Area 51: How do Acanthamoeba invade the central nervous system?

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[email protected] Department of Biological & Biomedical Sciences

Medical College, Pakistan

May 2011

Area 51: How do Acanthamoeba invade the central nervous system? Ruqaiyyah Siddiqui Aga Khan University

Richard Emes Hany Eisheikha Naveed Ahmed Khan Aga Khan University

Follow this and additional works at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs Part of the Nervous System Commons, and the Parasitology Commons Recommended Citation Siddiqui, R., Emes, R., Eisheikha, H., Khan, N. (2011). Area 51: How do Acanthamoeba invade the central nervous system?. Trends in Parasitology, 27(5), 185-189. Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/40

Opinion

Area 51: How do Acanthamoeba invade the central nervous system? Ruqaiyyah Siddiqui1, Richard Emes2, Hany Elsheikha2 and Naveed Ahmed Khan1,2 1 2

Aga Khan University, Stadium Road, Karachi, Pakistan School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington, United Kingdom

Acanthamoeba granulomatous encephalitis generally develops as a result of haematogenous spread, but it is unclear how circulating amoebae enter the central nervous system (CNS) and cause inflammation. At present, the mechanisms which Acanthamoeba use to invade this incredibly well-protected area of the CNS and produce infection are not well understood. In this paper, we propose two key virulence factors: mannose-binding protein and extracellular serine proteases as key players in Acanthamoeba traversal of the blood–brain barrier leading to neuronal injury. Both molecules should provide excellent opportunities as potential targets in the rational development of therapeutic interventions against Acanthamoeba encephalitis. Neuropathology due to Acanthamoeba Acanthamoeba granulomatous encephalitis (AGE; also known as granulomatous amoebic encephalitis) occurs in immunocompromised or debilitated patients and almost always leads to death (see Glossary). The predisposing factors include HIV infection, diabetes, immunosuppressive therapy, malignancies, malnutrition or alcoholism. The risk factors for patients suffering from the above diseases include exposure to contaminated water such as swimming pools, on beaches, working with garden soil and artificial man-made environments. Acanthamoeba attack the brain tissue and produce subacute necrotising haemorrhagic encephalitis leading to brain dysfunction and finally death [1–3]. Typically, encephalitis is of the granulomatous type composed of CD4 and CD8 T cells, B lymphocytes, plasma cells, macrophages and multinucleate giant cells [1–3]. However, in immunocompromised patients with severely impaired cellular immune response, granuloma formation can be minimal. Depending on the location of the lesions, Acanthamoeba encephalitis exhibits a broad spectrum of neurological signs and symptoms. In the majority of cases, lesions are most numerous in the temporal and parietal lobes. However, severely immunocompromised patients might not show such lesions and exhibit disseminated infections leading to death [1–3]. With the AIDS pandemic and the increasing numbers of debilitated patients, opportunistic pathogens such as Acanthamoeba are of growing concern. Worryingly, there is no recommended treatment against AGE and most of the cases are diagnosed postmortem. Thus, there is an urgent need to design therapeutic strategies against this often fatal Corresponding author: Khan, N.A. ([email protected]).

infection. In this paper, we describe mannose-binding protein (MBP) and extracellular serine proteases as important molecules in Acanthamoeba pathogenesis that could serve as potential targets in the development of therapeutic interventions against AGE. Invasion of the cerebral endothelium For the majority of microbial pathogens, the two probable routes of entry to the central nervous system (CNS) are through the olfactory neuroepithelium via the nasal passage or cerebral capillaries via the bloodstream. In humans, Acanthamoeba spp. are usually blood-borne, whereas other free-living amoeba, Naegleria spp., enter through the olfactory neuroepithelial route [4,5]. Although olfactory neuroepithelium is a possible route of entry into the CNS, it is commonly accepted that the routes of entry for Acanthamoeba include the respiratory tract, leading to amoebae invasion of the alveolar blood vessels, followed by haematogenous spread. Skin lesions can provide direct entry into the bloodstream, bypassing the lower respiratory tract. Amoebae entry into the CNS most probably occurs through the endothelial lining of cerebral capillaries [1,2]. In support, haematoxylin and eosin-stained sections of the brain tissue of Acanthamoeba encephalitis patients exhibit large numbers of amoebae in the perivascular space [2,6]. The fact that various tissues, other than the CNS, are

Glossary Aphasia: inability to comprehend language. Brain oedema: an excess accumulation of water in the intracellular or extracellular spaces of the brain. Clinical syndrome of Acanthamoeba encephalitis: the clinical symptoms resemble viral or bacterial meningitis and are characterised by headache, fever, stiff neck, nausea, sleepiness, mood swings, hemiparesis, aphasia, vomiting, acute confused state, cranial nerve palsies, increased intracranial pressure, seizures, brain oedema and finally lead to death. Cranial nerve palsies: damage to one or more of the cranial nerves where muscle becomes paralysed or someone loses control of it. Leukocytes extravasation: movement of leukocytes out of the circulatory system, towards the site of tissue damage or infection. Hemiparesis: partial paralysis of one side of the body. Host determinants that can contribute to Acanthamoeba encephalitis: interleukin alpha, interleukin beta, tumour necrosis factor alpha, gamma interferon, other mediators, immune cells and host cell apoptosis. Intracranial pressure: the pressure exerted by the cranium on brain tissue, cerebrospinal fluid and circulating blood volume of the brain. Meningitis: infection or inflammation of the meninges, that is membranes that enclose the brain and the spinal cord. Parasite-derived factors that can contribute to Acanthamoeba encephalitis: adhesins such as mannose-binding protein, laminin-binding protein, ectoATPases, neuraminidases, proteases, elastases, phospholipases and glycosidases.

1471-4922/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2011.01.005 Trends in Parasitology, May 2011, Vol. 27, No. 5

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Acanthamoeba

Immune cells

Cytokines (IL-α, β, TNFα, IFNγ)

Leukocyte extravasation

Adhesins (such as mannose binding protein)

Extracellular toxins (proteases and other hydrolytic enzymes)

Cerebral endothelium

Blood-side Blood-brain barrier perturbations Blood-brain barrier

CNS-side

Astrocytes and pericytes

Basement membrane TRENDS in Parasitology

Figure 1. Possible mechanisms of Acanthamoeba-mediated blood–brain barrier perturbations. The blood–brain barrier is composed of endothelial cells joined together by the presence of tight junctions, basement membrane, astrocytes and pericytes. This schematic diagram indicates the role of proinflammatory cytokines, leukocytes extravasation, amoeba adhesins and extracellular toxins in Acanthamoeba-mediated brain endothelial cell damage leading to its entry into the central nervous system to produce disease.

affected including skin, liver, lungs and kidneys further suggests haematogenous spread, but what attracts Acanthamoeba to the CNS or other ecological niches of the body remains unclear. Being an extracellular pathogen, Acanthamoeba transmigration of the cerebral endothelium most probably occurs via the paracellular route by disrupting tight junctions [7] and/or via the transcellular route by producing endothelial cell damage [8] which can potentially lead to blood–brain barrier perturbations [7]. Pathology: a joint result of pathogen and host factors The pathophysiology of AGE is multifactorial, that is Acanthamoeba-derived factors are not the only players but also host determinants contribute to Acanthamoeba penetration through various tissues leading to localised tissue degradation (Figure 1) [9]. This is supported with the finding that the characteristic granulomatous lesions in the CNS are a result of the host immune response and are most probably composed of CD4 and CD8 T cells, B lymphocytes, plasma cells, macrophages and Acanthamoeba [1,2]. The localisation of immune cells in the brain suggests the involvement of proinflammatory cytokines in protection as well as in pathophysiological complications including their probable role in the blood–brain barrier perturbations and disease development. Thus, one of the key factors in the disease state is a result of the imbalance in cytokine levels, that is overproduction of proinflammatory cytokines or deficiency of host-protective mechanisms, mediated by anti-inflammatory molecules, resulting in 186

immunosuppression, which can determine the susceptibility to and severity of Acanthamoeba encephalitis (reviewed in [10]). A complete understanding of pathogen–host interactions, Acanthamoeba survival in the bloodstream and traversal of the blood–brain barrier during the course of the disease will provide insights to its neuropathogenesis and could help the development of novel therapeutic interventions. For the latter, it is noteworthy that endothelial cells in vivo, that are part of the fully formed blood–brain barrier, are not always replicating and are under constant blood flow in comparison with the growing endothelial cell lines. The loss of ‘cell cycle control’ implies that adaptive mechanisms that maintain homeostasis or haemostasis might not be induced, and these aspects need investigation accordingly. For pathogen factors, studies have shown that Acanthamoeba adhesin, MBP, plays an important role in Acanthamoeba-mediated human blood–brain barrier dysfunction in vitro and requires further investigation as a therapeutic target against this often fatal infection [7,8,11,12]. Acanthamoeba mannose-binding protein The structure of MBP The domain architecture of MBP was determined using a simple modular architecture research tool (SMART) [13]; the MBP protein consists of a signal peptide (amino acids 1–21), an extracellular cysteine (C) rich region comprising 14 CxCxC repeats where x is any amino acid (Pfam PF03128 covering amino acid positions 274–615) and a

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CxCxC Motif Extracellular

Intracellular TRENDS in Parasitology

Figure 2. Schematic diagram of the domain architecture of mannose-binding protein (MBP). The domain architecture of MBP was determined using a simple modular architecture research tool (SMART) and indicated a signal peptide, an extracellular cysteine-rich region comprising 14 CxCxC repeats where x is any amino acid and a single predicted transmembrane region.

single predicted transmembrane region (amino acids 733– 755) [14,15,16] (Figure 2). The CxCxC repeat motif is found in several proteins. Pfam version 24.0 reports 157 occurrences of this motif in 37 unique sequences from 27 species (24 eukaryotes, 2 bacteria and 3 viruses). The motif is often associated with other domains comprising a total of 13 domain architectures. In total, 17 of these sequences feature co-occurrence between one and three CxCxC repeats and the PDGF domain (platelet-derived growth factor, Pfam PF00341) such as is seen in the vascular endothelial growth factor C (VEGF-C) proteins from varied vertebrate species. Although 22 of the 37 proteins, including the VEGF-C proteins, contain a signal peptide, they are predicted to be secreted from the expressing cell. MBP is the only reported CxCxC repeat containing protein which also contains a transmembrane domain, a finding that has also been biochemically verified [17]. This unique architecture results in the CxCxC repeat region extending to the extracellular space while remaining tethered to the surface of Acanthamoeba. Potential function of the MBP CxCxC repeat region Even as the function of this repeat region in MBP has not been determined, the conserved periodicity of the cysteines in the multiple CxCxC repeat motifs of MBP suggests a complex tertiary structure formed by disulfide crosslinking as seen in many cysteine rich proteins. Thus, the extracellular portion of MBP could potentially induce binding between individual protozoa to form aggregates as observed during cyst formation, to coordinate behaviour of local density of amoebae population as observed in quorum sensing, or simply act as an adhesin between Acanthamoeba and host glycoprotein(s). Alternatively, from studies of other CxCxC repeat motifs containing proteins such as VEGF-C, we might be able to infer a potential function.

Under normal conditions, the active lymphangiogenic VEGF-C protein is cleaved from a preprotein by removal of both the amino (N-)- and carboxy (C-)-terminal propeptide domains. The central PDGF domain containing portion is then secreted and forms a homodimer prior to binding to vascular endothelial growth factor receptor 2 (VEGFR2) [18]. However, engineered VEGF-C chimeras which maintain the C-terminal CxCxC repeat motifs (Cterminal cysteine rich silk domain [19]) result in increased angiogenesis [19], suggesting the potential importance of MBP in this process and its possible role in increased blood–brain barrier permeability. Conversely, the presence of the CxCxC repeat motif in the dipteran silk and Balbiani Ring 3 proteins suggest that the motif could have a structural or protein-binding role. The Balbiani ring 3 gene is expressed in the salivary glands from chromosome regions of intense transcription known as Balbiani rings. The genes are expressed in the fourth larval stage and encode a 185 kDa secretory protein [20]. The protein is used in the composition of a large water insoluble structure, the larval tube. It has been proposed that the Balbiani ring protein 3 binds to other proteins to prevent the formation of water insoluble fibres in the salivary gland lumen. The aforementioned functional properties have been implicated in cholesterol-mediated blood– brain barrier leakage [21,22]. Whether the protein-binding function or hydrophobic properties of MBP plays a role in Acanthamoeba-mediated blood–brain barrier perturbations requires further investigation. Evolution of the MBP gene A sequence similarity approach was used to investigate if the true ancestry of the MBP protein could be determined. Using a PSI-BLASTP search [23] with the full length MBP protein against the non-redundant database at NCBI, significant similarity is detected in several proteins including the dipteran proteins, the 185 kDa silk protein of Chironomus pallidivittatus (AAA99803.1), the 220 kDa silk protein of Chironomus thummi (AAA99804.1) and Balbiani ring protein 3 of Chironomus tentans, as described previously [14]. In addition, multiple significant hits are identified between the N terminus of MBP and various hypothetical bacterial and viral proteins. However, the known domain structure of MBP needs to be considered, as the region of sequence similarity is within the CxCxC motif region, which has low sequence complexity and is compositionally biased with many cysteine residues and hence a short region of similarity might not reflect a shared ancestry [24]. If the PSI-BLAST search is repeated with the full CxCxC motif (xxxxxxxxCxCxCx as reported by Pfam HMM PF03128) masked, no similarity to the silk or Balbiani ring proteins is detected after three PSI-BLAST iterations. Although the CxCxC motif containing proteins could share a common function associated with possession of the motif, it is potentially misleading to refer to them as homologues as their shared ancestry is unclear. Extracellular serine proteases and blood–brain barrier dysfunction Pathogenic Acanthamoeba have been shown to exhibit increased extracellular protease activities [25]. The link 187

Opinion between pathogenicity and the increased levels of extracellular proteases suggest that pathogenic Acanthamoeba utilise proteases to tease apart the host tissues. Using primary human brain microvascular endothelial cells which constitute the blood–brain barrier, the extracellular serine proteases of Acanthamoeba were shown to perturb the integrity of the host cell monolayer by degrading the tight junction proteins including occludin and zonula-occludens-1, resulting in increased permeability of the blood–brain barrier. These findings were supported using the serine protease inhibitor, phenylmethylsulfonyl fluoride (PMSF), which abolished protease-mediated human brain microvascular endothelial cells (HBMEC) permeability and loss of transendothelial electrical resistance [7]. Several extracellular serine proteases have been identified with approximate molecular weights ranging from 12 to 230 kDa [26–33]. Of these, the 133 kDa serine protease MIP133 is identified as a crucial component of the pathogenic cascade of Acanthamoeba. The MIP133 serine protease induces the apoptosis of keratocytes, iris ciliary body cells, the retinal pigment epithelial cells, corneal epithelial cells and the corneal endothelial cells. A direct functional role of serine proteases in Acanthamoeba infections is indicated by the observations that the intrastromal injections of Acanthamoeba conditioned medium produced corneal lesions in vivo, similar to those observed in Acanthamoeba keratitis patients, and this effect is inhibited by PMSF [32,34]. In addition, the chemically synthesised small interfering RNA that is highly specific and efficient in silencing the catalytic domain of the extracellular serine proteases of Acanthamoeba reduces protease activity and amoeba-mediated host cell cytotoxicity [35]. These results support the idea that extracellular serine proteases are directly involved in the pathogenesis and virulence of Acanthamoeba. Although Acanthamoeba is known to produce serine, cysteine and metalloproteases [9], it is noteworthy that only serine proteases are implicated in Acanthamoeba-mediated blood– brain barrier perturbations [7,12,36]. Proteases as drug targets Proteases are well-known virulence factors in the majority of viral, bacterial, protozoan and multicellular pathogens. Proteolytic enzymes play significant roles in the life cycle of protozoan pathogens or the pathogenesis of the diseases they produce. These comprise processing of the surface proteins of the host or parasite for the invasion of the host cells, digestion of the host proteins for nutrition and inactivation of the host immune defence mediators [37]. Different types of proteases are frequently expressed at different stages of the parasite life cycle to support parasite replication and metamorphosis. Thus, proteases are valuable drug targets in the treatment of infections caused by protozoa. For example, serine proteases in trypanosomatids, oligopeptidase B (OpdB) and prolyl oligopeptidase (POP) represent potential drug targets. During host cell entry, Trypanosoma cruzi OpdB is thought to generate a Ca2+-signalling agonist that mediates entry of the parasite into nonphagocytic cells [38]. Targeted deletion of OpdB impairs the ability of T. cruzi to invade host cells and attenuates virulence in vivo [39]. T. cruzi POP, which specifically hydrolyses human collagen (types I and IV) 188

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and fibronectin, has been implicated in the adhesion of the parasite to host cells and cell entry [40]. Studies have shown that the invasive capacity of T. cruzi is reduced in vitro in the presence of OpdB and POP inhibitors [39,41]. Likewise, cysteine proteases of African trypanosomes are shown to target G-protein-coupled receptors (known as protease activated receptors) on brain endothelium. This leads to the activation of Ca2+-signalling and increased permeability of the blood–brain barrier, suggesting the role of proteases to develop effective drug therapies [42,43]. The Leishmania OpdB gene has also been cloned and a structural homology model has been produced [44]. The serine protease inhibitors L-1-tosylamido-2-phenylethyl chloromethyl ketone, benzamidine and a sea anemonederived Kunitz-type inhibitor were found to have leishmanicidal activity against Leishmania amazonensis and induced changes in the ultrastructure of the flagellar pocket of the parasite [45]. Accordingly, proteases have been validated as targets in several parasitic infections. Despite challenges in developing drugs for new protease targets, they have been shown to be ‘druggable’ targets as evidenced by the widespread use of protease inhibitors as effective therapy for hypertension and AIDS, and the current clinical development of protease inhibitors for diabetes, cancer, thrombosis and osteoporosis. As long as issues such as the difficulty of achieving selectivity can be addressed through targeting allosteric sites, proteasebased drug therapy has tremendous potential in the treatment of many infectious diseases such as AGE. Perspective Overall, the adhesin, MBP and extracellular serine proteases of Acanthamoeba appear to play a key role in the pathogenesis of AGE. The availability of the Acanthamoeba genome [http://www.hgsc.bcm.tmc.edu/ microbial-detail.xsp?project_id=163], together with the recently developed transfection assays [46,47], and the RNA interference methods [35], will undoubtedly increase the pace of our understanding of this complex but fascinating organism and its neuropathogenesis. The availability of tools such as in vitro [7] and in vivo models of the blood– brain barrier [48] should allow us to test the role of specific virulence factors and their potent but nontoxic inhibitors in the penetration of tissue barriers to produce infection and then design strategies to intervene and/or prevent disease. Acknowledgments We apologise to authors we have not referenced owing to space limitations.

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