Legionella Pneumophila Infection Research Articles

Legionella Pneumophila Infections among Egyptian Patients: Updated Diagnostic Tests and Evaluation

Legionella Pneumophila Infections among Egyptian Patients: Updated Diagnostic Tests and Evaluation

Legionella pneumophila inhibits type I interferon signaling to avoid cell-intrinsic host cell defense.

The host type I interferon (IFN) response protects against Legionella pneumophila infections. Other bacterial pathogens inhibit type I IFN-mediated cell signaling; however, the interaction between this signaling pathway and L. pneumophila has not been well described. Here, we demonstrate that L. pneumophila inhibits the IFN-β signaling pathway but does not inhibit IFN-γ-mediated cell signaling. The addition of IFN-β to L. pneumophila-infected macrophages limited bacterial growth independently of NOS2 and reactive nitrogen species. The type IV secretion system of L. pneumophila is required to inhibit IFN-β-mediated cell signaling. Finally, we show that the inhibition of the IFN-β signaling pathway occurs downstream of STAT1 and STAT2 phosphorylation. In conclusion, our findings describe a novel host cell signaling pathway inhibited by L. pneumophila via its type IV secretion system.

Structural basis for the toxicity of Legionella pneumophila effector SidH

Legionella pneumophila (LP) secretes more than 300 effectors into the host cytosol to facilitate intracellular replication. One of these effectors, SidH, 253 kDa in size with no sequence similarity to proteins of known function is toxic when overexpressed in host cells. SidH is regulated by the LP metaeffector LubX which targets SidH for degradation in a temporal manner during LP infection. The mechanism underlying the toxicity of SidH and its role in LP infection are unknown. Here, we determined the cryo-EM structure of SidH at 2.7 Å revealing a unique alpha helical arrangement with no overall similarity to known protein structures. Surprisingly, purified SidH came bound to a E. coli EF-Tu/t-RNA/GTP ternary complex which could be modeled into the cryo-EM density. Mutation of residues disrupting the SidH-tRNA interface and SidH-EF-Tu interface abolish the toxicity of overexpressed SidH in human cells, a phenotype confirmed in infection of Acanthamoeba castellani. We also present the cryo-EM structure of SidH in complex with a U-box domain containing ubiquitin ligase LubX delineating the mechanism of regulation of SidH. Our data provide the basis for the toxicity of SidH and into its regulation by the metaeffector LubX.

Tools and mechanisms of vacuolar escape leading to host egress in Legionella pneumophila infection: Emphasis on bacterial phospholipases.

The phenomenon of host cell escape exhibited by intracellular pathogens is a remarkably versatile occurrence, capable of unfolding through lytic or non-lytic pathways. Among these pathogens, the bacterium Legionella pneumophila stands out, having adopted a diverse spectrum of strategies to disengage from their host cells. A pivotal juncture that predates most of these host cell escape modalities is the initial escape from the intracellular compartment. This critical step is increasingly supported by evidence suggesting the involvement of several secreted pathogen effectors, including lytic proteins. In this intricate landscape, L. pneumophila emerges as a focal point for research, particularly concerning secreted phospholipases. While nestled within its replicative vacuole, the bacterium deftly employs both its type II (Lsp) and type IVB (Dot/Icm) secretion systems to convey phospholipases into either the phagosomal lumen or the host cell cytoplasm. Its repertoire encompasses numerous phospholipases A (PLA), including three enzymes-PlaA, PlaC, and PlaD-bearing the GDSL motif. Additionally, there are 11 patatin-like phospholipases A as well as PlaB. Furthermore, the bacterium harbors three extracellular phospholipases C (PLCs) and one phospholipase D. Within this comprehensive review, we undertake an exploration of the pivotal role played by phospholipases in the broader context of phagosomal and host cell egress. Moreover, we embark on a detailed journey to unravel the established and potential functions of the secreted phospholipases of L. pneumophila in orchestrating this indispensable process.

4.3.1]Bicyclic FKBP Ligands Inhibit Legionella Pneumophila Infection by LpMip-Dependent and LpMip-Independent Mechanisms.

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.

TNF licenses macrophages to undergo rapid caspase-1, -11, and -8-mediated cell death that restricts Legionella pneumophila infection.

The inflammatory cytokine tumor necrosis factor (TNF) is necessary for host defense against many intracellular pathogens, including Legionella pneumophila. Legionella causes the severe pneumonia Legionnaires' disease and predominantly affects individuals with a suppressed immune system, including those receiving therapeutic TNF blockade to treat autoinflammatory disorders. TNF induces pro-inflammatory gene expression, cellular proliferation, and survival signals in certain contexts, but can also trigger programmed cell death in others. It remains unclear, however, which of the pleiotropic functions of TNF mediate control of intracellular bacterial pathogens like Legionella. In this study, we demonstrate that TNF signaling licenses macrophages to die rapidly in response to Legionella infection. We find that TNF-licensed cells undergo rapid gasdermin-dependent, pyroptotic death downstream of inflammasome activation. We also find that TNF signaling upregulates components of the inflammasome response, and that the caspase-11-mediated non-canonical inflammasome is the first inflammasome to be activated, with caspase-1 and caspase-8 mediating delayed pyroptotic death. We find that all three caspases are collectively required for optimal TNF-mediated restriction of bacterial replication in macrophages. Furthermore, caspase-8 is required for control of pulmonary Legionella infection. These findings reveal a TNF-dependent mechanism in macrophages for activating rapid cell death that is collectively mediated by caspases-1, -8, and -11 and subsequent restriction of Legionella infection.

Hiding in the yolk: A unique feature of Legionella pneumophila infection of zebrafish.

The zebrafish has become a powerful model organism to study host-pathogen interactions. Here, we developed a zebrafish model to dissect the innate immune response to Legionella pneumophila during infection. We show that L. pneumophila cause zebrafish larvae death in a dose dependent manner. Additionally, we show that macrophages are the first line of defence and cooperate with neutrophils to clear the infection. Immunocompromised humans have an increased propensity to develop pneumonia, similarly, when either macrophages or neutrophils are depleted, these "immunocompromised" larvae become lethally sensitive to L. pneumophila. Also, as observed in human infections, the adaptor signalling molecule Myd88 is not required to control disease in the larvae. Furthermore, proinflammatory cytokine genes il1β and tnf-α were upregulated during infection, recapitulating key immune responses seen in human infection. Strikingly, we uncovered a previously undescribed infection phenotype in zebrafish larvae, whereby bloodborne, wild type L. pneumophila invade and grow in the larval yolk region, a phenotype not observed with a type IV secretion system deficient mutant that cannot translocate effectors into its host cell. Thus, zebrafish larva represents an innovative L. pneumophila infection model that mimics important aspects of the human immune response to L. pneumophila infection and will allow the elucidation of mechanisms by which type IV secretion effectors allow L. pneumophila to cross host cell membranes and obtain nutrients from nutrient rich environments.

Legionella pneumophila Infections during a 7-Year Retrospective Analysis (2016-2022): Epidemiological, Clinical Features and Outcomes in Patients with Legionnaires' Disease.

Legionella pneumophila (LP) is one of the main causative agents of community-acquired pneumonia in Europe and its fifth bacterial cause in Italy (4.9%). We conducted a seven year retrospective analysis of LP infection serogroup 1 in Asti, Piedmont, between 2016 and 2022. Patients were included if they tested positive for the Legionella urinary antigen. Clinical, laboratory, and radiologic data were analyzed to describe the risk factors for mortality. Fifty patients with LD were collected, mainly male, with a median age of 69 years. The main comorbidities were cardiovascular diseases (50%), pulmonary diseases (26%), and neurological diseases (12%). The most common clinical presentations were fever, respiratory, gastrointestinal, and neurologic symptoms. Older age (p = 0.004), underlying cardiovascular diseases (p = 0.009), late diagnosis at admission (p = 0.035), and neurological symptoms at diagnosis (p = 0.046) were more common in the non-survivor group. Moreover, a septic-shock presentation or the need for non-invasive ventilation at admission were associated with a higher mortality. No considerable differences in the biochemical data were found between the two groups except for the median neutrophil count, lymphocyte count, neutrophil-to-lymphocyte ratio, and PCT value. We did not find any differences in mortality related to the choice of antibiotic regimen. Differences in outcome were associated with the median duration of treatment (p =< 0.001) but not to the choice of antibiotic regimen (mainly levofloxacin or azithromycin). In conclusion, early individuation of the wide spectrum of clinical characteristics of LP infection such as respiratory, cardiac, and neurological manifestations of the patient's comorbidities, and significant biochemical data should help clinicians flag high risk patients and potentially improve their outcome.

Proteomic analysis of Acanthamoeba castellanii response to Legionella pneumophila infection.

Legionella pneumophila is an opportunistic pathogen responsible for Legionnaires' disease or Legionellosis. This bacterium is found in the environment interacting with free-living amoebae such as Acanthamoeba castellanii. Until now, proteomic analyses have been done in amoebae infected with L. pneumophila but focused on the Legionella-containing vacuole. In this study, we propose a global proteomic analysis of the A. castellanii proteome following infection with L. pneumophila wild-type (WT) or with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We found that infection with L. pneumophila WT leads to reduced levels of A. castellanii proteins associated with lipid homeostasis/metabolism, GTPase regulation and kinase. The levels of organelle-associated proteins were also decreased during infection. L. pneumophila WT infection leads to increase levels of proteins associated with proteins polyubiquitination, folding or degradation and antioxidant activities. This study reinforces our knowledge of this too-little-explored but so fundamental interaction between L. pneumophila and A. castellanii, to understand how the bacterium could resist amoeba digestion.

Inflammasome activation and CCR2-mediated monocyte-derived dendritic cell recruitment restrict Legionella pneumophila infection.

Flagellin-induced NAIP/NLRC4 inflammasome activation and pyroptosis are critical events restricting Legionella pneumophila infection. However, the cellular and molecular dynamics of the in vivo responses against this bacterium are still unclear. We have found temporal coordination of two independent innate immunity pathways in controlling Legionella infection, the inflammasome activation and the CCR2-mediated Mo-DC recruitment. Inflammasome activation was an important player at the early stage of infection by lowering the numbers of bacteria for an efficient bacterial clearance conferred by the Mo-DC at the late stage of the infection. Mo-DC emergence highly depended on CCR2-signaling and dispensed inflammasome activation and pyroptosis. Also, Mo-DC compartment did not rely on the inflammasome machinery to deliver proper immune responses and was the most abundant cytokine-producing among the monocyte-derived cells in the infected lung. Importantly, when the CCR2- and NLRC4-dependent axes of response were simultaneously ablated, we observed an aggravated bacterial burden in the lung of infected mice. Taken together, we showed that inflammasome activation and CCR2-mediated immune response interplay in distinct pathways to restrict pulmonary bacterial infection. These findings extend our understanding of the in vivo integration and cooperation of different innate immunity arms in controlling infectious agents.

Hiding in the yolk: a unique feature of Legionella pneumophila infection of zebrafish

Hiding in the yolk: a unique feature of Legionella pneumophila infection of zebrafish

Lung Ultrasound Monitoring of Legionella Ventilator-Associated Pneumonia in an Extremely Low-Birth-Weight Infant.

Ventilator-associated pneumonia (VAP) is a common complication of different severe lung diseases that need to be treated with mechanical ventilation in newborn infants. However, VAP due to Legionella pneumophila infection is rarely reported in the literature, especially in extremely low-birth-weight (ELBW) infants. Lung ultrasound (LUS) has been used in the diagnosis of neonatal pneumonia, but there is no literature on the ultrasound characteristics of Legionella-VAP in ELBW infants. This paper introduced the typical LUS findings of Legionella-VAP in ELBW infants, which mainly includes severe and large-area lung consolidation and atelectasis in the bilateral lungs; whether there is blood supply in the consolidated area has an important reference value for predicting the prognosis. In addition, the treatment and management experience were also introduced together, thereby helping us to deepen the understanding of the disease and avoid missed diagnoses.

Tailored approach to study Legionella infection using a lattice light sheet microscope (LLSM).

Legionella is a genus of ubiquitous environmental pathogens found in freshwater systems, moist soil, and composted materials. More than four decades of Legionella research has provided important insights into Legionella pathogenesis. Although standard commercial microscopes have led to significant advances in understanding Legionella pathogenesis, great potential exists in the deployment of more advanced imaging techniques to provide additional insights. The lattice light sheet microscope (LLSM) is a recently developed microscope for 4D live cell imaging with high resolution and minimum photo-damage. We built a LLSM with an improved version for the optical layout with two path-stretching mirror sets and a novel reconfigurable galvanometer scanner (RGS) module to improve the reproducibility and reliability of the alignment and maintenance of the LLSM. We commissioned this LLSM to study Legionella pneumophila infection with a tailored workflow designed over instrumentation, experiments, and data processing methods. Our results indicate that Legionella pneumophila infection is correlated with a series of morphological signatures such as smoothness, migration pattern and polarity both statistically and dynamically. Our work demonstrates the benefits of using LLSM for studying long-term questions in bacterial infection. Our free-for-use modifications and workflow designs on the use of LLSM system contributes to the adoption and promotion of the state-of-the-art LLSM technology for both academic and commercial applications.

Association of Natural Waterways and Legionella pneumophila Infection in Eastern Wisconsin: A Case-Control Study.

Preliminary research has suggested possible associations between natural waterways and Legionella infection, and we previously explored these associations in eastern Wisconsin using positive L. pneumophila serogroup 1 urine antigen tests (LUAT) as diagnostic. This case-control study was a secondary analysis of home address data from patients who underwent LUAT at a single eastern Wisconsin health system from 2013 to 2017. Only zip codes within the health system's catchment area that registered ≥3 positive cases and ≥50 completed tests, as well as geographically adjacent zip codes with ≥2 positive cases and ≥50 tests, were included. A 1:3 ratio of cases to randomly selected controls was used. Home addresses were geocoded and mapped using ArcGIS software (Esri); nearest waterway and distance to home was identified. Distance to nearest waterway according to ArcGIS was verified/corrected using Google Maps incognito. Distances were analyzed using chi-squared and 2-sample t-tests. Overall, mean distance to nearest waterway did not differ between cases (2958 ± 2049 ft) and controls (2856 ± 2018 ft; P=0.701). However, in a subset of nonurban zip codes, cases were closer to nearest waterway than controls (1165 ± 905 ft vs 2113 ± 1710 ft; P=0.019). No association was found between cases and type of waterway. Further research is needed to investigate associations and differences between natural and built environmental water sources in relation to legionellosis.

A Patient With Stage III Locally Advanced Pancreatic Adenocarcinoma Treated With Intra-Arterial Infusion FOLFIRINOX: Impressive Tumoral Response and Death due to Legionella pneumophila Infection: A Unique Case Report.

Patients affected by pancreatic ductal adenocarcinoma (PDAC) have very poor prognosis, whereby at a follow-up of 5 years, the mortality rate is very similar to the incidence rate. Globally, around 10% of patients are amenable to radical surgery at the time of diagnosis, which represents the only chance of cure or long-term survival for these patients. Almost 40% of patients with PDAC show locally advanced pancreatic cancer (LAPC). LAPC is not a metastatic disease, although it is not amenable to radical surgery. For these patients, systemic induction chemotherapy with intravenous FOLFIRINOX (5-fluorouracil, folic acid, irinotecan, oxaliplatin) regimen is administered, with the aim of conversion to surgery, although the conversion rate remains low, at approximately 10% to 15%. Pancreatic arterial chemotherapy has been explored to overcome the intrinsic tumor pancreatic resistance to systemic chemotherapy, where an intra-arterial port-a-cath is placed by means of interventional oncology techniques under angiographic guidance in the operating theater. Here, we treated a patient with an intra-arterially modified FOLFIRINOX regimen. Three courses were administered, and the patient experienced no adverse events. At the end of the third course, the patient rapidly developed lung failure due to nosocomial Legionella pneumophila infection, despite the impressive pathological tumor response shown in the autopsy report. This is a first and unique report that demonstrates that pancreatic intra-arterial FOLFIRINOX can be safe and efficacious. We believe that this preliminary result will be confirmed in the next patients to be enrolled and that it provides a glimmer of hope for patients with this lethal disease.

Blockade of Cycloxygenase-2 ameliorates sepsis induced immune-suppression by regulating myeloid-derived suppressor cells

BackgroundMyeloid-derived suppressor cells (MDSCs) and cyclooxy-genase-2 (COX-2)/Prostaglandin E2 (PGE2) axis are important contributors to sepsis-induced immune-suppression. The purpose of present study is to explore whether COX-2 inhibitor can improve immunological disorder after sepsis via regulating MDSCs. MethodsA ‘‘two-hit’’ model reflecting clinical sepsis development was performed. Cecal ligation and puncture (CLP) and Legionella pneumophila infection were used as the first and the second hit, respectively. NS398, a selective COX-2 inhibitor, was utilized to treat septic mice. The motality, bacterial counts in the lung, systematic inflammatory reaction and CD4 + T cells response after sepsis were assessed, so as the frequency and function of MDSCs. In some experiments, the number of MDSCs was manipulated by adoptive transfer or neutralizing antibody before induction of secondary infection. ResultsMice surviving CLP showed a marked expansion and activation of MDSCs in spleen, accompanied by suppressed proliferating capability, impaired secreting functionand increased apoptosis of CD4 + T cells. Majority of CLP survivors became succumbed to L. pneumophila invasion, associated with defective bacteria elimination ability. NS398 treatment was found to ameliorate these adverse outcomes significantly. ConclusionMDSCs contribute greatly to the sepsis-induced immune dysfunction. Inhibiting COX-2 may become a promising therapy that targets MDSCs-induced immunosuppression.

Using Genomic Deletion Mutants to Investigate Effector-Triggered Immunity During Legionella pneumophila Infection.

Legionella pneumophila is an intracellular bacterial pathogen that uses a type IV secretion system (T4SS), termed Dot/Icm, to secrete more than 330 virulence effector proteins into the infected host cell. Many Dot/Icm effectors are involved in biogenesis of the Legionella-containing vacuole (LCV), which allows intracellular bacterial replication in environmental amoebae and alveolar macrophages. Through their activity, some effectors trigger the mammalian host immune response in a phenomenon termed effector-triggered immunity (ETI). Here, we describe a protocol to create and use L. pneumophila genome deletion mutants to identify effector(s) that alter pro-inflammatory cytokine production and bacterial clearance in the lungs of mice.

Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor.

Cytokines made by macrophages play a critical role in determining the course of Legionella pneumophila infection. Prior murine-based modeling indicated that this cytokine response is initiated upon recognition of L. pneumophila by a subset of Toll-like receptors, namely TLR2, TLR5, and TLR9. Through the use of shRNA/siRNA knockdowns and subsequently CRISPR/Cas9 knockouts (KO), we determined that TRIF, an adaptor downstream of endosomal TLR3 and TLR4, is required for full cytokine secretion by human primary and cell-line macrophages. By characterizing a further set of TLR KO’s in human U937 cells, we discerned that, contrary to the viewpoint garnered from murine-based studies, TLR3 and TLR4 (along with TLR2 and TLR5) are in fact vital to the macrophage response in the early stages of L. pneumophila infection. This conclusion was bolstered by showing that i) chemical inhibitors of TLR3 and TLR4 dampen the cytokine output of primary human macrophages and ii) transfection of TLR3 and TLR4 into HEK cells conferred an ability to sense L. pneumophila. TLR3- and TLR4-dependent cytokines promoted migration of human HL-60 neutrophils across an epithelial layer, pointing to the biological importance for the newfound signaling pathway. The response of U937 cells to L. pneumophila LPS was dependent upon TLR4, a further contradiction to murine-based studies, which had concluded that TLR2 is the receptor for Legionella LPS. Given the role of TLR3 in sensing nucleic acid (i.e., dsRNA), we utilized newly-made KO U937 cells to document that DNA-sensing by cGAS-STING and DNA-PK are also needed for the response of human macrophages to L. pneumophila. Given the lack of attention given them in the bacterial field, C-type lectin receptors were similarly examined; but, they were not required. Overall, this study arguably represents the most extensive, single-characterization of Legionella-recognition receptors within human macrophages.

Heart and Lung Involvement Detected by Cardiac Magnetic Resonance Imaging in a Patient with Legionella Pneumophila Infection: Case Report

Heart and Lung Involvement Detected by Cardiac Magnetic Resonance Imaging in a Patient with Legionella Pneumophila Infection: Case Report

Acute Myocarditis Associated with Legionella Infection: Usefulness of Layer-specific Two-dimensional Longitudinal Speckle-tracking Analysis.

Pneumonia is the most commonly described manifestation of Legionella pneumophila infection (legionellosis), and extrapulmonary manifestations are uncommon. There are a few descriptions of acute myocarditis associated with legionellosis. We present a case of acute myocarditis in a patient admitted for legionellosis with multisystemic involvement (lung, heart, and kidney). Left ventricular (LV) dysfunction was documented by cardiac magnetic resonance (CMR) and two-dimensional speckle-tracking echocardiography; layer-specific strain analyses were performed, which allowed to differentiate subendocardial or subepicardial contractile impairment. Layer-specific strain analyses by echocardiography demonstrated impairment of subepicardial deformation in the inferolateral wall, which mirrored CMR findings, showing late gadolinium enhancement in the subepicardium of the same LV segments. After initiation of appropriate antibiotic therapy with levofloxacin, LV systolic function rapidly improved as assessed by both CMR and strain analyses, with concomitant normalization of both clinical and biochemical abnormalities. The basic mechanisms of myocardial involvement during legionellosis are unclear; we discussed our findings according to the limited available evidence.