Clinically Approved Drugs against CNS Diseases as Potential Therapeutic Agents To Target Brain-Eating Amoebae.

Central nervous system (CNS) infections caused by free-living amoebae such as Acanthamoeba species and Naegleria fowleri are rare but fatal. A major challenge in the treatment against the infections caused by these amoebae is the discovery of novel compounds that can effectively cross the blood-brain barrier to penetrate the CNS. It is logical to test clinically approved drugs against CNS diseases for their potential antiamoebic effects since they are known for effective blood-brain barrier penetration and affect eukaryotic cell targets. The antiamoebic effects of clinically available drugs for seizures targeting gamma-amino butyric acid (GABA) receptor and ion channels were tested against Acanthamoeba castellanii belonging to the T4 genotype and N. fowleri. Three such drugs, namely, diazepam (Valium), phenobarbitone (Luminal), phenytoin (Dilantin), and their silver nanoparticles (AgNPs) were evaluated against both trophozoites and cysts stage. Drugs alone and drug conjugated silver nanoparticles were tested for amoebicidal, cysticidal, and host-cell cytotoxicity assays. Nanoparticles were synthesized by sodium borohydride reduction of silver nitrate with drugs as capping agents. Drug conjugated nanoconjugates were characterized by ultraviolet-visible (UV-vis) and Fourier transform infrared (FT-IR) spectroscopies and atomic force microscopy (AFM). In vitro moebicidal assay showed potent amoebicidal effects for diazepam, phenobarbitone, and phenytoin-conjugated AgNPs as compared to drugs alone against A. castellanii and N. fowleri. Furthermore, both drugs and drug conjugated AgNPs showed compelling cysticidal effects. Drugs conjugations with silver nanoparticles enhanced their antiacanthamoebic activity. Interestingly, amoeba-mediated host-cell cytotoxicity was also significantly reduced by drugs alone as well as their nanoconjugates. Since, these drugs are being used to target CNS diseases, their evaluation against brain-eating amoebae seems feasible due to advantages such as permeability of the blood-brain barrier, established pharmacokinetics and dynamics, and United States Food and Drug Administration (FDA) approval. Given the limited availability of effective drugs against brain-eating amoebae, the clinically available drugs tested here present potential for further in vivo studies.

Potential Value of Cellulose Synthesis Inhibitors Combined With PHMB in the Treatment of Acanthamoeba Keratitis.

PURPOSE: The aim of this study was to improve the cytopathic effect (CPE) of antiamebic agents by combining with cellulose synthesis inhibitor as an encystation inhibitor. METHODS: Cellulose synthesis inhibitors, 2,6-dichlorobenzonitrile (DCB) and isoxaben were used to block encystation of Acanthamoeba during cultivation. Cultured human corneal epithelial (HCE) cells and Acanthamoeba were treated with polyhexamethylene biguanide (PHMB) combined with cellulose synthesis inhibitors to evaluate the CPE as an antiamebic agent. RESULTS: 0.02% PHMB showed a 51.9% CPE on HCE cells within 30 minutes but exhibited significant toxic effects on Acanthamoeba. At a level of 0.00125%, PHMB had no significant CPEs on HCE cells, whereas 100 µM DCB and 10 µM isoxaben significantly inhibited the formation of the inner cyst wall of Acanthamoeba during encystation, and Acanthamoeba trophozoites failed to convert into mature cysts. Although a low concentration (0.00125%) of PHMB was used, the novel combinations with 100 µM DCB or 10 µM isoxaben had 23.4% or 18.7% additional amebicidal effects on Acanthamoeba. However, 100 µM DCB and 10 µM isoxaben had no CPEs on HCE cells. CONCLUSIONS: The combination of cellulose synthesis inhibitors with low concentrations of PHMB reduced the CPE on HCE cells and improved the amebicidal effect on Acanthamoeba by inhibition of encystation.

Proteomic characterization of the whole secretome of Legionella pneumophila and functional analysis of outer membrane vesicles.

Secretion of effector molecules is one of the major mechanisms by which the intracellular human pathogen Legionella pneumophila interacts with host cells during infection. Specific secretion machineries which are responsible for the subfraction of secreted proteins (soluble supernatant proteins [SSPs]) and the production of bacterial outer membrane vesicles (OMVs) both contribute to the protein composition of the extracellular milieu of this lung pathogen. Here we present comprehensive proteome reference maps for both SSPs and OMVs. Protein identification and assignment analyses revealed a total of 181 supernatant proteins, 107 of which were specific to the SSP fraction and 33 of which were specific to OMVs. A functional classification showed that a large proportion of the identified OMV proteins are involved in the pathogenesis of Legionnaires' disease. Zymography and enzyme assays demonstrated that the SSP and OMV fractions possess proteolytic and lipolytic enzyme activities which may contribute to the destruction of the alveolar lining during infection. Furthermore, it was shown that OMVs do not kill host cells but specifically modulate their cytokine response. Binding of immunofluorescently stained OMVs to alveolar epithelial cells, as visualized by confocal laser scanning microscopy, suggested that there is delivery of a large and complex group of proteins and lipids in the infected tissue in association with OMVs. On the basis of these new findings, we discuss the relevance of protein sorting and compartmentalization of virulence factors, as well as environmental aspects of the vesicle-mediated secretion.