Legionella Pneumophila Research Articles

Study of the effectiveness of microfiltration, solar disinfection, photocatalysis, and UV-C254 radiation on water contaminated by Legionella pneumophila

Legionella are opportunistic pathogens in artificial water systems and can cause a severe form of pneumonia called Legionnaires’ disease (LD), which is fatal in 5–50% of cases when contaminated aerosols are inhaled. LD emerged in the last half of the 20th century. Global challenges such as urbanization, aging populations, climate change, water scarcity, and circular economy approaches have led to an increase in the number of complex water systems and thus LD cases, which are expected to rise to around 2.5 billion by 2050. LD is a preventable disease if adequate measures are taken to control Legionella in water systems. Chlorine treatment and heat treatment are the methods of choice to sanitize a Legionella-contaminated water system. However, these two methods are not always effective in ensuring that the treated water is free of Legionella and safe for consumption and use. Therefore, we proposed to evaluate the efficacy of microfiltration, solar disinfection, photocatalysis, and UV-C254 irradiation on Legionella-contaminated water. Microfiltration was performed using a membrane filtration cell (SODIS). Solar disinfection and photocatalysis were performed with an experimental table consisting of six 1 L beakers, 3 for solar disinfection and 3 for photocatalysis. UV-C254 disinfection was also tested with 3 1 L beakers. For all four methods tested, the percentage of Legionella removal was over 99.99. The four disinfection systems tested provided satisfactory results and ensured that the treated water was free of Legionella and safe for consumption and use.

Size-selective sampler combined with an immunochromatographic assay for the rapid detection of airborne Legionella pneumophila

Airborne biological aerosols (also called bioaerosols) are found in various environmental and occupational settings. Among these, pathogenic bioaerosols can cause diseases such as legionellosis, influenza, measles, and tuberculosis. To prevent or minimize people's exposure to these pathogenic bioaerosols in the field, a rapid detection method is required. In this study, a size-selective bioaerosol (SSB) sampler was combined with the immunochromatographic assay (ICA). The SSB sampler can collect bioaerosols on the sampling swab and the lateral flow test kit used in ICA can rapidly detect the pathogens in bioaerosols collected on the swab. Before testing the combined method, the lower limit of detection (LOD) of the lateral flow test kit was determined. Legionella pneumophila (L. pneumophila) was used as a target pathogen. The results show that at least 1.3 × 103L. pneumophila cells are required to be detected by the lateral flow test kit. To test the developed method, L. pneumophila suspension was aerosolized in the sampling chamber and collected using two SSB samplers with different sampling times (10 and 20 min). The developed method could detect aerosolized L. pneumophila and also estimate the concentrations from the lower LOD, sampling time, and formation of a positive line on a test strip. When positive results were obtained from sampling for 10 min and 20 min, concentrations of respirable L. pneumophila were estimated ≥5.2 × 104 CFUresp/m3 and ≥2.6 × 104 CFUresp/m3, respectively. The conventional sampler Andersen impactor with colony counting was also used for comparison. In all cases, the estimated concentrations obtained by the developed method were higher than those obtained by the conventional method. These findings confirm that the developed method can overcome the limitations of conventional methods and eventually benefit environmental and occupational health by providing a better method for risk assessment.

CRP under 130 mg/L rules out the diagnosis of Legionella pneumophila serogroup 1 (URINELLA Study)

IntroductionIn case of pneumonia, some biological findings are suggestive for Legionnaire’s disease (LD) including C-reactive protein (CRP). A low level of CRP is predictive for negative Legionella Urinary-Antigen-Test (L-UAT).MethodObservational retrospective study in Nord-Franche‐Comté Hospital with external validation in Besançon University Hospital, France which included all adults with L-UAT performed during January 2018 to December 2022. The objective was to determine CRP optimal threshold to predict a L-UAT negative result.ResultsURINELLA included 5051 patients (83 with positive L-UAT). CRP optimal threshold was 131.9 mg/L, with a negative predictive value (NPV) at 100%, sensitivity at 100% and specificity at 58.0%. The AUC of the ROC-Curve was at 88.7% (95% CI, 86.3–91.1). External validation in Besançon Hospital patients showed an AUC at 89.8% (95% CI, 85.5–94.1) and NPV, sensitivity and specificity was respectively 99.9%, 97.6% and 59.1% for a CRP threshold at 131.9 mg/L; after exclusion of immunosuppressed patients, index sensitivity and NPV reached also 100%.ConclusionIn case of pneumonia suspicion with a CRP level under 130 mg/L (independently of the severity) L-UAT is useless in immunocompetent patients with a NPV at 100%. We must remain cautious in patients with symptoms onset less than 48 h before CRP dosage.

A novel hybridization capture method for direct whole genome sequencing of clinical specimens to inform Legionnaires' disease investigations.

The unprecedented precision and resolution of whole genome sequencing (WGS) can provide definitive identification of infectious agents for epidemiological outbreak tracking. WGS approaches, however, are frequently impeded by low pathogen DNA recovery from available primary specimens or unculturable samples. A cost-effective hybrid capture assay for Legionella pneumophila WGS analysis directly on primary specimens was developed. DNA from a diverse range of sputum and autopsy specimens PCR-positive for L. pneumophila serogroup 1 (LPSG1) was enriched with this method, and WGS was performed. All tested specimens were determined to be enriched for Legionella reads (up to 209,000-fold), significantly improving the discriminatory power to compare relatedness when no clinical isolate was available. We found the WGS data from some enriched specimens to differ by less than five single-nucleotide polymorphisms (SNPs) when compared to the WGS data of a matched culture isolate. This testing and analysis retrospectively provided previously unconfirmed links to environmental sources for clinical specimens of sputum and autopsy lung tissue. The latter provided the additional information needed to identify the source of these culture-negative cases associated with the South Bronx 2015 Legionnaires' disease (LD) investigation in New York City. This new method provides a proof of concept for future direct clinical specimen hybrid capture enrichment combined with WGS and bioinformatic analysis during outbreak investigations.IMPORTANCELegionnaires' disease (LD) is a severe and potentially fatal type of pneumonia primarily caused by inhalation of Legionella-contaminated aerosols from man-made water or cooling systems. LD remains extremely underdiagnosed as it is an uncommon form of pneumonia and relies on clinicians including it in the differential and requesting specialized testing. Additionally, it is challenging to obtain clinical lower respiratory specimens from cases with LD, and when available, culture requires specialized media and growth conditions, which are not available in all microbiology laboratories. In the current study, a method for Legionella pneumophila using hybrid capture by RNA baiting was developed, which allowed us to generate sufficient genome resolution from L. pneumophila serogroup 1 PCR-positive clinical specimens. This new approach offers an additional tool for surveillance of future LD outbreaks where isolation of Legionella is not possible and may help solve previously unanswered questions from past LD investigations.

Identification rate of Legionella species in non-purulent sputum culture is comparable to that in purulent sputum culture in Legionella pneumonia.

Many Legionella pneumonia patients do not produce sputum, and it is unknown whether purulent sputum is required for the identification of Legionella species. This study aimed to evaluate the identification rate of Legionella species based on sputum quality and the factors predictive of Legionella infection. This study included Legionella pneumonia patients at Kurashiki Central Hospital from November 2000 to December 2022. Sputum quality, based on gram staining, was classified as the following: Geckler 1/2, 3/6 and 4/5. Geckler 4/5 was defined as purulent sputum. The sputa of 104 of 124 Legionella pneumonia patients were cultured. Fifty-four patients (51.9%) were identified with Legionella species, most of which were Legionella pneumophila serogroup 1 (81.5%). The identification rates of Legionella species according to sputum quality were 57.1% (16/28) in Geckler 1/2 sputum, 50.0% (34/68) in Geckler 3/6 sputum, and 50.0% (4/8) in Geckler 4/5 sputum, which were not significantly different (P = 0.86). On multivariate analysis, pre-culture treatment with anti-Legionella antimicrobials (odds ratio [OR] 0.26, 95% confidence interval [CI] 0.06-0.91), Pneumonia Severity Index class ≥IV (OR 2.57 [95% CI 1.02-6.71]), and intensive care unit admission (OR 3.08, 95% CI 1.06-10.09) correlated with the ability to identify Legionella species, but sputum quality did not (OR 0.88, 95% CI 0.17-4.41). The identification rate of Legionella species in non-purulent sputum was similar to that in purulent sputum. For the diagnosis of Legionella pneumonia, sputum should be collected before administering anti-Legionella antibiotics and cultured regardless of sputum quality.

Legionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain

ADP-ribosylation is a reversible post-translational modification involved in various cellular activities. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. The pathogen Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. Here, we show that the Legionella macrodomain effector MavL regulates this pathway by reversing the arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by the modified ubiquitin. We determine the crystal structure of ADP-ribose-bound MavL, providing structural insights into recognition of the ADP-ribosyl group and catalytic mechanism of its removal. Further analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes whose representative members show unique selectivity for mono-ADP-ribosylated arginine residue in synthetic substrates. We find such enzymes are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. Collectively, our study reveals a new regulatory layer of SidE-catalyzed ubiquitination and expands the current understanding of macrodomain enzymes.

Risk Assessment and Detection of Legionella species in the Water System of A Luxury Hotel in Dhaka city, Bangladesh

Legionella cause Legionnaires’ disease and Pontiac fever, and, more rarely, extrapulmonary infections, collectively referred to as legionellosis. Legionella pneumophila causes serious pneumonic infections, and water and Air Conditioning systems are a common source. We performed this study focusing on identifying and assessing the potential sources of Legionella spp. in a luxury hotel water system in Dhaka, Bangladesh. As hotels often have complex water distribution systems that can harbor and facilitate the growth of these bacteria, understanding the risks and implementing effective control measures is crucial for public health. This study determines the presence of Legionella spp. and potential factors contributing to their proliferation within the hotel water systems. In total 42 water samples and six swabs from 7 different points were collected multiple times between March 2018 and April 2019 and were analyzed. Legionella spp. was detected in all samples (100%) tested. The highest colony counts were observed in water samples from the hot water tank and cooling system. The findings highlight the importance of regular monitoring, maintenance, disinfection, and flushing protocols to mitigate the risk of Legionella spp., contamination, and subsequent health issues among hotel guests and staff.
 Bangladesh J Microbiol, Volume 40, Number 1, June 2023, pp 33-39

The role of HDAC6 in enhancing macrophage autophagy via the autophagolysosomal pathway to alleviate legionella pneumophila-induced pneumonia

ABSTRACT Legionella pneumophila (L. pneumophila) is a prevalent pathogenic bacterium responsible for significant global health concerns. Nonetheless, the precise pathogenic mechanisms of L. pneumophila have still remained elusive. Autophagy, a direct cellular response to L. pneumophila infection and other pathogens, involves the recognition and degradation of these invaders in lysosomes. Histone deacetylase 6 (HDAC6), a distinctive member of the histone deacetylase family, plays a multifaceted role in autophagy regulation. This study aimed to investigate the role of HDAC6 in macrophage autophagy via the autophagolysosomal pathway, leading to alleviate L. pneumophila-induced pneumonia. The results revealed a substantial upregulation of HDAC6 expression level in murine lung tissues infected by L. pneumophila. Notably, mice lacking HDAC6 exhibited a protective response against L. pneumophila-induced pulmonary tissue inflammation, which was characterized by the reduced bacterial load and diminished release of pro-inflammatory cytokines. Transcriptomic analysis has shed light on the regulatory role of HDAC6 in L. pneumophila infection in mice, particularly through the autophagy pathway of macrophages. Validation using L. pneumophila-induced macrophages from mice with HDAC6 gene knockout demonstrated a decrease in cellular bacterial load, activation of the autophagolysosomal pathway, and enhancement of cellular autophagic flux. In summary, the findings indicated that HDAC6 knockout could lead to the upregulation of p-ULK1 expression level, promoting the autophagy-lysosomal pathway, increasing autophagic flux, and ultimately strengthening the bactericidal capacity of macrophages. This contributes to the alleviation of L. pneumophila-induced pneumonia.

ReporType: A Flexible Bioinformatics Tool for Targeted Loci Screening and Typing of Infectious Agents.

In response to the pressing need for continuous monitoring of emergence and circulation of pathogens through genomics, it is imperative to keep developing bioinformatics tools that can help in their rapid characterization and classification. Here, we introduce ReporType, a versatile bioinformatics pipeline designed for targeted loci screening and typing of infectious agents. Developed using the snakemake workflow manager, ReporType integrates multiple software for read quality control and de novo assembly, and then applies ABRicate for locus screening, culminating in the production of easily interpretable reports for the identification of pathogen genotypes and/or screening of specific genomic loci. The pipeline accommodates a range of input formats, from Illumina or Oxford Nanopore Technology (ONT) reads (FASTQ) to Sanger sequencing files (AB1), or FASTA files, making it flexible for application in multiple pathogens and with different purposes. ReporType is released with pre-prepared databases for some viruses and bacteria, yet it remains easily configurable to handle custom databases. ReporType performance and functionality were validated through proof-of-concept exercises, encompassing diverse pathogenic species, including viruses such as measles, Newcastle disease virus (NDV), Dengue virus (DENV), influenza, hepatitis C virus (HCV) and Human T-Cell Lymphotropic virus type 1 (HTLV-1), as well as bacteria like Chlamydia trachomatis and Legionella pneumophila. In summary, ReporType emerges as a simple, dynamic and pan-pathogen tool, poised to evolve in tandem with the ever-changing needs of the fields of pathogen genomics, infectious disease epidemiology, and one health bioinformatics. ReporType is freely available at GitHub.

Atypical pneumonia testing in transplant recipients.

The incidence of atypical pneumonia among immunocompromised patients is not well characterized. Establishing a diagnosis of atypical pneumonia is challenging as positive tests must be carefully interpreted. We aimed to assess the test positivity rate and incidence of atypical pneumonia in transplant recipients. A retrospective cohort study was conducted at the Yale New Haven Health System in Connecticut. Adults with solid organ transplant, hematopoietic stem cell transplant (HSCT), or chimeric antigen receptor T-cell, who underwent testing for atypical pathogens of pneumonia (Legionella pneumophilia, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Bordetella pertussis) between January 2016 and August 2022 were included. Positive results were adjudicated in a clinical context using pre-defined criteria. A cost analysis of diagnostic testing was performed. Note that, 1021 unique tests for atypical pathogens of pneumonia were performed among 481 transplant recipients. The testing positivity rate was 0.7% (n = 7). After clinical adjudication, there were three cases of proven Legionella and one case of possible Mycoplasma infection. All cases of legionellosis were in transplant recipients within 1-year post-transplantation with recently augmented immunosuppression and lymphopenia. The possible case of Mycoplasma infection was in an HSCT recipient with augmented immunosuppression. The cost of all tests ordered was $50,797.73. The positivity rate of tests for atypical pneumonia was very low in this transplant cohort. An algorithmic approach that targets testing for those with compatible host, clinical, radiographic, and epidemiologic factors, and provides guidance on test selection and test interpretation, may improve the diagnostic yield and lead to substantial cost savings.

Etiological characteristics of acute respiratory infections during the SARS-CoV-2 epidemic in Guizhou Province, China.

Acute respiratory infections are a major global public health concern. However, there are few epidemiological studies investigating pathogens associated with respiratory tract infections in Guizhou Province, China. We collected 17,850 blood samples from Guizhou Provincial People's Hospital between November 2018 and May 2023 to investigate the epidemiological characteristics of respiratory pathogens and their spread during the SARS-CoV-2 epidemic in Guizhou Province. We identified influenza virus and Mycoplasma pneumoniae as the predominant pathogens involved in acute respiratory infections in the study area. Immunoglobulin M positivity for respiratory syncytial virus, influenza virus, and M. pneumoniae showed a strong correlation with the clinical diagnosis of pneumonia. Seasonal epidemic patterns were observed for influenza A and B viruses. Following the SARS-CoV-2 outbreak, there was a significant decrease in the positive rates for most respiratory pathogens, particularly influenza A and B, Legionella pneumophila, and respiratory syncytial virus. This retrospective study contributes to the epidemiological evidence regarding respiratory pathogens in Guizhou Province, thereby enhancing the surveillance network for respiratory pathogens in China and providing valuable guidance for local hospitals.

Protocol for responding to the detection of Legionella pneumophila in drinking water distribution systems

AbstractA protocol for responding to Legionella pneumophila detections in distribution system samples is presented and justified. The protocol was developed using existing protocols for building water systems, vetted in workshops, and provided to utilities participating in a research project. The protocol provides action levels and actions that utilities can take when L. pneumophila is detected in distribution system samples. Action levels are based on the best available science and expert judgment and are similar to those in protocols for assessing building water system monitoring results. Action levels should be reassessed as additional data and knowledge on the occurrence and growth potential of L. pneumophila in distribution systems become available. All positive detections trigger assessments that focus first on conditions local to the sample location and then extend further into the distribution system. Higher concentration or more frequent detections initiate more assertive responses and communication than lower concentration and frequency detections. The protocol provides a starting point for the development of protocols for purposes such as L. pneumophila operational surveillance or within an outbreak investigation.

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

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

Comparison of pre-labelled primers and nucleotides as DNA labelling method for lateral flow detection of Legionella pneumophila amplicons

Labelling of nucleic acid amplicons during polymerase chain reaction (PCR) or isothermal techniques is possible by using both labelled primers and labelled nucleotides. While the former is the widely used method, the latter can offer significant advantages in terms of signal enhancement and improving the detection limit of an assay. Advantages and disadvantages of both methods depend on different factors, including amplification method, detection method and amplicon length. In this study, both methods for labelling PCR products for lateral flow assay (LFA) analysis (LFA-PCR) were analysed and compared. It was shown that labelling by means of nucleotides results in an increase in label incorporation rates. Nonetheless, this advantage is negated by the need for post-processing and competitive interactions. In the end, it was possible to achieve a detection limit of 3 cell equivalents for the detection of the Legionella-DNA used here via primer labelling. Labelling via nucleotides required genomic DNA of at least 3000 cell equivalents as starting material as well as an increased personnel and experimental effort.

A case of Legionella pneumonia after robot‐assisted radical prostatectomy

IntroductionPostoperative Legionella pneumonia is very rare.Case presentationA 71‐year‐old male patient with prostate cancer (cT2bN0M0) underwent a robotic‐assisted radical prostatectomy. On the 5th postoperative day, the patient developed chills and a fever of 39.2°C. Chest radiography revealed decreased permeability in the right middle lung field, leading to the diagnosis of postoperative pneumonia. Antimicrobial therapy was initiated immediately. Blood tests on postoperative day 10 revealed mild liver function abnormalities, electrolyte abnormalities, and a markedly elevated inflammatory response. Legionella pneumonia was suspected based on blood sample results and systemic symptoms, such as diarrhea and nausea. Furthermore, Legionella antigens were detected in the patient's urine, prompting further administration of levofloxacin. The patient's subsequent clinical course was favorable.ConclusionWhen bacterial pneumonia fails to respond to antimicrobial therapy and systemic symptoms develop, atypical pneumonia, caused by pathogens such as Legionella pneumophila, should be considered even in cases of postoperative pneumonia.

Legionella pneumophila usurps host cell lipids for vacuole expansion and bacterial growth.

Vacuolar pathogens reside in membrane-bound compartments within host cells. Maintaining the integrity of this compartment is paramount to bacterial survival and replication as it protects against certain host surveillance mechanisms that function to eradicate invading pathogens. Preserving this compartment during bacterial replication requires expansion of the vacuole membrane to accommodate the increasing number of bacteria, and yet, how this is accomplished remains largely unknown. Here, we show that the vacuolar pathogen Legionella pneumophila exploits multiple sources of host cell fatty acids, including inducing host cell fatty acid scavenging pathways, in order to promote expansion of the replication vacuole and bacteria growth. Conversely, when exogenous lipids are limited, the decrease in host lipid availability restricts expansion of the replication vacuole membrane, resulting in a higher density of bacteria within the vacuole. Modifying the architecture of the vacuole prioritizes bacterial growth by allowing the greatest number of bacteria to remain protected by the vacuole membrane despite limited resources for its expansion. However, this trade-off is not without risk, as it can lead to vacuole destabilization, which is detrimental to the pathogen. However, when host lipid resources become extremely scarce, for example by inhibiting host lipid scavenging, de novo biosynthetic pathways, and/or diverting host fatty acids to storage compartments, bacterial replication becomes severely impaired, indicating that host cell fatty acid availability also directly regulates L. pneumophila growth. Collectively, these data demonstrate dual roles for host cell fatty acids in replication vacuole expansion and bacterial proliferation, revealing the central functions for these molecules and their metabolic pathways in L. pneumophila pathogenesis.

Correlation between bacterial microbiome and Legionella species in water from public bath facilities by 16S rRNA gene amplicon sequencing.

Public bath facilities are major sources of sporadic cases and outbreaks of Legionella infections. Recently, 16S rRNA gene amplicon sequencing has been used to analyze bacterial characteristics in various water samples from both artificial and natural environments, with a particular focus on Legionella bacterial species. However, the relationship between the bacterial community and Legionella species in the water from public bath facilities remains unclear. In terms of hygiene management, it is important to reduce the growth of Legionella species by disinfecting the water in public bath facilities. Our findings contribute to the establishment of appropriate hygiene management practices and provide a basis for understanding the potential health effects of using bath and shower water available in public bath facilities.

Structural characterization of the Sel1-like repeat protein LceB from Legionella pneumophila.

Legionella are freshwater Gram-negative bacteria that in their normal environment infect protozoa. However, this adaptation also allows Legionella to infect human alveolar macrophages and cause pneumonia. Central to Legionella pathogenesis are more than 330 secreted effectors, of which there are nine core effectors that are conserved in all pathogenic species. Despite their importance, the biochemical function of several core effectors remains unclear. To address this, we have taken a structural approach to characterize the core effector of unknown function LceB, or Lpg1356, from Legionella pneumophila. Here, we solve an X-ray crystal structure of LceB using an AlphaFold model for molecular replacement. The experimental structure shows that LceB adopts a Sel1-like repeat (SLR) fold as predicted. However, the crystal structure captured multiple conformations of LceB, all of which differed from the AlphaFold model. A comparison of the predicted model and the experimental models suggests that LceB is highly flexible in solution. Additionally, the molecular analysis of LceB using its close structural homologs reveals sequence and structural motifs of known biochemical function. Specifically, LceB harbors a repeated KAAEQG motif that both stabilizes the SLR fold and is known to participate in protein-protein interactions with eukaryotic host proteins. We also observe that LceB forms several higher-order oligomers in solution. Overall, our results have revealed that LceB has conformational flexibility, self-associates, and contains a molecular surface for binding a target host-cell protein. Additionally, our data provides structural insights into the SLR family of proteins that remain poorly studied.

The intracellular growth of the vacuolar pathogen Legionella pneumophila is dependent on the acyl chain composition of host membranes

Legionella pneumophila is an accidental human bacterial pathogen that infects and replicates within alveolar macrophages causing a severe atypical pneumonia known as Legionnaires’ disease. As a prototypical vacuolar pathogen L. pneumophila establishes a unique endoplasmic reticulum (ER)-derived organelle within which bacterial replication takes place. Bacteria-derived proteins are deposited in the host cytosol and in the lumen of the pathogen-occupied vacuole via a type IVb (T4bSS) and a type II (T2SS) secretion system respectively. These secretion system effector proteins manipulate multiple host functions to facilitate intracellular survival of the bacteria. Subversion of host membrane glycerophospholipids (GPLs) by the internalized bacteria via distinct mechanisms feature prominently in trafficking and biogenesis of the Legionella-containing vacuole (LCV). Conventional GPLs composed of a glycerol backbone linked to a polar headgroup and esterified with two fatty acids constitute the bulk of membrane lipids in eukaryotic cells. The acyl chain composition of GPLs dictates phase separation of the lipid bilayer and therefore determines the physiochemical properties of biological membranes - such as membrane disorder, fluidity and permeability. In mammalian cells, fatty acids esterified in membrane GPLs are sourced endogenously from de novo synthesis or via internalization from the exogenous pool of lipids present in serum and other interstitial fluids. Here, we exploited the preferential utilization of exogenous fatty acids for GPL synthesis by macrophages to reprogram the acyl chain composition of host membranes and investigated its impact on LCV homeostasis and L. pneumophila intracellular replication. Using saturated fatty acids as well as cis- and trans- isomers of monounsaturated fatty acids we discovered that under conditions promoting lipid packing and membrane rigidification L. pneumophila intracellular replication was significantly reduced. Palmitoleic acid – a C16:1 monounsaturated fatty acid – that promotes membrane disorder when enriched in GPLs significantly increased bacterial replication within human and murine macrophages but not in axenic growth assays. Lipidome analysis of infected macrophages showed that treatment with exogenous palmitoleic acid resulted in membrane acyl chain reprogramming in a manner that promotes membrane disorder and live-cell imaging revealed that the consequences of increasing membrane disorder impinge on several LCV homeostasis parameters. Collectively, we provide experimental evidence that L. pneumophila replication within its intracellular niche is a function of the lipid bilayer disorder and hydrophobic thickness.

An acetyltransferase effector conserved across Legionella species targets the eukaryotic eIF3 complex to modulate protein translation.

The survival of Legionella spp. as intracellular pathogens relies on the combined action of protein effectors delivered inside their eukaryotic hosts by the Dot/Icm (defective in organelle trafficking/intracellular multiplication) type IVb secretion system. The specific repertoire of effector arsenals varies dramatically across over 60 known species of this genera with Legionella pneumophila responsible for most cases of Legionnaires' disease in humans encoding over 360 Dot/Icm effectors. However, a small subset of "core" effectors appears to be conserved across all Legionella species raising an intriguing question of their role in these bacteria's pathogenic strategy, which for most of these effectors remains unknown. L. pneumophila Lpg0103 effector, also known as VipF, represents one of the core effector families that features a tandem of Gcn5-related N-acetyltransferase (GNAT) domains. Here, we present the crystal structure of the Lha0223, the VipF representative from Legionella hackeliae in complex with acetyl-coenzyme A determined to 1.75 Å resolution. Our structural analysis suggested that this effector family shares a common fold with the two GNAT domains forming a deep groove occupied by residues conserved across VipF homologs. Further analysis suggested that only the C-terminal GNAT domain of VipF effectors retains the active site composition compatible with catalysis, whereas the N-terminal GNAT domain binds the ligand in a non-catalytical mode. We confirmed this by in vitro enzymatic assays which revealed VipF activity not only against generic small molecule substrates, such as chloramphenicol, but also against poly-L-lysine and histone-derived peptides. We identified the human eukaryotic translation initiation factor 3 (eIF3) complex co-precipitating with Lpg0103 and demonstrated the direct interaction between the several representatives of the VipF family, including Lpg0103 and Lha0223 with the K subunit of eIF3. According to our data, these interactions involve primarily the C-terminal tail of eIF3-K containing two lysine residues that are acetylated by VipF. VipF catalytic activity results in the suppression of eukaryotic protein translation in vitro, revealing the potential function of VipF "core" effectors in Legionella's pathogenic strategy.IMPORTANCEBy translocating effectors inside the eukaryotic host cell, bacteria can modulate host cellular processes in their favor. Legionella species, which includes the pneumonia-causing Legionella pneumophila, encode a widely diverse set of effectors with only a small subset that is conserved across this genus. Here, we demonstrate that one of these conserved effector families, represented by L. pneumophila VipF (Lpg0103), is a tandem Gcn5-related N-acetyltransferase interacting with the K subunit of human eukaryotic initiation factor 3 complex. VipF catalyzes the acetylation of lysine residues on the C-terminal tail of the K subunit, resulting in the suppression of eukaryotic translation initiation factor 3-mediated protein translation in vitro. These new data provide the first insight into the molecular function of this pathogenic factor family common across Legionellae.