Replication of <Emphasis Type="Italic">Legionella pneumophila</Emphasis> in Floating Biofilms

Biofilms are a major source of human pathogenic Legionella pneumophila in aquatic systems. In this study, we investigated the capacity of L. pneumophila to colonize floating biofilms and the impact of Acanthamoeba castellanii on the replication of biofilm-associated Legionella. Biofilms were grown in Petri dishes and consisted of Aeromonas hydrophila, Escherichia coli, Flavobacterium breve, and Pseudomonas aeruginosa. Six hours following inoculation, Legionella were detected in floating biofilms in mean concentrations of 1.4 × 10⁴ cells/cm² (real-time polymerase chain reaction) and 8.3 × 10² CFU/cm² (culture). Two-way analysis of variance tests and fluorescent in situ hybridization clearly proved that increased biofilm-associated L. pneumophila concentrations were the result of intracellular replication in A. castellanii. Forty-eight hours after the introduction of A. castellanii in the Petri dishes, 90 ± 0.8% of the amoebae (infection rate) were completely filled with highly metabolic active L. pneumophila (mean infection intensity).     

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.     

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.     

Identification and functional characterization of K+ transporters encoded by Legionella pneumophila kup genes

Legionnaires' disease is an emerging, severe, pneumonia‐like illness caused by the Gram‐negative intracellular bacteria Legionella pneumophila, which are able to infect and replicate intracellularly in macrophages. Little is known regarding the mechanisms used by intracellular L. pneumophila for the acquisition of specific nutrients that are essential for bacterial replication. Here, we investigate three L. pneumophila genes with high similarity to the Escherichia coli K⁺ transporters. These three genes were expressed by L. pneumophila and have been designated kupA, kupB and kupC. Investigation using the L. pneumophila kup mutants revealed that kupA is involved in K⁺ acquisition during axenic growth. The kupA mutants replicated efficiently in rich axenic media, but poorly in a chemically defined medium. The kupA mutants were defective in the recruitment of polyubiquitinated proteins to the Legionella‐containing vacuole that is formed in macrophages and displayed an intracellular multiplication defect during the replication in Acanthamoeba castellanii and in mouse macrophages. We found that bafilomycin treatment of macrophages was able to rescue the growth defects of kupA mutants, but itdid not influence the replication of wild‐type bacteria. The defects identified in kupA mutants of L. pneumophila were complemented by the expression E. coli trkD/Kup gene in trans, a bona fide K⁺ transporter encoded by E. coli. Collectively, our data indicate that KupA is a functional K⁺ transporter expressed by L. pneumophila that facilitates the bacterial replication intracellularly and in nutrient‐limited conditions.     

Amino Acid Uptake and Metabolism of Legionella pneumophila Hosted by Acanthamoeba castellanii

Legionella pneumophila survives and replicates within a Legionella-containing vacuole (LCV) of amoebae and macrophages. Less is known about the carbon metabolism of the bacteria within the LCV. We have now analyzed the transfer and usage of amino acids from the natural host organism Acanthamoeba castellanii to Legionella pneumophila under in vivo (LCV) conditions. For this purpose, A. castellanii was ¹³C-labeled by incubation in buffer containing [U-¹³C₆]glucose. Subsequently, these ¹³C-prelabeled amoebae were infected with L. pneumophila wild type or some mutants defective in putative key enzymes or regulators of carbon metabolism. ¹³C-Isotopologue compositions of amino acids from bacterial and amoebal proteins were then determined by mass spectrometry. In a comparative approach, the profiles documented the efficient uptake of Acanthamoeba amino acids into the LCV and further into L. pneumophila where they served as precursors for bacterial protein biosynthesis. More specifically, A. castellanii synthesized from exogenous [U-¹³C₆]glucose unique isotopologue mixtures of several amino acids including Phe and Tyr, which were also observed in the same amino acids from LCV-grown L. pneumophila. Minor but significant differences were only detected in the isotopologue profiles of Ala, Asp, and Glu from the amoebal or bacterial protein fractions, respectively, indicating partial de novo synthesis of these amino acids by L. pneumophila. The similar isotopologue patterns in amino acids from L. pneumophila wild type and the mutants under study reflected the robustness of amino acid usage in the LCV of A. castellannii.     

Effect of Bacteria on Survival and Growth of Acanthamoeba castellanii

The growth and survival of Acanthamoeba castellanii in the presence of Gram-negative bacteria such as Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, and Stenotrophomonas maltophilia varied with the densities and species of bacteria. All species of bacteria suspended in a buffered saline at densities of 10⁵ to 10⁶/ml supported the growth and survival of 10⁶/ml trophozoites of Acanthamoeba castellanii in a buffered saline solution. At densities of bacteria to amoebae of 100:1 or greater, growth and survival of A. castellanii were suppressed, particularly by P. aeruginosa. In an enrichment medium, the rapid growth of most co-inoculated bacteria inhibited the growth and survival of the amoeba. Received: 12 September 1996 / Accepted: 20 September 1996     

Diverse Legionella‐Like Bacteria Associated with Testate Amoebae of the Genus Arcella (Arcellinida: Amoebozoa)

Diverse species of Legionella and Legionella‐like amoebal pathogens (LLAPs) have been identified as intracellular bacteria in many amoeboid protists. There are, however, other amoeboid groups such as testate amoeba for which we know little about their potential to host such bacteria. In this study, we assessed the occurrence and diversity of Legionella spp. in cultures and environmental isolates of freshwater arcellinid testate amoebae species, Arcella hemispherica, Arcella intermedia, and Arcella vulgaris, via 16S rRNA gene sequence analyses and fluorescent in situ hybridization (FISH). Analysis of the 16S rRNA gene sequences indicated that A. hemispherica, A. intermedia, and A. vulgaris host Legionella‐like bacteria with 94–98% identity to other Legionella spp. based on NCBI BLAST search. Phylogenetic analysis placed Legionella‐like Arcella‐associated bacteria (LLAB) in three different clusters within a tree containing all other members of Legionella and LLAPs. The intracellular localization of the Legionella within Arcella hosts was confirmed using FISH with a Legionella‐specific probe. This study demonstrates that the host range of Legionella and Legionella‐like bacteria in the Amoebozoa extends beyond members of “naked” amoebae species, with members of the testate amoebae potentially serving an ecological role in the dispersal, protection, and replication of Legionella spp. in natural environments.     

Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

Legionella is ubiquitous in many environments. At least 50 species and 70 serogroups of the Gram-negative bacterium have been identified. Of the 50 species, 20 are pathogenic, and Legionella pneumophila is responsible for the great majority (approximately 90%) of the Legionnaires'' disease cases that occur. Furthermore, of the 15 L. pneumophila serogroups identified, O1 alone causes more than 84% of the Legionnaires'' disease cases that occur worldwide. Rapid and reliable assays for the detection and identification of L. pneumophila in water, environmental, and clinical samples are in great demand. L. pneumophila bacteria are traditionally identified by their O antigens by immunological methods. We have recently developed an O serogroup-specific DNA microarray for the detection of all 15 distinct O-antigen forms of L. pneumophila, including serogroups O1 to O15. A total of 35 strains were used to verify the specificity of the microarray, including 15 L. pneumophila O-antigen standard reference strains and seven L. pneumophila clinical isolates as target strains, seven reference strains of other non-pneumophila Legionella species as closely related strains, and six non-Legionella bacterial species as nonrelated strains. The detection sensitivity was 1 ng of genomic DNA or 0.4 CFU/ml in water samples with filter enrichment and plate culturing. This study demonstrated that the microarray allows specific, sensitive, and reproducible detection of L. pneumophila serogroups. To the best of our knowledge, this is the first report of a microarray serotyping method for all 15 distinct O-antigen forms of L. pneumophila.     

Specific Detection of Legionella pneumophila: Construction of a New 16S rRNA-Targeted Oligonucleotide Probe

Based on comparative sequence analysis, we have designed an oligonucleotide probe complementary to a region of 16S rRNA of Legionella pneumophila which allows the differentiation of L. pneumophila from other Legionella species without cultivation. The specificity of the new probe, LEGPNE1, was tested by in situ hybridization to a total of four serogroups of six strains of L. pneumophila, five different Legionella spp. and three nonlegionella species as reference strains. Furthermore, L. pneumophila cells could be easily distinguished from Legionella micdadei and Pseudomonas aeruginosa cells by using in situ hybridization with probes LEGPNE1, LEG705, and EUB338 after infection of the protozoan Acanthamoeba castellanii.     

LAMP-based method for a rapid identification of Legionella spp. and Legionella pneumophila

Legionella pneumophila is accounted for more than 80% of Legionella infection. However it is difficult to discriminate between the L. pneumophila and non-L. pneumophila species rapidly. In order to detect the Legionella spp. and distinguish L. pneumophila from Legionella spp., a real-time loop-mediated isothermal amplification (LAMP) platform that targets a specific sequence of the 16S rRNA gene was developed. LS-LAMP amplifies the fragment of the 16S rRNA gene to detect all species of Legionella genus. A specific sequence appears at the 16S rRNA gene of L. pneumophila, while non-L. pneumophila strains have a variable sequence in this site, which can be recognized by the primer of LP-LAMP. In the present study, 61 reference strains were used for the method verification. We found that the specificity was 100% for both LS-LAMP and LP-LAMP, and the sensitivity of LAMP assay for L. pneumophila detection was between 52 and 5.2 copies per reaction. In the environmental water samples detection, a total of 107 water samples were identified by the method. The culture and serological test were used as reference methods. The specificity of LS-LAMP and LP-LAMP for the samples detection were 91.59% (98/107) and 93.33% (56/60), respectively. The sensitivity of LS-LAMP and LP-LAMP were 100% (51/51) and 100% (18/18). The results suggest that real-time LAMP, as a new assay, provides a specific and sensitive method for rapid detection and differentiation of Legionella spp. and L. pneumophila and should be utilized to test environmental water samples for increased rates of detection.     

Intracellular parasitism,the driving force of evolution of Legionella pneumophila and the genus Legionella

Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia called Legionnaires' disease that is often fatal when not promptly diagnosed and treated. Legionella parasitize aquatic protozoa with which it co-evolved over an evolutionary long time. The close relationship between hosts and pathogens, their co-evolution, led to molecular interactions such as the exchange of genetic material through horizontal gene transfer (HGT). Genome sequencing of L. pneumophila and of the entire genus Legionella that comprises over 60 species revealed that Legionellae have co-opted genes and thus cellular functions from their eukaryotic hosts to a surprisingly high extent. Acquisition and loss of these eukaryotic-like genes and domains is an on-going process underlining the highly dynamic nature of the Legionella genomes. Although the large amount and diversity of HGT in Legionella seems to be unique in the prokaryotic world the analyses of more and more genomes from environmental organisms and symbionts of amoeba revealed that such genetic exchanges occur among all amoeba associated bacteria and also among the different microorganisms that infect amoeba. This dynamic reshuffling and gene-acquisition has led to the emergence of Legionella as human pathogen and may lead to the emergence of new human pathogens from the environment.     

Phylogenetic Study of <Emphasis Type="Italic">Legionella</Emphasis> Species in Pristine and Polluted Aquatic Samples from a Tropical Atlantic Forest Ecosystem

Legionella species are ubiquitous bacteria in aquatic environments. To examine the effect of anthropogenic impacts and physicochemical characteristics on the Legionellaceae population, we collected water from two sites in the Itanhaém River system in the Atlantic Forest of Brazil. One sample was collected from an upstream pristine region, the other from a downstream estuarine region moderately affected by untreated domestic sewage. Cultures on a selective medium failed to isolate Legionella species. Culture-independent methods showed that water from the estuarine aquatic habitat contained DNA sequences homologous to the 16S ribosomal DNA gene of Legionella pneumophila and non-pneumophila species. In pristine water, only two sequences related to L. pneumophila were detected. The results suggest that salinity and anthropogenic factors, such as wastewater discharge, favor a diversity of Legionella species, whereas pristine freshwater selects for Legionella pneumophila.     

Isolation of an Amoeba Naturally Harboring a Distinctive Legionella Species

There are numerous in vitro studies documenting the multiplication of Legionella species in free-living amoebae and other protozoa. It is believed that protozoa serve as host cells for the intracellular replication of certain Legionella species in a variety of environmental settings. This study describes the isolation and characterization of a bacterium initially observed within an amoeba taken from a soil sample. In the laboratory, the bacterium multiplied within and was highly pathogenic for Acanthamoeba polyphaga. Extracellular multiplication was observed on buffered charcoal yeast extract agar but not on a variety of conventional laboratory media. A 16S rRNA gene analysis placed the bacterium within the genus Legionella. Serological studies indicate that it is distinct from previously described species of the genus. This report also describes methods that should prove useful for the isolation and characterization of additional Legionella-like bacteria from free-living amoebae. In addition, the characterization of bacterial pathogens of amoebae has significant implications for understanding the ecology and identification of other unrecognized bacterial pathogens.     

Viewing Legionella pneumophila Pathogenesis through an Immunological Lens

Legionella pneumophila is the causative agent of the severe pneumonia Legionnaires' disease. L. pneumophila is ubiquitously found in freshwater environments, where it replicates within free-living protozoa. Aerosolization of contaminated water supplies allows the bacteria to be inhaled into the human lung, where L. pneumophila can be phagocytosed by alveolar macrophages and replicate intracellularly. The Dot/Icm type IV secretion system (T4SS) is one of the key virulence factors required for intracellular bacterial replication and subsequent disease. The Dot/Icm apparatus translocates more than 300 effector proteins into the host cell cytosol. These effectors interfere with a variety of cellular processes, thus enabling the bacterium to evade phagosome–lysosome fusion and establish an endoplasmic reticulum-derived Legionella-containing vacuole, which facilitates bacterial replication. In turn, the immune system has evolved numerous strategies to recognize intracellular bacteria such as L. pneumophila, leading to potent inflammatory responses that aid in eliminating infection. This review aims to provide an overview of L. pneumophila pathogenesis in the context of the host immune response.     

The Legionella pneumophila Collagen-Like Protein Mediates Sedimentation,Autoaggregation, and Pathogen-Phagocyte Interactions

Although only partially understood, multicellular behavior is relatively common in bacterial pathogens. Bacterial aggregates can resist various host defenses and colonize their environment more efficiently than planktonic cells. For the waterborne pathogen Legionella pneumophila, little is known about the roles of autoaggregation or the parameters which allow cell-cell interactions to occur. Here, we determined the endogenous and exogenous factors sufficient to allow autoaggregation to take place in L. pneumophila. We show that isolates from Legionella species which do not produce the Legionella collagen-like protein (Lcl) are deficient in autoaggregation. Targeted deletion of the Lcl-encoding gene (lpg2644) and the addition of Lcl ligands impair the autoaggregation of L. pneumophila. In addition, Lcl-induced autoaggregation requires divalent cations. Escherichia coli producing surface-exposed Lcl is able to autoaggregate and shows increased biofilm production. We also demonstrate that L. pneumophila infection of Acanthamoeba castellanii and Hartmanella vermiformis is potentiated under conditions which promote Lcl dependent autoaggregation. Overall, this study shows that L. pneumophila is capable of autoaggregating in a process that is mediated by Lcl in a divalent-cation-dependent manner. It also reveals that Lcl potentiates the ability of L. pneumophila to come in contact, attach, and infect amoebae.     

Effect of Bacterial Interference on Biofilm Development by Legionella pneumophila

In the ecology of Legionella pneumophila a crucial role may be played by its relationship with the natural flora; thus we investigated the interactions between Legionella and other aquatic bacteria, particularly within biofilms. Among 80 aquatic bacteria screened for the production of bacteriocin-like substances (BLSs), 66.2% of them were active against L. pneumophila. The possible effect of some of these aquatic bacteria on the development and stability of L. pneumophila biofilms was studied. Pseudomonas fluorescens, the best BLS producer, showed the greatest negative effect on biofilm formation and strongly enhanced the detachment of Legionella. Pseudomonas aeruginosa, Burkholderia cepacia, Pseudomonas putida, Aeromonas hydrophila, and Stenotrophomonas maltophilia, although producing BLSs at different levels, were less active in the biofilm experiments. Acinetobacter lwoffii did not produce any antagonistic compound and was the only one able to strongly enhance L. pneumophila biofilm. Our results highlight that BLS production may contribute to determining the fate of L. pneumophila within ecological niches. The interactions observed in this study are important features of L. pneumophila ecology, which knowledge may lead to more effective measures to control the persistance of the germ in the environment.     

Mycobacterium avium Bacilli Grow Saprozoically in Coculture with Acanthamoeba polyphaga and Survive within Cyst Walls

Protozoans are gaining recognition as environmental hosts for a variety of waterborne pathogens. We compared the growth of Mycobacterium avium, a human pathogen associated with domestic water supplies, in coculture with the free-living amoeba Acanthamoeba polyphaga with the growth of M. avium when it was separated from amoebae by a 0.1-μm-pore-size polycarbonate membrane (in a parachamber). Although viable mycobacteria were observed within amoebal vacuoles, there was no significant difference between bacterial growth in coculture and bacterial growth in the parachamber. This suggests that M. avium is able to grow saprozoically on products secreted by the amoebae. In contrast, Legionella pneumophila, a well-studied intracellular parasite of amoebae, multiplied only in coculture. A comparison of amoebae infected with L. pneumophila and amoebae infected with M. avium by electron microscopy demonstrated that there were striking differences in the locations of the bacteria within amoebal cysts. While L. pneumophila resided within the cysts, M. avium was found within the outer walls of the double-walled cysts of A. polyphaga. These locations may provide a reservoir for the bacteria when environmental conditions become unfavorable.