Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways

Coers, Jörn; Vance, Russell E.; Fontana, Mary F.; Dietrich, William F.

doi:10.1111/j.1462-5822.2007.00963.x

Cited by 133 publications

(152 citation statements)

References 62 publications

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“…On the other hand, full innate intracellular resistance to L. pneumophila (and certainly other intracellular bacteria) depends on many additional mechanisms such as types I and II IFN-as well as TNF-␣-mediated effects, and perhaps also on Nramp1, antimicrobial peptides, or immunity-related GTPases (13,15,(45)(46)(47)(48)(49)(50). It might thus be conceivable that in case of L. pneumophila infection, one of these last mentioned resistance mechanisms is less functional in human cells compared with mice resulting in an overall stronger replication of Legionella in human cells compared with, for example, C57BL/6 macrophages despite functional hNAIP and hIpaf.…”

Section: Discussionmentioning

confidence: 99%

NAIP and Ipaf Control Legionella pneumophila Replication in Human Cells

Vinzing

Eitel

Lippmann

et al. 2008

The Journal of Immunology

121

119

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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.

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Section: Discussionmentioning

confidence: 99%

NAIP and Ipaf Control Legionella pneumophila Replication in Human Cells

Vinzing

Eitel

Lippmann

et al. 2008

The Journal of Immunology

121

119

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“…Animal protocols were approved by the Institutional Animal Care and Use Committees at Duke University and the University of North Carolina, Chapel Hill. Bone marrow was collected from femurs as described (34). Details of macrophage differentiation procedures can be found in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Guanylate binding proteins promote caspase-11–dependent pyroptosis in response to cytoplasmic LPS

Pilla

Hagar

Haldar

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

294

364

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Significance A major component of the cell envelope of Gram-negative bacteria is LPS, also known as endotoxin. LPS produced during bacterial infections triggers inflammation, which can lead to septic shock and death. Our immune system can recognize LPS both outside and inside of cells. The recognition of extracellular and vacuolar LPS by LPS binding proteins is well described, but little is known about the recognition of cytoplasmic LPS. Here, we show that cytoplasmic LPS derived from the intracellular bacterial pathogen Legionella activated a proinflammatory immune response. We further identified host guanylate binding proteins as critical mediators of immunity triggered by cytoplasmic LPS. These findings are likely to advance our understanding of how cells can sense intracellular LPS.

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“…Experiments such as those shown in Figure 2 and Tables 1-3 performed with several infection ratios will allow assessment and optimization of conditions for use in future assays. Of note, for Legionella in particular, use of a strain background with natively lower host cell cytotoxicity (a flagellin mutant) 8 contributed significantly to the high assay robustness obtained.…”

Section: Discussionmentioning

confidence: 99%

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

Chiaraviglio

Kang

2016

JoVE

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Traditional measures of intracellular antimicrobial activity and eukaryotic cell cytotoxicity rely on endpoint assays. Such endpoint assays require several additional experimental steps prior to readout, such as cell lysis, colony forming unit determination, or reagent addition. When performing thousands of assays, for example, during high-throughput screening, the downstream effort required for these types of assays is considerable. Therefore, to facilitate high-throughput antimicrobial discovery, we developed a real-time assay to simultaneously identify inhibitors of intracellular bacterial growth and assess eukaryotic cell cytotoxicity. Specifically, real-time intracellular bacterial growth detection was enabled by marking bacterial screening strains with either a bacterial lux operon (1 st generation assay) or fluorescent protein reporters (2 nd generation, orthogonal assay). A non-toxic, cell membrane-impermeant, nucleic acid-binding dye was also added during initial infection of macrophages. These dyes are excluded from viable cells. However, non-viable host cells lose membrane integrity permitting entry and fluorescent labeling of nuclear DNA (deoxyribonucleic acid). Notably, DNA binding is associated with a large increase in fluorescent quantum yield that provides a solutionbased readout of host cell death. We have used this combined assay to perform a high-throughput screen in microplate format, and to assess intracellular growth and cytotoxicity by microscopy. Notably, antimicrobials may demonstrate synergy in which the combined effect of two or more antimicrobials when applied together is greater than when applied separately. Testing for in vitro synergy against intracellular pathogens is normally a prodigious task as combinatorial permutations of antibiotics at different concentrations must be assessed. However, we found that our real-time assay combined with automated, digital dispensing technology permitted facile synergy testing. Using these approaches, we were able to systematically survey action of a large number of antimicrobials alone and in combination against the intracellular pathogen, Legionella pneumophila.

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Restriction of Legionella pneumophila growth in macrophages requires the concerted action of cytokine and Naip5/Ipaf signalling pathways

Cited by 133 publications

References 62 publications

NAIP and Ipaf Control Legionella pneumophila Replication in Human Cells

NAIP and Ipaf Control Legionella pneumophila Replication in Human Cells

Guanylate binding proteins promote caspase-11–dependent pyroptosis in response to cytoplasmic LPS

High Throughput, Real-time, Dual-readout Testing of Intracellular Antimicrobial Activity and Eukaryotic Cell Cytotoxicity

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