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

Gomez-Valero L, Rusniok C, Rolando M, Neou M, Dervins-Ravault D, Demirtas J, Rouy Z, Moore RJ, Chen H, Petty NK, Jarraud S, Etienne J, Steinert M, Heuner K, Gribaldo S, Médigue C, Glöckner G, Hartland EL, Buchrieser C - Genome Biol. (2014)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

ABSTRACT

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.

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Intracellular replication ofL. hackeliae,L. micdadeiandL. fallonii(LLAP10). (A) THP-1 derived macrophages at 37°C. (B)A. castellanii culture at 20°C. (C)A. castellanii plate test at 37°C and 30°C. L. pneumophila strain Paris wild type (wt) and ∆dotA were used as positive and negative controls, respectively. Intracellular replication for each strain was determined by recording the number of colony-forming units (CFU) through plating on BCYE agar. Blue, L. pneumophila strain Paris; red, ∆dotA; orange, L. micdadei; violet, L. hackeliae; green, L. fallonii (LLAP10). Results are expressed as Log10 ratio CFU Tn/T0 and each point represents the mean ± standard deviation of two or three independent experiments. The error bars represent the standard deviation, but some were too small to clearly appear in the figure.
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Fig1: Intracellular replication ofL. hackeliae,L. micdadeiandL. fallonii(LLAP10). (A) THP-1 derived macrophages at 37°C. (B)A. castellanii culture at 20°C. (C)A. castellanii plate test at 37°C and 30°C. L. pneumophila strain Paris wild type (wt) and ∆dotA were used as positive and negative controls, respectively. Intracellular replication for each strain was determined by recording the number of colony-forming units (CFU) through plating on BCYE agar. Blue, L. pneumophila strain Paris; red, ∆dotA; orange, L. micdadei; violet, L. hackeliae; green, L. fallonii (LLAP10). Results are expressed as Log10 ratio CFU Tn/T0 and each point represents the mean ± standard deviation of two or three independent experiments. The error bars represent the standard deviation, but some were too small to clearly appear in the figure.

Mentions: Little to nothing is known about the environmental distribution and the virulence of different Legionella species for human cells. Similarly, it is not known why L. pneumophila and L. longbeachae are so predominant in human disease compared with other Legionella species. As a first step to understand these differences we analyzed the capacity of L. micdadei, L. hackeliae and L. fallonii to infect the protozoan species Acanthamoeba castellanii and the human monocytic cell line THP-1. As shown in Figure 1A, L. micdadei replicated in THP-1 cells, similar to L. pneumophila, while L. fallonii and L. hackeliae were unable to replicate in these cells, although they are phagocytosed efficiently as seen from the higher numbers entering the cells after one hour of infection (Figure 1A). In contrast, L. fallonii was able to replicate in A. castellanii (Figure 1B). However, neither L. hackeliae nor L. micdadei replicated in this amoeba. Thus, additional experiments are necessary to analyze whether A. castellani is their environmental host or not (Figure 1B). Similar results have been obtained using Dictyostelium discoideum as a host where L. micdadei can replicate in this model amoeba but L. hackeliae cannot [6]. In contrast, it was reported that L. micdadei is able to replicate in A. castellani [6,18]. Puzzled by these contradicting results we further analyzed the infection capacity of L. micdadei. Our infection assays had been carried out at 20°C whereas Hägele and colleagues [6] performed their infections at 30°C. We thought that the different results might be due to the different temperatures used. We thus carried out infection assays at 30°C and also used amoeba plate testing [19] at 37°C and 30°C (Figure 1C). Indeed, L. micdadei was able to replicate in A. castellani at 37°C and also at 30°C, although to a lesser extent compared with L. pneumophila (Additional file 1). This suggested that the replication capacity of L. micdadei in A. castellanii is temperature dependent.Figure 1


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

Gomez-Valero L, Rusniok C, Rolando M, Neou M, Dervins-Ravault D, Demirtas J, Rouy Z, Moore RJ, Chen H, Petty NK, Jarraud S, Etienne J, Steinert M, Heuner K, Gribaldo S, Médigue C, Glöckner G, Hartland EL, Buchrieser C - Genome Biol. (2014)

Intracellular replication ofL. hackeliae,L. micdadeiandL. fallonii(LLAP10). (A) THP-1 derived macrophages at 37°C. (B)A. castellanii culture at 20°C. (C)A. castellanii plate test at 37°C and 30°C. L. pneumophila strain Paris wild type (wt) and ∆dotA were used as positive and negative controls, respectively. Intracellular replication for each strain was determined by recording the number of colony-forming units (CFU) through plating on BCYE agar. Blue, L. pneumophila strain Paris; red, ∆dotA; orange, L. micdadei; violet, L. hackeliae; green, L. fallonii (LLAP10). Results are expressed as Log10 ratio CFU Tn/T0 and each point represents the mean ± standard deviation of two or three independent experiments. The error bars represent the standard deviation, but some were too small to clearly appear in the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4256840&req=5

Fig1: Intracellular replication ofL. hackeliae,L. micdadeiandL. fallonii(LLAP10). (A) THP-1 derived macrophages at 37°C. (B)A. castellanii culture at 20°C. (C)A. castellanii plate test at 37°C and 30°C. L. pneumophila strain Paris wild type (wt) and ∆dotA were used as positive and negative controls, respectively. Intracellular replication for each strain was determined by recording the number of colony-forming units (CFU) through plating on BCYE agar. Blue, L. pneumophila strain Paris; red, ∆dotA; orange, L. micdadei; violet, L. hackeliae; green, L. fallonii (LLAP10). Results are expressed as Log10 ratio CFU Tn/T0 and each point represents the mean ± standard deviation of two or three independent experiments. The error bars represent the standard deviation, but some were too small to clearly appear in the figure.
Mentions: Little to nothing is known about the environmental distribution and the virulence of different Legionella species for human cells. Similarly, it is not known why L. pneumophila and L. longbeachae are so predominant in human disease compared with other Legionella species. As a first step to understand these differences we analyzed the capacity of L. micdadei, L. hackeliae and L. fallonii to infect the protozoan species Acanthamoeba castellanii and the human monocytic cell line THP-1. As shown in Figure 1A, L. micdadei replicated in THP-1 cells, similar to L. pneumophila, while L. fallonii and L. hackeliae were unable to replicate in these cells, although they are phagocytosed efficiently as seen from the higher numbers entering the cells after one hour of infection (Figure 1A). In contrast, L. fallonii was able to replicate in A. castellanii (Figure 1B). However, neither L. hackeliae nor L. micdadei replicated in this amoeba. Thus, additional experiments are necessary to analyze whether A. castellani is their environmental host or not (Figure 1B). Similar results have been obtained using Dictyostelium discoideum as a host where L. micdadei can replicate in this model amoeba but L. hackeliae cannot [6]. In contrast, it was reported that L. micdadei is able to replicate in A. castellani [6,18]. Puzzled by these contradicting results we further analyzed the infection capacity of L. micdadei. Our infection assays had been carried out at 20°C whereas Hägele and colleagues [6] performed their infections at 30°C. We thought that the different results might be due to the different temperatures used. We thus carried out infection assays at 30°C and also used amoeba plate testing [19] at 37°C and 30°C (Figure 1C). Indeed, L. micdadei was able to replicate in A. castellani at 37°C and also at 30°C, although to a lesser extent compared with L. pneumophila (Additional file 1). This suggested that the replication capacity of L. micdadei in A. castellanii is temperature dependent.Figure 1

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus