Federico Iovino

Bacterial meningitis is a life‐threatening neurological disorder frequently caused by a Streptococcus pneumoniae (the pneumococcus) infection of the brain. Standard treatment consists of antibiotics to eliminate bacteria and dexamethasone to reduce inflammation. Despite this, mortality reaches 20% in treated individuals, and half of the survivors suffer long‐term neurological sequelae. This is largely due to the poor capacity of antibiotics to reach the brain and the lack of antimicrobial treatment capable of neutralizing the pneumococcal toxin pneumolysin (Ply). To address these limitations, we isolated extracellular vesicles (EVs) derived from human HEK293T cells and evaluated their therapeutic potential in pneumococcal meningitis. Alongside wild‐type EVs (WT.EVs), we bioengineered EVs to express RVG peptides (RVG.EV) for targeting neuronal acetylcholine receptors, signal incompetent IL‐6 signal transducer (IL‐6ST) decoy receptors (IL‐6.EV) to block the pro‐inflammatory signalling of IL‐6, or EVs expressing both RVG peptides and IL‐6ST (DB.EV). In vitro , all EVs reduced pneumococcal adhesion to neurons and mitigated cytotoxicity by binding and sequestering Ply. In a bacteremia‐derived pneumococcal meningitis model, EV treatment significantly increased the survival of the mice without affecting bacterial load in the brain or the periphery. Among all groups, RVG.EV treatment was most effective in reducing pro‐inflammatory cytokine release in the periphery and brain. These findings highlight the therapeutic potential of bioengineered EVs, particularly RVG peptides expressing EVs, as an adjunctive treatment for pneumococcal meningitis thanks to their (i) sequestration and neutralization of Ply, (ii) increased blood‐brain barrier crossing, and (iii) dampening of inflammation.

Streptococcus pneumoniae (pneumococcus) is a leading cause of bacterial meningitis worldwide and is associated with cerebrovascular complications and long-term neurological sequelae. These outcomes are largely driven by an excessive yet ineffective neuroinflammatory response. Although pneumococci express multiple virulence factors that enable immune evasion, how these factors modulate phagocytic responses in the central nervous system remains unclear. Here, we identify neuraminidase A (NanA) as a critical regulator of phagocytic activation in microglia and macrophages. Using murine and human microglia and RAW 264.7 macrophages, we show that NanA deficiency increases bacterial adhesion, indicating enhanced immune recognition, but paradoxically promotes intracellular bacterial survival. Despite elevated expression of the phagocytic marker CD68, microglia infected with NanA-deficient pneumococci fail to efficiently eliminate intracellular bacteria, as evidenced by unchanged levels of the lysosomal enzyme cathepsin 3 compared with NanA-expressing strains. Treatment with recombinant NanA restored phagocytic activation and significantly enhanced clearance of NanA-deficient bacteria. This effect was dependent on NanA sialidase activity, as enzymatically inactive NanA failed to restore immune sensing. Notably, this immunomodulatory function was specific to the secreted form of NanA produced by the invasive TIGR4 strain and was not observed with membrane-anchored NanA from D39-derived mutants. Together, these findings reveal an unexpected role for NanA in promoting effective innate immune activation and bacterial clearance, highlighting its potential as a therapeutic target for pneumococcal meningitis and invasive pneumococcal disease.

Streptococcus pneumoniae , the pneumococcus, is a cause of major illness globally. Invasive pneumococcal disease (IPD) is characterized by pneumococci invading blood (bacteremia), lungs (pneumonia), or brain and cerebrospinal fluid (meningitis). Meningitis remains an important global health concern because half of the survivors experience long-term neurological damage. The antibiotics commonly used to treat pneumococcal infections are β-lactams and macrolides, however, S. pneumoniae is nowadays often resistant to one or several antibiotics, therefore novel antimicrobials are needed. Here, we found that the bacteriophage-derived cpl-1 endolysin showed consistent antibacterial activity against β-lactam- and macrolide-resistant pneumococcal clinical strains grown in human blood and human cerebrospinal fluid. Exploiting synergistic and additive mechanisms, supplementation of cpl-1 to either penicillin or erythromycin, as representatives for β-lactam and macrolide antibiotics, rescued human neuronal cells from the cytotoxicity of antibiotic-resistant pneumococcal infections. Finally, systemic administration of cpl-1 supplemented to penicillin in mice infected with penicillin-resistant pneumococci successfully reduced bacteremia, and, thanks to the efficient penetration across the blood–brain barrier, abolished bacterial load in the brain, resulting in increased (89%) survival accompanied by an asymptomatic course of infection. These findings strongly suggest that cpl-1 can enhance antibiotic susceptibility in β-lactam- and macrolide-resistant S. pneumoniae , serving as a valuable adjunct therapy to standard-of-care antibiotics for multidrug-resistant IPD.

Streptococcus pneumoniae , the pneumococcus, is a cause of major illness globally. Invasive pneumococcal disease (IPD) is characterized by pneumococci invading blood (bacteremia), lungs (pneumonia), or brain and cerebrospinal fluid (meningitis). Meningitis remains an important global health concern because half of the survivors experience long-term neurological damage. The antibiotics commonly used to treat pneumococcal infections are β-lactams and macrolides, however, S. pneumoniae is nowadays often resistant to one or several antibiotics, therefore novel antimicrobials are needed. Here, we found that the bacteriophage-derived Cpl-1 endolysin showed consistent antibacterial activity against β-lactam– and macrolide-resistant pneumococcal clinical strains grown in human blood and human cerebrospinal fluid. Exploiting synergistic and additive mechanisms, supplementation of cpl-1 to either penicillin or erythromycin rescued human neuronal cells from the cytotoxicity of antibiotic-resistant pneumococcal infections. Finally, systemic administration of cpl-1 supplemented to penicillin in mice infected with penicillin-resistant pneumococci successfully reduced bacteremia, and, thanks to the efficient penetration across the blood-brain barrier, abolished bacterial load in the brain, resulting in increased (89%) survival accompanied by an asymptomatic course of infection. These findings strongly indicate that cpl-1 can restore antibiotic sensitivity against β-lactam– and macrolide-resistant S. pneumoniae , representing a fundamental adjunct therapy to standard-of-care antibiotics against multidrug-resistant IPD.

Summary: Streptococcus pneumoniae (the pneumococcus) is the major cause of bacterial meningitis globally, and pneumococcal meningitis is associated with increased risk of long-term neurological sequelae. These include several sensorimotor functions that are controlled by specific brain regions which, during bacterial meningitis, are damaged by the vast neuroinflammation and bacterial toxins. Little is known about the invasion pattern of the pneumococcus into the brain. Using a bacteremia-derived meningitis mouse model, we combined 3D whole brain imaging with brain microdissection to show that all brain regions were equally affected during disease progression, with pneumococci in close association to the microvasculature. In the hippocampus, the invasion provoked a dynamic microglial response, while the dentate gyrus showed a significant loss of neuroblasts. Our results indicate that, even before symptom occur, the bacterial load throughout the brain causes neuroinflammation and cell death, a pathological scenario which ultimately leads to a failing regeneration of new neurons.

The lack of solute drainage due to a dysfunctional glymphatic system leads to an increase of the neurotoxic bacterial material in the CSF compartments of the brain, ultimately leading to brain-wide neuroinflammation and neuronal damage with consequent impairment of neurological functions. The loss of function of the glymphatic system can therefore be a leading cause of the neurological sequelae developing post-bacterial meningitis.

Background: Pneumococcal meningitis, inflammation of the meninges due to an infection of the Central Nervous System caused by Streptococcus pneumoniae (the pneumococcus), is the most common form of community-acquired bacterial meningitis globally. The brain is separated from the systemic circulation by the blood-brain barrier (BBB), and meningitis triggers the host immune response increasing the BBB permeability, allowing peripheral immune cells to reach the cerebrospinal fluid (CSF), and increasing debris production. The glymphatic system is a glial-dependent clearance pathway that drives the exchange of compounds between the brain parenchyma and the CSF regulating the waste clearance away from the brain. Aquaporin-4 (AQP4)-water channels on astrocytic end feet regulate the solute transport of the glymphatic system. Methods: Wistar rats, either subjected to pneumococcal meningitis or to artificial-CSF (sham), received Evans blue albumin (EBA) intracisternal. Overall, the meningitis group presented a significant impairment of the glymphatic system by retaining the EBA in the brain without consistently releasing the EBA into the bloodstream compared to the sham non-infected group. Through western blot and immunofluorescence microscopy analysis using rat CSF and brain tissue sections, an increased accumulation of pneumococci was detected over time in the CSF, and because of a loss of drainage between CSF and brain interstitial space, such bacterial accumulation was not observed in the brain parenchyma. Western blot analysis for Iba1, TMEM119 and IFN-Ɣ in rat brain homogenates and NSE in serum showed increased neuroinflammation and neuronal damage in the brain over time during pneumococcal infection. Neurological impairment upon neuronal cell damage caused by meningitis with a malfunctioning glymphatic system was also demonstrated through open-field behavioral tests comparing rats from sham and meningitis groups. Lastly, protein expression analysis of AQP4 revealed no differences in AQP4 between the brains of the rats from the meningitis group and those from the sham non-infected rats. Importantly, confocal microscopy analysis showed a detachment of the astrocytic end feet from the BBB vascular endothelium with consequent misplacement of AQP4-water channels. Conclusions: These findings clearly indicate that pneumococcal meningitis decreases the glymphatic system’s functionality, increasing the neurotoxic waste debris in the brain ultimately leading to brain-wide neuroinflammation and neuronal damage. Finally, our results clearly showed that during pneumococcal meningitis, the glymphatic system does not function because of a detachment of the astrocytic end feet from the BBB vascular endothelium, which leads to a misplacement of AQP4 with consequent the loss of the AQP4-water channel’s functionality.

Editorial: Host-Pathogen Interaction in the Central Nervous System