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Streptococcus pneumoniae is the most common cause of severe bacterial meningitis in children, the elderly, and immunocompromised individuals. To identify virulence factors preferentially expressed during meningitis, we conducted niche-specific genome-wide in vivo transcriptomic analysis after intranasal infection of mice with serotype 4 or 6A pneumococci. The expression of 34 bacterial genes was substantially altered in brain tissue of mice infected with either of the 2 strains. Ten upregulated genes were common to both strains, 7 of which were evaluated for their role in the development of meningitis. One previously uncharacterized protein, α-glycerophosphate oxidase (GlpO), was cytotoxic for human brain microvascular endothelial cells (HBMECs) via generation of H(2)O(2). A glpO deletion mutant was defective in adherence to HBMECs in vitro as well as in progression from the blood to the brain in vivo. Mutant bacteria also induced markedly reduced meningeal inflammation and brain pathology compared with wild type, despite similar levels of bacteremia. Immunization of mice with GlpO protected against invasive pneumococcal disease and provided additive protection when formulated with pneumolysin toxoid. Our results provide the basis of a strategy that can be adapted to identify genes that contribute to the development of meningitis caused by other pathogens.

B7-H3, a new member of the B7 superfamily, plays a key role in the regulation of T cell-mediated immune responses. Our previous work showed that B7-H3 strongly augmented both LPS- and bacterial lipoprotein-induced NF-κB activation and inflammatory response, and soluble B7-H3 was elevated in CSF and plasma of patients with bacterial meningitis. MMP-9 has been implicated in blood–brain barrier disruption, inflammation, and vasculitis during the pathogenesis of bacterial meningitis. In this study, we report that in a murine model of pneumococcal meningitis, B7-H3 treatment enhances inflammatory response in the meninges, upregulates MMP-9 expression in cerebral parenchyma, and deteriorates clinical disease status indicated by weight loss and impaired movement ability. In vitro results showed that B7-H3 augmented MMP-9 secretion from Streptococcus pneumoniae -stimulated microglia cells. Thus, our data indicate that B7-H3 contributes to the development of pneumococcal meningitis by exaggerating inflammatory responses and upregulating MMP-9 activity in CNS, which ultimately lead to neuronal injury.

In experimental bacterial meningitis, matrix metalloproteinases (MMPs) and reactive oxygen species (ROS) contribute to brain damage. MMP-9 increases in cerebrospinal fluid (CSF) during bacterial meningitis and is associated with the brain damage that is a consequence of the disease. This study assesses the origin of MMP-9 in bacterial meningitis and how ROS modulate its activity. Rat brain-slice cultures and rat polymorphonuclear cells (PMNs) that had been challenged with capsule-deficient heat-inactivated Streptococcus pneumoniae R6 (hiR6) released MMP-9. Coincubation with either catalase, with the myeloperoxidase inhibitor azide, or with the hypochlorous acid scavenger methionine almost completely prevented activation, but not the release, of MMP-9, in supernatants of human PMNs stimulated with hiR6. Thus, in bacterial meningitis, both brain-resident cells and invading PMNs may act as sources of MMP-9, and stimulated PMNs may activate MMP-9 via an ROS-dependent pathway. MMP-9 activation by ROS may represent a target for therapeutic intervention in bacterial meningitis

Bacterial meningitis causes neuronal apoptosis in the hippocampal dentate gyrus, which is associated with learning and memory impairments after cured disease. The execution of the apoptotic program involves pathways that converge on activation of caspase-3, which is required for morphological changes associated with apoptosis. Here, the time course and the role of caspase-3 in neuronal apoptosis was assessed in an infant rat model of pneumococcal meningitis. During clinically asymptotic meningitis (0-12 h after infection), only minor apoptotic damage to the dentate gyrus was observed, while the acute phase (18-24 h) was characterized by a massive increase of apoptotic cells, which peaked at 36 h. In the subacute phase of the disease (36-72 h), the number of apoptotic cells decreased to control levels. Enzymatic caspase-3 activity was significantly increased in hippocampal tissue of infected animals compared to controls at 22 h. The activated enzyme was localized to immature cells of the dentate gyrus, and in vivo activity was evidenced by cleavage of the amyloid-beta precursor protein. Intracisternal administration of the caspase-3-specific inhibitor Ac-DEVD-CHO significantly reduced apoptosis in the hippocampal dentate gyrus. In contrast to a study where the decrease of hippocampal apoptosis after administration of a pan-caspase inhibitor was due to downmodulation of the inflammatory response, our data demonstrate that specific inhibition of caspase-3 did not affect inflammation assessed by TNF-alpha and IL-1beta concentrations in the cerebrospinal fluid space. Taken together, the present results identify caspase-3 as a key effector of neuronal apoptosis in pneumococcal meningitis.

The aim of the study was to determine the differential expression of nitric oxide (NO) synthase (NOS) isoforms and the pathophysiologic relevance of inducible NOS (iNOS) in experimental pneumococcal meningitis. By use of reverse transcription–polymerase chain reaction analysis, immunohistochemistry, and Western blotting, increased brain mRNA and increased protein levels of endothelial NOS (eNOS) and iNOS were detected 24 h after intracisternal pneumococcal inoculation. In iNOS-deficient mice, disruption of the blood-brain barrier (BBB) was significantly reduced, compared with that in wild-type mice. This beneficial effect of iNOS deficiency was associated with a lack of nitrotyrosine immunoreactivity. Furthermore, brain protein levels of interleukin (IL)–1β, IL-6, and tumor necrosis factor–α and brain mRNA levels of macrophage inflammatory protein (MIP)–1α and MIP-2 were significantly reduced in infected animals lacking iNOS. These findings suggest that (1) not only iNOS but also eNOS is up-regulated in the acute phase of experimental bacterial meningitis, and (2) iNOS-derived NO contributes to peroxynitrite formation and BBB breaching in this disease

Bacterial meningitis is associated with a high mortality and a high incidence of neurological sequelae. Parainfectious vasculitis leading to ischemic brain damage is a known complication of bacterial meningitis but its treatment is uncertain. We report the case of a 53-year-old man with pneumococcal meningitis who developed numerous ischemic lesions in the brainstem and basal ganglia caused by parainfectious vasculitis. Clinical and radiological improvement was observed after delayed corticosteroid initiation. Symptomatic vasculitis relapsed after steroid withdrawal and stabilized after reintroduction of the immunosuppressive therapy. Although the cerebrospinal fluid (CSF) contained high levels of MMP-9 at the time of symptomatic vasculitis, a significant decrease of the enzyme accompanied the introduction of corticotherapy and the regression of vasculitic symptoms. No relation between the level of MMP-9 and the white blood cell count in CSF could be found. Parainfectious vasculitis may respond to late corticosteroid treatment. MMP-9 level in CSF may be a marker of vasculitic complication in bacterial meningitis.

The present study assessed the role of PARP [poly(adenosine diphosphate-ribose) polymerase] activation in experimental pneumococcal meningitis. Mice with a targeted disruption of the PARP1 gene were protected against meningitis-associated central nervous system complications including blood-brain barrier breaching and increase in intracranial pressure. This beneficial effect was paralleled by a significant reduction in meningeal inflammation, as evidenced by significantly lower cerebrospinal fluid leukocyte counts and interleukin-1β, −6, and tumor necrosis factor-α concentrations in the brain (compared with infected wild-type mice). The reduction in inflammation and central nervous system complications was associated with an improved clinical status of infected, PARP1-deficient mice. A similar protective effect was achieved by PARP inhibition using 3-aminobenzamide, the pharmacologic efficacy of which was confirmed by a marked attenuation of meningitis-induced poly(ADP)ribose formation. When the rat brain-derived endothelial cell line GP8.3 was cocultured with macrophages, exposure to pneumococci induced endothelial cell death and was paralleled by PARP activation and a reduction in the oxidized form of cellular nicotinamide adenine dinucleotide content. Treatment with 3-aminobenzamide significantly attenuated cellular nicotinamide adenine dinucleotide depletion and pneumococci-induced cytotoxicity. Thus, PARP activation seems to play a crucial role in the development of meningitis-associated central nervous system complications and pneumococci-induced endothelial injury. Inhibitors of PARP activation could provide a potential therapy of acute bacterial meningitis.

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.

The costimulatory protein B7-H3 has been shown to play a contributory role in the development and progression of experimental pneumococcal meningitis by augmentation of the innate immunity-associated inflammatory response via a TLR2-dependent manner. This study aimed to clarify the component(s) of TLR2-mediated signal transduction pathways responsible for B7-H3-augmented inflammatory response and subsequent brain damage during experimental pneumococcal meningitis. Administration of B7-H3 did not augment expression of TLR2 and other TLR2 upstream components, but led to an enhanced formation of MyD88-IRAK immunocomplex in the brain of S. pneumoniae-infected mice. Furthermore, B7-H3 substantially augmented S. pneumoniae-induced activation of TLR2 downstream NF-κB p65 and MAPK p38 pathways in the brain of S. pneumoniae-infected mice. Notably, blockage of NF-κB p65 and/or MAPK p38 with their specific inhibitors strongly attenuated B7-H3-amplified inflammatory response with significantly reduced proinflammatory cytokine and chemokine production, and markedly ameliorated B7-H3-exacerbated disruption of blood-brain barrier and severity of disease status in S. pneumoniae-infected mice. These results indicate that targeting NF-κB p65 and/or MAPK p38 may represent a promising therapeutic option for amelioration of overwhelming inflammatory response-associated brain injury frequently observed during pneumococcal meningitis.

β-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of β-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking β-lactam resistance could benefit disease surveillance. Here we developed a classification system in which a pneumococcal isolate is assigned to a “PBP type” based on sequence signatures in the transpeptidase domains (TPDs) of the three critical penicillin-binding proteins (PBPs), PBP1a, PBP2b, and PBP2x. We identified 307 unique PBP types from 2,528 invasive pneumococcal isolates, which had known MICs to six β-lactams based on broth microdilution. We found that increased β-lactam MICs strongly correlated with PBP types containing divergent TPD sequences. The PBP type explained 94 to 99% of variation in MICs both before and after accounting for genomic backgrounds defined by multilocus sequence typing, indicating that genomic backgrounds made little independent contribution to β-lactam MICs at the population level. We further developed and evaluated predictive models of MICs based on PBP type. Compared to microdilution MICs, MICs predicted by PBP type showed essential agreement (MICs agree within 1 dilution) of >98%, category agreement (interpretive results agree) of >94%, a major discrepancy (sensitive isolate predicted as resistant) rate of <3%, and a very major discrepancy (resistant isolate predicted as sensitive) rate of <2% for all six β-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different β-lactam antibiotics in clinical pneumococcal isolates and serve as an accurate alternative to phenotypic susceptibility testing. IMPORTANCE The human pathogen Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide. β-Lactam antibiotics such as penicillin and ceftriaxone are the drugs of choice to treat pneumococcal infections. Some pneumococcal strains have developed β-lactam resistance through altering their penicillin-binding proteins (PBPs) and have become a major concern in choosing effective patient therapy. To systematically track and predict β-lactam resistance, we obtained the sequence signatures of PBPs from a large collection of clinical pneumococcal isolates using whole-genome sequencing data and found that these “PBP types” were predictive of resistance levels. Our findings can benefit the current era of strain surveillance when whole-genome sequencing data often lacks detailed resistance information. Using PBP positions that we found are always substituted within highly resistant strains may lead to further refinements. Sequence-based predictions are accurate and may lead to the ability to extract critical resistance information from nonculturable clinical specimens. The human pathogen Streptococcus pneumoniae is a leading cause of morbidity and mortality worldwide. β-Lactam antibiotics such as penicillin and ceftriaxone are the drugs of choice to treat pneumococcal infections. Some pneumococcal strains have developed β-lactam resistance through altering their penicillin-binding proteins (PBPs) and have become a major concern in choosing effective patient therapy. To systematically track and predict β-lactam resistance, we obtained the sequence signatures of PBPs from a large collection of clinical pneumococcal isolates using whole-genome sequencing data and found that these “PBP types” were predictive of resistance levels. Our findings can benefit the current era of strain surveillance when whole-genome sequencing data often lacks detailed resistance information. Using PBP positions that we found are always substituted within highly resistant strains may lead to further refinements. Sequence-based predictions are accurate and may lead to the ability to extract critical resistance information from nonculturable clinical specimens.