Ultrastructural, Cytochemical, and Comparative Genomic Evidence of Peroxisomes in Three Genera of Pathogenic Free-Living Amoebae, Including the First Morphological Data for the Presence of This Organelle in Heteroloboseans

Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins—which are essential for peroxisome biogenesis, proliferation, and protein import—are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.

Isolation and identification of Acanthamoeba genotypes and Naegleria spp. from the water samples of public swimming pools in Qazvin, Iran

Free-living amoeba (FLA), including Acanthamoeba and Naegleria are facultative parasites in humans. The amoeba have widespread distribution in various water sources. The aim of this study was isolation and molecular identification of Acanthamoeba and Naegleria isolated from swimming pools and also hot and cold tub waters in Qazvin province. The samples (166 water samples) were cultured to isolate and identify positive specimens. PCR (polymerase chain reaction) amplification, sequencing and phylogenetic analysis were conducted to confirm the isolated species and genotypes of amoeba. According to morphological characterizations, 18.6% of specimens were identified as FLA, which in 71% were Acanthamoeba by PCR method. Molecular analysis revealed that 36.3%, 18.1% and 4.5% of Acanthamoeba specimens were identified as T3, T4 and T11 Acanthamoeba genotypes, respectively. Protacanthamoeba bohemica (27.2%) and Acanthamoeba sp. (4.5%) were found among the specimens. The results of osmo-tolerance and thermo-tolerance assays demonstrated that 50% of T3 and 25% of T4 genotypes of Acanthamoeba were highly pathogenic parasites. The molecular approach showed the presence of Naegleria lovaniensis (9%) in hot tub water of swimming pools. This study demonstrated that the swimming pools and hot tub water in Qazvin province were contaminated with Acanthamoeba and Naegleria species.

Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein ofL. pneumophilapromotes intracellular infection ofAcanthamoeba castellaniiandHartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated byL. pneumophilaCas2 appeared to be distinct from this function, becausecas2mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the othercasgenes were not impaired in infection ability. We now report that the Cas2 protein ofL. pneumophilahas both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain ofL. pneumophilathat naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, acas2mutant was impaired for infection ofWillaertia magnabut notNaegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts.

The novel Legionella pneumophila type II secretion substrate NttC contributes to infection of amoebae Hartmannella vermiformis and Willaertia magna

The type II protein secretion (T2S) system of Legionella pneumophila secretes over 25 proteins, including novel proteins that have no similarity to proteins of known function. T2S is also critical for the ability of L. pneumophila to grow within its natural amoebal hosts, including Acanthamoeba castellanii, Hartmannella vermiformis and Naegleria lovaniensis. Thus, T2S has an important role in the natural history of legionnaires’ disease. Our previous work demonstrated that the novel T2S substrate NttA promotes intracellular infection of A. castellanii, whereas the secreted RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all promote infection of H. vermiformis and N. lovaniensis. In this study, we determined that another novel T2S substrate that is specific to Legionella, designated NttC, is unique in being required for intracellular infection of H. vermiformis but not for infection of N. lovaniensis or A. castellanii. Expanding our repertoire of amoebal hosts, we determined that Willaertia magna is susceptible to infection by L. pneumophila strains 130b, Philadelphia-1 and Paris. Furthermore, T2S and, more specifically, NttA, NttC and PlaC were required for infection of W. magna. Taken together, these data demonstrate that the T2S system of L. pneumophila is critical for infection of at least four types of aquatic amoebae and that the importance of the individual T2S substrates varies in a host cell-specific fashion. Finally, it is now clear that novel T2S-dependent proteins that are specific to the genus Legionella are particularly important for L. pneumophila infection of key, environmental hosts.

Multiple Legionella pneumophila Type II Secretion Substrates, Including a Novel Protein, Contribute to Differential Infection of the Amoebae Acanthamoeba castellanii, Hartmannella vermiformis, and Naegleria lovaniensis

ABSTRACTType II protein secretion (T2S) byLegionella pneumophilais required for intracellular infection of host cells, including macrophages and the amoebaeAcanthamoeba castellaniiandHartmannella vermiformis. Previous proteomic analysis revealed that T2S byL. pneumophila130b mediates the export of >25 proteins, including several that appeared to be novel. Following confirmation that they are unlike known proteins, T2S substrates NttA, NttB, and LegP were targeted for mutation.nttAmutants were impaired for intracellular multiplication inA. castellaniibut notH. vermiformisor macrophages, suggesting that novel exoproteins which are specific toLegionellaare especially important for infection. Because the importance of NttA was host cell dependent, we examined a panel of T2S substrate mutants that had not been tested before in more than one amoeba. As a result, RNase SrnA, acyltransferase PlaC, and metalloprotease ProA all proved to be required for optimal intracellular multiplication inH. vermiformisbut notA. castellanii. Further examination of anlspFmutant lacking the T2S apparatus documented that T2S is also critical for infection of the amoebaNaegleria lovaniensis. Mutants lacking SrnA, PlaC, or ProA, but not those deficient for NttA, were defective inN. lovaniensis. Based upon analysis of a double mutant lacking PlaC and ProA, the role of ProA inH. vermiformiswas connected to its ability to activate PlaC, whereas inN. lovaniensis, ProA appeared to have multiple functions. Together, these data document that the T2S system exports multiple effectors, including a novel one, which contribute in different ways to the broad host range ofL. pneumophila.