Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryotic-like proteins

doi:10.1016/j.mib.2005.12.009

Current Opinion in Microbiology

Volume 9, Issue 1, February 2006, Pages 86-94

https://doi.org/10.1016/j.mib.2005.12.009 Get rights and content

The intracellular pathogen Legionella pneumophila has evolved sophisticated mechanisms that enable it to subvert host functions, enter, survive and replicate in amoebae or alveolar macrophages, and to finally evade these hosts. Protozoa are essential for the growth of Legionella and the interaction with amoeba seems to be the driving force in the evolution of its pathogenicity. This is reflected in the genome of this pathogen, which encodes a high number and variety of eukaryotic-like proteins that are able to interfere in the various steps of the infectious cycle by mimicking functions of eukaryotic proteins. Central to the pathogenicity of L. pneumophila are the many secretion systems delivering these and other effectors to the host cell. Recent studies have highlighted the multi-functional role of these factors secreted by L. pneumophila, in host–pathogen interactions.

Introduction

Legionella pneumophila is the causative agent of the pneumonia-like Legionnaires’ disease. The bacterium's survival and spread depends on its ability to replicate inside eukaryotic phagocytic cells. Legionella has a dual host system allowing intracellular growth in protozoa, such as Acanthamoeba castellanii, Hartmanella sp. or Naeglaria sp., and in human alveolar macrophages. It has been speculated that the interaction of L. pneumophila with aquatic protozoa generated a pool of virulence traits during evolution that enables Legionella to also infect human cells. Upon internalization into the eukaryotic cell, L. pneumophila ensures its survival by manipulating host cell functions (e.g. disrupting vesicle trafficking), thereby reprogramming the endosomal-lysosomal degradation pathway of the phagocytic cell.

L. pneumophila research has increased during recent years as a result of the understanding that Legionella-host interactions reveal not only pivotal bacterial mechanisms of subverting host cell functions, but also gives valuable insight into the elementary cellular functions of phagocytic cells. Recent advances in Legionella research have been achieved by creating sophisticated experimental designs, such as specific two-hybrid screens [1^••] and Legionella gene expression in the yeast genetic system [2•, 3•], which identified new bacterial virulence traits, and by deciphering the genomes of three strains of L. pneumophila. These strains are Paris, Lens [4^••] and Philadelphia 1 [5]. Philadelphia 1 is derived from the original isolate [6] obtained from a patient who died from Legionnaires’ disease at the Legionnaires’ Convention in 1976. Paris is an abundant strain in France and Europe and it accounts for 12.7% of cases of legionellosis in France and 33% of those that occur in the Paris area [7]. It is associated with hospital- and community-acquired disease and is the only recognized endemic L. pneumophila strain; indicating it has particular adaptations to its environment or host. Lens is an epidemic strain, which, from November 2003 to January 2004, caused an outbreak in France, with 86 cases, resulting in 17 deaths [8], suggesting it is an adept disease-causing strain. It was isolated in January 2004 and sequenced in February 2004, thus minimizing possible changes to the genome between isolation and sequencing. The genome sequences have revealed a variety of features unique to Legionella, such as the array of eukaryotic-like proteins, likely to be candidates involved in interfering in phagocytic functions. However, despite recent progress, the precise understanding of how L. pneumophila manipulates host cell functions on the mechanistic level is still in its infancy.

Section snippets

Discovery of new virulence determinates of L. pneumophila

Upon entry into amoebae or macrophages, L. pneumophila has to act rapidly to prevent delivery of Legionella-containing phagosomes to lysosomes, which would result in bacterial degradation. Indeed, it has been shown that L. pneumophila is capable of altering its phagosomal environment within minutes of internalization [9^•]. It modifies the trafficking properties of the phagosome; avoiding entry into the host endocytic pathway by preventing immediate phagosome–lysosome fusion and by constructing

Central to pathogenesis — substrates of the Dot/Icm secretion system

In several pathogens, such as Agrobacterium, Bordetella, Helicobacter and the intracellular pathogens Chlamydia, Coxiella, Brucella and Legionella, type IV secretion systems are essential for delivery of host cell-modulating effectors. In the case of L. pneumophila, the type IV secretion system, designated Dot/Icm [10, 11], is indispensable for virulence and thus has been studied extensively. The Dot/Icm system appears to act during phagocytosis to prevent phagosome–lysosome fusion and to

Dot/Icm-independent secretion systems — implication in virulence

Until recently, the second L. pneumophila type IV secretion system, designated Lvh (for Legionella vir homologs), was poorly characterized. Its 11 genes are located on a genomic-island-like region, which appears to be horizontally acquired, as it has a different G+C content compared with the backbone chromosome. Although the Lvh system is dispensable for intracellular growth in HL-60-derived human macrophages and in A. castellanii [21], the system might play a role in the efficiency of host

Adaptation to the eukaryotic host — new putative virulence factors

Numerous proteins, other than those comprising the secretion systems and their substrates, play a role in the ability of L. pneumophila to replicate intracellularly and to cause disease. Such factors are involved in adherence, iron sequestration, hydrogen peroxide decomposition, and amino acid biosynthesis and transport. It is beyond the scope of this review to discuss all of these factors; however, a recently identified subfamily of major facilitator superfamily (MFS) transporters, called Pht

Conclusions and perspectives

The association between L. pneumophila and its hosts has been shaped by extensive co-evolution and offers a fascinating example of how pathogens can exploit host cell functions to their advantage. During recent years, there has been remarkable progress in our understanding of the complex interactions between L. pneumophila and its hosts. In particular, the recent analysis of the genome sequence of three L. pneumophila strains identified a high number of proteins that might be involved in these

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

This work received financial support from the Institut Pasteur. H Brüggemann is holder of a fellowship of the German Academy of Natural Scientists Leopoldina funded by the German Ministry of Education and Research (funding number BMBF-LPD9901/8-101) and C Cazalet is holder of a fellowship jointly financed by the (CNRS) France and VeoliaWater - Anjou Recherche.

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Notably, the finding that Ser serves as a carbon source for intracellular Lp at increasing rates during the late growth phase was partially in contrast to data from earlier in vitro experiments where Ser was the preferred carbon substrate during the early phase of growth (Gillmaier et al., 2016). However, this apparent discrepancy could be explained by the induction of a serine transport protein (Lpp2269) during the transmissive phase of intracellular growth (Brüggemann et al., 2006) resulting in the increased usage of 13C-Ser during the late phase of the intracellular life cycle. In the experiment with [U-13C6]glucose, the detected 13C-excess in 3-hydroxybutyrate from the F2 fraction (as the proxy for Lp-specific PHB) again indicated that the external 13C-glucose was transported into the LCV where it was used as a carbon source for bacterial metabolism (Fig. 5A, B, Supplemental Tables 1, 3, 5, 7, 9, 11, 25, 27).
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Legionella spp. then rely on their Dot/Icm (defective organelle trafficking/intracellular multiplication) type IV secretion system to translocate different effector proteins into host cells, some of which anchor to the Legionella containing vacuole by binding to phosphoinositide (PI) lipids (Hilbi et al., 2011; Haneburger and Hilbi, 2013). The effector proteins of Legionella spp. manipulate the host cell functions including the protozoan's phagocytic mechanisms and in this manner Legionella spp. alter the innate endosomal-lysosomal degradation pathway (Brüggemann et al., 2006) and avoid degradation by the amoeba (Clemens et al., 2000a, 2000b; Brüggemann et al., 2006). Literature pertaining to the Dot/Icm type IV secretion system of L. pneumophila is extensive, with more than 300 Dot/Icm substrates identified (Hubber and Roy, 2010; Lifshitz et al., 2013) and the mechanisms of 15% of these substrates have been characterized (Allombert et al., 2013; Dolinsky et al., 2014).
Legionella and Acanthamoeba spp. persist in harvested rainwater pasteurized at high temperatures (> 72 °C) and the interaction mechanisms exhibited between these organisms need to be elucidated. The resistance of two Legionella reference strains (Legionella pneumophila ATCC 33152 and Legionella longbeachae ATCC 33462), three environmental strains [Legionella longbeachae (env.), Legionella norrlandica (env.) and Legionella rowbothamii (env.)] and Acanthamoeba mauritaniensis ATCC 50676 to heat treatment (50–90 °C) was determined by monitoring culturability and viability [ethidium monoazide quantitative polymerase chain reaction (EMA-qPCR)]. The expression of metabolic and virulence genes of L. pneumophila ATCC 33152 (lolA, sidF, csrA) and L. longbeachae (env.) (lolA) in co-culture with A. mauritaniensis ATCC 50676 during heat treatment (50–90 °C) was monitored using relative qPCR. While the culturability (CFU/mL) and viability (gene copies/mL) of the Legionella strains reduced significantly (p < 0.05) following heat treatment (60–90 °C), L. longbeachae (env.) and L. pneumophila ATCC 33152 were culturable following heat treatment at 50–60 °C. Metabolically active trophozoites and dormant cysts of A. mauritaniensis ATCC 50676 were detected at 50 °C and 60–90 °C, respectively. For L. pneumophila ATCC 33152, lolA expression remained constant, sidF expression increased and the expression of csrA decreased during co-culture with A. mauritaniensis ATCC 50676. For L. longbeachae (env.), while lolA was up-regulated at 50–70 °C, expression was not detected at 80–90 °C and in co-culture. In conclusion, while heat treatment may reduce the number of viable Legionella spp. in monoculture, results indicate that the presence of A. mauritaniensis increases the virulence of L. pneumophila during heat treatment. The virulence of Legionella spp. in co-culture with Acanthamoeba spp. should thus be monitored in water distribution systems where temperature (heat) is utilized for treatment.

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Adaptation of Legionella pneumophila to the host environment: role of protein secretion, effectors and eukaryotic-like proteins

Introduction

Section snippets

Discovery of new virulence determinates of L. pneumophila

Central to pathogenesis — substrates of the Dot/Icm secretion system

Dot/Icm-independent secretion systems — implication in virulence

Adaptation to the eukaryotic host — new putative virulence factors

Conclusions and perspectives

References and recommended reading

Acknowledgements

Folia Histochem Cytobiol

Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer

Proc Natl Acad Sci USA

Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking

Proc Natl Acad Sci USA

A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system

Mol Microbiol

Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity

Nat Genet

The genomic sequence of the accidental pathogen Legionella pneumophila

Science

Legionnaires’ disease: description of an epidemic of pneumonia

N Engl J Med

Legionella pneumophila serogroup 1 strain paris: endemic distribution throughout France

J Clin Microbiol

A Community-wide outbreak of Legionnaires disease linked to industrial cooling towers — how far can contaminated aerosols spread?

J Infect Dis

Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae

Cell Microbiol

Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages

Proc Natl Acad Sci USA

Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila

Mol Microbiol

Activation of caspase-3 by the Dot/Icm virulence system is essential for arrested biogenesis of the Legionella-containing phagosome

Cell Microbiol

The Dot/lcm transporter of Legionella pneumophila: a bacterial conductor of vesicle trafficking that orchestrates the establishment of a replicative organelle in eukaryotic hosts

Int J Med Microbiol

Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake

Mol Microbiol

A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes

Science

LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway

Infect Immun

The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity

Mol Microbiol

Legionella effectors that promote nonlytic release from protozoa

Science

Identification of a novel adhesion molecule involved in the virulence of Legionella pneumophila

Infect Immun

Isolation of yeast mutants defective in protein targeting to the vacuole

Proc Natl Acad Sci USA 83