Melissa Jamerson

Free-living amoebae, Legionella and Mycobacterium in tap water supplied by a municipal drinking water utility in the USA

Legionella and Mycobacterium can proliferate within free-living amoebae (FLA) where they are protected from disinfectants at concentrations that can kill bacteria but not protozoa. Despite effective treatment of drinking water, microbes can enter water utility distribution systems (DS) and hence the plumbing within building premises. Additionally, biofilm formation may account for the persistence of microbes in the DS. In the present study a domestic water tap in north-central United States (USA) was sampled in March and September 2007 and analysed for FLA, Legionella and Mycobacterium. Identification of organisms was determined by growth on specific culture media, light and electron microscopy, and amplification of DNA probes specific for each organism. In both the spring and fall samples, amoebae, Legionella and Mycobacterium were detected. However, Acanthamoeba was prominent in the spring sample whereas Vahlkampfia and Naegleria were the amoebae detected in the autumn. Bacterial proliferation in laboratory cultures was noticeably enhanced in the presence of amoebae and biofilms rapidly formed in mixed amoebae and bacteria cultures. It is hypothesized that temperature affected the dynamics of FLA species population structure within the DS and that pathogenic bacteria that proliferate within FLA, which are themselves opportunistic pathogens, pose dual public health risks.

The interaction between the amoeba Balamuthia mandrillaris and extracellular matrix glycoproteins in vitro.

Balamuthia mandrillaris, a soil amoeba, is the causative agent of Balamuthia granulomatous amoebic encephalitis, a life-threatening brain infection. This amoeba is acquired from contaminated soil and may enter the host through cutaneous lesions or through nasal passages, migrating to the lungs or brain. During invasion, B. mandrillaris has access to components of the extracellular matrix (ECM) of the host. Therefore, we investigated the interaction of B. mandrillaris with 3 ECM glycoproteins (collagen-I, fibronectin and laminin-1) that are encountered in host connective tissues and at the basal lamina. Using optical microscopy, amoeba association on ECM-coated surfaces was examined. Binding of amoebae on laminin was greater than that on collagen or fibronectin. Laminin-adhered B. mandrillaris exhibited elongated and spread forms, distinctive from those observed for amoebae on a plastic surface. Collagen and fibronectin-adhered B. mandrillaris presented elongated shapes with cellular expansions. Binding to collagen, fibronectin, or laminin was inhibited when amoebae were pre-treated with sialic acid. Treatment with galactose resulted in diminished binding of amoebae on laminin, while mannose increased binding in all coating conditions tested. Dependence of divalent cations on amoeba binding was demonstrated for laminin-amoeba interaction. Collectively, the results indicate that B. mandrillaris recognizes specific glycoproteins of the mammalian extracellular matrix.

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. 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. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

Acanthamoeba interaction with extracellular matrix glycoproteins: biological and biochemical characterization and role in cytotoxicity and invasiveness.

ABSTRACT. Acanthamoeba are free‐living amoebae that are dispersed in most environments. Occasionally, Acanthamoeba cause serious human infections, such as keratitis and encephalitis. During the infection process, amoebic adhesion to, and degradation of, host cells and their extracellular matrix (ECM) appear to be important requirements. We examined the interaction of Acanthamoeba with the ECM, and related this event to host cell destruction and tissue invasion. Pathogenic Acanthamoeba culbertsoni differentially attached on the ECM glycoproteins laminin‐1, collagen‐I, and fibronectin, as compared with non‐pathogenic Acanthamoeba astronyxis. Binding to collagen‐I and laminin‐1 induced A. culbertsoni to become flattened and elongated. Because attachment on laminin‐1 was higher in A. culbertsoni, laminin‐1 was chosen for further analysis. A 55‐kDa laminin‐binding protein was identified in pathogenic amoebae, but it was not found in non‐pathogenic amoebae. No differential cytotoxicity against distinct cell types was observed between A. culbertsoni incubated with or without ECM. On the other hand, binding on collagen‐I or matrigel scaffolds induced a differential effect where A. culbertsoni invaded collagen‐I matrices more rapidly. These results indicate that ECM recognition, as an antecedent to tissue invasion, may be a trait characteristic of pathogenic Acanthamoeba.

Acanthamoeba culbertsoni: analysis of amoebic adhesion and invasion on extracellular matrix components collagen I and laminin-1.

Acanthamoeba are free-living amoebae found in most environments that can cause brain and corneal infections. To infect humans, these pathogens must interact with host cells and the extracellular matrix (ECM). In order to define the mode by which amoebae recognize ECM components and process this recognition, we analyzed Acanthamoeba culbertsoni attachment and invasion, respectively, on collagen I and laminin-1 and on tridimensional collagen I and matrigel matrices. We determined that amoebae surface proteins are involved in adhesion, that exogenous sugars can decrease adhesion and invasion, and that adhesion and invasion are dependent on microfilament reorganization. In addition, we determined the role of serine- and metallo-proteases on invasion and found that adhesion was blocked when amoebae were treated with a metallo-protease inhibitor. Collectively, these results suggest that adhesion and invasion are protease- and microfilament-dependent events in which amoebic surface proteins play a pivotal role.

Cannabinoid inhibits HIV-1 Tat-stimulated adhesion of human monocyte-like cells to extracellular matrix proteins

AIMS The aim of this study was to assess the effect of select cannabinoids on human immunodeficiency virus type 1 (HIV-1) transactivating (Tat) protein-enhanced monocyte-like cell adhesion to proteins of the extracellular matrix (ECM). MAIN METHODS Collagen IV, laminin, or an ECM gel was used to construct extracellular matrix layers. Human U937 monocyte-like cells were exposed to Tat in the presence of ∆(9)-tetrahydrocannabinol (THC), CP55,940, and other select cannabinoids. Cell attachment to ECM proteins was assessed using an adhesion assay. KEY FINDINGS THC and CP55,940 inhibited Tat-enhanced attachment of U937 cells to ECM proteins in a mode that was linked to the cannabinoid receptor type 2 (CB2R). The cannabinoid treatment of Tat-activated U937 cells was associated with altered β1-integrin expression and distribution of polymerized actin, suggesting a modality by which these cannabinoids inhibited adhesion to the ECM. SIGNIFICANCE The blood-brain barrier (BBB) is a complex structure that is composed of cellular elements and an extracellular matrix (ECM). HIV-1 Tat promotes transmigration of monocytes across this barrier, a process that includes interaction with ECM proteins. The results indicate that cannabinoids that activate the CB2R inhibit the ECM adhesion process. Thus, this receptor has potential to serve as a therapeutic agent for ablating neuroinflammation associated with HIV-elicited influx of monocytes across the BBB.

Identification of Peptidases in Highly Pathogenic vs. Weakly Pathogenic Naegleria fowleri Amebae

Naegleria fowleri, a free‐living ameba, is the causative agent of Primary Amebic Meningoencephalitis. Highly pathogenic mouse‐passaged amebae (Mp) and weakly pathogenic axenically grown (Ax) N. fowleri were examined for peptidase activity. Zymography and azocasein peptidase activity assays demonstrated that Mp and Ax N. fowleri exhibited a similar peptidase pattern. Prominent for whole cell lysates, membranes and conditioned medium (CM) from Mp and Ax amebae was the presence of an activity band of approximately 58 kDa that was sensitive to E64, a cysteine peptidase inhibitor. However, axenically grown N. fowleri demonstrated a high level of this peptidase activity in membrane preparations. The inhibitor E64 also reduced peptidase activity in ameba‐CM consistent with the presence of secreted cysteine peptidases. Exposure of Mp amebae to E64 reduced their migration through matrigel that was used as an extracellular matrix, suggesting a role for cysteine peptidases in invasion of the central nervous system (CNS). The collective results suggest that the profile of peptidases is not a discriminative marker for distinguishing Mp from Ax N. fowleri. However, the presence of a prominent level of activity for cysteine peptidases in N. fowleri membranes and CM, suggests that these enzymes may serve to facilitate passage of the amebae into the CNS.

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.

Marijuana use and brain immune mechanisms.

The recreational smoking of marijuana, or Cannabis sativa, has become widespread, including among adolescents. Marijuana contains a class of compounds known as phytocannabinoids that include cannabidiol (CBD) and Δ(9)-tetrahydrocannabinol (THC). THC is the major psychoactive component in marijuana, but also exhibits immunosuppressive activity. CBD, while not psychotropic, also modulates immune function, but its mechanism of action appears to differ from that of THC. Since both compounds are highly lipophilic, they readily passage the blood-brain barrier and access the central nervous system. Since CBD is not psychotropic, it has been considered as a candidate therapeutic compound for ablating neuropathological processes characterized by hyperinflammation. However, an unresolved question centers around the impact of these compounds on immune-competent cells within the CNS in relation to susceptibility to infection. There are accumulating data indicating that THC inhibits the migratory capability of macrophage-like cells resident in the CNS, such as microglia, toward nodes of microbial invasion. Furthermore, phytocannabinoids have been reported to exert developmental and long-term effects on the immune system suggesting that exposure to these substances during an early stage in life has the potential to alter the fundamental neuroimmune response to select microbial agents in the adult.

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.