Vinicius José Maschio

Infection in a rat model reactivates attenuated virulence after long-term axenic culture of Acanthamoeba spp

Prolonged culturing of many microorganisms leads to the loss of virulence and a reduction of their infective capacity. However, little is known about the changes in the pathogenic strains of Acanthamoeba after long culture periods. Our study evaluated the effect of prolonged culturing on the invasiveness of different isolates of Acanthamoeba in an in vivo rat model. ATCC strains of Acanthamoeba, isolates from the environment and clinical cases were evaluated. The in vivo model was effective in establishing the infection and differentiating the pathogenicity of the isolates and re-isolates. The amoebae cultured in the laboratory for long periods were less virulent than those that were recently isolated, confirming the importance of passing Acanthamoeba strains in animal models.

IDENTIFICATION OF Pseudomonas spp. AS AMOEBA-RESISTANT MICROORGANISMS IN ISOLATES OF Acanthamoeba

Acanthamoeba is a “Trojan horse” of the microbial world. The aim of this study was to identify the presence of Pseudomonas as an amoeba-resistant microorganism in 12 isolates of Acanthamoeba. All isolates showed the genus Pseudomonas spp. as amoeba-resistant microorganisms. Thus, one can see that the Acanthamoeba isolates studied are hosts of Pseudomonas.

Characterization of Isolates of Acanthamoeba from the Nasal Mucosa and Cutaneous Lesions of Dogs

AbstractAcanthamoeba spp. are free-living amoebae that are ubiquitously distributed in the environment and can cause encephalomyelitis in animals and humans. The factors that contribute to Acanthamoeba infections include parasite biology, genetic diversity, environmental spread, and host susceptibility. The aim of the present study was to characterize isolates of Acanthamoeba from the nasal mucosa and cutaneous lesions of dogs in order to access the occurence and pathogenicity of these organisms in this animal group. We studied 13 isolates of Acanthamoeba confirmed by polymerase chain reaction. They were sequenced, the genotype was determined, and their potential of pathogenicity was evaluated.

Acanthamoeba T4, T5 and T11 Isolated From Mineral Water Bottles in Southern Brazil

Acanthamoeba is a protist potential pathogen, capable of causing a blinding keratitis in contact lens wearers and disseminated infection, leading to granulomatous amebic encephalitis in immunocompromised individuals. This amoeba is a ubiquitous organism that has been isolated from various domestic water systems, such as cooling towers and hospital water networks. The objective of this work was to investigate the presence of Acanthamoeba in mineral water bottles marketed in Porto Alegre, southern Brazil. Positive samples were further classified at the genotype level after sequencing the ASA.S1 region of 18S rDNA gene. Six of the eight isolates belonged to T5 genotype, one to T4 genotype, and one was T11. Several genotypes have been reported worldwide as causative of pathologies in humans, including genotypes T4, T5 and T11. Overall, the widespread distribution of potentially pathogenic Acanthamoeba strains in the studied source demands more awareness within the public and health professionals, because this pathogen is emerging as a risk for human health worldwide.

Proteomic profiling of the infective trophozoite stage of Acanthamoeba polyphaga.

Acanthamoeba polyphaga is a free-living protozoan pathogen, whose infective trophozoite form is capable of causing a blinding keratitis and fatal granulomatous encephalitis in humans. The damage caused by A. polyphaga trophozoites in human corneal or brain infections is the result of several different pathogenic mechanisms that have not yet been elucidated at the molecular level. We performed a comprehensive analysis of the proteins expressed by A. polyphaga trophozoites, based on complementary 2-DE MS/MS and gel-free LC-MS/MS approaches. Overall, 202 non-redundant proteins were identified. An A. polyphaga proteomic map in the pH range 3-10 was produced, with protein identification for 184 of 370 resolved spots, corresponding to 142 proteins. Additionally, 94 proteins were identified by gel-free LC-MS/MS. Functional classification revealed several proteins with potential importance for pathogen survival and infection of mammalian hosts, including surface proteins and proteins related to defense mechanisms. Our study provided the first comprehensive proteomic survey of the trophozoite infective stage of an Acanthamoeba species, and established foundations for prospective, comparative and functional studies of proteins involved in mechanisms of survival, development, and pathogenicity in A. polyphaga and other pathogenic amoebae.

Evaluation of the immunodiagnostic potential of a recombinant surface protein domain from Acanthamoeba castellanii

Acanthamoeba spp. are free-living protists widely distributed in environment, able to cause keratitis, encephalitis and skin lesions in humans and animals. Acanthamoeba spp. exist in two forms: an infective trophozoite and a dormant cyst. Several factors contribute to the pathogenesis of Acanthamoeba spp. The parasite adhesion to the host cell is the primary step for infection and is mediated by a mannose binding-protein, expressed in the surface and considered the main pathogenicity factor in Acanthamoeba spp. So far, there was no evidence of another surface protein of Acanthamoeba spp. relevant for host invasion or infection by these organisms. The aims of this study were to identify and characterize an Acanthamoeba castellanii surface protein and to evaluate its diagnostic potential. In silico predictions of surface proteins allowed to identify the A. castellanii calreticulin as a possible surface antigen. The coding sequence of a predicted extracellular domain of A. castellanii calreticulin was cloned by in vivo homologous recombination and the recombinant polypeptide (AcCRT29-130) was produced. Its immunodiagnostic potential was assessed in a recombinant antigen-based ELISA with sera from experimentally infected rats that developed keratitis and encephalitis, and sera from patients with encephalitis. The AcCRT29-130 was significantly more recognized by sera from encephalitis infected rats in comparison with the non-infected controls. Human sera from encephalitis patients, however presented no significant response. These results showed the AcCRT29-130 potential for A. castellanii infection immunodiagnosis in animals, with further studies being required for assessment of its use for human infections.

Identification of Paenibacillus as a Symbiont in Acanthamoeba.

Amoebae of the genus Acanthamoeba occur worldwide and in addition to being pathogens, are important vehicles for microorganisms with clinical and environmental importance. This study aimed to evaluate the profiling of endosymbionts in 12 isolates of Acanthamoeba using V3 region of 16S rDNA denaturing gradient gel electrophoresis (DGGE) and sequencing. The DGGE enabled us to characterize the endosymbionts diversity in isolates of Acanthamoeba, and to identify Paenibacillus sp., an emerging pathogen, as an amoebic endosymbiont. The results of this study demonstrated that Acanthamoeba is capable of transporting a large number of endosymbionts. This is the first study that reports, the presence of Paenibacillus sp. as amebic symbiont.

Box Behnken design of siRNA-loaded liposomes for the treatment of a murine model of ocular keratitis caused by Acanthamoeba

Acanthamoeba keratitis is an ophthalmic disease with no specific treatment that specially affects contact lens users. The silencing of serine phosphatase (SP) and glycogen phosphorylase (GP) proteins produced by Acanthamoeba has been shown to significantly reduce the cytopathic effect, although no vehicle was proposed yet to deliver the siRNA sequences to the trophozoites. In this study, PEGylated cationic liposomes were proposed and optimized using Box-Behnken design. The influence of DOTAP:DOPE ratio, DSPE-PEG concentration, and siRNA/DOTAP charge ratio were evaluated over both biological response and physicochemical properties of liposomes. The ratio of DOTAP:DOPE had an effect in the trophozoite activity whereas the charge ratio influenced both size and protease activity. The predicted values were very close to the observed values, yielding a formulation with good activity and toxicity profile, which was used in the following experiments. A murine model of ocular keratitis was treated with siGP + siSP-loaded liposomes, as well as their respective controls, and combined treatment of liposomes and chlorhexidine. After 15 days of eight daily administrations, the liposomal complex combined with chlorhexidine was the only treatment able to reverse the more severe lesions associated with keratitis. There was 60% complete regression in corneal damage, with histological sections demonstrating the presence of an integral epithelium, without lymphocytic infiltrate. The set of results demonstrate the efficacy of a combined therapy based on siRNA with classical drugs for a better prognosis of keratitis caused by Acanthamoeba.

Comparative proteomic analysis of soluble and surface-enriched proteins from Acanthamoeba castellanii trophozoites

Acanthamoeba castellanii is a free-living organism widely distributed in the environment that may cause disease. This protozoan exists in two forms, an infective trophozoite and a dormant cyst. The trophozoites are able to cause keratitis and granulomatous amoebic encephalitis in humans. Keratitis is an acute, sight threatening infection of cornea with potential to cause permanent blindness without prompt treatment. However, the lack of suspicion and the low awareness about these amoebae besides of the absence of commercially available immunodiagnostic tests may delay an accurate diagnosis. The identification of proteins with potential for use in immunodiagnosis may improve the parasite detection more quickly and specifically. The amoeba adhesion to the host cell is the primary step for infection but there is no full understanding of A. castellanii proteins relevant for host invasion or infection. In this study, an assessment of soluble and surface-enriched protein fractions expressed by A. castellanii trophozoites, based on complementary LC-MS/MS approach using peptides from SDS-PAGE excised bands, was performed. Our proteomic analysis allowed identification of a total of 503 proteins, of which 308 proteins were exclusively identified in the soluble fraction, 119 in surface-enriched fraction and 76 in both. In silico analysis of functional classification revealed several proteins involved in many biological mechanisms in A. castellanii, including pathogen survival and infection of mammalian hosts. The analysis of predicted antigenic peptides allowed the identification of proteins with potential for immunodiagnostic assays.

Virulent T4 Acanthamoeba causing keratitis in a patient after swimming while wearing contact lenses in Southern Brazil

Several strains of free-living amoebae belonging to the genus Acanthamoeba can cause a painful sight-threatening disease of the cornea known as Acanthamoeba keratitis (AK). The numbers of AK cases keep rising worldwide mainly due to an increase in contact lens wearers and lack of hygiene in the maintenance of contact lenses and their cases. We report a case of AK in a healthy young woman admitted to the Hospital de Clinicas in Porto Alegre, southern Brazil. Corneal scrapings were examined for the presence of Acanthamoeba strains. The initial isolate was characterized by morphological and genotypic properties. The isolate belonged to group III according to Pussard and Pons’ cyst morphology. Analysis of its 18S rDNA sequence identified the isolate as genotype T4. The T4 genotype is the most commonly reported among keratitis isolates and the most common in environmental samples.