Enterococci are commensals of the intestinal tract of many animals and humans. Of the various known and still unnamed new enterococcal species, only isolates of Enterococcus faecium and Enterococcus faecalis have received increased medical and public health attention. According to textbook knowledge, the majority of infections are caused by E. faecalis. In recent decades, the number of enterococcal infections has increased, with the increase being exclusively associated with a rising number of nosocomial E. faecium infections. This increase has been accompanied by the dissemination of certain hospital-acquired strain variants and an alarming progress in the development of antibiotic resistance namely vancomycin resistance. With this review we focus on a description of the specific situation of vancomycin resistance among clinical E. faecium isolates in Germany over the past 30 years. The present review describes three VRE episodes in Germany, each of which is framed by the beginning and end of the respective decade. The first episode is specified by the first appearance of VRE in 1990 and a country-wide spread of specific vanA-type VRE strains (ST117/CT24) until the late 1990s. The second decade was initially marked by regional clusters and VRE outbreaks in hospitals in South-Western Germany in 2004 and 2005, mainly caused by vanA-type VRE of ST203. Against the background of a certain "basic level" of VRE prevalence throughout Germany, an early shift from the vanA genotype to the vanB genotype in clinical isolates already occurred at the end of the 2000s without much notice. With the beginning of the third decade in 2010, VRE rates in Germany have permanently increased, first in some federal states and soon after country-wide. Besides an increase in VRE prevalence, this decade was marked by a sharp increase in vanB-type resistance and a dominance of a few, novel strain variants like ST192 and later on ST117 (CT71, CT469) and ST80 (CT1065). The largest VRE outbreak, which involved about 2,900 patients and lasted over three years, was caused by a novel and until that time, unknown strain type of ST80/CT1013 (vanB). Across all periods, VRE outbreaks were mainly oligoclonal and strain types varied over space (hospital wards) and time. The spread of VRE strains obviously respects political borders; for instance, both vancomycin-variable enterococci which were highly prevalent in Denmark and ST796 VRE which successfully disseminated in Australia and Switzerland, were still completely absent among German hospital patients, until to date.

The Toll/IL-1 receptor (TIR) domains are crucial innate immune signaling modules. Microbial TIR domain-containing proteins inhibit Toll-like receptor (TLR) signaling through molecular mimicry. The TIR domain-containing protein TcpB from Brucella inhibits TLR signaling through interaction with host adaptor proteins TIRAP/Mal and MyD88. To characterize the microbial mimicry of host proteins, we have determined the X-ray crystal structures of the TIR domains from the Brucella protein TcpB and the host adaptor protein TIRAP. We have further characterized homotypic interactions of TcpB using hydrogen/deuterium exchange mass spectrometry and heterotypic TcpB and TIRAP interaction by co-immunoprecipitation and NF-κB reporter assays. The crystal structure of the TcpB TIR domain reveals the microtubule-binding site encompassing the BB loop as well as a symmetrical dimer mediated by the DD and EE loops. This dimerization interface is validated by peptide mapping through hydrogen/deuterium exchange mass spectrometry. The human TIRAP TIR domain crystal structure reveals a unique N-terminal TIR domain fold containing a disulfide bond formed by Cys(89) and Cys(134). A comparison between the TcpB and TIRAP crystal structures reveals substantial conformational differences in the region that encompasses the BB loop. These findings underscore the similarities and differences in the molecular features found in the microbial and host TIR domains, which suggests mechanisms of bacterial mimicry of host signaling adaptor proteins, such as TIRAP.

Successful bacterial pathogens produce an array of virulence factors that allow subversion of the immune system and persistence within the host. For example, uropathogenic Escherichia coli strains, such as CFT073, express Toll/IL-1 receptor–containing (TIR-containing) protein C (TcpC), which impairs TLR signaling, thereby suppressing innate immunity in the urinary tract and enhancing persistence in the kidneys. Here, we have reported that TcpC also reduces secretion of IL-1β by directly interacting with the NACHT leucin-rich repeat PYD protein 3 (NLRP3) inflammasome, which is crucial for recognition of pathogens within the cytosol. At a low MOI, IL-1β secretion was minimal in CFT073-infected macrophages; however, IL-1β release was markedly increased in macrophages infected with CFT073 lacking tcpC. Induction of IL-1β secretion by CFT073 and tcpC–deficient CFT073 required the NLRP3 inflammasome. TcpC attenuated activation of the NLRP3 inflammasome by binding both NLRP3 and caspase-1 and thereby preventing processing and activation of caspase-1. Moreover, in a murine urinary tract infection model, CFT073 infection rapidly induced expression of the NLRP3 inflammasome in the bladder mucosa; however, the presence of TcpC in WT CFT073 reduced IL-1β levels in the urine of infected mice. Together, these findings illustrate how uropathogenic E. coli use the multifunctional virulence factor TcpC to attenuate innate immune responses in the urinary tract.

During the ongoing COVID-19 pandemic, PCR testing and antigen tests have proven critical for helping to stem the spread of its causative agent, SARS-CoV-2. However, these methods suffer from either general applicability and/or sensitivity. Moreover, the emergence of variant strains creates the need for flexibility to correctly and efficiently diagnose the presence of substrains. To address these needs we developed the diagnostic test ADESSO (Accurate Detection of Evolving SARS-CoV-2 through SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) Optimization) which employs Cas13 to diagnose patients in 1 h without sophisticated equipment. Using an extensive panel of clinical samples, we demonstrate that ADESSO correctly identifies infected individuals at a sensitivity and specificity comparable to RT-qPCR on extracted RNA and higher than antigen tests for unextracted samples. Altogether, ADESSO is a fast, sensitive and cheap method that can be applied in a point of care setting to diagnose COVID-19 and can be quickly adjusted to detect new variants.

Activation of TLR7 and TLR9 by endogenous RNA- or DNA-containing ligands, respectively, is thought to contribute to the complicated pathophysiology of systemic lupus erythematosus (SLE). These ligands induce the release of type-I interferons by plasmacytoid dendritic cells and autoreactive antibodies by B-cells, both responses being key events in perpetuating SLE. We recently described the development of inhibitory oligonucleotides (INH-ODN), which are characterized by a phosphorothioate backbone, a CC(T)XXX_3–5GGG motif and a chemical modification of the G-quartet to avoid the formation of higher order structures via intermolecular G-tetrads. These INH-ODNs were equally or significantly more efficient to impair TLR7- and TLR9-stimulated murine B-cells, macrophages, conventional and plasmacytoid dendritic cells than the parent INH-ODN 2088, which lacks G-modification. Here, we evaluate the inhibitory/therapeutic potential of our set of G-modified INH-ODN on human immune cells. We report the novel finding that G-modified INH-ODNs efficiently inhibited the release of IFN-α by PBMC stimulated either with the TLR7-ligand oligoribonucleotide (ORN) 22075 or the TLR9-ligand CpG-ODN 2216. G-modification of INH-ODNs significantly improved inhibition of IL-6 release by PBMCs and purified human B-cells stimulated with the TLR7-ligand imiquimod or the TLR9-ligand CpG-ODN 2006. Furthermore, inhibition of B-cell activation analyzed by expression of activation markers and intracellular ATP content was significantly improved by G-modification. As observed with murine B-cells, high concentrations of INH-ODN 2088 but not of G-modified INH-ODNs stimulated IL-6 secretion by PBMCs in the absence of TLR-ligands thus limiting its blocking efficacy. In summary, G-modification of INH-ODNs improved their ability to impair TLR7- and TLR9-mediated signaling in those human immune cells which are considered as crucial in the pathophysiology of SLE.

The TIR-containing protein C (TcpC) of uropathogenic Escherichia coli strains is a powerful virulence factor by impairing the signaling cascade of Toll-like receptors (TLRs). Several other bacterial pathogens like Salmonella, Yersinia, Staphylococcus aureus but also non-pathogens express similar proteins. We discuss here the pathogenic potential of TcpC and its interaction with TLRs and TLR-adapter proteins on the molecular level and compare its activity with the activity of other bacterial TIR-containing proteins. Finally, we analyze and compare the structure of bacterial TIR-domains with the TIR-domains of TLRs and TLR-adapters.