated fungal cell wall targets. Although macrophage MPPs had more intense binding when compared to the amoeba receptors, the inhibition of this pathway affects fungal internalization and survival in both phagocytes. Mass spectrometry identified several MPPs in both models, and in silico alignment showed highly conserved regions between spotted amoeboid receptors (MBP and MBP1) and immune receptors (Mrc1 and Mrc2) and potential molecular mimicry, pointing to a possible convergent evolution of pathogen recognition mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ferreira, Mendoza, Gonçalves, Rodríguez-de la Noval, Honorato, Nimrichter, Ramos, Nogueira, Domont, Peralta and Guimarães.)

In the present study, the effect of sublethal chlorine-induced oxidative stress on the subsequent interaction of Salmonella enterica serovars Enteritidis and Typhimurium with Acanthamoeba castellanii and A. polyphaga was evaluated. Sublethal chlorine concentration was determined using the lag phase extension information and used to prepare chlorine-stressed Salmonella cells. Coculture experiments of Acanthamoeba and Salmonella cells were performed in Page's amoeba saline (PAS) at 25 °C for 2 h. The results showed that the chlorine-stressed Salmonella cells were significantly more engulfed by A. castellanii and A. polyphaga trophozoites than the non-stressed cells. The uptake rates of the chlorine-stressed and non-stressed Salmonella cells were in the range of 14.17-27.34 and 6.51-11.52% for A. castellanii, and in the range of 8.32-17.76 and 2.28-6.12% for A. polyphaga trophozoites, respectively. Moreover, intracystic survival time of chlorine-stressed cells of S. Enteritidis and S. Typhimurium was significantly longer than that of non-stressed cells. While, non-stressed Salmonella cells survived within A. castellanii and A. polyphaga cysts for 13-20 and 8-15 days, chlorine-stressed cells were recovered from A. castellanii and A. polyphaga cysts after 22-32 and 15-24 days, respectively. These results underscore the importance of bacterial exposure to sublethal chlorine concentrations in their interaction with free-living amoebae, and may lead to a better understanding of the parameters affecting the persistence of Salmonella enterica serovars in food-related environments., (Copyright © 2021. Published by Elsevier Ltd.)

The ubiquitous unicellular eukaryote, Acanthamoeba , is known to play a role in the survival and dissemination of Campylobacter jejuni. C. jejuni is the leading cause of bacterial foodborne gastroenteritis world-wide and is a major public health problem. The ability of C. jejuni to interact and potentially invade epithelial cells is thought to be key for disease development in humans. We examined C. jejuni grown under standard laboratory conditions, 11168H CBA with that harvested from within Acanthamoeba castellanii (11168H AC/CBA ) or Acanthamoeba polyphaga (11168H AP/CBA ), and compared their ability to invade different cell lines. C. jejuni harvested from within amoebae had a ~3.7-fold increase in invasiveness into T84 human epithelial cells and a striking ~11-fold increase for re-entry into A. castellanii cells. We also investigated the invasiveness and survivability of six diverse representative C. jejuni strains within Acanthamoeba spp., our results confirm that invasion and survivability is likely host-cell-dependent. Our survival assay data led us to conclude that Acanthamoeba spp. are a transient host for C. jejuni and that survival within amoebae pre-adapts C. jejuni and enhances subsequent cell invasion. This study provides new insight into C. jejuni interactions with amoebae and its increased invasiveness potential in mammalian hosts.

Mobbing, group attack of prey on predator, is a behavior seen in many animal species in which prey animals use numbers and coordination to counter individually superior predators. We studied attack behavior of Pseudomonas aeruginosa toward the bacterivore Acanthamoeba castellanii. This behavior consists of directed motility toward and specific adhesion to the predator cells, enacted in seconds and responding to both prey and predator population densities. Attack coordination relies on remote sensing of the predator and the use of the Pseudomonas quinolone signal (PQS), a P. aeruginosa species-specific quorum sensing molecule. Mutants unable to produce the PQS show unspecific adhesion and reduced survival, and a corresponding increase in predator population occurs as a result of predation. The addition of an external PQS restored some predator-specific adherence within seconds, suggesting a novel response mechanism to this quorum sensing (QS) signal. Fast behavioral response of P. aeruginosa to PQS is also supported by the rate of signal accumulation in the culture, reaching relevant concentrations within minutes, enabling bacteria response to self population density in these short timescales. These results portray a well-regulated group attack of the bacteria against their predator, reacting within seconds to environmental cues and species-specific signaling, which is analogous in many ways to animal mobbing behavior. IMPORTANCE Pseudomonas aeruginosa was shown previously to attack amoebae and other predators by adhering to them and injecting them with virulent substances. In this work, we show that an active, coordinated group behavior is enacted by the bacteria to utilize these molecular components, responding to both predator and bacterial population density. In addition to their ecological significance, immediate behavioral changes observed in response to PQS suggest the existence of a fast QS signal cascade, which is different from canonical QS that relies on slow-to-respond gene regulation. Similar regulatory circuits may drive other bacterial adaptations and pathogenicity mechanisms and may have important clinical implications.

The bacterial pathogen Salmonella enterica , which causes enteritis, has a broad host range and extensive environmental longevity. In water and soil, Salmonella interacts with protozoa and multiplies inside their phagosomes. Although this relationship resembles that between Salmonella and mammalian phagocytes, the interaction mechanisms and bacterial genes involved are unclear. Here, we characterized global gene expression patterns of S. enterica serovar Typhimurium within Acanthamoeba castellanii at the early stage of infection by Cappable-Seq. Gene expression features of S. Typhimurium within A. castellanii were presented with downregulation of glycolysis-related, and upregulation of glyoxylate cycle-related genes. Expression of Salmonella Pathogenicity Island-1 (SPI-1), chemotaxis system, and flagellar apparatus genes was upregulated. Furthermore, expression of genes mediating oxidative stress response and iron uptake was upregulated within A. castellanii as well as within mammalian phagocytes. Hence, global S. Typhimurium gene expression patterns within A. castellanii help better understand the molecular mechanisms of Salmonella adaptation to an amoeba cell and intracellular persistence in protozoa inhabiting water and soil ecosystems.

Legionella pneumophila possesses a unique intracellular lifecycle featuring distinct morphological stages that include replicative forms and transmissive cyst forms. Expression of genes associated with virulence traits and cyst morphogenesis is concomitant, and governed by a complex stringent response based-regulatory network and the stationary phase sigma factor RpoS. In Pseudomonas spp., rpoS expression is controlled by the autorepressor PsrA, and orthologs of PsrA and RpoS are required for cyst formation in Azotobacter. Here we report that the L. pneumophila psrA ortholog, expressed as a leaderless monocistronic transcript, is also an autorepressor, but is not a regulator of rpoS expression. Further, the binding site sequence recognized by L. pneumophila PsrA is different from that of Pseudomonas PsrA, suggesting a repertoire of target genes unique to L. pneumophila. While PsrA was dispensable for growth in human U937-derived macrophages, lack of PsrA affected bacterial intracellular growth in Acanthamoeba castellanii protozoa, but also increased the quantity of poly-3-hydroxybutyrate (PHB) inclusions in matured transmissive cysts. Interestingly, overexpression of PsrA increased the size and bacterial load of the replicative vacuole in both host cell types. Taken together, we report that PsrA is a host-specific requirement for optimal temporal progression of L. pneumophila intracellular lifecycle in A. castellanii., (© 2021 John Wiley & Sons Ltd.)

Rhodococcus equi is a soil saprophytic bacterium and intracellular pathogen that causes pneumonia in foals. Strains of R. equi that are virulent in foals contain a plasmid that encodes a virulence-associated protein A (VapA) necessary for replication in macrophages. Because other intracellular pathogens survive and replicate inside amoebae, we postulated that the VapA-bearing plasmid (pVAPA) confers a survival advantage for R. equi against environmental predators like amoebae. To test this hypothesis, we compared phagocytosis by and survival in Acanthamoeba castellanii of isogenic strains of pVAPA-positive and pVAPA-negative R. equi. Phagocytosis of the pVAPA-negative strain by A. castellanii was significantly (P < 0.0001) greater than the pVAPA-positive strain. Intracellular replication of the pVAPA-positive strain in A. castellanii was significantly (P < 0.0001) greater than the pVAPA-negative strain during both 48 h and 9 days. These results indicate that the presence of the VapA plasmid reduces uptake and aids replication of R. equi in A. castellanii., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Amoeboid predators, such as amoebae, are proposed to select for survival traits in soil microbes such as Cryptococcus neoformans ; these traits can also function in animal virulence by defeating phagocytic immune cells, such as macrophages. Consistent with this notion, incubation of various fungal species with amoebae enhanced their virulence, but the mechanisms involved are unknown. In this study, we exposed three strains of C. neoformans (1 clinical and 2 environmental) to predation by Acanthamoeba castellanii for prolonged times and then analyzed surviving colonies phenotypically and genetically. Surviving colonies comprised cells that expressed either pseudohyphal or yeast phenotypes, which demonstrated variable expression of traits associated with virulence, such as capsule size, urease production, and melanization. Phenotypic changes were associated with aneuploidy and DNA sequence mutations in some amoeba-passaged isolates, but not in others. Mutations in the gene encoding the oligopeptide transporter (CNAG&#95;03013; OPT1 ) were observed among amoeba-passaged isolates from each of the three strains. Isolates derived from environmental strains gained the capacity for enhanced macrophage toxicity after amoeba selection and carried mutations on the CNAG&#95;00570 gene encoding Pkr1 (AMP-dependent protein kinase regulator) but manifested reduced virulence in mice because they elicited more effective fungal-clearing immune responses. Our results indicate that C. neoformans survival under constant amoeba predation involves the generation of strains expressing pleiotropic phenotypic and genetic changes. Given the myriad potential predators in soils, the diversity observed among amoeba-selected strains suggests a bet-hedging strategy whereby variant diversity increases the likelihood that some will survive predation. IMPORTANCE Cryptococcus neoformans is a ubiquitous environmental fungus that is also a leading cause of fatal fungal infection in humans, especially among immunocompromised patients. A major question in the field is how an environmental yeast such as C. neoformans becomes a human pathogen when it has no need for an animal host in its life cycle. Previous studies showed that C. neoformans increases its pathogenicity after interacting with its environmental predator amoebae. Amoebae, like macrophages, are phagocytic cells that are considered an environmental training ground for pathogens to resist macrophages, but the mechanism by which C. neoformans changes its virulence through interactions with protozoa is unknown. Our study indicates that fungal survival in the face of amoeba predation is associated with the emergence of pleiotropic phenotypic and genomic changes that increase the chance of fungal survival, with this diversity suggesting a bet-hedging strategy to ensure that some forms survive., (Copyright © 2021 Fu et al.)

Legionella pneumophila is an opportunistic pathogen that survives and proliferates within protists such as Acanthamoeba spp. in environment. However, intracellular pathogenic endosymbiosis and its implications within Acanthamoeba spp. remain poorly understood. In this study, RNA sequencing analysis was used to investigate transcriptional changes in A. castellanii in response to L. pneumophila infection. Based on RNA sequencing data, we identified 1,211 upregulated genes and 1,131 downregulated genes in A. castellanii infected with L. pneumophila for 12 hr. After 24 hr, 1,321 upregulated genes and 1,379 downregulated genes were identified. Gene ontology (GO) analysis revealed that L. pneumophila endosymbiosis enhanced hydrolase activity, catalytic activity, and DNA binding while reducing oxidoreductase activity in the molecular function (MF) domain. In particular, multiple genes associated with the GO term 'integral component of membrane' were downregulated during endosymbiosis. The endosymbiont also induced differential expression of various methyltransferases and acetyltransferases in A. castellanii. Findings herein are may significantly contribute to understanding endosymbiosis of L. pneumophila within A. castellanii.

Background: Trichophyton rubrum (Tr) is the main aetiological agent of human dermatophytosis, being isolated from the environment and keratinised tissues. In the environment, Tr can interact with other organisms, such as free-living amoebas (FLA), which can act as an alternative host system to study the interaction between microbes and phagocytic cells., Objectives: To characterise the Acanthamoeba castellanii (ALX)-Tr interaction., Methods: Interaction was characterised in three conditions: trophozoites (PYG), late (PYG/NES) and early (NES) encystation stimulus, evaluating encystation kinetics, phagocytosis, exocytosis and fungicidal activity dynamics., Results: Tr was able to induce ALX encystation and be internalised by ALX. The number of internalised conidia was high at 1 hour, and ALX presented fungicidal activity with increased intracellular ROS production and exocytosis. In PYG/NES, phagocytosis and ROS production were reduced, with decreased ALX's fungicidal activity. However, in NES there was an increased fungal engulfment, and a reduced ROS production and higher fungal burden. Furthermore, exogenous mannose decreased phagocytosis of Tr conidia, and divalent cations induced ROS production and increased ALX's fungicidal activity. Interestingly, phagocytosis was reduced in the presence of cytoskeleton inhibitor, but exocytosis was increased, suggesting that Tr conidia may have alternative pathways to escape ALX's cells., Conclusion: A castellanii is a proper model for studying Tr-FLA interaction, since ALX can engulf, produce ROS and kill Tr, and all these parameters are influenced by an encystation stimulus and divalent cations. Moreover, this interaction is likely to occur in the environment implicating in the adaptation to environmental stressful conditions in both organisms., (© 2020 Wiley-VCH GmbH.)

Verona-Integron-encoded-Metallo-β-lactamase-positive Pseudomonas aeruginosa (VIM-PA) is a cause of hard-to-treat nosocomial infections, and can colonize hospital water networks alongside Acanthamoeba. We developed an in-vitro disinfection model to examine whether Acanthamoeba castellanii can harbour VIM-PA intracellularly, allowing VIM-PA to evade being killed by currently used hospital disinfectants. We observed that A. castellanii presence resulted in significantly increased survival of VIM-PA after exposure to chlorine for 30 s or for 2 min. This undesirable effect was not observed after disinfection by 70% alcohol or 24% acetic acid. Confocal microscopy confirmed the presence of VIM-PA within A. castellanii pseudocysts. Our data indicate that A. castellanii contributes to persistent VIM-PA colonization of water systems after chlorine treatment., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Mycobacterium abscessus (MAB) comprise rapidly growing, often multidrug-resistant (MDR), nontuberculous mycobacteria responsible for pulmonary and other infections in susceptible hosts. Antimicrobial peptides (APs) are natural and synthetic antimicrobials active against a range of microorganisms including mycobacteria. We evaluated APs activity against MAB reference and clinical strains. We observed minimal inhibitory concentrations of 1.6 to >50 μg/mL. Further work with the most active AP demonstrated protection of Acanthamoeba castellanii (AC) from killing by ingested MAB including MDR MAB strains. Antimicrobial peptides offer an attractive potential option for treatment of drug resistant treatment-refractory MAB., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Published by Elsevier Masson SAS.)

Amoebic bacterial interactions are the most ancient form of host pathogen interactions. Here, we investigate the fate of Salmonella typhimurium and Acanthamoeba castellanii T4 genotype upon mutual interactions in a nutrition free environment. The role of type 1 fimbriae and motility of S. typhimurium during interactions with A. castellanii has also been investigated. Deletion of genes encoding the type 1 fimbriae subunit FimA, type 1 fimbriae tip protein FimH, chemotaxis regulatory proteins CheA and CheY and major flagella subunits FliC and FljB was performed through homologous recombination. In vitro association, invasion and survival assays of S. typhimurium wild-type and mutant strains were performed upon co-incubation of bacteria with A. castellanii trophozoites in a nutrition free environment. The deletion gene encoding type 1 fimbriae subunit FimA reduced, whereas the deletion of genes encoding flagella subunits FliC and FljB of flagella enhanced the association capability of S. typhimurium with A. castellanii. Invasion of A. castellanii by Salmonella was significantly reduced upon the loss of type 1 fimbriae subunit FimA and type 1 fimbriae tip protein FimH. Co-incubation of S. typhimurium with A. castellanii in phosphate buffered saline medium stimulated the growth of S. typhimurium wild-type and mutant strains. Viable A. castellanii trophozoites count became significantly reduced upon co-incubation with S. typhimurium within 48 h. Type 1 fimbriae play a pivotal role in the adherence of S. typhimurium to the A. castellanii cell surface. Subsequently, this interaction provides S. typhimurium an advantage in growth.

Cryptococcus neoformans and Cryptococcus gattii are pathogenic fungi that cause significant morbidity and mortality. Cell surface hydrophobicity (CSH) is a biophysical parameter that influences the adhesion of fungal cells or spores to biotic and abiotic surfaces. C. neoformans is encased by polysaccharide capsule that is highly hydrophilic and is a critical determinant of virulence. In this study, we report large differences in the CSH of some C. neoformans and C. gattii strains. The capsular polysaccharides of C. neoformans strains differ in repeating motifs and therefore vary in the number of hydroxyl groups, which, along with higher-order structure of the capsule, may contribute to the variation in hydrophobicity that we observed. We found that cell wall composition, in the context of chitin-chitosan content, does not influence CSH. For C. neoformans , CSH correlated with phagocytosis by natural soil predator Acanthamoeba castellanii Furthermore, capsular binding of the protective antibody (18B7), but not the nonprotective antibody (13F1), altered the CSH of C. neoformans strains. Variability in CSH could be an important characteristic in comparing the biological properties of cryptococcal strains. IMPORTANCE The interaction of a microbial cell with its environment is influenced by the biophysical properties of a cell. The affinity of the cell surface for water, defined by the cell surface hydrophobicity (CSH), is a biophysical parameter that varies among different strains of Cryptococcus neoformans The CSH influences the phagocytosis of the yeast by its natural predator in the soil, the amoeba. Studying variation in biophysical properties like CSH gives us insight into the dynamic host-predator interaction and host-pathogen interaction in a damage-response framework., (Copyright © 2020 Vij et al.)

Temporin-SHa (SHa) is a small cationic host defence peptide (HDP) produced in skin secretions of the Sahara frog Pelophylax saharicus. This peptide has a broad-spectrum activity, efficiently targeting bacteria, parasites and viruses. Noticeably, SHa has demonstrated an ability to kill Leishmania infantum parasites (amastigotes) within macrophages. Recently, an analog of SHa with an increased net positive charge, named [K 3 ]SHa, has been designed to improve those activities. SHa and [K 3 ]SHa were both shown to exhibit leishmanicidal activity mainly by permeabilization of cell membranes but could also induce apoptotis-like death. Temporins are usually poorly active against Gram-negative bacteria whereas many of these species are of public health interest. Among them, Legionella pneumophila, the etiological agent of Legionnaire's disease, is of major concern. Indeed, this bacterium adopts an intracellular lifestyle and replicate inside alveolar macrophages likewise inside its numerous protozoan hosts. Despite several authors have studied the antimicrobial activity of many compounds on L. pneumophila released from host cells, nothing is known about activity on intracellular L. pneumophila within their hosts, and subsequently mechanisms of action that could be involved. Here, we showed for the first time that SHa and [K 3 ]SHa were active towards several species of Legionella. Both peptides displayed bactericidal activity and caused a loss of the bacterial envelope integrity leading to a rapid drop in cell viability. Regarding amoebae and THP-1-derived macrophages, SHa was less toxic than [K 3 ]SHa and exhibited low half maximal lethal concentrations (LC 50 ). When used at non-toxic concentration (6.25 µM), SHa killed more than 90% L. pneumophila within amoebae and around 50% within macrophages. Using SHa labeled with the fluorescent dye Cy5, we showed an evenly diffusion within cells except in vacuoles. Moreover, SHa was able to enter the nucleus of amoebae and accumulate in the nucleolus. This subcellular localization seemed specific as macrophages nucleoli remained unlabeled. Finally, no modifications in the expression of cytokines and HDPs were recorded when macrophages were treated with 6.25 µM SHa. By combining all data, we showed that temporin-SHa decreases the intracellular L. pneumophila load within amoebae and macrophages without being toxic for eukaryotic cells. This peptide was also able to reach the nucleolus of amoebae but was not capable to penetrate inside vacuoles. These data are in favor of an indirect action of SHa towards intracellular Legionella and make this peptide a promising template for further developments.

Background: Sporotrichosis is a group of zoonotic subcutaneous mycoses, found worldwide and caused by fungi belonging to the genus Sporothrix. Protozoans of the genus Acanthamoeba are widely distributed, and some species may be pathogenic and/or opportunistic. These organisms coexist in the same environment and may interact., Objectives: This study determined the profile of interactions of S schenckii sensu stricto and S brasiliensis with A castellanii, using an in vitro co-culture model to evaluate the intrinsic characteristics of the two Sporothrix species and A castellanii., Methods: We compared the rate of phagocytosis of S schenckii sensu stricto and S brasiliensis by A castellanii; the viability of S schenckii sensu stricto and S brasiliensis after contact with A castellanii; the viability of the amoeba after contact with a fungal species; and the influence of S schenckii sensu stricto and S brasiliensis on the encystment process of A castellanii., Results: The analyses indicated that A castellanii phagocytised both S schenckii and S brasiliensis, with significantly more S schenckii than S brasiliensis in the first two hours of contact. Our results showed a significant increase in conidia and hyphae count after 72 hours of co-culture of A castellanii with S brasiliensis, and the amoebae lysed after they ingested the fungi, indicating that the fungi probably used the amoebae as a source of nutrition., Conclusions: Our results were obtained in vitro and these organisms may not behave similarly in vivo; in vivo studies of co-infections are necessary in order to gain a thorough understanding of this relationship., (© 2019 Blackwell Verlag GmbH.)

Acanthamoeba castellanii is a free-living amoeba found mainly in humid environments and Arcobacter butzleri is an emerging zoonotic pathogen, both can establish in vitro endosymbiotic relationships in the absence of bacterial replication. We analyzed the localization of A. butzleri within A. castellanii establishing their association with endoplasmic reticulum vesicles and mitochondria. Through confocal microscopy, we observed that during the early stages of endosymbiosis, there is not colocalization between amoebic vacuoles containing A. butzleri and mitochondria or ER vesicles of A. castellanii. Considering that energy production of this bacterium occurs via metabolism of amino acids or the tricarboxylic acid cycle, these results contribute to explain the absence of bacterial replication, since A. butzleri would not have access to the nutrients found in endoplasmic reticulum vesicles and mitochondria. In addition, we observe that A. butzleri induces significantly the actin polymerization of A. castellanii during the early stages of endosymbiosis.

It was previously determined that the type II secretion system (T2SS) promotes the ability of Legionella pneumophila to grow in coculture with amoebae. Here, we discerned the stage of intracellular infection that is potentiated by comparing the wild-type and T2SS mutant legionellae for their capacity to parasitize Acanthamoeba castellanii Whereas the mutant behaved normally for entry into the host cells and subsequent evasion of degradative lysosomes, it was impaired in the ability to replicate, with that defect being first evident at approximately 9 h postentry. The replication defect was initially documented in three ways: by determining the numbers of CFU recovered from the lysates of the infected monolayers, by monitoring the levels of fluorescence associated with amoebal monolayers infected with green fluorescent protein (GFP)-expressing bacteria, and by utilizing flow cytometry to quantitate the amounts of GFP-expressing bacteria in individual amoebae. By employing confocal microscopy and newer imaging techniques, we further determined the progression in volume and shape of the bacterial vacuoles and found that the T2SS mutant grows at a decreased rate and does not attain maximally sized phagosomes. Overall, the entire infection cycle (i.e., entry to egress) was considerably slower for the T2SS mutant than it was for the wild-type strain, and the mutant's defect was maintained over multiple rounds of infection. Thus, the T2SS is absolutely required for L. pneumophila to grow to larger numbers in its intravacuolar niche within amoebae. Combining these results with those of our recent analysis of macrophage infection, T2SS is clearly a major component of L. pneumophila intracellular infection., (Copyright © 2019 American Society for Microbiology.)

Free-living amoebae (FLAs) are major reservoirs for a variety of bacteria, viruses, and fungi. The most studied mycophagic FLA, Acanthamoeba castellanii (Ac), is a potential environmental host for endemic fungal pathogens such as Cryptococcus spp., Histoplasma capsulatum, Blastomyces dermatitides, and Sporothrix schenckii. However, the mechanisms involved in this interaction are poorly understood. The aim of this work was to characterize the molecular instances that enable Ac to interact with and ingest fungal pathogens, a process that could lead to selection and maintenance of possible virulence factors. The interaction of Ac with a variety of fungal pathogens was analysed in a multifactorial evaluation that included the role of multiplicity of infection over time. Fungal binding to Ac surface by living image consisted of a quick process, and fungal initial extrusion (vomocytosis) was detected from 15 to 80 min depending on the organism. When these fungi were cocultured with the amoeba, only Candida albicans and Cryptococcus neoformans were able to grow, whereas Paracoccidioides brasiliensis and Sporothrix brasiliensis displayed unchanged viability. Yeasts of H. capsulatum and Saccharomyces cerevisiae were rapidly killed by Ac; however, some cells remained viable after 48 hr. To evaluate changes in fungal virulence upon cocultivation with Ac, recovered yeasts were used to infect Galleria mellonella, and in all instances, they killed the larvae faster than control yeasts. Surface biotinylated extracts of Ac exhibited intense fungal binding by FACS and fluorescence microscopy. Binding was also intense to mannose, and mass spectrometry identified Ac proteins with affinity to fungal surfaces including two putative transmembrane mannose-binding proteins (MBP, L8WXW7 and MBP1, Q6J288). Consistent with interactions with such mannose-binding proteins, Ac-fungi interactions were inhibited by mannose. These MBPs may be involved in fungal recognition by amoeba and promotes interactions that allow the emergence and maintenance of fungal virulence for animals., (© 2019 John Wiley & Sons Ltd.)

Arcobacter butzleri is an emerging foodborne zoonotic pathogen that has been isolated from environmental water sources. This pathogen establishes in vitro endosymbiotic relationships with Acanthamoeba castellanii, a free-living amoeba found in environmental matrices such as soil and water. The principal aim of this study was to analyse the transcriptional pattern of flagellar (flaA-flaB-flgH-motA) and other putative virulence genes (ciaB-cadF-mviN-pldA) of A. butzleri during its interaction with A. castellanii by quantitative real-time PCR. The transcriptional analysis showed up-regulation of all genes analysed before A. butzleri became established as an endocytobiont of A. castellanii. In contrast, while A. butzleri remains an endocytobiont, a significant and sustained decrease in the transcription of all analysed genes was observed. Our findings suggest that A. butzleri requires a biphasic transcriptional pattern of flagellar and other putative virulence genes to establish an endosymbiotic relationship with A. castellanii.

Shigella species and Acanthamoeba castellanii share the same ecological niches, and their interaction has been addressed in a limited number of research. However, there are still uncertain aspects and discrepant findings of this interaction. In the present study, the effects of the bacterial growth phase, cocultivation temperature and the type of culture media on the interaction of A. castellanii with Shigella dysenteriae, Shigella sonnei and Shigella flexneri were evaluated. In nutrient-poor page's amoeba saline (PAS) medium, the number of recovered bacteria and the uptake rates were significantly higher in stationary phase cells than logarithmic phase cells. However, no significant differences were observed in the number of recovered bacteria and the uptake rates between logarithmic and stationary phase cells in nutrient-rich peptone-yeast extract-glucose (PYG) medium. While the number of recovered bacteria was significantly higher in nutrient-rich than nutrient-poor media, in all the three Shigella species, the bacterial uptake rates were significantly higher in nutrient-poor than nutrient-rich media at both cocultivation temperatures. In both nutrient-poor and nutrient-rich media and at both cocultivation temperatures, the number of viable Shigella species after 24 h incubation were not influenced by the presence of A. castellanii. Although Shigella species did not proliferate in A. castellanii trophozoites, a considerable number of bacteria were survived in the trophozoites up to 15 days. From the public health perspective, the results of this study are important for further understanding of the nature of the interaction of these organisms and to deal with Shigella species in the environment., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication-permissive compartment termed the Mycobacterium-containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid-borne genes. Fluorescence microscopy of M. marinum-infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol-3-phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V-ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape., (© 2019 John Wiley & Sons Ltd.)

Legionella pneumophila is an important opportunistic pathogen for which environmental reservoirs are crucial for the infection of humans. In the environment, free-living amoebae represent key hosts providing nutrients and shelter for highly efficient intracellular proliferation of L. pneumophila , which eventually leads to lysis of the protist. However, the significance of other bacterial players for L. pneumophila ecology is poorly understood. In this study, we used a ubiquitous amoeba and bacterial endosymbiont to investigate the impact of this common association on L. pneumophila infection. We demonstrate that L. pneumophila proliferation was severely suppressed in Acanthamoeba castellanii harboring the chlamydial symbiont Protochlamydia amoebophila The amoebae survived the infection and were able to resume growth. Different environmental amoeba isolates containing the symbiont were equally well protected as different L. pneumophila isolates were diminished, suggesting ecological relevance of this symbiont-mediated defense. Furthermore, protection was not mediated by impaired L. pneumophila uptake. Instead, we observed reduced virulence of L. pneumophila released from symbiont-containing amoebae. Pronounced gene expression changes in the presence of the symbiont indicate that interference with the transition to the transmissive phase impedes the L. pneumophila infection. Finally, our data show that the defensive response of amoebae harboring P. amoebophila leaves the amoebae with superior fitness reminiscent of immunological memory. Given that mutualistic associations between bacteria and amoebae are widely distributed, P. amoebophila and potentially other amoeba endosymbionts could be key in shaping environmental survival, abundance, and virulence of this important pathogen, thereby affecting the frequency of human infection. IMPORTANCE Bacterial pathogens are generally investigated in the context of disease. To prevent outbreaks, it is essential to understand their lifestyle and interactions with other microbes in their natural environment. Legionella pneumophila is an important human respiratory pathogen that survives and multiplies in biofilms or intracellularly within protists, such as amoebae. Importantly, transmission to humans occurs from these environmental sources. Legionella infection generally leads to rapid host cell lysis. It was therefore surprising to observe that amoebae, including fresh environmental isolates, were well protected during Legionella infection when the bacterial symbiont Protochlamydia amoebophila was also present. Legionella was not prevented from invading amoebae but was impeded in its ability to develop fully virulent progeny and were ultimately cleared in the presence of the symbiont. This study highlights how ecology and virulence of an important human pathogen is affected by a defensive amoeba symbiont, with possibly major consequences for public health., (Copyright © 2019 König et al.)

Legionella pneumophila resides in multispecies biofilms, where it infects and replicates in environmental protozoa such as Acanthamoeba castellanii. Studies on L. pneumophila physiology and host-pathogen interactions are frequently conducted using clonal bacterial populations and population level analysis, overlooking the remarkable differences in single cell behavior. The fastidious nutrient requirements of extracellular L. pneumophila and the extraordinary motility of Acanthamoeba castellanii hamper an analysis at single cell resolution. In this chapter, we describe a method to study L. pneumophila and its natural host A. castellanii at single cell level by using an agarose embedment assay. Agarose-embedded bacteria and infected cells can be monitored over several hours up to several days. Using properly adapted flow chambers, agarose-embedded specimens can be subjected to a wide range of fluctuating conditions.

The gammaproteobacterium Legionella pneumophila is the causative agent of Legionnaires' disease, an atypical pneumonia that manifests itself with severe lung damage. L. pneumophila , a common inhabitant of freshwater environments, replicates in free-living amoebae and persists in biofilms in natural and man-made water systems. Its environmental versatility is reflected in its ability to survive and grow within a broad temperature range as well as its capability to colonize and infect a wide range of hosts, including protozoa and humans. Peptidyl-prolyl- cis/trans -isomerases (PPIases) are multifunctional proteins that are mainly involved in protein folding and secretion in bacteria. In L. pneumophila the surface-associated PPIase Mip was shown to facilitate the establishment of the intracellular infection cycle in its early stages. The cytoplasmic PpiB was shown to promote cold tolerance. Here, we set out to analyze the interrelationship of these two relevant PPIases in the context of environmental fitness and infection. We demonstrate that the PPIases Mip and PpiB are important for surfactant-dependent sliding motility and adaptation to suboptimal temperatures, features that contribute to the environmental fitness of L. pneumophila Furthermore, they contribute to infection of the natural host Acanthamoeba castellanii as well as human macrophages and human explanted lung tissue. These effects were additive in the case of sliding motility or synergistic in the case of temperature tolerance and infection, as assessed by the behavior of the double mutant. Accordingly, we propose that Mip and PpiB are virulence modulators of L. pneumophila with compensatory action and pleiotropic effects., (Copyright © 2018 American Society for Microbiology.)

In our previous report, we had shown that the free-living amoeba Acanthamoeba castellanii influenced the abundance, competiveness, and virulence of Mesorhizobium loti NZP2213, the microsymbiont of agriculturally important plants of the genus Lotus . The molecular basis of this phenomenon; however, had not been explored. In the present study, we demonstrated that oatB , the O -acetyltransferase encoding gene located in the lipopolysaccharide (LPS) synthesis cluster of M. loti , was responsible for maintaining the protective capacity of the bacterial cell envelope, necessary for the bacteria to fight environmental stress and survive inside amoeba cells. Using co-culture assays combined with fluorescence and electron microscopy, we showed that an oatB mutant, unlike the parental strain, was efficiently destroyed after rapid internalization by amoebae. Sensitivity and permeability studies of the oatB mutant, together with topography and nanomechanical investigations with the use of atomic force microscopy (AFM), indicated that the incomplete substitution of lipid A-core moieties with O-polysaccharide (O-PS) residues rendered the mutant more sensitive to hydrophobic compounds. Likewise, the truncated LPS moieties, rather than the lack of O -acetyl groups, made the oatB mutant susceptible to the bactericidal mechanisms (nitrosative stress and the action of lytic enzymes) of A. castellanii .

Shiga toxin-producing Escherichia coli (STEC) are a diverse group of strains that are implicated in over 270,000 cases of human illness annually in the United States alone. Shiga toxin (Stx), encoded by a resident temperate lambdoid bacteriophage, is the main STEC virulence factor. Although the population structure of E. coli O157:H7, the most common disease-causing STEC strain, is highly homogenous, the range of clinical illness caused by this strain varies by dramatically outbreak, suggesting that human virulence is evolving. However, the factors governing this variation in disease severity are poorly understood. STEC evolved from an O55:H7-like progenitor into a human pathogen. In addition to causing human disease, Stx released from STEC kill bacterivorous protist predators and enhance bacterial survival in the face of protist predation. Cattle are the primary reservoir for STEC and protists and bacteria occur together within the ruminant intestinal tract. Cattle associated STEC are not highly pathogenic to humans. These observations suggest that disease causing STEC strains evolved from cattle-associated "antipredator" STEC strains. To test this idea and to gain insight into the features that govern the evolution of STEC from a commensal strain of ruminants strain to virulent human pathogen, we compared the predation resistance of STEC strains isolated from asymptomatic infected cows and human patients. We find that STEC O157:H7 progenitor lineages and clades are more effective than human associated ones at killing the types of protist predators. In addition, our results indicate that the presence of Stx2c-containing bacteriophage is associated with more efficient amoeba killing. Also, these phage apparently also encode Q21-like version of the Q antitermination protein, the protein that controls expression of Stx., (Copyright © 2018 Elsevier GmbH. All rights reserved.)

Vibrio cholerae, which causes the diarrheal disease cholera, is a species of bacteria commonly found in aquatic habitats. Within such environments, the bacterium must defend itself against predatory protozoan grazers. Amoebae are prominent grazers, with Acanthamoeba castellanii being one of the best-studied aquatic amoebae. We previously showed that V. cholerae resists digestion by A. castellanii and establishes a replication niche within the host's osmoregulatory organelle. In this study, we decipher the molecular mechanisms involved in the maintenance of V. cholerae's intra-amoebal replication niche and its ultimate escape from the succumbed host. We demonstrate that minor virulence features important for disease in mammals, such as extracellular enzymes and flagellum-based motility, have a key role in the replication and transmission of V. cholerae in its aqueous environment. This work, therefore, describes new mechanisms that provide the pathogen with a fitness advantage in its primary habitat, which may have contributed to the emergence of these minor virulence factors in the species V. cholerae.

Pathogenic bacteria share their natural habitat with many other organisms such as animals, plants, insects, parasites and amoeba. Interactions between these organisms influence not only the life style of the host organisms, but also modulate bacterial physiology. Adaptation can include biofilm formation, capsule formation, and production of virulence factors. Although biofilm formation is a dominant mode of bacterial life in environmental settings, its role in host-pathogen interactions is not extensively studied. In this work, we investigated the role of molecular pathways involved in rdar biofilm formation in the interaction of Salmonella typhimurium with the Acanthamoeba castellanii genotype T4. Genes coding for the rdar biofilm activator CsgD, the cellulose synthase BcsA, and curli fimbriae subunits CsgBA were deleted from the genome of S. typhimurium. Assessment of interactions of wild-type and mutant strains of S. typhimurium with A. castellanii revealed that deletion of the cellulose synthase BcsA promoted association and uptake by A. castellanii, whereas the interactions with csgD and csgBA mutants were not changed. Our findings suggest that cellulose synthase BcsA inhibits the capabilities of S. typhimurium to associate with and invade into A. castellanii.

Legionella pneumophila genes encoding LapA, LapB, and PlaC were identified as the most highly upregulated type II secretion (T2S) genes during infection of Acanthamoeba castellanii , although these genes had been considered dispensable on the basis of the behavior of mutants lacking either lapA and lapB or plaC A plaC mutant showed even higher levels of lapA and lapB transcripts, and a lapA lapB mutant showed heightening of plaC mRNA levels, suggesting that the role of the LapA/B aminopeptidase is compensatory with respect to that of the PlaC acyltransferase. Hence, we made double mutants and found that lapA plaC mutants have an ~50-fold defect during infection of A. castellanii These data revealed, for the first time, the importance of LapA in any sort of infection; thus, we purified LapA and defined its crystal structure, activation by another T2S-dependent protease (ProA), and broad substrate specificity. When the amoebal infection medium was supplemented with amino acids, the defect of the lapA plaC mutant was reversed, implying that LapA generates amino acids for nutrition. Since the LapA and PlaC data did not fully explain the role of T2S in infection, we identified, via proteomic analysis, a novel secreted protein (NttD) that promotes infection of A. castellanii A lapA plaC nttD mutant displayed an even greater (100-fold) defect, demonstrating that the LapA, PlaC, and NttD data explain, to a significant degree, the importance of T2S. LapA-, PlaC-, and NttD-like proteins had distinct distribution patterns within and outside the Legionella genus. LapA was notable for having as its closest homologue an A. castellanii protein. IMPORTANCE Transmission of L. pneumophila to humans is facilitated by its ability to grow in Acanthamoeba species. We previously documented that type II secretion (T2S) promotes L. pneumophila infection of A. castellanii Utilizing transcriptional analysis and proteomics, double and triple mutants, and crystal structures, we defined three secreted substrates/effectors that largely clarify the role of T2S during infection of A. castellanii Particularly interesting are the unique functional overlap between an acyltransferase (PlaC) and aminopeptidase (LapA), the broad substrate specificity and eukaryotic-protein-like character of LapA, and the novelty of NttD. Linking LapA to amino acid acquisition, we defined, for the first time, the importance of secreted aminopeptidases in intracellular infection. Bioinformatic investigation, not previously applied to T2S, revealed that effectors originate from diverse sources and distribute within the Legionella genus in unique ways. The results of this study represent a major advance in understanding Legionella ecology and pathogenesis, bacterial secretion, and the evolution of intracellular parasitism., (Copyright © 2018 White et al.)

Shigella flexneri and Shigella sonnei bacteria cause the majority of all shigellosis cases worldwide. However, their distributions differ, with S. sonnei predominating in middle- and high-income countries and S. flexneri predominating in low-income countries. One proposed explanation for the continued range expansion of S. sonnei is that it can survive in amoebae, which could provide a protective environment for the bacteria. In this study, we demonstrate that while both S. sonnei and S. flexneri can survive coculture with the free-living amoebae Acanthamoebae castellanii , bacterial growth is predominantly extracellular. All isolates of Shigella were degraded following phagocytosis by A. castellanii , unlike those of Legionella pneumophila , which can replicate intracellularly. Our data suggest that S. sonnei is not able to use amoebae as a protective host to enhance environmental survival. Therefore, alternative explanations for S. sonnei emergence need to be considered. IMPORTANCE The distribution of Shigella species closely mirrors a country's socioeconomic conditions. With the transition of many populous nations from low- to middle-income countries, S. sonnei infections have emerged as a major public health issue. Understanding why S. sonnei infections are resistant to improvements in living conditions is key to developing methods to reduce exposure to this pathogen. We show that free-living amoebae are not likely to be environmental hosts of S. sonnei , as all Shigella strains tested were phagocytosed and degraded by amoebae. Therefore, alternative scenarios are required to explain the emergence and persistence of S. sonnei infections., (© Crown copyright 2018.)

Environmental bacteria of the genus Legionella naturally parasitize free-living amoebae. Upon inhalation of bacteria-laden aerosols, the opportunistic pathogens grow intracellularly in alveolar macrophages and can cause a life-threatening pneumonia termed Legionnaires' disease. Intracellular replication in amoebae and macrophages takes place in a unique membrane-bound compartment, the Legionella -containing vacuole (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system, which translocates literally hundreds of "effector" proteins into host cells, where they modulate crucial cellular processes for the pathogen's benefit. The mechanism of LCV formation appears to be evolutionarily conserved, and therefore, amoebae are not only ecologically significant niches for Legionella spp., but also useful cellular models for eukaryotic phagocytes. In particular, Acanthamoeba castellanii and Dictyostelium discoideum emerged over the last years as versatile and powerful models. Using genetic, biochemical and cell biological approaches, molecular interactions between amoebae and Legionella pneumophila have recently been investigated in detail with a focus on the role of phosphoinositide lipids, small and large GTPases, autophagy components and the retromer complex, as well as on bacterial effectors targeting these host factors.

Tuberculosis remains a serious threat to human health world-wide, and improved efficiency of medical treatment requires a better understanding of the pathogenesis and the discovery of new drugs. In the present study, we performed a whole-cell based screen in order to complete the characterization of 168 compounds from the GlaxoSmithKline TB-set. We have established and utilized novel previously unexplored host-model systems to characterize the GSK compounds, i.e. the amoeboid organisms D. discoideum and A. castellanii, as well as a microglial phagocytic cell line, BV2. We infected these host cells with Mycobacterium marinum to monitor and characterize the anti-infective activity of the compounds with quantitative fluorescence measurements and high-content microscopy. In summary, 88.1% of the compounds were confirmed as antibiotics against M. marinum, 11.3% and 4.8% displayed strong anti-infective activity in, respectively, the mammalian and protozoan infection models. Additionally, in the two systems, 13-14% of the compounds displayed pro-infective activity. Our studies underline the relevance of using evolutionarily distant pathogen and host models in order to reveal conserved mechanisms of virulence and defence, respectively, which are potential "universal" targets for intervention. Subsequent mechanism of action studies based on generation of over-expresser M. bovis BCG strains, generation of spontaneous resistant mutants and whole genome sequencing revealed four new molecular targets, including FbpA, MurC, MmpL3 and GlpK.

Acanthamoeba castellanii, a ubiquitous organism in water environments, is pathogenic toward humans and also is a host for bacteria of the genus Legionella, a causative agent of legionellosis. Fragrance ingredients were investigated for their antibacterial activity against planktonic Legionella pneumophila, amoebicidal activity against A. castellanii, and inhibitory effect against L. pneumophila uptake into A. castellanii. Helional ® exhibited relatively high antibacterial activity [minimum inhibitory concentration (MIC) , 32.0 μg/mL] . Anis aldehyde, canthoxal, helional ® and vanillin exhibited amoebicidal activity (IC 50 values, 58.4±2.0, 71.2±14.7, 66.8±8.3 and 49.1±2.5μg/mL, respectively) . L. pneumophila pretreatment with sub-MICs (0.25×MIC) of anis aldehyde, canthoxal, cortex aldehyde ® 50 percent or vanillin evidently reduced L. pneumophila uptake into A. castellanii (p < 0.01) . Thus, fragrance ingredients were good candidates for disinfectant against L. pneumophila and A. castellanii.

Legionella pneumophila, the causative agent of Legionnaires' disease, is widely distributed throughout natural and artificial water systems and can replicate in macrophages and amoebae. Amoebae are the natural hosts of L. pneumophila, whereas macrophages are incidentally infected. The life cycle of L. pneumophila comprises a replicative phase within the Legionella-containing vacuole (LCV) and a transmissive phase during which bacterial cells become motile and are released via killing of the host. Although the host death mechanisms induced by L. pneumophila have been studied, the expression patterns of related L. pneumophila genes have not been reported. The present study compared the expression patterns of host cell death-associated genes in L. pneumophila grown in the human monocytic cell line THP-1 and Acanthamoeba castellanii. Notably, when L. pneumophila was grown in THP-1, expression of the gene flaA, which is involved in the induction of pyroptosis, was downregulated during the course of infection. In contrast, sdhA associated indirectly with host death, was upregulated. Expression of the genes vipD and sidF, which are involved in the induction and suppression of apoptosis, changed by less than 2-fold. Notably, a lower percentage of pyroptotic cells was observed among infected THP-1 cells relative to uninfected cells, and the latter exhibited stronger expression of caspase-1. A different pattern was observed when L. pneumophila was grown in A. castellanii: flaA and vipD were activated, whereas sdhA and sidF were downregulated during the later stage of replication. The percentage of non-viable (annexin-V + PI + or annexin-V + PI - ) A. castellanii organisms increased with Legionella infection, and the expression of metacaspase-1, which is involved in encystation was up-regulated at late infection time. In summary, L. pneumophila can multiply intracellularly in both amoebae and macrophages to induce cell death and secondary infection, and this characteristic is essential for its survival in water and the lungs. The gene expression profiles observed in this study indicated the increased cytotoxicity of L. pneumophila in A. castellanii, suggesting an increased adaptation of Legionella to this host.

We used an amoeba model to study the intracellular growth and cytotoxicity of clinical strains of Mycobacterium abscessus subsp. massiliense (Mabsm) isolated from 2 patients (one with cystic fibrosis, the other one with idiopathic bronchiectasis) during the early (smooth colonies) and late stage (rough colonies) of chronic pulmonary infection. Acanthamoeba castellanii were infected with Mabsm (MOI 100) and samples collected every 24 h for 72 h. Results showed Mabsm is able to survive in trophozoites and persist in cysts for at least 7 days. Late Mabsm demonstrated higher cytotoxicity toward A. castellanii when compared to early strains. A. castellanii is a useful in vitro host model to study infection of Mabsm clinical isolates., (Published by Elsevier Masson SAS.)

Infections with filamentous fungi are common to all animals, but attention is rising especially due to the increasing incidence and high mortality rates observed in immunocompromised human individuals. Here, Aspergillus fumigatus and other members of its genus are the leading causative agents. Attributes like their saprophytic life-style in various ecological niches coupled with nutritional flexibility and a broad host range have fostered the hypothesis that environmental predators could have been the actual target for some of their virulence determinants. In this mini review, we have merged the recent findings focused on the potential dual-use of fungal defense strategies against innate immune cells and soil amoebae as natural phagocytes. Well-established virulence attributes like the melanized surface of fungal conidia or their capacity to produce toxic secondary metabolites have also been found to be protective against the model amoeba Dictyostelium discoideum . Some of the recent advances during interaction studies with human cells have further promoted the adaptation of other amoeba infection models, including the wide-spread generalist Acanthamoeba castellanii , or less prominent representatives like Vermamoeba vermiformis . We further highlight prospects and limits of these natural phagocyte models with regard to the infection biology of filamentous fungi and in comparison to the phagocytes of the innate immune system.

Bacterial infections have remained significant despite our advances in the development of a plethora of disinfectants as well as antimicrobial chemotherapy. This is in part due to our incomplete understanding of the prevalence of bacterial pathogens in the environmental and clinical settings. Several lines of evidence suggest that Acanthamoeba is one of the most ubiquitous/resilient protists that also acts as a host/reservoir for pathogenic microbes. Thus targeting the hardy host, which harbour microbial pathogens, offer a potential avenue to counter infection transmission, particularly hospital/community-acquired infections. This will complement existing approach of applying disinfectants that are targeted against bacterial pathogens directly., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Acanthamoeba castellanii is a free-living amoeba commonly found in aquatic environment. It feeds on bacteria even if some bacteria resist amoebal digestion. Thus, A. castellanii is described as a Trojan horse able to harbor pathogenic bacteria. L. pneumophila is one of the amoeba-resisting bacteria able to avoid host degradation by phagocytosis and to multiply inside the amoeba. When infecting its host, L. pneumophila injects hundreds of effectors via a type IV secretion system that change physiology of the amoeba to its profit. In this study, we assess mobility of A. castellanii upon infection with L. pneumophila. Electron-microscopy analysis of amoebae revealed a reduction of acanthopodia on cells infected with L. pneumophila. Analysis of velocity showed that migration of A. castellanii infected with L. pneumophila was significantly impaired compare to uninfected cells. Taken together, infection with L. pneumophila could prevent formation of cytoplasmic extensions such as acanthopodia with consequences on the shape, adherence and mobility of A. castellanii., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Protozoan predation is one of the main environmental factors constraining bacterial growth in aquatic environments, and thus has led to the evolution of a number of defence mechanisms that protect bacteria from predation. These mechanisms may also function as virulence factors in infection of animal and human hosts. Whole transcriptome shotgun sequencing of Vibrio cholerae biofilms during predation by the amoebae, Acanthamoeba castellanii, revealed that 131 transcripts were significantly differentially regulated when compared to the non-grazed control. Differentially regulated transcripts included those involved in biosynthetic and metabolic pathways. The transcripts of genes involved in tyrosine metabolism were down-regulated in the grazed population, which indicates that the tyrosine metabolic regulon may have a role in the response of V. cholerae biofilms to A. castellanii predation. Homogentisate 1, 2-dioxygenase (HGA) is the main intermediate of the normal L-tyrosine catabolic pathway which is known to auto-oxidize, leading to the formation of the pigment, pyomelanin. Indeed, a pigmented mutant, disrupted in hmgA, was more resistant to amoebae predation than the wild type. Increased grazing resistance was correlated with increased production of pyomelanin and thus reactive oxygen species (ROS), suggesting that ROS production is a defensive mechanism used by bacterial biofilms against predation by amoebae A. castellanii., (© FEMS 2017.)