Wulf Schneider-Brachert

Background: Staphylococcus aureus ( S. aureus ) is an increasingly recognized intracellular pathogen, yet infection outcomes vary with bacterial isolate and host cell type. Many mechanisms underlying these differences remain poorly understood. This study investigated how distinct intracellular S. aureus isolates influence host signaling programs and infection outcomes by modulating cell death pathways and TNF-R1 dependent regulation of host cell fates across different human cell lines. Methods: The intracellular localization of four S. aureus isolates was analyzed via transmission electron microscopy (TEM), structured illumination microscopy (SIM), serial block-face scanning electron microscopy (SBF-SEM), and imaging flow cytometry. The transcriptional reprogramming of infected U937 monocytes was examined via mRNA sequencing. Infection outcomes and early TNF-R1 surface expression were characterized and compared with those of A549 and SaOS-2 cell lines employing flow cytometry and Western blot analysis, characterizing host cell death mechanisms in both wild-type and TNF-R1‒deficient backgrounds. Results: All S. aureus isolates localized to the endo-lysosomal and cytosolic compartments but were also frequently detected in nucleo-proximal regions. In U937 monocytes, infection induced a conserved stress signature alongside isolate‑specific transcriptional programs divergently affecting inflammation, metabolism, and cell fate, which was markedly attenuated in response to the chronic‑infection isolate EDCC 5464. Similarly, cell death outcomes were isolate‑ and cell type-dependent, involved caspase activation consistent with intrinsic and extrinsic apoptosis signaling, and caspase-1 activation at distinct temporal dynamics. TNF‑R1 loss initially delayed but later increased later isolate-independent cytotoxicity. Higher TNF-R1 surface expression was triggered in U937 monocytes by all viable isolates, but only by EDCC 5464 in SaOS-2 and A549 cells. Viability of the latter was only marginally influenced by isolate or TNF-R1 loss. Conclusions: These results highlight the multilayered determinants governing intracellular S. aureus survival, host cell susceptibility, and noncanonical intracellular localization that warrants further investigation. The TNF/TNF-R1 axis appears to critically influence regulated host defenses during early infection stages in a tissue-specific manner, though its mechanistic contribution remains to be fully elucidated. Together with distinct isolate-driven gene expression profiles of U937 monocytes, infection risks under TNF-targeted therapies and the contribution of S. aureus heterogeneity should be considered in the design of future host-directed treatment strategies. Graphical Isolate- and cell type- specific host responses to internalized S. aureus across subcellular localization, transcriptional programs, host fates, and the role of TNF-R1 signaling in infection outcomes of U937 monocytes. In U937 _wt , SaOS-2 _wt , and A549 _wt cells, intracellular S. aureus resides in (frequently LAMP-1–decorated) membrane-enclosed compartments or directly in the cytosol at 2 hpi, but also close to host nuclear structures across cell lines. TNF-R1 surface expression is increased by all viable isolates in U937 _wt cells, but only by EDCC 5464 in A549 _wt and SaOS-2 _wt cells. At 4 hpi, S. aureus triggers differential gene expression in (A) U937 _wt cells to an isolate-specific extent, with both unique and shared transcriptomic signatures across the four isolates. This response is weakest for the chronic infection isolate EDCC 5464. Isolate-dependent cell death (marked by Annexin V/7-AAD staining; not shown) of U937 _wt cells involves heterogenous activation of caspase-8, caspase-9 (EDCC 5055 only), and variable effector caspase-3/-7 activity in earlier stages of infection (6 hpi). The extent of activation then barely further increases until 24 hpi and includes changes in the mitochondrial trans-membrane potential (ΔΨm) and additional caspase-1 activation, consistent with engagement of apoptotic signaling alongside other pathways at distinct stages of infection. S. aureus -induced cell death and caspase activation is abolished in (B) U937 _ΔTNF-R1 at 6 hpi, but is significantly reinforced across the tested effectors differences at 24 hpi, whereas isolate-specific differences observed in U937 _wt are lost. (C) SaOS-2 _wt cells show moderate caspase-3/-7 and -1 activation, while infection induces detachment of (D) A549 _wt cells that correlates with Annexin V/-7-AAD staining (not shown) but is accompanied by minimal caspase activation. The number of coloured arrows indicate the mean proportion of effector-positive cells (↑ ~20-40%, ↑ ↑ 40-60%, ↑ ↑ ↑ >60%), the number of coloured plus-signs (+) indicate a trend for the extent of the effect; colours represent each S. aureus isolate: SH1000 (green), EDCC 5055 (blue), EDCC 5458 (orange), EDCC 5464 (yellow). Created with BioRender.com.

Today, there is a continuous worldwide battle against antimicrobial resistance (AMR) and that includes vancomycin-resistant enterococci (VRE). Methods that can adequately and quickly detect transmission chains in outbreaks are needed to trace and manage this problem fast and cost-effectively. In this study, DNA-microarray-based technology was developed for this purpose. It commenced with the bioinformatic design of specific oligonucleotide sequences to obtain amplification primers and hybridization probes. Microarrays were manufactured using these synthesized oligonucleotides. A highly parallel and stringent labeling and hybridization protocol was developed and employed using isolated genomic DNA from previously sequenced (referenced) clinical VRE strains for optimal sensitivity and specificity. Microarray results showed the detection of virulence, resistance, and species-specific genes in the VRE strains. Theoretical predictions of the microarray results were also derived from the sequences of the same VRE strain and were compared to array results while optimizing protocols until the microarray result and theoretical predictions were a match. The study concludes that DNA microarray technology can be used to quickly, accurately, and economically detect specifically and massively parallel target genes in enterococci.

Purpose: Community-acquired methicillin-resistant Staphylococcus aureus strains (CA-MRSA) are spread worldwide and often cause recurring and persistent infections in humans. CA-MRSA strains frequently carry Panton–Valentine leukocidin (PVL) as a distinctive virulence factor. This study investigates the molecular epidemiology, antibiotic resistance and clinical characteristics of PVL-positive MRSA strains in Northern Bavaria, Germany, isolated over an eight-year period. Methods: Strains were identified by MALDI-TOF MS and antibiotic susceptibility was tested by automated microdilution (VITEK 2) or disk diffusion. PVL-encoding genes and mecA were detected by PCR. MRSA clonal complexes (CC) and lineages were assigned by genotyping via DNA microarray and spa-typing. Results: In total, 131 PVL-positive MRSA were collected from five hospital sites between 2009 and 2016. Predominant lineages were CC8-MRSA-[IV+ACME], USA300 (27/131; 20.6%); CC30-MRSA-IV, Southwest Pacific Clone (26/131; 19.8%) and CC80-MRSA-IV (25/131; 19.1%). Other CCs were detected less frequently. Resistance against erythromycin and clindamycin was prevalent, whereas all strains were sensitive towards vancomycin and linezolid. In total, 100 cases (76.3%) were causally linked to an infection. The majority (102/131; 77.9%) of isolates were detected in skin swabs or swabs from surgical sites. Conclusions: During the sample period we found an increase in the PVL-positive MRSA lineages CC30 and CC1. Compared to less-abundant lineages CC1 or CC22, the predominant lineages CC8, CC30 and CC80 harbored a broader resistance spectrum. Furthermore, these lineages are probably associated with a travel and migration background. In the spatio-temporal setting we investigated, these were arguably drivers of diversification and change in the landscape of PVL-positive MRSA.

Millions of people use public transportation daily worldwide and frequently touch surfaces, thereby producing a reservoir of microorganisms on surfaces increasing the risk of transmission. Constant occupation makes sufficient cleaning difficult to achieve. Thus, an autonomous, permanent, antimicrobial coating (AMC) could keep down the microbial burden on such surfaces. A photodynamic AMC was applied to frequently touched surfaces in buses. The microbial burden (colony forming units, cfu) was determined weekly and compared to equivalent surfaces in buses without AMC (references). The microbial burden ranged from 0–209 cfu/cm² on references and from 0–54 cfu/cm² on AMC. The means were 13.4 ± 29.6 cfu/cm² on references and 4.5 ± 8.4 cfu/cm² on AMC (p < 0.001). The difference in microbial burden on AMC and references was almost constant throughout the study. Considering a hygiene benchmark of 5 cfu/cm², the data yield an absolute risk reduction of 22.6% and a relative risk reduction of 50.7%. In conclusion, photodynamic AMC kept down the microbial burden, reducing the risk of transmission of microorganisms. AMC permanently and autonomously contributes to hygienic conditions on surfaces in public transportation. Photodynamic AMC therefore are suitable for reducing the microbial load and closing hygiene gaps in public transportation.

Signaling via death receptor family members such as TNF-R1 mediates pleiotropic biological outcomes ranging from inflammation and proliferation to cell death. Pro-survival signaling is mediated via TNF-R1 complex I at the cellular plasma membrane. Cell death induction requires complex IIa/b or necrosome formation, which occurs in the cytoplasm. In many cell types, full apoptotic or necroptotic cell death induction requires the internalization of TNF-R1 and receptosome formation to properly relay the signal inside the cell. We interrogated the role of the enzyme A disintegrin and metalloprotease 17 (ADAM17)/TACE (TNF-α converting enzyme) in death receptor signaling in human hematopoietic cells, using pharmacological inhibition and genetic ablation. We show that in U937 and Jurkat cells the absence of ADAM17 does not abrogate, but rather increases TNF mediated cell death. Likewise, cell death triggered via DR3 is enhanced in U937 cells lacking ADAM17. We identified ADAM17 as the key molecule that fine-tunes death receptor signaling. A better understanding of cell fate decisions made via the receptors of the TNF-R1 superfamily may enable us, in the future, to more efficiently treat infectious and inflammatory diseases or cancer.

Background: A variety of rapid molecular PCR tests has been developed and commercialised that interrogate the junction site between the staphylococcal core genome, and the mobile genetic element (SCC mec ) which harbours the gene responsible for methicillin-/beta-lactam-resistance. Aim: The purpose of the present study was to investigate why a clinical MRSA isolate yielded false negative test results with widely used, commercial orfX /SCC mec junction site PCR tests. Methods: A collection of isolates that belonged to the same epidemic strain as the index isolate were investigated with commercial MRSA assays and all isolates were sequenced in order to explain this observation. Results: It was found that isolates of the epidemic “European CC1-MRSA-IV” clone, which likely originated in South-Eastern Europe and subsequently spread to Western Europe, generally exhibit this behaviour. The failure of the assays was attributable to a characteristic large insertion in the orfX /SCC mec integration site presumably targeted by such tests. In contrast to MW2 (GenBank BA000033.2 , a CC1 “USA400” strain which also harbours SCC mec IVa), the European CC1 clone harbours an insertion of ca . 5,350 nucleotides adjacent to orfX . This sequence starts with a novel SCC terminal sequence alternate to dcs and encodes six different hypothetical proteins (E7MHX1, ydiL 2, C5QAP8, A8YYX4, npd- SCC, H4AYD7; nucleotide positions 280,690–286,024 of GenBank RBVO000005.1). An SCC mec element with the same insertion was previously found in a Staphylococcus epidermidis isolate (GenBank MH188467.1 ) suggesting transfer between staphylococcal species. Conclusion: In order to ensure the reliability of molecular MRSA tests, it is vital to monitor the emergence of new SCC mec junction sites, not only in Staphylococcus aureus but also in coagulase-negative staphylococci.

Death receptors were initially recognised as potent inducers of apoptotic cell death and soon ambitious attempts were made to exploit selective ignition of controlled cellular suicide as therapeutic strategy in malignant diseases. However, the complexity of death receptor signalling has increased substantially during recent years. Beyond activation of the apoptotic cascade, involvement in a variety of cellular processes including inflammation, proliferation and immune response was recognised. Mechanistically, these findings raised the question how multipurpose receptors can ensure selective activation of a particular pathway. A growing body of evidence points to an elegant spatiotemporal regulation of composition and assembly of the receptor-associated signalling complex. Upon ligand binding, receptor recruitment in specialized membrane compartments, formation of receptor-ligand clusters and internalisation processes constitute key regulatory elements. In this review, we will summarise the current concepts of death receptor trafficking and its implications on receptor-associated signalling events.

Predominant T-helper 1 (Th1) responses with increased gamma interferon (IFN-γ) levels have been proposed to play an important role inHelicobacter pylori-induced gastritis and peptic ulceration. However, bacterial factors contributing to the initiation of Th1 polarization ofH. pylori-specific immune responses have not been characterized in detail thus far. We report here on the identification ofHelicobactercysteine-rich protein A (HcpA) as a novel proinflammatory and Th1-promoting protein. The capacity of HcpA to induce immune activation was studied in splenocyte cultures of naiveH. pylori-negative mice. HcpA stimulated the release of high concentrations of the proinflammatory and Th1-promoting cytokines interleukin-6 (IL-6) and IFN-γ, in addition to significant levels of IL-12, tumor necrosis factor alpha, and IL-10. The observed cytokine profile was comparable to that induced by lipopolysaccharide but differed in the kinetics and maximum levels of cytokine production. In addition, HcpA-induced cytokine release resembled that observed upon incubation withH. pyloriexcept for IL-10, which was only moderately released upon HcpA stimulation. Both HcpA- andH. pylori-mediated IFN-γ production was drastically reduced by a neutralizing antibody against IL-12 but not by an anti-IL-2 antibody. Thus, HcpA seems to represent a novel bacterial virulence factor triggering the release of a concerted set of cytokines to instruct the adaptive immune system for the initiation of proinflammatory and Th1-biased immunity.

Background: Tumor necrosis factor (TNF) is a pleiotropic cytokine involved in a broad spectrum of inflammatory and immune responses including proliferation, differentiation, and cell death. The biological effects of TNF are mediated via two cell surface TNF receptors: p55TNFR (TNFR1; CD120a) and p75TNFR (TNFR2; CD120b). Soluble forms of these two receptors consisting of the extracellular domains are proteolytically cleaved from the membrane and act as inhibitors. A novel p75TNFR isoform generated by the use of an additional transcriptional start site has been described and was termed hicp75TNFR. We focused on the characterization of this new isoform as this protein may be involved in chronic inflammatory processes.Methods: Cell lines were retroviraly transduced with hp75TNFR isoforms. Subcellular localization and colocalization studies with TNF were performed using fluorescence microscopy including exhaustive photon reassignment software, flow cytometry, and receptosome isolation by magnetic means. Biochemical properties of the hicp75TNFR were determined by affinity chromatography, ELISA, and western blot techniques.Results: We describe the localization and activation of a differentially spliced and mainly intracellularly expressed isoform of human p75TNFR, termed hicp75TNFR. Expression studies with hicp75TNFR cDNA in different cell types showed the resulting protein mostly retained in the trans-Golgi network and in endosomes and colocalizes with endogenous TNF. Surface expressed hicp75TNFR behaves like hp75TNFR demonstrating susceptibility for TACE-induced shedding and NFκB activation after TNF binding.Conclusion: Our data demonstrate that intracellular hicp75TNFR is not accessible for exogenously provided TNF but colocalizes with endogenously produced TNF. These findings suggest a possible intracellular activation mechanism of hicp75TNFR by endogenous TNF. Subsequent NFκB activation might induce anti-apoptotic mechanisms to protect TNF-producing cells from cytotoxic effects of TNF. In addition, the intracellular and not TACE-accessible splice form of the hp75TNFR could serve as a pool of preformed, functional hp75TNFR.