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Abstract
The neritid snail Theodoxus fluviatilis has formed regional subgroups in northern Europe, where it appears in both freshwater (FW) and brackish water (BW) in coastal areas of the Baltic Sea. These ecotypes show clear differences in osmotolerance and in the modes of accumulating organic osmolytes under hyperosmotic stress. We reasoned that the expression patterns of soluble proteins in the two ecotypes may differ as well. BW snails have to deal with a higher salinity (up to 20‰) than FW snails (0.5‰) and also cope with frequent fluctuations in environmental salinity that occur after heavy rains or evaporation caused by extended periods of intense sunshine. Therefore, the protein expression patterns of specimens collected at five different FW and BW sites were analyzed using 2D SDS‐PAGE, mass spectrometry, and sequence comparisons based on a transcriptome database for Theodoxus fluviatilis. We identified 89 differentially expressed proteins. The differences in the expression between FW and BW snails may be due to phenotypic plasticity, but may also be determined by local genetic adaptations. Among the differentially expressed proteins, 19 proteins seem to be of special interest as they may be involved in mediating the higher tolerance of BW animals towards environmental change compared with FW animals.
Background and Objectives: Vaccine induced thrombotic thrombocytopenia (VITT) may occur after COVID-19 vaccination with recombinant adenoviral vector-based vaccines. VITT can present as cerebral sinus and venous thrombosis (CSVT), often complicated by intracranial hemorrhage. Today it is unclear, how long symptomatic VITT can persist. Here, we report the complicated long-term course of a VITT patient with extremely high titers of pathogenic anti-platelet factor 4 (PF4)-IgG antibodies. Methods: Clinical and laboratory findings are presented, including the course of platelet counts, D-Dimer levels, clinical presentation, imaging, SARS-CoV-2-serological and immunological, platelet activating anti-PF4-IgG, as well as autopsy findings. Results: The patient presented with extended superior sagittal sinus thrombosis with accompanying bifrontal intracerebral hemorrhage. Repeated treatment with intravenous immune globuline (IVIG) resolved recurrent episodes of thrombocytopenia. Moreover, the patient’s serum remained strongly positive for platelet-activating anti-PF4-IgG over three months. After a period of clinical stabilization, the patient suffered a recurrent and fatal intracranial hemorrhage. Conclusions: Complicated VITT with extremely high anti-PF4-IgG titers over three months can induce recurrent thrombocytopenia despite treatment with IVIG and anticoagulation. Plasma exchange, immunoadsorption, and /or immunosuppressive treatment may be considered in complicated VITT to reduce extraordinarily high levels of anti-PF4-IgG. Long-term therapy in such cases must take the individual bleeding risk and CSVT risk into account.
Introduction: The environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients.
Methods and results: In this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio.
Conclusion: Our study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.
Introduction
Respiratory tract infections are a worldwide health problem for humans and animals. Different cell types produce lipid mediators in response to infections, which consist of eicosanoids like hydroxyeicosatetraenoic acids (HETEs) or oxylipins like hydroxydocosahexaenoic acids (HDHAs). Both substance classes possess immunomodulatory functions. However, little is known about their role in respiratory infections.
Objectives
Here, we aimed to analyze the lipid mediator imprint of different organs of C57BL/6J mice after intranasal mono-infections with Streptococcus pneumoniae (pneumococcus), Staphylococcus aureus or Influenza A virus (IAV) as wells as pneumococcal-IAV co-infection.
Methods
C57BL/6J mice were infected with different pathogens and lungs, spleen, and plasma were collected. Lipid mediators were analyzed using HPLC-MS/MS. In addition, spatial-distribution of sphingosine 1-phosphate (S1P) and ceramide 1-phosphates (C1P) in tissue samples was examined using MALDI-MS-Imaging. The presence of bacterial pathogens in the lung was confirmed via immunofluorescence staining.
Results
We found IAV specific changes for different HDHAs and HETEs in mouse lungs as well as enhanced levels of 20-HETE in severe S. aureus infection. Moreover, MALDI-MS-Imaging analysis showed an accumulation of C1P and a decrease of S1P during co-infection in lung and spleen. Long chain C1P was enriched in the red and not in the white pulp of the spleen.
Conclusions
Lipid mediator analysis showed that host synthesis of bioactive lipids is in part specific for a certain pathogen, in particular for IAV infection. Furthermore, MS-Imaging displayed great potential to study infections and revealed changes of S1P and C1P in lungs and spleen of co-infected animals, which was not described before.
Invasion of the bacterial pathogen Listeria monocytogenes into human host cells requires specialized surface molecules for attachment and induction of phagocytosis. However, efficient invasion is also dependent on factors with house-keeping functions, such as SecA2-dependent secretion of autolysins for post-divisional segregation of daughter cells. Mutations in this pathway prevent degradation of peptidoglycan cross-walls, so that long cell chains are formed that cannot be phagocytosed. The extreme chaining of such mutants manifests as rough colony phenotype. One rough clone was isolated from a transposon library with a transposon insertion in the uncharacterized lmo0720 gene (lftS) together with a spontaneous point mutation in the secA2 gene. We separated both mutations and demonstrated that this point mutation in the intramolecular regulator 2 domain of SecA2 was sufficient to inactivate the protein. In contrast, lftS deletion did not cause a ΔsecA2-like phenotype. lftS is located in an operon with lftR (lmo0719), encoding a PadR-like transcriptional regulator, and lftR deletion affected growth, invasion and day-light dependent coordination of swarming. Inactivation of lftS partially suppressed these phenotypes, suggesting a functional relationship between LftR and LftS. However, the invasion defect of the ΔlftR mutant was only marginally suppressed by lftS removal. LftR regulates expression of the lmo0979–0980 (lieAB) operon, encoding a putative multidrug resistance transporter and lieAB transcription was strongly upregulated in the absence of LftR. Deletion of lieAB in the ΔlftR background restores wild type-like invasion levels. Hence, we conclude that tight transcriptional repression of the lieAB operon is essential for efficient listerial host cell invasion.
Summary
This study aimed to establish a robust and reliable metaproteomics protocol for an in‐depth characterization of marine particle‐associated (PA) bacteria. To this end, we compared six well‐established protein extraction protocols together with different MS‐sample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In the final optimized workflow, proteins are extracted using a combination of SDS‐containing lysis buffer and cell disruption by bead‐beating, separated by SDS‐PAGE, in‐gel digested and analysed by LC–MS/MS, before MASCOT search against a metagenome‐based database and data processing/visualization with the in‐house‐developed bioinformatics tools Prophane and Paver. As an application example, free‐living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9354 and 5034 identified protein groups for FL and PA bacteria, respectively. Our data suggest that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. Furthermore, our data points to functional differences including proteins involved in polysaccharide degradation, sugar‐ and phosphorus uptake, adhesion, motility, and stress response.
In food chain, Pseudomonas spp. cause spoilage by reducing shelf life of fresh products, especially during cold storage, with a high economic burden for industries. However, recent studies have shed new light on health risks occurring when they colonize immunocompromised patient tissues. Likewise to P. aeruginosa, they exhibit antibiotic resistance and biofilm formation, responsible for their spread and persistence in the environment. Biofilm formation might be induced by environmental stresses, such as temperature fluctuations causing physiological and metabolic changes exacerbating food spoilage (by protease and pigment synthesis), and the production of adhesion molecules, chemotactic or underestimated virulence factors. In order to provide a new insight into phenotypic biodiversity of Pseudomonas spoilers isolated from cold stored cheese, in this work 19 Pseudomonas spp. were investigated for biofilm, pigments, exopolysaccharide production and motility at low temperature. Only nine strains showed these phenotypic traits and the blue pigmenting cheese strain P. fluorescens ITEM 17298 was the most distinctive. In addition, this strain decreased the survival probability of infected Galleria mellonella larvae, showing, for the first time, a pathogenic potential. Genomic and proteomic analyses performed on the ITEM 17298 planktonic cells treated or not with lactoferrin derived antibiofilm peptides allowed to reveal specific biofilm related-pathways as well as proteins involved in pathogenesis. Indeed, several genes were found related to signaling system by cGMP-dependent protein kinases, cellulose, rhamnolipid and alginate synthesis, antibiotic resistance, adhesion and virulence factors. The proteome of the untreated ITEM 17298, growing at low temperature, showed that most of the proteins associated with biofilm regulation, pigmentation motility, antibiotic resistance and pathogenecity were repressed, or decreased their levels in comparison to that of the untreated cultures. Thus, the results of this work shed light on the complex pathways network allowing psychrotrophic pseudomonads to adapt themselves to food-refrigerated conditions and enhance their spoilage. In addition, the discovery of virulence factors and antibiotic resistance determinants raises some questions about the need to deeper investigate these underestimated bacteria in order to increase awareness and provide input to update legislation on their detection limits in foods.