The unique Legionella longbeachae capsule favors intracellular replication and immune evasion.

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.

Phylogenomic, structural, and cell biological analyses reveal that Stenotrophomonas maltophilia replicates in acidified Rab7A-positive vacuoles of Acanthamoeba castellanii.

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.

A time-resolved multi-omics atlas of Acanthamoeba castellanii encystment.

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.

Recognition of Cell Wall Mannosylated Components as a Conserved Feature for Fungal Entrance, Adaptation and Survival Within Trophozoites of Acanthamoeba castellanii and Murine Macrophages.

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.

Transient internalization of Campylobacter jejuni in Amoebae enhances subsequent invasion of human cells.

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, 11168HCBA with that harvested from within Acanthamoeba castellanii (11168HAC/CBA) or Acanthamoeba polyphaga (11168HAP/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.

Chlorine-stressed Salmonella cells are significantly more engulfed by Acanthamoeba trophozoites and have a longer intracystic survival than the non-stressed cells.

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.

Autorepressor PsrA is required for optimal Legionella pneumophila growth in Acanthamoeba castellanii protozoa.

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.

Amoeba Predation of Cryptococcus neoformans Results in Pleiotropic Changes to Traits Associated with Virulence.

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_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_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.IMPORTANCECryptococcus 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.

Acanthamoeba castellanii as an alternative interaction model for the dermatophyte Trichophyton rubrum.

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.

In vitro and intracellular activities of frog skin temporins against Legionella pneumophila and its eukaryotic hosts.

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 [K3]SHa, has been designed to improve those activities. SHa and [K3]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 [K3]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 [K3]SHa and exhibited low half maximal lethal concentrations (LC50). 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.

Distinct Mycobacterium marinum phosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol.

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.

Peptidyl-Prolyl-cis/trans-Isomerases Mip and PpiB of Legionella pneumophila Contribute to Surface Translocation, Growth at Suboptimal Temperature, and Infection.

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.

Acanthamoeba and Dictyostelium as Cellular Models for Legionella Infection.

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.

Survival of pathogenic Mycobacterium abscessus subsp. massiliense in Acanthamoeba castellanii.

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.

Differential expression of virulence genes in Legionella pneumophila growing in Acanthamoeba and human monocytes.

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.

Identification of Conserved ABC Importers Necessary for Intracellular Survival of Legionella pneumophila in Multiple Hosts.

It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term "protozoan-priming." We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC) transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication.

Acanthamoeba castellanii interactions with Streptococcus pneumoniae and Streptococcus pyogenes.

Among the genus Streptococcus, S. pyogenes and S. pneumoniae are the major causes of pharyngitis, impetigo, pneumonia and meningitis in humans. Streptococcus spp. are facultative anaerobes that are nutritionally fastidious, yet survive in the environment and target the predisposed population. Antibacterial disinfectants have been partially effective only, indicating the need for novel preventative measures and to understand mechanisms of bacterial resistance. Acanthamoeba is a free-living protist that is known to harbour microbial pathogens, provide shelter, and assist in their transmission to susceptible population. The overall aim of this study was to determine whether S. pyogenes and S. pneumoniae can interact with A. castellanii by associating, invading, and surviving inside trophozoites and cysts. It was observed that both S. pyogenes and S. pneumoniae were able to associate as well as invade and/or taken up by the phagocytic A. castellanii trophozoite. Notably, S. pyogenes and S. pneumoniae survived the encystation process, avoided phagocytosis, multiplied, and exhibited higher recovery from the mature cysts, compared with the trophozoite stage (approximately 2 bacteria per amoebae ratio for cyst stage versus 0.02 bacteria per amoeba ration for trophozoite stage). As Acanthamoeba cysts are resilient and can disperse through the air, A. castellanii can act as a vector in providing shelter, facilitating growth and possibly genetic exchanges. In addition, these interactions may contribute to S. pyogenes and S. pneumoniae survival in harsh environments, and transmission to susceptible population and possibly affecting their virulence. Future studies will determine the molecular mechanisms associated with Acanthamoeba interactions with Streptococcus and the evolution of pathogenic bacteria and in turn expedite the discovery of novel therapeutic and/or preventative measures.

Impact of Acanthamoeba Cysts on Stress Resistance of Salmonella enterica Serovar Typhimurium, Yersinia enterocolitica 4/O:3, Listeria monocytogenes 1/2a, and Escherichia coli O:26.

The formation of robust resting cysts enables Acanthamoeba to resist harsh environmental conditions. This study investigated to what extent these cysts are resistant to physical and chemical stresses as applied in food industry cleaning and disinfection procedures. Moreover, it was assessed whether certain intracystic meat-borne bacterial pathogens are more stress resistant than free-living bacterial monocultures and if intracystic passage and subsequent association with trophozoites induces cross-tolerance toward other stressors. Several physical and chemical stressors (NaCl, H2O2, benzalkonium chloride, 55°C, heating until boiling, ethanol, dishwashing detergent, and sodium hypochlorite) frequently used in domestic and industrial food-related environments were tested against (i) Acanthamoeba castellanii cysts, (ii) single strains of bacterial monocultures, (iii) intracystic bacteria, and (iv) bacteria after intracystic passage (cyst-primed bacteria). Only heating until boiling and hypochlorite treatment were cysticidal. After boiling, no viable trophozoites could be recovered from the cysts, and hypochlorite treatment caused a 1.34- to 4.72-log10 cells/ml reduction in cyst viability. All treatments were effective in reducing or even eliminating the tested bacterial monocultures, whereas bacteria residing inside cysts were more tolerant toward these stressors. All cyst-primed bacteria exhibited an increased tolerance toward subsequent H2O2 (>92% decrease in median log10 CFU/ml reduction) and 70% ethanol (>99% decrease) treatments. Moreover, intracystic passage significantly increased the survival of Yersinia enterocolitica (74% decrease in median log10 reduction), Escherichia coli (58%), and Salmonella enterica (48%) after NaCl treatment and of E. coli (96%), S. enterica (99%), and Listeria monocytogenes (99%) after sodium hypochlorite treatment compared with that of nonprimed bacteria.IMPORTANCE The results from this study demonstrated that both viable and nonviable amoebal cysts can protect internalized bacteria against stressful conditions. Moreover, cyst passage can induce cross-tolerance in bacteria, increasing their survival when exposed to selected stressors. These findings underscore the potential importance of free-living amoebae in food-related environments and their impact on the persistence of meat-borne bacterial pathogens.

Role of the LuxR family transcriptional regulator Lpg2524 in the survival of Legionella pneumophila in water.

The water-borne Gram-negative bacterium Legionella pneumophila (Lp) is the causative agent of Legionnaires' disease. Lp is typically transmitted to humans from water systems, where it grows inside amoebae. Survival of Lp in water is central to its transmission to humans. A transcriptomic study previously identified many genes induced by Lp in water. One such gene, lpg2524, encodes a putative LuxR family transcriptional regulator. It was hypothesized that this gene could be involved in the survival of Lp in water. Deletion of lpg2524 does not affect the growth of Lp in rich medium, in the amoeba Acanthamoeba castellanii, or in human macrophage-like THP-1 cells, showing that Lpg2524 is not required for growth in vitro and in vivo. Nevertheless, deletion of lpg2524 results in a faster colony-forming unit (CFU) reduction in an artificial freshwater medium, Fraquil, indicating that Lpg2524 is important for Lp to survive in water. Overexpression of Lpg2524 also results in a survival defect, suggesting that a precise level of this transcriptional regulator is essential for its function. However, our result shows that Lpg2524 is dispensable for survival in water when Lp is at a high cell density (109 CFU/mL), suggesting that its regulon is regulated by another regulator activated at high cell density.

Protochlamydia phocaeensis sp. nov., a new Chlamydiales species with host dependent replication cycle.

Chlamydiae are pathogenic and symbiotic bacteria, which form an important part of amoeba-associated microorganisms. In this paper, we report the isolation, developmental cycle and genome analysis of Protochlamydia phocaeensis sp. nov., an obligate intracellular parasite with a large host spectrum, able to infect Acanthamoeba castellanii, Acanthamoeba polyphaga, and Vermamoeba vermiformis. The genome size is 3,424,182 bp with a GC content of 42%. This bacterium displayed a particular developmental cycle depending on the infected host. The P. phocaeensis showed typical inclusion vacuoles in A. castellanii, while these were absent in V. vermiformis. Since "Chlamydiae-amoebae" interactions are supposed to depend on the chlamydial species, our findings speculate that variations in the developmental cycle of certain Chlamydiae are also host dependent.