Free-living freshwater amoebae differ in their susceptibility to the pathogenic bacterium Legionella pneumophila

Legionella pneumophila is known as a facultative intracellular parasite of free-living soil and freshwater amoebae, of which several species have been shown to support the growth of the pathogenic bacteria. We report for the first time the behaviour of two strains (c2c and Z503) of the amoeba Willaertia magna towards different strains of L. pneumophila serogroup 1 and compared it with Acanthamoeba castellanii and Hartmannella vermiformis , known to be L. pneumophila permissive. In contrast to the results seen with other amoebae, W. magna c2c inhibited the growth of one strain of Legionella ( L. pneumophila , Paris), but not of others belonging to the same serogroup ( L. pneumophila , Philadelphia and L. pneumophila , Lens). Also, the different L. pneumophila inhibited cell growth and induced cell death in A. castellanii, H. vermiformis and W. magna Z503 within 3–4 days while W. magna c2c strain remained unaffected even up to 7 days. Electron microscopy demonstrated that the formation of numerous replicative phagosomes observed within Acanthamoeba and Hartmannella is rarely seen in W. magna c2c cocultured with L. pneumophila . Moreover, the morphological differences were observed between L. pneumophila cultured either with Willaertia or other amoebae. These observations show that amoebae are not all equally permissive to L. pneumophila and highlight W. magna c2c as particularly resistant towards some strains of this bacterium.     

Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellanii.

Survival and distribution of legionellae in the environment are assumed to be associated with their multiplication in amoebae, whereas the ability to multiply in macrophages is usually regarded to correspond to pathogenicity. Since most investigations focused on Legionella pneumophila serogroup 1, we examined the intracellular multiplication of different Legionella species in Mono Mac 6 cells, which express phenotypic and functional features of mature monocytes, and in Acanthamoeba castellanii, an environmental host of Legionella spp. According to the bacterial doubling time in Mono Mac 6 cells and in A. castellanii, seven clusters of legionellae could be defined which could be split further with regard to finer differences. L. longbeachae serogroup 1, L. jordanis, and L. anisa were not able to multiply in either A. castellanii or Mono Mac 6 cells and are members of the first cluster. L. dumoffi did not multiply in Mono Mac 6 cells but showed a delayed multiplication in A. castellanii 72 h after infection and is the only member of the second cluster. L. steigerwaltii, L. gormanii, L. pneumophila serogroup 6 ATCC 33215, L. bozemanii, and L. micdadei showed a stable bacterial count in Mono Mac 6 cells after infection but a decreasing count in amoebae. They can be regarded as members of the third cluster. As the only member of the fourth cluster, L. oakridgensis was able to multiply slight in Mono Mac 6 cells but was killed within amoebae. A strain of L. pneumophila serogroup 1 Philadelphia obtained after 30 passages on SMH agar and a strain of L. pneumophila serogroup 1 Philadelphia obtained after intraperitoneal growth in guinea pigs are members of the fifth cluster, which showed multiplication in Mono Mac 6 cells but a decrease of bacterial counts in A. castellanii. The sixth cluster is characterized by intracellular multiplication in both host cell systems and consists of several strains of L. pneumophila serogroup 1 Philadelphia, a strain of L. pneumophila serogroup 2, and a fresh clinical isolate of L. pneumophila serogroup 6. Members of the seventh cluster are a strain of agar-adapted L. pneumophila serogroup 1 Bellingham and a strain of L. pneumophila serogroup 1 Bellingham which was passaged fewer than three times on BCYE alpha agar after inoculation and intraperitoneal growth in guinea pigs. In comparison to members of the sixth cluster, both strains showed a slightly enhanced multiplication in Mono Mac 6 cells but a reduced multiplication in amoebae. From our investigations, we could demonstrate a correlation between prevalence of a given Legionella species and their intracellular multiplication in Mono Mac 6 cells. Multiplication of members of the genus Legionella in A. castellanii seems to be dependent on mechanisms different from those in monocytes.     

Influence of Acanthamoeba castellanii on intracellular growth of different Legionella species in human monocytes

Previous studies using a murine model of coinhalation of Legionella pneumophila and Hartmannella vermiformis have shown a significantly enhanced intrapulmonary growth of L. pneumophila in comparison to inhalation of legionellae alone (J. Brieland, M. McClain, L. Heath, C. Chrisp, G. Huffnagle, M. LeGendre, M. Hurley, J. Fantone, and C. Engleberg, Infect. Immun. 64:2449-2456, 1996). In this study, we introduce an in vitro coculture model of legionellae, Mono Mac 6 cells (MM6) and Acanthamoeba castellanii, using a cell culture chamber system which separates both cell types by a microporous polycarbonate membrane impervious to bacteria, amoebae, and human cells. Whereas L. pneumophila has shown a maximal 4-log-unit multiplication within MM6, which could not be further increased by coculture with Acanthamoeba castellanii, significantly enhanced replication of L. gormanii, L. micdadei, L. steigerwaltii, L. longbeachae, and L. dumoffii was seen after coculture with amoebae. This effect was seen only with uninfected amoebae, not with Legionella-infected amoebae. The supporting effect for intracellular multiplication in MM6 could be reproduced in part by addition of a cell-free coculture supernatant obtained from a coincubation experiment with uninfected A. castellanii and Legionella-infected MM6, suggesting that amoeba-derived effector molecules are involved in this phenomenon. This coculture model allows investigations of molecular and biochemical mechanisms which are responsible for the enhancement of intracellular multiplication of legionellae in monocytic cells after interaction with amoebae.     

Replication of Legionella pneumophila in biofilms of water distribution pipes

Biofilms similar to those present in water distribution pipes of anthropogenic aquatic systems were simulated in a rotating annular reactor using a non-Legionella community consisting of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve and Pseudomonas aeruginosa. The impact of this community and Acanthamoeba castellanii on the replication of Legionella pneumophila was investigated. Despite the presence of 10(7) non-Legionella bacteria, culture and real-time polymerase chain reaction (PCR) results clearly showed that biofilm-associated Legionella bacteria only increased after intracellular replication in A. castellanii. Fluorescent in situ hybridization (FISH) staining of biofilm samples revealed that 48 h after addition of amoebae to the reactor, the amoeba population was lysing and replicated Legionella bacteria were released into the bulk water. This study demonstrated that amoebae like A. castellanii can play a crucial role in the increase and spread of L. pneumophila in anthropogenic aquatic systems and thus in the occurrence of Legionnaires' disease.     

Temperature-dependent parasitic relationship between Legionella pneumophila and a free-living amoeba (Acanthamoeba castellanii)

We analyzed the effects of temperature on the interaction of Legionella pneumophila with Acanthamoeba castellanii. At <20 degrees C, overexpression of type 1 metacaspase, a stimulator of A. castellanii encystation, was associated with a reduced number of bacteria within amoeba. At low temperatures, A. castellanii seems to eliminate L. pneumophila by encystation and digestion.     

Long-Term Survival of Legionella pneumophila in the Viable But Nonculturable State After Monochloramine Treatment

Legionella pneumophila, a facultative intracellular human pathogen, can persist for long periods in natural and artificial aquatic environments. Eradication of this bacterium from plumbing systems is often difficult. We tested L. pneumophila survival after monochloramine treatment. Survival was monitored using the BacLight Bacterial Viability Kit (Molecular Probes), ChemChrome V6 Kit (Chemunex), quantitative polymerase chain reaction and culturability on buffered charcoal-yeast extract agar. In nonculturable samples, regain of culturability was obtained after addition of the amoeba Acanthamoeba castellanii, and esterase activity and membrane integrity were observed after >4 months after treatment. These results demonstrate for the first time that L. pneumophila could persist for long periods in biofilms into the viable but nonculturable (VBNC) state. Monitoring L. pneumophila in water networks is generally done by enumeration on standard solid medium. This method does not take into account VBNC bacteria. VBNC L. pneumophila could persist for long periods and should be resuscitated by amoeba. These cells constitute potential sources of contamination and should be taken into account in monitoring water networks.     

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii.

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.     

Development of a real-time PCR assay for quantification of Acanthamoeba trophozoites and cysts

Free-living amoebae have been found to be a reservoir for various pathogenic bacteria in aquatic environments. For example, the Acanthamoeba genus renders possible the intracellular multiplication of Legionella pneumophila, which is responsible for legionellosis. It consequently matters to quantify Acanthamoeba cells and thereby enhance our assessment of the risk of contamination. The classical microbiological method of quantification relies on amoebae growth and most probable number calculation. We have developed a real-time PCR assay based on a TaqMan probe that hybridizes onto 18S rDNA. This probe is specific to the Acanthamoeba genus. The assay was successful with both the trophozoite and the cyst forms of Acanthamoeba. Highly sensitive, it proved to permit detection of fewer than 10 cells, even those that are not easily cultivable, such as the cyst forms.     

Lgt: a family of cytotoxic glucosyltransferases produced by Legionella pneumophila

Legionella pneumophila is a facultative intracellular pathogen responsible for severe lung disease in humans, known as legionellosis or Legionnaires' disease. Previously, we reported on the approximately 60-kDa glucosyltransferase (Lgt1) from Legionella pneumophila, which modified eukaryotic elongation factor 1A. In the present study, using L. pneumophila Philadelphia-1, Lens, Paris, and Corby genome databases, we identified several genes coding for proteins with considerable sequence homology to Lgt1. These new enzymes form three subfamilies, termed Lgt1 to -3, glucosylate mammalian elongation factor eEF1A at serine-53, inhibit its activity, and subsequently kill target eukaryotic cells. Expression studies on L. pneumophila grown in broth medium or in Acanthamoeba castellanii revealed that production of Lgt1 was maximal at stationary phase of broth culture or during the late phase of Legionella-host cell interaction, respectively. In contrast, synthesis of Lgt3 peaked during the lag phase of liquid culture and at early steps of bacterium-amoeba interaction. Thus, the data indicate that members of the L. pneumophila glucosyltransferase family are differentially regulated, affect protein synthesis of host cells, and represent potential virulence factors of Legionella.     

Receptor-mediated uptake of Legionella pneumophila by Acanthamoeba castellanii and Naegleria lovaniensis

AIMS: Investigation of the attachment and uptake of Legionella pneumophila by Acanthamoeba castellanii and Naegleria lovaniensis, as these are two critical steps in the subsequent bacterial survival in both amoeba hosts. METHODS AND RESULTS: Initially, the mode of Legionella uptake was examined using inhibitors of microfilament-dependent and receptor-mediated uptake phagocytosis. Secondly, the minimum saccharide structure to interfere with L. pneumophila uptake was determined by means of selected saccharides. Bacterial attachment and uptake by each of the amoeba species occurred through a receptor-mediated endocytosis, which required de novo synthesis of host proteins. Legionella pneumophila showed a high affinity to the alpha1-3D-mannobiose domain of the mannose-binding receptor located on A. castellanii. In contrast, L. pneumophila bacteria had a high affinity for the GalNAcbeta1-4Gal domain of the N-acetyl-D-galactosamine receptor of N. lovaniensis. CONCLUSIONS: Our data pointed to a remarkable adaptation of L. pneumophila to invade different amoeba hosts, as the uptake by both amoeba species is mediated by two different receptor families. SIGNIFICANCE AND IMPACT OF THE STUDY: The fact that L. pneumophila is taken up by two different amoeba species using different receptor families adds further complexity to the host-parasite interaction process, as 14 amoeba species are known to be appropriate Legionella hosts.     

The Legionella pneumophila response regulator LqsR promotes host cell interactions as an element of the virulence regulatory network controlled by RpoS and LetA

Legionella pneumophila is an opportunistic human pathogen that replicates within environmental amoebae including Acanthamoeba castellanii and Dictyostelium discoideum. The Icm/Dot type IV secretion system promotes phagocytosis and intracellular replication of L. pneumophila in an endoplasmic reticulum-derived 'Legionella-containing vacuole' (LCV). L. pneumophila adopts a biphasic life cycle consisting of a replicative growth phase and a transmissive (stationary) phase, the latter of which is characterized by the preferential expression of genes required for motility and virulence. A bioinformatic analysis of the L. pneumophila genome revealed a gene cluster homologous to the Vibrio cholerae cqsAS genes, encoding a putative quorum sensing autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a novel response regulator (lqsR). We report here that an L. pneumophila lqsR deletion mutant grew in broth with the same rate as wild-type bacteria, but entered the replicative growth phase earlier. Overexpression of lqsR led to an elongated morphology of the bacteria. The lqsR mutant strain was found to be more salt-resistant and impaired for intracellular growth in A. castellanii, D. discoideum and macrophages, formation of the ER-derived LCV and toxicity. Moreover, L. pneumophila lacking LqsR, as well as strains lacking the stationary sigma factor RpoS or the two-component response regulator LetA, were phagocytosed less efficiently by A. castellanii, D. discoideum or macrophages. The expression of lqsR was dependent on RpoS and, to a lesser extent, also on LetA. DNA microarray experiments revealed that lqsR regulates the expression of genes involved in virulence, motility and cell division, consistent with a role for LqsR in the transition from the replicative to the transmissive (virulent) phase. Our findings indicate that LqsR is a novel pleiotropic regulator involved in RpoS- and LetA-controlled interactions of L. pneumophila with phagocytes.     

Viability of Listeria monocytogenes in co-culture with Acanthamoeba spp.

Listeria monocytogenes is a human pathogen, ubiquitous in the environment, and can grow and survive under a wide range of environmental conditions. It contaminates foods via raw materials or food-processing environments. However, the current knowledge of its ecology and, in particular, the mode of environmental survival and transmission of this intracellular pathogen remains limited. Research has shown that several intracellular pathogens are able to survive or replicate within free-living amoebae. To examine the viability of L. monocytogenes in interaction with Acanthamoeba spp., bacteria were co-cultured with three freshly isolated amoebae, namely Acanthamoeba polyphaga, Acanthamoeba castellanii and Acanthamoeba lenticulata . The survival of bacteria and amoebae was determined using culture techniques and microscopy. Under the experimental conditions used, all amoebae were able to eliminate bacteria irrespective of the hly gene. Bacteria did not survive or replicate within amoeba cells. However, extra-amoebic bacteria grew saprophytically on materials released from amoebae, which may play an important role in the survival of bacteria under extreme environmental conditions.     

Immunolocalization of the Mip protein of intracellularly and extracellularly grown Legionella pneumophila

The macrophage infectivity potentiator (Mip) protein is an important factor in the optimal intracellular survival of Legionella pneumophila in protozoa and human cell lines. In this study we have localized the Mip protein in Legionella grown on buffered charcoal yeast extract (BCYE) agar as well as in Legionella which were ingested by Acanthamoeba castellanii. Immunogold techniques have shown that Mip is exposed on the cell surface of extracellularly grown bacteria. In A. castellanii infected with Legionella the Mip protein was also detected on host membranes which exhibited a multilamellar structure. The morphology of these structures is similar to that of respirable vesicles of amoebas by which live legionellas may be transmitted to humans. It can be assumed that the accumulation of Mip protein in the multilamellar host membranes increases the infection potential.     

Balamuthia mandrillaris, free-living ameba and opportunistic agent of encephalitis, is a potential host for Legionella pneumophila bacteria

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as Acanthamoeba and Hartmannella, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease, Legionella pneumophila. Infection of amebas by L. pneumophila enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of B. mandrillaris by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host L. pneumophila bacteria. Our experiments showed that L. pneumophila could initiate uptake by B. mandrillaris and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a dotA mutant, known to be unable to propagate in Acanthamoeba castellanii, also did not replicate within B. mandrillaris. Approaching completion of the intracellular cycle, L. pneumophila wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a L. pneumophila-infected B. mandrillaris ameba resembled those described for other amebas hosting Legionella bacteria. We hence speculate that B. mandrillaris might serve as a host for bacteria in its natural environment.     

Studies on the uptake and intracellular replication of Legionella pneumophila in protozoa and in macrophage-like cells

Abstract Legionella pneumophila strains isolated from different sources were tested for their host range in the protists Acanthamoeba castellanii, Hartmannella vermiformis and Entamoeba histolytica . It has been shown that A. castellanii and H. vermiformis but not E. histolytica support the intracellular replication of L. pneumophila . Furthermore it could be demonstrated that in vivo virulence in the guinea pig and the intracellular growth in U937 cells coincides with the capability to replicate intracellularly in A. castellanii at 37°C. The infectivity of L. pneumophila that had sustained a 48 hours nutrient deprivation was not significantly different from that of legionellae grown to log-phase on BCYE plates. In contrast the nutrient limitation on A. castellanii increased the amount of intracellular legionellae at the beginning of infection. An initial opsonin independent attachement stage of legionellae to U937 cells was demonstrated by scanning electron microscopy. In contrast, L. pneumophila's capability of stable or long term attachmennt to A. castellanii was shown to be inefficient.