A COMPARATIVE STUDY OF A PATHOGENIC VERSUS A NONPATHOGENIC NAEGLERIA SPECIES

Naegleria fowleri (N. fowleri) and Naegleria lovaniensis (N. lovaniensis) are closely related amebae found in the environment. N. fowleri causes Primary Amebic Meningoencephalitis (PAM), a fatal disease of the central nervous system, while N. lovaniensis is nonpathogenic. N. fowleri infection occurs when amebae enter the nasal passages, and migrate to the brain. The molecular mechanisms involved in the pathogenesis of PAM are not well-defined. Therefore, the purpose of this study was to define phenotypic characteristics that may be functionally linked to the pathogenicity associated with N. fowleri. Studies revealed that N. fowleri has a faster growth rate and is more resistant to complement-mediated lysis when compared to N. lovaniensis. Additionally, contact-independent cytotoxicity was observed only for N. fowleri. The ability to invade tissues can be a characteristic that distinguishes pathogens from nonpathogens. Therefore, adhesion to extracellular matrix components (ECM), laminin-1, fibronectin, and collagen I, was assessed. N. fowleri exhibited a higher level of adhesion to ECM components and was shown to invade tri-dimensional ECM scaffolds (matrigel and collagen I) to a greater extent than N. lovaniensis. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Attachment of N. fowleri to ECM components was decreased significantly when amebae were pretreated with trypsin, suggesting a role for a surface protein in this process. Pretreatment of N. fowleri amebae with periodate, a sugar oxidant, led to a decrease in attachment to laminin-1 and fibronectin suggesting that the surface component contained a sugar moiety. Western immunoblotting revealed two integrin-like proteins for both species. However, one with a molecular mass of approximately 70 kDa, was detected at a higher level for N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. An anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Zymographic analysis demonstrated differential expression of proteases occurs when N. fowleri and N. lovaniensis invade ECM components using an in vitro invasion assay. These results indicate a distinction in adhesion to, and invasion of, extracellular matrix proteins between N. fowleri and N. lovaniensis

Identification of Naegleria fowleri proteins linked to primary amoebic meningoencephalitis

Naegleria fowleri (N. fowleri) causes primary amoebic meningoencephalitis, a rapidly fatal disease of the central nervous system. N. fowleri can exist in cyst, flagellate or amoebic forms, depending on environmental conditions. The amoebic form can invade the brain following introduction into the nasal passages. When applied intranasally to a mouse model, cultured N. fowleri amoebae exhibit low virulence. However, upon serial passage in mouse brain, the amoebae acquire a highly virulent state. In the present study, a proteomics approach was applied to the identification of N. fowleri amoeba proteins whose expression was associated with the highly virulent state in mice. Mice were inoculated intranasally with axenically cultured amoebae or with mouse-passaged amoebae. Examination by light and electron microscopy revealed no morphological differences. However, mouse-passaged amoebae were more virulent in mice as indicated by exhibiting a two log10 titre decrease in median infective dose 50 (ID50). Scatter plot analysis of amoebic lysates revealed a subset of proteins, the expression of which was associated with highly virulent amoebae. MS-MS indicated that this subset contained proteins that shared homology with those linked to cytoskeletal rearrangement and the invasion process. Invasion assays were performed in the presence of a select inhibitor to expand on the findings. The collective results suggest that N. fowleri gene products linked to cytoskeletal rearrangement and invasion may be candidate targets in the management of primary amoebic meningoencephalitis

SJL Mice Infected with Acanthamoeba castellanii Develop Central Nervous System Autoimmunity through the Generation of Cross-Reactive T Cells for Myelin Antigens

We recently reported that Acanthamoeba castellanii (ACA), an opportunistic pathogen of the central nervous system (CNS) possesses mimicry epitopes for proteolipid protein (PLP) 139–151 and myelin basic protein 89–101, and that the epitopes induce experimental autoimmune encephalomyelitis (EAE) in SJL mice reminiscent of the diseases induced with their corresponding cognate peptides. We now demonstrate that mice infected with ACA also show the generation of cross-reactive T cells, predominantly for PLP 139–151, as evaluated by T cell proliferation and IAs/dextramer staining. We verified that PLP 139–151-sensitized lymphocytes generated in infected mice contained a high proportion of T helper 1 cytokine-producing cells, and they can transfer disease to naïve animals. Likewise, the animals first primed with suboptimal dose of PLP 139–151 and later infected with ACA, developed EAE, suggesting that ACA infection can trigger CNS autoimmunity in the presence of preexisting repertoire of autoreactive T cells. Taken together, the data provide novel insights into the pathogenesis of Acanthamoeba infections, and the potential role of infectious agents with mimicry epitopes to self-antigens in the pathogenesis of CNS diseases such as multiple sclerosis

Use of a Climate and Culture Survey to Assess Diversity, Equity, and Inclusion Initiatives at the VCU School of Social Work

The project aimed to create a survey to assess the effectiveness of previously implemented diversity, equity and inclusion (DEI) initiatives, specifically racial equity at the VCU School of Social Work. The VCU School of Social Work has been actively working with their faculty and staff to address DEI at their school and encourage them to become active in DEI initiatives in other areas in the community. School of Social Work leadership was looking for a way to assess if those DEI initiatives met their intended purpose. This project researched ways to assess the effectiveness of DEI initiatives. The result was the creation of an electronic survey distributed to School of Social Work faculty and staff in the fall of 2021

SJL Mice Infected with \u3ci\u3eAcanthamoeba castellanii\u3c/i\u3e Develop Central Nervous System Autoimmunity through the Generation of Cross-Reactive T Cells for Myelin Antigens

We recently reported that Acanthamoeba castellanii (ACA), an opportunistic pathogen of the central nervous system (CNS) possesses mimicry epitopes for proteolipid protein (PLP) 139–151 and myelin basic protein 89–101, and that the epitopes induce experimental autoimmune encephalomyelitis (EAE) in SJL mice reminiscent of the diseases induced with their corresponding cognate peptides. We now demonstrate that mice infected with ACA also show the generation of crossreactive T cells, predominantly for PLP 139–151, as evaluated by T cell proliferation and IAs/dextramer staining. We verified that PLP 139–151-sensitized lymphocytes generated in infected mice contained a high proportion of T helper 1 cytokineproducing cells, and they can transfer disease to naı¨ve animals. Likewise, the animals first primed with suboptimal dose of PLP 139–151 and later infected with ACA, developed EAE, suggesting that ACA infection can trigger CNS autoimmunity in the presence of preexisting repertoire of autoreactive T cells. Taken together, the data provide novel insights into the pathogenesis of Acanthamoeba infections, and the potential role of infectious agents with mimicry epitopes to self-antigens in the pathogenesis of CNS diseases such as multiple sclerosis

ACA infection alters susceptibility to EAE in mice primed with suboptimal doses of PLP 139–151.

<p>Groups of SJL mice were immunized with or without PLP 139–151 (2.5 or 10 µg/mouse) in CFA. On day 7 post-immunization, the animals were infected with ACA (1×10³) and the animals were monitored for clinical signs of EAE and scored. Data represent mean EAE scores for a group of mice pooled from two to three individual experiments.</p

Histologic evaluation of EAE in mice that received lymphocytes from ACA-infected mice.

†<p>represents mice that showed histological disease.</p

Antigen-sensitized lymphocytes from ACA<i>-</i>infected mice contain predominantly Th1 cytokine-producing cells.

<p>Groups of SJL mice were infected with ACA (1×10³). After 21 days, mice were killed, and lymphocytes were prepared. Cells were stimulated with PLP 139–151 or ACA 83–95 for two days and the cultures were maintained in IL-2 medium. Cells harvested on day 5 from the above cultures or those obtained from naïve mice were stimulated with PMA (20 ng/ml) and ionomycin (300 ng/ml) for ∼5 hours in the presence of 2 mM monensin followed by staining with anti-CD4 and 7-AAD. After fixation and permeabilization, cells were stained with cytokine antibodies and acquired by FC. Frequencies of cytokine-producing cells were then analyzed in the live (7-AAD⁻) CD4 subset. <u>Inset:</u> The combined frequencies of Th1 (IFN-γ), Th2 (IL-4+ IL-10) and Th17 (IL-17A+IL-17F+IL-22) cytokine-producing cells were calculated and compared between groups. Mean ± SEM values obtained from five independent experiments involving two mice in each are shown.</p

ACA infection leads to the generation of PLP 139–151-reactive T cells.

<p><u>Top panels:</u> Groups of SJL mice were infected intranasally with ACA trophozoites (5×10³) and after 30 days, the mice were killed and spleens were harvested to prepare single cell suspensions. Cells were stimulated with PLP 139–151 or ACA 83–95 (top left panel), MBP 89–101 or NAD 108–120 (top right panel) for two days followed by pulsing with ³[H] thymidine, the incorporation of which was measured as cpm 16 hours later. TMEV 70–86, control. Mean ± SEM values from three individual experiments involving one mouse in each are shown. <u>Middle and bottom panels:</u> Lymphocytes were obtained from infected animals on day 10 post-infection and CD4 or CD8 T cells were then purified. Cells were stimulated with PLP 139–151 or ACA 83–95 (middle- and bottom left panels), MBP 89–101 or NAD 108–120 (middle- and bottom right panels) in the presence of irradiated APCs, and proliferative responses were measured as above. TMEV 70–86, control. Mean ± SEM values from one of the four individual experiments involving a group of five mice in each are shown.</p