Can free chlorine residuals entering building plumbing systems really be maintained to prevent microbial growth?

Secondary disinfection aims to prevent microbial regrowth during distribution by maintaining disinfectant residuals in water systems. However, multi-factorial interactions contribute to free chlorine decay in distribution systems, and even more so in building plumbing. Assembling 1737 samples from nine large institutional buildings, a meta-analysis was conducted to determine whether building managers can actively rely on incoming free chlorine residuals to prevent in-building microbial amplification. Findings showed that free chlorine concentrations in first draws met the 0.2 mg/L common guide level in respectively 26 %, 6 % and 2 % of cold, tepid and hot water samples, whereas flushing for 2-60 min only significantly increased this ratio in cold water (83 %), without reaching background levels found in service lines. Free chlorine was significantly but weakly (R≤ 0.2) correlated to adenosine triphosphate, heterotrophic plate count and total and intact cell counts, thus evidencing that residuals contributed to decreased culturable and viable biomass. Detection of culturable Legionella pneumophila spanning over a 4-log distribution solely occurred when free chlorine levels were below 0.2 mg/L, but no such trend could be distinguished clearly for culturable Pseudomonas aeruginosa. Water temperatures below 20 °C and >60 °C also completely prevented L. pneumophila detection. Overall, the majority of elevated microbial counts were measured in distal sites and in tepid and hot water, where free chlorine is less likely to be present due to stagnation and increased temperature. Therefore, building managers cannot solely rely on this chemical barrier to mitigate bacterial growth in bulk water.

Structural and Functional Characterization of Legionella pneumophila Effector MavL.

Legionella pneumophila is a Gram-negative intracellular pathogen that causes Legionnaires' disease in elderly or immunocompromised individuals. This bacterium relies on the Dot/Icm (Defective in organelle trafficking/Intracellular multiplication) Type IV Secretion System (T4SS) and a large (>330) set of effector proteins to colonize the host cell. The structural variability of these effectors allows them to disrupt many host processes. Herein, we report the crystal structure of MavL to 2.65 Å resolution. MavL adopts an ADP-ribosyltransferase (ART) fold and contains the distinctive ligand-binding cleft of ART proteins. Indeed, MavL binds ADP-ribose with Kd of 13 µM. Structural overlay of MavL with poly-(ADP-ribose) glycohydrolases (PARGs) revealed a pair of aspartate residues in MavL that align with the catalytic glutamates in PARGs. MavL also aligns with ADP-ribose "reader" proteins (proteins that recognize ADP-ribose). Since no glycohydrolase activity was observed when incubated in the presence of ADP-ribosylated PARP1, MavL may play a role as a signaling protein that binds ADP-ribose. An interaction between MavL and the mammalian ubiquitin-conjugating enzyme UBE2Q1 was revealed by yeast two-hybrid and co-immunoprecipitation experiments. This work provides structural and molecular insights to guide biochemical studies aimed at elucidating the function of MavL. Our findings support the notion that ubiquitination and ADP-ribosylation are global modifications exploited by L. pneumophila.

SdhA blocks disruption of the Legionella-containing vacuole by hijacking the OCRL phosphatase.

Legionella pneumophila grows intracellularly within a replication vacuole via action of Icm/Dot-secreted proteins. One such protein, SdhA, maintains the integrity of the vacuolar membrane, thereby preventing cytoplasmic degradation of bacteria. We show here that SdhA binds and blocks the action of OCRL (OculoCerebroRenal syndrome of Lowe), an inositol 5-phosphatase pivotal for controlling endosomal dynamics. OCRL depletion results in enhanced vacuole integrity and intracellular growth of a sdhA mutant, consistent with OCRL participating in vacuole disruption. Overexpressed SdhA alters OCRL function, enlarging endosomes, driving endosomal accumulation of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), and interfering with endosomal trafficking. SdhA interrupts Rab guanosine triphosphatase (GTPase)-OCRL interactions by binding to the OCRL ASPM-SPD2-Hydin (ASH) domain, without directly altering OCRL 5-phosphatase activity. The Legionella vacuole encompassing the sdhA mutant accumulates OCRL and endosomal antigen EEA1 (Early Endosome Antigen 1), consistent with SdhA blocking accumulation of OCRL-containing endosomal vesicles. Therefore, SdhA hijacking of OCRL is associated with blocking trafficking events that disrupt the pathogen vacuole.

Autorepressor PsrA is required for optimal Legionella pneumophila growth in Acanthamoeba castellanii protozoa.

Legionella pneumophila possesses a unique intracellular lifecycle featuring distinct morphological stages that include replicative forms and transmissive cyst forms. Expression of genes associated with virulence traits and cyst morphogenesis is concomitant, and governed by a complex stringent response based-regulatory network and the stationary phase sigma factor RpoS. In Pseudomonas spp., rpoS expression is controlled by the autorepressor PsrA, and orthologs of PsrA and RpoS are required for cyst formation in Azotobacter. Here we report that the L. pneumophila psrA ortholog, expressed as a leaderless monocistronic transcript, is also an autorepressor, but is not a regulator of rpoS expression. Further, the binding site sequence recognized by L. pneumophila PsrA is different from that of Pseudomonas PsrA, suggesting a repertoire of target genes unique to L. pneumophila. While PsrA was dispensable for growth in human U937-derived macrophages, lack of PsrA affected bacterial intracellular growth in Acanthamoeba castellanii protozoa, but also increased the quantity of poly-3-hydroxybutyrate (PHB) inclusions in matured transmissive cysts. Interestingly, overexpression of PsrA increased the size and bacterial load of the replicative vacuole in both host cell types. Taken together, we report that PsrA is a host-specific requirement for optimal temporal progression of L. pneumophila intracellular lifecycle in A. castellanii.

Dynamic remodeling of host membranes by self-organizing bacterial effectors.

During infection, intracellular bacterial pathogens translocate a variety of effectors into host cells that modify host membrane trafficking for their benefit. We found a self-organizing system consisting of a bacterial phosphoinositide kinase and its opposing phosphatase that formed spatiotemporal patterns, including traveling waves, to remodel host cellular membranes. The Legionella effector MavQ, a phosphatidylinositol (PI) 3-kinase, was targeted to the endoplasmic reticulum (ER). MavQ and the Legionella PI 3-phosphatase SidP, even in the absence of other bacterial components, drove rapid PI 3-phosphate turnover on the ER and spontaneously formed traveling waves that spread along ER subdomains inducing vesicle and tubule budding. Thus, bacteria can exploit a self-organizing membrane-targeting mechanism to hijack host cellular structures for survival.

Dictyostelium lacking the single atlastin homolog Sey1 shows aberrant ER architecture, proteolytic processes and expansion of the Legionella-containing vacuole.

Dictyostelium discoideum Sey1 is the single ortholog of mammalian atlastin 1-3 (ATL1-3), which are large homodimeric GTPases mediating homotypic fusion of endoplasmic reticulum (ER) tubules. In this study, we generated a D. discoideum mutant strain lacking the sey1 gene and found that amoebae deleted for sey1 are enlarged, but grow and develop similarly to the parental strain. The ∆sey1 mutant amoebae showed an altered ER architecture, and the tubular ER network was partially disrupted without any major consequences for other organelles or the architecture of the secretory and endocytic pathways. Macropinocytic and phagocytic functions were preserved; however, the mutant amoebae exhibited cumulative defects in lysosomal enzymes exocytosis, intracellular proteolysis, and cell motility, resulting in impaired growth on bacterial lawns. Moreover, ∆sey1 mutant cells showed a constitutive activation of the unfolded protein response pathway (UPR), but they still readily adapted to moderate levels of ER stress, while unable to cope with prolonged stress. In D. discoideum ∆sey1 the formation of the ER-associated compartment harbouring the bacterial pathogen Legionella pneumophila was also impaired. In the mutant amoebae, the ER was less efficiently recruited to the "Legionella-containing vacuole" (LCV), the expansion of the pathogen vacuole was inhibited at early stages of infection and intracellular bacterial growth was reduced. In summary, our study establishes a role of D. discoideum Sey1 in ER architecture, proteolysis, cell motility and intracellular replication of L. pneumophila.

High-polyphenol extracts from Sorghum bicolor attenuate replication of Legionella pneumophila within RAW 264.7 macrophages.

Polyphenols derived from a variety of plants have demonstrated antimicrobial activity against diverse microbial pathogens. Legionella pneumophila is an intracellular bacterial pathogen that opportunistically causes a severe inflammatory pneumonia in humans, called Legionnaires' Disease, via replication within macrophages. Previous studies demonstrated that tea polyphenols attenuate L. pneumophila intracellular replication within mouse macrophages via increased tumor necrosis factor (TNF) production. Sorghum bicolor is a sustainable cereal crop that thrives in arid environments and is well-suited to continued production in warming climates. Sorghum polyphenols have anticancer and antioxidant properties, but their antimicrobial activity has not been evaluated. Here, we investigated the impact of sorghum polyphenols on L. pneumophila intracellular replication within RAW 264.7 mouse macrophages. Sorghum high-polyphenol extract (HPE) attenuated L. pneumophila intracellular replication in a dose-dependent manner but did not impair either bacterial replication in rich media or macrophage viability. Moreover, HPE treatment enhanced both TNF and IL-6 secretion from L. pneumophila infected macrophages. Thus, polyphenols derived from sorghum enhance macrophage restriction of L. pneumophila, likely via increased pro-inflammatory cytokine production. This work reveals commonalities between plant polyphenol-mediated antimicrobial activity and provides a foundation for future evaluation of sorghum as an antimicrobial agent.

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

Legionella 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 antagonise 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 destabilise the replication vacuole, EEA1 and Rab11FIP1 showed increased density about the sdhA mutant vacuole compared with the wild type (WT) vacuole. Depletion of Rab5 isoforms or Rab11b reduced this aberrant redistribution. These findings are consistent with SdhA interfering with both endocytic and recycling membrane trafficking events that act to destabilise vacuole integrity during infection.

Combinatorial selection in amoebal hosts drives the evolution of the human pathogen Legionella pneumophila.

Virulence mechanisms typically evolve through the continual interaction of a pathogen with its host. In contrast, it is poorly understood how environmentally acquired pathogens are able to cause disease without prior interaction with humans. Here, we provide experimental evidence for the model that Legionella pathogenesis in humans results from the cumulative selective pressures of multiple amoebal hosts in the environment. Using transposon sequencing, we identify Legionella pneumophila genes required for growth in four diverse amoebae, defining universal virulence factors commonly required in all host cell types and amoeba-specific auxiliary genes that determine host range. By comparing genes that promote growth in amoebae and macrophages, we show that adaptation of L. pneumophila to each amoeba causes the accumulation of distinct virulence genes that collectively allow replication in macrophages and, in some cases, leads to redundancy in this host cell type. In contrast, some bacterial proteins that promote replication in amoebae restrict growth in macrophages. Thus, amoebae-imposed selection is a double-edged sword, having both positive and negative impacts on disease. Comparing the genome composition and host range of multiple Legionella species, we demonstrate that their distinct evolutionary trajectories in the environment have led to the convergent evolution of compensatory virulence mechanisms.

TOLLIP deficiency is associated with increased resistance to Legionella pneumophila pneumonia.

Legionella pneumophila (Lp) is a flagellated, intracellular bacterium that can cause Legionnaires' disease (LD). Lp activates multiple innate immune receptors, and TOLLIP dampens MyD88-dependent signaling and may influence susceptibility to LD. We evaluated the effect of TOLLIP on innate immunity, pneumonia severity, and LD susceptibility in mouse lungs and human populations. To accomplish this, we evaluated the effect of TOLLIP on lung-specific Lp control and immune response and associated a common functional TOLLIP variant with Lp-induced innate immune responses and LD susceptibility in humans. After aerosol Lp infection, Tollip-/- mice demonstrated significantly fewer bacterial colony-forming unit and increased cytokine responses from BAL fluid. Tollip-/- macrophages also suppressed intracellular Lp replication in a flagellin-independent manner. The presence of a previously characterized, functionally active SNP associated with decreased TOLLIP mRNA transcript in monocytes was associated with increased TNF and IL-6 secretion after Lp stimulation of PBMC ex vivo. This genotype was separately associated with decreased LD susceptibility (309 controls, 88 cases, p = 0.008, OR 0.36, 95% CI 0.16-0.76) in a candidate gene association study. These results suggest that TOLLIP decreases lung-specific TLR responses to increase LD susceptibility in human populations. Better understanding of TOLLIP may lead to novel immunomodulatory therapies.

Legionella-Containing Vacuoles Capture PtdIns(4)P-Rich Vesicles Derived from the Golgi Apparatus.

Legionella pneumophila is the causative agent of a pneumonia termed Legionnaires' disease. The facultative intracellular bacterium employs the Icm/Dot type IV secretion system (T4SS) and a plethora of translocated "effector" proteins to interfere with host vesicle trafficking pathways and establish a replicative niche, the Legionella-containing vacuole (LCV). Internalization of the pathogen and the events immediately ensuing are accompanied by host cell-mediated phosphoinositide (PI) lipid changes and the Icm/Dot-controlled conversion of the LCV from a PtdIns(3)P-positive vacuole into a PtdIns(4)P-positive replication-permissive compartment, which tightly associates with the endoplasmic reticulum. The source and formation of PtdIns(4)P are ill-defined. Using dually labeled Dictyostelium discoideum amoebae and real-time high-resolution confocal laser scanning microscopy (CLSM), we show here that nascent LCVs continuously capture and accumulate PtdIns(4)P-positive vesicles from the host cell. Trafficking of these PtdIns(4)P-positive vesicles to LCVs occurs independently of the Icm/Dot system, but their sustained association requires a functional T4SS. During the infection, PtdIns(3)P-positive membranes become compacted and segregated from the LCV, and PtdIns(3)P-positive vesicles traffic to the LCV but do not fuse. Moreover, using eukaryotic and prokaryotic PtdIns(4)P probes (2×PHFAPP-green fluorescent protein [2×PHFAPP-GFP] and P4CSidC-GFP, respectively) along with Arf1-GFP, we show that PtdIns(4)P-rich membranes of the trans-Golgi network associate with the LCV. Intriguingly, the interaction dynamics of 2×PHFAPP-GFP and P4CSidC-GFP are spatially separable and reveal the specific PtdIns(4)P pool from which the LCV PI originates. These findings provide high-resolution real-time insights into how L. pneumophila exploits the cellular dynamics of membrane-bound PtdIns(4)P for LCV formation.IMPORTANCE The environmental bacterium Legionella pneumophila causes a life-threatening pneumonia termed Legionnaires' disease. The bacteria grow intracellularly in free-living amoebae as well as in respiratory tract macrophages. To this end, L. pneumophila forms a distinct membrane-bound compartment called the Legionella-containing vacuole (LCV). Phosphoinositide (PI) lipids are crucial regulators of the identity and dynamics of host cell organelles. The PI lipid PtdIns(4)P is a hallmark of the host cell secretory pathway, and decoration of LCVs with this PI is required for pathogen vacuole maturation. The source, dynamics, and mode of accumulation of PtdIns(4)P on LCVs are largely unknown. Using Dictyostelium amoebae producing different fluorescent probes as host cells, we show here that LCVs rapidly acquire PtdIns(4)P through the continuous interaction with PtdIns(4)P-positive host vesicles derived from the Golgi apparatus. Thus, the PI lipid pattern of the secretory pathway contributes to the formation of the replication-permissive pathogen compartment.

The Legionella Effector Kinase LegK7 Hijacks the Host Hippo Pathway to Promote Infection.

The intracellular pathogen Legionella pneumophila encodes translocated effector proteins that modify host cell processes to support bacterial survival and growth. Here, we show that the L. pneumophila effector protein LegK7 hijacks the conserved Hippo signaling pathway by molecularly mimicking host Hippo kinase (MST1 in mammals), which is the key regulator of pathway activation. LegK7, like Hippo/MST1, phosphorylates the scaffolding protein MOB1, which triggers a signaling cascade resulting in the degradation of the transcriptional regulators TAZ and YAP1. Transcriptome analysis revealed that LegK7-mediated targeting of TAZ and YAP1 alters the transcriptional profile of mammalian macrophages, a key cellular target of L. pneumophila infection. Specifically, genes targeted by the transcription factor PPARγ, which is regulated by TAZ, displayed altered expression, and continuous interference with PPARγ activity rendered macrophages less permissive to L. pneumophila intracellular growth. Thus, a conserved L. pneumophila effector kinase exploits the Hippo pathway to promote bacterial growth and infection.

A unique cytoplasmic ATPase complex defines the Legionella pneumophila type IV secretion channel.

Type IV secretion systems (T4SSs) are complex machines used by bacteria to deliver protein and DNA complexes into target host cells1-5. Conserved ATPases are essential for T4SS function, but how they coordinate their activities to promote substrate transfer remains poorly understood. Here, we show that the DotB ATPase associates with the Dot-Icm T4SS at the Legionella cell pole through interactions with the DotO ATPase. The structure of the Dot-Icm apparatus was solved in situ by cryo-electron tomography at 3.5 nm resolution and the cytoplasmic complex was solved at 3.0 nm resolution. These structures revealed a cell envelope-spanning channel that connects to the cytoplasmic complex. Further analysis revealed a hexameric assembly of DotO dimers associated with the inner membrane complex, and a DotB hexamer associated with the base of this cytoplasmic complex. The assembly of a DotB-DotO energy complex creates a cytoplasmic channel that directs the translocation of substrates through the T4SS. These data define distinct stages in Dot-Icm machine biogenesis, advance our understanding of channel activation, and identify an envelope-spanning T4SS channel.

LotA, a Legionella deubiquitinase, has dual catalytic activity and contributes to intracellular growth.

The intracellular bacterial pathogen, Legionella pneumophila, establishes the replicative niche as a result of the actions of a large array of effector proteins delivered via the Legionella Type 4 secretion system. Many effector proteins are expected to be involved in biogenesis and regulation of the Legionella-containing vacuole (LCV) that is highly decorated with ubiquitin. Here, we identified a Legionella deubiquitinase, designated LotA, by carrying out a genome analysis to find proteins resembling the eukaryotic ovarian tumour superfamily of cysteine proteases. LotA exhibits a dual ability to cleave ubiquitin chains that is dependent on 2 distinctive catalytic cysteine residues in the eukaryotic ovarian tumour domains. One cysteine dominantly contributes to the removal of ubiquitin from the LCVs by its polyubiquitin cleavage activity. The other specifically cleaves conjugated Lys6-linked ubiquitin. After delivered by the Type 4 secretion system, LotA localises on the LCVs via its PI(3)P-binding domain. The lipid-binding ability of LotA is crucial for ubiquitin removal from the vacuoles. We further analysed the functional interaction of the protein with the recently reported noncanonical ubiquitin ligases of L. pneumophila, revealing that the effector proteins are involved in coordinated regulation that contributes to bacterial growth in the host cells.

Differential expression of virulence genes in Legionella pneumophila growing in Acanthamoeba and human monocytes.

Legionella pneumophila, the causative agent of Legionnaires' disease, is widely distributed throughout natural and artificial water systems and can replicate in macrophages and amoebae. Amoebae are the natural hosts of L. pneumophila, whereas macrophages are incidentally infected. The life cycle of L. pneumophila comprises a replicative phase within the Legionella-containing vacuole (LCV) and a transmissive phase during which bacterial cells become motile and are released via killing of the host. Although the host death mechanisms induced by L. pneumophila have been studied, the expression patterns of related L. pneumophila genes have not been reported. The present study compared the expression patterns of host cell death-associated genes in L. pneumophila grown in the human monocytic cell line THP-1 and Acanthamoeba castellanii. Notably, when L. pneumophila was grown in THP-1, expression of the gene flaA, which is involved in the induction of pyroptosis, was downregulated during the course of infection. In contrast, sdhA associated indirectly with host death, was upregulated. Expression of the genes vipD and sidF, which are involved in the induction and suppression of apoptosis, changed by less than 2-fold. Notably, a lower percentage of pyroptotic cells was observed among infected THP-1 cells relative to uninfected cells, and the latter exhibited stronger expression of caspase-1. A different pattern was observed when L. pneumophila was grown in A. castellanii: flaA and vipD were activated, whereas sdhA and sidF were downregulated during the later stage of replication. The percentage of non-viable (annexin-V+ PI+ or annexin-V+PI-) A. castellanii organisms increased with Legionella infection, and the expression of metacaspase-1, which is involved in encystation was up-regulated at late infection time. In summary, L. pneumophila can multiply intracellularly in both amoebae and macrophages to induce cell death and secondary infection, and this characteristic is essential for its survival in water and the lungs. The gene expression profiles observed in this study indicated the increased cytotoxicity of L. pneumophila in A. castellanii, suggesting an increased adaptation of Legionella to this host.

KKL-35 Exhibits Potent Antibiotic Activity against Legionella Species Independently of trans-Translation Inhibition.

trans-Translation is a ribosome-rescue system that is ubiquitous in bacteria. Small molecules defining a new family of oxadiazole compounds that inhibit trans-translation have been found to have broad-spectrum antibiotic activity. We sought to determine the activity of KKL-35, a potent member of the oxadiazole family, against the human pathogen Legionella pneumophila and other related species that can also cause Legionnaires' disease (LD). Consistent with the essential nature of trans-translation in L. pneumophila, KKL-35 inhibited the growth of all tested strains at submicromolar concentrations. KKL-35 was also active against other LD-causing Legionella species. KKL-35 remained equally active against L. pneumophila mutants that have evolved resistance to macrolides. KKL-35 inhibited the multiplication of L. pneumophila in human macrophages at several stages of infection. No resistant mutants could be obtained, even during extended and chronic exposure. Surprisingly, KKL-35 was not synergistic with other ribosome-targeting antibiotics and did not induce the filamentation phenotype observed in cells defective for trans-translation. Importantly, KKL-35 remained active against L. pneumophila mutants expressing an alternate ribosome-rescue system and lacking transfer-messenger RNA, the essential component of trans-translation. These results indicate that the antibiotic activity of KKL-35 is not related to the specific inhibition of trans-translation and its mode of action remains to be identified. In conclusion, KKL-35 is an effective antibacterial agent against the intracellular pathogen L. pneumophila with no detectable resistance development. However, further studies are needed to better understand its mechanism of action and to assess further the potential of oxadiazoles in treatment.

Identification of Conserved ABC Importers Necessary for Intracellular Survival of Legionella pneumophila in Multiple Hosts.

It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L.p. are expressed by responding to stimuli generated by the protozoan host specifically; a process we term "protozoan-priming." We sought to identify L.p. virulence factors that were required for replication in amoebae in order to highlight the genes necessary for production of the most infectious form of the bacterium. Using a transposon mutagenesis screen, we successfully identified 12 insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages, revealing attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 were required for survival and replication in amoebae and macrophage host cells. Both genes are conserved among numerous important human pathogenic bacteria that can persist or replicate in amoebae. Each gene encodes a component of an ATP binding cassette (ABC) transport complex of unknown function. We demonstrate the lpg0730 ortholog in Francisella tularensis subsp. novicida to be essential for colonization of both protozoan and mammalian host cells, highlighting conserved survival mechanisms employed by bacteria that utilize protozoa as an environmental reservoir for replication.

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila.

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER-associated compartment termed the Legionella-containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule-resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant-negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P-positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1-dependent aggregation of purified, ER-positive LCVs in vitro Thus, Sey1/Atl3-dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.

Intracellular Growth and Morphological Characteristics of Legionella pneumophila during Invasion and Proliferation in Different Cells.

Various studies have been made to attempt to study the interaction between Legionella pneumophila and the host cells. In this research, we successfully constructed a L. pneumophila mutant strain that stably expressed high levels of green fluorescent protein and used this strain to evaluate the adherence, invasion and proliferation of L. pneumophila in association with several cell lines, including seven cell lines [human macrophage-like cell lines (U937, THP-1), murine macrophage-like cell lines (J774.1A, Raw264.7), human bronchial epithelial cell lines (16HBE, Beas-2B) and human cerrical cancer cell line (HeLa)] which have been used as the host models of L. pneumophila, and two breast carcinoma cell lines (MCF-7 and MDA-MB-231). Our results showed that the two newly tested cell lines are able to support the intracellular proliferation of L. pneumophila, and there were some morphological variations during the invasion and intracellular replication of L. pneumophila in different cell lines. These results can help us find out the common and special patterns of invasion and proliferation of L. pneumophila within different hosts. This is conducive to our knowledge on the relationship and interaction between bacteria and host.

Role of the LuxR family transcriptional regulator Lpg2524 in the survival of Legionella pneumophila in water.

The water-borne Gram-negative bacterium Legionella pneumophila (Lp) is the causative agent of Legionnaires' disease. Lp is typically transmitted to humans from water systems, where it grows inside amoebae. Survival of Lp in water is central to its transmission to humans. A transcriptomic study previously identified many genes induced by Lp in water. One such gene, lpg2524, encodes a putative LuxR family transcriptional regulator. It was hypothesized that this gene could be involved in the survival of Lp in water. Deletion of lpg2524 does not affect the growth of Lp in rich medium, in the amoeba Acanthamoeba castellanii, or in human macrophage-like THP-1 cells, showing that Lpg2524 is not required for growth in vitro and in vivo. Nevertheless, deletion of lpg2524 results in a faster colony-forming unit (CFU) reduction in an artificial freshwater medium, Fraquil, indicating that Lpg2524 is important for Lp to survive in water. Overexpression of Lpg2524 also results in a survival defect, suggesting that a precise level of this transcriptional regulator is essential for its function. However, our result shows that Lpg2524 is dispensable for survival in water when Lp is at a high cell density (109 CFU/mL), suggesting that its regulon is regulated by another regulator activated at high cell density.