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.     

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.     

Comparative analyses of Legionella species identifies genetic features of strains causing Legionnaires’ disease

Background

The genus Legionella comprises over 60 species. However, L. pneumophila and L. longbeachae alone cause over 95% of Legionnaires’ disease. To identify the genetic bases underlying the different capacities to cause disease we sequenced and compared the genomes of L. micdadei, L. hackeliae and L. fallonii (LLAP10), which are all rarely isolated from humans.

Results

We show that these Legionella species possess different virulence capacities in amoeba and macrophages, correlating with their occurrence in humans. Our comparative analysis of 11 Legionella genomes belonging to five species reveals highly heterogeneous genome content with over 60% representing species-specific genes; these comprise a complete prophage in L. micdadei, the first ever identified in a Legionella genome. Mobile elements are abundant in Legionella genomes; many encode type IV secretion systems for conjugative transfer, pointing to their importance for adaptation of the genus. The Dot/Icm secretion system is conserved, although the core set of substrates is small, as only 24 out of over 300 described Dot/Icm effector genes are present in all Legionella species. We also identified new eukaryotic motifs including thaumatin, synaptobrevin or clathrin/coatomer adaptine like domains.

Conclusions

Legionella genomes are highly dynamic due to a large mobilome mainly comprising type IV secretion systems, while a minority of core substrates is shared among the diverse species. Eukaryotic like proteins and motifs remain a hallmark of the genus Legionella. Key factors such as proteins involved in oxygen binding, iron storage, host membrane transport and certain Dot/Icm substrates are specific features of disease-related strains.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0505-0) contains supplementary material, which is available to authorized users.     

Saccamoeba lacustris,sp. nov. (Amoebozoa: Lobosea: Hartmannellidae), a new lobose amoeba,parasitized by the novel chlamydia ‘Candidatus Metachlamydia lacustris’ (Chlamydiae: Parachlamydiaceae)

An amoeba isolated from an aquatic biotope, identified morphologically as Saccamoeba limax, was found harbouring mutualistic rod-shaped gram-negative bacteria. During their cultivation on agar plates, a coinfection also by lysis-inducing chlamydia-like organisms was found in some subpopulations of that amoeba. .Here we provide a molecular-based identification of both the amoeba host and the two bacterial endosymbionts. Analysis of the 18S rRNA gene revealed that this strain is the sister-group to Glaeseria, for which we proposed the name Saccamoeba lacustris. The rod-shaped endosymbiont was identified as a member of Variovorax paradoxus group (Comamonadaceae, Beta-Proteobacteria). No growth on bacteriological agars was recorded, hence this symbiont might be strictly intracellular. The chlamydia-like parasite was unable to infect Acanthamoeba and other amoebae in coculture, showing high host specificity. Phylogenetic analysis based on the 16S rDNA indicated that it is a new member of the family Parachlamydiaceae (order Chlamydiales), for which we proposed the name ‘Candidatus Metachlamydia lacustris’.     

Members of the Cytophaga–Flavobacterium–Bacteroides phylum as intracellular bacteria of acanthamoebae: proposal of 'Candidatus Amoebophilus asiaticus'

Three Gram-negative, rod-shaped bacteria that were found intracellularly in two environmental and one clinical Acanthamoeba sp. isolates were analysed. Two endocytobionts showing a parasitic behaviour were propagated successfully outside their amoebal host cells and were identified subsequently by comparative 16S rRNA sequence analysis as being most closely affiliated with Flavobacterium succinicans (99% 16S rRNA sequence similarity) or Flavobacterium johnsoniae (98% 16S rRNA sequence similarity). One endocytobiont could neither be cultivated outside its original Acanthamoeba host ( Acanthamoeba sp. TUMSJ-321) nor transferred into other amoebae. Electron microscopy revealed that the amoebal trophozoites and cysts were almost completely filled with cells of this endosymbiont which are surrounded by a host-derived membrane. According to 16S rRNA sequence analysis, this endosymbiont could also be assigned to the Cytophaga – Flavobacterium – Bacteroides (CFB) phylum, but was not closely affiliated to any recognized species within this phylogenetic group (less than 82% 16S rRNA sequence similarity). Identity and intracellular localization of this endosymbiont were confirmed by application of a specific fluorescently labelled 16S rRNA-targeted probe. Based on these findings, we propose classification of this obligate Acanthamoeba endosymbiont as ' Candidatus Amoebophilus asiaticus'. Comparative 18S rRNA sequence analysis of the host of ' Candidatus Amoebophilus asiaticus' revealed its membership with Acanthamoeba 18S rDNA sequence type T4 that comprises the majority of all Acanthamoeba isolates.     

Biodiversity of Amoebae and Amoeba-Resisting Bacteria in a Hospital Water Network

Free-living amoebae (FLA) are ubiquitous organisms that have been isolated from various domestic water systems, such as cooling towers and hospital water networks. In addition to their own pathogenicity, FLA can also act as Trojan horses and be naturally infected with amoeba-resisting bacteria (ARB) that may be involved in human infections, such as pneumonia. We investigated the biodiversity of bacteria and their amoebal hosts in a hospital water network. Using amoebal enrichment on nonnutrient agar, we isolated 15 protist strains from 200 (7.5%) samples. One thermotolerant Hartmannella vermiformis isolate harbored both Legionella pneumophila and Bradyrhizobium japonicum. By using amoebal coculture with axenic Acanthamoeba castellanii as the cellular background, we recovered at least one ARB from 45.5% of the samples. Four new ARB isolates were recovered by culture, and one of these isolates was widely present in the water network. Alphaproteobacteria (such as Rhodoplanes, Methylobacterium, Bradyrhizobium, Afipia, and Bosea) were recovered from 30.5% of the samples, mycobacteria (Mycobacterium gordonae, Mycobacterium kansasii, and Mycobacterium xenopi) were recovered from 20.5% of the samples, and Gammaproteobacteria (Legionella) were recovered from 5.5% of the samples. No Chlamydia or Chlamydia-like organisms were recovered by amoebal coculture or detected by PCR. The observed strong association between the presence of amoebae and the presence of Legionella (P < 0.001) and mycobacteria (P = 0.009) further suggests that FLA are a reservoir for these ARB and underlines the importance of considering amoebae when water control measures are designed.     

Presence of Legionella and Free-Living Amoebae in Composts and Bioaerosols from Composting Facilities

Several species of Legionella cause Legionnaires’ disease (LD). Infection may occur through inhalation of Legionella or amoebal vesicles. The reservoirs of Legionella are water, soil, potting soil and compost. Some species of free-living amoebae (FLA) that are naturally present in water and soil were described as hosts for Legionella. This study aimed to understand whether or not the composting facilities could be sources of community-acquired Legionella infections after development of bioaerosols containing Legionella or FLA. We looked for the presence of Legionella (by co-culture) and FLA (by culture) in composts and bioaerosols collected at four composting facilities located in southern Switzerland. We investigated the association between the presence of Legionella and compost and air parameters and presence of FLA. Legionella spp. (including L. pneumophila) were detected in 69.3% (61/88) of the composts and FLA (mainly Acanthamoeba, Vermamoeba, Naegleria and Stenamoeba) in 92.0% (81/88). L. pneumophila and L. bozemanii were most frequently isolated. FLA as potential host for Legionella spp. were isolated from 40.9% (36/88) of the composts in all facilities. In Legionella-positive samples the temperature of compost was significantly lower (P = 0.012) than in Legionella-negative samples. Of 47 bioaerosol samples, 19.1% (9/47) were positive for FLA and 10.6% (5/47) for L. pneumophila. Composts (62.8%) were positive for Legionella and FLA contemporaneously, but both microorganisms were never detected simultaneously in bioaerosols. Compost can release bioaerosol containing FLA or Legionella and could represent a source of infection of community-acquired Legionella infections for workers and nearby residents.     

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 Common Drinking Water Disinfectants,Chlorine and Heat,on Free Legionella and Amoebae-Associated Legionella

Chlorine and thermal treatments are the most commonly used procedures to control and prevent Legionella proliferation in drinking water systems of large buildings. However, cases of legionellosis still occur in facilities with treated water. The purpose of this work was to model the effect of temperature and free chlorine applied in similar exposure conditions as in drinking water systems on five Legionella spp. strains and two amoebal strains of the genera Acanthamoeba. Inactivation models obtained were used to determine the effectiveness of the treatments applied which resulted more effective against Legionella than Acanthamoeba, especially those in cystic stages. Furthermore, to determine the influence of the relationship between L. pneumophila and Acanthamoeba spp. on the treatment effectiveness, inactivation models of the bacteria-associated amoeba were also constructed and compared to the models obtained for the free living bacteria state. The Legionella-amoeba association did not change the inactivation models, but it reduced the effectiveness of the treatments applied. Remarkably, at the lowest free chlorine concentration, 0.5 mg L^-1, as well as at the lowest temperatures, 50°C and 55°C, the influence of the Legionella-amoeba associate state was the strongest in reducing the effectiveness of the treatments compared to the free Legionella state. Therefore, the association established between L. pneumophila and amoebae in the water systems indicate an increased health risk in proximal areas of the system (close to the tap) where lower free chlorine concentrations and lower temperatures are commonly observed.     

Support for the coevolution of Neoparamoeba and their endosymbionts,Perkinsela amoebae-like organisms

Some of the species from the genus Neoparamoeba, for example N. perurans have been shown to be pathogenic to aquatic animals and thus have economic significance. They all contain endosymbiont, Perkinsela amoebae like organisms (PLOs). In this study we investigated phylogenetic ambiguities within the Neoparamoeba taxonomy and phylogenetic congruence between PLOs and their host Neoparamoeba to confirm the existence of a single ancient infection/colonisation that led to cospeciation between all PLOs and their host Neoparamoeba. DNA was extracted and rRNA genes from host amoeba and endosymbiont were amplified using PCR. Uncertainties in the Neoparamoeba phylogeny were initially resolved by a secondary phylogenetic marker, the internal transcribed spacer 2 (ITS2). The secondary structure of ITS2 was reconstructed for Neoparamoeba. The ITS2 was phylogenetically informative, separating N. pemaquidensis and N. aestuarina into distinct monophyletic clades and designating N. perurans as the most phylogenetically divergent Neoparamoeba species. The new phylogenetic data were used to verify the tree topologies used in cophylogenetic analyses that revealed strict phylogenetic congruence between endosymbiotic PLOs with their host Neoparamoeba. Strict congruence in the phylogeny of all PLOs and their host Neoparamoeba was demonstrated implying that PLOs are transmitted vertically from parent to daughter cell.     

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.     

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.     

The destruction of Schistosoma mansoni mother sporocysts in vitro by amoebae isolated from Biomphalaria glabrata: an ultrastructural study

Some of the ultrastructural features of the in vitro destruction of the mother sporocyst of Schistosoma mansoni by amoebae (Nuclearia sp.) isolated from the pericardial sacs of Biomphalaria glabrata are described. The interaction involves (1) the attachment of the amoeba plasma membrane to the tegumental membrane of the sporocyst, (2) penetration of the tegument by a single cell process containing only a finely granular cytoplasm, (3) the lytic destruction of the sporocyst tissues, and (4) their removal by phagocytosis. Since the amoebae retain their lysosomal complement, the mechanisms of sporocyst penetration and lysis are unknown. Myelin figures and multivesiculate and residual bodies are frequently seen in amoebal secondary lysosomes. Cell organelles and body components of the sporocysts are phagocytosed by amoebae attached to their surfaces, located inside the body or elsewhere in the vicinity of the disintegrating sporocyst.     

Legionella Species Diversity in an Acidic Biofilm Community in Yellowstone National Park

Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30°C) and higher (35 to 38°C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.     

Infection of Acanthamoeba polyphaga with Simkania negevensis and S. negevensis Survival within Amoebal Cysts

Simkania negevensis, a novel microorganism belonging to the family Simkaniaceae in the order Chlamydiales, has an intracellular developmental cycle during which two morphological entities, elementary bodies (EB) and reticulate bodies (RB), are seen by electron microscopy. Rates of seropositivity to the organism are high in certain population groups, and S. negevensis has been associated with respiratory illness in humans. This study reports for the first time the ability of S. negevensis to survive and grow inside Acanthamoeba polyphaga in addition to its known ability to grow in cell cultures of human or simian origin. Infectivity of S. negevensis and growth in amoebae were monitored by immunoperoxidase assays. Long-term persistence and exponential growth of S. negevensis in amoebal trophozoites were demonstrated by infectivity assays and by electron microscopy. EB and dividing RB of S. negevensis were observed within inclusion bodies inside A. polyphaga. When S. negevensis-infected A. polyphaga amoebae were exposed to adverse conditions resulting in encystation of the amoebae, several possible outcomes were observed: cysts containing both normal amoebic cytoplasm and S. negevensis; cysts in which S. negevensis cells were relegated to the space between cyst walls; and cysts containing S. negevensis, but apparently lacking amoebal cytoplasm. S. negevensis within dried amoebal cysts was capable of long-term survival. The possibility that amoebae may have a role in natural transmission of S. negevensis needs to be investigated.     

Schistosome sporocyst-killing Amoebae isolated from Biomphalaria glabrata.

Explants and swabs from the pericardium and mantle of three strains of Biomphalaria glabrata, two of them resistant to infection with Schistosoma mansoni, have yielded small amoebae, 3–5μm in diameter, in culture. These amoebae have been grown axenically through > 50 passages to date. The amoebae form cysts in dense cultures. When mixed with S. mansoni mother sporocysts in vitro, the amoebae adhere to and kill the trematodes within several hours. For 1–2 days thereafter, the amoebae proliferate rapidly at a generation time of about 5 hr, then return to normal growth. Sonically disrupted sporocysts also induce proliferation. Live sporocysts do not attract the amoebae or emit soluble substances which influence amoebal growth. Amoebae also adhered to and killed S. mansoni daughter sporocysts and cells derived from B. glabrata embryos; however, they did not harm S. mansoni cercariae or rediae of other trematode species. The proportion of mantle explants yielding amoebae was significantly higher (P<0.05) in one of the resistant snail strains than in the susceptible strain; however, whether amoebae contribute to snail resistance is unknown. Exposure of snails to S. mansoni miracidia did not influence the proportion of snails yielding amoebae.     

Impact of Non-Legionella Bacteria on the Uptake and Intracellular Replication of Legionella pneumophila in Acanthamoeba castellanii and Naegleria lovaniensis

In aquatic environments, Legionella pneumophila survives, in association with other bacteria, within biofilms by multiplying in free-living amoebae. The precise mechanisms underlying several aspects of the uptake and intracellular replication of L. pneumophila in amoebae, especially in the presence of other bacteria, remain unknown. In the present study, we examined the competitive effect of selected non-Legionella bacteria (Escherichia coli, Aeromonas hydrophila, Flavobacterium breve, and Pseudomonas aeruginosa) on the uptake of L. pneumophila serogroup 1 by the amoebae Acanthamoeba castellanii and Naegleria lovaniensis. We also investigated their possible influence on the intracellular replication of L. pneumophila in both amoeba species. Our results showed that the non-Legionella bacteria did not compete with L. pneumophila for uptake, suggesting that the amoeba hosts took in L. pneumophila through a specific and presumably highly efficient uptake mechanism. Living and heat-inactivated P. aeruginosa best supported the replication of L. pneumophila in N. lovaniensis and A. castellanii, respectively, whereas for both amoeba species, E. coli yielded the lowest number of replicated L. pneumophila. Furthermore, microscopic examination showed that 100% of the A. castellanii and only 2% of the N. lovaniensis population were infected with L. pneumophila at the end of the experiment. This study clearly shows the influence of some non-Legionella bacteria on the intracellular replication of L. pneumophila in A. castellanii and N. lovaniensis. It also demonstrates the different abilities of the two tested amoeba species to serve as a proper host for the replication and distribution of the human pathogen in man-made aquatic environments such as cooling towers, shower heads, and air conditioning systems with potential serious consequences for human health.     

Bodo saltans (Kinetoplastida) is dependent on a novel Paracaedibacter-like endosymbiont that possesses multiple putative toxin-antitoxin systems

Bacterial endosymbiosis has been instrumental in eukaryotic evolution, and includes both mutualistic, dependent and parasitic associations. Here we characterize an intracellular bacterium inhabiting the flagellated protist Bodo saltans (Kinetoplastida). We present a complete bacterial genome comprising a 1.39 Mb circular chromosome with 40.6% GC content. Fluorescent in situ hybridisation confirms that the endosymbiont is located adjacent to the nuclear membrane, and a detailed model of its intracellular niche is generated using serial block-face scanning electron microscopy. Phylogenomic analysis shows that the endosymbiont belongs to the Holosporales, most closely related to other α-proteobacterial endosymbionts of ciliates and amoebae. Comparative genomics indicates that it has a limited metabolism and is nutritionally host-dependent. However, the endosymbiont genome does encode diverse symbiont-specific secretory proteins, including a type VI secretion system and three separate toxin-antitoxin systems. We show that these systems are actively transcribed and hypothesize they represent a mechanism by which B. saltans becomes addicted to its endosymbiont. Consistent with this idea, attempts to cure Bodo of endosymbionts led to rapid and uniform cell death. This study adds kinetoplastid flagellates to ciliates and amoebae as hosts of Paracaedibacter-like bacteria, suggesting that these antagonistic endosymbioses became established very early in Eukaryotic evolution.Subject terms: Microbial ecology, Bacteria     

A Distinct and Divergent Lineage of Genomic Island-Associated Type IV Secretion Systems in Legionella

Legionella encodes multiple classes of Type IV Secretion Systems (T4SSs), including the Dot/Icm protein secretion system that is essential for intracellular multiplication in amoebal and human hosts. Other T4SSs not essential for virulence are thought to facilitate the acquisition of niche-specific adaptation genes including the numerous effector genes that are a hallmark of this genus. Previously, we identified two novel gene clusters in the draft genome of Legionella pneumophila strain 130b that encode homologues of a subtype of T4SS, the genomic island-associated T4SS (GI-T4SS), usually associated with integrative and conjugative elements (ICE). In this study, we performed genomic analyses of 14 homologous GI-T4SS clusters found in eight publicly available Legionella genomes and show that this cluster is unusually well conserved in a region of high plasticity. Phylogenetic analyses show that Legionella GI-T4SSs are substantially divergent from other members of this subtype of T4SS and represent a novel clade of GI-T4SSs only found in this genus. The GI-T4SS was found to be under purifying selection, suggesting it is functional and may play an important role in the evolution and adaptation of Legionella. Like other GI-T4SSs, the Legionella clusters are also associated with ICEs, but lack the typical integration and replication modules of related ICEs. The absence of complete replication and DNA pre-processing modules, together with the presence of Legionella-specific regulatory elements, suggest the Legionella GI-T4SS-associated ICE is unique and may employ novel mechanisms of regulation, maintenance and excision. The Legionella GI-T4SS cluster was found to be associated with several cargo genes, including numerous antibiotic resistance and virulence factors, which may confer a fitness benefit to the organism. The in-silico characterisation of this new T4SS furthers our understanding of the diversity of secretion systems involved in the frequent horizontal gene transfers that allow Legionella to adapt to and exploit diverse environmental niches.