Hyaluronic Acid Receptor CD44 Deficiency Is Associated with Decreased Cryptococcus neoformans Brain Infection
2012; Elsevier BV; Volume: 287; Issue: 19 Linguagem: Inglês
10.1074/jbc.m112.353375
ISSN1083-351X
AutoresAmbrose Jong, Chunhua Wu, Ignacio Gonzales-Gomez, Kyung J. Kwon‐Chung, Yun C. Chang, Hsiang-Kuang Tseng, Wen-Long Cho, Sheng‐He Huang,
Tópico(s)Antifungal resistance and susceptibility
ResumoCryptococcus neoformans is a pathogenic yeast that can invade the brain and cause meningoencephalitis. Our previous in vitro studies suggested that the interaction between C. neoformans hyaluronic acid and human brain endothelial CD44 could be the initial step of brain invasion. In this report, we used a CD44 knock-out (KO or CD44−/−) mouse model to explore the importance of CD44 in C. neoformans brain invasion. Our results showed that C. neoformans-infected CD44 KO mice survived longer than the infected wild-type mice. Consistent with our in vitro results, the brain and cerebrospinal fluid fungal burden was reduced in CD44-deficient mice. Histopathological studies showed smaller and fewer cystic lesions in the brains of CD44 KO mice. Interestingly, the cystic lesions contained C. neoformans cells embedded within their polysaccharide capsule and were surrounded by host glial cells. We also found that a secondary hyaluronic acid receptor, RHAMM (receptor of hyaluronan-mediated motility), was present in the CD44 KO mice. Importantly, our studies demonstrated an in vivo blocking effect of simvastatin. These results suggest that the CD44 and RHAMM receptors function on membrane lipid rafts during invasion and that simvastatin may have a potential therapeutic role in C. neoformans infections of the brain. Cryptococcus neoformans is a pathogenic yeast that can invade the brain and cause meningoencephalitis. Our previous in vitro studies suggested that the interaction between C. neoformans hyaluronic acid and human brain endothelial CD44 could be the initial step of brain invasion. In this report, we used a CD44 knock-out (KO or CD44−/−) mouse model to explore the importance of CD44 in C. neoformans brain invasion. Our results showed that C. neoformans-infected CD44 KO mice survived longer than the infected wild-type mice. Consistent with our in vitro results, the brain and cerebrospinal fluid fungal burden was reduced in CD44-deficient mice. Histopathological studies showed smaller and fewer cystic lesions in the brains of CD44 KO mice. Interestingly, the cystic lesions contained C. neoformans cells embedded within their polysaccharide capsule and were surrounded by host glial cells. We also found that a secondary hyaluronic acid receptor, RHAMM (receptor of hyaluronan-mediated motility), was present in the CD44 KO mice. Importantly, our studies demonstrated an in vivo blocking effect of simvastatin. These results suggest that the CD44 and RHAMM receptors function on membrane lipid rafts during invasion and that simvastatin may have a potential therapeutic role in C. neoformans infections of the brain. IntroductionCryptococcus neoformans invades the brain and causes meningoencephalitis, primarily in immunocompromised patients and sporadically in normal hosts. It is the most common fungal infection of the central nervous system and is one of the major causes of death in AIDS patients (1Bicanic T. Harrison T.S. Cryptococcal meningitis.Br. Med Bull. 2004; 72: 99-118Crossref PubMed Scopus (293) Google Scholar, 2Mitchell T.G. Perfect J.R. Cryptococcosis in the era of AIDS: 100 years after the discovery of Cryptococcus neoformans.Clin Microbiol. Rev. 1995; 8: 515-548Crossref PubMed Google Scholar). C. neoformans is encompassed by a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM) 3The abbreviations used are: GXMglucuronoxylomannanCSFcerebrospinal fluidBMECbrain microvascular endothelial cellGFAPglial fibrillary acidic proteinHAhyaluronic acid or hyaluronanHBMEChuman brain microvascular endothelial cellRHAMMreceptor of hyaluronan-mediated motilityYPDyeast extract, peptone, and dextrose. (3Zaragoza O. Rodrigues M.L. De Jesus M. Frases S. Dadachova E. Casadevall A. The capsule of the fungal pathogen Cryptococcus neoformans.Adv. Appl. Microbiol. 2009; 68: 133-216Crossref PubMed Scopus (298) Google Scholar); this capsule is known to be the major virulence factor of this yeast (4Chang Y.C. Kwon-Chung K.J. Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence.Mol. Cell. Biol. 1994; 14: 4912-4919Crossref PubMed Scopus (390) Google Scholar, 5McFadden D. Zaragoza O. Casadevall A. The capsular dynamics of Cryptococcus neoformans.Trends Microbiol. 2006; 14: 497-505Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 6Yauch L.E. Lam J.S. Levitz S.M. Direct inhibition of T-cell responses by the Cryptococcus capsular polysaccharide glucuronoxylomannan.PLoS Pathog. 2006; 2: e120Crossref PubMed Scopus (83) Google Scholar). As such, the role and biogenesis of the capsule has become an area of focus for research in this pathogen (7Casadevall A. Nosanchuk J.D. Williamson P. Rodrigues M.L. Vesicular transport across the fungal cell wall.Trends Microbiol. 2009; 17: 158-162Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Previously, we characterized the C. neoformans gene CPS1 (capsule polysaccharide synthase 1) (8Chang Y.C. Jong A. Huang S. Zerfas P. Kwon-Chung K.J. CPS1, a homolog of the Streptococcus pneumoniae type 3 polysaccharide synthase gene, is important for the pathobiology of Cryptococcus neoformans.Infect. Immun. 2006; 74: 3930-3938Crossref PubMed Scopus (53) Google Scholar). Deletion of CPS1 from C. neoformans cells causes alterations in the ultrastructure between the cell wall and capsule, and the deletant shows a reduced ability to associate with human brain microvascular endothelial cells (HBMEC). Polysaccharide hyaluronic acid (HA; also known as hyaluronan) can be detected in the wild-type C. neoformans strain but not in cps1Δ cells. Testing of C. neoformans strains with different concentrations of HA demonstrated that the ability of yeast to bind to HBMEC is proportional to their HA content. Subsequent analysis indicated that the CPS1 gene encodes an HA synthase (9Jong A. Wu C.H. Chen H.M. Luo F. Kwon-Chung K.J. Chang Y.C. Lamunyon C.W. Plaas A. Huang S.H. Identification and characterization of CPS1 as a hyaluronic acid synthase contributing to the pathogenesis of C. neoformans infection.Eukaryot. Cell. 2007; 6: 1486-1496Crossref PubMed Scopus (72) Google Scholar). Taken together, these findings indicate that the C. neoformans CPS1 gene product, HA, plays a role as an adhesion molecule during interaction with endothelial cells.Several HA-binding proteins (or HA receptors) localized in the membranes, such as CD44, RHAMM, Ivd4, LEC receptor, and others, have been identified in different cell types (10Turley E.A. Noble P.W. Bourguignon L.Y. Signaling properties of hyaluronan receptors.J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar, 11Bono P. Rubin K. Higgins J.M. Hynes R.O. Layilin, a novel integral membrane protein, is a hyaluronan receptor.Mol. Biol. Cell. 2001; 12: 891-900Crossref PubMed Scopus (114) Google Scholar). The most common HA receptor is CD44, which plays different roles in different cell types. As CD44 is a major membrane HA receptor, it is conceivable that C. neoformans HA directly engages with the HBMEC CD44 as a part of its invasion mechanism. Indeed, we have demonstrated that C. neoformans HA interacts with CD44 on HBMEC, a primary receptor in C. neoformans infection (12Jong A. Wu C.H. Shackleford G.M. Kwon-Chung K.J. Chang Y.C. Chen H.M. Ouyang Y. Huang S.H. Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells.Cell. Microbiol. 2008; 10: 1313-1326Crossref PubMed Scopus (79) Google Scholar). For example, an anti-CD44 neutralizing antibody treatment significantly reduces C. neoformans association with HBMEC. Association of C. neoformans with HBMEC is also found to be considerably impaired either in CD44 knockdown HBMEC or HA-deficient C. neoformans strains. Likewise, overexpression of CD44 in HBMEC increases the association of C. neoformans with HBMEC. Furthermore, confocal microscopic images show that CD44 is enriched at and around the C. neoformans adhesion sites. Finally, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and β-actin are translocated to the host membrane lipid rafts. Our results highlight the interactions between C. neoformans HA and host CD44. The dynamic results of these interactions may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts by eliciting downstream events of the entry process at HBMEC membrane rafts. Subsequently, membrane signaling may relay through CD44 to the actin cytoskeleton inside the HBMEC cells. Scanning electron microscopic images reveal the course of invasion: internalization by the microvilli embrace of C. neoformans, followed by a zipper-like mechanism in which the host cell plasma membrane encompasses the invading yeasts (13Chang Y.C. Stins M.F. McCaffery M.J. Miller G.F. Pare D.R. Dam T. Paul-Satyaseela M. Kim K.S. Kwon-Chung K.J. Cryptococcal yeast cells invade the central nervous system via transcellular penetration of the blood-brain barrier.Infect. Immun. 2004; 72: 4985-4995Crossref PubMed Scopus (186) Google Scholar). This mechanism requires C. neoformans cell-induced HBMEC cytoskeletal rearrangements for the accumulation of actin at the site of C. neoformans entry. The C. neoformans cell may then be drawn progressively into the host cell.Pathogens usually use multiple mechanisms to invade their host(s). Several studies have suggested that C. neoformans may use different routes to enter the brain. For example, transmission electron microscopic images showed that C. neoformans cells embedded inside macrophages could be found in the crossing site of the brain microcapillaries, suggesting a “Trojan horse” mechanism (14Chrétien F. Lortholary O. Kansau I. Neuville S. Gray F. Dromer F. Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia.J. Infect. Dis. 2002; 186: 522-530Crossref PubMed Scopus (163) Google Scholar). It has also been reported that urease is required for brain invasion (15Shi M. Li S.S. Zheng C. Jones G.J. Kim K.S. Zhou H. Kubes P. Mody C.H. Real-time imaging of trapping and urease-dependent transmigration of Cryptococcus neoformans in mouse brain.J. Clin. Invest. 2010; 120: 1683-1693Crossref PubMed Scopus (148) Google Scholar, 16Olszewski M.A. Noverr M.C. Chen G.H. Toews G.B. Cox G.M. Perfect J.R. Huffnagle G.B. Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion.Am. J. Pathol. 2004; 164: 1761-1771Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The roles of Ure1 and CPS1 in brain invasion are not necessarily mutually exclusive. However, their relevant functions are yet to be elucidated, and our understanding of their complicated invasion process has just begun. Recently, it has been documented that the lipid raft-dependent endocytosis process mediates C. neoformans internalization into HBMEC and that the CD44 protein of the hosts, cytoskeleton, and intracellular kinase DYRK3 are involved in this process (17Huang S.H. Long M. Wu C.H. Kwon-Chung K.J. Chang Y.C. Chi F. Lee S. Jong A. Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3).J. Biol. Chem. 2011; 286: 34761-34769Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Taken together, a transcellular mechanism seems to be the predominant route for C. neoformans brain invasion (13Chang Y.C. Stins M.F. McCaffery M.J. Miller G.F. Pare D.R. Dam T. Paul-Satyaseela M. Kim K.S. Kwon-Chung K.J. Cryptococcal yeast cells invade the central nervous system via transcellular penetration of the blood-brain barrier.Infect. Immun. 2004; 72: 4985-4995Crossref PubMed Scopus (186) Google Scholar, 17Huang S.H. Long M. Wu C.H. Kwon-Chung K.J. Chang Y.C. Chi F. Lee S. Jong A. Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3).J. Biol. Chem. 2011; 286: 34761-34769Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Previously, we had used the in vitro blood-brain barrier model to demonstrate that the C. neoformans HA-HBMEC CD44 interaction plays an initial role during the fungal invasion across the blood-brain barrier (12Jong A. Wu C.H. Shackleford G.M. Kwon-Chung K.J. Chang Y.C. Chen H.M. Ouyang Y. Huang S.H. Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells.Cell. Microbiol. 2008; 10: 1313-1326Crossref PubMed Scopus (79) Google Scholar). The CPS1 deletion strain (C559) is temperature-sensitive (unable to grow at 37 °C), precluding in vivo studies. In this report, we used a mouse model to investigate C. neoformans brain invasion in vivo. In the virulent studies, the CD44 knock-out (KO) mice survived longer than the wild-type mice. Compared with the wild-type mice, the fungal load in the brain and CSF was lower in infected CD44 KO mice. Similarly, the CD44−/− mouse brain contained smaller and fewer cystic lesions. These results suggested that host CD44 is required for C. neoformans brain invasion. Immunofluorescence microscopic images of the brains sections showed that a mass of C. neoformans cells within the lesions are imbedded in their capsule material; these lesions were surrounded by many reactive astrocytes displaying a unique pattern. We also found that, in both the wild-type and CD44−/− mice, a secondary HA receptor, RHAMM, was present in the brain. Finally, we demonstrated the in vivo blocking effect of the hypolipidemic drug simvastatin. Our results suggest that simvastatin may remove receptor function on the membrane lipid raft or yet identified action(s), providing a cue for a novel anticryptococcal intervention.DISCUSSIONC. neoformans has a predilection for the brain and causes devastating cryptococcal meningoencephalitis. However, the mechanism of its brain invasion, resulting in central nervous system dysfunction, is still largely unknown. From our previous in vitro studies (12Jong A. Wu C.H. Shackleford G.M. Kwon-Chung K.J. Chang Y.C. Chen H.M. Ouyang Y. Huang S.H. Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells.Cell. Microbiol. 2008; 10: 1313-1326Crossref PubMed Scopus (79) Google Scholar), we found that C. neoformans uses the host CD44 as the receptor and HA as the ligand in their association. To verify this finding in vivo, we used CD44−/− mice to explore the role of CD44 during C. neoformans invasion. Our results show that CD44−/− mice are more resistant to C. neoformans infection (Fig. 1). This is most likely because C. neoformans cells are less effective in crossing the blood-brain barrier in CD44−/− mice. In support of this hypothesis, the fungal loads of the brain as well as the cfu counts of CSF are lower in CD44−/− mice than in wild-type mice (Fig. 2). Thus, the CD44−/− mouse model is useful for in vivo C. neoformans brain infection studies. As a control, we used a pathogenic Escherichia coli E44 strain to perform the experiments; we observed no difference in invasion (data not shown). Thus, CD44 specifically is required for C. neoformans brain invasion. In complement, several CD44 functions have been documented in CD44−/− murine models from various studies. For example, studies with CD44−/− mice demonstrate that CD44 is a macrophage binding site for Mycobacterium tuberculosis (23Leemans J.C. Florquin S. Heikens M. Pals S.T. van der Neut R. Van Der Poll T. CD44 is a macrophage binding site for Mycobacterium tuberculosis that mediates macrophage recruitment and protective immunity against tuberculosis.J. Clin. Invest. 2003; 111: 681-689Crossref PubMed Scopus (104) Google Scholar). It has also been demonstrated that CD44 plays a critical role in the progression of atherosclerosis through the recruitment of macrophages to the atherosclerotic lesion (24Cuff C.A. Kothapalli D. Azonobi I. Chun S. Zhang Y. Belkin R. Yeh C. Secreto A. Assoian R.K. Rader D.J. Puré E. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation.J. Clin. Invest. 2001; 108: 1031-1040Crossref PubMed Scopus (254) Google Scholar). Moreover, CD44−/− mice injected with ConA exhibited a more severely acute suppurative hepatitis, suggesting that activated T cells use CD44 to undergo apoptosis and that dysregulation in this pathway could lead to increased pathogenesis in hepatitis (25Chen D. McKallip R.J. Zeytun A. Do Y. Lombard C. Robertson J.L. Mak T.W. Nagarkatti P.S. Nagarkatti M. CD44-deficient mice exhibit enhanced hepatitis after concanavalin A injection: evidence for involvement of CD44 in activation-induced cell death.J. Immunol. 2001; 166: 5889-5897Crossref PubMed Scopus (92) Google Scholar). Roles for CD44 in the resolution of lung injury (26Teder P. Vandivier R.W. Jiang D. Liang J. Cohn L. Puré E. Henson P.M. Noble P.W. Resolution of lung inflammation by CD44.Science. 2002; 296: 155-158Crossref PubMed Scopus (567) Google Scholar) and E. coli urinary tract infection (27Rouschop K.M. Sylva M. Teske G.J. Hoedemaeker I. Pals S.T. Weening J.J. van der Poll T. Florquin S. Urothelial CD44 facilitates Escherichia coli infection of the murine urinary tract.J. Immunol. 2006; 177: 7225-7232Crossref PubMed Scopus (41) Google Scholar) have also been demonstrated. Our studies with CD44−/− mice also clearly demonstrate that CD44 plays a role during C. neoformans invasion (FIGURE 1, FIGURE 2, FIGURE 3). We further used anti-GFAP antibodies to examine the distribution of glial cells in infected brain sections. Interestingly, the staining of GFAP-positive glial cells (or reactive astrocytes) with prominent cytoplasmic processes showed a unique pattern, i.e. surrounding a mass of C. neoformans cells (Fig. 4). On the other hand, inflammation is not evident when C. neoformans invades the brain, which substantiates how C. neoformans, in general, could cause chronic meningoencephalitis.However, the use of a CD44−/− murine model is not without limitations. One puzzling occurrence is that there still remain some C. neoformans cells in the brains of CD44−/− mice (Fig. 2). Although CD44 is known to play a wide variety of roles, mice deficient in CD44 do not exhibit defects in development or neurological functions (28Schmits R. Filmus J. Gerwin N. Senaldi G. Kiefer F. Kundig T. Wakeham A. Shahinian A. Catzavelos C. Rak J. Furlonger C. Zakarian A. Simard J.J. Ohashi P.S. Paige C.J. Gutierrez-Ramos J.C. Mak T.W. CD44 regulates hematopoietic progenitor distribution, granuloma formation, and tumorigenicity.Blood. 1997; 90: 2217-2233Crossref PubMed Google Scholar). One possibility is that a second HA receptor may at least partially compensate for CD44 functions in the CD44−/− mice. Indeed, we have observed that another HA receptor, RHAMM, is present in the CD44−/− brain. The expression level of RHAMM is approximately the same in both wild-type and CD44−/−mouse brains (Fig. 5), and its ability to associate with C. neoformans is weaker than that of CD44 in vitro (Fig. 6). Similar observations of RHAMM compensation have been documented in a collagen-induced arthritis CD44 knock-out mouse model (21Nedvetzki S. Gonen E. Assayag N. Reich R. Williams R.O. Thurmond R.L. Huang J.F. Neudecker B.A. Wang F.S. Turley E.A. Naor D. RHAMM, a receptor for hyaluronan-mediated motility, compensates for CD44 in inflamed CD44-knockout mice: a different interpretation of redundancy.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 18081-18086Crossref PubMed Scopus (158) Google Scholar). Based on the knowledge gained from our studies, we favor the possibility that CD44 is a primary HA receptor and that RHAMM, in the case of a CD44−/− background, partially compensates for the lack of CD44 (Fig. 6). The complexity of in vivo models may hinder a further pursuit. For example, a double KO of CD44 and RHAMM may induce expression of another HA receptor or may even cause a lethal phenotype. Additionally, pathogens may use multiple mechanisms to invade hosts and thus complicate the issue further. For example, it has been reported that the Trojan horse mechanism is another possible route for C. neoformans brain invasion (14Chrétien F. Lortholary O. Kansau I. Neuville S. Gray F. Dromer F. Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia.J. Infect. Dis. 2002; 186: 522-530Crossref PubMed Scopus (163) Google Scholar). However, ample evidence shows that a transcellular mechanism may be the major one for C. neoformans brain invasion, as a free C. neoformans cell can be detected inside the brain a short time after i.v. injection (∼0.5 h) (13Chang Y.C. Stins M.F. McCaffery M.J. Miller G.F. Pare D.R. Dam T. Paul-Satyaseela M. Kim K.S. Kwon-Chung K.J. Cryptococcal yeast cells invade the central nervous system via transcellular penetration of the blood-brain barrier.Infect. Immun. 2004; 72: 4985-4995Crossref PubMed Scopus (186) Google Scholar). Although macrophages containing C. neoformans cells near the capillary in the brain parenchyma have been reported, it is unclear whether the inclusion of C. neoformans cells in macrophages occurred after C. neoformans crossed the blood-brain barrier. Macrophages or monocytes alter their morphology significantly when crossing the BMEC, and thus the Trojan horse mechanism may be suitable for viruses but not appropriate for the large size of C. neoformans (>5 μm in diameter plus its rigid cell wall). Overall, the complexity of the in vivo model prompts us to inquire, under certain pathophysiology conditions, as to what is the main mechanism of C. neoformans invasion rather than what specific mechanism(s) C. neoformans uses to invade the brain.Despite the limitations of the in vivo CD44−/− model, it is evident that HA receptors may exert their function(s) on surface membrane rafts. Sufficient evidence indicates that the CD44 (and RHAMM) function on membrane lipid rafts (12Jong A. Wu C.H. Shackleford G.M. Kwon-Chung K.J. Chang Y.C. Chen H.M. Ouyang Y. Huang S.H. Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells.Cell. Microbiol. 2008; 10: 1313-1326Crossref PubMed Scopus (79) Google Scholar) and the endocytic pathway (17Huang S.H. Long M. Wu C.H. Kwon-Chung K.J. Chang Y.C. Chi F. Lee S. Jong A. Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3).J. Biol. Chem. 2011; 286: 34761-34769Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) are crucial for C. neoformans brain invasion. Blocking this pathway can be an effective way of attenuating the fungal infection. Indeed, our results (Fig. 7) show that simvastatin significantly reduced the brain fungal load. One possible mechanism is that simvastatin may exert its role on the membrane lipid rafts, disturbing CD44 and RHAMM functions and consequently attenuating cryptococcal meningitis (Fig. 7, B and C). Notably, it has been shown that statins have other actions (29Miida T. Hirayama S. Nakamura Y. Cholesterol-independent effects of statins and new therapeutic targets: ischemic stroke and dementia.J. Atheroscler. Thromb. 2004; 11: 253-264Crossref PubMed Scopus (95) Google Scholar); for example, administration of simvastatin in injured rat brains improves the outcome through attenuating the NF-κB-mediated inflammatory response (30Chen G. Zhang S. Shi J. Ai J. Qi M. Hang C. Simvastatin reduces secondary brain injury caused by cortical contusion in rats: possible involvement of TLR4/NF-κB pathway.Exp. Neurol. 2009; 216: 398-406Crossref PubMed Scopus (118) Google Scholar). Thus, the exact mechanisms by which simvastatin could offer protection against C. neoformans brain invasion requires further investigation. In any case, simvastatin is an FDA-approved drug with low toxicity. It is significant that simvastatin can drastically reduce the C. neoformans brain burden after treatment. Taken together, our studies support the view that inhibitors of lipid rafts (Ref. 12Jong A. Wu C.H. Shackleford G.M. Kwon-Chung K.J. Chang Y.C. Chen H.M. Ouyang Y. Huang S.H. Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells.Cell. Microbiol. 2008; 10: 1313-1326Crossref PubMed Scopus (79) Google Scholar and Fig. 7), or other regulators of endocytic function (17Huang S.H. Long M. Wu C.H. Kwon-Chung K.J. Chang Y.C. Chi F. Lee S. Jong A. Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3).J. Biol. Chem. 2011; 286: 34761-34769Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) administered in combination with antifungal drugs, could be an effective anti-cryptococcal intervention. IntroductionCryptococcus neoformans invades the brain and causes meningoencephalitis, primarily in immunocompromised patients and sporadically in normal hosts. It is the most common fungal infection of the central nervous system and is one of the major causes of death in AIDS patients (1Bicanic T. Harrison T.S. Cryptococcal meningitis.Br. Med Bull. 2004; 72: 99-118Crossref PubMed Scopus (293) Google Scholar, 2Mitchell T.G. Perfect J.R. Cryptococcosis in the era of AIDS: 100 years after the discovery of Cryptococcus neoformans.Clin Microbiol. Rev. 1995; 8: 515-548Crossref PubMed Google Scholar). C. neoformans is encompassed by a polysaccharide capsule composed mainly of glucuronoxylomannan (GXM) 3The abbreviations used are: GXMglucuronoxylomannanCSFcerebrospinal fluidBMECbrain microvascular endothelial cellGFAPglial fibrillary acidic proteinHAhyaluronic acid or hyaluronanHBMEChuman brain microvascular endothelial cellRHAMMreceptor of hyaluronan-mediated motilityYPDyeast extract, peptone, and dextrose. (3Zaragoza O. Rodrigues M.L. De Jesus M. Frases S. Dadachova E. Casadevall A. The capsule of the fungal pathogen Cryptococcus neoformans.Adv. Appl. Microbiol. 2009; 68: 133-216Crossref PubMed Scopus (298) Google Scholar); this capsule is known to be the major virulence factor of this yeast (4Chang Y.C. Kwon-Chung K.J. Complementation of a capsule-deficient mutation of Cryptococcus neoformans restores its virulence.Mol. Cell. Biol. 1994; 14: 4912-4919Crossref PubMed Scopus (390) Google Scholar, 5McFadden D. Zaragoza O. Casadevall A. The capsular dynamics of Cryptococcus neoformans.Trends Microbiol. 2006; 14: 497-505Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 6Yauch L.E. Lam J.S. Levitz S.M. Direct inhibition of T-cell responses by the Cryptococcus capsular polysaccharide glucuronoxylomannan.PLoS Pathog. 2006; 2: e120Crossref PubMed Scopus (83) Google Scholar). As such, the role and biogenesis of the capsule has become an area of focus for research in this pathogen (7Casadevall A. Nosanchuk J.D. Williamson P. Rodrigues M.L. Vesicular transport across the fungal cell wall.Trends Microbiol. 2009; 17: 158-162Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). Previously, we characterized the C. neoformans gene CPS1 (capsule polysaccharide synthase 1) (8Chang Y.C. Jong A. Huang S. Zerfas P. Kwon-Chung K.J. CPS1, a homolog of the Streptococcus pneumoniae type 3 polysaccharide synthase gene, is important for the pathobiology of Cryptococcus neoformans.Infect. Immun. 2006; 74: 3930-3938Crossref PubMed Scopus (53) Google Scholar). Deletion of CPS1 from C. neoformans cells causes alterations in the ultrastructure between the cell wall and capsule, and the deletant shows a reduced ability to associate with human brain microvascular endothelial cells (HBMEC). Polysaccharide hyaluronic acid (HA; also known as hyaluronan) can be detected in the wild-type C. neoformans strain but not in cps1Δ cells. Testing of C. neoformans strains with different concentrations of HA demonstrated that the ability of yeast to bind to HBMEC is proportional to their HA content. Subsequent analysis indicated that the CPS1 gene encodes an HA synthase (9Jong A. Wu C.H. Chen H.M. Luo F. Kwon-Chung K.J. Chang Y.C. Lamunyon C.W. Plaas A. Huang S.H. Identification and characterization of CPS1 as a hyaluronic acid synthase contributing to the pathogenesis of C. neoformans infection.Eukaryot. Cell. 2007; 6: 1486-1496Crossref PubMed Scopus (72) Google Scholar). Taken together, these findings indicate that the C. neoformans CPS1 gene product, HA, plays a role as an adhesion molecule during interaction with endothelial cells.Several HA-binding proteins (or HA receptors) localized in the membranes, such as CD44, RHAMM, Ivd4, LEC receptor, and others, have been identified in different cell types (10Turley E.A. Noble P.W. Bourguignon L.Y. Signaling properties of hyaluronan receptors.J. Biol. Chem. 2002; 277: 4589-4592Abstract Full Text Full Text PDF PubMed Scopus (861) Google Scholar, 11Bono P. Rubin K. Higgins J.M. Hynes R.O. Layilin, a novel integral membrane protein, is a hyaluronan receptor.Mol. Biol. Cell. 2001; 12: 891-900Crossref PubMed Scopus (114) Google Scholar). The most common HA receptor is CD44, which plays different roles in different cell types. As CD44 is a major membrane HA receptor, it is conceivable that C. neoformans HA directly engages with the HBMEC CD44 as a part of its invasion mechanism. Indeed, we have demonstrated that C. neoformans HA interacts with CD44 on HBMEC, a primary receptor in C. neoformans infection (12Jong A. Wu C.H. Shackleford G.M. Kwon-Chung K.J. Chang Y.C. Chen H.M. Ouyang Y. Huang S.H. Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells.Cell. Microbiol. 2008; 10: 1313-1326Crossref PubMed Scopus (79) Google Scholar). For example, an anti-CD44 neutralizing antibody treatment significantly reduces C. neoformans association with HBMEC. Association of C. neoformans with HBMEC is also found to be considerably impaired either in CD44 knockdown HBMEC or HA-deficient C. neoformans strains. Likewise, overexpression of CD44 in HBMEC increases the association of C. neoformans with HBMEC. Furthermore, confocal microscopic images show that CD44 is enriched at and around the C. neoformans adhesion sites. Finally, upon C. neoformans and HBMEC engagement, a subpopulation of CD44 and β-actin are translocated to the host membrane lipid rafts. Our results highlight the interactions between C. neoformans HA and host CD44. The dynamic results of these interactions may represent events during the adhesion and entry of C. neoformans at HBMEC membrane rafts by eliciting downstream events of the entry process at HBMEC membrane rafts. Subsequently, membrane signaling may relay through CD44 to the actin cytoskeleton inside the HBMEC cells. Scanning electron microscopic images reveal the course of invasion: internalization by the microvilli embrace of C. neoformans, followed by a zipper-like mechanism in which the host cell plasma membrane encompasses the invading yeasts (13Chang Y.C. Stins M.F. McCaffery M.J. Miller G.F. Pare D.R. Dam T. Paul-Satyaseela M. Kim K.S. Kwon-Chung K.J. Cryptococcal yeast cells invade the central nervous system via transcellular penetration of the blood-brain barrier.Infect. Immun. 2004; 72: 4985-4995Crossref PubMed Scopus (186) Google Scholar). This mechanism requires C. neoformans cell-induced HBMEC cytoskeletal rearrangements for the accumulation of actin at the site of C. neoformans entry. The C. neoformans cell may then be drawn progressively into the host cell.Pathogens usually use multiple mechanisms to invade their host(s). Several studies have suggested that C. neoformans may use different routes to enter the brain. For example, transmission electron microscopic images showed that C. neoformans cells embedded inside macrophages could be found in the crossing site of the brain microcapillaries, suggesting a “Trojan horse” mechanism (14Chrétien F. Lortholary O. Kansau I. Neuville S. Gray F. Dromer F. Pathogenesis of cerebral Cryptococcus neoformans infection after fungemia.J. Infect. Dis. 2002; 186: 522-530Crossref PubMed Scopus (163) Google Scholar). It has also been reported that urease is required for brain invasion (15Shi M. Li S.S. Zheng C. Jones G.J. Kim K.S. Zhou H. Kubes P. Mody C.H. Real-time imaging of trapping and urease-dependent transmigration of Cryptococcus neoformans in mouse brain.J. Clin. Invest. 2010; 120: 1683-1693Crossref PubMed Scopus (148) Google Scholar, 16Olszewski M.A. Noverr M.C. Chen G.H. Toews G.B. Cox G.M. Perfect J.R. Huffnagle G.B. Urease expression by Cryptococcus neoformans promotes microvascular sequestration, thereby enhancing central nervous system invasion.Am. J. Pathol. 2004; 164: 1761-1771Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The roles of Ure1 and CPS1 in brain invasion are not necessarily mutually exclusive. However, their relevant functions are yet to be elucidated, and our understanding of their complicated invasion process has just begun. Recently, it has been documented that the lipid raft-dependent endocytosis process mediates C. neoformans internalization into HBMEC and that the CD44 protein of the hosts, cytoskeleton, and intracellular kinase DYRK3 are involved in this process (17Huang S.H. Long M. Wu C.H. Kwon-Chung K.J. Chang Y.C. Chi F. Lee S. Jong A. Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3).J. Biol. Chem. 2011; 286: 34761-34769Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Taken together, a transcellular mechanism seems to be the predominant route for C. neoformans brain invasion (13Chang Y.C. Stins M.F. McCaffery M.J. Miller G.F. Pare D.R. Dam T. Paul-Satyaseela M. Kim K.S. Kwon-Chung K.J. Cryptococcal yeast cells invade the central nervous system via transcellular penetration of the blood-brain barrier.Infect. Immun. 2004; 72: 4985-4995Crossref PubMed Scopus (186) Google Scholar, 17Huang S.H. Long M. Wu C.H. Kwon-Chung K.J. Chang Y.C. Chi F. Lee S. Jong A. Invasion of Cryptococcus neoformans into human brain microvascular endothelial cells is mediated through the lipid rafts-endocytic pathway via the dual specificity tyrosine phosphorylation-regulated kinase 3 (DYRK3).J. Biol. Chem. 2011; 286: 34761-34769Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar).Previously, we had used the in vitro blood-brain barrier model to demonstrate that the C. neoformans HA-HBMEC CD44 interaction plays an initial role during the fungal invasion across the blood-brain barrier (12Jong A. Wu C.H. Shackleford G.M. Kwon-Chung K.J. Chang Y.C. Chen H.M. Ouyang Y. Huang S.H. Involvement of human CD44 during Cryptococcus neoformans infection of brain microvascular endothelial cells.Cell. Microbiol. 2008; 10: 1313-1326Crossref PubMed Scopus (79) Google Scholar). The CPS1 deletion strain (C559) is temperature-sensitive (unable to grow at 37 °C), precluding in vivo studies. In this report, we used a mouse model to investigate C. neoformans brain invasion in vivo. In the virulent studies, the CD44 knock-out (KO) mice survived longer than the wild-type mice. Compared with the wild-type mice, the fungal load in the brain and CSF was lower in infected CD44 KO mice. Similarly, the CD44−/− mouse brain contained smaller and fewer cystic lesions. These results suggested that host CD44 is required for C. neoformans brain invasion. Immunofluorescence microscopic images of the brains sections showed that a mass of C. neoformans cells within the lesions are imbedded in their capsule material; these lesions were surrounded by many reactive astrocytes displaying a unique pattern. We also found that, in both the wild-type and CD44−/− mice, a secondary HA receptor, RHAMM, was present in the brain. Finally, we demonstrated the in vivo blocking effect of the hypolipidemic drug simvastatin. Our results suggest that simvastatin may remove receptor function on the membrane lipid raft or yet identified action(s), providing a cue for a novel anticryptococcal intervention.
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