Mechanisms of invasion of macrophages and lung epithelial cells by filamentous Legionella pneumophila

Legionella species are causative agents of legionellosis, a severe form of pneumonia. These bacteria are pleomorphic and different morphologies have varying virulence potential. Although filamentous forms of Legionella have been reported in patient samples since the first description of legionellosis, their role in disease remains unknown. This work examined the virulence potential of filamentous Legionella pneumophila (FLp) and provides the first description of the invasion mechanisms used by FLp to invade human lung epithelial cells (LECs) and macrophages, and the intracellular differentiation and replication of FLp to produce infectious progeny. FLp were successfully internalized by macrophages and engulfment occurred along the long axis of the filament. However, this uptake process deviated from the canonical phagocytic pathway. Filamentous morphology resulted in the formation of a long-lasting tubular phagocytic cup (T-PC) stage that modulated the outcome of phagocytosis, allowing FLp to escape phagolysosomal killing in a length-dependent manner. Investigation of the mechanism responsible for this revealed that despite being unsealed compartments, the T-PCs fused with endosomes and lysosomes, events linked to the maturation of phagosomes. Nevertheless, these T-PCs were `leaky' and failed to become hydrolytic prior to sealing, impeding their microbicidal capacity. This allowed FLp to secrete effectors while in a non-hydrolytic T-PC and modify their intracellular compartments. FLp attachment to LECs was mediated by E-cadherin and β1 integrin receptors and induced the formation of actin-enriched membrane surface structures that we designated `hooks' and `membrane wraps'. These structures entrapped the filament at the cell surface, and through a variation of the zipper mechanism of invasion, facilitated the actomyosin-dependent internalization of FLp. A supply of E-cadherins from intracellular recycling pathway and β1 integrins released by focal adhesion turnover sustained this internalization process. Collectively, these results demonstrate that FLp can invade both LECs and macrophages, and could play an important role in pathogenesis.Ph.D.2017-06-30 00:00:0

Rab GTPases in Immunity and Inflammation

Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation

Survival of intracellular pathogens in response to mTORC1- or TRPML1-TFEB-induced xenophagy

Intracellular pathogens establish persistent infections by generating reservoirs that protect them from the action of antibiotics and the host immune response. Novel therapeutics should then target the host pathways exploited by the pathogens to form these intracellular niches. An attractive strategy to achieve this is inducing xenophagy, the selective autophagy that recognizes and targets invading pathogens for degradation. However, some bacteria have evolved mechanisms to co-opt xenophagy for their own benefit. Therefore, in this study we determine the effect of inducing xenophagy by different pathways, namely the inhibition of MTOR or through TRPML1-TFEB activation, on the fate of pathogens that are either susceptible to, evade or require autophagy for intracellular survival. We identified a dose of rapamycin that exclusively induces autophagy through MTOR inhibition and used ML-SA1 to activate the TRPML1-TFEB pathway, which also increases lysosomal biogenesis. We found that ML-SA1 induced greater autophagy flux than rapamycin. By performing in vitro infections with H. pylori, S. Typhimurium, S. flexneri, L. monocytogenes and S. aureus, we established that ML-SA1 had a more potent effect than rapamycin in restricting the growth of pathogens susceptible to xenophagy. In the case of pathogens that produce effectors to block xenophagy, ML-SA1, but not rapamycin, resulted in bacterial killing. During S. aureus infection, which depends on autophagy for intracellular survival, ML-SA1 administration potentiated bacterial growth. We suggest that while targeting the xenophagy pathway holds promise for treatment of intracellular pathogens, a precision approach to select the correct target to induce effective bacterial killing is warranted. Abbreviations: 3-MA: 3-methyladenine, ATG: autophagy-related protein, Baf: bafilomycin A1; Ca2+: calcium, CFU: colony-forming units, DMSO: dimethyl sulfoxide, h: hour, Hp: Helicobacter pylori, hpi: hours post-infection, Lamp1: lysosomal-associated membrane protein 1, LC3: microtubule-associated protein 1A/1B-light chain, Lm: Listeria monocytogenes, LSD: lysosomal storage disorder, min: minutes, mTOR: mechanistic target of rapamycin; mTORC1: mechanistic target of rapamycin complex 1, MEF: mouse embryonic fibroblast, μM: micromolar, moi: multiplicity of infection, nM: nanomolar, OD: optical density, PBS: phosphate buffer saline, Sa: Staphylococcus aureus, SCV: Salmonella containing vacuole, Sifs: Salmonella-induced filaments, Sf: Shigella flexneri, SLAPs: Spacious Listeria containing phagosomes, St: Salmonella Typhimurium TFEB: transcription factor EB, TRPML1: transient receptor potential membrane channel 1, VacA: vacuolating cytotoxin, wt: wild-type

Small Rho GTPases and the Effector VipA Mediate the Invasion of Epithelial Cells by Filamentous Legionella pneumophila

Legionella pneumophila (Lp) exhibits different morphologies with varying degrees of virulence. Despite their detection in environmental sources of outbreaks and in respiratory tract secretions and lung autopsies from patients, the filamentous morphotype of Lp remains poorly studied. We previously demonstrated that filamentous Lp invades lung epithelial cells (LECs) and replicates intracellularly in a Legionella containing vacuole. Filamentous Lp activates β1integrin and E-cadherin receptors at the surface of LECs leading to the formation of actin-rich cell membrane structures we termed hooks and membrane wraps. These structures entrap segments of an Lp filament on host cell surface and mediate bacterial internalization. Here we investigated the molecular mechanisms responsible for the actin rearrangements needed for the formation and elongation of these membrane wraps and bacterial internalization. We combined genetic and pharmacological approaches to assess the contribution of signaling downstream of β1integrin and E-cadherin receptors, and Lp Dot/Icm secretion system- translocated effectors toward the invasion process. Our studies demonstrate a multi-stage mechanism of LEC invasion by filamentous Lp. Bacterial attachment to host cells depends on signaling downstream of β1integrin and E-cadherin activation, leading to Rho GTPases-dependent activation of cellular actin nucleating proteins, Arp2/3 and mDia. This mediates the formation of primordial membrane wraps that entrap the filamentous bacteria on the cell surface. Following this, in a second phase of the invasion process the Dot/Icm translocated effector VipA mediates rapid membrane wrap elongation, leading to the engulfment of the filamentous bacteria by the LECs. Our findings provide the first description of Rho GTPases and a Dot/Icm effector VipA regulating the actin dynamics needed for the invasion of epithelial cells by Lp

Essential Roles and Regulation of the <em>Legionella pneumophila</em> Collagen-Like Adhesin during Biofilm Formation

<div><p>Legionellosis is mostly caused by <em>Legionella pneumophila</em> (<em>Lp</em>) and is defined by a severe respiratory illness with a case fatality rate ranging from 5 to 80%. In a previous study, we showed that a glycosaminoglycan (GAG)-binding adhesin of <em>Lp</em>, named Lcl, is produced during legionellosis and is unique to the <em>L. pneumophila</em> species. Importantly, a mutant depleted in Lcl (<em>Δlpg2644</em>) is impaired in adhesion to GAGs and epithelial cells and in biofilm formation. Here, we examine the molecular function(s) of Lcl and the transcriptional regulation of its encoding gene during different stages of the biofilm development. We show that the collagen repeats and the <em>C</em>-terminal domains of Lcl are crucial for the production of biofilm. We present evidence that Lcl is involved in the early step of surface attachment but also in intercellular interactions. Furthermore, we address the relationship between Lcl gene regulation during biofilm formation and quorum sensing (QS). In a static biofilm assay, we show that Lcl is differentially regulated during growth phases and biofilm formation. Moreover, we show that the transcriptional regulation of <em>lpg2644,</em> mediated by a prototype of QS signaling homoserine lactone (3OC12-HSL), may play a role during the biofilm development. Thus, transcriptional down-regulation of <em>lpg2644</em> may facilitate the dispersion of Lp to reinitiate biofilm colonization on a distal surface.</p> </div

pH of endophagosomes controls association of their membranes with Vps34 and PtdIns(3)P levels

Phagocytosis of filamentous bacteria occurs through tubular phagocytic cups (tPCs) and takes many minutes to engulf these filaments into phagosomes. Contravening the canonical phagocytic pathway, tPCs mature by fusing with endosomes. Using this model, we observed the sequential recruitment of early and late endolysosomal markers to the elongating tPCs. Surprisingly, the regulatory early endosomal lipid phosphatidylinositol-3-phosphate (PtdIns(3)P) Persists on tPCs as long as their luminal pH remains neutral. Interestingly, by manipulating cellular pH, we determined that PtdIns(3) P behaves similarly in canonical phagosomes as well as endosomes. We found that this is the product of a pH-based mechanism that induces the dissociation of the Vps34 class III phosphatidylinositol-3-kinase from these organelles as they acidify. The detachment of Vps34 stops the production of PtdIns(3)P, allowing for the turnover of this lipid by PIKfyve. Given that PtdIns(3)P-dependent signaling is important for multiple cellular pathways, this mechanism for pH-dependent regulation of Vps34 could be at the center of many PtdIns(3)P-dependent cellular processes. </p

Adherence and persistence of <i>L. pneumophila</i> in a continuous-flow chamber system.

<p>Fluorescence micrographs of GFP-expressing <i>L.pneumophila</i> strains Lp02, Lp02 <i>Δlpg2644</i> and chromosomal complemented mutant (Lp02 <i>Δlpg2644/clpg2644</i>) in a continuous-flow chamber system operated at 5.5 µl/mn with BYE medium (magnification 100 X). During the initial attachment (0 h), Lp02 and Lp02 <i>Δlpg2644/clpg2644</i> strains formed a monolayer on abiotic surface whereas Lp02 <i>Δlpg2644</i> was unable to efficiently attach to the flow chambers and detached in a flow dependent manner (2 h to 48 h). Data are representative of three independent replicates. Scale bar, 5 µm.</p

Transcriptional regulation of <i>lpg2644</i> and synthesis of Lcl in broth cultures, biofilm and in presence of 3OC₁₂-HSL homoserine lactone.

<p>Amounts of <i>lpg2644</i> transcripts and synthesis of Lcl were respectively measured by (A) qRT-PCR and (B) anti-Lcl immunoblot analysis of cell pellet (c) supernatant (s) and sessile cells/matrix (sm) fractions in Lp02 harvested from exponential (E), post-exponential (PE), mid-stationary (MS), late stationary (LS) broth cultures and biofilms (2 and 6 days old). Quantitative RT-PCR values are means+/−s.d. from three individual experiments. # two-tailed Student’s t-test P-value ≤0.01 versus MS phase, * two-tailed Student’s t-test P-value ≤0.001 versus MS phase. (C) Effect of 3OC12-HSL homoserine lactone on <i>lpg2644</i> transcription and (D) Lcl synthesis. Lp02 was grown in absence or in presence of 3OC12-HSL (10 µM) for 24 h. Transcriptional level of <i>lpg2644</i> was determined by qRT-PCR analysis. Data are representative of 3 independent experiments.* Student’s t-test P-value ≤0,001 versus untreated Lp02. (D) Synthesis of Lcl was estimated by anti-Lcl immunoblot of cell pellets (c) and supernatant fractions (s) of Lp02 and Lp02 <i>Δlpg2644</i> p<i>lpg2644</i>. The positions of the molecular size markers are indicated on the left of the blot in kDa.</p