Role of the Putative Histidine Kinase BP1092 in Bordetella pertussis Virulence Regulation and Intracellular Survival.

Bordetella pertussis persists inside host cells, and virulence factors are crucial for intracellular adaptation. The regulation of B. pertussis virulence factor transcription primarily occurs through the modulation of the two-component system (TCS) known as BvgAS. However, additional regulatory systems have emerged as potential contributors to virulence regulation. Here, we investigate the impact of BP1092, a putative TCS histidine kinase that shows increased levels after bacterial internalization by macrophages, on B. pertussis proteome adaptation under nonmodulating (Bvg+) and modulating (Bvg-) conditions. Using mass spectrometry, we compare B. pertussis wild-type (wt), a BP1092-deficient mutant (ΔBP1092), and a ΔBP1092 trans-complemented strain under both conditions. We find an altered abundance of 10 proteins, including five virulence factors. Specifically, under nonmodulating conditions, the mutant strain showed decreased levels of FhaB, FhaS, and Cya compared to the wt. Conversely, under modulating conditions, the mutant strain exhibited reduced levels of BvgA and BvgS compared to those of the wt. Functional assays further revealed that the deletion of BP1092 gene impaired B. pertussis ability to survive within human macrophage THP-1 cells. Taken together, our findings allow us to propose BP1092 as a novel player involved in the intricate regulation of B. pertussis virulence factors and thus in adaptation to the intracellular environment. The data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD041940.

BCL11B depletion induces the development of highly cytotoxic innate T cells out of IL-15 stimulated peripheral blood αß CD8+ T cells.

BCL11B, an essential transcription factor for thymopoiesis, regulates also vital processes in post-thymic lymphocytes. Increased expression of BCL11B was recently correlated with the maturation of NK cells, whereas reduced BCL11B levels were observed in native and induced T cell subsets displaying NK cell features. We show that BCL11B-depleted CD8+ T cells stimulated with IL-15 acquired remarkable innate characteristics. These induced innate CD8+ (iiT8) cells expressed multiple innate receptors like NKp30, CD161, and CD16 as well as factors regulating migration and tissue homing while maintaining their T cell phenotype. The iiT8 cells effectively killed leukemic cells spontaneously and neuroblastoma spheroids in the presence of a tumor-specific monoclonal antibody mediated by CD16 receptor activation. These iiT8 cells integrate the innate natural killer cell activity with adaptive T cell longevity, promising an interesting therapeutic potential. Our study demonstrates that innate T cells, albeit of limited clinical applicability given their low frequency, can be efficiently generated from peripheral blood and applied for adoptive transfer, CAR therapy, or combined with therapeutic antibodies.

Bordetella pertussis outer membrane vesicles as virulence factor vehicles that influence bacterial interaction with macrophages.

Gram-negative pathogenic bacteria constitutively shed outer membrane vesicles (OMVs) which play a significant role in the host-pathogen interaction, eventually determining the outcome of the infection. We previously found that Bordetella pertussis, the etiological agent of whooping cough, survives the innate interaction with human macrophages remaining alive inside these immune cells. Adenylate cyclase (CyaA), one of the main toxins of this pathogen, was found involved in the modulation of the macrophage defense response, eventually promoting bacterial survival within the cells. We here investigated whether B. pertussis OMVs, loaded with most of the bacterial toxins and CyaA among them, modulate the macrophage response to the bacterial infection. We observed that the pre-incubation of macrophages with OMVs led to a decreased macrophage defense response to the encounter with the bacteria, in a CyaA dependent way. Our results suggest that CyaA delivered by B. pertussis OMVs dampens macrophages protective function by decreasing phagocytosis and the bactericidal capability of these host cells. By increasing the chances of bacterial survival to the innate encounter with the macrophages, B. pertussis OMVs might play a relevant role in the course of infection, promoting bacterial persistence within the host and eventually, shaping the whole infection process.

Comparative analysis of ChAdOx1 nCoV-19 and Ad26.COV2.S SARS-CoV-2 vector vaccines.

Vector-based SARS-CoV-2 vaccines have been associated with vaccine- induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. A much smaller amount of impurities was found in Ad26.COV2.S. Platelet factor 4 formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S. These differences in impurities together with EDTAinduced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.

Insights in ChAdOx1 nCoV-19 vaccine-induced immune thrombotic thrombocytopenia.

SARS-CoV-2 vaccine ChAdOx1 nCoV-19 (AstraZeneca) causes a thromboembolic complication termed vaccine-induced immune thrombotic thrombocytopenia (VITT). Using biophysical techniques, mouse models, and analysis of VITT patient samples, we identified determinants of this vaccine-induced adverse reaction. Super-resolution microscopy visualized vaccine components forming antigenic complexes with platelet factor 4 (PF4) on platelet surfaces to which anti-PF4 antibodies obtained from VITT patients bound. PF4/vaccine complex formation was charge-driven and increased by addition of DNA. Proteomics identified substantial amounts of virus production-derived T-REx HEK293 proteins in the ethylenediaminetetraacetic acid (EDTA)-containing vaccine. Injected vaccine increased vascular leakage in mice, leading to systemic dissemination of vaccine components known to stimulate immune responses. Together, PF4/vaccine complex formation and the vaccine-stimulated proinflammatory milieu trigger a pronounced B-cell response that results in the formation of high-avidity anti-PF4 antibodies in VITT patients. The resulting high-titer anti-PF4 antibodies potently activated platelets in the presence of PF4 or DNA and polyphosphate polyanions. Anti-PF4 VITT patient antibodies also stimulated neutrophils to release neutrophil extracellular traps (NETs) in a platelet PF4-dependent manner. Biomarkers of procoagulant NETs were elevated in VITT patient serum, and NETs were visualized in abundance by immunohistochemistry in cerebral vein thrombi obtained from VITT patients. Together, vaccine-induced PF4/adenovirus aggregates and proinflammatory reactions stimulate pathologic anti-PF4 antibody production that drives thrombosis in VITT. The data support a 2-step mechanism underlying VITT that resembles the pathogenesis of (autoimmune) heparin-induced thrombocytopenia.

Major Determinants of Airway Epithelial Cell Sensitivity to S. aureus Alpha-Toxin: Disposal of Toxin Heptamers by Extracellular Vesicle Formation and Lysosomal Degradation.

Alpha-toxin is a major virulence factor of Staphylococcus aureus. Monomer binding to host cell membranes results in the formation of heptameric transmembrane pores. Among human model airway epithelial cell lines, A549 cells were most sensitive toward the toxin followed by 16HBE14o- and S9 cells. In this study we investigated the processes of internalization of pore-containing plasma membrane areas as well as potential pathways for heptamer degradation (lysosomal, proteasomal) or disposal (formation of exosomes/micro-vesicles). The abundance of toxin heptamers upon applying an alpha-toxin pulse to the cells declined both in extracts of whole cells and of cellular membranes of S9 cells, but not in those of 16HBE14o- or A549 cells. Comparisons of heptamer degradation rates under inhibition of lysosomal or proteasomal degradation revealed that an important route of heptamer degradation, at least in S9 cells, seems to be the lysosomal pathway, while proteasomal degradation appears to be irrelevant. Exosomes prepared from culture supernatants of toxin-exposed S9 cells contained alpha-toxin as well as low amounts of exosome and micro-vesicle markers. These results indicate that lysosomal degradation of internalized toxin heptamers may be the most important determinant of toxin-resistance of some types of airway epithelial cells.

Tuning the Mycobacterium tuberculosis Alternative Sigma Factor SigF through the Multidomain Regulator Rv1364c and Osmosensory Kinase Protein Kinase D.

Bacterial alternative sigma factors are mostly regulated by a partner-switching mechanism. Regulation of the virulence-associated alternative sigma factor SigF of Mycobacterium tuberculosis has been an area of intrigue, with SigF having more predicted regulators than other sigma factors in this organism. Rv1364c is one such predicted regulator, the mechanism of which is confounded by the presence of both anti-sigma factor and anti-sigma factor antagonist functions in a single polypeptide. Using protein binding and phosphorylation assays, we demonstrate that the anti-sigma factor domain of Rv1364c mediates autophosphorylation of its antagonist domain and binds efficiently to SigF. Furthermore, we identified a direct role for the osmosensor serine/threonine kinase PknD in regulating the SigF-Rv1364c interaction, adding to the current understanding about the intersection of these discrete signaling networks. Phosphorylation of SigF also showed functional implications in its DNA binding ability, which may help in activation of the regulon. In M. tuberculosis, osmotic stress-dependent induction of espA, a SigF target involved in maintaining cell wall integrity, is curtailed upon overexpression of Rv1364c, showing its role as an anti-SigF factor. Overexpression of Rv1364c led to induction of another target, pks6, involved in lipid metabolism. This induction was, however, curtailed in the presence of osmotic stress conditions, suggesting modulation of SigF target gene expression via Rv1364c. These data provide evidence that Rv1364c acts an independent SigF regulator that is sensitive to the osmosensory signal, mediating the cross talk of PknD with the SigF regulon.IMPORTANCEMycobacterium tuberculosis, capable of latently infecting the host and causing aggressive tissue damage during active tuberculosis, is endowed with a complex regulatory capacity built of several sigma factors, protein kinases, and phosphatases. These proteins regulate expression of genes that allow the bacteria to adapt to various host-derived stresses, like nutrient starvation, acidic pH, and hypoxia. The cross talk between these systems is not well understood. SigF is one such regulator of gene expression that helps M. tuberculosis to adapt to stresses and imparts virulence. This work provides evidence for its inhibition by the multidomain regulator Rv1364c and activation by the kinase PknD. The coexistence of negative and positive regulators of SigF in pathogenic bacteria reveals an underlying requirement for tight control of virulence factor expression.

Metaproteogenomic Profiling of Microbial Communities Colonizing Actively Venting Hydrothermal Chimneys.

At hydrothermal vent sites, chimneys consisting of sulfides, sulfates, and oxides are formed upon contact of reduced hydrothermal fluids with oxygenated seawater. The walls and surfaces of these chimneys are an important habitat for vent-associated microorganisms. We used community proteogenomics to investigate and compare the composition, metabolic potential and relative in situ protein abundance of microbial communities colonizing two actively venting hydrothermal chimneys from the Manus Basin back-arc spreading center (Papua New Guinea). We identified overlaps in the in situ functional profiles of both chimneys, despite differences in microbial community composition and venting regime. Carbon fixation on both chimneys seems to have been primarily mediated through the reverse tricarboxylic acid cycle and fueled by sulfur-oxidation, while the abundant metabolic potential for hydrogen oxidation and carbon fixation via the Calvin-Benson-Bassham cycle was hardly utilized. Notably, the highly diverse microbial community colonizing the analyzed black smoker chimney had a highly redundant metabolic potential. In contrast, the considerably less diverse community colonizing the diffusely venting chimney displayed a higher metabolic versatility. An increased diversity on the phylogenetic level is thus not directly linked to an increased metabolic diversity in microbial communities that colonize hydrothermal chimneys.