Abstract Citrate is a central metabolite linking tricarboxylic acid (TCA) cycle activity to energy and lipid metabolism and supports the synthesis of inflammatory mediators, including itaconate, in macrophages. While citrate is primarily generated endogenously, extracellular citrate levels are elevated under pathological conditions such as citrate transporter disorder. Cells import extracellular citrate through SLC13 transporters, including the sodium-dependent citrate transporter NaCT (encoded by SLC13A5 ). However, whether macrophages take up extracellular citrate and how this affects metabolism and function remains unclear. Here, we combined mass spectrometry and tracing approaches to investigate the metabolic fate of citrate in human macrophage cell lines, primary, and iPSC-derived macrophages. We demonstrate that cells take up extracellular citrate, which was enhanced under metabolic stress conditions. Exogenous citrate was not substantially utilized as a carbon source but selectively altered glutamine metabolism and responses to bacterial infection with Salmonella enterica Typhimurium and Legionella pneumophila Corby. Our work identifies extracellular citrate as a context-dependent regulator in macrophages that decouples uptake from metabolic utilization. Highlights Macrophages import extracellular citrate via SLC13 transporters Extracellular citrate accumulates under hypoxia and inflammatory activation Extracellular citrate does not fuel central carbon metabolism in human macrophages Citrate modulates glutamine immunometabolism and modulates immune responses eTOC blurb Voß-Willenbockel et al. demonstrate that human macrophages accumulate extracellular citrate without using it as a major carbon source. Instead, citrate modulates glutamine utilization, inflammatory responses, and host-pathogen interactions revealing a context-dependent regulatory role for extracellular metabolites in immune cell function.

Bumble bees are important pollinators of crops in the field and greenhouses. They are naturally exposed to a combination of interacting stressors, e.g., loss of flowering resources and exposure to plant protection products. Mass-flowering crops are important resources for bees, but they may result in unbalanced nutrition due to different nutritional values. In this study, a semi-field experiment was conducted to evaluate the response of Bombus terrestris colonies after the application of a tank mixture containing the insecticide chlorantraniliprole and the fungicide prochloraz, either in monofloral-managed lupin (Lupinus albus) as high pollen protein resource or in presence of an additional polyfloral flower mixture. Our results demonstrate an evident effect on worker mortality after application of the tank mixture. Higher worker mortality in polyfloral treatments compared to the untreated control was observed. The number of young brood and pupae in colonies in polyfloral control were significantly higher than in monofloral treatment. However, no long-term effects on the number or weight of new queens were found. Furthermore, flowering resources, but not pesticide exposure, affected colony weight. Exposure to the tank mixture resulted in declining nectar yeasts abundance and an increasing proportion of phylloplane yeasts in forager guts. In conclusion, diverse flowering resources are important for a bumble bee colony’s development. Even in a high pollen protein crop, low flower diversity may act as an additional stressor. Thus, we suggest further maintaining and promoting flowering strips or flowering fields in agricultural landscapes, even near high pollen protein crops, to enhance bee health.

Ash dieback is a tree disease caused by the fungal pathogen Hymenoscyphus fraxineus. Since its introduction into Europe, it has caused widespread and significant losses of the European ash, Fraxinus excelsior. Inoculations of F. excelsior with a low virulent H. fraxineus isolate were assessed as a promising method for reducing symptoms associated with ash dieback, presumably by triggering systemic induced resistance. Two strains of H. fraxineus were chosen based on observations of high and low in planta virulence. Crude extracts obtained from cultures of the highly virulent strain were more phytotoxic in a leaf puncture assay than ones obtained from the low virulent strain. UHPLC-DAD-MS/MS data identified the phytotoxin viridiol and the potential phytotoxin hyfraxin A in both cultures. However, the production of these compounds in vitro did not correspond with virulence in planta. To test the effects of priming, saplings of F. excelsior were first inoculated with the low virulent strain and subsequently with the highly virulent strain. On average, necrosis expansion on the stems was reduced by 53

European ash trees (Fraxinus excelsior) are threatened by the non-native fungal pathogen Hymenoscyphus fraxineus, which causes crown dieback and root collar necroses. The exploitation of fungal endophytes, which naturally colonise trees asymptomatically and can produce bioactive metabolites, may provide opportunities as biocontrol agents to reduce symptom development in F. excelsior. We focused our investigations on isolates of four genera which fulfil these criteria and selected six promising candidates for greenhouse experiments: Diaporthe oncostoma (DSM 116298), Pezicula abietina (DSM 5141), Pezicula cf. ericae (DSM 110620), Nemania diffusa (DSM 116299), Hypoxylon perforatum (MUCL 54174) and Hypoxylon rubiginosum (DSM 106870). A detailed analysis of the secondary metabolomes by NMR (nuclear magnetic resonance) and HRMS (high resolution mass spectrometry) data identified known metabolites from these endophytes: mycorrhizin A produced by P. abietina, CJ-17,572 from P. cf. ericae, phomopsidin from Hyp. rubiginosum and cytochalasin E from N. diffusa as key anti-fungal agents. Besides detecting multiple potential cytochalasins, the new compound 9-epi-xylaolide A was isolated from D. oncostoma. A greenhouse experiment was conducted to determine whether the endophytes could reduce symptoms of ash dieback in planta. When two-year old F. excelsior saplings were artificially-inoculated with each of the six endophytes alone, no significant symptoms of disease developed. For two of the six endophytes, we observed significantly reduced necrotic lesion development when saplings were subsequently inoculated with a H. fraxineus strain of low virulence, compared to saplings inoculated only with the pathogen. In combinations of the six biocontrol candidates and a H. fraxineus strain of higher virulence, lesion development was initially inhibited, however inhibition was not significant and decreased at different rates over the monitoring period. Mortality was delayed in dual-inoculated saplings compared to saplings inoculated with either of the H. fraxineus strains alone. These results indicate symptoms and mortality associated with H. fraxineus infection can be reduced by endophytes.

The feeding behavior of free-living nematodes is shaped by several factors, including food choice and food consumption. The latter is commonly investigated by determining the pumping rate of the pharynx. However, whether the pharyngeal pumping activity also reflects food selection, i.e. whether the pumping rate for preferred food is increased, is largely unknown. The aim of this study was to investigate the relation between pumping rate and food choice. For this, the preference of the nematodes Diploscapter coronatus, Diploscapter pachys, Plectus similis and Plectus sp. were examined in binary choice assays. The bacteria Bacillus subtilis, Kitasatospora sp., Sphingomonas trueperi, Pseudomonas fluorescens and two Legionella pneumophila strains were tested against Escherichia coli OP50. The nematode pumping rates were determined for each bacterial diet. Additionally, the effects of bacterial species and size, and of nematode species and genus, size of buccal cavity and pharynx morphology, were investigated.The nematodes showed a preference for P. fluorescens (except D. pachys) and avoidance of B. subtilis, Kitasatospora sp. and L. pneumophila. The determinants of food choice were nematode species, bacterial species and size, while the pumping rates varied with genus and pharynx proportion. The food preference determined by the choice assay was not necessarily associated with an upregulation of pharyngeal pumping, e.g. Plectus preferred P. fluorescens over E. coli OP50 when having a choice but pumped significantly faster when feeding on the latter. Although the pumping rate changed with bacterial species, there was no clear pattern that allowed a correlation with food choice across the tested nematode taxa, suggesting that pumping frequency is no indicator for food preference. Relating these results to nematode foraging strategy, food choice was determined by bacterial traits associated with diet quality and search, while food consumption was linked to nematode traits affecting diet handling.

Legionnaires' disease is a severe pneumonia caused by the aquatic bacterium Legionella pneumophila. In recent years, the number of cases has continuously increased according to the distribution of aerosol-producing technologies, global warming, and demographic ageing, which elevates bacterial transmission, environmental cell counts and host susceptibility. Despite the availability of an antibiotic therapy, this treatment cannot eradicate the risk of irreversible symptoms or high mortality rates. The bacterial zinc metalloprotease ProA significantly contributes to life-threatening tissue damage in patients and hence promotes progression of the disease. We hypothesize that additional application of an inhibitory agent against this protease may reduce serious complications and the risk of respiratory failure until successful bacterial clearance. Here we present a newly designed set of zinc-binding compounds and evaluated their inhibitory effects on the versatile physiological activity of ProA during infection. We identified a potent phosphonate inhibitor, which reduces ProA-dependent cleavage of host collagen IV and bacterial flagellin, immune evasion from the TLR5-NF-κB pathway and PMN-mediated inflammation in human lung tissue explants (HLTEs). Based on efficacy at the biochemical, cellular and tissue levels and the results of ProA co-crystallizations, we conclude that the selected inhibitors represent promising lead structures for the future development of clinically applicable pathoblockers against Legionnaires' disease.

Biopesticides, having as active ingredients viruses, bacteria, or fungi, are developed to substitute or reduce the use of chemical plant protection products in different agrosystems. Though the application of mixtures containing several products is a common practice, interactions between microbial biopesticides and related effects on bees as non-target organisms have not been studied yet. In the current study, we exposed winter bees to five different microbial-based products and their combinations at the maximum recommended application rate to assess their responses. Laboratory oral exposure tests (acute/chronic) to single or binary products were conducted. Survival and food consumption of the tested bees were evaluated over the experimental duration. Our results show that some product combinations have potential additive or synergistic effects on bees, whereas others did not affect the bee’s survival compared to the control. Exposure of tested bees to the most critical combination of products containing Bacillus thuringiensis aizawai ABTS-1857 and B. amyloliquefaciens QST 713 strongly resulted in a median lifespan of 4.5 days compared to 8.0 and 8.5 days after exposure to the solo products, respectively. The exposure to inactivated microorganisms by autoclaving them did not differ from their respective uncontaminated negative controls, indicating effects on bee mortality might originate in the treatment with the different microorganisms or their metabolites. Further investigations should be conducted under field conditions to prove the magnitude of observed effects on bee colonies and other bee species.

Myxobacteria are Gram-negative bacteria renowned for their valuable production of secondary metabolites; however, their diverse protease enzymes remain less explored. In this investigation, a novel strain was isolated from mangrove soil that produces alkaline protease. Using 16S rRNA sequence analysis, the strain was identified as Myxococcus virescens species. The extracellular alkaline protease MvCP of strain S2-2 was produced using growth media containing agro-industrial by-products such as soybean meal and tofu dregs. MvCP exhibited optimum activity within a pH range from 9 to 10 and temperatures ranging from 30 °C to 50 °C. MvCP activity was improved with Ca2⁺, Mg2⁺, and Zn2⁺ ions, whereas EDTA and β-mercaptoethanol at concentrations of 0.5 and 1.25 mM had no significant effect. MvCP was stable in chloroform, acetone, Triton X-100, Tween-20, and Tween 80. MvCP can be considered an eco-friendly option for use in household cleaning products and chitin extraction processes.

Biofilms in cooling towers represent a common habitat for the human pathogen Legionella pneumophila. Within the biofilm consortium, frequent interactions with protozoa, i.e. amoebae and ciliates, were reported, while nematodes have only recently been considered as potential environmental reservoir for the pathogenic bacteria. This study is the first approach to investigate the biotic interactions between L. pneumophila and bacterial-feeding nematodes in a semi-natural biofilm model. The species were Diploscapter coronatus, Diploscapter pachys, Plectus similis and Plectus sp., which all co-occur with L. pneumophila in the environment. Biofilms derived from cooling towers were either inoculated with mCherry-labeled L. pneumophila solely or in combination with GFP-labeled Escherichia coli. All experiments were conducted in single-species set-ups and multi-species (D. coronatus and P. similis) set-ups, to account for interspecific competition. Bacterial ingestion was assessed after 24 and 96 h as fluorescence patterns in the digestive tract of the nematodes using confocal laser scanning microscopy. L. pneumophila cells were ingested by all nematode species, with D. coronatus having the highest pathogen load. The fluorescence intensity (i.e. bacterial load) varied between compartments within the digestive tract and was independent of incubation time. Bacterial cells accumulated mostly around the cardia and in the intestine, while less cells were found within stoma and pharynx. Interspecific competition changed the pattern, i.e. with incubation of D. coronatus and P. similis in the same biofilm a significantly higher pathogen load occurred in the intestine of D. coronatus than P. similis after 24 h and 96 h. Remarkably, when given a choice between L. pneumophila and E. coli, P. similis was the only nematode species containing both bacteria after incubation for 24 h. None of the other nematode species contained E. coli after 24 h and 96 h incubation, while L. pneumophila was present. This study thus provides the first evidence, that under environmental conditions L. pneumophila is a frequent diet of bacterial-feeding nematodes, highlighting their potential as pathogen vectors or even host in cooling tower habitats.

Ash dieback, caused by the fungal pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), is threatening the existence of the European ash, Fraxineus excelsior. During our search for biological control agents for this devastating disease, endophytic fungi were isolated from healthy plant tissues and co-cultivated with H. fraxineus to assess their antagonistic potential. Among the strains screened, Penicillium cf. manginii DSM 104493 most strongly inhibited the pathogen. Initially, DSM 104493 showed promise in planta as a biocontrol agent. Inoculation of DSM 104493 into axenically cultured ash seedlings greatly decreased the development of disease symptoms in seedlings infected with H. fraxineus. The fungus was thus cultivated on a larger scale in order to obtain sufficient material to identify active metabolites that accounted for the antibiosis observed in dual culture. We isolated PF1140 (1) and identified it as the main active compound in the course of a bioassay-guided isolation strategy. Furthermore, its derivative 2, the mycotoxin citreoviridin (3), three tetramic acids of the vancouverone type (4-6), and penidiamide (7) were isolated by preparative chromatography. The structures were elucidated mainly by NMR spectroscopy and high-resolution mass spectrometry (HRMS), of which compounds 2 and 6 represent novel natural products. Of the compounds tested, not only PF1140 (1) strongly inhibited H. fraxineus in an agar diffusion assay but also showed phytotoxic effects in a leaf puncture assay. Unfortunately, both the latent virulent attributes of DSM 104493 observed subsequent to these experiments in planta and the production of mycotoxins exclude strain Penicillium cf. manginii DSM 104493 from further development as a safe biocontrol agent. IMPORTANCE Environmentally friendly measures are urgently needed to control the causative agent of ash dieback, Hymenoscyphus fraxineus. Herein, we show that the endophyte DSM 104493 exhibits protective effects in vitro and in planta. We traced the activity of DSM 104493 to the antifungal natural product PF1140, which unfortunately also showed phytotoxic effects. Our results have important implications for understanding plant-fungal interactions mediated by secondary metabolites, not only in the context of ash dieback but also generally in plant-microbial interactions.

Honey bees (Apis mellifera) have to withstand various environmental stressors alone or in combination in agriculture settings. Plant protection products are applied to achieve high crop yield, but residues of their active substances are frequently detected in bee matrices and could affect honey bee colonies. In addition, intensified agriculture could lead to resource limitation for honey bees. This study aimed to compare the response of full-sized and nucleus colonies to the combined stressors of fungicide exposure and resource limitation. A large-scale field study was conducted simultaneously at five different locations across Germany, starting in spring 2022 and continuing through spring 2023. The fungicide formulation Pictor® Active (active ingredients boscalid and pyraclostrobin) was applied according to label instructions at the maximum recommended rate on oil seed rape crops. Resource limitation was ensured by pollen restriction using a pollen trap and stressor responses were evaluated by assessing colony development, brood development, and core gut microbiome alterations. Furthermore, effects on the plant nectar microbiome were assessed since nectar inhabiting yeast are beneficial for pollination. We showed, that honey bee colonies were able to compensate for the combined stressor effects within six weeks. Nucleus colonies exposed to the combined stressors showed a short-term response with a less favorable brood to bee ratio and reduced colony development in May. No further impacts were observed in either the nucleus colonies or the full-sized colonies from July until the following spring. In addition, no fungicide-dependent differences were found in core gut and nectar microbiomes, and these differences were not distinguishable from local or environmental effects. Therefore, the provision of sufficient resources is important to increase the resilience of honey bees to a combination of stressors.

Abstract Background The first 24 h of infection represent a critical time window in interactions between pathogens and host tissue. However, it is not possible to study such early events in human lung during natural infection due to lack of clinical access to tissue this early in infection. We, therefore, applied RNA sequencing to ex vivo cultured human lung tissue explants (HLTE) from patients with emphysema to study global changes in small noncoding RNA, mRNA, and long noncoding RNA (lncRNA, lincRNA) populations during the first 24 h of infection with influenza A virus (IAV), Mycobacterium bovis Bacille Calmette-Guerin (BCG), and Pseudomonas aeruginosa. Results Pseudomonas aeruginosa caused the strongest expression changes and was the only pathogen that notably affected expression of microRNA and PIWI-associated RNA. The major classes of long RNAs (> 100 nt) were represented similarly among the RNAs that were differentially expressed upon infection with the three pathogens (mRNA 77–82%; lncRNA 15–17%; pseudogenes 4–5%), but lnc-DDX60-1, RP11-202G18.1, and lnc-THOC3-2 were part of an RNA signature (additionally containing SNX10 and SLC8A1) specifically associated with IAV infection. IAV infection induced brisk interferon responses, CCL8 being the most strongly upregulated mRNA. Single-cell RNA sequencing identified airway epithelial cells and macrophages as the predominant IAV host cells, but inflammatory responses were also detected in cell types expressing few or no IAV transcripts. Combined analysis of bulk and single-cell RNAseq data identified a set of 6 mRNAs (IFI6, IFI44L, IRF7, ISG15, MX1, MX2) as the core transcriptomic response to IAV infection. The two bacterial pathogens induced qualitatively very similar changes in mRNA expression and predicted signaling pathways, but the magnitude of change was greater in P. aeruginosa infection. Upregulation of GJB2, VNN1, DUSP4, SerpinB7, and IL10, and downregulation of PKMYT1, S100A4, GGTA1P, and SLC22A31 were most strongly associated with bacterial infection. Conclusions Human lung tissue mounted substantially different transcriptomic responses to infection by IAV than by BCG and P. aeruginosa, whereas responses to these two divergent bacterial pathogens were surprisingly similar. This HLTE model should prove useful for RNA-directed pathogenesis research and tissue biomarker discovery during the early phase of infections, both at the tissue and single-cell level.

The pathogenicity of L. pneumophila, the causative agent of Legionnaires' disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted "protein sociology" of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

Background Legionella pneumophila is water-based bacterium causing Legionnaires' disease (LD). We describe the first documented case of nosocomial LD caused by L. pneumophila sequence type (ST) 461 and serogroup 6. The etiology of LD was confirmed by culturing the bronchoalveolar lavage sample retrieving L. pneumophila strain ALAW1. A 7-days treatment of the LD patient with Azithromycin and Levofloxacin allowed complete recovery. Methods In details, we sequenced the whole genome of the L. pneumophila ALAW1 using Illumina HiSeq platform. The sequence of ALAW1 was aligned with the genome sequence from the closely related reference strain Alcoy 2300/99 and a whole-genome phylogeny based on single nucleotide polymorphisms (SNPs) was created using Parsnp software. Also, the TYGS web-server was used in order to compare the genome with type strain. Results An analysis of the population structure by SNP and TYGS comparison clustered ALAW1 with the reference genome Alcoy 2300/99. Blastp analysis of the type IV secretion Dot/Icm system genes showed that these genes were highly conserved with (<= 25%) structural differences at the protein level. Conclusions Overall, this study provides insights into detailed genome structure and demonstrated the value of whole-genome sequencing as the ultimate typing tool for Legionella.

The invasive ascomycete Hymenoscyphus fraxineus is the causative agent for ash dieback on the European species Fraxinus excelsior and Fraxinus angustifolia, and there is concern that it is going to replace the native, closely related and nonpathogenic Hymenoscyphus albidus . Fungal management in forests is limited, and alternative approaches for control are needed. Within the scope of the project “FraxForFuture”, several strategies are being investigated. One idea comprises the use of a viral hyperparasite, which can induce a reduced virulence in the fungal host H. fraxineus in an antagonist-like system. This phenomenon, the reduction of fungal virulence by a viral infection, is known as hypovirulence, and a similar method has already been established to control the Chestnut Blight in Europe. We examined 34 isolates of H. fraxineus for both their virulence and presence of a viral infection. Although a predominant number of isolates were found to be infected with Hymenoscyphus mitovirus 1 (HfMV1), no additional viruses were detected, and our data did not indicate a link to reduced virulence. The search for a viral infection was extended to one isolate of H. albidus in which we found and characterized a novel mycovirus. Based on phylogenetic analysis and sequence properties, it was assigned to the genus Victorivirus in the family of Totiviridae and was tentatively denominated as Hymenoscyphus albidus victorivirus 1. This novel and native mycovirus might be suitable for inducing hypovirulence in H. fraxineus as a biocide .

Background:Honeybees are one of the three most important animals for mankind. In order to be safe and increase number of bee colonies for pollination, the breeding of queens is necessary. For several decades, bees were selected on economic and behavioral aspects. With the appearance of the neozootic mite Varroa destructor beekeepers were forced to adapt their methods. Varroa destructor can act as a vector for many different bee pathogenic viruses and by this potentiates its devastating impact.Aim:Methods of rearing queens were not evaluated since the mites' appearance. Besides scientific approaches, viruses received too little attention in regard to the rearing process of honeybee queens. Herein, we present a detailed analysis of virus abundances [Aparavirus, acute bee paralysis virus (ABPV); Triatovirus, black queen cell virus (BQCV); Cripavirus, chronic bee paralysis virus (CBPV); and Iflaviruses, deformed wings virus (DWV), Sacbrood virus (SBV), VDV-1] in breeding hives, donating first instar larvae, hives that are nursing these larvae until the pupa stage, and on queens of Apis mellifera in a breeding apiary.Methods:Nurse and donor colonies of the queen-rearing process were sampled in the year 2020 and analyzed by RT qPCR. Virus quantifications were correlated with queen mortalities and seasonal effects.Results:Virus detections increased in reared queens, however, the elevated virus titers did not increase the mortality of the queens until their exclosure. Moreover, we observed a lower interrelation between virus abundance in queens and their original donor colonies, than between nurse hives and their nursed queens.Conclusion:The bee pathogenic viruses ABPV, BQCV, CBPV, DWV, SBV, and VDV-1 do not influence the mortality of bee queens during the rearing process. Whether respective virus loads result in sublethal or long-term effects remains to be elucidated.

Free-living nematodes harbor and disseminate various soil-borne bacterial pathogens. Whether they function as vectors or environmental reservoirs for the aquatic L. pneumophila, the causative agent of Legionnaires’ disease, is unknown. A survey screening of biofilms of natural (swimming lakes) and technical (cooling towers) water habitats in Germany revealed that nematodes can act as potential reservoirs, vectors or grazers of L. pneumophila in cooling towers. Consequently, the nematode species Plectus similis and L. pneumophila were isolated from the same cooling tower biofilm and taken into a monoxenic culture. Using pharyngeal pumping assays, potential feeding relationships between P. similis and different L. pneumophila strains and mutants were examined and compared with Plectus sp., a species isolated from a L. pneumophila-positive thermal source biofilm. The assays showed that bacterial suspensions and supernatants of the L. pneumophila cooling tower isolate KV02 decreased pumping rate and feeding activity in nematodes. However, assays investigating the hypothesized negative impact of Legionella’s major secretory protein ProA on pumping rate revealed opposite effects on nematodes, which points to a species-specific response to ProA. To extend the food chain by a further trophic level, Acanthamoebae castellanii infected with L. pneumphila KV02 were offered to nematodes. The pumping rates of P. similis increased when fed with L. pneumophila-infected A. castellanii, while Plectus sp. pumping rates were similar when fed either infected or non-infected A. castellanii. This study revealed that cooling towers are the main water bodies where L. pneumophila and free-living nematodes coexist and is the first step in elucidating the trophic links between coexisting taxa from that habitat. Investigating the Legionella–nematode–amoebae interactions underlined the importance of amoebae as reservoirs and transmission vehicles of the pathogen for nematode predators.

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.

A Legionella pneumophila bacterium is ubiquitous in water distribution systems, including dental unit waterlines (DUWLs). Legionellosis is atypical pneumonia, including Legionnaires’ disease (LD) and the less acute form of Pontiac fever. Legionellosis occurs as a result of inhalation/aspiration of aerosolized Legionella-contaminated water by susceptible patients, health workers, and dentists. In this study, we undertook to determine the prevalence of Legionella in water and biofilm samples from Tap and DUWLs collected from five sites of dental clinics and faculties across the West Bank. Water samples were tested for physical and chemical parameters. The study samples included 185 samples, 89 (48%) water samples, and 96 (52%) biofilm swabs, which were analyzed by cultivation-dependent analysis (CDA) and by the cultivation-independent technique (CIA). Also, partial sequencing of the 16S rRNA gene for fifteen L. pneumophila isolates was performed for quality assurance and identification. L. pneumophila was isolated from 28 (15%) of 185 samples using CDA and was detected in 142 (77%) of 185 samples using CIA. The abundance of culturable L. pneumophila was low in DUWL of the sampling sites (range: 27–115 CFU/Liter). PCR was 5× more sensitive than the culture technique. L. pneumophila Sg 1 was detected in (75%) of the isolates, while (25%) isolates were L. pneumophila Sg 2–14. All fifteen sequenced Legionella isolates were identified as L. pneumophila ≥ 94.5%. The analysis of phylogenetic tree showed that L. pneumophila branch clearly identified and distinguished from other branches. These results show that DUWLs of the examined dental clinics and faculties are contaminated with L. pneumophila. This finding reveals a serious potential health risk for infection of immunocompromised patients and dentists’ post-exposure.