Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires

Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A.; Pupko, Tal; Shuman, Howard A.; Segal, G. M.

doi:10.1038/ng.3481

Cited by 243 publications

(411 citation statements)

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“…In both amoebae and macrophages, the bacterium evades the host's degradative lysosomal pathway and replicates to large numbers within a membrane-bound vacuole, the Legionella-containing vacuole (LCV) (15,16). The Dot/Icm type IV secretion (T4S) system of L. pneumophila plays a major role in pathogenesis (17)(18)(19). Indeed, the Dot/Icm apparatus translocates myriad proteins across the LCV membrane and into the host cell cytoplasm, and then the delivered effectors proceed to modulate a wide variety of host processes.…”

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Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

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2016

Infect Immun

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Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages by Legionella pneumophila. In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replication between 4 and 8 h postentry and to grow to large numbers in the Legionella-containing vacuole (LCV), as evident at 12 h. At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum (ER)-derived vesicles to LCVs. The mutant did not lose expression or translocation of six type IV secretion effectors (e.g., SidM) that are well known for mediating Rab1B association with the LCV, indicating that T2S promotes the interaction between the LCV and Rab1B via a novel mechanism. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA (shRNA) treatment, indicating that T2S also potentiates events beyond Rab1B association. In support of this, a sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant (and a sidM mutant) and showed a growth difference of as much as a 400-fold compared to the wild type. Together, these data reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes. L egionella pneumophila is widespread in natural and man-made water systems and is best known as the etiologic agent of Legionnaires' disease (1-3). Recently, the incidence of pneumonias caused by Gram-negative L. pneumophila has roughly tripled in the United States and elsewhere (4-6). In water, L. pneumophila grows primarily as an intracellular parasite of amoebae (7-11). Human infection occurs mainly by the inhalation of contaminated water droplets originating from a wide range of aerosolgenerating devices (12,13). Yet the first case of person-to-person spread was reported in 2016 (14). In the lungs, L. pneumophila invades and grows in the resident macrophage population and ultimately triggers severe inflammation (3). In both amoebae and macrophages, the bacterium evades the host's degradative lysosomal pathway and replicates to large numbers within a membrane-bound vacuole, the Legionella-containing vacuole (LCV) (15, 16). The Dot/Icm type IV secretion (T4S) system of L. pneumophila plays a major role in pathogenesis (17-19). Indeed, the Dot/Icm apparatus translocates myriad proteins across the LCV membrane and into the host cell cytoplasm, and then the delivered effectors proceed to modulate a wide variety of host processes. One of the earliest and most notable events that has been ascribed to the Dot/Icm system is the recruitment of host GTPases and endoplasmic reticulu...

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Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

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2016

Infect Immun

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“…Interestingly, L. adelaidensis and L. londiniensis, which form a separate phylogenetic cluster with L. oakridgensis (32), are also flagellar regulon negative and still exhibit the presence of fliA and flgM genes within their genomes. Since L. longbeachae and L. hackeliae are also nonflagellated and negative for most of the genes of the flagellar regulon (8, 10, 34), representing another major clade of the Legionella genus, the loss of genes of the flagellar regulon did not take place as a single event during the evolution of legionellae.…”

Section: Discussionmentioning

confidence: 99%

Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis

et al. 2017

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Legionella oakridgensis causes Legionnaires' disease but is known to be less virulent than Legionella pneumophila. L. oakridgensis is one of the Legionella species that is nonflagellated. The genes of the flagellar regulon are absent, except those encoding the alternative sigma-28 factor (FliA) and its anti-sigma-28 factor (FlgM). Similar to L. oakridgensis, Legionella adelaidensis and Legionella londiniensis, located in the same phylogenetic clade, have no flagellar regulon, although both are positive for fliA and flgM. Here, we investigated the role and function of both genes to better understand the role of FliA, the positive regulator of flagellin expression, in nonflagellated strains. We demonstrated that the FliA gene of L. oakridgensis encodes a functional sigma-28 factor that enables the transcription start from the sigma-28-dependent promoter site. The investigations have shown that FliA is necessary for full fitness of L. oakridgensis. Interestingly, expression of FliA-dependent genes depends on the growth phase and temperature, as already shown for L. pneumophila strains that are flagellated. In addition, we demonstrated that FlgM is a negative regulator of FliA-dependent gene expression. FlgM seems to be degraded in a growth-phase-and temperature-dependent manner, instead of being exported into the medium as reported for most bacteria. The degradation of FlgM leads to an increase of FliA activity.IMPORTANCE A less virulent Legionella species, L. oakridgensis, causes Legionnaires' disease and is known to not have flagella, even though L. oakridgensis has the regulator of flagellin expression (FliA). This protein has been shown to be involved in the expression of virulence factors. Thus, the strain was chosen for use in this investigation to search for FliA target genes and to identify putative virulence factors of L. oakridgensis. One of the five major target genes of FliA identified here encodes the anti-FliA sigma factor FlgM. Interestingly, in contrast to most homologs in other bacteria, FlgM in L. oakridgensis seems not to be transported from the cell so that FliA gets activated. In L. oakridgensis, FlgM seems to be degraded by protease activities.KEYWORDS sigma-28 factor, anti-sigma factor, FliA, FlgM, flagella, Legionella oakridgensis L egionella oakridgensis is less virulent than L. pneumophila and is pathogenic for guinea pigs (1, 2). Recently, two human cases of Legionnaires' disease caused by L. oakridgensis were reported in France (3), showing that the bacteria are able to replicate inside human cells (1,(4)(5)(6)(7). Moreover, it was demonstrated that L. oakridgensis is able to multiply inside amoebae (4) and that the type IV secretion system is a key virulence factor; new virulence factors were also described (4). In general, no additional cysteine is needed to grow L. oakridgensis on agar plates and extracel-

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“…As described above (Fig. 2), about 20 Legionella species belonging to the L. micdadei clade (27) examined its effect on the levels of expression of two PmrA-regulated effectorencoding genes, ceg19 and ceg21. The expression of the L. micdadei ptsN gene in L. pneumophila resulted in a reduction in the levels of expression of both the ceg19 and ceg21 lacZ fusions, and the reduction strengthened as the concentration of the added IPTG increased (Fig.…”

Section: Figmentioning

confidence: 99%

“…Therefore, we decided to examine the possibility that one of these systems is regulated by the PTS Ntr and leads to the intracellular growth phenotype observed. To this end, genes known to be regulated by these two-component regulatory systems were examined: (i) an effectorencoding gene (cegC3) and an Icm/Dot-encoding gene (icmR) activated by the CpxRA (27). For each species, the presence (gray) or absence (white) of the ptsP, ptsO, and ptsN genes of the PTS Ntr is indicated.…”

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The Legionella pneumophila Incomplete Phosphotransferase System Is Required for Optimal Intracellular Growth and Maximal Expression of PmrA-Regulated Effectors

et al. 2017

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The nitrogen phosphotransferase system (PTS Ntr ) is a regulatory cascade present in many bacteria, where it controls different functions. This system is usually composed of three basic components: enzyme I Ntr (EI Ntr ), NPr, and EIIA Ntr (encoded by the ptsP, ptsO, and ptsN genes, respectively). In Legionella pneumophila, as well as in many other Legionella species, the EIIA Ntr component is missing. However, we found that deletion mutations in both ptsP and ptsO are partially attenuated for intracellular growth. Furthermore, these two PTS Ntr components were found to be required for maximal expression of effector-encoding genes regulated by the transcriptional activator PmrA. Genetic analyses which include the construction of single and double deletion mutants and overexpression of wild-type and mutated forms of EI Ntr , NPr, and PmrA indicated that the PTS Ntr components affect the expression of PmrA-regulated genes via PmrA and independently from PmrB and that EI Ntr and NPr are part of the same cascade and require their conserved histidine residues in order to function. Furthermore, expression of the Legionella micdadei EII Ntr component in L. pneumophila resulted in a reduction in the levels of expression of PmrAregulated genes which was completely dependent on the L. pneumophila PTS components and the L. micdadei EII Ntr conserved histidine residue. Moreover, reconstruction of the L. pneumophila PTS in vitro indicated that EI Ntr is phosphorylated by phosphoenolpyruvate (PEP) and transfers its phosphate to NPr. Our results demonstrate that the L. pneumophila incomplete PTS Ntr is functional and involved in the expression of effector-encoding genes regulated by PmrA.

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Genomic analysis of 38 Legionella species identifies large and diverse effector repertoires

Cited by 243 publications

References 83 publications

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms

Functional Analysis of the Alternative Sigma-28 Factor FliA and Its Anti-Sigma Factor FlgM of the Nonflagellated Legionella Species L. oakridgensis

The Legionella pneumophila Incomplete Phosphotransferase System Is Required for Optimal Intracellular Growth and Maximal Expression of PmrA-Regulated Effectors

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