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Abstract Conventional thin section electron microscopy of viral pathogens, such as the pandemic SARS-CoV-2, can provide structural information on the virus particle phenotype and its evolution. We recorded about 900 transmission electron microscopy images of different SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2) and Omicron BA.2 (B.1.1.529) and determined various morphometric parameters, such as maximal diameter and spike number, using a previously published measurement method. The datasets of the evolved virus variants were supplemented with images and measurements of the early SARS-CoV-2 isolates Munich929 and Italy-INMI1 to allow direct comparison. Infected Vero cell cultures were cultivated under comparable conditions to produce the viruses for imaging and morphometric analysis. The images and measurements can be used as a basis to analyse the morphometric changes of further evolving viruses at the particle level or for developing automated image processing workflows and analysis.

Abstract Mutations in LRBA, a BEACH domain protein, cause severe immune deficiency in humans. LRBA is expressed in many tissues and organs according to biochemical analysis, but little is known about its cellular and subcellular localization, and its deficiency phenotype outside the immune system. By LacZ histochemistry of Lrba gene-trap mice, we performed a comprehensive survey of LRBA expression in numerous tissues, detecting it in many if not all epithelia, in exocrine and endocrine cells, and in subpopulations of neurons. Immunofluorescence microscopy of the exocrine and endocrine pancreas, salivary glands, and intestinal segments, confirmed these patterns of cellular expression and provided information on the subcellular localizations of the LRBA protein. Immuno-electron microscopy demonstrated that in neurons and endocrine cells, which co-express LRBA and its closest relative, neurobeachin, both proteins display partial association with endomembranes in complementary, rather than overlapping, subcellular distributions. Prominent manifestations of human LRBA deficiency, such as inflammatory bowel disease or endocrinopathies, are believed to be primarily due to immune dysregulation. However, as essentially all affected tissues also express LRBA, it is possible that LRBA deficiency enhances their vulnerability and contributes to the pathogenesis.

ABSTRACT The egress of intracellular bacteria from host cells and cellular tissues is a critical process during the infection cycle. This process is essential for bacteria to spread inside the host and can influence the outcome of an infection. For the obligate intracellular Gram-negative zoonotic bacterium Chlamydia psittaci, little is known about the mechanisms resulting in bacterial egress from the infected epithelium. Here, we describe and characterize Chlamydia-containing spheres (CCSs), a novel and predominant type of non-lytic egress utilized by Chlamydia spp. CCSs are spherical, low-phase contrast structures surrounded by a phosphatidylserine-exposing membrane with specific barrier functions. They contain infectious progeny and morphologically impaired cellular organelles. CCS formation is a sequential process starting with the proteolytic cleavage of a DEVD tetrapeptide-containing substrate that can be detected inside the chlamydial inclusions, followed by an increase in the intracellular calcium concentration of the infected cell. Subsequently, blebbing of the plasma membrane begins, the inclusion membrane destabilizes, and the proteolytic cleavage of a DEVD-containing substrate increases rapidly within the whole infected cell. Finally, infected, blebbing cells detach and leave the monolayer, thereby forming CCS. This sequence of events is unique for chlamydial CCS formation and fundamentally different from previously described Chlamydia egress pathways. Thus, CCS formation represents a major, previously uncharacterized egress pathway for intracellular pathogens that could be linked to Chlamydia biology in general and might influence the infection outcome in vivo.IMPORTANCEHost cell egress is essential for intracellular pathogens to spread within an organism and for host-to-host transmission. Here, we characterize Chlamydia-containing sphere (CCS) formation as a novel and predominant non-lytic egress pathway of the intracellular pathogens Chlamydia psittaci and Chlamydia trachomatis. CCS formation is fundamentally different from extrusion formation, the previously described non-lytic egress pathway of C. trachomatis. CCS formation is a unique sequential process, including proteolytic activity, followed by an increase in intracellular calcium concentration, inclusion membrane destabilization, plasma membrane blebbing, and the final detachment of a whole phosphatidylserine-exposing former host cell. Thus, CCS formation represents an important and previously uncharacterized egress pathway for intracellular pathogens that could possibly be linked to Chlamydia biology, including host tropism, protection from host cell defense mechanisms, or bacterial pathogenicity.

Abstract Background SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2. Methods Suspensions from swab samples with a high load of virus RNA (Ct

Infection with SARS-CoV-2 has been linked with substantive inflammation, lung pathology and development of COVID-19. Here the authors spatially associate CCL18 and CCL21 in distinct tissue niches with lung pathology of severe COVID-19.

Human alveolar epithelial lentivirus immortalized cells are very permissive for human coronaviruses and influenza A viruses, provided a suitable model for such infections of the lower respiratory tract as shown by reduced propagation of the SARS-CoV-2 Omicron variant.

Abstract Background Porcine endogenous retroviruses (PERVs) can infect human cells and pose a risk for xenotransplantation when pig cells, tissues or organs are transplanted to human recipients. Xenotransplantation holds great promise to overcome the shortage of human donor organs after solving the problems of rejection, functionality and virus safety. We recently described the transmission of a human-tropic recombinant PERV-A/C, designated PERV-F, from peripheral blood mononuclear cells (PBMCs) of a Göttingen Minipig (GöMP) to human 293 cells (Krüger et al., in Viruses 12(1):38, 2019). The goal of this study was to characterize PERV-F in more detail and to analyze the probability of virus isolation from other animals. Methods The recombination site in the envelope (env) gene, the long terminal repeats (LTR), the proteins and the morphology of the recombinant PERV-F were characterized by polymerase chain reaction (PCR), sequencing, Western blot analysis, immunofluorescence, and transmissible electron microscopy. Mitogen-stimulated PBMCs from 47 additional pigs, including 17 new GöMP, were co-cultured with highly susceptible human 293 T cells, and the PERV-A/C prevalence and PERV transmission was analyzed by PCR. Results PERV-F, isolated from a GöMP, is an infectious human-tropic PERV-A/C virus with a novel type of recombination in the env gene. The length of the LTR of PERV-F increased after passaging on human cells. In a few minipigs, but not in German landrace pigs, PERV-A/C were found. There was no transmission of human-tropic PERV-A/C from additional 47 pigs, including 17 GöMP, to human cells. Conclusion These data show that human-tropic recombinant PERV-A/C proviruses can only be found in a very small number of minipigs, but not in other pigs, and that their isolation as infectious virus able to replicate on human cells is an extremely rare event, even when using highly susceptible 293 cells.

Dippity Pig Syndrome (DPS) is a well-known but rare complex of clinical signs affecting minipigs, which has not been thoroughly investigated yet. Clinically affected animals show acute appearance of red, exudating lesions across the spine. The lesions are painful, evidenced by arching of the back (dipping), and the onset of clinical signs is generally sudden. In order to understand the pathogenesis, histological and virological investigations were performed in affected and unaffected Göttingen Minipigs (GöMPs). The following DNA viruses were screened for using PCR-based methods: Porcine cytomegalovirus (PCMV), which is a porcine roseolovirus (PCMV/PRV), porcine lymphotropic herpesviruses (PLHV-1, PLHV-2, PLHV-3), porcine circoviruses (PCV1, PCV2, PCV3, PCV4), porcine parvovirus 1 (PPV1), and Torque Teno sus viruses (TTSuV1, TTSuV2). Screening was also performed for integrated porcine endogenous retroviruses (PERV-A, PERV-B, PERV-C) and recombinant PERV-A/C and their expression as well as for the RNA viruses hepatitis E virus (HEV) and SARS-CoV-2. Eight clinically affected and one unaffected GöMPs were analyzed. Additional unaffected minipigs had been analyzed in the past. The analyzed GöMPs contained PERV-A and PERV-B integrated in the genome, which are present in all pigs and PERV-C, which is present in most, but not all pigs. In one affected GöMPs recombinant PERV-A/C was detected in blood. In this animal a very high expression of PERV mRNA was observed. PCMV/PRV was found in three affected animals, PCV1 was found in three animals with DPS and in the unaffected minipig, and PCV3 was detected in two animals with DPS and in the unaffected minipig. Most importantly, in one animal only PLHV-3 was detected. It was found in the affected and unaffected skin, and in other organs. Unfortunately, PLHV-3 could not be studied in all other affected minipigs. None of the other viruses were detected and using electron microscopy, no virus particles were found in the affected skin. No porcine virus RNA with exception of PERV and astrovirus RNA were detected in the affected skin by next generation sequencing. This data identified some virus infections in GöMPs with DPS and assign a special role to PLHV-3. Since PCMV/PRV, PCV1, PCV3 and PLHV-3 were also found in unaffected animals, a multifactorial cause of DPS is suggested. However, elimination of the viruses from GöMPs may prevent DPS.

Bacillus cereus biovar anthracis (Bcbva) is an untypical pathogen causing a fatal anthrax-like disease in a variety of wildlife species in African rainforest areas. In contrast to Bacillus anthracis and most species of the B. cereus group, all strains of the Bcbva cluster contain a 22 kb insertion in the sigK gene which encodes the essential late sporulation sigma factor σK. This insertion is excised during sporulation in a site-specific recombination process resulting in an intact sigK gene and a circular molecule. The sporulation kinetics of two strains each of Bcbva and B. anthracis were compared by the expression analysis of eight sporulation-associated genes, including sigK, using reverse transcriptase quantitative real-time PCR. In addition, morphological sporulation stages were analyzed and quantified by electron microscopy. Our results indicated that the necessary excision of the insertion in Bcbva neither delayed nor inhibited its sporulation. In two spontaneous mutants of Bcbva, the excision of the sigK insertion and sporulation were impeded due to mutations in the spo0A and spoVG regulator genes, respectively. The spo0A frameshift mutation was overcome by intragenic suppression in a revertant which was able to sporulate normally, despite an M171S amino acid exchange in the global regulator Spo0A. A screening of the NCBI database identified further strains of the B. cereus group which possess unrelated insertions in the sigK gene, and two strains containing almost identical insertions at the same gene position. Some of the sigK insertions encode putative prophages, whereas the Bcbva insertion encoded a type I restriction–modification system. The function of these insertions and if they are possibly essential for sporulation remains to be assessed.

Summary: Background: Autopsy studies have provided valuable insights into the pathophysiology of COVID-19. Controversies remain about whether the clinical presentation is due to direct organ damage by SARS-CoV-2 or secondary effects, such as overshooting immune response. SARS-CoV-2 detection in tissues by RT-qPCR and immunohistochemistry (IHC) or electron microscopy (EM) can help answer these questions, but a comprehensive evaluation of these applications is missing. Methods: We assessed publications using IHC and EM for SARS-CoV-2 detection in autopsy tissues. We systematically evaluated commercially available antibodies against the SARS-CoV-2 proteins in cultured cell lines and COVID-19 autopsy tissues. In a multicentre study, we evaluated specificity, reproducibility, and inter-observer variability of SARS-CoV-2 IHC. We correlated RT-qPCR viral tissue loads with semiquantitative IHC scoring. We used qualitative and quantitative EM analyses to refine criteria for ultrastructural identification of SARS-CoV-2. Findings: Publications show high variability in detection and interpretation of SARS-CoV-2 abundance in autopsy tissues by IHC or EM. We show that IHC using antibodies against SARS-CoV-2 nucleocapsid yields the highest sensitivity and specificity. We found a positive correlation between presence of viral proteins by IHC and RT-qPCR-determined SARS-CoV-2 viral RNA load (N= 35; r=-0.83, p-value

The biotechnology- and medicine-relevant fungus Aspergillus niger is a common colonizer of indoor habitats such as the International Space Station (ISS). Being able to colonize and biodegrade a wide range of surfaces, A. niger can ultimately impact human health and habitat safety. Surface contamination relies on two key-features of the fungal colony: the fungal spores, and the vegetative mycelium, also known as biofilm. Aboard the ISS, microorganisms and astronauts are shielded from extreme temperatures and radiation, but are inevitably affected by spaceflight microgravity. Knowing how microgravity affects A. niger colony growth, in particular regarding the vegetative mycelium (biofilm) and spore production, will help prevent and control fungal contaminations in indoor habitats on Earth and in space. Because fungal colonies grown on agar can be considered analogs for surface contamination, we investigated A. niger colony growth on agar in normal gravity (Ground) and simulated microgravity (SMG) conditions by fast-clinorotation. Three strains were included: a wild-type strain, a pigmentation mutant (ΔfwnA), and a hyperbranching mutant (ΔracA). Our study presents never before seen scanning electron microscopy (SEM) images of A. niger colonies that reveal a complex ultrastructure and biofilm architecture, and provide insights into fungal colony development, both on ground and in simulated microgravity. Results show that simulated microgravity affects colony growth in a strain-dependent manner, leading to thicker biofilms (vegetative mycelium) and increased spore production. We suggest that the Rho GTPase RacA might play a role in A. niger’s adaptation to simulated microgravity, as deletion of ΔracA leads to changes in biofilm thickness, spore production and total biomass. We also propose that FwnA-mediated melanin production plays a role in A. niger’s microgravity response, as ΔfwnA mutant colonies grown under SMG conditions showed increased colony area and spore production. Taken together, our study shows that simulated microgravity does not inhibit A. niger growth, but rather indicates a potential increase in surface-colonization. Further studies addressing fungal growth and surface contaminations in spaceflight should be conducted, not only to reduce the risk of negatively impacting human health and spacecraft material safety, but also to positively utilize fungal-based biotechnology to acquire needed resources in situ.

Abstract SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV.

A novel tool for the presentation of peptides and small proteins on the surface of human cells has been developed. Our tANCHOR system utilizes tetraspanin anchors containing heterologous amino acid sequences inserted instead of the large extracellular loop. This technology allows a highly effective extracellular display of epitopes for antibody binding studies and many other potential applications.

Insect olfactory sensilla house olfactory sensory neurons (OSNs) and supports cells (SCs). The olfactory sensory processes require, besides the odorant receptors (ORs), insect-specific members of the CD36 family, named sensory neuron membrane proteins (SNMPs). While SNMP1 is considered to act as a coreceptor in the OR-mediated detection of pheromones, SNMP2 was found to be expressed in SCs; however, its function is unknown. For the desert locust, Schistocerca gregaria, we previously visualized mRNA for SNMP1 in OSNs and SNMP2 mRNA in cells associated with OSN clusters. Towards an understanding of their functional implication, it is imperative to explore the cellular and the subcellular localization the SNMP proteins. Therefore, we have generated polyclonal antibodies against SNMP1 and SNMP2 and used fluorescence immunohistochemistry (FIHC) to visualize the SNMP proteins. We found SNMP1 in the somata and respective dendrites of all OSNs in trichoid sensilla and in subsets of OSNs in basiconic sensilla. Notably, SNMP1 was also detected in SCs of these sensilla types. In contrast, SNMP2 protein was only visualized in SCs of basiconic and coeloconic sensilla, but not of trichoid sensilla. Exploring the subcellular localization by electron microscopy using anti-SNMP1-ab and anti-SNMP2-ab revealed an immunogold labelling of SC microvilli bordering the sensillum lymph. Together our findings suggest a dual role of SNMP1 in the antenna of S. gregaria, in some OSN subpopulations in odor detection as well as in functions of some SCs, whereas the role of SNMP2 is limited to the functions of support cells.

Enteroviruses (EV) are implicated in an extensive range of clinical manifestations, such as pancreatic failure, cardiovascular disease, hepatitis, and meningoencephalitis. We recently reported on the biochemical properties of the highly conserved cysteine residue at position 38 (C38) of enteroviral protein 3A and demonstrated a C38-mediated homodimerization of the Coxsackievirus B3 protein 3A (CVB3-3A) that resulted in its profound stabilization. Here, we show that residue C38 of protein 3A supports the replication of CVB3, a clinically relevant member of the enterovirus genus. The infection of HeLa cells with protein 3A cysteine 38 to alanine mutants (C38A) attenuates virus replication, resulting in comparably lower virus particle formation. Consistently, in a mouse infection model, the enhanced virus propagation of CVB3-3A wt in comparison to the CVB3-3A[C38A] mutant was confirmed and found to promote severe liver tissue damage. In contrast, infection with the CVB3-3A[C38A] mutant mitigated hepatic tissue injury and ameliorated the signs of systemic inflammatory responses, such as hypoglycemia and hypothermia. Based on these data and our previous report on the C38-mediated stabilization of the CVB3-3A protein, we conclude that the highly conserved amino acid C38 in protein 3A enhances the virulence of CVB3.

Abstract A large German outbreak in 2011 was caused by a locus of enterocyte effacement (LEE)-negative enterohemorrhagic E. coli (EHEC) strain of the serotype O104:H4. This strain harbors markers that are characteristic of both EHEC and enteroaggregative E. coli (EAEC), including aggregative adhesion fimbriae (AAF) genes. Such rare EHEC/EAEC hybrids are highly pathogenic due to their possession of a combination of genes promoting severe toxicity and aggregative adhesion. We previously identified novel EHEC/EAEC hybrids and observed that one strain exhibited aggregative adherence but had no AAF genes. In this study, a genome sequence analysis showed that this strain belongs to the genoserotype O23:H8, MLST ST26, and harbors a 5.2 Mb chromosome and three plasmids. One plasmid carries some EAEC marker genes, such as aatA and genes with limited protein homology (11–61%) to those encoding the bundle-forming pilus (BFP) of enteropathogenic E. coli. Due to significant protein homology distance to known pili, we designated these as aggregate-forming pili (AFP)-encoding genes and the respective plasmid as pAFP. The afp operon was arranged similarly to the operon of BFP genes but contained an additional gene, afpA2, which is homologous to afpA. The deletion of the afp operon, afpA, or a nearby gene (afpR) encoding an AraC-like regulator, but not afpA2, led to a loss of pilin production, piliation, bacterial autoaggregation, and importantly, a >80% reduction in adhesion and cytotoxicity toward epithelial cells. Gene sets similar to the afp operon were identified in a variety of aatA-positive but AAF-negative intestinal pathogenic E. coli. In summary, we characterized widely distributed and novel fimbriae that are essential for aggregative adherence and cytotoxicity in a LEE-negative Shiga-toxigenic hybrid.

Abstract The emergence of Variola virus-like viruses by natural evolution of zoonotic Orthopoxviruses, like Cowpox virus (CPXV), is a global health threat. The proteasome is essential for poxvirus replication, making the viral components interacting with the ubiquitin-proteasome system attractive antiviral targets. We show that proteasome inhibition impairs CPXV replication by prevention of uncoating, suggesting that uncoating is mediated by proteasomal degradation of viral core proteins. Although Orthopoxvirus particles contain considerable amounts of ubiquitin, distinct modification sites are largely unknown. Therefore, for the first time, we analyzed globally ubiquitination sites in CPXV mature virion proteins using LC-MS/MS. Identification of 137 conserved sites in 54 viral proteins among five CPXV strains revealed extensive ubiquitination of structural core proteins. Moreover, since virions contained primarily K48-linked polyubiquitin, we hypothesized that core proteins are modified accordingly. However, quantitative analysis of ubiquitinated CPXV proteins early in infection showed no proteasomal degradation of core proteins. Instead, our data indicate that the recently suggested proteasomal regulation of the uncoating factor E5 is a prerequisite for uncoating. Expanding our understanding of poxvirus uncoating and elucidating a multitude of novel ubiquitination sites in poxvirus proteins, the present study verifies the major biological significance of ubiquitin in poxvirus infection.

Available treatments for hepatitis B can control the virus but are rarely curative. This led to a global initiative to design new curative therapies for the 257 million patients affected. Discovery and development of these new therapies is contingent upon functional in vitro and in vivo hepatitis B virus (HBV) infection models. However, low titer and impurity of conventional HBV stocks reduce significance of in vitro infections and moreover limit challenge doses in current in vivo models. Therefore, there is a critical need for a robust, simple and reproducible protocol to generate high-purity and high-titer infectious HBV stocks. Here, we outline a three-step protocol for continuous production of high-quality HBV stocks from supernatants of HBV-replicating cell lines. This purification process takes less than 6 h, yields to high-titer stocks (up to 1 × 1011 enveloped, DNA-containing HBV particles/mL each week), and is with minimal equipment easily adaptable to most laboratory settings.

The exceptional toxicity of botulinum neurotoxins (BoNTs) is mediated by high avidity binding to complex polysialogangliosides and intraluminal segments of synaptic vesicle proteins embedded in the presynaptic membrane. One peculiarity is an exposed hydrophobic loop in the toxin's cell binding domain HC, which is located between the ganglioside- and protein receptor-binding sites, and that is particularly pronounced in the serotypes BoNT/B, DC, and G sharing synaptotagmin as protein receptor. Here, we provide evidence that this HC loop is a critical component of their tripartite receptor recognition complex. Binding to nanodisc-embedded receptors and toxicity were virtually abolished in BoNT mutants lacking residues at the tip of the HC loop. Surface plasmon resonance experiments revealed that only insertion of the HC loop into the lipid-bilayer compensates for the entropic penalty inflicted by the dual-receptor binding. Our results represent a new paradigm of how BoNT/B, DC, and G employ ternary interactions with a protein, ganglioside, and lipids to mediate their extraordinary neurotoxicity.

The ability to form endospores allows certain Gram-positive bacteria (e.g. Bacillus subtilis) to challenge the limits of microbial resistance and survival. Thus, B. subtilis is able to tolerate many environmental extremes by transitioning into a dormant state as spores, allowing survival under otherwise unfavorable conditions. Despite thorough study of spore resistance to external stresses, precisely how long B. subtilis spores can lie dormant while remaining viable, a period that potentially far exceeds the human lifespan; is not known although convincing examples of long term spore survival have been recorded. In this study, we report the first data from a 500-year microbial experiment, which started in 2014 and will finish in 2514. A set of vials containing a defined concentration of desiccated B. subtilis spores is opened and tested for viability every two years for the first 24 years and then every 25 years until experiment completion. Desiccated baseline spore samples were also exposed to environmental stresses, including X-rays, 254 nm UV-C, 10% H2O2, dry heat (120°C) and wet heat (100°C) to investigate how desiccated spores respond to harsh environmental conditions after long periods of storage. Data from the first 2 years of storage show no significant decrease in spore viability. Additionally, spores of B. subtilis were subjected to various short-term storage experiments, revealing that space-like vacuum and high NaCl concentration negatively affected spore viability.

Formaldehyde (FA) fixation of infectious samples is a well-established protocol in diagnostic electron microscopy of viruses. However, published experimental data that demonstrate virus inactivation by these fixation procedures are lacking. Usually, fixation is performed immediately before the sample preparation for microscopy. The fixation procedure should transform viruses in a non–infectious but nonetheless structurally intact form in order to allow a proper diagnosis based on morphology. FA provides an essential advantage in comparison to other disinfectants, because it preserves the ultrastructure of biological material without interfering significantly with the preparation (i.e., the negative staining) and the detection of viruses. To examine the efficiency of FA inactivation, we used Vaccinia virus, Human adenovirus and Murine norovirus as models and treated them with FA under various conditions. Critical parameters for the inactivation efficiency were the temperature, the duration of the FA treatment, and the resistance of the virus in question. Our results show that FA inactivation at low temperature (4 °C) bears a high risk of incomplete inactivation. Higher temperatures (25 °C) are more efficient, although they still require rather long incubation times to fully inactivate a complex and highly robust virus like Vaccinia. A protocol, which applied 2% buffered FA for 60 min and a temperature–shift from 25 to 37 °C after 30 min was efficient for the complete inactivation of all test viruses, and therefore has the potential to improve both biosafety and speed of diagnostic electron microscopy.

Xenotransplantation using pig tissues and organs is under development in order to alleviate the increasing shortage of human transplants. Since xenotransplantation may be associated with the transmission of porcine microorganisms to the human recipient, the donor pigs should be carefully analyzed, especially for the presence of potentially zoonotic viruses. Göttingen Minipigs (GöMP) are potential donors of islet cells for the treatment of diabetes. Despite the fact that all animals produced at Ellegaard Göttingen Minipigs A/S carry porcine endogenous retroviruses (PERVs) in their genome and that very few animals were infected with porcine cytomegalovirus (PCMV), hepatitis E virus (HEV) and porcine lymphotropic herpesvirus (PLHV), no transmission of these viruses was observed in a preclinical trial transplanting GöMP islet cells into cynomolgus monkeys. Using a new comprehensive strategy, we then analyzed an isolated subpopulation of Göttingen Minipigs which remained at the University of Göttingen. We concentrated on 11 xenotransplantation-relevant viruses and combined co-incubation assays with susceptible human target cells and molecular biological methods to evaluate the risk posed by PERV. All animals in Göttingen carry PERV-A, PERV-B, and PERV-C in their genome but they are not infected with PCMV, PLHV and HEV. The difference may be explained by selection of negative animals and/or de novo infection. The PERV copy number was established using ddPCR (93 copies) and a human-tropic PERV-A/C was found released from PBMCs of one animal with a high expression of PERV-C.

Current research on wound infections is primarily conducted on animal models, which limits direct transferability of these studies to humans. Some of these limitations can be overcome by using-otherwise discarded-skin from cosmetic surgeries. Superficial wounds are induced in fresh ex vivo skin, followed by intradermal injection of Pseudomonas aeruginosa under the wound. Subsequently, the infected skin is incubated for 20 hours at 37°C and the CFU/wound are determined. Within 20 hours, the bacteria count increased from 107 to 109 bacteria per wound, while microscopy revealed a dense bacterial community in the collagen network of the upper wound layers as well as numerous bacteria scattered in the dermis. At the same time, IL-1alpha and IL-1beta amounts increased in all infected wounds, while-due to bacteria-induced cell lysis-the IL-6 and IL-8 concentrations rose only in the uninfected samples. High-dosage ciprofloxacin treatment resulted in a decisive decrease in bacteria, but consistently failed to eradicate all bacteria. The main benefits of the ex vivo wound model are the use of healthy human skin, a quantifiable bacterial infection, a measureable donor-dependent immune response and a good repeatability of the results. These properties turn the ex vivo wound model into a valuable tool to examine the mechanisms of host-pathogen interactions and to test antimicrobial agents.

In its natural habitat, the soil bacterium Bacillus subtilis often has to cope with fluctuating osmolality and nutrient availability. Upon nutrient depletion it can form dormant spores, which can revive to form vegetative cells when nutrients become available again. While the effects of salt stress on spore germination have been analyzed previously, detailed knowledge on the salt stress response during the subsequent outgrowth phase is lacking. In this study we investigated the changes in gene expression during B. subtilis outgrowth in the presence of 1.2 M NaCl using RNA sequencing. In total, 402 different genes were upregulated and 632 genes were downregulated during 90 min of outgrowth in the presence of salt. The salt stress response of outgrowing spores largely resembled the osmospecific response of vegetative cells exposed to sustained high salinity and included strong upregulation of genes involved in osmoprotectant uptake and compatible solute synthesis. The σB-dependent general stress response typically triggered by salt shocks was not induced, whereas the σW regulon appears to play an important role for osmoadaptation of outgrowing spores. Furthermore, high salinity induced many changes in the membrane protein and transporter transcriptome. Overall, salt stress seemed to slow down the complex molecular reorganization processes (‘ripening’) of outgrowing spores by exerting detrimental effects on vegetative functions such as amino acid metabolism.

Orthopoxvirus species like cowpox, vaccinia and monkeypox virus cause zoonotic infections in humans worldwide. Infections often occur in rural areas lacking proper diagnostic infrastructure as exemplified by monkeypox, which is endemic in Western and Central Africa. While PCR detection requires demanding equipment and is restricted to genome detection, the evidence of virus particles can complement or replace PCR. Therefore, an easily distributable and manageable antigen capture enzyme-linked immunosorbent assay (ELISA) for the detection of orthopoxviruses was developed to facilitate particle detection. By comparing the virus particle binding properties of polyclonal antibodies developed against surface-exposed attachment or fusion proteins, the surface protein A27 was found to be a well-bound, highly immunogenic and exposed target for antibodies aiming at virus particle detection. Subsequently, eight monoclonal anti-A27 antibodies were generated and characterized by peptide epitope mapping and surface plasmon resonance measurements. All antibodies were found to bind with high affinity to two epitopes at the heparin binding site of A27, toward either the N- or C-terminal of the crucial KKEP-segment of A27. Two antibodies recognizing different epitopes were implemented in an antigen capture ELISA. Validation showed robust detection of virus particles from 11 different orthopoxvirus isolates pathogenic to humans, with the exception of MVA, which is apathogenic to humans. Most orthopoxviruses could be detected reliably for viral loads above 1 × 103 PFU/mL. To our knowledge, this is the first solely monoclonal and therefore reproducible antibody-based antigen capture ELISA able to detect all human pathogenic orthopoxviruses including monkeypox virus, except variola virus which was not included. Therefore, the newly developed antibody-based assay represents important progress towards feasible particle detection of this important genus of viruses.

C. albicans is a commensal yeast of the mucous membranes in healthy humans that can also cause disseminated candidiasis, mainly originating from the digestive tract, in vulnerable patients. It is necessary to understand the cellular and molecular mechanisms of the interaction of C. albicans with enterocytes to better understand the basis of commensalism and pathogenicity of the yeast and to improve the management of disseminated candidiasis. In this study, we investigated the kinetics of tight junction (TJ) formation in parallel with the invasion of C. albicans into the Caco-2 intestinal cell line. Using invasiveness assays on Caco-2 cells displaying pharmacologically altered TJ (i.e. differentiated epithelial cells treated with EGTA or patulin), we were able to demonstrate that TJ protect enterocytes against invasion of C. albicans. Moreover, treatment with a pharmacological inhibitor of endocytosis decreased invasion of the fungus into Caco-2 cells displaying altered TJ, suggesting that facilitating access of the yeast to the basolateral side of intestinal cells promotes endocytosis of C. albicans in its hyphal form. These data were supported by SEM observations of differentiated Caco-2 cells displaying altered TJ, which highlighted membrane protrusions engulfing C. albicans hyphae. We furthermore demonstrated that Als3, a hypha-specific C. albicans invasin, facilitates internalization of the fungus by active penetration and induced endocytosis by differentiated Caco-2 cells displaying altered TJ. However, our observations failed to demonstrate binding of Als3 to E-cadherin as the trigger mechanism of endocytosis of C. albicans into differentiated Caco-2 cells displaying altered TJ.

Bacteria live primarily in microbial communities (biofilms), where they exhibit considerably higher biocide tolerance than their planktonic counterparts. Current standardized efficacy testing protocols of disinfectants, however, employ predominantly planktonic bacteria. In order to test the efficacy of biocides on biofilms in a standardized manner, a new assay was developed and optimized for easy-handling, quickness, low running costs, and above all-repeatability. In this assay, 5 mm glass- or polytetrafluoroethylene beads in 24 well microtiter plates served as substrate for Pseudomonas aeruginosa biofilms. After optimizing result-relevant steps, the actual performance of the assay was explored by treating P. aeruginosa biofilms with glutaraldehyde, isopropanol, or peracetic acid in predefined concentrations. The aspired 5 log10 reduction in CFU counts was achieved by glutaraldehyde at 5% (30 min), and by peracetic acid at 0.3% (10 min). In contrast, 80% isopropanol (30 min) failed to meet the reduction goal. However, the main accomplishment of this study was to unveil the potential of the array itself; most noteworthy here, a reliable repeatability of the results. The new bead assay for biofilms is a robust, quick and cost-effective method for assessing the efficacy of biocides against biofilms.

Chlamydia trachomatis is an important human pathogen that replicates inside the infected host cell in a unique vacuole, the inclusion. The formation of this intracellular bacterial niche is essential for productive Chlamydia infections. Despite its importance for Chlamydia biology, a holistic view on the protein composition of the inclusion, including its membrane, is currently missing. Here we describe the host cell-derived proteome of isolated C. trachomatis inclusions by quantitative proteomics. Computational analysis indicated that the inclusion is a complex intracellular trafficking platform that interacts with host cells' antero- and retrograde trafficking pathways. Furthermore, the inclusion is highly enriched for sorting nexins of the SNX-BAR retromer, a complex essential for retrograde trafficking. Functional studies showed that in particular, SNX5 controls the C. trachomatis infection and that retrograde trafficking is essential for infectious progeny formation. In summary, these findings suggest that C. trachomatis hijacks retrograde pathways for effective infection.

Giardiasis, a gastrointestinal disease caused by Giardia duodenalis, is currently treated mainly with nitroimidazoles, primarily metronidazole (MTZ). Treatment failure rates of up to 20 percent reflect the compelling need for alternative treatment options. Here, we investigated whether orlistat, a drug approved to treat obesity, represents a potential therapeutic agent against giardiasis. We compared the growth inhibitory effects of orlistat and MTZ on a long-term in vitro culture adapted G. duodenalis strain, WB-C6, and on a new isolate, 14-03/F7, from a patient refractory to MTZ treatment using a resazurin assay. The giardiacidal concentration of the drugs and their combined in vitro efficacy was determined by median-effect analysis. Morphological changes after treatment were analysed by light and electron microscopy. Orlistat inhibited the in vitro growth of G. duodenalis at low micromolar concentrations, with isolate 14-03/F7 (IC50(24h) = 2.8 µM) being more sensitive than WB-C6 (IC50(24h) = 6.2 µM). The effect was significantly more potent compared to MTZ (IC50(24h) = 4.3 µM and 11.0 µM, respectively) and led to specific undulated morphological alterations on the parasite surface. The giardiacidal concentration of orlistat was >14 µM for 14-03/F7 and >43 µM for WB-C6, respectively. Importantly, the combination of both drugs revealed no interaction on their inhibitory effects. We demonstrate that orlistat is a potent inhibitor of G. duodenalis growth in vitro and kills parasites at concentrations achievable in the gut by approved treatment regimens for obesity. We therefore propose to investigate orlistat in controlled clinical studies as a new drug in giardiasis.

Caries and periodontitis are important human diseases associated with formation of multi-species biofilms. The involved bacteria are intensively studied to understand the molecular basis of the interactions in such biofilms. This study established a basic in vitro single and mixed-species culture model for oral bacteria combining three complimentary methods. The setup allows a rapid screening for effects in the mutual species interaction. Furthermore, it is easy to handle, inexpensive, and reproducible. Streptococcus mitis, S. salivarius and S. sanguinis, typical inhabitants of the healthy oral cavity, S. mutans as main carriogenic species, and Porphyromonas gingivalis, Fusobacterium nucleatum, Parvimonas micra, S. intermedius and Aggregatibacter actinomycetemcomitans as periodontitis-associated bacteria, were investigated for their biofilm forming ability. Different liquid growth media were evaluated. Safranin-staining allowed monitoring of biofilm formation under the chosen conditions. Viable counts and microscopy permitted investigation of biofilm behavior in mixed-species and transwell setups. S. mitis, F. nucleatum, P. gingivalis and P. micra failed to form biofilm structures. S. mutans, S. sanguinis, S. intermedius and S. salivarius established abundant biofilm masses in CDM/sucrose. A. actinomycetemcomitans formed patchy monolayers. For in depth analysis S. mitis, S. mutans and A. actinomycetemcomitans were chosen, because i) they are representatives of the physiological-, cariogenic and periodontitis-associated bacterial flora, respectively and ii) their difference in their biofilm forming ability. Microscopic analysis confirmed the results of safranin staining. Investigation of two species combinations of S. mitis with either S. mutans or A. actinomycetemcomitans revealed bacterial interactions influencing biofilm mass, biofilm structure and cell viability. This setup shows safranin staining, microscopic analysis and viable counts together are crucial for basic examination and evaluation of biofilms. Our experiment generated meaningful results, exemplified by the noted S. mitis influence, and allows a fast decision about the most important bacterial interactions which should be investigated in depth.

Iron sequestration by host iron-binding proteins is an important mechanism of resistance to microbial infections. Inside oral epithelial cells, iron is stored within ferritin, and is therefore not usually accessible to pathogenic microbes. We observed that the ferritin concentration within oral epithelial cells was directly related to their susceptibility to damage by the human pathogenic fungus, Candida albicans. Thus, we hypothesized that host ferritin is used as an iron source by this organism. We found that C. albicans was able to grow on agar at physiological pH with ferritin as the sole source of iron, while the baker's yeast Saccharomyces cerevisiae could not. A screen of C. albicans mutants lacking components of each of the three known iron acquisition systems revealed that only the reductive pathway is involved in iron utilization from ferritin by this fungus. Additionally, C. albicans hyphae, but not yeast cells, bound ferritin, and this binding was crucial for iron acquisition from ferritin. Transcriptional profiling of wild-type and hyphal-defective C. albicans strains suggested that the C. albicans invasin-like protein Als3 is required for ferritin binding. Hyphae of an Deltaals3 null mutant had a strongly reduced ability to bind ferritin and these mutant cells grew poorly on agar plates with ferritin as the sole source of iron. Heterologous expression of Als3, but not Als1 or Als5, two closely related members of the Als protein family, allowed S. cerevisiae to bind ferritin. Immunocytochemical localization of ferritin in epithelial cells infected with C. albicans showed ferritin surrounding invading hyphae of the wild-type, but not the Deltaals3 mutant strain. This mutant was also unable to damage epithelial cells in vitro. Therefore, C. albicans can exploit iron from ferritin via morphology dependent binding through Als3, suggesting that this single protein has multiple virulence attributes.