Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.

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Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori, associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1β- and TNF-α-dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, and mutant H. pylori strains identified the H. pylori metabolite D-glycero-β-D-manno-heptose 1,7-bisphosphate (βHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation depend on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as a core innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen's T4SS.

BackgroundThe anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups.ResultsCulture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, β-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures.ConclusionsOur proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates. Thus, our data presents a rich resource for guiding much-needed investigations on P. acnes virulence factors and host interacting properties.

The gastric pathogen Helicobacter pylori activates the NF‐κB pathway in human epithelial cells via the recently discovered α‐kinase 1 TRAF‐interacting protein with forkhead‐associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7‐bisphosphate (HBP), an LPS intermediate produced in gram‐negative bacteria that represents a new pathogen‐associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF‐κB activation. Instead, we identified ADP‐glycero‐β‐D‐manno‐heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram‐negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the β‐form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori–induced NF‐κB activation in epithelial cells, but it acts as a general gram‐negative bacterial PAMP.—Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen‐associated molecular pattern identified in Helicobacter pylori. FASEB J. 33, 9087–9099 (2019). http://www.fasebj.org

The primary aim of this study was to determine whether native speakers of German living in either Canada or the Netherlands are perceived to have a foreign accent in their native German speech. German monolingual listeners (n = 19) assessed global foreign accent of 34 L1 German speakers in Anglophone Canada, 23 L1 German speakers in the Dutch Netherlands, and five German monolingual controls in Germany. The experimental subjects had moved to either Canada or the Netherlands at an average age of 27 years and had resided in their country of choice for an average of 37 years. The results revealed that the German listeners were more likely to perceive a global foreign accent in the German speech of the consecutive bilinguals in Anglophone Canada and the Dutch Netherlands than in the speech of the control group and that nine immigrants to Canada and five immigrants to the Netherlands were clearly perceived to be non-native speakers of German. Further analysis revealed that quality and quantity of contact with the native German language had a more significant effect on predicting global foreign accent in native speech than age of arrival or length of residence. Two types of contact were differentiated: (i) C−M represented communicative settings in which little code-mixing between the L1 and L2 was expected to occur, and (ii) C+M represented communicative settings in which code-mixing was expected to be more likely. The variable C−M had a significant impact on predicting foreign accent in native speech, whereas the variable C+M did not. The results suggest that contact with the L1 through communicative settings in which code-mixing is inhibited is especially conducive to maintaining the stability of native language pronunciation in consecutive bilinguals living in a migrant context.

No abstract

This study investigates constraints on ultimate attainment in second language (L2) pronunciation in a direct comparison of perceived foreign accent of 40 late L2 learners and 40 late first language (L1) attriters of German. Both groups were compared with 20 predominantly monolingual controls. Contrasting participants who acquired the target language from birth (monolinguals, L1 attriters) with late L2 learners, on the one hand, and bilinguals (L1 attriters, L2ers) with monolinguals, on the other hand, allowed us to disentangle the impacts of age of onset and bilingualism in speech production. At the group level, the attriters performed indistinguishably from controls, and both differed from the L2 group. However, 80% of all L2ers scored within the native (attriter) range. Correlational analyses with background factors further found some effects of use and language aptitude. These results show that acquiring a language from birth is not sufficient to guarantee nativelike pronunciation, and late acquisition does not necessarily prevent it. The results are discussed in the light of models on the role of age and cross-linguistic influence in L2 acquisition.Studies on age effects in second language (L2) acquisition show that pronunciation accuracy in the target language is one of the most difficult skills to acquire for late learners. Such investigations consistently demonstrate that postpuberty learners across different acquisition contexts are detectably different in speech production from monolingual native speakers and from early L2 learners (L2ers). The most robust finding is that foreign accent ratings show a negative correlation with age of acquisition (AOA); that is, the later an L2er is exposed to the L2, the stronger the foreign accent tends to be at the endstate of the acquisition process