Escherichia coli K1 Modulates Peroxisome Proliferator-Activated Receptor γ and Glucose Transporter 1 at the Blood-Brain Barrier in Neonatal Meningitis.

Escherichia coli K1 meningitis continues to be a major threat to neonatal health. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with endothelial cell glycoprotein 96 (Ecgp96) in the blood-brain barrier to enter the central nervous system. Here we show that the interaction between OmpA and Ecgp96 downregulates peroxisome proliferator-activated receptor γ (PPAR-γ) and glucose transporter 1 (GLUT-1) levels in human brain microvascular endothelial cells, causing disruption of barrier integrity and inhibition of glucose uptake. The suppression of PPAR-γ and GLUT-1 by the bacteria in the brain microvessels of newborn mice causes extensive pathophysiology owing to interleukin 6 production. Pretreatment with partial or selective PPAR-γ agonists ameliorate the pathological outcomes of infection by suppressing interleukin 6 production in the brain. Thus, inhibition of PPAR-γ and GLUT-1 by E. coli K1 is a novel pathogenic mechanism in meningitis, and pharmacological upregulation of PPAR-γ and GLUT-1 levels may provide novel therapeutic avenues.

The interaction of N-glycans in Fcγ receptor I α-chain with Escherichia coli K1 outer membrane protein A for entry into macrophages: experimental and computational analysis.

Neonatal meningitis, caused by Escherichia coli K1, is a serious central nervous system disease. We have established that macrophages serve as permissive niches for E. coli K1 to multiply in the host and for attaining a threshold level of bacterial load, which is a prerequisite for the onset of the disease. Here, we demonstrate experimentally that three N-glycans in FcγRIa interact with OmpA of E. coli K1 for binding to and entering the macrophages. Adoptive transfer of FcγRIa(-/-) bone marrow-derived macrophages transfected with FcγRIa into FcγRIa(-/-) newborn mice renders them susceptible to E. coli K1-induced meningitis. In contrast, mice that received bone marrow-derived macrophages transfected with FcγRIa in which N-glycosylation sites 1, 4, and 5 are mutated to alanines exhibit resistance to E. coli K1 infection. Our molecular dynamics and simulation studies predict that N-glycan 5 exhibits strong binding at the barrel site of OmpA formed by loops 3 and 4, whereas N-glycans 1 and 4 interact with loops 1, 3, and 4 of OmpA at tip regions. Molecular modeling data also suggest no role for the IgG binding site in the invasion process. In agreement, experimental mutations in IgG binding site had no effect on the E. coli K1 entry into macrophages in vitro or on the onset of meningitis in newborn mice. Together, this integration of experimental and computational studies reveals how the N-glycans in FcγRIa interact with the OmpA of E. coli K1 for inducing the disease pathogenesis.

Retinoid agonist Am80-enhanced neutrophil bactericidal activity arising from granulopoiesis in vitro and in a neutropenic mouse model.

Despite advances in the therapeutic use of recombinant granulocyte colony-stimulating factor (G-CSF) to promote granulopoiesis of human hematopoietic stem cells (HSCs), neutropenia remains one of the most serious complications of cancer chemotherapy. We discovered that retinoid agonist Am80 (tamibarotene) is more potent than G-CSF in coordinating neutrophil differentiation and immunity development. Am80-induced neutrophils (AINs) either in vitro or in neutropenic mouse model displayed strong bactericidal activities, similar to those of human peripheral blood neutrophils (PBNs) or mouse peripheral blood neutrophils (MPBNs) but markedly greater than did G-CSF­induced neutrophils (GINs). In contrast to GINs but similar to PBNs, the enhanced bacterial killing by AINs accompanied both better granule maturation and greater coexpression of CD66 antigen with the integrin ß2 subunit CD18. Consistently, anti-CD18 antibody neutralized Am80-induced bactericidal activities of AINs. These studies demonstrate that Am80 is more effective than G-CSF in promoting neutrophil differentiation and bactericidal activities, probably through coordinating the functional interaction of CD66 with CD18 to enhance the development of neutrophil immunity during granulopoiesis. Our findings herein suggest a molecular rationale for developing new therapy against neutropenia using Am80 as a cost-effective treatment option.

Angiotensin II receptor type 1--a novel target for preventing neonatal meningitis in mice by Escherichia coli K1.

The increasing incidence of Escherichia coli K1 meningitis due to escalating antibiotic resistance warrants alternate treatment options to prevent this deadly disease. We screened a library of small molecules from the National Institutes of Health clinical collection and identified telmisartan, an angiotensin II receptor type 1 (AT1R) blocker, as a potent inhibitor of E. coli invasion into human brain microvascular endothelial cells (HBMECs). Immunoprecipitation studies revealed that AT1R associates with endothelial cell gp96, the receptor in HBMECs for E. coli outer membrane protein A. HBMECs pretreated with telmisartan or transfected with AT1R small interfering RNA were resistant to E. coli invasion because of downregulation of protein kinase C-α phosphorylation. Administration of a soluble derivative of telmisartan to newborn mice before infection with E. coli prevented the onset of meningitis and suppressed neutrophil infiltration and glial cell migration in the brain. Therefore, telmisartan has potential as an alternate treatment option for preventing E. coli meningitis.

Regulation of Toll-like receptor 2 interaction with Ecgp96 controls Escherichia coli K1 invasion of brain endothelial cells.

The interaction of outer membrane protein A (OmpA) with its receptor, Ecgp96 (a homologue of Hsp90ß), is critical for the pathogenesis of Escherichia coli K1 meningitis. Since Hsp90 chaperones Toll-like receptors (TLRs), we examined the role of TLRs in E. coli K1 infection. Herein, we show that newborn TLR2(-/-) mice are resistant to E. coli K1 meningitis, while TLR4(-/-) mice succumb to infection sooner. In vitro, OmpA+ E. coli infection selectively upregulates Ecgp96 and TLR2 in human brain microvascular endothelial cells (HBMEC), whereas OmpA- E. coli upregulates TLR4 in these cells. Furthermore, infection with OmpA+ E. coli causes Ecgp96 and TLR2 translocate to the plasma membrane of HBMEC as a complex. Immunoprecipitation studies of the plasma membrane fractions from infected HBMEC reveal that the C termini of Ecgp96 and TLR2 are critical for OmpA+ E. coli invasion. Knockdown of TLR2 using siRNA results in inefficient membrane translocation of Ecgp96 and significantly reduces invasion. In addition, the interaction of Ecgp96 andTLR2 induces a bipartite signal, one from Ecgp96 through PKC-α while the other from TLR2 through MyD88, ERK1/2 and NF-κB. This bipartite signal ultimately culminates in the efficient production of NO, which in turn promotes E. coli K1 invasion of HBMEC.

Attenuation of biopterin synthesis prevents Escherichia coli K1 invasion of brain endothelial cells and the development of meningitis in newborn mice.

Elevated levels of pterins and nitric oxide (NO) are observed in patients with septic shock and bacterial meningitis. We demonstrate that Escherichia coli K1 infection of human brain microvascular endothelial cells (HBMECs) induces the expression of guanosine triphosphate cyclohydrolase (GCH1), the rate-limiting enzyme in pterin synthesis, thereby elevating levels of biopterin. DAHP (2,4-diamino hydroxyl pyrimidine), a specific inhibitor of GCH1, prevented biopterin and NO production and invasion of E. coli K1 in HBMECs. GCH1 interaction with Ecgp96, the receptor for outer membrane protein A of E. coli K1, also increases on infection, and suppression of Ecgp96 expression prevents GCH1 activation and biopterin synthesis. Pretreatment of newborn mice with DAHP prevented the production of biopterin and the development of meningitis. These results suggest a novel role for biopterin synthesis in the pathogenesis of E. coli K1 meningitis.

SR-like RNA-binding protein Slr1 affects Candida albicans filamentation and virulence.

Candida albicans causes both mucosal and disseminated infections, and its capacity to grow as both yeast and hyphae is a key virulence factor. Hyphal formation is a type of polarized growth, and members of the SR (serine-arginine) family of RNA-binding proteins influence polarized growth of both Saccharomyces cerevisiae and Aspergillus nidulans. Therefore, we investigated whether SR-like proteins affect filamentous growth and virulence of C. albicans. BLAST searches with S. cerevisiae SR-like protein Npl3 (ScNpl3) identified two C. albicans proteins: CaNpl3, an apparent ScNpl3 ortholog, and Slr1, another SR-like RNA-binding protein with no close S. cerevisiae ortholog. Whereas ScNpl3 was critical for growth, deletion of NPL3 in C. albicans resulted in few phenotypic changes. In contrast, the slr1Δ/Δ mutant had a reduced growth rate in vitro, decreased filamentation, and impaired capacity to damage epithelial and endothelial cells in vitro. Mice infected intravenously with the slr1Δ/Δ mutant strain had significantly prolonged survival compared to that of mice infected with the wild-type or slr1Δ/Δ mutant complemented with SLR1 (slr1Δ/Δ+SLR1) strain, without a concomitant decrease in kidney fungal burden. Histopathology, however, revealed differential localization of slr1Δ/Δ hyphal and yeast morphologies within the kidney. Mice infected with slr1Δ/Δ cells also had an increased brain fungal burden, which correlated with increased invasion of brain, but not umbilical vein, endothelial cells in vitro. The enhanced brain endothelial cell invasion was likely due to the increased surface exposure of the Als3 adhesin on slr1Δ/Δ cells. Our results indicate that Slr1 is an SR-like protein that influences C. albicans growth, filamentation, host cell interactions, and virulence.

Mechanisms of Candida albicans trafficking to the brain.

During hematogenously disseminated disease, Candida albicans infects most organs, including the brain. We discovered that a C. albicans vps51Δ/Δ mutant had significantly increased tropism for the brain in the mouse model of disseminated disease. To investigate the mechanisms of this enhanced trafficking to the brain, we studied the interactions of wild-type C. albicans and the vps51Δ/Δ mutant with brain microvascular endothelial cells in vitro. These studies revealed that C. albicans invasion of brain endothelial cells is mediated by the fungal invasins, Als3 and Ssa1. Als3 binds to the gp96 heat shock protein, which is expressed on the surface of brain endothelial cells, but not human umbilical vein endothelial cells, whereas Ssa1 binds to a brain endothelial cell receptor other than gp96. The vps51Δ/Δ mutant has increased surface expression of Als3, which is a major cause of the increased capacity of this mutant to both invade brain endothelial cells in vitro and traffic to the brain in mice. Therefore, during disseminated disease, C. albicans traffics to and infects the brain by binding to gp96, a unique receptor that is expressed specifically on the surface of brain endothelial cells.

IQGAP1 mediates the disruption of adherens junctions to promote Escherichia coli K1 invasion of brain endothelial cells.

The transcellular entry of Escherichia coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain oedema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96, to induce PKC-α phosphorylation, adherens junction (AJ) disassembly (by dislodging ß-catenin from VE-cadherin), and remodelling of actin in HBMEC. We report here that IQGAP1 mediates ß-catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C-terminal truncated IQGAP1 (IQΔC) that cannot bind ß-catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho-PKC-α interacts with the C-terminal portion of Ecgp96 as well as with VE-cadherin after IQGAP1-mediated AJ disassembly. HBMEC overexpressing either C-terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction of phospho-PKC-α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC-α phosphorylation and association of phospho-PKC-α with Ecgp96, and then signals IQGAP1 to detach ß-catenin from AJs. Subsequently, IQGAP1/ß-catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion.

Recruitment of dendritic cells is responsible for intestinal epithelial damage in the pathogenesis of necrotizing enterocolitis by Cronobacter sakazakii.

Cronobacter sakazakii is a Gram-negative pathogen associated with the cases of necrotizing enterocolitis (NEC) that result from formula contamination. In a mouse model of NEC, we demonstrate that C. sakazakii infection results in epithelial damage by recruiting greater numbers of dendritic cells (DCs) than macrophages and neutrophils in the gut and suppresses DC maturation, which requires outer membrane protein A (OmpA) expression in C. sakazakii. Pretreatment of intestinal epithelial cell monolayers with supernatant from OmpA(+) C. sakazakii/DC culture markedly enhanced membrane permeability and enterocyte apoptosis, whereas OmpA(-) C. sakazakii/DC culture supernatant had no effect. Analysis of OmpA(+) C. sakazakii/DC coculture supernatant revealed significantly greater TGF-ß production compared with the levels produced by OmpA(-) C. sakazakii infection. TGF-ß levels were elevated in the intestinal tissue of mice infected with OmpA(+) C. sakazakii. Cocultures of CaCo-2 cells and DCs in a "double-layer" model followed by infection with OmpA(+) C. sakazakii significantly enhanced monolayer leakage by increasing TGF-ß production. Elevated levels of inducible NO synthase (iNOS) were also observed in the double-layer infection model, and abrogation of iNOS expression prevented the C. sakazakii-induced CaCo-2 cell monolayer permeability despite the presence of DCs or OmpA(+) C. sakazakii/DC supernatant. Blocking TGF-ß activity using a neutralizing Ab suppressed iNOS production and prevented apoptosis and monolayer leakage. Depletion of DCs in newborn mice protected against C. sakazakii-induced NEC, whereas adoptive transfer of DCs rendered the animals susceptible to infection. Therefore, C. sakazakii interaction with DCs in intestine enhances the destruction of the intestinal epithelium and the onset of NEC due to increased TGF-ß production.

Deciphering the roles of outer membrane protein A extracellular loops in the pathogenesis of Escherichia coli K1 meningitis.

Outer membrane protein A (OmpA) has been implicated as an important virulence factor in several gram-negative bacterial infections such as Escherichia coli K1, a leading cause of neonatal meningitis associated with significant mortality and morbidity. In this study, we generated E. coli K1 mutants that express OmpA in which three or four amino acids from various extracellular loops were changed to alanines, and we examined their ability to survive in several immune cells. We observed that loop regions 1 and 2 play an important role in the survival of E. coli K1 inside neutrophils and dendritic cells, and loop regions 1 and 3 are needed for survival in macrophages. Concomitantly, E. coli K1 mutants expressing loop 1 and 2 mutations were unable to cause meningitis in a newborn mouse model. Of note, mutations in loop 4 of OmpA enhance the severity of the pathogenesis by allowing the pathogen to survive better in circulation and to produce high bacteremia levels. These results demonstrate, for the first time, the roles played by different regions of extracellular loops of OmpA of E. coli K1 in the pathogenesis of meningitis and may help in designing effective preventive strategies against this deadly disease.

Fcγ receptor I alpha chain (CD64) expression in macrophages is critical for the onset of meningitis by Escherichia coli K1.

Neonatal meningitis due to Escherichia coli K1 is a serious illness with unchanged morbidity and mortality rates for the last few decades. The lack of a comprehensive understanding of the mechanisms involved in the development of meningitis contributes to this poor outcome. Here, we demonstrate that depletion of macrophages in newborn mice renders the animals resistant to E. coli K1 induced meningitis. The entry of E. coli K1 into macrophages requires the interaction of outer membrane protein A (OmpA) of E. coli K1 with the alpha chain of Fcγ receptor I (FcγRIa, CD64) for which IgG opsonization is not necessary. Overexpression of full-length but not C-terminal truncated FcγRIa in COS-1 cells permits E. coli K1 to enter the cells. Moreover, OmpA binding to FcγRIa prevents the recruitment of the γ-chain and induces a different pattern of tyrosine phosphorylation of macrophage proteins compared to IgG2a induced phosphorylation. Of note, FcγRIa(-/-) mice are resistant to E. coli infection due to accelerated clearance of bacteria from circulation, which in turn was the result of increased expression of CR3 on macrophages. Reintroduction of human FcγRIa in mouse FcγRIa(-/-) macrophages in vitro increased bacterial survival by suppressing the expression of CR3. Adoptive transfer of wild type macrophages into FcγRIa(-/-) mice restored susceptibility to E. coli infection. Together, these results show that the interaction of FcγRI alpha chain with OmpA plays a key role in the development of neonatal meningitis by E. coli K1.

Human isolates of Cronobacter sakazakii bind efficiently to intestinal epithelial cells in vitro to induce monolayer permeability and apoptosis.

BACKGROUND: Cronobacter sakazakii (CS) is an emerging opportunistic pathogen that causes life-threatening infections in infants. This pathogen has been implicated in the outbreaks of necrotizing enterocolitis (NEC) with associated rates of high mortality and morbidity. In this study, we compared the abilities of CS strains isolated from human and environmental sources to bind to intestinal epithelial cells and trigger apoptosis. MATERIALS AND METHODS: CS strains were isolated from human and environmental sources and their abilities to bind to intestinal epithelial cells were determined. Monolayer permeability was determined by transepithelial electrical resistance (TEER) and horseradish peroxidase (HRP) leakage. Apoptosis was examined by ApoTag and AnnexinV-7AAD staining. PKC activation was evaluated by non-radioactive PepTag assay. RESULTS: Human isolates of CS bind to rat and human enterocytes more efficiently than environmental strains. Additionally, these strains induced increased enterocyte monolayer permeability as indicated by a decrease in TEER and an increase in transcellular leakage of exogenously added HRP. Human isolates also caused tight junction disruption and significant apoptosis of enterocytes compared with environmental strains due to increased production of inducible nitric oxide. We also observed that human CS isolates caused 2-fold increase in the activation of phosphokinase C (PKC) than environmental strains. Blocking the PKC activity in enterocytes by an inhibitor, Gö 6983, suppressed CS-mediated tight junction disruption, monolayer permeability, and apoptosis of the cells. CONCLUSION: These results suggest that human isolates of CS more efficiently bind to and cause damage to intestinal epithelial cells compared with environmental strains.

Role of neutrophils and macrophages in the pathogenesis of necrotizing enterocolitis caused by Cronobacter sakazakii.

BACKGROUND: Cronobacter sakazakii (CS) is a highly virulent gram-negative opportunistic pathogen that has been implicated in clinical outbreaks of necrotizing enterocolitis (NEC). The role of mucosal immune cells in CS infection is not well understood. In this study, we sought to elucidate the role of neutrophils (polymorphonuclear leukocytes; PMNs) and macrophages in the pathogenesis of NEC induced by CS using a novel newborn mouse model. MATERIALS AND METHODS: PMNs and macrophages were depleted in newborn mice using Gr-1 antibody and carrageenan, respectively, and then infected with 10(3) CFU of CS. The development of NEC in these mice was assessed by a pathologist based on the morphologic changes in the intestine. Cytokine production was determined in the serum and intestinal homogenates of infected mice by enzyme-linked immunosorbent assay (ELISA). Inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production was determined by flow cytometry and Greiss method, respectively. RESULTS: Depletion of PMNs and macrophages in newborn mice led to increased recruitment of dendritic cells (DCs) in the intestine compared with wild-type mice upon infection with CS. PMN- and macrophage-depleted mice showed increased bacterial load, production of pro-inflammatory cytokines, iNOS expression, and NO production in the intestines in comparison to wild-type mice fed with CS. In addition, depletion of PMNs and macrophages prior to infection in mice resulted in severe inflammation, villus destruction, and enhanced enterocyte apoptosis in the intestines compared with CS-infected wild-type mice. CONCLUSIONS: Our data suggest that depletion of PMNs and macrophages from the lamina propria (LP) exacerbates experimental NEC, indicating that both of these immunocytes play an important role in the clearance of CS during the initial stages of infection. The increased mucosal cytokine response and NO production in the absence of these immunocytes may be responsible for the observed increase in mucosal injury. Understanding how CS manipulates these cells, employing novel mouse model of NEC reported in this study, will provide significant insights for the development of novel therapeutic and preventive strategies to combat NEC.

Escherichia coli K1 promotes the ligation of CD47 with thrombospondin-1 to prevent the maturation of dendritic cells in the pathogenesis of neonatal meningitis.

Dendritic cells (DCs) are professional APCs providing a critical link between adaptive and innate immune responses. Our previous studies have shown that Escherichia coli K1 internalization of myeloid DCs suppressed the maturation of the cells for which outer membrane protein A (OmpA) expression is essential. In this study, we demonstrate that infection of DCs with OmpA(+) E. coli significantly upregulates the expression of CD47, an integrin-associated protein, and its natural ligand thrombospondin 1 (TSP-1). Pretreatment of DCs with anti-CD47 blocking Ab or knocking down the expression of CD47 or TSP-1, but not signal regulatory protein alpha by small interfering RNA, abrogated the suppressive effect of E. coli K1. Ligation of CD47 with a mAb prevented the maturation and cytokine production by DCs upon stimulation with LPS similar to the inhibitory effect induced by OmpA(+) E. coli. In agreement with the in vitro studies, suppression of CD47 or TSP-1 expression in newborn mice by a novel in vivo small interfering RNA technique protected the animals against E. coli K1 meningitis. Reconstitution of CD47 knockdown mice with CD47(+) DCs renders the animals susceptible to meningitis by E. coli K1, substantiating the role of CD47 expression in DCs for the occurrence of meningitis. Our results demonstrate a role for CD47 for the first time in bacterial pathogenesis and may be a novel target for designing preventive approaches for E. coli K1 meningitis.

Experimental validation of the predicted binding site of Escherichia coli K1 outer membrane protein A to human brain microvascular endothelial cells: identification of critical mutations that prevent E. coli meningitis.

Escherichia coli K1, the most common cause of meningitis in neonates, has been shown to interact with GlcNAc1-4GlcNAc epitopes of Ecgp96 on human brain microvascular endothelial cells (HBMECs) via OmpA (outer membrane protein A). However, the precise domains of extracellular loops of OmpA interacting with the chitobiose epitopes have not been elucidated. We report the loop-barrel model of these OmpA interactions with the carbohydrate moieties of Ecgp96 predicted from molecular modeling. To test this model experimentally, we generated E. coli K1 strains expressing OmpA with mutations of residues predicted to be critical for interaction with the HBMEC and tested E. coli invasion efficiency. For these same mutations, we predicted the interaction free energies (including explicit calculation of the entropy) from molecular dynamics (MD), finding excellent correlation (R(2) = 90%) with experimental invasion efficiency. Particularly important is that mutating specific residues in loops 1, 2, and 4 to alanines resulted in significant inhibition of E. coli K1 invasion in HBMECs, which is consistent with the complete lack of binding found in the MD simulations for these two cases. These studies suggest that inhibition of the interactions of these residues of Loop 1, 2, and 4 with Ecgp96 could provide a therapeutic strategy to prevent neonatal meningitis due to E. coli K1.

P-glycoprotein induction by breast milk attenuates intestinal inflammation in experimental necrotizing enterocolitis.

P-glycoprotein (Pgp), a product of the multi-drug resistance gene MDR1a, is a broad specificity efflux ATP cassette transmembrane transporter that is predominantly expressed in epithelial tissues. Because mdr1a(-/-) mice tend to develop spontaneous colitis in bacteria-dependent manner, Pgp is believed to have a role in protection of the intestinal epithelium from luminal bacteria. Here we demonstrate that levels of Pgp in the small intestine of newborn rodents dramatically increase during breastfeeding, but not during formula feeding (FF). In rats and mice, levels of intestinal Pgp peak on days 3-7 and 1-5 of breastfeeding, respectively. The mdr1a(-/-) neonatal mice subjected to FF, hypoxia, and hypothermia have significantly higher incidence and pathology, as well as significantly earlier onset of necrotizing enterocolitis (NEC) than congenic wild type mice. Breast-fed mdr1a(-/-) neonatal mice are also more susceptible to intestinal damage caused by the opportunistic pathogen Cronobacter sakazakii that has been associated with hospital outbreaks of NEC. Breast milk, but not formula, induces Pgp expression in enterocyte cell lines in a dose- and time-dependent manner. High levels of ectopically expressed Pgp protect epithelial cells in vitro from apoptosis induced by C. sakazakii. Taken together, these results show that breast milk-induced expression of Pgp may have a role in the protection of the neonatal intestinal epithelium from injury associated with nascent bacterial colonization.

Inhibition of inducible nitric oxide controls pathogen load and brain damage by enhancing phagocytosis of Escherichia coli K1 in neonatal meningitis.

Escherichia coli K1 is a leading cause of neonatal meningitis in humans. In this study, we sought to determine the pathophysiologic relevance of inducible nitric oxide (iNOS) in experimental E. coli K1 meningitis. By using a newborn mouse model of meningitis, we demonstrate that E. coli infection triggered the expression of iNOS in the brains of mice. Additionally, iNOS-/- mice were resistant to E. coli K1 infection, displaying normal brain histology, no bacteremia, no disruption of the blood-brain barrier, and reduced inflammatory response. Treatment with an iNOS specific inhibitor, aminoguanidine (AG), of wild-type animals before infection prevented the development of bacteremia and the occurrence of meningitis. The infected animals treated with AG after the development of bacteremia also completely cleared the pathogen from circulation and prevented brain damage. Histopathological and micro-CT analysis of brains revealed significant damage in E. coli K1-infected mice, which was completely abrogated by AG administration. Peritoneal macrophages and polymorphonuclear leukocytes isolated from iNOS-/- mice or pretreated with AG demonstrated enhanced uptake and killing of the bacteria compared with macrophages and polymorphonuclear leukocytes from wild-type mice in which E. coli K1 survive and multiply. Thus, NO produced by iNOS may be beneficial for E. coli to survive inside the macrophages, and prevention of iNOS could be a therapeutic strategy to treat neonatal E. coli meningitis.