Metabolic adaption of Legionella pneumophila during intracellular growth in Acanthamoeba castellanii.

The metabolism of Legionella pneumophila strain Paris was elucidated during different time intervals of growth within its natural host Acanthamoeba castellanii. For this purpose, the amoebae were supplied after bacterial infection (t =0 h) with 11 mM [U-13C6]glucose or 3 mM [U-13C3]serine, respectively, during 0-17 h, 17-25 h, or 25-27 h of incubation. At the end of these time intervals, bacterial and amoebal fractions were separated. Each of these fractions was hydrolyzed under acidic conditions. 13C-Enrichments and isotopologue distributions of resulting amino acids and 3-hydroxybutyrate were determined by gas chromatography - mass spectrometry. Comparative analysis of the labelling patterns revealed the substrate preferences, metabolic pathways, and relative carbon fluxes of the intracellular bacteria and their amoebal host during the time course of the infection cycle. Generally, the bacterial infection increased the usage of exogenous glucose via glycolysis by A. castellanii. In contrast, carbon fluxes via the amoebal citrate cycle were not affected. During the whole infection cycle, intracellular L. pneumophila incorporated amino acids from their host into the bacterial proteins. However, partial bacterial de novo biosynthesis from exogenous 13C-Ser and, at minor rates, from 13C-glucose could be shown for bacterial Ala, Asp, Glu, and Gly. More specifically, the catabolic usage of Ser increased during the post-exponential phase of intracellular growth, whereas glucose was utilized by the bacteria throughout the infection cycle and not only late during infection as assumed on the basis of earlier in vitro experiments. The early usage of 13C-glucose by the intracellular bacteria suggests that glucose availability could serve as a trigger for replication of L. pneumophila inside the vacuoles of host cells.

Severe Acute Respiratory Syndrome Coronavirus 2 Outbreak Related to a Nightclub, Germany, 2020.

We report an outbreak of coronavirus disease with 74 cases related to a nightclub in Germany in March 2020. Staff members were particularly affected (attack rate 56%) and likely caused sustained viral transmission after an event at the club. This outbreak illustrates the potential for superspreader events and corroborates current club closures.

IFNs Modify the Proteome of Legionella-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid.

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria.

Analysis of two choir outbreaks acting in concert to characterize long- range transmission risks through SARS-CoV-2, Berlin, Germany, 2020.

BACKGROUND: Superspreading events are important drivers of the SARS-CoV-2 pandemic and long-range (LR) transmission is believed to play a major role. We investigated two choir outbreaks with different attack rates (AR) to analyze the contribution of LR transmission and highlight important measures for prevention. METHODS: We conducted two retrospective cohort studies and obtained demographic, clinical, laboratory and contact data, performed SARS-CoV-2 serology, whole genome sequencing (WGS), calculated LR transmission probabilities, measured particle emissions of selected choir members, and calculated particle air concentrations and inhalation doses. RESULTS: We included 65 (84%) and 42 (100%) members of choirs 1 and 2, respectively, of whom 58 (89%) and 10 (24%) became cases. WGS confirmed strain identity in both choirs. Both primary cases transmitted presymptomatically. Particle emission rate when singing was 7 times higher compared to talking. In choir 1, the median concentration of primary cases' emitted particles in the room was estimated to be 8 times higher, exposure at least 30 minutes longer and room volume smaller than in choir 2, resulting in markedly different estimated probabilities for LR transmission (mode: 90% vs. 16%, 95% CI: 80-95% vs. 6-36%). According to a risk model, the first transmission in choir 1 occurred likely after 8 minutes of singing. CONCLUSIONS: The attack rate of the two choirs differed significantly reflecting the differences in LR transmission risks. The pooled proportion of cases due to LR transmission was substantial (81%; 55/68 cases) and was facilitated by likely highly infectious primary cases, high particle emission rates, and indoor rehearsing for an extended time. Even in large rooms, singing of an infectious person may lead to secondary infections through LR exposure within minutes. In the context of indoor gatherings without mask-wearing and waning or insufficient immunity, these results highlight the ongoing importance of non-pharmaceutical interventions wherever aerosols can accumulate.

The Pathometabolism of Legionella Studied by Isotopologue Profiling.

Metabolic pathways and fluxes can be analyzed under in vivo conditions by incorporation experiments using general 13C-labelled precursors. On the basis of the isotopologue compositions in amino acids or other metabolites, the incorporation rates of the supplied precursors and the pathways of their utilization can be studied in considerable detail. In this chapter, the method of isotopologue profiling is illustrated with recent work on the metabolism of intracellular living Legionella pneumophila.