Virulence factors encoded by Legionella longbeachae identified on the basis of the genome sequence analysis of clinical isolate D-4968

Abstract

Legionella longbeachae causes most cases of legionellosis in Australia and may be underreported worldwide due to the lack of L. longbeachae-specific diagnostic tests. L. longbeachae displays distinctive differences in intracellular trafficking, caspase 1 activation, and infection in mouse models compared to Legionella pneumophila, yet these two species have indistinguishable clinical presentations in humans. Unlike other legionellae, which inhabit freshwater systems, L. longbeachae is found predominantly in moist soil. In this study, we sequenced and annotated the genome of an L. longbeachae clinical isolate from Oregon, isolate D-4968, and compared it to the previously published genomes of L. pneumophila. The results revealed that the D-4968 genome is larger than the L. pneumophila genome and has a gene order that is different from that of the L. pneumophila genome. Genes encoding structural components of type II, type IV Lvh, and type IV Icm/Dot secretion systems are conserved. In contrast, only 42/140 homologs of genes encoding L. pneumophila Icm/Dot substrates have been found in the D-4968 genome. L. longbeachae encodes numerous proteins with eukaryotic motifs and eukaryote-like proteins unique to this species, including 16 ankyrin repeat-containing proteins and a novel U-box protein. We predict that these proteins are secreted by the L. longbeachae Icm/Dot secretion system. In contrast to the L. pneumophila genome, the L. longbeachae D-4968 genome does not contain flagellar biosynthesis genes, yet it contains a chemotaxis operon. The lack of a flagellum explains the failure of L. longbeachae to activate caspase 1 and trigger pyroptosis in murine macrophages. These unique features of L. longbeachae may reflect adaptation of this species to life in soil.

FIG. 1.

Comparison of the genomes of L. longbeachae and L. pneumophila: Mauve alignment of two scaffolds of the L. longbeachae D-4968 genome with the genomes of L. pneumophila strains Corby, Lens, Paris, and Philadelphia-1 presented horizontally. The colored boxes represent homologous segments completely free of genomic rearrangements. These homologous regions are connected by lines between genomes. Blocks below the center line indicate regions with inverse orientation. Regions outside blocks lack homology between genomes. Within each block there is a similarity profile of the DNA sequences, and white areas indicate the sequences specific to a genome. The Corby strain has more large-scale genomic rearrangements than the Lens, Paris, and Philadelphia-1 strains, which are almost colinear. The D-4968 genome composition is substantially different from that of all L. pneumophila genomes.

FIG. 2.

Model for the regulatory network of L. longbeachae. This model is based on proposed L. pneumophila regulatory cascades (1, 44, 49, 64, 69, 78, 85) and the availability of homologs of L. pneumophila regulators in the D-4968 genome. When nutrients are abundant inside the cell, CsrA represses transmission traits and promotes replication. Nutrient starvation stimulates the RelA and SpoT enzymes to produce the alarmone ppGpp. A high alarmone concentration activates the major regulator RpoS, which in turn activates the two-component LetA/S system. When stimulated, LetA/S activates transcription of the small noncoding RNAs RsmY and RsmZ, which sequester CsrA and stop its repressor activity, resulting in expression of transmission traits. Specifically, elimination of CsrA repression activates the master regulator FleQ, which, together with the alternative sigma factor RpoN, activates expression of fleN and fleSR. FleN may be an anti-activator of FleQ. FleS/R is a two-component regulatory system in which the sensor kinase FleS activates the response regulator FleR by phosphorylation. In turn, FleR∼P, together with RpoN, induces expression of the fliA and flgM genes. The alternative sigma factor FliA may positively regulate expression of genes involved in cell invasion (49, 63). High levels of ppGpp may also lead to activation of the Dot/Icm type IV secretion system regulators PmrA and CpxR.

FIG. 3.

Type IVA Lvh secretion system: schematic diagram of the integrated form of the putative plasmid-like element. The gray boxes indicate the predicted att sites, and the arrows indicate the open reading frames flanking these sites. The black rectangle indicates the position of the lvh locus.

FIG. 4.

Organization and open reading frames of the Icm/Dot genomic regions in L. longbeachae (Llb) (regions I, II, and III) and L. pneumophila (Lp) (regions I and II). The icm/dot genes shared by the two species are indicated by black arrows. Functional homologs ligB and icmR are indicated by striped arrows. Sequences adjacent to the Icm/Dot genes that are homologous in the two species are indicated by gray arrows, whereas genes that do not have a homolog in this vicinity are indicated by open arrows. The sequence encoding the 370-amino-acid region specific to L. longbeachae IcmE (see text) is indicated by a striped box. The LLB_2545 and LLB_2546 genes encoding a putative sensor kinase and a response regulator, respectively, of a two-component signal transduction system are proposed to be involved in icm/dot regulation and are indicated by checkered arrows.

FIG. 5.

Comparison of the genomic regions containing flagellar operons in L. pneumophila (Lp) with the corresponding regions in L. longbeachae (Llb). Flagellar genes shared by the two species are indicated by black arrows. L. pneumophila flagellar genes that do not have counterparts in L. longbeachae are indicated by striped arrows. Sequences adjacent to flagellar operon genes that are homologous in the two species are indicated by gray arrows, whereas genes that do not have homologs in this vicinity are indicated by open arrows. The enhA homologs LLB_0870 and LLB_3030, which are proposed to constitute the FliA regulon in D-4968 (see text), are indicated by bold type.

FIG. 6.

Organization and open reading frames of the L. longbeachae chemotaxis operon and comparison of the genomic region containing this operon with the homologous genomic region in L. pneumophila. Chemotaxis genes are indicated by striped arrows. Sequences adjacent to chemotaxis operon genes that are homologous in the two species are indicated by gray arrows, whereas genes that do not have a homolog in this vicinity are indicated by open arrows. MCP, methyl accepting chemotaxis protein. LLB_2002 and LLB_2292 that encode CheY-like proteins and may function together with the gene products of the chemotaxis operon are indicated by checkered arrows.