Interactions of free-living amoebae with the rice fungal pathogen, Rhizoctonia solani.

OBJECTIVE: Rhizoctonia solani is a soil-borne fungal pathogen of many important crop plants. In rice, R. solani causes sheath blight disease, which results in devastating grain yield and quality losses. Few methods are available to control this pathogen and classic single gene resistance mechanisms in rice plants have not been identified. We hypothesize that alternate means of control are available in the environment including free-living amoebae. Amoebae are soil-, water- and air-borne microorganisms that are predominantly heterotrophic. Many amoeba species are mycophagous, and several harm their prey using mechanisms other than phagocytosis. Here, we used light and scanning electron microscopy to survey the interactions of R. solani with four amoeba species, with the goal of identifying amoebae species with potential for biocontrol. RESULTS: We observed a wide range of responses during interactions of R. solani with four different free-living amoebae. Two Acanthamoeba species encyst in co-cultures with R. solani at higher rates than medium without R. solani. Vermamoeba vermiformis (formerly Hartmanella vermiformis) attach to R. solani mycelium and are associated with mycelial shriveling and perforations of fungal cell walls, indicating an antagonistic interaction. No phenotypic changes were observed in co-cultures of Dictyostelium discoideum and R. solani.

Vermamoeba vermiformis: a Free-Living Amoeba of Interest.

Free-living amoebae are protists that are widely distributed in the environment including water, soil, and air. Although the amoebae of the genus Acanthamoeba are still the most studied, other species, such as Vermamoeba vermiformis (formerly Hartmannella vermiformis), are the subject of increased interest. Found in natural or man-made aquatic environments, V. vermiformis can support the multiplication of other microorganisms and is able to harbor and potentially protect pathogenic bacteria or viruses. This feature is to be noted because of the presence of this thermotolerant amoeba in hospital water networks. As a consequence, this protist could be implicated in health concerns and be indirectly responsible for healthcare-related infections. This review highlights, among others, the consequences of V. vermiformis relationships with other microorganisms and shows that this free-living amoeba species is therefore of interest for public health.

From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella.

The 1976 outbreak of Legionnaires' disease led to the discovery of the intracellular bacterial pathogen Legionella pneumophila. Given their impact on human health, Legionella species and the mechanisms responsible for their replication within host cells are often studied in alveolar macrophages, the primary human cell type associated with disease. Despite the potential severity of individual cases of disease, Legionella are not spread from person-to-person. Thus, from the pathogen's perspective, interactions with human cells are accidents of time and space-evolutionary dead ends with no impact on Legionella's long-term survival or pathogenic trajectory. To understand Legionella as a pathogen is to understand its interaction with its natural hosts: the polyphyletic protozoa, a group of unicellular eukaryotes with a staggering amount of evolutionary diversity. While much remains to be understood about these enigmatic hosts, we summarize the current state of knowledge concerning Legionella's natural host range, the diversity of Legionella-protozoa interactions, the factors influencing these interactions, the importance of avoiding the generalization of protozoan-bacterial interactions based on a limited number of model hosts and the central role of protozoa to the biology, evolution, and persistence of Legionella in the environment.

Molecular detection of Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Hartmannella) vermiformis as vectors for Legionella spp. in untreated and solar pasteurized harvested rainwater.

BACKGROUND: Legionella spp. employ multiple strategies to adapt to stressful environments including the proliferation in protective biofilms and the ability to form associations with free-living amoeba (FLA). The aim of the current study was to identify Legionella spp., Acanthamoeba spp., Vermamoeba (Hartmannella) vermiformis and Naegleria fowleri that persist in a harvested rainwater and solar pasteurization treatment system. METHODS: Pasteurized (45 °C, 65 °C, 68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples were screened for Legionella spp. and the heterotrophic plate count was enumerated. Additionally, ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR) was utilized for the quantification of viable Legionella spp., Acanthamoeba spp., V. vermiformis and N. fowleri in pasteurized (68 °C, 74 °C, 84 °C and 93 °C) and unpasteurized tank water samples, respectively. RESULTS: Of the 82 Legionella spp. isolated from unpasteurized tank water samples, Legionella longbeachae (35 %) was the most frequently isolated, followed by Legionella norrlandica (27 %) and Legionella rowbothamii (4 %). Additionally, a positive correlation was recorded between the heterotrophic plate count vs. the number of Legionella spp. detected (ρ = 0.710, P = 0.048) and the heterotrophic plate count vs. the number of Legionella spp. isolated (ρ = 0.779, P = 0.0028) from the tank water samples collected. Solar pasteurization was effective in reducing the gene copies of viable V. vermiformis (3-log) and N. fowleri (5-log) to below the lower limit of detection at temperatures of 68-93 °C and 74-93 °C, respectively. Conversely, while the gene copies of viable Legionella and Acanthamoeba were significantly reduced by 2-logs (P = 0.0024) and 1-log (P = 0.0015) overall, respectively, both organisms were still detected after pasteurization at 93 °C. CONCLUSIONS: Results from this study indicate that Acanthamoeba spp. primarily acts as the vector and aids in the survival of Legionella spp. in the solar pasteurized rainwater as both organisms were detected and were viable at high temperatures (68-93 °C).

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 of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella 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 by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has 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 of L. pneumophila that 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, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria 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.

Endosymbiotic Mycobacterium chelonae in a Vermamoeba vermiformis strain isolated from the nasal mucosa of an HIV patient in Lima, Peru.

In March 2010, a 35 year-old HIV/AIDS female patient was admitted to hospital to start treatment with Highly Active Antiretroviral Therapy (HAART) since during a routine control a dramatic decrease in the CD4(+) levels was detected. At this stage, a nasal swab from each nostril was collected from the patient to include it in the samples for the case study mentioned above. Moreover, it is important to mention that the patient was diagnosed in 2009 with invasive pneumococcal disease, acute cholecystitis, pancreatitis and pulmonary tuberculosis. The collected nasal swabs from both nostrils were positive for Vermamoeba vermiformis species which was identified using morphological and PCR/DNA sequencing approaches. Basic Local Alignment Search Tool (BLAST) homology and phylogenetic analysis confirmed the amoebic strain to belong to V.vermiformis species. Molecular identification of the Mycobacterium strain was carried out using a bacterial universal primer pair for the 16S rDNA gene at the genus level and the rpoB gene was amplified and sequenced as previously described to identify the Mycobacterium species (Shin et al., 2008; Sheen et al., 2013). Homology and phylogenetic analyses of the rpoB gene confirmed the species as Mycobacterium chelonae. In parallel, collected swabs were tested by PCR and were positive for the presence of V.vermiformis and M.chelonae. This work describes the identification of an emerging bacterial pathogen,M.chelonae from a Free-Living Amoebae (FLA) strain belonging to the species V.vermiformis that colonized the nasal cavities of an HIV/AIDS patient, previously diagnosed with TB. Awareness within clinicians and public health professionals should be raised, as pathogenic agents such as M.chelonae may be using FLA to propagate and survive in the environment.

Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae.

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named 'Candidatus Nucleicultrix amoebiphila', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. 'Candidatus Nucleicultrix amoebiphila' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96-120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.

Hartmannella vermiformis inhibition of Legionella pneumophila cultivability.

Hartmannella vermiformis and Acanthamoeba polyphaga are frequently isolated from drinking water and permissive to Legionella pneumophila parasitization. In this study, extracellular factor(s) produced by H. vermiformis and A. polyphaga were assessed for their effects on cultivability of L. pneumophila. Page's amoeba saline (PAS) was used as an encystment medium for H. vermiformis and A. polyphaga monolayers, and the culture supernatants (HvS and ApS, respectively) were assessed against L. pneumophila growth. Compared to PAS and ApS, HvS significantly inhibited L. pneumophila strain Philadelphia-1 (Ph-1) cultivability by 3 log(10) colony forming unit (CFU) mL(-1) after 3 days of exposure compared to <0.5 log(10) CFU mL(-1) reduction of strain Lp02 (P < 0.001). Flow cytometric analysis revealed changes in the percentage and cultivability of three bacterial subpopulations: intact/slightly damaged membrane (ISM), undefined membrane status (UD), and mixed type (MT). After 3 days of HvS exposure, the MT subpopulation decreased significantly (31.6 vs 67.2 %, respectively, P < 0.001), while the ISM and UD subpopulations increased (+26.7 and +6.9 %, respectively) with the ISM subpopulation appearing as viable but nonculturable (VBNC) cells. HvS was separated into two fractions based on molecular weight, with more than 99 % of the L. pneumophila inhibition arising from the <5 kDa fraction (P < 0.001). Liquid chromatography indicated the inhibitory molecule(s) are likely polar and elute from a Novapak C18 column between 6 and 15 min. These results demonstrate that H. vermiformis is capable of extracellular modulation of L. pneumophila cultivability and probably promote the VBNC state for this bacterium.

Cooccurrence of free-living amoebae and nontuberculous Mycobacteria in hospital water networks, and preferential growth of Mycobacterium avium in Acanthamoeba lenticulata.

The incidence of lung and other diseases due to nontuberculous mycobacteria (NTM) is increasing. NTM sources include potable water, especially in households where NTM populate pipes, taps, and showerheads. NTM share habitats with free-living amoebae (FLA) and can grow in FLA as parasites or as endosymbionts. FLA containing NTM may form cysts that protect mycobacteria from disinfectants and antibiotics. We first assessed the presence of FLA and NTM in water and biofilm samples collected from a hospital, confirming the high prevalence of NTM and FLA in potable water systems, particularly in biofilms. Acanthamoeba spp. (genotype T4) were mainly recovered (8/17), followed by Hartmannella vermiformis (7/17) as well as one isolate closely related to the genus Flamella and one isolate only distantly related to previously described species. Concerning mycobacteria, Mycobacterium gordonae was the most frequently found isolate (9/17), followed by Mycobacterium peregrinum (4/17), Mycobacterium chelonae (2/17), Mycobacterium mucogenicum (1/17), and Mycobacterium avium (1/17). The propensity of Mycobacterium avium hospital isolate H87 and M. avium collection strain 104 to survive and replicate within various FLA was also evaluated, demonstrating survival of both strains in all amoebal species tested but high replication rates only in Acanthamoeba lenticulata. As A. lenticulata was frequently recovered from environmental samples, including drinking water samples, these results could have important consequences for the ecology of M. avium in drinking water networks and the epidemiology of disease due to this species.

The CRISPR-associated gene cas2 of Legionella pneumophila is required for intracellular infection of amoebae.

UNLABELLED: Recent studies have shown that the clustered regularly interspaced palindromic repeats (CRISPR) array and its associated (cas) genes can play a key role in bacterial immunity against phage and plasmids. Upon analysis of the Legionella pneumophila strain 130b chromosome, we detected a subtype II-B CRISPR-Cas locus that contains cas9, cas1, cas2, cas4, and an array with 60 repeats and 58 unique spacers. Reverse transcription (RT)-PCR analysis demonstrated that the entire CRISPR-Cas locus is expressed during 130b extracellular growth in both rich and minimal media as well as during intracellular infection of macrophages and aquatic amoebae. Quantitative reverse transcription-PCR (RT-PCR) further showed that the levels of cas transcripts, especially those of cas1 and cas2, are elevated during intracellular growth relative to exponential-phase growth in broth. Mutants lacking components of the CRISPR-Cas locus were made and found to grow normally in broth and on agar media. cas9, cas1, cas4, and CRISPR array mutants also grew normally in macrophages and amoebae. However, cas2 mutants, although they grew typically in macrophages, were significantly impaired for infection of both Hartmannella and Acanthamoeba species. A complemented cas2 mutant infected the amoebae at wild-type levels, confirming that cas2 is required for intracellular infection of these host cells. IMPORTANCE: Given that infection of amoebae is critical for L. pneumophila persistence in water systems, our data indicate that cas2 has a role in the transmission of Legionnaires' disease. Because our experiments were done in the absence of added phage, plasmid, or nucleic acid, the event that is facilitated by Cas2 is uniquely distinct from current dogma concerning CRISPR-Cas function.

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.

Type II protein secretion (T2S) by Legionella pneumophila is required for intracellular infection of host cells, including macrophages and the amoebae Acanthamoeba castellanii and Hartmannella vermiformis. Previous proteomic analysis revealed that T2S by L. pneumophila 130b 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. nttA mutants were impaired for intracellular multiplication in A. castellanii but not H. vermiformis or macrophages, suggesting that novel exoproteins which are specific to Legionella are 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 in H. vermiformis but not A. castellanii. Further examination of an lspF mutant lacking the T2S apparatus documented that T2S is also critical for infection of the amoeba Naegleria lovaniensis. Mutants lacking SrnA, PlaC, or ProA, but not those deficient for NttA, were defective in N. lovaniensis. Based upon analysis of a double mutant lacking PlaC and ProA, the role of ProA in H. vermiformis was connected to its ability to activate PlaC, whereas in N. 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 of L. pneumophila.

Differential Legionella spp. survival between intracellular and extracellular forms in thermal spring environments.

Legionella are commonly found in natural and man-made aquatic environments and are able to inhabit various species of protozoa. The relationship between the occurrence of Legionella spp. within protozoa and human legionellosis has been demonstrated; however, the proportions of intracellular and extracellular Legionella spp. in the aquatic environment were rarely reported. In this study, we developed a new method to differentiate intracellular and extracellular Legionella spp. in the aquatic environment. Water samples from three thermal spring recreational areas in southeastern Taiwan were collected and analyzed. For each water sample, concurrent measurements were performed for Legionella spp. and their free-living amoebae hosts. The overall detection rate was 32 % (16/50) for intracellular Legionella spp. and 12 % (6/50) for extracellular Legionella spp. The most prevalent host of Legionella spp. was Hartmannella vermiformis. The identified Legionella spp. differed substantially between intracellular and extracellular forms. The results showed that it may be necessary to differentiate intracellular and extracellular forms of Legionella spp.

Hartmannella vermiformis can promote proliferation of Candida spp. in tap-water.

Candida yeasts colonize humans' oral cavities as commensals or opportunistic pathogens. They may be isolated from water circulating in dental unit waterlines mixed with saliva traces mainly because of dysfunction of anti-retraction valves. Free-living amoebae (FLA), like Hartmannella vermiformis, are frequently found in aquatic environments and they have also been already isolated from dental unit waterlines. They can be implicated as reservoir for pathogens or directly in infections. This work deals with the survival of three species of Candida (Candida albicans, Candida glabrata and Candida parapsilosis), in co-cultivation with FLA in tap-water. One strain of each Candida species was studied. Microbiological and microscopic approaches were used; amoebae-yeasts co-cultivation assays were performed at different temperatures of incubation. Results have shown that H. vermiformis were able to internalize Candida yeasts and promote their proliferation in tap-water with or without saliva traces (2% v/v). Amoebae interact differently with Candida depending on the temperature used and the studied species of yeasts. In conclusion, this study emphasizes the survival of yeasts and/or FLA in tap-water. The ability of yeasts and amoebae to interact should be taken into account because it could lead to candidiasis infection for the frailest patients after a dental treatment.

Differential growth of Legionella pneumophila strains within a range of amoebae at various temperatures associated with in-premise plumbing.

AIMS: The potential effect of in-premise plumbing temperatures (24, 32, 37 and 41°C) on the growth of five different Legionella pneumophila strains within free-living amoebae (Acanthamoeba polyphaga, Hartmannella vermiformis and Naegleria fowleri) was examined. METHODS AND RESULTS: Compared with controls that actively fed on Escherichia coli prey, when Leg. pneumophila was used as prey, strains Lp02 and Bloomington-2 increased in growth at 30, 32 and 37°C while strains Philadelphia-1 and Chicago 2 did not grow at any temperature within A. polyphaga. Strains Lp02, Bloomington-2 and Dallas 1E did not proliferate in the presence of H. vermiformis nor did strain Philadelphia-1 in the presence of N. fowleri. Yet, strain Bloomington-2 grew at all temperatures examined within N. fowleri, while strain Lp02 proliferated at all temperatures except 41°C. More intriguing, strain Chicago 2 only grew at 32°C within H. vermiformis and N. fowleri suggesting a limited temperature growth range for this strain. CONCLUSIONS: Identifying the presence of pathogenic legionellae may require the use of multiple host amoebae and incubation temperatures. SIGNIFICANCE AND IMPACT OF THE STUDY: Temperature conditions and species of amoeba host supported in drinking water appear to be important for the selection of human-pathogenic legionellae and point to future research required to better understand Legionella ecology.

Cytochrome c4 is required for siderophore expression by Legionella pneumophila, whereas cytochromes c1 and c5 promote intracellular infection.

A panel of cytochrome c maturation (ccm) mutants of Legionella pneumophila displayed a loss of siderophore (legiobactin) expression, as measured by both the chrome azurol S assay and a Legionella-specific bioassay. These data, coupled with the finding that ccm transcripts are expressed by wild-type bacteria grown in deferrated medium, indicate that the Ccm system promotes siderophore expression by L. pneumophila. To determine the basis of this newfound role for Ccm, we constructed and tested a set of mutants specifically lacking individual c-type cytochromes. Whereas ubiquinol-cytochrome c reductase (petC) mutants specifically lacking cytochrome c(1) and cycB mutants lacking cytochrome c(5) had normal siderophore expression, cyc4 mutants defective for cytochrome c(4) completely lacked legiobactin. These data, along with the expression pattern of cyc4 mRNA, indicate that cytochrome c(4) in particular promotes siderophore expression. In intracellular infection assays, petC mutants and cycB mutants, but not cyc4 mutants, had a reduced ability to infect both amoebae and macrophage hosts. Like ccm mutants, the cycB mutants were completely unable to grow in amoebae, highlighting a major role for cytochrome c(5) in intracellular infection. To our knowledge, these data represent both the first direct documentation of the importance of a c-type cytochrome in expression of a biologically active siderophore and the first insight into the relative importance of c-type cytochromes in intracellular infection events.

Detection of protozoan hosts for Legionella pneumophila in engineered water systems by using a biofilm batch test.

Legionella pneumophila proliferates in aquatic habitats within free-living protozoa, 17 species of which have been identified as hosts by using in vitro experiments. The present study aimed at identifying protozoan hosts for L. pneumophila by using a biofilm batch test (BBT). Samples (600 ml) collected from 21 engineered freshwater systems, with added polyethylene cylinders to promote biofilm formation, were inoculated with L. pneumophila and subsequently incubated at 37°C for 20 days. Growth of L. pneumophila was observed in 16 of 18 water types when the host protozoan Hartmannella vermiformis was added. Twelve of the tested water types supported growth of L. pneumophila or indigenous Legionella anisa without added H. vermiformis. In 12 of 19 BBT flasks H. vermiformis was indicated as a host, based on the ratio between maximum concentrations of L. pneumophila and H. vermiformis, determined with quantitative PCR (Q-PCR), and the composition of clone libraries of partial 18S rRNA gene fragments. Analyses of 609 eukaryotic clones from the BBTs revealed that 68 operational taxonomic units (OTUs) showed the highest similarity to free-living protozoa. Forty percent of the sequences clustering with protozoa showed ≥99.5% similarity to H. vermiformis. None of the other protozoa serving as hosts in in vitro studies were detected in the BBTs. In several tests with growth of L. pneumophila, the protozoa Diphylleia rotans, Echinamoeba thermarum, and Neoparamoeba sp. were identified as candidate hosts. In vitro studies are needed to confirm their role as hosts for L. pneumophila. Unidentified protozoa were implicated as hosts for uncultured Legionella spp. grown in BBT flasks at 15°C.

Legionella pneumophila secretes an endoglucanase that belongs to the family-5 of glycosyl hydrolases and is dependent upon type II secretion.

Examination of cell-free culture supernatants revealed that Legionella pneumophila strains secrete an endoglucanase activity. Legionella pneumophila lspF mutants were deficient for this activity, indicating that the endoglucanase is secreted by the bacterium's type II protein secretion (T2S) system. Inactivation of celA, encoding a member of the family-5 of glycosyl hydrolases, abolished the endoglucanase activity in L. pneumophila culture supernatants. The cloned celA gene conferred activity upon recombinant Escherichia coli. Thus, CelA is the major secreted endoglucanase of L. pneumophila. Mutants inactivated for celA grew normally in protozoa and macrophage, indicating that CelA is not required for the intracellular phase of L. pneumophila. The CelA endoglucanase is one of at least 25 proteins secreted by the type II system of L. pneumophila and the 17th type of enzyme effector associated with this pathway. Only a subset of the other Legionella species tested expressed secreted endoglucanase activity, suggesting that the T2S output differs among the different legionellae. Overall, this study represents the first documentation of an endoglucanase (EC 3.2.1.4) being produced by a strain of Legionella.

A type II secreted RNase of Legionella pneumophila facilitates optimal intracellular infection of Hartmannella vermiformis.

Type II protein secretion plays a role in a wide variety of functions that are important for the ecology and pathogenesis of Legionella pneumophila. Perhaps most dramatic is the critical role that this secretion pathway has in L. pneumophila intracellular infection of aquatic protozoa. Recently, we showed that virulent L. pneumophila strain 130b secretes RNase activity through its type II secretion system. We now report the cloning and mutational analysis of the gene (srnA) encoding that novel type of secreted activity. The SrnA protein was defined as being a member of the T2 family of secreted RNases. Supernatants from mutants inactivated for srnA completely lacked RNase activity, indicating that SrnA is the major secreted RNase of L. pneumophila. Although srnA mutants grew normally in bacteriological media and human U937 cell macrophages, they were impaired in their ability to grow within Hartmannella vermiformis amoebae. This finding represents the second identification of a L. pneumophila type II effector being necessary for optimal intracellular infection of amoebae, with the first being the ProA zinc metalloprotease. Newly constructed srnA proA double mutants displayed an even larger infection defect that appeared to be the additive result of losing both SrnA and ProA. Overall, these data represent the first demonstration of a secreted RNase promoting an intracellular infection event, and support our long-standing hypothesis that the infection defects of L. pneumophila type II secretion mutants are due to the loss of multiple secreted effectors.

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.