Klaus Heuner

Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila

Adaptation to the host environment and exploitation of host cell functions are critical to the success of intracellular pathogens. Here, insight to these virulence mechanisms was obtained for the first time from the transcriptional program of the human pathogen Legionella pneumophila during infection of its natural host, Acanthamoeba castellanii. The biphasic life cycle of L. pneumophila was reflected by a major shift in gene expression from replicative to transmissive phase, concerning nearly half of the genes predicted in the genome. However, three different L. pneumophila strains showed similar in vivo gene expression patterns, indicating that common regulatory mechanisms govern the Legionella life cycle, despite the plasticity of its genome. During the replicative phase, in addition to components of aerobic metabolism and amino acid catabolism, the Entner‐Doudoroff pathway, a NADPH producing mechanism used for sugar and/or gluconate assimilation, was expressed, suggesting for the first time that intracellular L. pneumophila may also scavenge host carbohydrates as nutrients and not only proteins. Identification of genes only upregulated in vivo but not in vitro, may explain higher virulence of in vivo grown L. pneumophila. Late in the life cycle, L. pneumophila upregulates genes predicted to promote transmission and manipulation of a new host cell, therewith priming it for the next attack. These including substrates of the Dot/Icm secretion system, other factors associated previously with invasion and virulence, the motility and the type IV pilus machineries, and > 90 proteins not characterized so far. Analysis of a fliA (σ28) deletion mutant identified genes coregulated with the flagellar regulon, including GGDEF/EAL regulators and factors that promote host cell entry and survival.

Molecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells

BackgroundLegionella pneumophila is the causative agent of human Legionnaires disease. During infection, the bacterium invades macrophages and lung epithelial cells, and replicates intracellularly. However, little is known about its interaction with T cells. We investigated the ability of L. pneumophila to infect and stimulate the production of interleukin-8 (IL-8) in T cells. The objective of this study was to assess whether L. pneumophila interferes with the immune system by interacting and infecting T cells.ResultsWild-type L. pneumophila and flagellin-deficient Legionella, but not L. pneumophila lacking a functional type IV secretion system Dot/Icm, replicated in T cells. On the other hand, wild-type L. pneumophila and Dot/Icm-deficient Legionella, but not flagellin-deficient Legionella or heat-killed Legionella induced IL-8 expression. L. pneumophila activated an IL-8 promoter through the NF-κB and AP-1 binding regions. Wild-type L. pneumophila but not flagellin-deficient Legionella activated NF-κB, p38 mitogen-activated protein kinase (MAPK), Jun N-terminal kinase (JNK), and transforming growth factor β-associated kinase 1 (TAK1). Transfection of dominant negative mutants of IκBα, IκB kinase, NF-κB-inducing kinase, TAK1, MyD88, and p38 MAPK inhibited L. pneumophila-induced IL-8 activation. Inhibitors of NF-κB, p38 MAPK, and JNK blocked L. pneumophila-induced IL-8 expression. In addition, c-Jun, JunD, cyclic AMP response element binding protein, and activating transcription factor 1, which are substrates of p38 MAPK and JNK, bound to the AP-1 site of the IL-8 promoter.ConclusionsTaken together, L. pneumophila induced a flagellin-dependent activation of TAK1, p38 MAPK, and JNK, as well as NF-κB and AP-1, which resulted in IL-8 production in human T cells, presumably contributing to the immune response in Legionnaires disease.

Dictyostelium as host model for pathogenesis

The haploid social soil amoeba Dictyostelium discoideum has been established as a host model for several pathogens including Pseudomonas aeruginosa, Cryptococcus neoformans, Mycobacterium spp. and Legionella pneumophila. The research areas presently pursued include (i) the use of Dictyostelium wild‐type cells as screening system for virulence of extracellular and intracellular pathogens and their corresponding mutants, (ii) the use of Dictyostelium mutant cells to identify genetic host determinants of susceptibility and resistance to infection and (iii) the use of reporter systems in Dictyostelium cells which allow the dissection of the complex host‐pathogen cross‐talk. The body of information presented in this review demonstrates that the availability of host cell markers, the knowledge of cell signalling pathways, the completion of the genome sequencing project and the tractability for genetic studies qualifies Dictyostelium for the study of fundamental cellular processes of pathogenesis.

Treponema brennaborense sp. nov., a novel spirochaete isolated from a dairy cow suffering from digital dermatitis.

A novel Treponema species was isolated from an ulcerative lesion of a cow suffering from digital dermatitis (DD), a disease which causes painful ulcerations along the coronary band. Among other anaerobic bacteria, high numbers of spirochaetes have been regularly found in DD lesions. Here data are presented of a spirochaete isolated from a DD ulcer. By chemotaxonomy, protein analysis and comparative 16S rDNA sequence analysis this isolate was classified as a treponeme that differed from all Treponema species described previously. The only isolate, DD5/3T, for which the name Treponema brennaborense is proposed, is designated the type strain of the novel species. The strain is a small, highly motile spirochaete that has two periplasmic flagella, one flagellum being attached at each cell pole. Strain DD5/3T exhibits alpha-glucosidase and N-acetyl-beta-glucosaminidase activity and growth is inhibited by rabbit serum. T. brennaborense was phylogenetically most closely related (89.5% 16S rRNA similarity) to Treponema maltophilum, an oral spirochaete isolated from a periodontitis patient.

The flagellum of Legionella pneumophila and its link to the expression of the virulent phenotype

Legionalla pneumophila is a human pathogen causing atypical pneumonia. It is a monopolar flagellated gram-negative bacterium. Flagellation of L. pneumophila is life cycle dependent and the expression of flagella is genetically linked to the virulence phenotype. Non-flagellated mutants of L. pneumophila are less infectious for macrophages and amoebae compared to the wild type. The flagellar operon is expressed in a hierarchical manner, and different sigma factors and transcriptional regulators are involved in this cascade of gene regulation. The genome sequence of L. pneumophila was used to identify putative regulatory elements of various flagellar operons. Preliminary reports about regulators which are involved in the link between virulence gene regulation and flagellation are discussed.

Influence of the Alternative σ28 Factor on Virulence and Flagellum Expression of Legionella pneumophila

ABSTRACT The fliA gene of Legionella pneumophila encoding the alternative σ28 factor was inactivated by introducing a kanamycin resistance cassette. Electron microscopy and Western blot analysis revealed that the fliA mutant strain is aflagellate and expresses no flagellin. Reporter gene assays indicated that the flaA promoter is not active in the fliA mutant strain. The fliA mutant strain multiplied less effectively in coculture with amoebae than the wild-type strain and was not able to replicate in coculture with Dictyostelium discoideum.

First indication for a functional CRISPR/Cas system in Francisella tularensis

Francisella tularensis is a zoonotic agent and the subspecies novicida is proposed to be a water-associated bacterium. The intracellular pathogen F. tularensis causes tularemia in humans and is known for its potential to be used as a biological threat. We analyzed the genome sequence of F. tularensis subsp. novicida U112 in silico for the presence of a putative functional CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system. CRISPR/Cas systems are known to encode an RNA-guided adaptive immunity-like system to protect bacteria against invading genetic elements like bacteriophages and plasmids. In this work, we present a first indication that F. tularensis subsp. novicida encodes a functional CRISPR/Cas defence system. Additionally, we identified various spacer DNAs homologous to a putative phage present within the genome of F. tularensis subsp. novicida-like strain 3523. CRISPR/Cas is also present in F. tularensis subsp. tularensis, holarctica, and mediasiatica, but these systems seem to be non-functional.

Characterization of the Alternative Sigma Factor σ54 and the Transcriptional Regulator FleQ of Legionella pneumophila, Which Are Both Involved in the Regulation Cascade of Flagellar Gene Expression

We cloned and analyzed Legionella pneumophila Corby homologs of rpoN (encoding sigma(54)) and fleQ (encoding sigma(54) activator protein). Two other genes (fleR and pilR) whose products have a sigma(54) interaction domain were identified in the genome sequence of L. pneumophila. An rpoN mutant strain was nonflagellated and expressed very small amounts of the FlaA (flagellin) protein. Like the rpoN mutant, the fleQ mutant strain of L. pneumophila was also nonflagellated and expressed only small amounts of FlaA protein compared to the amounts expressed by the wild type. In this paper we show that the sigma(54) factor and the FleQ protein are involved in regulation of flagellar gene operons in L. pneumophila. RpoN and FleQ positively regulate the transcription of FliM and FleN, both of which have a sigma(54)-dependent promoter consensus sequence. However, they seemed to be dispensable for transcription of flaA, fliA, or icmR. Our results confirmed a recently described model of the flagellar gene regulation cascade in L. pneumophila (K. Heuner and M. Steinert, Int. J. Med. Microbiol. 293:133-145, 2003). Flagellar gene regulation was found to be different from that of Enterobacteriaceae but seems to be comparable to that described for Pseudomonas or Vibrio spp.

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

BackgroundThe 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.ResultsWe 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.ConclusionsLegionella 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.

Control of Flagellar Gene Regulation in Legionella pneumophila and Its Relation to Growth Phase

The bacterial pathogen Legionella pneumophila responds to environmental changes by differentiation. At least two forms are well described: replicative bacteria are avirulent; in contrast, transmissive bacteria express virulence traits and flagella. Phenotypic analysis, Western blotting, and electron microscopy of mutants of the regulatory genes encoding RpoN, FleQ, FleR, and FliA demonstrated that flagellin expression is strongly repressed and that the mutants are nonflagellated in the transmissive phase. Transcriptome analyses elucidated that RpoN, together with FleQ, enhances transcription of 14 out of 31 flagellar class II genes, which code for the basal body, hook, and regulatory proteins. Unexpectedly, FleQ independent of RpoN enhances the transcription of fliA encoding sigma 28. Expression analysis of a fliA mutant showed that FliA activates three out of the five remaining flagellar class III genes and the flagellar class IV genes. Surprisingly, FleR does not induce but inhibits expression of at least 14 flagellar class III genes on the transcriptional level. Thus, we propose that flagellar class II genes are controlled by FleQ and RpoN, whereas the transcription of the class III gene fliA is controlled in a FleQ-dependent but RpoN-independent manner. However, RpoN and FleR might influence flagellin synthesis on a posttranscriptional level. In contrast to the commonly accepted view that enhancer-binding proteins such as FleQ always interact with RpoN to fullfill their regulatory functions, our results strongly indicate that FleQ regulates gene expression that is RpoN dependent and RpoN independent. Finally, FliA induces expression of flagellar class III and IV genes leading to the complete synthesis of the flagellum.