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A Europe-wide outbreak of invasive pediatric group A streptococcal infections (iGAS) began in fall 2022. Here, we report the evolution of GAS hospitalizations in children and adolescents during the second outbreak year in 2023–2024 at a tertiary center in Switzerland. Using prospective monitoring of all in-patient GAS cases below 16 years of age, including those with iGAS, we compared case frequencies and clinical characteristics in three time periods (2013–2020; 2022–2023; 2023–2024). Annual GAS hospitalizations increased from a median of 25 cases (range 11–28) in 2013–2020 to 89 and 63 cases, respectively, in 2022–2023 and 2023–2024. iGAS cases evolved similarly (2013–2020, 4 cases (3–8); 2022–2023, 32 cases; 2023–2024, 21 cases). The decline in cases from 2022–2023 to 2023–2024 included all types of GAS organ involvement, except suppurative infections in the head area, which remained largely unchanged (48 vs. 45 cases). Pleural empyema declined from 13 to 7 cases, possibly explained by a poor overlap of the GAS and influenza curves, respectively, in 2023–2024 compared to 2022–2023. These data document the prolongation of the GAS outbreak into its second winter season in 2023–2024.

For dosimetry of radiopharmaceutical therapies, it is essential to determine the volume of relevant structures exposed to therapeutic radiation. For many radiopharmaceuticals, the kidneys represent an important organ-at-risk. To reduce the time required for kidney segmentation, which is often still performed manually, numerous approaches have been presented in recent years to apply deep learning-based methods for CT-based automated segmentation. While the automatic segmentation methods presented so far have been based solely on CT information, the aim of this work is to examine the added value of incorporating PSMA-PET data in the automatic kidney segmentation. Methods: A total of 108 PET/CT examinations (53 [68Ga]Ga-PSMA-I&T and 55 [18F]F-PSMA-1007 examinations) were grouped to create a reference data set of manual segmentations of the kidney. These segmentations were performed by a human examiner. For each subject, two segmentations were carried out: one CT-based (detailed) segmentation and one PET-based (coarser) segmentation. Five different u-net based approaches were applied to the data set to perform an automated segmentation of the kidney: CT images only, PET images only (coarse segmentation), a combination of CT and PET images, a combination of CT images and a PET-based coarse mask, and a CT image, which had been pre-segmented using a PET-based coarse mask. A quantitative assessment of these approaches was performed based on a test data set of 20 patients, including Dice score, volume deviation and average Hausdorff distance between automated and manual segmentations. Additionally, a visual evaluation of automated segmentations for 100 additional (i.e., exclusively automatically segmented) patients was performed by a nuclear physician. Results: Out of all approaches, the best results were achieved by using CT images which had been pre-segmented using a PET-based coarse mask as input. In addition, this method performed significantly better than the segmentation based solely on CT, which was supported by the visual examination of the additional segmentations. In 80% of the cases, the segmentations created by exploiting the PET-based pre-segmentation were preferred by the nuclear physician. Conclusion: This study shows that deep-learning based kidney segmentation can be significantly improved through the addition of a PET-based pre-segmentation. The presented method was shown to be especially beneficial for kidneys with cysts or kidneys that are closely adjacent to other organs such as the spleen, liver or pancreas. In the future, this could lead to a considerable reduction in the time required for dosimetry calculations as well as an improvement in the results.

Abstract Giant viruses (Nucleocytoviricota) are significant lethality agents of various eukaryotic hosts. Although metagenomics indicates their ubiquitous distribution, available giant virus isolates are restricted to a very small number of protist and algal hosts. Here we report on the first viral isolate that replicates in the amoeboflagellate Naegleria. This genus comprises the notorious human pathogen Naegleria fowleri, the causative agent of the rare but fatal primary amoebic meningoencephalitis. We have elucidated the structure and infection cycle of this giant virus, Catovirus naegleriensis (a.k.a. Naegleriavirus, NiV), and show its unique adaptations to its Naegleria host using fluorescence in situ hybridization, electron microscopy, genomics, and proteomics. Naegleriavirus is only the fourth isolate of the highly diverse subfamily Klosneuvirinae, and like its relatives the NiV genome contains a large number of translation genes, but lacks transfer RNAs (tRNAs). NiV has acquired genes from its Naegleria host, which code for heat shock proteins and apoptosis inhibiting factors, presumably for host interactions. Notably, NiV infection was lethal to all Naegleria species tested, including the human pathogen N. fowleri. This study expands our experimental framework for investigating giant viruses and may help to better understand the basic biology of the human pathogen N. fowleri.

Brachyspira pilosicoli (B. pilosicoli) is a pathogen in pigs, poultry, and humans causing colitis, diarrhea, and poor growth rates. Its role as a canine pathogen is controversial, and the seroprevalence of specific IgG antibodies against B. pilosicoli in dogs is unknown. A further, not yet officially recognized Brachyspira species in dogs is “Brachyspira canis” (“B. canis“), which is proposed to be apathogenic. This study evaluates enzyme-linked immunosorbent assays (ELISAs) measuring serum IgG antibodies specific for B. pilosicoli or “B. canis” and investigates levels of specific IgG antibodies against B. pilosicoli and “B. canis” in a cohort of clinical patients presented at an animal referral clinic. These ELISAs use detergent-extracted antigens from B. pilosicoli and “B. canis”. To increase analytic specificity, we precipitated the antigens with trichloroacetic acid (TCA) to isolate and concentrate the respective protein fraction. Our results indicate that a large number of serum IgG antibodies bind to shared epitopes of detergent-extracted antigens of the two spirochaetes. Our data also suggest that dogs might not only carry B. pilosicoli but also have “B. canis”-specific serum IgG antibodies.

The purpose of the European Green Deal and the regulation associated with the so-called EU Taxonomy for Sustainable Activities is to lead capital flows towards sustainable investments. According to the European Commission this is necessary to finance solutions for the immense challenges ahead, such as climate change, social inequality, and loss of biodiversity. However, academic research raises concerns that sustainable investments may earn lower risk-adjusted returns in the long run. The European Supervisory Authorities identify greenwashing risks and pronounce the limited ability of retail investors to make informed investment decisions. The aim of this perspective article is to provide suggestions for a regulation on relevant information on sustainable investments provided by financial service providers to retail investors. Therefore, the existing regulations are put in relation with recent research on investments considering ESG issues. To enable retail investors' self-determined decision-making, financial service providers such as banks, mutual funds, and financial advisors must provide easily accessible, clear, and easily understandable information regarding the ESG-conformity of the offered financial products. In addition, financial service providers must inform retail investors about some specific risks that can arise from a narrow focus on ESG assets such as under-diversification, an overweight of large stocks, and returns that can be lower than the market return.

ObjectiveTo compare machine learning (ML)-based CT-derived fractional flow reserve (CT-FFR) in patients before transcatheter aortic valve replacement (TAVR) by observers with differing training and to assess influencing factors.BackgroundCoronary computed tomography angiography (cCTA) can effectively exclude CAD, e.g. prior to TAVR, but remains limited by its specificity. CT-FFR may mitigate this limitation also in patients prior to TAVR. While a high reliability of CT-FFR is presumed, little is known about the reproducibility of ML-based CT-FFR.MethodsConsecutive patients with obstructive CAD on cCTA were evaluated with ML-based CT-FFR by two observers. Categorization into hemodynamically significant CAD was compared against invasive coronary angiography. The influence of image quality and coronary artery calcium score (CAC) was examined.ResultsCT-FFR was successfully performed on 214/272 examinations by both observers. The median difference of CT-FFR between both observers was −0.05(−0.12-0.02) (p 0.13).ConclusionDifferences between CT-FFR values increased in values below the cut-off, having little clinical impact. Categorization into CAD differed in several patients, but ultimately only had a moderate influence on diagnostic accuracy. This was independent of image quality or CAC.

Abstract This study analyzes whether stocks of companies with environmental social governance (ESG) rating show lower idiosyncratic risk. The main analysis covers 898,757 company-month observations of US stocks in the period from 1991 to 2018 and controls for stocks’ exposure to liquidity, mispricing, innovations in volatility risk, investor sentiment, and analysts’ forecast divergence. The main finding is that the receipt of an ESG rating decreases idiosyncratic stock risk. The effect is stronger for stocks that receive a higher ESG rating. Nevertheless, even when companies receive a lower ESG rating, they show significantly lower idiosyncratic risk than stocks without an ESG rating. Furthermore, stocks subject to a negative screen show lower idiosyncratic risk during recessions than comparable stocks with an ESG rating but without a negative screen. The results support the notion that the receipt of an ESG rating decreases uncertainty regarding future stock risk and return and show that ESG ratings and negative screens individually influence stock risk and, therefore, should be considered separately.

The International Committee on Systematics of Prokaryotes (ICSP) discussed and rejected in 2020 a proposal to modify the International Code of Nomenclature of Prokaryotes to allow the use of gene sequences as type for naming prokaryotes. An alternative nomenclatural code, the Code of Nomenclature of Prokaryotes Described from Sequence Data (SeqCode), which considers genome sequences as type material for naming species, was published in 2022. Members of the ICSP subcommittee for the taxonomy of the phylum Chlamydiae (Chlamydiota) consider that the use of gene sequences as type would benefit the taxonomy of microorganisms that are difficult to culture such as the chlamydiae and other strictly intracellular bacteria. We recommend the registration of new names of uncultured prokaryotes in the SeqCode registry.

Introduction Two next-generation pneumococcal conjugate vaccines (PCVs), a 15- and a 20-valent PCV (PCV15 and PCV20), have recently been licensed for use in adults, and PCV15 has also been licensed in children. We developed a dynamic transmission model specific for Germany, with the aim to predict carriage prevalence and invasive pneumococcal disease (IPD) burden for serotypes included in these vaccines. Methods The model allows to follow serotype distributions longitudinally both in the absence and presence of PCV vaccinations. We considered eight age cohorts and seven serotype groups according to the composition of different pneumococcal vaccines. This comprises the additional serotypes contained in PCV15 and PCV20 but not in PCV13. Results The model predicted that by continuing the current vaccine policy (standard vaccination with PCV13 in children and with PPSV23 in adults) until 2031, IPD case counts due to any serotype in children Conclusions We conclude that introduction of next-generation PCVs for adults may prevent a substantial and increasing proportion of adult IPDs, with PCV20 having the potential to provide the broadest protection against pneumococcal disease.

ABSTRACT Free-living bacteria have evolved multiple times to become host-restricted endosymbionts. The transition from a free-living to a host-restricted lifestyle comes with a number of different genomic changes, including a massive loss of genes. In host-restricted endosymbionts, gene inactivation and genome reduction are facilitated by mobile genetic elements, mainly insertion sequences (ISs). ISs are small autonomous mobile elements, and one of, if not the most, abundant transposable elements in bacteria. Proliferation of ISs is common in some facultative endosymbionts, and is likely driven by the transmission bottlenecks, which increase the level of genetic drift. In this study, we present a manually curated genome annotation for a Cardinium endosymbiont of the dwarf spider Oedothorax gibbosus. Cardinium species are host-restricted endosymbionts that, similarly to ColbachiaWolbachia spp., include strains capable of manipulating host reproduction. Through the focus on mobile elements, the annotation revealed a rampant spread of ISs, extending earlier observations in other Cardinium genomes. We found that a large proportion of IS elements are pseudogenized, with many displaying evidence of recent inactivation. Most notably, we describe the lineage-specific emergence and spread of a novel IS-derived Miniature Inverted repeat Transposable Element (MITE), likely being actively maintained by intact copies of its parental IS982-family element. This study highlights the relevance of manual curation of these repeat-rich endosymbiont genomes for the discovery of novel MITEs, as well as the possible role these understudied elements might play in genome streamlining. IMPORTANCE Cardinium bacteria, a widespread symbiont lineage found across insects and nematodes, have been linked to reproductive manipulation of their hosts. However, the study of Cardinium has been hampered by the lack of comprehensive genomic resources. The high content of mobile genetic elements, namely, insertion sequences (ISs), has long complicated the analyses and proper annotations of these genomes. In this study, we present a manually curated annotation of the Cardinium symbiont of the spider Oedothorax gibbosus. Most notably, we describe a novel IS-like element found exclusively in this strain. We show that this mobile element likely evolved from a defective copy of its parental IS and then spread throughout the genome, contributing to the pseudogenization of several other mobile elements. We propose this element is likely being maintained by the intact copies of its parental IS element and that other similar elements in the genome could potentially follow this route.

Chlamydiae are strictly intracellular bacteria that exist in a wide variety of environments but the diversity of the phylum is not well described. Here, the authors analyze 82 metagenome-assembled genomes, identify seven new families, and describe genomic signals of metabolic diversity.

The obligate intracellular bacteria Chlamydia trachomatis store glycogen in the lumen of the vacuoles in which they grow. Glycogen catabolism generates glucose-1-phosphate (Glc1P), while the bacteria can take up only glucose-6-phosphate (Glc6P). We tested whether the conversion of Glc1P into Glc6P could be catalyzed by a phosphoglucomutase (PGM) of host or bacterial origin. We found no evidence for the presence of the host PGM in the vacuole. Two C. trachomatis proteins, CT295 and CT815, are potential PGMs. By reconstituting the reaction using purified proteins, and by complementing PGM deficient fibroblasts, we demonstrated that only CT295 displayed robust PGM activity. Intriguingly, we showed that glycogen accumulation in the lumen of the vacuole of a subset of Chlamydia species (C. trachomatis, C. muridarum, C. suis) correlated with the presence, in CT295 orthologs, of a secretion signal recognized by the type three secretion (T3S) machinery of Shigella. C. caviae and C. pneumoniae do not accumulate glycogen, and their CT295 orthologs lack T3S signals. In conclusion, we established that the conversion of Glc1P into Glc6P was accomplished by a bacterial PGM, through the acquisition of a T3S signal in a “housekeeping” protein. Acquisition of this signal likely contributed to shaping glycogen metabolism within Chlamydiaceae.

Abstract Background Individualization and patient-specific optimization of treatment is a major goal of modern health care. One way to achieve this goal is the application of high-resolution diagnostics together with the application of targeted therapies. However, the rising number of different treatment modalities also induces new challenges: Whereas randomized clinical trials focus on proving average treatment effects in specific groups of patients, direct conclusions at the individual patient level are problematic. Thus, the identification of the best patient-specific treatment options remains an open question. Systems medicine, specifically mechanistic mathematical models, can substantially support individual treatment optimization. In addition to providing a better general understanding of disease mechanisms and treatment effects, these models allow for an identification of patient-specific parameterizations and, therefore, provide individualized predictions for the effect of different treatment modalities. Results In the following we describe a software framework that facilitates the integration of mathematical models and computer simulations into routine clinical processes to support decision-making. This is achieved by combining standard data management and data exploration tools, with the generation and visualization of mathematical model predictions for treatment options at an individual patient level. Conclusions By integrating model results in an audit trail compatible manner into established clinical workflows, our framework has the potential to foster the use of systems-medical approaches in clinical practice. We illustrate the framework application by two use cases from the field of haematological oncology.

In this work, we study the characteristics of femtosecond-filament-laser–matter interactions and laser-induced periodic surface structures (LIPSS) at a beam-propagation distance up to 55 m. The quantification of the periodicity of filament-induced self-organized surface structures was accomplished by SEM and AFM measurements combined with the use of discrete two-dimensional fast Fourier transform (2D-FFT) analysis, at different filament propagation distances. The results show that the size of the nano-scale surface features increased with ongoing laser filament processing and, further, periodic ripples started to form in the ablation-spot center after irradiation with five spatially overlapping pulses. The effective number of irradiating filament pulses per spot area affected the developing surface texture, with the period of the low spatial frequency LIPSS reducing notably at a high pulse number. The high regularity of the filament-induced ripples was verified by the demonstration of the angle-of-incidence-dependent diffraction of sunlight. This work underlines the potential of long-range femtosecond filamentation for energy delivery at remote distances, with suppressed diffraction and long depth focus, which can be used in biomimetic laser surface engineering and remote-sensing applications.

Abstract Free-living amoebae are well known for their role in controlling microbial community composition through grazing, but some groups, namely Acanthamoeba species, also frequently serve as hosts for bacterial symbionts. Here we report the first identification of a bacterial symbiont in the testate amoeba Cochliopodium. The amoeba was isolated from a cooling tower water sample and identified as C. minus. Fluorescence in situ hybridization and transmission electron microscopy revealed intracellular symbionts located in vacuoles. 16S rRNA-based phylogenetic analysis identified the endosymbiont as member of a monophyletic group within the family Coxiellaceae (Gammaprotebacteria; Legionellales), only moderately related to known amoeba symbionts. We propose to tentatively classify these bacteria as ‘Candidatus Cochliophilus cryoturris’. Our findings add both, a novel group of amoeba and a novel group of symbionts, to the growing list of bacteria-amoeba relationships.

ABSTRACT Legionella pneumophila is an important opportunistic pathogen for which environmental reservoirs are crucial for the infection of humans. In the environment, free-living amoebae represent key hosts providing nutrients and shelter for highly efficient intracellular proliferation of L. pneumophila, which eventually leads to lysis of the protist. However, the significance of other bacterial players for L. pneumophila ecology is poorly understood. In this study, we used a ubiquitous amoeba and bacterial endosymbiont to investigate the impact of this common association on L. pneumophila infection. We demonstrate that L. pneumophila proliferation was severely suppressed in Acanthamoeba castellanii harboring the chlamydial symbiont Protochlamydia amoebophila. The amoebae survived the infection and were able to resume growth. Different environmental amoeba isolates containing the symbiont were equally well protected as different L. pneumophila isolates were diminished, suggesting ecological relevance of this symbiont-mediated defense. Furthermore, protection was not mediated by impaired L. pneumophila uptake. Instead, we observed reduced virulence of L. pneumophila released from symbiont-containing amoebae. Pronounced gene expression changes in the presence of the symbiont indicate that interference with the transition to the transmissive phase impedes the L. pneumophila infection. Finally, our data show that the defensive response of amoebae harboring P. amoebophila leaves the amoebae with superior fitness reminiscent of immunological memory. Given that mutualistic associations between bacteria and amoebae are widely distributed, P. amoebophila and potentially other amoeba endosymbionts could be key in shaping environmental survival, abundance, and virulence of this important pathogen, thereby affecting the frequency of human infection. IMPORTANCE Bacterial pathogens are generally investigated in the context of disease. To prevent outbreaks, it is essential to understand their lifestyle and interactions with other microbes in their natural environment. Legionella pneumophila is an important human respiratory pathogen that survives and multiplies in biofilms or intracellularly within protists, such as amoebae. Importantly, transmission to humans occurs from these environmental sources. Legionella infection generally leads to rapid host cell lysis. It was therefore surprising to observe that amoebae, including fresh environmental isolates, were well protected during Legionella infection when the bacterial symbiont Protochlamydia amoebophila was also present. Legionella was not prevented from invading amoebae but was impeded in its ability to develop fully virulent progeny and were ultimately cleared in the presence of the symbiont. This study highlights how ecology and virulence of an important human pathogen is affected by a defensive amoeba symbiont, with possibly major consequences for public health.

The subject of the present study is the reproduction of a submersible electric pump impeller through reverse engineering and additive manufacturing. All of the phases commonly envisaged in the reproduction of an existing piece were carried out. The aim of the study is to show how the chosen pump component can be effectively re-engineered and produced with the selective laser melting technique, obtaining a final product that is comparable if not even better than the starting one. To achieve this goal, the original piece was redesigned and a new model was created and analyzed. The whole process has been split into three main phases: (i) realization of the three-dimensional model from an existing piece using reverse engineering techniques; (ii) finite element analysis for the optimization of the use of the material; and (iii) 3D printing of a concept model in polyethylene terephthalate by using the fused deposition modeling technology and of the functional model in AISI 316 stainless steel with selective laser melting technology.

High amounts of aerial pollutants like dust and microorganisms can pose serious health hazards to animals and humans. The aim of the current study therefore was, to assess the efficiency of UVC irradiation combined to air filtration in reducing airborne microorganisms at laboratory scale. In a second part, a UVC-combined recirculating air filtration module (UVC module) was implemented in a small animal facility in order to assess its improvement of air quality with regard to airborne bacteria and dust. Tests at laboratory scale were performed using aerosols of Staphylococcus (S.) aureus, Actinobacillus pleuropneumoniae, porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus. We varied relative humidity (RH) to evaluate its effect on UVC irradiation efficiency. In addition, viability of pathogens inside the filter material was determined over up to six months. UVC-combined air filtration resulted in a more than 99% reduction of viral and bacterial particles. RH had no influence on UVC efficiency. Viability in the filter matter varied depending on the pathogen used and RH with S. aureus and PPV being most resistant. In our small pig facility consisting of two separated barns, weekly air measurements were conducted over a period of 13 weeks (10 piglets) and 16 weeks (11 piglets), respectively. Airborne bacterial numbers were significantly lower in the barn equipped with the UVC module compared to the reference barn. On average a reduction to 37% of reference values could be achieved for bacteria, whereas the amount of total dust was reduced to a much lesser extent (i.e. to 78% of reference values). Measures taken in front of and behind the UVC module revealed a reduction of 99.4% for airborne bacteria and 95.0% for total dust. To conclude, recirculating air filtration combined to UVC provided efficient reduction of pathogens at laboratory and experimental scale. The implementation of such devices might improve the overall environmental quality in animal facilities.

ABSTRACT Chlamydiae are obligate intracellular bacteria comprising well-known human pathogens and ubiquitous symbionts of protists, which are characterized by a unique developmental cycle. Here we comprehensively analyzed gene expression dynamics of Protochlamydia amoebophila during infection of its Acanthamoeba host by RNA sequencing. This revealed a highly dynamic transcriptional landscape, where major transcriptional shifts are conserved among chlamydial symbionts and pathogens. Our data served to propose a time-resolved model for type III protein secretion during the developmental cycle, and we provide evidence for a biphasic metabolism of P. amoebophila during infection, which involves energy parasitism and amino acids as the carbon source during initial stages and a postreplicative switch to endogenous glucose-based ATP production. This fits well with major transcriptional changes in the amoeba host, where upregulation of complex sugar breakdown precedes the P. amoebophila metabolic switch. The biphasic chlamydial metabolism represents a unique adaptation to exploit eukaryotic host cells, which likely contributed to the evolutionary success of this group of microbes. IMPORTANCE Chlamydiae are known as major bacterial pathogens of humans, causing the ancient disease trachoma, but they are also frequently found in the environment where they infect ubiquitous protists such as amoebae. All known chlamydiae require a eukaryotic host cell to thrive. Using the environmental chlamydia Protochlamydia amoebophila within its natural host, Acanthamoeba castellanii, we investigated gene expression dynamics in vivo and throughout the complete chlamydial developmental cycle for the first time. This allowed us to infer how a major virulence mechanism, the type III secretion system, is regulated and employed, and we show that the physiology of chlamydiae undergoes a complete shift regarding carbon metabolism and energy generation. This study provides comprehensive insights into the infection strategy of chlamydiae and reveals a unique adaptation to life within a eukaryotic host cell.

Genomic heterogeneity of bacterial species is observed and studied in experimental evolution experiments and clinical diagnostics, and occurs as micro-diversity of natural habitats. The challenge for genome research is to accurately capture this heterogeneity with the currently used short sequencing reads. Recent advances in NGS technologies improved the speed and coverage and thus allowed for deep sequencing of bacterial populations. This facilitates the quantitative assessment of genomic heterogeneity, including low frequency alleles or haplotypes. However, false positive variant predictions due to sequencing errors and mapping artifacts of short reads need to be prevented. We therefore created VarCap, a workflow for the reliable prediction of different types of variants even at low frequencies. In order to predict SNPs, InDels and structural variations, we evaluated the sensitivity and accuracy of different software tools using synthetic read data. The results suggested that the best sensitivity could be reached by a union of different tools, however at the price of increased false positives. We identified possible reasons for false predictions and used this knowledge to improve the accuracy by post-filtering the predicted variants according to properties such as frequency, coverage, genomic environment/localization and co-localization with other variants. We observed that best precision was achieved by using an intersection of at least two tools per variant. This resulted in the reliable prediction of variants above a minimum relative abundance of 2%. VarCap is designed for being routinely used within experimental evolution experiments or for clinical diagnostics. The detected variants are reported as frequencies within a VCF file and as a graphical overview of the distribution of the different variant/allele/haplotype frequencies. The source code of VarCap is available at https://github.com/ma2o/VarCap. In order to provide this workflow to a broad community, we implemeted VarCap on a Galaxy webserver, which is accessible at http://galaxy.csb.univie.ac.at.

The giant tubeworm Riftia pachyptila lives in symbiosis with the chemoautotrophic gammaproteobacterium Cand. Endoriftia persephone. Symbionts are released back into the environment upon host death in high-pressure experiments, while microbial fouling is not involved in trophosome degradation. Therefore, we examined the antimicrobial effect of the tubeworm's trophosome and skin. The growth of all four tested Gram-positive, but only of one of the tested Gram-negative bacterial strains was inhibited by freshly fixed and degrading trophosome (incubated up to ten days at either warm or cold temperature), while no effect on Saccharomyces cerevisiae was observed. The skin did not show antimicrobial effects. A liquid chromatography-mass spectrometric analysis of the ethanol supernatant of fixed trophosomes lead to the tentative identification of the phospholipids 1-palmitoleyl-2-lyso-phosphatidylethanolamine, 2-palmitoleyl-1-lyso-phosphatidylethanolamine and the free fatty acids palmitoleic, palmitic and oleic acid, which are known to have an antimicrobial effect. As a result of tissue autolysis, the abundance of the free fatty acids increased with longer incubation time of trophosome samples. This correlated with an increasing growth inhibition of Bacillus subtilis and Listeria welshimeri, but not of the other bacterial strains. Therefore, the free fatty acids produced upon host degradation could be the cause of inhibition of at least these two bacterial strains.

The Chlamydiae are a highly successful group of obligate intracellular bacteria, whose members are remarkably diverse, ranging from major pathogens of humans and animals to symbionts of ubiquitous protozoa. While their infective developmental stage, the elementary body (EB), has long been accepted to be completely metabolically inert, it has recently been shown to sustain some activities, including uptake of amino acids and protein biosynthesis. In the current study, we performed an in-depth characterization of the metabolic capabilities of EBs of the amoeba symbiont Protochlamydia amoebophila. A combined metabolomics approach, including fluorescence microscopy-based assays, isotope-ratio mass spectrometry (IRMS), ion cyclotron resonance Fourier transform mass spectrometry (ICR/FT-MS), and ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was conducted, with a particular focus on the central carbon metabolism. In addition, the effect of nutrient deprivation on chlamydial infectivity was analyzed. Our investigations revealed that host-free P. amoebophila EBs maintain respiratory activity and metabolize D-glucose, including substrate uptake as well as host-free synthesis of labeled metabolites and release of labeled CO2 from (13)C-labeled D-glucose. The pentose phosphate pathway was identified as major route of D-glucose catabolism and host-independent activity of the tricarboxylic acid (TCA) cycle was observed. Our data strongly suggest anabolic reactions in P. amoebophila EBs and demonstrate that under the applied conditions D-glucose availability is essential to sustain metabolic activity. Replacement of this substrate by L-glucose, a non-metabolizable sugar, led to a rapid decline in the number of infectious particles. Likewise, infectivity of Chlamydia trachomatis, a major human pathogen, also declined more rapidly in the absence of nutrients. Collectively, these findings demonstrate that D-glucose is utilized by P. amoebophila EBs and provide evidence that metabolic activity in the extracellular stage of chlamydiae is of major biological relevance as it is a critical factor affecting maintenance of infectivity.

The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydiaouter membrane proteins, PomS (pc1489) and PomT (pc1077), are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts.

The fundamental role of programmed cell death in host defense is highlighted by the multitude of anti-apoptotic strategies evolved by various microbes, including the well-known obligate intracellular bacterial pathogens Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae. As inhibition of apoptosis is assumed to be essential for a successful infection of humans by these chlamydiae, we analyzed the anti-apoptotic capacity of close relatives that occur as symbionts of amoebae and might represent emerging pathogens. While Simkania negevensis was able to efficiently replicate within insect cells, which served as model for metazoan-derived host cells, the Parachlamydiaceae (Parachlamydia acanthamoebae and Protochlamydia amoebophila) displayed limited intracellular growth, yet these bacteria induced typical features of apoptotic cell death, including formation of apoptotic bodies, nuclear condensation, internucleosomal DNA fragmentation, and effector caspase activity. Induction of apoptosis was dependent on bacterial activity, but not bacterial de novo protein synthesis, and was detectable already at very early stages of infection. Experimental inhibition of host cell death greatly enhanced parachlamydial replication, suggesting that lack of potent anti-apoptotic activities in Parachlamydiaceae may represent an important factor compromising their ability to successfully infect non-protozoan hosts. These findings highlight the importance of the evolution of anti-apoptotic traits for the success of chlamydiae as pathogens of humans and animals.

Epitheliocystis, a disease characterised by cytoplasmic bacterial inclusions (cysts) in the gill and less commonly skin epithelial cells, has been reported in many marine and freshwater fish species and may be associated with mortality. Previously, molecular and ultrastructural analyses have exclusively associated members of the Chlamydiae with such inclusions. Here we investigated a population of farmed Atlantic salmon from the west coast of Norway displaying gill epitheliocystis. Although 'Candidatus Piscichlamydia salmonis', previously reported to be present in such cysts, was detected by PCR in most of the gill samples analysed, this bacterium was found to be a rare member of the gill microbiota, and not associated with the observed cysts as demonstrated by fluorescence in situ hybridization assays. The application of a broad range 16 S rRNA targeted PCR assay instead identified a novel betaproteobacterium as an abundant member of the gill microbiota. Fluorescence in situ hybridization demonstrated that this bacterium, tentatively classified as 'Candidatus Branchiomonas cysticola', was the cyst-forming agent in these samples. While histology and ultrastructure of 'Ca. B. cysticola' cysts revealed forms similar to the reticulate and intermediate bodies described in earlier reports from salmon in seawater, no elementary bodies typical of the chlamydial developmental cycle were observed. In conclusion, this study identified a novel agent of epitheliocystis in sea-farmed Atlantic salmon and demonstrated that these cysts can be caused by bacteria phylogenetically distinct from the Chlamydiae.

Terrestrial arthropods are commonly infected with maternally inherited bacterial symbionts that cause cytoplasmic incompatibility (CI). In CI, the outcome of crosses between symbiont-infected males and uninfected females is reproductive failure, increasing the relative fitness of infected females and leading to spread of the symbiont in the host population. CI symbionts have profound impacts on host genetic structure and ecology and may lead to speciation and the rapid evolution of sex determination systems. Cardinium hertigii, a member of the Bacteroidetes and symbiont of the parasitic wasp Encarsia pergandiella, is the only known bacterium other than the Alphaproteobacteria Wolbachia to cause CI. Here we report the genome sequence of Cardinium hertigii cEper1. Comparison with the genomes of CI-inducing Wolbachia pipientis strains wMel, wRi, and wPip provides a unique opportunity to pinpoint shared proteins mediating host cell interaction, including some candidate proteins for CI that have not previously been investigated. The genome of Cardinium lacks all major biosynthetic pathways but harbors a complete biotin biosynthesis pathway, suggesting a potential role for Cardinium in host nutrition. Cardinium lacks known protein secretion systems but encodes a putative phage-derived secretion system distantly related to the antifeeding prophage of the entomopathogen Serratia entomophila. Lastly, while Cardinium and Wolbachia genomes show only a functional overlap of proteins, they show no evidence of laterally transferred elements that would suggest common ancestry of CI in both lineages. Instead, comparative genomics suggests an independent evolution of CI in Cardinium and Wolbachia and provides a novel context for understanding the mechanistic basis of CI.

BackgroundThe hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained.Methodology/principal findingsThis study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias.ConclusionsReciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization.

The type III secretion system (TTSS) is a key mechanism for host cell interaction used by a variety of bacterial pathogens and symbionts of plants and animals including humans. The TTSS represents a molecular syringe with which the bacteria deliver effector proteins directly into the host cell cytosol. Despite the importance of the TTSS for bacterial pathogenesis, recognition and targeting of type III secreted proteins has up until now been poorly understood. Several hypotheses are discussed, including an mRNA-based signal, a chaperon-mediated process, or an N-terminal signal peptide. In this study, we systematically analyzed the amino acid composition and secondary structure of N-termini of 100 experimentally verified effector proteins. Based on this, we developed a machine-learning approach for the prediction of TTSS effector proteins, taking into account N-terminal sequence features such as frequencies of amino acids, short peptides, or residues with certain physico-chemical properties. The resulting computational model revealed a strong type III secretion signal in the N-terminus that can be used to detect effectors with sensitivity of approximately 71% and selectivity of approximately 85%. This signal seems to be taxonomically universal and conserved among animal pathogens and plant symbionts, since we could successfully detect effector proteins if the respective group was excluded from training. The application of our prediction approach to 739 complete bacterial and archaeal genome sequences resulted in the identification of between 0% and 12% putative TTSS effector proteins. Comparison of effector proteins with orthologs that are not secreted by the TTSS showed no clear pattern of signal acquisition by fusion, suggesting convergent evolutionary processes shaping the type III secretion signal. The newly developed program EffectiveT3 (http://www.chlamydiaedb.org) is the first universal in silico prediction program for the identification of novel TTSS effectors. Our findings will facilitate further studies on and improve our understanding of type III secretion and its role in pathogen-host interactions.

Energy parasitism by ATP/ADP transport proteins is an essential, common feature of intracellular bacteria such as chlamydiae and rickettsiae, which are major pathogens of humans. Although several ATP/ADP transport proteins have so far been characterized, some fundamental questions regarding their function remained unaddressed. In this study, we focused on the detailed biochemical analysis of a representative ATP/ADP transporter (PamNTT1), from the amoeba symbiont Protochlamydia amoebophila (UWE25) to further clarify the principle of energy exploitation. We succeeded in the purification of the first bacterial nucleotide transporter (NTT) and its functional reconstitution into artificial lipid vesicles. Reconstituted PamNTT1 revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Latter preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibits a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. The data presented here comprehensively explain the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using the nucleotide transporter PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, and the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional reconstitution of this essential chlamydial protein paves the way for high-throughput uptake studies in order to screen for specific inhibitors potentially suitable as anti-chlamydial drugs.