Cysteinyl leukotrienes as novel host factors facilitating Cryptococcus neoformans penetration into the brain

Cryptococcus neoformas infection of the central nervous system (CNS) continues to be an important cause of mortality and morbidity, and a major contributing factor is our incomplete knowledge of the pathogenesis of this disease. Here, we provide the first direct evidence that C. neoformans exploits host cysteinyl leukotrienes (LTs), formed via LT biosynthetic pathways involving cytosolic phospholipase A2α (cPLA2α) and 5â lipoxygenase (5â LO) and acting via cysteinyl leukotriene type 1 receptor (CysLT1), for penetration of the bloodâ brain barrier. Gene deletion of cPLA2α and 5â LO and pharmacological inhibition of cPLA2α, 5â LO and CysLT1 were effective in preventing C. neoformans penetration of the bloodâ brain barrier in vitro and in vivo. A CysLT1 antagonist enhanced the efficacy of an antiâ fungal agent in therapy of C. neoformans CNS infection in mice. These findings demonstrate that host cysteinyl LTs, dependent on the actions of cPLA2α and 5â LO, promote C. neoformans penetration of the bloodâ brain barrier and represent novel targets for elucidating the pathogenesis and therapeutic development of C. neoformans CNS infection.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/136343/1/cmi12661_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/136343/2/cmi12661.pd

Sphingosine 1-Phosphate Activation of EGFR as a Novel Target for Meningitic \u3cem\u3eEscherichia coli\u3c/em\u3e Penetration of the Blood-Brain Barrier

Central nervous system (CNS) infection continues to be an important cause of mortality and morbidity, necessitating new approaches for investigating its pathogenesis, prevention and therapy. Escherichia coli is the most common Gram-negative bacillary organism causing meningitis, which develops following penetration of the blood–brain barrier (BBB). By chemical library screening, we identified epidermal growth factor receptor (EGFR) as a contributor to E. coli invasion of the BBB in vitro. Here, we obtained the direct evidence that CNS-infecting E. coli exploited sphingosine 1-phosphate (S1P) for EGFR activation in penetration of the BBB in vitro and in vivo. We found that S1P was upstream of EGFR and participated in EGFR activation through S1P receptor as well as through S1P-mediated up-regulation of EGFR-related ligand HB-EGF, and blockade of S1P function through targeting sphingosine kinase and S1P receptor inhibited EGFR activation, and also E. coli invasion of the BBB. We further found that both S1P and EGFR activations occurred in response to the same E. coli proteins (OmpA, FimH, NlpI), and that S1P and EGFR promoted E. coli invasion of the BBB by activating the downstream c-Src. These findings indicate that S1P and EGFR represent the novel host targets for meningitic E. coli penetration of the BBB, and counteracting such targets provide a novel approach for controlling E. coli meningitis in the era of increasing resistance to conventional antibiotics

Effects of polymyxin B on the outer membranes of Aeromonas species

The effect of polymyxin B on the outer membranes of the Gram negative bacteria Aeromonas spp. was examined and compared with the outer membranes prepared by breaking cells by the conventional method of osmotic shock and subsequent solubilization with sodium lauryl sarcosinate (Sarkosyl). SDS profiles of the two preparations revealed differences but immunoblot analysis showed close-to-similar profiles. The striking difference which the preparations revealed was the absence of smearing in the polymyxin treated ones. The same was also revealed when the two methods were appled on a slightly larger scale to a survey analysis done with 14 strains of the three species of Aeromonas - A. hydrophila, A. sobria and A. caviae. The results of this study indicate that apart from being a fast and convenient method for extracting outer membranes, application of polymyxin B is useful in removing lipopolysaccharide from outer membranes when applied to viable cells

NlpI Contributes to Escherichia coli K1 Strain RS218 Interaction with Human Brain Microvascular Endothelial Cells▿

Escherichia coli K1 is the most common Gram-negative bacillary organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMECs) is a prerequisite for its traversal of the blood-brain barrier (BBB) and penetration into the brain. In the present study, we identified NlpI as a novel bacterial determinant contributing to E. coli K1 interaction with HBMECs. The deletion of nlpI did not affect the expression of the known bacterial determinants involved in E. coli K1-HBMEC interaction, such as type 1 fimbriae, flagella, and OmpA, and the contribution of NlpI to HBMECs binding and invasion was independent of those bacterial determinants. Previous reports have shown that the nlpI mutant of E. coli K-12 exhibits growth defect at 42°C at low osmolarity, and its thermosensitive phenotype can be suppressed by a mutation on the spr gene. The nlpI mutant of strain RS218 exhibited similar thermosensitive phenotype, but additional spr mutation did not restore the ability of the nlpI mutant to interact with HBMECs. These findings suggest the decreased ability of the nlpI mutant to interact with HBMECs is not associated with the thermosensitive phenotype. NlpI was determined as an outer membrane-anchored protein in E. coli, and the nlpI mutant was defective in cytosolic phospholipase A2α (cPLA2α) phosphorylation compared to the parent strain. These findings illustrate the first demonstration of NlpI's contribution to E. coli K1 binding to and invasion of HBMECs, and its contribution is likely to involve cPLA2α

Extracellular Loops of the Eschericia coli Outer Membrane Protein A Contribute to the Pathogenesis of Meningitis

Neonatal meningitis by Eschericia coli RS218 occurs due to bacteremia and its transmigration across the blood-brain barrier. Although the outer membrane protein A (OmpA), a molecule with extracellular loops has been shown to contribute to the above phenomenon, we do not know the exact the role of these individual loops. Using bacterial strains whose individual loops have been removed, we demonstrated that whereas Loops1 and 2 contribute to 70%–80% bacterial survival in serum, bacterial entry into human brain microvascular endothelial cells (HBMEC) is governed by Loops1, 2, and 3. Cellular invasion was shown to require activation of host cytosolic phospholipase A2 (cPLA2α) by Loops1 and 2 but not 3. This suggests 2 distinct pathways for bacterial entry into host cells. Loop 4 played no role in either serum survival, cellular entry, or cPLA2α signaling. These findings demonstrate for the first time the different contributions of extracellular loops of OmpA to the pathogenesis of E. coli meningitis

SphK2-S1P-S1P₂ mediates meningitic <i>E</i>. <i>coli</i> penetration of the BBB <i>in vitro</i> and <i>in vivo</i>.

<p>(A) S1P generation was significantly higher in HBMEC incubated with wild-type RS218 compared with the triple mutant deleted of <i>ompA</i>, <i>fimH</i> and <i>nlpI</i>. Correspondingly, the sphingosine level was lower in HBMEC incubated with wild-type RS218 than those incubated with the triple deletion mutant. * <i>p</i><0.05. (B) Structures of (<i>S</i>)-FTY720-vinylphosphonae (SphK1 and SphK2 inhibitor), (<i>R</i>)-FTY720-methyl ether (selective SphK2 inhibitor), RB-032 and RB-033 (selective SphK1 inhibitors), and RB-034 (inactive analogue). (C) Both (<i>S</i>)-FTY720-vinylphosphonae (SphK1 and SphK2 inhibitor, shown as (<i>S</i>)-FTY-Pn) and (<i>R</i>)-FTY720-methyl ether (SphK2 inhibitor, shown as ROME) significantly inhibited RS218 invasion of HBMEC, while the SphK1 inhibitors (RB-032 and RB-033) and inactive analogue (RB-034) did not exhibit any inhibition. ** <i>p</i><0.01. The inhibitors were all used at 10 μM. (D) (<i>R</i>)-FTY720-methyl ether inhibited <i>E</i>. <i>coli</i> RS218 invasion of HBMEC in a dose-dependent manner. ** <i>p</i><0.01. (E) <i>E</i>. <i>coli</i> RS218 activated SphK2 in a time-dependent manner in HBMEC, while such activation was abolished by pretreatment with 10 μM (<i>R</i>)-FTY720-methyl ether. ** <i>p</i><0.01. (F) <i>E</i>. <i>coli</i> penetration into the brain was significantly less in SphK2 ^−/− mice compared with wild-type mice. In contrast, the levels of bacteremia did not differ between the two groups of mice. (G) JTE-013 (S1P₂ antagonist) significantly inhibited <i>E</i>. <i>coli</i> invasion of HBMEC, while VPC23019 (S1P₁ and S1P₃ antagonist) did not exhibit any inhibition. ** <i>p</i><0.01. (H) The mutants with deletion of <i>ompA</i>, <i>fimH</i>, or <i>nlpI</i> as well as the triple mutant (<i>ΔompAΔfimHΔnlpI</i>) induced significantly lower levels of SphK2 activation in HBMEC, compared with wild-type RS218. ** <i>p</i><0.01.</p