key: cord-0883748-ae07ljtz
authors: Donnelly, Sheila; Dalton, John P.; Robinson, Mark W.
title: How Pathogen-Derived Cysteine Proteases Modulate Host Immune Responses
date: 2011-05-19
journal: Cysteine Proteases of Pathogenic Organisms
DOI: 10.1007/978-1-4419-8414-2_12
sha: 3984c124e4ccf3d9d03ce4c539c2e8695d1230ef
doc_id: 883748
cord_uid: ae07ljtz

In mammals, cysteine proteases are essential for the induction and development of both innate and adaptive immune responses. These proteases play a role in antigen-and pathogen-recognition and elimination, signal processing and cell homeostasis. Many pathogens also secrete cysteine proteases that often act on the same target proteins as the mammalian proteases and thereby can modulate host immunity from initial recognition to effector mechanisms. Pathogen-derived proteases range from nonspecific proteases that degrade multiple proteins involved in the immune response to enzymes that are very specific in their mode of action. Here, we overview current knowledge of pathogen-derived cysteine proteases that modulate immune responses by altering the normal function of key receptors or pathways in the mammalian immune system.

It is the role of the immune system to synthesise and release effector molecules that detect, implement or orchestrate an appropriate response to a potential threat or danger signal. To regulate many aspects of this process, cells of the immune system utilise protease activity. One major group of cysteine proteases, termed cathepsins, are common constituents of the endolysosomal compartments of immune cells where they have critical roles in events such as antigen presentation and zymogen processing. Despite a widely held belief that the cathepsins were enzymes that function only at the acidic pH within the lysosomal compartment, it is now evident that they also operate in the cytosolic and nuclear compartments of cells, as well as the extracellular space. 1 As a result of this widespread localisation, cathepsins can exert diverse effects on the development and regulation of immune responses by playing key roles in cytokine regulation, cell GHYHORSPHQW LQGXFWLRQ RI DSRSWRVLV DQG LQÀXHQFLQJ 7ROOOLNH UHFHSWRU 7/5 VLJQDOOLQJ 1, 2 Many pathogenic organisms synthesize proteases that resemble host cathepsins (Table  1) , both functionally and structurally and have fundamental roles in the life-cycle and survival of the pathogen. For example, parasitic helminths (worms) secrete proteases in large quantities to degrade the extracellular matrix during host invasion, 3, 4 intracellular protozoans require proteases for invasion, nutrition and exit from host cells, 5 bacterial FDWKHSVLQV SHUIRUP KRXVHNHHSLQJ UROHV OLNH DPLQR DFLG XSWDNH DQG ¿PEULDH PDWXUDWLRQ 6, 7 and viral proteases are involved in processing of viral gene products. 8,9 A number of the target amino acid sequences for pathogen cathepsins are also present in key molecules involved in regulating the host immune response including immunoglobulins (Ig), cytokines, TLRs and ubiquitin (Ub). It has been suggested that adaptation of pathogen proteases to target these structures has occurred in an evolutionary process to increase pathogenicity. 2,10 7KLV KDV EHHQ LQÀXHQFHG E\ WKH XQLTXH ELRFKHPLFDO FKDOOHQJHV SUHVHQWHG to each pathogenic organism and is linked, in part, to their localisation as extracellular or intracellular organisms and therefore their exposure to different cellular structures and macromolecules. Here, we review the functions that have been attributed to pathogen cysteine proteases in the evasion, suppression and modulation of the host immune response.

The immunity and pathology occurring in response to infection with any pathogen is predominantly mediated by T-lymphocytes. In general, control of infection and healing is associated with a polarized Th1 type response whereas the induction of interleukin (IL)-4-dominated Th2 responses are largely suboptimal against a number of pathogens.

In certain protozoan infections, cysteine proteases are critical for the induction of Th2 type immune responses. Leishmania are obligate intracellular parasites that live as nonmotile amastigotes within cells of the mononuclear phagocyte lineage of their mammalian hosts. The outcome of infection in leishmaniasis is determined by the Th1 versus Th2 nature of the effector response with parasites successfully establishing infection by driving a Th2 immune response. 11 Inhibition of Leishmania mexicana cathepsin B, ZLWK D VSHFL¿F LQKLELWRU FDXVHG D VZLWFK LQ WKH SRODULVDWLRQ RI 7FHOO GLIIHUHQWLDWLRQ IURP a Th2 to a protective Th1 phenotype in mice. 12, 13 In addition, mutants of L. mexicana lacking cysteine protease activity induced less IL-4 and IgE in BALB/c mice compared to wild type parasites. 14 Finally, the delivery of recombinant cysteine proteases derived from either L. mexicana, or Trypanosoma cruzi HOLFLWHG VWURQJ 7KW\SH UHVSRQVHV W\SL¿HG by enhanced mRNA expression and production of Th2 cytokines (IL-4, IL-5 and IL-13) from the draining lymph nodes and also a polarised splenocyte response toward a Th2 bias in response to stimulation with anti-CD3. 15, 16 The ability of cysteine proteases to induce Th2-type immune responses is dependent on their enzymatic activity 12-17 DQG DOVR RQ WKHLU VXEVWUDWH VSHFL¿FLW\ DV RQO\ F\VWHLQH proteases belonging to clan CA demonstrate an ability to promote the differentiation of Th2. 18 Injection of the plant-derived cysteine protease papain to mice was shown to promote  the activation of basophils, which then present antigen to CD4 T-cells and induce Th2 cell  responses through the secretion of IL-4 and thymic stromal lymphopoietin. 17 Therefore, it was suggested that basophils detect the presence of protease products which in turn activates these cells to induce a Th2 response. 18 This strategy of immune recognition is distinct from typical recognition by cells of the innate system since it is not dependent on the detection of pathogen molecular structures. Instead, it requires the detection of enzymatic activity associated with the presence of a pathogen or antigen. The immune outcome closely resembles the type of reaction that is triggered by helminths and has led to the suggestion that the innate immune system has evolved a detection mechanism based on sensing the abnormal protease activity associated with helminth infection. 18 This idea has support in studies showing that the secreted proteases of the hookworm Necator americanus induce Type 2 cytokine production by basophils. 19 Another means by which cysteine proteases could modulate host immune responses towards a Th2 environment may be associated with their ability to inhibit Th1 immune responses. In support of this idea, it was shown that the predominant secreted product of the human and animal helminth pathogen Fasciola hepatica, a cathepsin L1 cysteine protease, suppressed the onset of protective Th1 immune responses in mice to infections with the respiratory microbe Bordetella pertussis, making them more susceptible to disease. In addition, injection of the cysteine protease immediately prior to immunization of mice with a B. pertussis whole-cell pertussis vaccine prevented the development of a Th1 response to the vaccine. [20] [21] [22] Studies with L. mexicana cathepsin B suggest that cleavage of CD25 from the surface of T-cells may prevent the development of Th1 responses. 14 Infection with the helminth Schistosoma mansoni may provide another example of how cysteine proteases suppress Th1 responses. The acute stages of schistosomiasis leads to the development of a weak Th1 response, but this switches to a potent Th2 response when female worms become fecund and release eggs that get trapped in host liver and intestinal tissues. 23 Secretions from the trapped eggs are responsible for the induction of this Th2 response. The major schistosome proteases, cathepsin B and cathepsin L, are expressed and secreted by the egg stage of the parasite 24 and, therefore, may facilitate this immune switching.

Substantial evidence supports the view that cysteine proteases secreted by a range RI SDWKRJHQV VSHFL¿FDOO\ SUHYHQW FHOOV RI WKH LQQDWH LPPXQH UHVSRQVH SURPRWLQJ Th1-adaptive immune responses. 10, [25] [26] [27] The mechanisms are varied and depend on the VXEVWUDWH VSHFL¿FLW\ RI WKH SURWHDVH DQG RI WKH ORFDWLRQ RI WKH SDWKRJHQ ZLWKLQ WKH KRVW 7KH LQQDWH LPPXQH V\VWHP FRQVWLWXWHV WKH ¿UVW OLQH RI KRVW GHIHQFH GXULQJ LQIHFWLRQ DQG plays a crucial role in the early recognition of invading pathogens. Unlike the adaptive immune UHVSRQVH WKH LQQDWH LPPXQH UHVSRQVH LV UHODWLYHO\ QRQVSHFL¿F UHO\LQJ RQ WKH UHFRJQLWLRQ of evolutionarily conserved structures on pathogens, termed pathogen-associated molecular patterns (PAMPs), through a limited number of pattern recognition receptors (PRRs). 28 A number of different PRR families have been described but the best characterised is the IDPLO\ RI 7/5V 7R GDWH 7/5V KDYH EHHQ LGHQWL¿HG LQ KXPDQV DQG DUH GLVWLQJXLVKHG by their recognition of distinct PAMPs derived from various pathogens. 29 Interaction between a PAMP and its corresponding TLR, present either at the cell surface or intracellularly, leads to the recruitment of an adaptor molecule, followed by the activation of downstream signal transduction pathways. 28, 29 These signaling SDWKZD\V DUH FODVVL¿HG RQ WKH EDVLV RI WKHLU XWLOL]DWLRQ RI GLIIHUHQW DGDSWRU PROHFXOHV i.e., MyD88 or TIR domain-containing adaptor inducing IFN` (TRIF) and, additionally, their respective activation of individual kinases and transcription factors. [30] [31] Three major signaling pathways mediating TLR-induced responses have been described (i) NFgB, (ii) mitogen-activated protein kinases (MAPKs) and (iii) IFN regulatory factors (IRFs). NFg% DQG 0$3.V DUH HVVHQWLDO LQ WKH LQGXFWLRQ RI D SURLQÀDPPDWRU\ UHVSRQVH ZKHUHDV IRFs are required for stimulation of IFN production. [30] [31] [32] Ultimately, TLR-induced signal transduction pathways result in the activation of gene expression and synthesis of a range of molecules such as cytokines, chemokines and immunoreceptors. 28, 30 Together, these coordinate the immediate host response to infection and provide an essential link to the adaptive immune response. Given that the containment and eradication of pathogens is GHSHQGHQW RQ HI¿FLHQW UHFRJQLWLRQ DQG VLJQDOLQJ WKURXJK 355V LW LV SHUKDSV QRW VXUSULVLQJ that some pathogens have evolved strategies to interfere with this process.

Among the various bacterial species associated with the development of periodontitis, the Gram negative anaerobic bacterium Porphyromonas gingivalis is suspected to be one of the most important causative agents of the chronic form of this disease. 33 Arg-and Lys-gingipain cysteine proteases are the main endopeptidases produced by P. gingivalis and are considered to be important virulence factors 34 as both proteases inhibit CD14-dependent monocyte activation. CD14 is a 55-kDa glycosylphosphatidylinositol (GPI)-anchored membrane protein, which functions in the detection of many Gram-negative bacteria by cells of the innate immune response. Presentation of CD14-associated bacterial lipopolysaccharide (LPS) to TLR4 and the DFFHVVRU\ PROHFXOH 0' OHDGV WR WKH UHOHDVH RI SURWHFWLYH LQÀDPPDWRU\ IDFWRUV E\ macrophages and dendritic cells. 35 %RWK $UJ DQG /\V VSHFL¿F JLQJLSDLQV IURP P. gingivalis preferentially cleave CD14, but not TLR4, from the surface of macrophages, 36, 37 making the cells hyporesponsive to LPS stimulation. The gingipains display a preference for cleaving CD14 over other polypeptides on human monocytes which can be explained by the high frequency of Arg-X and Lys-X peptide bonds in the amino acid sequence of CD14. 38 This preference is further demonstrated by the observation that gingipains also VSHFL¿FDOO\ UHPRYH &' IURP WKH VXUIDFH RI JLQJLYDO ¿EUREODVWV 39 As a consequence of this proteolysis, macrophage recognition of the bacterium is attenuated thus neutrophil GHJUDQXODWLRQ DQG UHVSLUDWRU\ EXUVW PHGLDWHG E\ ¿EUREODVWVHFUHWHG ,/ LV UHGXFHG Therefore bacterial survival in the periodontal tissues is promoted. 40 Like the gingipains, the major cysteine protease of F. hepatica, cathepsin L1, is secreted into the extracellular environment during infection and also inhibits macrophage recognition of bacterial products. However, this inactivation is not mediated by cleavage of cell surface CD14. Instead, cathepsin L1 is internalised by the host's phagocytic PDFURSKDJHV DQG WUDI¿FNHG WR WKH HQGRO\VRVRPDO FRPSDUWPHQWV ZKHUH LW VSHFL¿FDOO\ degrades TLR3. 25 7KLV YDULDWLRQ LQ SURWHRO\WLF DFWLYLW\ UHÀHFWV FOHDU GLIIHUHQFHV LQ VXEVWUDWH VSHFL¿FLW\ EHWZHHQ WKH $UJ/\VVSHFL¿F JLQJLSDLQV DQG FDWKHSVLQ / ZKLFK prefers hydrophobic residues such as Leu, Phe and Ala. 41, 42 Considering both CD14 and members of the Toll receptor family contain multiple leucine-rich repeat motifs 43 it is perhaps surprising that cathepsin L does not cleave either CD14 or TLR4 from the surface RI PDFURSKDJHV \HW VSHFL¿FDOO\ GHJUDGHV LQWUDFHOOXODU 7/5 8QOLNH WKH VXUIDFH 7ROO UHFHSWRUV 7/5 XQGHUJRHV FRQIRUPDWLRQDO FKDQJHV LQ UHVSRQVH WR O\VRVRPDO DFLGL¿FDWLRQ 44 which may make it susceptible to cleavage by cathepsin L, a protease that is stable and functional over a broad pH range. 41 In general, macromolecules internalized by macrophages are degraded into antigenic peptides by the range of endogenous cathepsins resident in the endolysosomal compartments. However, F. hepatica cathepsin L1 is resistant to this endosomal degradation and data shows that the mature enzyme is highly resistant to proteolytic degradation by various endopeptidases. 42 It has also been reported that F. hepatica cathepsin L1 can degrade cystatins/serpins such as SCCA1 and SCCA2 45 which may protect the enzyme from inhibition by cystatins within the lysosome that are known to regulate the activity of resident endolysosomal cathepsins. 46 During schistosomiasis, the eggs released by S. mansoni suppress the maturation of dendritic cells in response to the bacterial products poly-I:C and LPS. 47 Similar to F. hepatica cathepsin L1, immature dendritic cells rapidly internalized egg antigens and targeted them to endolysosomal compartments. 48 The egg antigens contain cysteine proteases cathepsin B and cathepsin L which, like F. hepatica cathepsin L1, may cleave TLRs within the endosome thus preventing the dendritic cells from maturing in response to activation signals. 25 This in turn would inhibit Th1 responses and allow the promotion of Th2 responses which correlates with the immune switching during egg deposition.

Several studies have demonstrated that eradication of the intramacrophage-dwelling Leishmania sp. requires the induction of Th1 cells. Prolonged survival of these pathogens is associated with the parasites ability to regulate IL-12 production by macrophages and therefore control the production of protective IFN-a. [50] [51] [52] [53] [54] Most mouse strains are resistant to infection with L. major but develop nonhealing lesions following infection with L. mexicana. 20 The inability of these mouse strains to heal following infection with L. mexicana is associated with a higher level of parasite cysteine protease B activity and subsequently lower induction of IL-12. 55 7KLV UROH IRU WKH SDUDVLWH SURWHDVH ZDV FRQ¿UPHG by the observation that amastigotes of protease deletion mutants of L. mexicana had limited ability to inhibit IL-12 production and unlike the wild type parasites, were unable to suppress a Type 1 adaptive immune response. 55 The mechanism of action appears to be WKH VSHFL¿F SURWHRO\WLF GHJUDGDWLRQ RI 1)gB, which did not affect the nuclear translocation of this transcription factor, but did prevent it binding to DNA and therefore inhibited its ability to induce IL-12 gene expression. 56 The infection of cells with the obligate intracellular protozoan parasite, Toxoplasma gondii also results in the inhibition of IL-12 expression via interference with NFgB activation. In this case the termination of NF-gB activity was associated with a reduction of the phosphorylation of p65/RelA, an event required for the translocation of NF-gB to the nucleus. 57 :KLOH D QXPEHU RI F\VWHLQH SURWHDVHV KDYH EHHQ LGHQWL¿HG IURP T. gondii, 58 the possibility of their involvement in this immune-modulatory effect has not been investigated.

Following interaction between a PAMP and its corresponding PRR, the activation of downstream signal transduction pathways is dependent on the ubiquitination of protein components of the signalling cascade. 30, 31 Ubiquitination is an enzyme-mediated process by which ubiquitin (an 8 kDa protein) is covalently attached to lysine residues of target proteins. While this alteration to proteins does not mediate degradation it has functional FRQVHTXHQFHV IRU WKH PRGL¿HG SURWHLQ VXFK DV FKDQJHV LQ WKHLU FRQIRUPDWLRQ VXEFHOOXODU ORFDOL]DWLRQ RU FDWDO\WLF DFWLYLW\ +RZHYHU XELTXLWLQDWLRQ LV D UHYHUVLEOH PRGL¿FDWLRQ DQG the rapid removal of ubiquitin from substrates is catalysed by de-ubiquitinating enzymes (DUBs) which are predominantly classed as cysteine proteases. The human genome is predicted to encode almost 500 proteins that are known to recognize ubiquitin and attach LW WR VSHFL¿F VXEVWUDWHV DQG RYHU '8%V WKDW UHYHUVH WKDW UHDFWLRQ 60 During virus replication, the innate immune response is activated, resulting in the production of several hundred antiviral proteins converting the intracellular environment into a suboptimal context for replication. 60 In response to this selective environment, evidence suggests that a number of viral proteases have adapted a ubiquitin-removal VSHFL¿FLW\ DV D PHFKDQLVP WR GLVDEOH WKH KRVW LPPXQH V\VWHP DQG RSWLPL]H WKH LQWUDFHOOXODU HQYLURQPHQW IRU HI¿FLHQW YLUXV UHSOLFDWLRQ DQG UHOHDVH 10 For example, the nonstructural protein of severe acute respiratory syndrome (SARS) corona virus, nsp3, known to be involved in the processing of replicase polyproteins, has recently been shown to carry a conserved deubiquitinase (DUB) motif within its papain-like protease (PLpro) domain. 61, 62 7KLV SURWHLQ HI¿FLHQWO\ LQKLELWV ,5) XELTXLWLQDWLRQ LQ D SURWHDVH GHSHQGHQW PHFKDQLVP 63 IRF3 is a critical transcription factor for the activation of antiviral IFN and requires ubiquitination to translocate to the nucleus. Its inhibition by a viral DUB could explain why cultured cells infected with Coronaviridae characteristically produce very low levels of IFNs. [64] [65] [66] Indeed, most viruses, including all highly pathogenic human viruses, attempt to modulate this aspect of the innate immune response early in infection. 66, 67 Virus-encoded DUBs from a variety of virus families have been described, 10 including UL36 of herpesviruses such as herpes simplex virus Type 1, Epstein-Barr virus and mouse and human cytomegalovirus; 68-70 the adenain protease of adenovirus; 71 and the PLP domains from Coronaviridae. 72 These proteases are unique to the viral pathogens and quite distinct from host-encoded DUBs. While a pathogenic role has not been assigned to many of these viral DUBs, it is tempting to speculate that the prevention of ubiquitination of essential transcription factors is a common mechanism to suppress antiviral IFN production.

Despite having no intrinsic ubiquitin system, several bacterial strains have been found WR FRQWDLQ XELTXLWLQVSHFL¿F F\VWHLQH SURWHDVHV /LNH WKH YLUDO '8%V WKHVH SURWHDVHV interfere with innate cell signalling, inhibiting activation of antibacterial responses by VSHFL¿FDOO\ WDUJHWLQJ HOHPHQWV RI WKH 1)gB pathway. For example, YopJ secreted by Yersinia sp. ZDV ¿UVW GHVFULEHG DV D '8% SUHYHQWLQJ XELTXLWLQDWLRQ RI WKH 7/5DGDSWRU proteins TRAF2, TRAF3 and TRAF6. 73, 74 It has also been reported that YopJ can de-ubiquitinate the transcription factor Ig%Į WKHUHE\ SUHYHQWLQJ LWV GHJUDGDWLRQ DQG WKH subsequent translocation of NF-gB to the nucleus. 75 Similarly, AvrA, a protease secreted by Salmonella sp. stabilises Ig%Į E\ SUHYHQWLQJ LWV XELTXLWLQDWLRQ DQG WKHUHE\ LQKLELWV NF-g% DFWLYDWHG LQÀDPPDWRU\ UHVSRQVHV RI WKH KRVW 75 De-ubiquinitase activity has also been attributed to cysteine proteases secreted by Chlamydia trachomatis (ChlaDub1 and ChlaDub2) 77 and Escherichia coli (ElaD) 78 

Bioinformatic analyses have predicted that a number of medically-relevant parasitic protozoa encode putative DUBs. 79 However, only three have been functionally assessed for their ability to bind to ubiquitin. Plasmodium falciparum (the causative agent of malaria) expresses two DUBs, namely PfUCHL3 and PfUCH54 80 and a third, TgUCHL3, a homologue of PfUCHL3, is expressed by T. gondii. 81 It has been hypothesized that these proteases may only function within the parasite itself and not target host proteins. There is currently no information on their physiological effects.

Cells of the innate and acquired immune systems communicate via the release of cytokines. These proteins are released in response to the presence of pathogens or to FRPSRQHQWV RI GDPDJHG WLVVXH DQG DUH HVVHQWLDO IRU WKH LQGXFWLRQ RI DQ LQÀDPPDWRU\ response as well as its regulation and resolution. Interruption of this communication network profoundly impacts on the outcome of infectious disease. One method of disruption of signalling, performed promiscuously by cysteine proteases derived from P. gingivalis, RFFXUV WKURXJK WKH SURWHRO\WLF PRGL¿FDWLRQ RI F\WRNLQHV ,/ ,/5D ,/ 71)_, IFN-a) and their receptors (IL-6R). 27 A second method of interference by pathogen proteases is mimicking the activity of host proteases used in the regulation of cytokine activity. Unlike most other cytokines, IL-18 and IL-1` ODFN D VLJQDO SHSWLGH DQG DUH ¿UVW V\QWKHVL]HG DV ELRORJLFDOO\ LQDFWLYH precursors (proIL-18 and proIL-1`). These precursors are cleaved by caspase-1 (IL-1`-converting enzyme [ICE]), between Asp-116 and Ala-117 82,83 to produce the mature active cytokines. Cysteine proteases isolated from Streptococcus pyogenes (SpeB) and Entamoeba histolytica both convert Pro-IL-1` to a mature cytokine, targeting cleavage sites 1 and 5 amino acids from the caspase-1 site of action, respectively. 84, 85 In both cases the resultant cytokine retains biological activity which leads to the augmentation of an LQÀDPPDWRU\ UHVSRQVH )RU E. histolytica this action may facilitate its spread beyond cells in direct contact with amoebic trophozoites. 84 In addition, the ICE-like activity of pathogen cysteine proteases suggests a mechanism which could activate caspases within infected cells and thus induce cell death by apoptosis.

Another cysteine protease from E. histolytica cleaves pro-IL-18. 86 However, the proteolytic action removes Glu-42, a key residue for biological activity of IL-18, 87 and therefore, the cleavage product is an inactive protein. It was suggested that the secondary structure of IL-18 may contribute to the choice of cleavage site, as the amino acid sequence GRHV QRW FRUUHODWH ZLWK NQRZQ SHSWLGH VXEVWUDWH VSHFL¿FLWLHV 87

Chemokines are a distinct, large superfamily of cytokines encompassing small structurally-related proteins. Physiologically, chemokines have an important role in the UHFUXLWPHQW RI OHXNRF\WHV GXULQJ DQ DFXWH LQÀDPPDWRU\ UHVSRQVH +RVW SURWHDVHV WLJKWO\ UHJXODWH WKLV FHOOXODU UHFUXLWPHQW E\ PRGXODWLQJ WKH DFWLYLW\ RI FKHPRNLQHV YLD VSHFL¿F cleavage of their N-and C-termini. 88, 89 The proteolytic cleavage of the N-terminus enhances DFWLYLW\ IRU VHYHUDO &;& LQÀDPPDWRU\ FKHPRNLQHV LQFOXGLQJ WKH PDLQ QHXWURSKLODWWUDFWLQJ chemokine CXCL8 (IL-8). This function has been appropriated by the gingipains of P. gingivalis ZKLFK HI¿FLHQWO\ FOHDYH ,/ DW WKH 1WHUPLQXV HQKDQFLQJ LWV DFWLYLW\ 90 an DFWLRQ ZKLFK FRUUHODWHV ZLWK WKH PDVVLYH LQ¿OWUDWLRQ RI QHXWURSKLOV REVHUYHG DW VLWHV RI periodontitis. However, when associated with bacterial outer membrane vesicles the same proteases can also degrade IL-8. It has been suggested that this dual role of enhancing DQG LQKLELWRU\ DFWLYLW\ UHVXOWV LQ WKH DVVRFLDWLRQ RI SUR DQG DQWLLQÀDPPDWRU\ UHDFWLRQV to distal and proximal positions of the bacterial plaque, respectively, and explains why there is no elimination of infection despite the accumulation of neutrophils. 90 Despite the fact that mammalian hosts infected with the helminth F. hepatica develop VSHFL¿F DQWLERGLHV 91 and that the major protein isolated from eosinophils is highly toxic to newly excysted juvenile (NEJ) worms, 92 no evidence exists of antibody-mediated eosinophil damage to NEJs in nonpermissive bovine hosts. 93 While effector cells readily adhered to NEJs in the presence of immune sera, they failed to adhere if the parasite's excretory/secretory (ES) products were added which indicated that the contents of ES were preventing interaction between immune sera and eosinophils. In the presence of leupeptin, the effector cells remained attached to the NEJs, suggesting a role for cysteine proteases. 94 Subsequently, in vitro studies have demonstrated that the papain-like cathepsin L1 and L2 proteases secreted by F. hepatica cleaved all human IgG subclasses in the hinge region. Despite clear evidence that these proteases have distinct peptide bond preferences, both cathepsin L1 and L2 cleaved each of the IgG molecules at the same peptide bond. 95 The ability to degrade human IgG subclasses is not exclusive to the cathepsin Ls of Fasciola 6LPLODU SURWHRO\WLF FOHDYDJH VLWHV DQG VSHFL¿FLW\ IRU KXPDQ ,J* VXEFODVVHV have been shown for the major secreted cysteine proteases of other helminths, such as Paragonimus westermani, 96 Spirometra mansoni 97 and Schistosoma mansoni 98 which DOO GHJUDGH KRVW ,J* LQ YLWUR DQG SUHYHQW SDUDVLWH VSHFL¿F DQWLERG\PHGLDWHG HRVLQRSKLO activation. 99 Cruzipain, the cysteine protease secreted by the protozoan parasite T. cruzi also exhibits cathepsin-like activity, cleaving all human IgG subclasses at the hinge region in a similar but not identical region to cathepsins L1 and L2 of F. hepatica. 100 The fact that the main cleavage sites for this range of proteases all exist within the hinge UHJLRQV RI ,J* VXJJHVWV WKDW WKH FRQIRUPDWLRQ RI WKH DQWLERG\ PROHFXOHV LQÀXHQFHV WKH DFFHVVLELOLW\ RI HQ]\PHV DQG WKHUHIRUH GHWHUPLQHV WKH VSHFL¿FLW\ RI FOHDYDJH LUUHVSHFWLYH of the amino acid preference of each protease active site.

In all cases, the end result of proteolytic cleavage by these parasite proteases is the release of intact monomeric fragments of Fab. 95, 98, 100 The Fab fragments retain their capacity to bind to surface antigens, a term called 'fabulation'. Surface epitopes are therefore masked from intact and functional antibodies, while the loss of Fc fragments eliminates the interaction of IgG with effector cells and prevents resultant antibody-dependent cytotoxicity. In addition, the Fc fragments produced by initial proteolyic cleavage at the hinge region are further degraded by proteases from F. hepatica and T. gondii cruzipain in the CH2 region producing Fc-like-fragments of 14 kDa composed of the CH3 domain. 95 ,100 $V WKH FRPSOHPHQW IDFWRU &T VSHFL¿FDOO\ ELQGV WR WKH &+ UHJLRQ RI ,J* UHPRYDO RI this would prevent the initiation of the classical pathway.

Despite lacking sequence identity to the helminth papain-like proteases, the cysteine protease (IdeS) secreted by the pathogenic bacterium S. pyogenes adopts a canonical papain fold 101 104 However, the proteolytic activity of SpeB towards immunoglobulins is not restricted to IgG. This protease also degrades the carboxy-terminal of the heavy chains of IgA, IgD, IgE and IgM. 103 6SHFL¿F,J$ SURWHDVH DFWLYLW\ LV D ZHOOHVWDEOLVKHG IHDWXUH RI PDQ\ KXPDQ LQIHFWLRXV diseases that take place at, or originate from, mucosal surfaces. 105, 106 For bacterial pathogens WKH ,J$ SURWHDVHV KDYH EHHQ SULPDULO\ FODVVL¿HG DV VHULQH RU PHWDOORW\SH SURWHDVHV cleaving only the IgAl subclass of antibody, because the susceptible site is one of the Pro-Ser or Pro-Thr peptide bonds located within a 12-amino acid proline rich sequence in the hinge region of IgAl but absent from IgA2. 106, 107 However, the cathepsin B-like cysteine protease (EhCp5) secreted by the protozoan E. histolytica with a preference for Arg-Arg residues displays an ability to cleave both IgA1 and IgA2 degrading the antibody structures at positions 245 and 250 of the hinge region. 108, 109 Similar to the proteolytic cleavage of IgG described above, release of Fab fragments inhibits antigen disposal as immunogenic determinants are masked by fabulation. Moreover, removal of the Fc region eliminates the ability of IgA to agglutinate, preventing opsonophagocytosis thus facilitating survival of pathogenic organisms at the mucosal surface.

The complement system is composed of three distinct pathways: (1) classical pathway, activated by antigen-antibody complexes; (2) lectin pathway, activated by carbohydrate arrays found on microbial surfaces; and (3) alternative, activated by C3 binding to the surface of micro-organisms. Central to all pathways of activation is the formation of C3 and C5 convertase complexes. In particular, the C3 protein and its activated form, C3b, is an integral component of the C5 convertase complex whichever pathway of activation has been engaged. Cleavage of these molecules produces the C3a and C5a fragments which are responsible for attracting white blood cells to the site of infection and therefore form a link between the innate and adaptive immune systems.

Of the members of the complement cascade, cysteine proteases secreted by pathogens primarily target components of the C3 and C5 convertase complex. A number of bacterial proteases, including the SpeB cysteine protease of S. pyogenes cleave C3 and thus inactivate or prevent the formation of the C5 convertase complex. 110, 111 Similarly, the cysteine protease from E. histolytica prevents the formation of the membrane attack FRPSOH[ 0$& DQG WKH UHOHDVH RI WKH SURLQÀDPPDWRU\ PHGLDWRU &D E\ GHJUDGLQJ both C5a and C3a directly. 109, 112 The refractoriness of P. gingivalis to the immune response has been attributed to the ELSKDVLF HIIHFWV RI JLQJLSDLQV RQ WKH FRPSOHPHQW V\VWHP 7KH O\VLQHVSHFL¿F JLQJLSDLQ LV FDSDEOH RI GHJUDGLQJ WKH &D UHFHSWRU EXW PRUH LPSRUWDQWO\ WKH DUJLQLQHVSHFL¿F HQ]\PHV cleave C3, C4 and C5. 113 6KDULQJ VXEVWUDWH VSHFL¿FLW\ ZLWK WKH SURWHDVHV UHTXLUHG IRU complement activation, the gingipains activate C3, C4 and C5 by cleaving them at their activation sites. However, at high concentrations the Arg-gingipains completely degrade and thus inactivate the complement proteins. 113 This has led to the suggestion that early infections by P. gingivalis may actually stimulate complement activation to cause an LQÀDPPDWRU\ VWDWH WKDW LV DGYDQWDJHRXV WR WKH EDFWHULXP +RZHYHU DV LQIHFWLRQV EHFRPH chronic the higher number of bacteria lead to the degradation of complement. This idea is supported by data showing that the cysteine protease, Interpain A, secreted by the periodontal bacterium, Prevotella intermedia, which co-aggregates with P. gingivalis, activates the C1 complex in serum, causing deposition of C1q on bacterial surfaces and UHVXOWLQJ LQ D ORFDO LQÀDPPDWRU\ UHDFWLRQ GXULQJ WKH LQLWLDO VWDJHV RI LQIHFWLRQ 114 Activation of the complement cascade involves a number of proteases which are tightly regulated by cystatins and serpins. The StcE cysteine protease of E. coli uniquely SUHYHQWV FRPSOHPHQW DFWLYDWLRQ YLD WKH FODVVLFDO SDWKZD\ E\ VSHFL¿FDOO\ FOHDYLQJ WKH serpin, C1-inhibitor, which regulates the proteases of the initiating C1 complex of the classical pathway. 115 

The immune system has evolved many different strategies to control many types of infections and thus prevent associated disease. Equally, pathogenic organisms appear to have developed processes to evade, suppress or subvert the immune response with pathogen-derived cysteine proteases emerging as key molecules. Whilst some of these proteases share a common origin with mammalian-encoded proteases, most of them have ancient intrinsic functions, such as processing pathogen protein components and may have DFTXLUHG WKH VSHFL¿FLW\ IRU KRVW SURWHLQ WDUJHWV E\ LQWHUDFWLRQ ZLWK WKHLU KRVW ¶V LPPXQH system over time. As many of these proteases have evolved distinct biochemical features from their mammalian counterparts they remain attractive as targets for new antimicrobial drugs. In addition, some of these proteases may be useful as novel therapeutics for the WUHDWPHQW RI 7K LQÀDPPDWRU\ GLVRUGHUV SDUWLFXODUO\ LQ OLJKW RI WKH FXUUHQW VWUDWHJ\ RI developing antagonists of innate immune responses as immunotherapeutics for sepsis and autoimmune disease.

Diverse regulatory roles for lysosomal proteases in the immune response

Endolysosomal proteases and their inhibitors in immunity

An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: proteins associated with invasion and infection of the mammalian host

Helminth pathogen cathepsin proteases: it's a family affair

Proteases in parasitic diseases

The role of gingipains in the pathogenesis of periodontal disease

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Emerging patterns and molecular principles

T helper (h) 1/Th2 and Leishmania: paradox rather than paradigm

Cathepsin B-inhibitor promotes the development of Th1 type protective T-cells in mice infected with Leishmania major

Switch of CD4 T-cell differentiation from Th2 to Th1 by treatment with cathepsin B inhibitor in experimental leishmaniasis

Expression of multiple CPB genes encoding cysteine proteases is required for Leishmania mexicana virulence in vivo

The Leishmania mexicana cysteine protease, CPB2.8, induces potent Th2 responses

Cruzipain, a major Trypanosoma cruzi antigen, conditions the host immune response in favor of parasite

A mechanism for the initiation of allergen-induced T helper type 2 responses

Basophils function as antigen-presenting cells for an allergen-induced T helper type 2 response

SRVXUH WR proteases from helminths and house dust mites

Fasciola hepatica suppresses a protective Th1 response against Bordetella pertussis

Fasciola hepatica infection downregulates Th1 responses in mice

Fasciola hepatica cathepsin L cysteine proteinase suppresses

The immunobiology of schistosomiasis

Helminth cysteine proteases inhibit TRIF-dependent activation of macrophages via degradation of TLR3

Major secretory antigens of the helminth Fasciola hepatica activate a suppressive dendritic cell phenotype that attenuates Th17 cells but fails to activate Th2 immune responses

Corruption of innate immunity by bacterial proteases

Pathogen recognition and innate immunity

The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors

Toll-like receptor signalling

Signaling to NF-kappaB by Toll-like receptors

Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in human periodontal disease: occurrence and treatment

Virulence factors of Porphyromonas gingivalis

Regulation of interactions of Gram-negative bacterial endotoxins with mammalian cells

Proteolysis of human monocyte CD14 by cysteine proteinases (gingipains) from Porphyromonas gingivalis leading to lipopolysaccharide hyporesponsiveness

Loss of lipopolysaccharide receptor CD14 from the surface of human macrophage-like cells mediated by Porphyromonas gingivalis outer membrane vesicles

Crystal structure of CD14 and its implications for lipopolysaccharide signaling

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The role of gingipains in the pathogenesis of periodontal disease

The importance of pH in regulating the function of the Fasciola hepatica cathepsin L1 cysteine protease

DVFLROD hepatica: a Leu-12 to Pro-12 replacement in the nonconserved C-terminal region of the prosegment SUHYHQWV FRPSOHWH HQ

CD14 is a member of the family of leucine-rich proteins and is encoded by a gene syntenic with multiple receptor genes

Biochemical and Functional Analyses of the Human Toll-like Receptor 3 Ectodomain

Squamous cell carcinoma antigen 1 is an inhibitor of parasite-derived cysteine proteases

The role of cystatins in cells of the immune system

Helminth antigens modulate TLR-initiated dendritic cell activation

Schistosoma mansoni soluble egg antigens are internalized by human dendritic cells through multiple C-type lectins and suppress TLR-induced dendritic cell activation

Leishmania species: models of intracellular parasitism

Leishmania promastigotes evade interleukin-12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4 T-cells during initiation of infection

Leishmania promastigotes selectively inhibit interleukin-12 induction in bone marrow-derived macrophages from susceptible and resistant mice

Recombinant interleukin 12 cures mice infected with Leishmania major

Resolution of cutaneous leishmaniasis: interleukin-12 initiates a protective T helper type 1 immune response

The adjuvant effect of interleukin-12 in a vaccine against Leishmania major

CPB cysteine proteases of Leishmania mexicana inhibit host Th1 responses and protective immunity

Inhibition of lipopolysaccharide-induced macrophage IL-12 production by Leishmania mexicana amastigotes: the role of cysteine peptidases and the NF-kappaB signaling pathway

Initiation and termination of NF-kappaB signaling by the intracellular protozoan parasite Toxoplasma gondii

New roles for perforins and proteases in apicomplexan egress

A genomic and functional inventory of deubiquitinating enzymes

The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion

The Papain like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

Severe acute respiratory syndrome coronavirus papain-like protease ubiquitin-like domain and catalytic domain regulate antagonism of IRF3 and NF-kappaB signaling

SARS coronavirus and innate immunity

Mouse hepatitis virus does not induce Beta interferon synthesis and does not inhibit its induction by doublestranded RNA

Group 2 coronaviruses prevent immediate early interferon induction by protection of viral RNA from host cell recognition

Viruses know it all: new insights into IFN networks

A deubiquitinating enzyme encoded by HSV-1 belongs to a family of cysteine proteases that is conserved across the family Herpesviridae

A deubiquitinating activity is conserved in the large tegument protein of the Herpesviridae

High-molecular-weight protein (pUL48) of human cytomegalovirus is a competent deubiquitinating protease: mutant viruses altered in its activesite cysteine or histidine are viable

Deubiquitinating function of adenovirus proteinase

The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

YopJ targets TRAF proteins to inhibit TLR-mediated NF-kappaB, MAPK and IRF3 signal transduction

Yersinia virulence factor YopJ acts as a deubiquitinase to inhibit NF-kappa B activation

Disruption of signaling by Yersinia effector YopJ, a ubiquitin-like protein protease

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Immunodiagnosis of Fasciola hepatica infection (fascioliasis)

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Cathepsin L proteinase secreted by Fasciola hepatica in vitro prevents antibody-mediated eosinophil attachment to newly excysted juveniles

Fasciola hepatica: parasite-secreted proteinases degrade all human

Activities of different cysteine proteases of Paragonimus westermani in cleaving human IgG

Cleavage of immunoglobulin G by excretory-secretory cathepsin S-like protease of Spirometra mansoni plerocercoid

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IdeS and SpeB: immunoglobulin-degrading cysteine proteinases of Streptococcus pyogenes

Effect of SpeB and EndoS from Streptococcus pyogenes on human immunoglobulins

The IgAl proteases of pathogenic bacteria

Defense mechanisms involving Fc-dependent functions of immunoglobulin A and their subversion by bacterial immunoglobulin A proteases

Degradation of human secretory IgA1 and IgA2 by Entamoeba histolytica surface-associated proteolytic activity

Cysteine proteinases and the pathogenesis of amebiasis

Degradation of complement 3 by streptococcal pyrogenic exotoxin B inhibits complement activation and neutrophil opsonophagocytosis

Group A streptococcal cysteine protease degrades C3 (C3b) and contributes to evasion of innate immunity

The extracellular neutral cysteine proteinase of Entamoeba histolytica degrades anaphylatoxins C3a and C5a

Biphasic effect of gingipains from Porphyromonas gingivalis on the human complement system

Interpain A, a cysteine proteinase from Prevotella intermedia, inhibits complement by degrading complement factor C3

Potentiation of C1 esterase inhibitor by StcE, a metalloprotease secreted by Escherichia coli O157:H7