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PubMed2004

Cytochrome c maturation proteins are critical for in vivo growth of Legionella pneumophila.

Legionella pneumophila, an intracellular parasite of macrophages and protozoa, requires iron for extra- and intracellular growth. In a new screen of a mutant library of L. pneumophila for strains defective for growth on agar media lacking supplemental iron, seven mutants were obtained. All of the mutants had a disruption in the cytochrome c maturation (ccm) locus; two had insertions in ccmB, two in ccmC, and three in ccmF. The ccm mutants were unable to multiply within macrophage-like cells (i.e., U937 and THP-1 cells) and Hartmannella vermiformis amoebae. A competition assay in A/J mice revealed that ccm mutants are severely defective for growth within the lung. Taken together, these data confirm that ccm and cytochrome c maturation proteins are required for L. pneumophila growth in low iron, intracellular infection, and virulence.

Animals↗
PubMed1999

The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila.

Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.

Animals↗
PubMed2000

Impact of silver and copper on the survival of amoebae and ciliated protozoa in vitro.

The efficacy of 1:10 silver/copper combinations for inactivation of Hartmannella vermiformis amoebas and the ciliated protozoan Tetrahymena pyriformis in vitro was studied. Tetrahymena and Hartmannella/isolate 19 were inactivated for 2 log steps by 100 + 1000 micrograms/l Ag + Cu. Hartmannella/isolate 21 was more resistant. 500 + 5000 micrograms/l produced only a 0.6 log reduction. The investigations clearly showed that levels within the limit of the German drinking water regulation (10 + 100 micrograms/l Ag + Cu) could not inactivate these protozoas in vitro.

Amebiasis↗
PubMed2001

Role of biofilms in the survival of Legionella pneumophila in a model potable-water system.

Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6.7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist.

Animals↗
PubMed2002

Legionella pneumophila genes that encode lipase and phospholipase C activities.

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular parasite of aquatic protozoans and human macrophages. The type II protein secretion system of the Gram-negative Legionella organism promotes intracellular infection. A lipase activity and a p-nitrophenylphosphorylcholine (pNPPC) hydrolytic activity are two of the factors that are diminished in L. pneumophila type II secretion mutants. The Legionella lipase activity was found to include free fatty acid release from di- and triacylglycerol substrates, in addition to the previously reported cleavage of monoacylglycerol. In a number of other bacterial systems, the release of p-nitrophenol from pNPPC is due to a phospholipase C. In an attempt to identify exoproteins that potentiate intracellular infection, three genes were identified and mutated in L. pneumophila strain 130b that were predicted to encode either a secreted lipase or a phospholipase C. The first two genes, which were designated lipA and lipB, encoded proteins containing the lipase consensus sequence [LIV]-X-[LIVFY]-[LIVMST]-G-[HYWV]-S-X-G-[GSTAC]. Mutations in lipA in particular reduced supernatant activity against mono- and triacylglycerols. However, loss of lipA and/or lipB did not impair the ability of L. pneumophila to infect Hartmannella amoebae or U937 cell macrophages. The third L. pneumophila gene, which was denoted plcA, encoded a protein that was highly homologous with a phospholipase C from Pseudomonas fluorescens. Inactivation of plcA diminished secreted pNPPC hydrolase activity but did not influence Legionella infection of host cells. Taken together, these data indicate that L. pneumophila has multiple lipases and possibly several phospholipase C enzymes but that LipA, LipB and PlcA are not among those exoproteins required for optimal intracellular infection.

Amino Acid Sequence↗
PubMed1977

Excystment of axenically prepared cysts of Hartmanella culbertsoni.

Axenically prepared cysts of Hartmannella culbertsoni readily excysted in the presence of heat stable factors prepared from Escherichia coli, Klebsiella aerogenes, Staphylococcus aureus, Sarcina lutea, Bacillus subtilis, Bacillus megaterium and several fungi. Peptone, proteose peptone, tryptone or amino acids also promoted excystment. Crowding of the cysts and dilution of bacterial extracts adversely affected the excystment. Continual presence of the factors in the medium was essential for excystment.

Amino Acids↗
PubMed1995

Non-culturable Legionella pneumophila associated with Acanthamoeba castellanii: detection of the bacterium using DNA amplification and hybridization.

The intracellular localization of Legionella pneumophila serogroup 1 within Acanthamoeba castellanii rendered the bacteria non-culturable on supplemented BCYE agar. DNA amplification, using two 19-mer primers, and hybridization using a 25-mer oligonucleotide probe, permitted detection of Leg. pneumophila in approximately 81% (29/36) of samples where the bacteria could not be detected using culture. A combination of co-cultivation of samples with Leg. pneumophila-naive A. polyphaga or Hartmannella vermiformis, incubation in a defined liquid medium or use of catalase indicated that approximately 31% (9/29) of the samples contained Leg. pneumophila which were viable although not culturable.

Acanthamoeba↗
PubMed1998

Heterogeneity in the attachment and uptake mechanisms of the Legionnaires' disease bacterium, Legionella pneumophila, by protozoan hosts.

Invasion and intracellular replication of Legionella pneumophila within protozoa in the environment plays a major role in the transmission of Legionnaires' disease. Intracellular replication of L. pneumophila within protozoa occurs in a rough endoplasmic reticulum (RER)-surrounded phagosome (Y. Abu Kwaik, Appl. Environ. Microbiol. 62:2022-2028, 1996). Since the subsequent fate of many intracellular pathogens is determined by the route of entry, we compared the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoa Hartmannella vermiformis and Acanthamoeba polyphaga. Our data provide biochemical and genetic evidence that the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoan hosts are, in part, different. First, uptake of L. pneumophila by H. vermiformis is completely blocked by the monovalent sugars galactose and N-acetyl-D-galactosamine, but these sugars partially blocked A. polyphaga. Second, attachment of L. pneumophila to H. vermiformis is associated with a time-dependent and reversible tyrosine dephosphorylation of multiple host proteins. In contrast, only a slight dephosphorylation of a 170-kDa protein of A. polyphaga is detected upon infection. Third, synthesis of H. vermiformis proteins but not of A. polyphaga proteins is required for uptake of L. pneumophila. Fourth, we have identified L. pneumophila mutants that are severely defective in attachment to A. polyphaga but which exhibit minor reductions in attachment to H. vermiformis and, thus, provide a genetic basis for the difference in mechanisms of attachment to both protozoa. The data indicate a remarkable adaptation of L. pneumophila to attach and invade different protozoan hosts by different mechanisms, yet invasion is followed by a remarkably similar intracellular replication within a RER-surrounded phagosome and subsequent killing of the host cell.

Acanthamoeba↗
PubMed2003

Gamma-proteobacteria Aquicella lusitana gen. nov., sp. nov., and Aquicella siphonis sp. nov. infect protozoa and require activated charcoal for growth in laboratory media.

Several isolates, belonging to two new species of the same novel genus of gamma-proteobacteria, were recovered from drilled well (borehole) and spa water at São Gemil in central Portugal. These organisms are phylogenetically most closely related to the strictly intracellular uncultured species of the genus Rickettsiella, which cause disease in arthropods, and to the facultatively intracellular species of the genus Legionella, some of which cause Legionnaires' disease and Pontiac fever. The São Gemil strains grew only on media containing charcoal, as is also true of the species of the genus LEGIONELLA: Unlike the vast majority of Legionella isolates, the new isolates did not require L-cysteine or ferric pyrophosphate for growth but like the legionellae had an absolute requirement for alpha-ketoglutarate. Strains SGT-39(T) and SGT-56 grew consistently between 30 and 43 degrees C, while strains SGT-108(T) and SGT-109 grew between 30 and 40 degrees C. The pH ranges for growth of these organisms were surprisingly narrow: strains SGT-39(T) and SGT-56 grew between pH 6.3 and 7.3, while strains SGT-108(T) and SGT-109 grew between pH 6.3 and 7.0. Both organisms proliferated in the amoeba Hartmannella vermiformis but did not grow in U937 human cells. Based on 16S rRNA gene sequence analysis and physiological, biochemical, and chemical analysis we describe two new species of one novel genus; one species is represented by strain SGT-39(T), for which we propose the name Aquicella lusitana, while strain SGT-108(T) represents a second species of the same genus, for which we propose the name Aquicella siphonis.

Animals↗
PubMed1990

Virulence of a Legionella anisa strain associated with Pontiac fever: an evaluation using protozoan, cell culture, and guinea pig models.

Legionella anisa and the amoeba Hartmannella vermiformis were isolated from an indoor fountain implicated as the infectious reservoir in an outbreak of Pontiac fever. We evaluated the ability of this strain of L. anisa to multiply in cultures of an amoeba (H. vermiformis), a ciliated protozoan (Tetrahymena pyriformis), and human mononuclear cells and to infect guinea pigs. These bacteria multiplied in the culture of H. vermiformis but failed to infect guinea pigs or the cultures of T. pyriformis and human mononuclear cells. These findings suggest that some Legionella spp. may multiply only in specific protozoan hosts. The inability of this strain of L. anisa to multiply in human phagocytic cells may be related to the development of Pontiac fever rather than pneumonic legionellosis in exposed individuals. Further studies are necessary to determine whether the ability of legionellae to infect certain host cells can be correlated to differences in human disease.

Animals↗
PubMed1991

Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells.

A cloned and axenically cultured strain of Hartmannella vermiformis was used as a model to study intracellular multiplication of Legionella pneumophila in amoebae. The growth of L. pneumophilia in both H. vermiformis and a human monocyte-like cell line (U937) was investigated with cytoskeletal and metabolic inhibitors. L. pneumophila replicated only intracellularly in these cellular models, and electron microscopy showed ultrastructural similarities in the initial phase of multiplication. Treatment of amoebae with an inhibitor of microfilament-dependent phagocytosis (cytochalasin D, 0.5 or 1.0 micrograms/ml) did not inhibit intracellular growth of L. pneumophila; however, intracellular multiplication was inhibited by treatment of U937 monocytes with the same concentrations of cytochalasin D. Methylamine (10 to 100 mM), an inhibitor of adsorptive pinocytosis, inhibited the replication of L. pneumophila in amoebae in a dose-dependent manner. All doses of methylamine tested (10 to 50 mM) inhibited growth of L. pneumophila in U937 monocytes. Cytochalasin D and methylamine had no effect on the multiplication of L. pneumophila in culture medium or on the viability of amoebae or U937 monocytes. Intracellular replication of L. pneumophila in H. vermiformis may be accomplished by a cytochalasin D-independent mechanism, such as adsorptive pinocytosis. In contrast, both cytochalasin D- and methylamine-sensitive mechanisms may be essential for the intracellular multiplication of L. pneumophila in U937 monocytes.

Analysis of Variance↗
PubMed1995

Characterization of a Legionella micdadei mip mutant.

The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human macrophages and freshwater protozoa. Southern hybridization and immunoblot analyses demonstrated that mip sequences were present and expressed within a panel of virulent L. micdadei strains. Using allelic exchange mutagenesis, we then constructed an L. micdadei strain that completely and specifically lacked Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip- organism displayed a 24-fold reduction in survivability immediately after its entry into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei.

Animals↗
PubMed2002

Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection.

In order to determine the role of ferrous iron transport in Legionella pathogenesis, we identified and mutated the feoB gene in virulent Legionella pneumophila strain 130b. As it is in Escherichia coli, the L. pneumophila feoB gene was contained within a putative feoAB operon. L. pneumophila feoB insertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss of L. pneumophila FeoB. However, the L. pneumophila feoB mutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition of feoB in trans. In unsupplemented buffered yeast extract broth, the feoB mutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2'-dipyridyl. The feoB mutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of the feoB mutant was noted within iron-depleted Hartmannella vermiformis amoebae and human U937 cell macrophages. The reduced intracellular infectivity of the feoB mutant was complemented by the introduction of a plasmid containing feoAB. The L. pneumophila feoB gene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate that L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.

Animals↗
PubMed1987

Pathogenic and non-pathogenic Naegleria and Acanthamoeba spp.: a new autochthonous isolate from an Italian thermal area.

We performed an epidemiological survey of 17 thermal baths and the same number of mud-basins. This study aimed to ascertain the presence and incidence of small free-living amoebae, particularly species and/or strains of Naegleria and Acanthamoeba spp., occasional etiological agents of fatal meningoencephalitis and/or ocular infections in man. Over 51 samples of water and mud incubated at 37 degrees C and at 45 degrees C, 34 (66.7%) became positive at 37 degrees C and 33 (64.7%) at 45 degrees C. We isolated 7 (6%) strains of Naegleria spp., 6 (5.2%) of Acanthamoeba spp., 39 (33.6%) of Vahlkampfia spp., 28 (24.1%) of Hartmannella spp. and 36 (31.1%) strains of other species of free-living amoebae. 4 strains of Naegleria spp. and 6 of Acanthamoeba spp. proved pathogenic both in vivo, after experimental infection (meningoencephalitis) in the albino mouse, and in vitro, having previously contaminated monolayers of Vero cell line (cytopathic effect). Within the isolated pathogenic strains of Naegleria spp., a new strain of N. australiensis s.sp. italica was typified from an immunochemical point of view. This should be added to previous isolations reported by us.

Amoeba↗
PubMed1984

[Acanthamoebae, Naegleria and other free-living Amoebae in cooling and rinsing water of dental treatment units].

215 water samples were taken from 49 dental treatment units and investigated for the existence of free-living amoebae. In all water-carrying systems of the dental treatment units it was possible to verify the incidence of one or more amoeba species. In 8.2 per cent of the units Naegleria species was found and in 12.2 per cent Acanthamoeba species was present. Seven Naegleria and six Acanthamoeba strains (2 A. castellanii and 4 A. polyphaga) were isolated. From samples originating from 12 dental treatment units (DTU) another 42 amoeba strains were isolated which consisted of 14 different species within 9 classes. Among them Vannella mira (in 19 per cent of samples) and Hartmannella vermiformis (10.6 per cent) were found to be the most frequent species, followed by H. cantabrigensis (9.5 per cent), V. platypodia, Platyamoeba stenopodia and V. simplex (7.1 per cent each). In 10 per cent of samples monotrichous and bitrichous flagellates such as the Bodo species were found, whereas two samples contained ova, larvae and adult free-living nematodes. Among the isolated Naegleria strains no thermophilic strain was present. Consequently they belong to the N. gruberi complex. Among the Acanthamoebae five of the six strains were thermophilic. All strains were investigated for pathogenic properties by means of the mice inoculation test. Two strains proved pathogenic - it was possible to isolate them from the brain and lung of dead mice. Another two strains proved to have invasive properties because they were isolated from the brain of infected animals; however, they did not give rise to disease or death of the respective animals. Supplementary microbiological tests demonstrated the existence of bacteria and fungi in 84 per cent of dental treatment units. Pseudomonas spec. were detected in 75% of dental units, Serratia marcescens in 2% and fungi in nearly 3%. 58.3% of all water samples contained total germ counts of more than 100/ml.

Amebiasis↗
PubMed1982

Incidence of free-living amoebae in the nasal passages of local population in Zaria, Nigeria.

Following the observation of cases of primary amoebic meningoencephalitis (PAME) during the dusty harmattan period in Zaria, a survey was carried out in randomly selected local populations of Zaria, to find out the incidence of free-living amoebae in the nasal passages. The times of sampling were spaced so as to cover both the rainy (non-harmattan) abd dry (harmattan) seasons. In all 1250 individuals were sampled, and were grouped in the three age groups of above 18 years, between 4 and 18 years and below 4 years. The overall incidence was 4.2% (52 out of 1250). There was no marked difference in the three age groups studied. The incidence rate in males was 4.8% (30 of 630) and that in females was 3.5% (22 of 620). Nine different species of free-living amoebae were isolated. Six belonged to the Genus Hartmannella, two to the genus Naegleria, and one to the genus Schizopyrenus. Three species were found to be pathogenic for mice: H. culbertsoni, H. rhysodes and N. fowleri. It was observed from this study that a significant percentage of the Zaria population carry free-living amoebae in the nasal passages. The monthly incidence rate in population ranged from 1.8 to 3.1% during the rainy (non-harmattan) season whereas in the dry (harmattan) season it ranged from 4.2 to 7.9%. The highest incidence rate coincided with the peak of the dry (harmattan) season. The possible role of harmattan winds on the nasal carriage as well as the necessity to investigate fully the disease PAME in this environment is discussed.

Adolescent↗
PubMedDate not supplied

[Nuclear division in 15 species of free-living amoebae].

It was made possible to study the mode of nuclear division during all the stage of mitotic division of the free-living amoebae belonging to the families Schizopyrenidae and Hartmannellidae, when the massive number of them were fixed and stained. While the four phases of nuclear division vary in different genera, in the amoebae belonging to the same genus the mitotic division was similar. In the family Schizopyrenidae, the nucleus contains a central Feulgen-negative nucleolus which during mitosis form "polar masses". In the family Hartmannellidae, the resting nucleus has either a single Feulgen-negative nucleolus or several nucleoli. During mitosis the nucleolus or nucleoli disappear and a spindle with chromosomes arranged as an equatorial plate develops. The mitotic apparatus is an essential criterion in the differentiation of the genera Hartmannella and Naegleria.

Amoeba↗
PubMed1995

[Acanthamoeba, naturally intracellularly infected with Pseudomonas aeruginosa, after their isolation from a microbiologically contaminated drinking water system in a hospital].

The drinking water system of a new hospital building that was highly contaminated with bacteria before opening was investigated too for the prevalence of small free living amoebae. Germ counts resulted in > 100 CFU/ml in 100% of the cold water samples, that showed also growth of P. aeruginosa, whereas E. coli and coliforme bacteria could not be identified. The investigation of 37 water samples for protozoa revealed growth of small freeliving amoebae in 20 samples (54%) belonging to 10 species of the genus Acanthamoeba, Naegleria, Hartmannella, Echinamoeba among others. In addition 2 Ciliate- and 2 Microflagellate-species could be observed. While all Naegleria strains isolated belonged to the N. gruberi-complex two of 16 Acanthamoeba-isolates proved to be pathogenic for laboratory mice. From 7 watersamples positive with P. aeruginosa 5 Acanthamoeba- and 2 Echinamoeba strains could be isolated which revealed intracellular multiplication of P. aeruginosa. Because of their well known resistances against chlorine, the amoebae and their cysts are considered to be vectors for these intracellular bacteria. A complete sanitation of the incriminated drinking water system was accomplished by combined chemical and thermic disinfection measures.

Acanthamoeba↗