Impact of temperature on Legionella pneumophila, its protozoan host cells, and the microbial diversity of the biofilm community of a pilot cooling tower

ABSTRACTLegionella pneumophila (Lp) is a waterborne bacterium known for causing Legionnaires’ Disease, a severe pneumonia. Cooling towers are a major source of outbreaks, since they provide ideal conditions for Lp growth and produce aerosols. In such systems, Lp typically grow inside protozoan hosts. Several abiotic factors such as water temperature, pipe material and disinfection regime affect the colonization of cooling towers by Lp. The local physical and biological factors promoting the growth of Lp in water systems and its spatial distribution are not well understood. Therefore, we built a lab-scale cooling tower to study the dynamics of Lp colonization in relationship to the resident microbiota and spatial distribution. The pilot was filled with water from an operating cooling tower harboring low levels of Lp. It was seeded with Vermamoeba vermiformis, a natural host of Lp, and then inoculated with Lp. After 92 days of operation, the pilot was disassembled, the water was collected, and biofilm was extracted from the pipes. The microbiome was studied using 16S rRNA and 18S rRNA genes amplicon sequencing. The communities of the water and of the biofilm were highly dissimilar. The relative abundance of Legionella in water samples reached up to 11% whereas abundance in the biofilm was extremely low (≤0.5 %). In contrast, the host cells were mainly present in the biofilm. This suggest that Lp grows in host cells associated with biofilm and is then released back into the water following host cell lysis. In addition, water temperature shaped the bacterial and eukaryotic community of the biofilm, indicating that different parts of the systems may have different effects on Legionella growth.

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Presence ofLegionellaspp. in cooling towers: the role of microbial diversity,Pseudomonas, and continuous chlorine application

10.1101/540302 ◽

2019 ◽

Cited By ~ 3

Author(s):

Kiran Paranjape ◽

Émilie Bédard ◽

Lyle G. Whyte ◽

Jennifer Ronholm ◽

Michèle Prévost ◽

...

Keyword(s):

Bacterial Community ◽

Microbial Diversity ◽

Legionella Pneumophila ◽

Cooling Tower ◽

Severe Pneumonia ◽

Water Source ◽

Amplicon Sequencing ◽

Physical Parameters ◽

Cooling Towers ◽

Strong Negative Correlation

ABSTRACTLegionnaire’s Disease (LD) is a severe pneumonia caused byLegionella pneumophila. Cooling towers are the main source ofL. pneumophiladuring large outbreaks. Colonization, survival, and proliferation ofL. pneumophilain cooling towers are necessary for outbreaks to occur. These steps are affected by chemical and physical parameters of the cooling tower environment. We hypothesize that the bacterial community residing in the cooling tower could also affect the presence ofL. pneumophila. A16S rRNAtargeted amplicon sequencing approach was used to study the bacterial community of cooling towers and its relationship with theLegionella spp.andL. pneumophilacommunities. The results indicated that the water source shaped the bacterial community of cooling towers. Several taxa were enriched and positively correlated withLegionella spp.andL. pneumophila. In contrast,Pseudomonasshowed a strong negative correlation withLegionella spp.and several other genera. Most importantly, continuous chlorine application reduced microbial diversity and promoted the presence ofPseudomonascreating a non-permissive environment forLegionella spp. This suggests that disinfection strategies as well as the resident microbial population influences the ability ofLegionella spp.to colonize cooling towers.

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Efficient cooling tower operation at alkaline pH for the control of Legionella pneumophila and other pathogenic genera

Water Research ◽

10.1016/j.watres.2021.117047 ◽

2021 ◽

pp. 117047

Author(s):

I.S.M. Pinel ◽

P.M. Hankinson ◽

D.H. Moed ◽

L.J. Wyseure ◽

J.S. Vrouwenvelder ◽

...

Keyword(s):

Legionella Pneumophila ◽

Cooling Tower ◽

Alkaline Ph

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Legionella pneumophila DotU and IcmF Are Required for Stability of the Dot/Icm Complex

Infection and Immunity ◽

10.1128/iai.72.10.5983-5992.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 5983-5992 ◽

Cited By ~ 56

Author(s):

Jessica A. Sexton ◽

Jennifer L. Miller ◽

Aki Yoneda ◽

Thomas E. Kehl-Fie ◽

Joseph P. Vogel

Keyword(s):

Cell Surface ◽

Legionella Pneumophila ◽

Bacterial Species ◽

Wide Distribution ◽

Host Cells ◽

Growth Defect ◽

Intracellular Growth ◽

Homologous Proteins ◽

Type Iv ◽

Cell Surface Structure

ABSTRACT Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a ΔdotU ΔicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the ΔdotU ΔicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.

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Subinhibitory Concentrations of Antimicrobial Agents Reduce the Uptake of Legionella pneumophila into Acanthamoeba castellanii and U937 Cells by Altering the Expression of Virulence-Associated Antigens

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.42.11.2870 ◽

1998 ◽

Vol 42 (11) ◽

pp. 2870-2876 ◽

Cited By ~ 8

Author(s):

P. Christian Lück ◽

Jürgen W. Schmitt ◽

Arne Hengerer ◽

Jürgen H. Helbig

Keyword(s):

Monoclonal Antibodies ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Legionella Pneumophila ◽

Antimicrobial Agents ◽

Acanthamoeba Castellanii ◽

Host Cells ◽

Uncharacterized Protein ◽

Subinhibitory Concentrations

ABSTRACT We determined the MICs of ampicillin, ciprofloxacin, erythromycin, imipenem, and rifampin for two clinical isolates of Legionella pneumophila serogroup 1 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay and by quantitative culture. To test the influence of subinhibitory concentrations (sub-MICs) of antimicrobial agents on Legionella uptake into Acanthamoeba castellanii and U937 macrophage-like cells, both strains were pretreated with 0.25 MICs of the antibiotics for 24 h. In comparison to that for the untreated control, subinhibitory concentrations of antibiotics significantly reducedLegionella uptake into the host cells. Measurement of the binding of monoclonal antibodies against several Legionellaantigens by enzyme-linked immunoassays indicated that sub-MIC antibiotic treatment reduced the expression of the macrophage infectivity potentiator protein (Mip), the Hsp 60 protein, the outer membrane protein (OmpM), an as-yet-uncharacterized protein of 55 kDa, and a few lipopolysaccharide (LPS) epitopes. In contrast, the expression of some LPS epitopes recognized by monoclonal antibodies 8/5 and 30/4 as well as a 45-kDa protein, a 58-kDa protein, and the major outer membrane protein (OmpS) remained unaffected.

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Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector of Legionella pneumophila

Journal of Experimental Medicine ◽

10.1084/jem.20100771 ◽

2010 ◽

Vol 207 (8) ◽

pp. 1713-1726 ◽

Cited By ~ 97

Author(s):

Christopher T.D. Price ◽

Tasneem Al-Quadan ◽

Marina Santic ◽

Snake C. Jones ◽

Yousef Abu Kwaik

Keyword(s):

Legionella Pneumophila ◽

Biological Function ◽

Host Cells ◽

Dependent Manner ◽

Protein Farnesyltransferase ◽

Type Iv ◽

Eukaryotic Proteins ◽

Bacterial Effectors

Farnesylation involves covalent linkage of eukaryotic proteins to a lipid moiety to anchor them into membranes, which is essential for the biological function of Ras and other proteins. A large cadre of bacterial effectors is injected into host cells by intravacuolar pathogens through elaborate type III–VII translocation machineries, and many of these effectors are incorporated into the pathogen-containing vacuolar membrane by unknown mechanisms. The Dot/Icm type IV secretion system of Legionella pneumophila injects into host cells the F-box effector Ankyrin B (AnkB), which functions as platforms for the docking of polyubiquitinated proteins to the Legionella-containing vacuole (LCV) to enable intravacuolar proliferation in macrophages and amoeba. We show that farnesylation of AnkB is indispensable for its anchoring to the cytosolic face of the LCV membrane, for its biological function within macrophages and Dictyostelium discoideum, and for intrapulmonary proliferation in mice. Remarkably, the protein farnesyltransferase, RCE-1 (Ras-converting enzyme-1), and isoprenyl cysteine carboxyl methyltransferase host farnesylation enzymes are recruited to the LCV in a Dot/Icm-dependent manner and are essential for the biological function of AnkB. In conclusion, this study shows novel localized recruitment of the host farnesylation machinery and its anchoring of an F-box effector to the LCV membrane, and this is essential for biological function in vitro and in vivo.

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Lipopolysaccharide Architecture of Legionella pneumophila Grown in Broth and Host Cells

Legionella ◽

10.1128/9781555815660.ch64 ◽

2014 ◽

pp. 261-264 ◽

Cited By ~ 1

Author(s):

Jürgen H. Helbig ◽

Esteban Fernandez-Moreira ◽

Enno Jacobs ◽

Paul Christian Lück ◽

Martin Witt

Keyword(s):

Legionella Pneumophila ◽

Host Cells

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Identification of Translocated Substrates of the Legionella pneumophila Dot/Icm System without the use of Eukaryotic Host Cells

Legionella ◽

10.1128/9781555815660.ch45 ◽

2014 ◽

pp. 167-176

Author(s):

Ralph R. Isberg ◽

Matthias Machner

Keyword(s):

Legionella Pneumophila ◽

Host Cells

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Molecular architecture of theLegionellaDot/Icm type IV secretion system

10.1101/312009 ◽

2018 ◽

Cited By ~ 3

Author(s):

Debnath Ghosal ◽

Yi-Wei Chang ◽

Kwang Cheol Jeong ◽

Joseph P. Vogel ◽

Grant J. Jensen

Keyword(s):

Legionella Pneumophila ◽

Cell Envelope ◽

Secretion System ◽

Host Cells ◽

Molecular Architecture ◽

Intact Cells ◽

Type Iv ◽

Type Ivb Secretion ◽

Electron Cryotomography ◽

First Time

AbstractLegionella pneumophilasurvives and replicates inside host cells by secreting ~300 effectors through the Dot/Icm type IVB secretion system (T4BSS). Understanding this machine’s structure is challenging because of its large number of components (27) and integration into all layers of the cell envelope. Previously we overcame this obstacle by imaging the Dot/Icm T4BSS in its native state within intact cells through electron cryotomography. Here we extend our observations by imaging a stabilized mutant that yielded a higher resolution map. We describe for the first time the presence of a well-ordered central channel that opens up into a windowed large (~32 nm wide) secretion chamber with an unusual 13-fold symmetry. We then dissect the complex by matching proteins to densities for many components, including all those with periplasmic domains. The placement of known and predicted structures of individual proteins into the map reveals the architecture of the T4BSS and provides a roadmap for further investigation of this amazing specialized secretion system.

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Components of the endocytic and recycling trafficking pathways interfere with the integrity of the Legionella-containing vacuole

10.1101/781849 ◽

2019 ◽

Author(s):

Ila S. Anand ◽

Won Young Choi ◽

Ralph R. Isberg

Keyword(s):

Membrane Trafficking ◽

Legionella Pneumophila ◽

Host Cells ◽

Rab Gtpases ◽

Intracellular Growth ◽

Mutant Proteins ◽

Host Cytoplasm ◽

Downstream Effectors ◽

Cell Death Response ◽

Host Membrane

SummaryLegionella pneumophila requires the Dot/Icm translocation system to replicate in a vacuolar compartment within host cells. Strains lacking the translocated substrate SdhA form a permeable vacuole during residence in the host cell, exposing bacteria to the host cytoplasm. In primary macrophages, mutants are defective for intracellular growth, with a pyroptotic cell death response mounted due to bacterial exposure to the cytosol. To understand how SdhA maintains vacuole integrity during intracellular growth, we performed high-throughput RNAi screens against host membrane trafficking genes to identify factors that antagonize vacuole integrity in the absence of SdhA. Depletion of host proteins involved in endocytic uptake and recycling resulted in enhanced intracellular growth and lower levels of permeable vacuoles surrounding the ΔsdhA mutant. Of interest were three different Rab GTPases involved in these processes: Rab11b, Rab8b and Rab5 isoforms, that when depleted resulted in enhanced vacuole integrity surrounding the sdhA mutant. Proteins regulated by these Rabs are responsible for interfering with proper vacuole membrane maintenance, as depletion of the downstream effectors EEA1, Rab11FIP1, or VAMP3 rescued vacuole integrity and intracellular growth of the sdhA mutant. To test the model that specific vesicular components associated with these effectors could act to destabilize the replication vacuole, EEA1 and Rab11FIP1 showed enhanced colocalization with the vacuole surrounding the sdhA mutant compared with the WT vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant colocalization. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilize vacuole integrity during infection.

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