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Importance: Antibiotic resistance and tolerance are substantial healthcare-related problems, hampering effective treatment of bacterial infections. Mutations in the phosphodiesterase GdpP, which degrades cyclic di-3', 5'-adenosine monophosphate (c-di-AMP), have recently been associated with resistance to beta-lactam antibiotics in clinical Staphylococcus aureus isolates. In this study, we show that high c-di-AMP levels decreased the cell size and increased the cell wall thickness in S. aureus mutant strains. As a consequence, an increase in resistance to cell wall targeting antibiotics, such as oxacillin and fosfomycin as well as in tolerance to ceftaroline, a cephalosporine used to treat methicillin-resistant S. aureus infections, was observed. These findings underline the importance of investigating the role of c-di-AMP in the development of tolerance and resistance to antibiotics in order to optimize treatment in the clinical setting., Competing Interests: The authors declare no conflict of interest.

Staphylococcus aureus can cause infections that are often chronic and difficult to treat, even when the bacteria are not antibiotic resistant because most antibiotics act only on metabolically active cells. Subpopulations of persister cells are metabolically quiescent, a state associated with delayed growth, reduced protein synthesis, and increased tolerance to antibiotics. Serine-threonine kinases and phosphatases similar to those found in eukaryotes can fine-tune essential bacterial cellular processes, such as metabolism and stress signaling. We found that acid stress-mimicking conditions that S. aureus experiences in host tissues delayed growth, globally altered the serine and threonine phosphoproteome, and increased threonine phosphorylation of the activation loop of the serine-threonine protein kinase B (PknB). The deletion of stp , which encodes the only annotated functional serine-threonine phosphatase in S. aureus , increased the growth delay and phenotypic heterogeneity under different stress challenges, including growth in acidic conditions, the intracellular milieu of human cells, and abscesses in mice. This growth delay was associated with reduced protein translation and intracellular ATP concentrations and increased antibiotic tolerance. Using phosphopeptide enrichment and mass spectrometry-based proteomics, we identified targets of serine-threonine phosphorylation that may regulate bacterial growth and metabolism. Together, our findings highlight the importance of phosphoregulation in mediating bacterial quiescence and antibiotic tolerance and suggest that targeting PknB or Stp might offer a future therapeutic strategy to prevent persister formation during S. aureus infections.

Regulated cell death (RCD) and the concomitant release of extracellular traps by neutrophils (NETs) constitute an important antibacterial effector response. Usually, the dynamic processes of RCD and NETs release are assessed independently of each other by either unspecific or time-consuming methods. Here, we describe a flow cytometry-based high-throughput analysis method incorporating neutrophil RCD and NETs release with visual live-imaging conformation upon ex vivo bacterial challenge. This combined approach allows to quantify and closely follow the kinetics of the dynamic neutrophil effector response towards bacterial infection., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

COVID-19 displays diverse disease severities and symptoms including acute systemic inflammation and hypercytokinemia, with subsequent dysregulation of immune cells. Bacterial superinfections in COVID-19 can further complicate the disease course and are associated with increased mortality. However, there is limited understanding of how SARS-CoV-2 pathogenesis and hypercytokinemia impede the innate immune function against bacterial superinfections. We assessed the influence of COVID-19 plasma hypercytokinemia on the functional responses of myeloid immune cells upon bacterial challenges from acute-phase COVID-19 patients and their corresponding recovery-phase. We show that a severe hypercytokinemia status in COVID-19 patients correlates with the development of bacterial superinfections. Neutrophils and monocytes derived from COVID-19 patients in their acute-phase showed an impaired intracellular microbicidal capacity upon bacterial challenges. The impaired microbicidal capacity was reflected by abrogated MPO and reduced NETs production in neutrophils along with reduced ROS production in both neutrophils and monocytes. Moreover, we observed a distinct pattern of cell surface receptor expression on both neutrophils and monocytes, in line with suppressed autocrine and paracrine cytokine signaling. This phenotype was characterized by a high expression of CD66b, CXCR4 and low expression of CXCR1, CXCR2 and CD15 in neutrophils and low expression of HLA-DR, CD86 and high expression of CD163 and CD11b in monocytes. Furthermore, the impaired antibacterial effector function was mediated by synergistic effect of the cytokines TNF-α, IFN-γ and IL-4. COVID-19 patients receiving dexamethasone showed a significant reduction of overall inflammatory markers in the plasma as well as exhibited an enhanced immune response towards bacterial challenge ex vivo. Finally, broad anti-inflammatory treatment was associated with a reduction in CRP, IL-6 levels as well as length of ICU stay and ventilation-days in critically ill COVID-19 patients. Our data provides insights into the transient functional dysregulation of myeloid immune cells against subsequent bacterial infections in COVID-19 patients and describe a beneficial role for the use of dexamethasone in these patients., Competing Interests: The authors have declared that no competing interests exist.

Objectives: Critically ill coronavirus disease 2019 (COVID-19) patients are characterised by a severely dysregulated cytokine profile and elevated neutrophil counts, impacting disease severity. However, it remains unclear how neutrophils contribute to pathophysiology during COVID-19. Here, we assessed the impact of the dysregulated cytokine profile on the regulated cell death (RCD) programme of neutrophils., Methods: Regulated cell death phenotype of neutrophils isolated from critically ill COVID-19 patients or healthy donors and stimulated with COVID-19 or healthy plasma ex vivo was assessed by flow cytometry, time-lapse microscopy and cytokine multiplex analysis. Immunohistochemistry of COVID-19 patients and control biopsies were performed to assess the in situ neutrophil RCD phenotype. Plasma cytokine levels of COVID-19 patients and healthy donors were measured by multiplex analysis. Clinical parameters were correlated to cytokine levels of COVID-19 patients., Results: COVID-19 plasma induced a necroptosis-sensitive neutrophil phenotype, characterised by cell lysis, elevated release of damage-associated molecular patterns (DAMPs), increased receptor-interacting serine/threonine-protein kinase (RIPK) 1 levels and mixed lineage kinase domain-like pseudokinase (MLKL) involvement. The occurrence of neutrophil necroptosis MLKL axis was further confirmed in COVID-19 thrombus and lung biopsies. Necroptosis was induced by the tumor necrosis factor receptor 1 (TNFRI)/TNF-α axis. Moreover, reduction of soluble Fas ligand (sFasL) levels in COVID-19 patients and hence decreased signalling to Fas directly increased RIPK1 levels, exacerbated TNF-driven necroptosis and correlated with disease severity, which was abolished in patients treated with glucocorticoids., Conclusion: Our results suggest a novel role for sFasL signalling in the TNF-α-induced RCD programme in neutrophils during COVID-19 and a potential therapeutic target to curb inflammation and thus influence disease severity and outcome., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Clinical & Translational Immunology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

Antibody-mediated opsonic phagocytosis (OP) of Plasmodium falciparum blood-stage merozoites has been associated with protection against malaria. However, the precise contribution of different peripheral blood phagocytes in the OP mechanism remains unknown. Here, we developed an in vitro OP assay using peripheral blood leukocytes that allowed us to quantify the contribution of each phagocytic cell type in the OP of merozoites. We found that CD14  +   + CD16 - monocytes were the dominant phagocytic cells at very low antibody levels and Fc gamma receptor (FcγR) IIA plays a key role. At higher antibody levels however, neutrophils were the main phagocytes in the OP of merozoites with FcγRIIIB acting synergistically with FcγRIIA in the process. We found that OP activity by neutrophils was strongly associated with protection against febrile malaria in longitudinal cohort studies performed in Ghana and India. Our results demonstrate that peripheral blood neutrophils are the main phagocytes of P. falciparum blood-stage merozoites., (© 2021. The Author(s).)