Antje Flieger

NAIP and Ipaf Control Legionella pneumophila Replication in Human Cells

In mice, different alleles of the mNAIP5 (murine neuronal apoptosis inhibitory protein-5)/mBirc1e gene determine whether macrophages restrict or support intracellular replication of Legionella pneumophila, and whether a mouse is resistant or (moderately) susceptible to Legionella infection. In the resistant mice strains, the nucleotide-binding oligomerization domain (Nod)-like receptor (NLR) family member mNAIP5/mBirc1e, as well as the NLR protein mIpaf (murine ICE protease-activating factor), are involved in recognition of Legionella flagellin and in restriction of bacterial replication. Human macrophages and lung epithelial cells support L. pneumophila growth, and humans can develop severe pneumonia (Legionnaires disease) after Legionella infection. The role of human orthologs to mNAIP5/mBirc1e and mIpaf in this bacterial infection has not been elucidated. Herein we demonstrate that flagellin-deficient L. pneumophila replicate more efficiently in human THP-1 macrophages, primary monocyte-derived macrophages, and alveolar macrophages, and in A549 lung epithelial cells compared with wild-type bacteria. Additionally, we note expression of the mNAIP5 ortholog hNAIP in all cell types examined, and expression of hIpaf in human macrophages. Gene silencing of hNAIP or hIpaf in macrophages or of hNAIP in lung epithelial cells leads to an enhanced bacterial growth, and overexpression of both molecules strongly reduces Legionella replication. In contrast to experiments with wild-type L. pneumophila, hNAIP or hIpaf knock-down affects the (enhanced) replication of flagellin-deficient Legionella only marginally. In conclusion, hNAIP and hIpaf mediate innate intracellular defense against flagellated Legionella in human cells.

IFNβ responses induced by intracellular bacteria or cytosolic DNA in different human cells do not require ZBP1 (DLM-1/DAI)

Intracellular bacteria and cytosolic stimulation with DNA activate type I IFN responses independently of Toll‐like receptors, most Nod‐like receptors and RIG‐like receptors. A recent study suggested that ZBP1 (DLM‐1/DAI) represents the long anticipated pattern recognition receptor which mediates IFNα/β responses to cytosolic DNA in mice. Here we show that Legionella pneumophila infection, and intracellular challenge with poly(dA‐dT), but not with poly(dG‐dC), induced expression of IFNβ, full‐length hZBP1 and a prominent splice variant lacking the first Zα domain (hZBP1ΔZα) in human cells. Overexpression of hZBP1 but not hZBP1ΔZα slightly amplified poly(dA‐dT)‐stimulated IFNβ reporter activation in HEK293 cells, but had no effect on IFNβ and IL‐8 production induced by bacteria or poly(dA‐dT) in A549 cells. We found that mZBP1 siRNA impaired poly(dA‐dT)‐induced IFNβ responses in mouse L929 fibroblasts at a later time point, while multiple hZBP1 siRNAs did not suppress IFNβ or IL‐8 expression induced by poly(dA‐dT) or bacterial infection in human cells. In contrast, IRF3 siRNA strongly impaired the IFNβ responses to poly(dA‐dT) or bacterial infection. In conclusion, intracellular bacteria and cytosolic poly(dA‐dT) activate IFNβ responses in different human cells without requiring human ZBP1.

EHEC/EAEC O104:H4 strain linked with the 2011 German outbreak of haemolytic uremic syndrome enters into the viable but non-culturable state in response to various stresses and resuscitates upon stress relief

Various non-spore forming bacteria, including Escherichia coli, enter a dormant-like state, the viable but non-culturable (VBNC) state, characterized by the presence of viable cells but the inability to grow on routine laboratory media. Upon resuscitation, these VBNC cells recover both culturability and pathogenicity. In 2011, a large outbreak involving more than 3000 cases of bloody diarrhoea and haemolytic uremic syndrome was caused by an E. coli O104:H4 strain expressing genes characteristic of both enterohaemorrhagic (EHEC) and enteroaggregative E. coli (EAEC). The ability of the outbreak strain to enter the VBNC state may have complicated its detection in the suspected sources. In this paper, we investigated the ability of the outbreak strain to enter and subsequently recover from the VBNC state. We found that in a nutrient-poor micro-environment, various stresses such as toxic concentrations of copper ions or certain types of tap water are able to render the bacteria unculturable within a few days. Without copper ion stress, the majority of cells remained culturable for at least 40 days. Incubation with the stressors at 23°C compared with 4°C hastened this observed loss of culturability. The integrity of a considerable fraction of copper ion- and tap water 1-stressed bacteria was demonstrated by live/dead staining and microscopy. Relieving stress by copper-ion chelation facilitated resuscitation of these bacteria while preserving their fitness, major virulence gene markers (stx2, aggR, aggA genes) and specific phenotypes (ESBL resistance, autoaggregation typical for EAEC strains).

Histone Acetylation and Flagellin Are Essential for Legionella pneumophila-Induced Cytokine Expression

Legionella pneumophila causes severe pneumonia. Acetylation of histones is thought to be an important regulator of gene transcription, but its impact on L. pneumophila-induced expression of proinflammatory cytokines is unknown. L. pneumophila strain 130b induced the expression of the important chemoattractant IL-8 and genome-wide histone modifications in human lung epithelial A549 cells. We analyzed the IL-8-promoter and found that histone H4 was acetylated and H3 was phosphorylated at Ser10 and acetylated at Lys14, followed by transcription factor NF-κB. Recruitment of RNA polymerase II to the IL-8 promoter corresponded with increases in gene transcription. Histone modification and IL-8 release were dependent on p38 kinase and NF-κB pathways. Legionella-induced IL-8 expression was decreased by histone acetylase (HAT) inhibitor anacardic acid and enhanced by histone deacetylase (HDAC) inhibitor trichostatin A. After Legionella infection, HATs p300 and CREB-binding protein were time-dependently recruited to the IL-8 promoter, whereas HDAC1 and HDAC5 first decreased and later reappeared at the promoter. Legionella specifically induced expression of HDAC5 but not of other HDACs in lung epithelial cells, but knockdown of HDAC1 or 5 did not alter IL-8 release. Furthermore, Legionella-induced cytokine release, promoter-specific histone modifications, and RNA polymerase II recruitment were reduced in infection with flagellin-deletion mutants. Legionella-induced histone modification as well as HAT-/HDAC-dependent IL-8 release could also be shown in primary lung epithelial cells. In summary, histone acetylation seems to be important for the regulation of proinflammatory gene expression in L. pneumophila infected lung epithelial cells. These pathways may contribute to the host response in Legionnaires’ disease.

Temporal resolution of two-tracked NF-kappa B activation by Legionella pneumophila

The intracellular pathogen Legionella pneumophila activates the transcription factor NF‐κB in macrophages and human epithelial cells, contributing to cytokine production and anti‐apoptosis. The former is important for the innate immune response to infection, the latter for intracellular replication by securing host cell survival. Here, we demonstrate biphasic activation of NF‐κB by L. pneumophila in human epithelial cells, using a p65‐GFP expressing variant of A549 cells. Early in infection, a strong but transient nuclear translocation of p65 was observed. Only flagellin‐deficient (ΔfliA and ΔflaA) mutants could not induce this first, TLR5 and MyD88‐dependent activation. The second p65 translocation event, however, is a long‐term activation, independent of flagellin, TLR5 and MyD88, and marked by permanent nuclear localization of p65‐GFP without oscillation for 30 h. Persistent p65 translocation also involved degradation of IκBα and upregulation of anti‐apoptotic genes. L. pneumophila mutants lacking a functional Dot/Icm secretion system (ΔdotA; ΔicmB/dotO), Dot/Icm effectors (ΔsdbA; ΔlubX) and two bacterial effector mutants (ΔenhC; ΔptsP) could not induce persistent p65 translocation. Strikingly, all these mutants were deficient in intracellular replication in A549 cells. Our data underline the strong connection between NF‐κB activation and intracellular replication and hints at an active interference of NF‐κB signalling by L. pneumophila.

Induction of human β-defensin-2 in pulmonary epithelial cells by Legionella pneumophila: involvement of TLR2 and TLR5, p38 MAPK, JNK, NF-κB, and AP-1

Legionella pneumophila is an important causative agent of severe pneumonia in humans. Human alveolar epithelium is an effective barrier for inhaled microorganisms and actively participates in the initiation of innate host defense. Induction of antimicrobial peptide human β-defensin-2 (hBD-2) by various stimuli in epithelial cells has been reported. However, the mechanisms by which bacterial infections enhance hBD-2 expression remain poorly understood. In this study, we investigated the effect of the pulmonary pathogen L. pneumophila on induction of hBD-2 in human pulmonary epithelial cells. Infection with L. pneumophila markedly increased hBD-2 production, and the response was attenuated in Toll-like receptor (TLR) 2 and TLR5 transient knockdown cells. Furthermore, pretreatment with SB-202190 (an inhibitor of p38 MAPK) and JNK II (an inhibitor of c-Jun NH(2)-terminal kinase), but not U0126 (an inhibitor of ERK), reduced L. pneumophila-induced hBD-2 release in A549 cells. L. pneumophila-induced hBD-2 liberation was mediated via recruitment of NF-κB and AP-1 to the hBD-2 gene promoter. Additionally, we showed that exo- and endogenous hBD-2 elicited a strong antimicrobial effect towards L. pneumophila. Together, these results suggest that L. pneumophila induces hBD-2 release in A549 cells, and the induction seems to be mediated through TLR2 and TLR5 as well as activation of p38 MAPK, JNK, NF-κB, and AP-1.

Automated Pipeline for Purification, Biophysical and X-Ray Analysis of Biomacromolecular Solutions

Small angle X-ray scattering (SAXS), an increasingly popular method for structural analysis of biological macromolecules in solution, is often hampered by inherent sample polydispersity. We developed an all-in-one system combining in-line sample component separation with parallel biophysical and SAXS characterization of the separated components. The system coupled to an automated data analysis pipeline provides a novel tool to study difficult samples at the P12 synchrotron beamline (PETRA-3, EMBL/DESY, Hamburg).

DivIVA affects secretion of virulence-related autolysins in Listeria monocytogenes.

DivIVA is a well‐conserved coiled‐coil protein present in most Gram‐positive bacteria and has been implicated in division site selection, peptidoglycan biosynthesis and sporulation. DivIVA proteins bind lipid membranes and characteristically accumulate at curved membrane areas, i.e. the cell poles and the division site, to which they recruit various interaction partners. We have studied the role of this morphogen in the human pathogen Listeria monocytogenes and our results suggest a novel mechanism by which DivIVA contributes to cell division. Contrary to expectation a ΔdivIVA mutant exhibited a pronounced chaining phenotype rather than a defect in cell division which we attributed to reduced extracellular levels of the autolytic enzymes p60 and MurA. We demonstrate that this is due to a malfunction in secretion of these autolysins and phenotypic comparison of the ΔdivIVA strain with a ΔsecA2 mutant suggests that DivIVA influences the activity of the SecA2 secretion route in L. monocytogenes. Also from the phenotypic analysis it was clear that divIVA affected swarming motility, biofilm formation, invasiveness and cell‐to‐cell spread in cell culture infection models. Thus, our experiments show that DivIVA is an important factor for various listerial traits that are essential for the pathogenicity of this organism.

The Legionella pneumophila Dot/Icm-secreted effector PlcC/CegC1 together with PlcA and PlcB promotes virulence and belongs to a novel zinc metallophospholipase C family present in bacteria and fungi

Background: It is unclear whether Legionella pneumophila possesses phospholipase C (PLC) activity and thereby generates 1,2-diacylglycerol. Results: L. pneumophila possesses three secreted enzymes with PLC activity, PlcA, PlcB, and PlcC, and a plcABC mutant was attenuated in host killing. Conclusion: L. pneumophila encodes three members of a novel PLC family contributing to virulence. Significance: We determined PLC activity for L. pneumophila and defined the characteristics of a novel PLC family present in Legionella, Pseudomonas, and fungi. Legionella pneumophila is a water-borne bacterium that causes pneumonia in humans. PlcA and PlcB are two previously defined L. pneumophila proteins with homology to the phosphatidylcholine-specific phospholipase C (PC-PLC) of Pseudomonas fluorescens. Additionally, we found that Lpg0012 shows similarity to PLCs and has been shown to be a Dot/Icm-injected effector, CegC1, which is designated here as PlcC. It remained unclear, however, whether these L. pneumophila proteins exhibit PLC activity. PlcC expressed in Escherichia coli hydrolyzed a broad phospholipid spectrum, including PC, phosphatidylglycerol (PG), and phosphatidylinositol. The addition of Zn2+ ions activated, whereas EDTA inhibited, PlcC-derived PLC activity. Protein homology search revealed that the three Legionella enzymes and P. fluorescens PC-PLC share conserved domains also present in uncharacterized fungal proteins. Fifteen conserved amino acids were essential for enzyme activity as identified via PlcC mutagenesis. Analysis of defined L. pneumophila knock-out mutants indicated Lsp-dependent export of PG-hydrolyzing PLC activity. PlcA and PlcB exhibited PG-specific activity and contain a predicted Sec signal sequence. In line with the reported requirement of host cell contact for Dot/Icm-dependent effector translocation, PlcC showed cell-associated PC-specific PLC activity after bacterial growth in broth. A PLC triple mutant, but not single or double mutants, exhibited reduced host killing in a Galleria mellonella infection model, highlighting the importance of the three PLCs in pathogenesis. In summary, we describe here a novel Zn2+-dependent PLC family present in Legionella, Pseudomonas, and fungi with broad substrate preference and function in virulence.

Characterisation of Legionella pneumophila phospholipases and their impact on host cells

Phospholipases are a diverse class of enzymes produced both by eukaryotic hosts and their pathogens. Major insights into action pathways of bacterial phospholipases have been provided during the last years. On the one hand bacterial phospholipases act as potent membrane destructors and on the other hand they manipulate and initiate host signalling paths, such as chemokine expression or the inflammatory cascade. Reaction products of bacterial phospholipases may potentially influence many more host cell processes, such as cell respreading, lamellopodia formation, cell migration and membrane traffic. Phospholipases play a dominant role in the biology of the lung pathogen Legionella pneumophila. So far, 15 different phospholipase A-encoding genes have been identified in the L. pneumophila genome. These phospholipases can be divided into three major groups, the GDSL, the patatin-like and the PlaB-like enzymes. The first two lipase families are also found in higher plants (such as flowering plants) and the second family shows similarities to eukaryotic cytosolic phospholipases A. Therefore, when those enzymes are injected or secreted by the bacterium into the host cell they may mimic eukaryotic phospholipases. The current knowledge on L. pneumophila phospholipases is summarised here with emphasis on their activity, mode of secretion, localisation, expression and importance for host cell infections.