Lysosomal permeabilization by Group A Streptococcus releases proteins into the macrophage cytosol 2256825

MARCO is an IFN-restricted immunometabolic decoy LPS receptor 2308885

Cytosolic sensing of bacterial lipopolysaccharide (LPS) is mediated by caspase-11 (caspase-4/5 in humans), which triggers non-canonical inflammasome activation. This leads to gasdermin D cleavage, pyroptosis, and release of pro-inflammatory cytokines. Severe gram-negative bacterial infections cause endotoxic septic shock, with systemic inflammation, organ failure, and potential death. Bacterial derived LPS gains access to the cytosol through extracellular vesicles and CD14 receptor mediated endocytosis. However, how this process is controlled during infection to maintain homeostasis is unknown. We performed an unbiased proteomic screen with biotinylated LPS and identified MARCO as an LPS-binding protein by mass spectrometry. Using macrophages from transgenic mice, we defined the mechanism by which MARCO facilitates LPS clearance to limit activation of the non-canonical inflammasome. MARCO is a scavenger receptor expressed on tissue resident macrophages (TRMs) and is known to promote clearance of pathogens and apoptotic debris. However, there is a lack of genetic studies and a paucity in our understanding of the functional role MARCO plays in innate immunity and how MARCO expression is regulated on TRMs. Here, we show that activation of the non-canonical inflammasome is preceded by a priming step by which type I interferon (IFN) suppresses MARCO expression on TRMs to allow for optimal recognition of LPS and activation of caspase-11. Remarkably, the metabolite itaconate and transcription factor NRF2 mediate MARCO expression. IFN signaling inhibits NRF2 stabilization, reducing surface MARCO levels. In vivo, MARCO-deficient mice show increased sensitivity to LPS-induced septic shock due to enhanced caspase-11 and non-canonical inflammasome activation. IFN-mediated suppression of MARCO expression is a previously unknown innate checkpoint that facilitates the recognition of LPS by caspase-11, activation of the non-canonical inflammasome, and progression of septic shock. n/a Innate Immune Responses and Host Defense: Molecular Mechanisms (INM)

Surface-associated phosphoglycerate kinase (PGK) of Staphylococcus aureus binds human plasminogen in a partially lysine-independent manner.

Findings suggest that PGK may function as a moonlighting protein, playing an essential role in the interaction of S. aureus with its host through plasminogen binding.

Exploring the role of complement in extracellular vesicle production: Insights into host-pathogen interactions 2258111

Increased intravascular coagulation is a molecular hallmark of sepsis and we showed that extracellular vesicles (EVs) from human neutrophils challenged with bacteria activate the coagulation cascade. Discovery of the EV biogenesis pathway may lead to the ability to interfere with EV production and reduce coagulation. To investigate the mechanism of EV biogenesis, we performed proteomics on three EV subsets: those produced spontaneously, in response to fMLF, and after staphylococcal challenge. After protein abundances were normalized, we compared fold changes between subsets. We validated the proteomic results with immunoblotting. The role of nSmase was evaluated using GW4869, an nSmase inhibitor. Although the production of some neutrophil EV subsets relies on membrane curvature caused by neutral sphingomyelinase (nSmase), the production of EVs in response to staphylococcal challenge was nSmase-independent. Annexins and proteins involved in the formation of the membrane attack complex (C5b-9) were enriched in EVs produced after staphylococcal challenge. Based on these results, we propose that complement-mediated bystander activation produces membrane damage, which initiates Annexin-mediated repair and EV production. To test this, we opsonized bacteria with C9-sufficient or deficient serum, and measured production of EVs by monitoring Flotillin-1. In the absence of C9, less Flotillin-1 was observed compared to the control. These data suggest a role for complement in EV production. The Frost Fund in the Cal Poly Bailey College of Science and Mathematics Innate Immune Responses and Host Defense: Cellular Mechanisms (INC)

Extracellular pneumolysin enhances the activation of cytosolic pattern-recognition receptor NOD2 through its pore-forming activity

ABSTRACT Streptococcus pneumoniae is the leading cause of community-acquired pneumonia, meningitis, and sepsis. This bacterium produces pneumolysin, a cholesterol-dependent cytolysin that forms oligomeric transmembrane pores in the host cells. Although pneumolysin is known to exhibit proinflammatory properties, the mechanisms by which it activates innate immune responses remain to be investigated. Here, we show that extracellular pneumolysin enhances the activation of nucleotide-binding oligomerization domain 2 (NOD2), a cytosolic receptor that recognizes bacterial peptidoglycans. Experiments using HEK-Blue cell lines expressing specific pattern recognition receptors revealed that pneumolysin does not directly activate Toll-like receptors or other innate immune receptors. In contrast, our findings suggest that pneumolysin-generated membrane pores may facilitate the cytosolic entry of peptidoglycan, thereby contributing to an elevated activation of NOD2. Furthermore, pneumolysin enhances NOD1 activation in cells stimulated with a NOD1 ligand, suggesting a broader role for pore-forming toxins in innate immunity. These findings shed light on S. pneumoniae-modulated immune modulation and highlight toxin-induced immune pathways as potential therapeutic targets. IMPORTANCE The mechanisms by which the pneumococcal pore-forming toxin pneumolysin activates innate immune responses have not been fully understood. Specifically, it remains unclear whether pneumolysin is directly sensed by Toll-like receptor 4 (TLR4) or activates the NLRP3 inflammasome. Here, we show that pneumolysin is not a direct ligand for pattern-recognition receptors. Instead, pneumolysin forms membrane pores that increase plasma membrane permeabilization, thereby amplifying innate immune signaling through multiple pathways. These pores may provide a route for the cytosolic entry of pneumococcal peptidoglycan, contributing to enhanced activation of the cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2). Additionally, membrane pores may promote the extracellular release of damage-associated molecular patterns, such as high mobility group box 1 (HMGB1), providing a mechanistic explanation for previously reported pneumolysin-induced TLR4 activation. Furthermore, pore-induced ion efflux provides a framework to explain previously reported NLRP3 inflammasome activation. Together, our findings establish membrane permeabilization as a central mechanism by which pneumolysin modulates innate immune sensing during pneumococcal infection. The mechanisms by which the pneumococcal pore-forming toxin pneumolysin activates innate immune responses have not been fully understood. Specifically, it remains unclear whether pneumolysin is directly sensed by Toll-like receptor 4 (TLR4) or activates the NLRP3 inflammasome. Here, we show that pneumolysin is not a direct ligand for pattern-recognition receptors. Instead, pneumolysin forms membrane pores that increase plasma membrane permeabilization, thereby amplifying innate immune signaling through multiple pathways. These pores may provide a route for the cytosolic entry of pneumococcal peptidoglycan, contributing to enhanced activation of the cytosolic receptor nucleotide-binding oligomerization domain 2 (NOD2). Additionally, membrane pores may promote the extracellular release of damage-associated molecular patterns, such as high mobility group box 1 (HMGB1), providing a mechanistic explanation for previously reported pneumolysin-induced TLR4 activation. Furthermore, pore-induced ion efflux provides a framework to explain previously reported NLRP3 inflammasome activation. Together, our findings establish membrane permeabilization as a central mechanism by which pneumolysin modulates innate immune sensing during pneumococcal infection.

TARGETING THE CAPSULE OF KLEBSIELLA PNEUMONIAE WITH A CATIONIC CR3-BINDING PROTEIN ENHANCES PHAGOCYTOSIS AND PROMOTES BACTERIAL CLEARANCE AND SURVIVAL IN A MOUSE SEPSIS MODEL

Platelet Factor 4 (PF4), a cationic antimicrobial peptide, serves as a ligand for the myeloid-specific phagocytic receptor CR3 (Mac-1, CD11b/CD18). We previously demonstrated that recombinant dimeric PF4 (rdPF4) functions as a bacterial opsonin, enhancing phagocytosis of Gram-positive Staphylococcus aureus and facilitating clearance of both antibiotic-susceptible and methicillin-resistant S. aureus in a mouse model of infectious peritonitis. In this study, we examined whether rdPF4 is pathogen-agnostic by assessing its effect on phagocytosis of Gram-negative encapsulated Klebsiella pneumoniae, a WHO Bacterial Priority Pathogen. We demonstrate that rdPF4 enhances CR3-mediated phagocytosis of both live and heat-inactivated high-virulence K2 and low-virulence K3 strains of K. pneumoniae by various mouse and human macrophage cell lines, as well as primary neutrophils and macrophages. It also increased phagocytosis of carbapenem-resistant K. pneumoniae. rdPF4 did not directly kill bacteria but acted as an opsonin binding to the negatively charged bacterial capsule and creating recognition sites for CR3 on leukocytes. In a mouse sepsis model, a single dose of rdPF4 significantly enhanced bacterial clearance from the lungs, liver, and peritoneum and reduced bacteremia. Histological analyses showed that rdPF4 provided substantial protection to lung and liver tissues against K. pneumoniae-induced damage. Consistent with these findings, rdPF4 treatment increased the survival rates of infected mice. These results show that rdPF4 effectively targets the capsule, a key virulence factor of K. pneumoniae, thereby reducing the bacterium’s ability to evade the host immune response. Overall, the data suggest a common mechanism in which cationic rdPF4, by binding to the negatively charged surfaces of both Gram-negative and Gram-positive bacteria, diminishes their antiphagocytic properties.

Leishmanial GP63 acts as a protease for the small pore forming toxin aerolysin