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1.
Proc Natl Acad Sci U S A ; 117(35): 21658-21666, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32817434

RESUMO

Symbiosis with microbes is a ubiquitous phenomenon with a massive impact on all living organisms, shaping the world around us today. Theoretical and experimental studies show that vertical transmission of symbionts leads to the evolution of mutualistic traits, whereas horizontal transmission facilitates the emergence of parasitic features. However, these studies focused on phenotypic data, and we know little about underlying molecular changes at the genomic level. Here, we combined an experimental evolution approach with infection assays, genome resequencing, and global gene expression analysis to study the effect of transmission mode on an obligate intracellular bacterial symbiont. We show that a dramatic shift in the frequency of genetic variants, coupled with major changes in gene expression, allow the symbiont to alter its position in the parasitism-mutualism continuum depending on the mode of between-host transmission. We found that increased parasitism in horizontally transmitted chlamydiae residing in amoebae was a result of processes occurring at the infectious stage of the symbiont's developmental cycle. Specifically, genes involved in energy production required for extracellular survival and the type III secretion system-the symbiont's primary virulence mechanism-were significantly up-regulated. Our results identify the genomic and transcriptional dynamics sufficient to favor parasitic or mutualistic strategies.


Assuntos
Chlamydia/genética , Interações entre Hospedeiro e Microrganismos/genética , Simbiose/genética , Amoeba/metabolismo , Amoeba/microbiologia , Animais , Bactérias/genética , Evolução Biológica , Chlamydia/metabolismo , Genoma Bacteriano/genética , Parasitos/genética , Virulência
2.
Parasitol Res ; 119(3): 925-934, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32048025

RESUMO

The Rozellomycota form a lineage basal or sister to the Fungi, ancestor of Microsporidia. Their biodiversity is very rich but remains poorly characterized. The few known species are all parasites, whether of water molds and algae (Rozella), crustaceans (Mitosporidium), or as endonuclear parasites of amoebae (Nucleophaga, Paramicrosporidium). Since the nineteenth century, intracytoplasmic parasites of various protozoa have been described as species of the same genus Sphaerita. However, it was later thought possible to separate these parasites into at least two distinct groups, those forming flagellated zoospores, prevalent in Euglena and other flagellates, and those forming immobile spores, found mainly in free-living and endozoic amoebae. Herein, we report the recovery of a strain of the free-living amoeba species Saccamoeba lacustris, naturally infected by an intracytoplasmic parasite, which under light microscope has a morphology consistent with that of Sphaerita. Biomolecular analyses were thus performed. Our results show that the intracytoplasmic parasite of Saccamoeba belongs to the same subgroup of Mitosporidium and that it forms a new genus within Rozellomycota, Morellospora, that corresponds to the former spore-forming Sphaerita-like parasites of amoebae.


Assuntos
Amoeba/microbiologia , Fungos/classificação , Animais , Biodiversidade , Fungos/isolamento & purificação , Filogenia
3.
Nat Microbiol ; 5(4): 599-609, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31988381

RESUMO

Virulence mechanisms typically evolve through the continual interaction of a pathogen with its host. In contrast, it is poorly understood how environmentally acquired pathogens are able to cause disease without prior interaction with humans. Here, we provide experimental evidence for the model that Legionella pathogenesis in humans results from the cumulative selective pressures of multiple amoebal hosts in the environment. Using transposon sequencing, we identify Legionella pneumophila genes required for growth in four diverse amoebae, defining universal virulence factors commonly required in all host cell types and amoeba-specific auxiliary genes that determine host range. By comparing genes that promote growth in amoebae and macrophages, we show that adaptation of L. pneumophila to each amoeba causes the accumulation of distinct virulence genes that collectively allow replication in macrophages and, in some cases, leads to redundancy in this host cell type. In contrast, some bacterial proteins that promote replication in amoebae restrict growth in macrophages. Thus, amoebae-imposed selection is a double-edged sword, having both positive and negative impacts on disease. Comparing the genome composition and host range of multiple Legionella species, we demonstrate that their distinct evolutionary trajectories in the environment have led to the convergent evolution of compensatory virulence mechanisms.


Assuntos
Amoeba/microbiologia , Coevolução Biológica , Interações Hospedeiro-Patógeno/genética , Legionella pneumophila/genética , Legionella pneumophila/patogenicidade , Macrófagos/microbiologia , Fatores de Virulência/genética , Adaptação Fisiológica , Amoeba/classificação , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular , Feminino , Especificidade de Hospedeiro , Humanos , Legionella pneumophila/classificação , Legionella pneumophila/crescimento & desenvolvimento , Camundongos , Filogenia , Cultura Primária de Células , Seleção Genética , Virulência , Fatores de Virulência/classificação , Fatores de Virulência/metabolismo
4.
Int J Food Microbiol ; 318: 108477, 2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-31855786

RESUMO

Vegetables are one of the sources from which Helicobacter pylori can be acquired. This bacterium infects >50% of the global population and is a recognized type I human carcinogen. H. pylori enters into the viable but non-culturable state when it is in the environment, and therefore the use of molecular techniques is much convenient for its detection. Free-living amoebae (FLA) are protozoans found in vegetables. They are transmission vehicles for amoeba-resistant bacteria, among which H. pylori is included. The aim of this study is to study the occurrence and viability of H. pylori from lettuce samples, H. pylori internalized into FLA and the microbiome of FLA isolated from these samples. Special focus was pointed to human pathogenic bacteria. H. pylori was not directly detected in any lettuce sample by means of molecular techniques and neither by culture. However, intra-amoebic H. pylori DNA was detected by means of PMA-qPCR in 55% of the samples and viable intra-amoebic H. pylori cells in 25% of the samples by means of DVC-FISH technique. When FLA microbiome was studied, 21 bacterial genera were part of FLA microbiome in all samples. Helicobacter genus was detected as part of the FLA microbiome in two samples. Other bacteria of public health interest such as Aeromonas sp., Arcobacter sp., Legionella sp., Mycobacterium sp., Pseudomonas sp. and Salmonella sp. were detected as part of FLA microbiome along the analysed samples. This study demonstrates for the first time that H. pylori is internalized as well as alive inside FLA isolated from vegetables. Moreover, this study shows that FLA promote H. pylori detection in environmental samples. In addition, as far as we are aware, this is the first study which studies the microbiome of FLA isolated from vegetables. Among the FLA microbiome, bacteria of public health interest were detected, pointing out that FLA are carriers of these pathogens which can reach humans and cause a public health concern.


Assuntos
Amoeba/microbiologia , Helicobacter pylori/isolamento & purificação , Alface/microbiologia , Alface/parasitologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Microbiologia de Alimentos , Parasitologia de Alimentos , Helicobacter pylori/genética , Humanos , Microbiota/genética , Saúde Pública , Reação em Cadeia da Polimerase em Tempo Real
5.
Microb Pathog ; 140: 103942, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31881258

RESUMO

Understanding polymicrobial interactions involving fungi in the environment and the human mycobiome is necessary to address environmental and medically related problems such as drought or antimicrobial resistance. The diversity of these interactions highlights the complexity of fungi, considering how some interactions can be antagonistic, while others synergistic. Over the years, an increase in studies on the mycobiome have revealed similarities between the human and environmental hosts. More recently, studies have focused on microbial commensal relationships and identifying causative agents of human disease. The overlap of some of these interactions is impossible to ignore, indicating that there are areas for medical exploitation that need to be further investigated. This review provides the latest advances in polymicrobial interactions involving fungi and discusses the importance of the fungal lifestyle in the environment and in human disease.


Assuntos
Fungos , Interações Microbianas , Amoeba/microbiologia , Bactérias , Biofilmes , Doença , Água Potável/microbiologia , Meio Ambiente , Microbioma Gastrointestinal , Humanos , Pulmão/microbiologia , Micobioma , Micoses/microbiologia , Doenças das Plantas/microbiologia
6.
PLoS Pathog ; 15(11): e1008069, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31703112

RESUMO

Free-living amoebae are thought to represent an environmental niche in which amoeba-resistant bacteria may evolve towards pathogenicity. To get more insights into factors playing a role for adaptation to intracellular life, we characterized the transcriptomic activities of the emerging pathogen Mycobacterium abscessus in amoeba and murine macrophages (Mϕ) and compared them with the intra-amoebal transcriptome of the closely related, but less pathogenic Mycobacterium chelonae. Data on up-regulated genes in amoeba point to proteins that allow M. abscessus to resist environmental stress and induce defense mechanisms, as well as showing a switch from carbohydrate carbon sources to fatty acid metabolism. For eleven of the most upregulated genes in amoeba and/or Mϕ, we generated individual gene knock-out M. abscessus mutant strains, from which ten were found to be attenuated in amoeba and/or Mϕ in subsequence virulence analyses. Moreover, transfer of two of these genes into the genome of M. chelonae increased the intra-Mϕ survival of the recombinant strain. One knock-out mutant that had the gene encoding Eis N-acetyl transferase protein (MAB_4532c) deleted, was particularly strongly attenuated in Mϕ. Taken together, M. abscessus intra-amoeba and intra-Mϕ transcriptomes revealed the capacity of M. abscessus to adapt to an intracellular lifestyle, with amoeba largely contributing to the enhancement of M. abscessus intra-Mϕ survival.


Assuntos
Amoeba/genética , Macrófagos/metabolismo , Infecções por Mycobacterium não Tuberculosas/genética , Mycobacterium abscessus/patogenicidade , Transcriptoma , Fatores de Virulência/genética , Virulência/genética , Amoeba/crescimento & desenvolvimento , Amoeba/microbiologia , Animais , Proteínas de Bactérias/genética , Macrófagos/microbiologia , Camundongos , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/genética , Mycobacterium abscessus/isolamento & purificação
7.
Nat Commun ; 10(1): 5216, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31740681

RESUMO

The facultative intracellular bacterium Legionella pneumophila replicates in environmental amoebae and in lung macrophages, and causes Legionnaires' disease. Here we show that L. pneumophila reversibly forms replicating and nonreplicating subpopulations of similar size within amoebae. The nonreplicating bacteria are viable and metabolically active, display increased antibiotic tolerance and a distinct proteome, and show high virulence as well as the capacity to form a degradation-resistant compartment. Upon infection of naïve or interferon-γ-activated macrophages, the nonreplicating subpopulation comprises ca. 10% or 50%, respectively, of the total intracellular bacteria; hence, the nonreplicating subpopulation is of similar size in amoebae and activated macrophages. The numbers of nonreplicating bacteria within amoebae are reduced in the absence of the autoinducer synthase LqsA or other components of the Lqs quorum-sensing system. Our results indicate that virulent, antibiotic-tolerant subpopulations of L. pneumophila are formed during infection of evolutionarily distant phagocytes, in a process controlled by the Lqs system.


Assuntos
Legionella/patogenicidade , Legionelose/microbiologia , Macrófagos/microbiologia , Percepção de Quorum , Amoeba/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Espaço Intracelular/microbiologia , Legionella/crescimento & desenvolvimento , Camundongos , Viabilidade Microbiana , Proteoma/metabolismo , Vacúolos/microbiologia , Virulência
8.
Emerg Infect Dis ; 25(11): 2104-2107, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31625866

RESUMO

Legionellosis was diagnosed in an immunocompromised 3-year-old girl in Canada. We traced the source of the bacterium through co-culture with an ameba collected from a hot tub in her home. We identified Legionella pneumophila serogroup 6, sequence type 185, and used whole-genome sequencing to confirm the environmental and clinical isolates were of common origin.


Assuntos
Amoeba/microbiologia , Legionella pneumophila/isolamento & purificação , Doença dos Legionários/epidemiologia , Doença dos Legionários/microbiologia , Canadá/epidemiologia , Técnicas de Cocultura , Surtos de Doenças , Genoma Bacteriano , Humanos , Legionella pneumophila/classificação , Legionella pneumophila/genética , Filogenia , Vigilância em Saúde Pública , Sequenciamento Completo do Genoma
9.
PLoS Negl Trop Dis ; 13(10): e0007742, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31589617

RESUMO

Paracoccidioides spp. are thermodimorphic fungi that cause a neglected tropical disease (paracoccidioidomycosis) that is endemic to Latin America. These fungi inhabit the soil, where they live as saprophytes with no need for a mammalian host to complete their life cycle. Despite this, they developed sophisticated virulence attributes allowing them not only to survive in host tissues but also to cause disease. A hypothesis for selective pressures driving the emergence or maintenance of virulence of soil fungi is their interaction with soil predators such as amoebae and helminths. We evaluated the presence of environmental amoeboid predators in soil from armadillo burrows where Paracoccidioides had been previously detected and tested if the interaction of Paracoccidioides with amoebae selects for fungi with increased virulence. Nematodes, ciliates, and amoebae-all potential predators of fungi-grew in cultures from soil samples. Microscopical observation and ITS sequencing identified the amoebae as Acanthamoeba spp, Allovahlkampfia spelaea, and Vermamoeba vermiformis. These three amoebae efficiently ingested, killed and digested Paracoccidioides spp. yeast cells, as did laboratory adapted axenic Acanthamoeba castellanii. Sequential co-cultivation of Paracoccidioides with A. castellanii selected for phenotypical traits related to the survival of the fungus within a natural predator as well as in murine macrophages and in vivo (Galleria mellonella and mice). These changes in virulence were linked to the accumulation of cell wall alpha-glucans, polysaccharides that mask recognition of fungal molecular patterns by host pattern recognition receptors. Altogether, our results indicate that Paracoccidioides inhabits a complex environment with multiple amoeboid predators that can exert selective pressure to guide the evolution of virulence traits.


Assuntos
Amoeba/fisiologia , Interações Hospedeiro-Patógeno/fisiologia , Paracoccidioides/fisiologia , Microbiologia do Solo , Acanthamoeba castellanii/fisiologia , Amoeba/citologia , Amoeba/microbiologia , Animais , Tatus , Cilióforos , Técnicas de Cocultura , Modelos Animais de Doenças , Fungos , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Nematoides , Paracoccidioides/patogenicidade , Paracoccidioidomicose/microbiologia , Fagocitose , Solo , Virulência , Fatores de Virulência/fisiologia
10.
Parasitol Res ; 118(11): 3191-3194, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31511977

RESUMO

Aeromonas hydrophila, considered as an emerging pathogen, is increasingly involved in opportunistic human infections. This bacterium, mainly present in aquatic environments, can therefore develop relationships with the free-living amoeba Vermamoeba vermiformis in hospital water networks. We showed in this study that the joint presence of V. vermiformis and A. hydrophila led to an increased bacterial growth in the first 48 h of contact and moreover to the protection of the bacteria in adverse conditions even after 28 days. These results highlight the fact that strategies should be implemented to control the development of FLA in hospital water systems.


Assuntos
Aeromonas hydrophila/crescimento & desenvolvimento , Amoeba/microbiologia , Antibiose/fisiologia , Lobosea/microbiologia , Hospitais/estatística & dados numéricos , Humanos , Água/análise , Microbiologia da Água
11.
Genes Immun ; 20(5): 394-402, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31053752

RESUMO

Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia called Legionnaires' disease that is often fatal when not promptly diagnosed and treated. However, L. pneumophila is mainly an environmental pathogen of protozoa. This bacterium parasitizes free-living amoeba and other aquatic protozoa with which it co-evolved over an evolutionary long time. Due to the close relationship between hosts and pathogens, their co-evolution leads to molecular interactions such as the exchange of genetic material through horizontal gene transfer (HGT). Those genes that confer an advantage to the bacteria were fixed in their genomes and help these pathogens to subvert host functions to their advantage. Genome sequencing of L. pneumophila and recently of the entire genus Legionella that comprises over 60 species revealed that Legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts to a surprisingly high extent never observed before for an prokaryotic organism. Acquisition and loss of these eukaryotic-like genes and eukaryotic domains is an ongoing process underlining the highly dynamic nature of the Legionella genomes. Although the large amount and diversity of HGT that occurred between Legionella and their protozoan hosts seems to be unique in the prokaryotic world, the analyses of more and more genomes from environmental organisms and symbionts of amoeba revealed that such genetic exchanges occur among all amoeba-associated bacteria and also among the different microorganisms that infect amoeba such as viruses. This dynamic reshuffling and gene-acquisition has led to the emergence of major human pathogens such as Legionella and may lead to the emergence of new human pathogens from the environment.


Assuntos
Evolução Molecular , Interações Hospedeiro-Patógeno/genética , Legionella pneumophila/patogenicidade , Amoeba/genética , Amoeba/microbiologia , Transferência Genética Horizontal , Legionella pneumophila/genética
12.
BMC Microbiol ; 19(1): 91, 2019 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-31072343

RESUMO

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


Assuntos
Amoeba/microbiologia , Núcleo Celular/microbiologia , Gammaproteobacteria/fisiologia , Interações entre Hospedeiro e Microrganismos , Monócitos/microbiologia , Citoplasma/microbiologia , Gammaproteobacteria/ultraestrutura , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Monócitos/ultraestrutura , Simbiose , Células THP-1 , Células U937
13.
ISME J ; 13(8): 2068-2081, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31019270

RESUMO

Symbiotic associations impact and are impacted by their surrounding ecosystem. The association between Burkholderia bacteria and the soil amoeba Dictyostelium discoideum is a tractable model to unravel the biology underlying symbiont-endowed phenotypes and their impacts. Several Burkholderia species stably associate with D. discoideum and typically reduce host fitness in food-rich environments while increasing fitness in food-scarce environments. Burkholderia symbionts are themselves inedible to their hosts but induce co-infections with secondary bacteria that can serve as a food source. Thus, Burkholderia hosts are "farmers" that carry food bacteria to new environments, providing a benefit when food is scarce. We examined the ability of specific Burkholderia genotypes to induce secondary co-infections and assessed host fitness under a range of co-infection conditions and environmental contexts. Although all Burkholderia symbionts intracellularly infected Dictyostelium, we found that co-infections are predominantly extracellular, suggesting that farming benefits are derived from extracellular infection of host structures. Furthermore, levels of secondary infection are linked to conditional host fitness; B. agricolaris infected hosts have the highest level of co-infection and have the highest fitness in food-scarce environments. This study illuminates the phenomenon of co-infection induction across Dictyostelium associated Burkholderia species and exemplifies the contextual complexity of these associations.


Assuntos
Amoeba/microbiologia , Burkholderia/fisiologia , Dictyostelium/microbiologia , Interação Gene-Ambiente , Simbiose , Animais , Bactérias , Fenômenos Fisiológicos Bacterianos , Burkholderia/genética , Ecossistema , Genótipo , Fenótipo
14.
Environ Microbiol ; 21(5): 1809-1820, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30868709

RESUMO

Size and diverse morphologies pose a primary challenge for phagocytes such as innate immune cells and predatory amoebae when encountering fungal prey. Although filamentous fungi can escape phagocytic killing by pure physical constraints, unicellular spores and yeasts can mask molecular surface patterns or arrest phagocytic processing. Here, we show that the fungivorous amoeba Protostelium aurantium was able to adjust its killing and feeding mechanisms to these different cell shapes. Yeast-like fungi from the major fungal groups of basidiomycetes and ascomycetes were readily internalized by phagocytosis, except for the human pathogen Candida albicans whose mannoprotein coat was essential to escape recognition by the amoeba. Dormant spores of the filamentous fungus Aspergillus fumigatus also remained unrecognized, but swelling and the onset of germination induced internalization and intracellular killing by the amoeba. Mature hyphae of A. fumigatus were mostly attacked from the hyphal tip and killed by an actin-mediated invasion of fungal filaments. Our results demonstrate that predatory pressure imposed by amoebae in natural environments selects for distinct survival strategies in yeast and filamentous fungi but commonly targets the fungal cell wall as a crucial molecular pattern associated to prey and pathogens.


Assuntos
Amoeba/microbiologia , Fungos/fisiologia , Leveduras/fisiologia , Amoeba/fisiologia , Parede Celular , Fungos/genética , Fungos/crescimento & desenvolvimento , Fungos/isolamento & purificação , Hifas/genética , Hifas/crescimento & desenvolvimento , Fagocitose , Leveduras/genética , Leveduras/crescimento & desenvolvimento , Leveduras/isolamento & purificação
15.
Methods Mol Biol ; 1921: 55-78, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30694485

RESUMO

Predation by protozoa provides a strong selective pressure for Legionella to develop and maintain mechanisms conferring resistance to digestion and ability to replicate within both amoebae and mammalian macrophages. Here we describe how to isolate environmental protozoa that prey on virulent Legionella. These protists are extremely useful models to study the cellular mechanisms employed by Legionellae to survive and grow in its natural environment. We present here procedures that are available to study the interactions between environmental protozoa and Legionella and thus increase our current understanding of Legionella virulence and the infection process.


Assuntos
Microbiologia Ambiental , Interações Hospedeiro-Patógeno , Legionella/fisiologia , Característica Quantitativa Herdável , Seleção Genética , Virulência , Amoeba/microbiologia , Amoeba/fisiologia , Amoeba/ultraestrutura , Técnicas de Cocultura , Resistência à Doença , Legionelose/microbiologia , Virulência/genética
16.
Methods Mol Biol ; 1921: 347-370, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30694504

RESUMO

The professional phagocyte Dictyostelium discoideum is a well-established model organism to study host-pathogen interactions. Dictyostelium amoebae grow as separate, independent cells; they divide by binary fission and take up bacteria and yeast via phagocytosis. In the year 2000, D. discoideum was described by two groups as a novel system for genetic analysis of host-pathogen interactions for the intracellular pathogen Legionella pneumophila. Since then additional microbial pathogens that can be studied in D. discoideum have been reported. The organism has various advantages for the dissection of the complex cross-talk between a host and a pathogen. A fully sequenced and well-curated genome is available, there are excellent molecular genetic tools on the market, and the generation of targeted multiple gene knock-outs as well as the realization of untargeted genetic screens is generally straightforward. Dictyostelium also offers easy cultivation, and the cells are suitable for cell biological studies, which in combination with in vivo expression of fluorescence-tagged proteins allows the investigation of the dynamics of bacterial uptake and infection. Furthermore, a large mutant collection is available at the Dictyostelium stock center, favoring the identification of host resistance or susceptibility genes. Here, we briefly describe strategies to identify host cell factors important during an infection, followed by protocols for cell culture and storage, uptake and infection, and confocal microscopy of infected cells.


Assuntos
Dictyostelium/microbiologia , Interações Hospedeiro-Patógeno , Legionella/fisiologia , Legionelose/microbiologia , Amoeba/microbiologia , Dictyostelium/genética , Dictyostelium/imunologia , Citometria de Fluxo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Microscopia Confocal , Mutação , Fagocitose
17.
Methods Mol Biol ; 1921: 421-428, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30694507

RESUMO

The presence of Legionella spp. in natural and man-made water systems is a great public health concern and heavily depends on the presence of free-living amoebae. Taking advantage of the development and affordability of next-generation sequencing technology, we present here a method to characterize the whole bacterial community directly from water samples, as well as from isolated free-living amoebae.


Assuntos
Amoeba/microbiologia , Metagenoma , Metagenômica , Microbiota , Microbiologia da Água , Biodiversidade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metagenômica/métodos , RNA Ribossômico 16S
18.
Cell Microbiol ; 21(6): e13008, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30656819

RESUMO

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.


Assuntos
Citosol/metabolismo , Mycobacterium marinum/crescimento & desenvolvimento , Fagossomos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Vacúolos/metabolismo , Acanthamoeba castellanii/microbiologia , Adenosina Trifosfatases/metabolismo , Amoeba/microbiologia , Animais , Proteínas de Bactérias/metabolismo , Dictyostelium/metabolismo , Dictyostelium/microbiologia , Interações Hospedeiro-Patógeno/genética , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos , Microscopia de Fluorescência , Mycobacterium marinum/enzimologia , Mycobacterium marinum/genética , Mycobacterium marinum/patogenicidade , Proteínas Tirosina Fosfatases/metabolismo , Células RAW 264.7 , Vacúolos/microbiologia
19.
Cell Microbiol ; 21(1): e12971, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30370624

RESUMO

Legionella pneumophila is a ubiquitous environmental bacterium that has evolved to infect and proliferate within amoebae and other protists. It is thought that accidental inhalation of contaminated water particles by humans is what has enabled this pathogen to proliferate within alveolar macrophages and cause pneumonia. However, the highly evolved macrophages are equipped with more sophisticated innate defence mechanisms than are protists, such as the evolution of phagotrophic feeding into phagocytosis with more evolved innate defence processes. Not surprisingly, the majority of proteins involved in phagosome biogenesis (~80%) have origins in the phagotrophy stage of evolution. There are a plethora of highly evolved cellular and innate metazoan processes, not represented in protist biology, that are modulated by L. pneumophila, including TLR2 signalling, NF-κB, apoptotic and inflammatory processes, histone modification, caspases, and the NLRC-Naip5 inflammasomes. Importantly, L. pneumophila infects haemocytes of the invertebrate Galleria mellonella, kill G. mellonella larvae, and proliferate in and kill Drosophila adult flies and Caenorhabditis elegans. Although coevolution with protist hosts has provided a substantial blueprint for L. pneumophila to infect macrophages, we discuss the further evolutionary aspects of coevolution of L. pneumophila and its adaptation to modulate various highly evolved innate metazoan processes prior to becoming a human pathogen.


Assuntos
Amoeba/metabolismo , Amoeba/microbiologia , Evasão da Resposta Imune , Imunidade Inata , Legionella pneumophila/fisiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Animais , Caenorhabditis elegans/imunologia , Caenorhabditis elegans/microbiologia , Drosophila/imunologia , Drosophila/microbiologia , Lepidópteros/imunologia , Lepidópteros/microbiologia
20.
Mol Ecol ; 28(4): 847-862, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30575161

RESUMO

The establishment of symbioses between eukaryotic hosts and bacterial symbionts in nature is a dynamic process. The formation of such relationships depends on the life history of both partners. Bacterial symbionts of amoebae may have unique evolutionary trajectories to the symbiont lifestyle, because bacteria are typically ingested as prey. To persist after ingestion, bacteria must first survive phagocytosis. In the social amoeba Dictyostelium discoideum, certain strains of Burkholderia bacteria are able to resist amoebal digestion and maintain a persistent relationship that includes carriage throughout the amoeba's social cycle that culminates in spore formation. Some Burkholderia strains allow their host to carry other bacteria, as food. This carried food is released in new environments in a trait called farming. To better understand the diversity and prevalence of Burkholderia symbionts and the traits they impart to their amoebae hosts, we first screened 700 natural isolates of D. discoideum and found 25% infected with Burkholderia. We next used a multilocus phylogenetic analysis and identified two independent transitions by Burkholderia to the symbiotic lifestyle. Finally, we tested the ability of 38 strains of Burkholderia from D. discoideum, as well as strains isolated from other sources, for traits relevant to symbiosis in D. discoideum. Only D. discoideum native isolates belonging to the Burkholderia agricolaris, B. hayleyella, and B. bonniea species were able to form persistent symbiotic associations with D. discoideum. The Burkholderia-Dictyostelium relationship provides a promising arena for further studies of the pathway to symbiosis in a unique system.


Assuntos
Amoeba/microbiologia , Burkholderia/genética , Burkholderia/fisiologia , Burkholderia/classificação , Dictyostelium/classificação , Dictyostelium/genética , Dictyostelium/fisiologia , Filogenia , Simbiose/genética , Simbiose/fisiologia
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