Currently working towards unearthing the anomalies responsible for Aspirin intolerant Asthma (AERD). It is known that every 10th person with asthma is sensitive to aspirin and similar drugs, and upon ingestion leads to severe bronchoconstriction and acute eosinophilia. But very little is known about the cause of aspirin intolerance in asthmatic patients. I am trying to answer this by studying prostaglandins and leukotrienes during respiratory infections, host response and onset of Asthma.
Type 2 immunopathology is a cardinal feature of allergic diseases, and involves cooperation betwe... more Type 2 immunopathology is a cardinal feature of allergic diseases, and involves cooperation between adaptive immunity and innate effector responses. Virtually all cell types relevant to this pathology generate leukotriene and/or prostaglandin mediators that derive from arachidonic acid, and/or express receptors for such mediators. Recent studies highlight prominent functions for these mediators in communication between innate and adaptive immune systems, as well as amplification or suppression of type 2 effector responses. This review focuses on recent advances and insights, and highlights existing and potential therapeutic applications of drugs that target these mediators or their receptors, with a special emphasis on their regulation of the innate and adaptive lymphocytes relevant to type 2 immunopathology.
Group V phospholipase A2 (Pla2g5) is a lipid-generating enzyme necessary for macrophage effector ... more Group V phospholipase A2 (Pla2g5) is a lipid-generating enzyme necessary for macrophage effector functions in pulmonary inflammation. However, the lipid mediators involved and their cellular targets have not been identified. Mice lacking Pla2g5 showed markedly reduced lung ILC2 activation and eosinophilia following repetitive Alternaria Alternata inhalation. While Pla2g5-null mice had Wt levels of immediate IL-33 release after one Alternaria dose, they failed to upregulate IL-33 in macrophages following repeated Alternaria administration. Unexpectedly, while adoptive transfer of bone marrow-derived (BM)-macrophages restored ILC2 activation and eosinophilia in Alternaria-exposed Pla2g5-null mice, exogenous IL-33 did not. Conversely, transfers of Pla2g5-null BM-macrophages reduced inflammation in Alternaria-exposed Wt mice. Mass spectrometry analysis of free fatty acids (FFAs) demonstrated significantly reduced FFAs (including linoleic acid (LA) and oleic acid (OA)) in lung and BM-macrophages lacking Pla2g5. Exogenous administration of LA or LA+OA to Wt mice sharply potentiated IL-33-induced lung eosinophilia and ILC2 expansion in vitro and in vivo. In contrast, OA potentiated IL-33-induced inflammation and ILC2 expansion in Pla2g5-null mice, but LA was inactive both in vivo and in vitro. Notably, Pla2g5-null ILC2s showed significantly reduced expression of the FFA-receptor-1 compared to Wt ILC2s. Thus, macrophage-associated Pla2g5 contributes significantly to type-2 immunity through regulation of IL-33 induction and FFA-driven ILC2 activation.
When activated through toll-like receptors (TLRs), macro-phages generate IL-33, an IL-1 family me... more When activated through toll-like receptors (TLRs), macro-phages generate IL-33, an IL-1 family member that induces innate immune responses through ST2 signaling. LPS, a TLR4 ligand, induces macrophages to generate prostaglandin E 2 (PGE 2) through inducible COX-2 and microsomal PGE 2 syn-thase 1 (mPGES-1) (1). We demonstrate that IL-33 production by bone marrow-derived murine macrophages (bmMFs) requires the generation of endogenous PGE 2 and the intrinsic expression of EP 2 receptors to amplify NF-B-dependent, LPS-induced IL-33 expression via exchange protein activated by cAMP (EPAC). Compared with WT cells, bmMFs lacking either mPGES-1 or EP 2 receptors displayed reduced LPS-induced IL-33 levels. A selective EP 2 agonist and, to a lesser extent, EP 4 receptor agonist potentiated LPS-induced IL-33 generation from both mPGES-1-null and WT bmMFs, whereas EP 1 and EP 3 receptor agonists were inactive. The effects of PGE 2 depended on cAMP, were mimicked by an EPAC-selective agonist, and were attenuated by EPAC-selective antagonism and knockdown. LPS-induced p38 MAPK and NF-B activations were necessary for both IL-33 production and PGE 2 generation, and exogenous PGE 2 partly reversed the suppression of IL-33 production caused by p38 MAPK and NF-B inhibition. Mice lacking mPGES-1 showed lower IL-33 levels and attenuated lung inflammation in response to repetitive Alternaria inhalation challenges. Cumulatively, our data demonstrate that endogenous PGE 2 , EP 2 receptors, and EPAC are prerequisites for maximal LPS-induced IL-33 expression and that exogenous PGE 2 can amplify IL-33 production via EP 2 and EP 4 receptors. The ubiquitous induction of mPGES-1-dependent PGE 2 may be crucial for innate immune system activation during various IL-33 driven pathologic disorders. IL-33 is a recently discovered member of the IL-1 cytokine family that binds to TLR/IL1R 2 superfamily receptor ST2 (2– 4). IL-33 is constitutively expressed in the nuclei of epithe-lial cells at barrier sites such as the skin, lung, and intestine and is also expressed by fibroblastic reticular cells and endothelial cells within lymphoid tissues (5). It interacts with histones, promotes chromatin compaction, and retards NF-B transcrip-tional activity (5). It can also be released in response to cellular damage or activation and, after proteolytic processing to an active cytokine, can bind to a heterotrimeric receptor composed of T1/ST2 and IL-1RAcP, influencing both innate and adaptive type 2 immune responses (2). In addition to barrier cells, macrophages and dendritic cells can inducibly express IL-33 and contribute to the amplification of inflammatory responses in sepsis, cardiovascular disease, and allergy (6 –9). However, little is known about the mechanisms that control such inducible IL-33 expression. Among the resident hematopoietic cells involved in innate immunity, macrophages are noteworthy for inducible expression of IL-33 in response to LPS in vitro (10 –12) and also upon infection with H3N1 or by TLR7/8 agonist stimulation (13). These same stimuli also induce macrophages to generate pros-taglandin E 2 (PGE 2), a lipid mediator that plays critical roles in pyrexia, pain sensation, and inflammation. LPS-stimulated macrophages generate PGE 2 from endogenous arachidonic acid that is converted sequentially to prostaglandin H 2 (PGH 2) and then to PGE 2 , primarily by the inducible enzymes COX-2 and microsomal PGE 2 synthase 1 (mPGES-1), respectively (1). When released, PGE 2 acts through four types of E prostanoid receptors (EP 1-EP 4) and can function as both autocrine as well as paracrine mediators (14). In addition to generating PGE 2 , macrophages also express EP receptors and respond to endog-enous and exogenous PGE 2 ex vivo. In particular, by eliciting signaling through cAMP and PKA-dependent transcription factors, PGE 2 can amplify the production of IL-6 by macro-phages and of IL-23 by dendritic cells but can also suppress the generation of IL-23 and IL-12 by human monocytes. Although exogenous PGE 2 can potentiate IL-33 expression by dendritic
Background: The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been know... more Background: The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung.
Methodology/Principal Findings: During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage.
Conclusions/Significance: While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide’s binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.
Intercellular adhesion molecules (ICAMs) belong to the immunoglobulin superfamily and
participate... more Intercellular adhesion molecules (ICAMs) belong to the immunoglobulin superfamily and participate in diverse cellular processes including host–pathogen interactions. ICAM-1 is expressed on various cell types including macrophages, whereas ICAM-4 is restricted to red blood cells. Here we report the identification of an 11-kDa synthetic protein, M5, that binds to human ICAM-1 and ICAM-4, as shown by in vitro interaction studies, surface plasmon resonance and immunolocalization. M5 greatly inhibits the invasion of macrophages and erythrocytes by Mycobacterium tuberculosis and Plasmodium falciparum, respectively. Pharmacological and siRNA-mediated inhibition of ICAM-1 expression also results in reduced M. tuberculosis invasion of macrophages. ICAM-4 binds to P. falciparum merozoites, and the addition of recombinant ICAM-4 to parasite cultures blocks invasion of erythrocytes by newly released merozoites. Our results indicate that ICAM-1 and ICAM-4 play roles in host cell invasion by M. tuberculosis and P. falciparum, respectively, either as receptors or as crucial accessory molecules.
Tuberculosis (TB) is a huge global burden, with new and resistant strains emerging at an alarming... more Tuberculosis (TB) is a huge global burden, with new and resistant strains emerging at an alarming rate, necessitating an urgent need for a new class of drug candidates. Here, we report that SL3, a novel 33-amino acid peptide, causes debilitating effects on mycobacterial morphology. Treatment with SL3 drastically inhibits the growth of Mycobacterium tuberculosis in vitro as well as in a pre-clinical mouse model for M.tb infection. Microarray analysis of SL3-expressing strain demonstrates wide-scale transcriptional disruption in M.tb. We therefore believe that SL3 and similar peptides may herald a new approach towards discovering new molecules for TB therapy.
Background: Tuberculosis (TB) is one of the most prevalent infectious diseases affecting millions... more Background: Tuberculosis (TB) is one of the most prevalent infectious diseases affecting millions worldwide. The currently available anti-TB drugs and vaccines have proved insufficient to contain this scourge, necessitating an urgent need for identification of novel drug targets and therapeutic strategies. The disruption of crucial protein-protein interactions, especially those that are responsible for virulence in Mycobacterium tuberculosis – for example the ESAT-6: CFP10 complex – are a worthy pursuit in this direction.
Background: Protein-protein interactions play a crucial role in enabling a pathogen to survive wi... more Background: Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.
Background: The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms... more Background: The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms facilitating the intra-macrophage survival of Mycobacterium tuberculosis are as yet not entirely understood. However, there is evidence showing the involvement of host cell cytoskeleton in every step of establishment and persistence of mycobacterial infection.
Type 2 immunopathology is a cardinal feature of allergic diseases, and involves cooperation betwe... more Type 2 immunopathology is a cardinal feature of allergic diseases, and involves cooperation between adaptive immunity and innate effector responses. Virtually all cell types relevant to this pathology generate leukotriene and/or prostaglandin mediators that derive from arachidonic acid, and/or express receptors for such mediators. Recent studies highlight prominent functions for these mediators in communication between innate and adaptive immune systems, as well as amplification or suppression of type 2 effector responses. This review focuses on recent advances and insights, and highlights existing and potential therapeutic applications of drugs that target these mediators or their receptors, with a special emphasis on their regulation of the innate and adaptive lymphocytes relevant to type 2 immunopathology.
Group V phospholipase A2 (Pla2g5) is a lipid-generating enzyme necessary for macrophage effector ... more Group V phospholipase A2 (Pla2g5) is a lipid-generating enzyme necessary for macrophage effector functions in pulmonary inflammation. However, the lipid mediators involved and their cellular targets have not been identified. Mice lacking Pla2g5 showed markedly reduced lung ILC2 activation and eosinophilia following repetitive Alternaria Alternata inhalation. While Pla2g5-null mice had Wt levels of immediate IL-33 release after one Alternaria dose, they failed to upregulate IL-33 in macrophages following repeated Alternaria administration. Unexpectedly, while adoptive transfer of bone marrow-derived (BM)-macrophages restored ILC2 activation and eosinophilia in Alternaria-exposed Pla2g5-null mice, exogenous IL-33 did not. Conversely, transfers of Pla2g5-null BM-macrophages reduced inflammation in Alternaria-exposed Wt mice. Mass spectrometry analysis of free fatty acids (FFAs) demonstrated significantly reduced FFAs (including linoleic acid (LA) and oleic acid (OA)) in lung and BM-macrophages lacking Pla2g5. Exogenous administration of LA or LA+OA to Wt mice sharply potentiated IL-33-induced lung eosinophilia and ILC2 expansion in vitro and in vivo. In contrast, OA potentiated IL-33-induced inflammation and ILC2 expansion in Pla2g5-null mice, but LA was inactive both in vivo and in vitro. Notably, Pla2g5-null ILC2s showed significantly reduced expression of the FFA-receptor-1 compared to Wt ILC2s. Thus, macrophage-associated Pla2g5 contributes significantly to type-2 immunity through regulation of IL-33 induction and FFA-driven ILC2 activation.
When activated through toll-like receptors (TLRs), macro-phages generate IL-33, an IL-1 family me... more When activated through toll-like receptors (TLRs), macro-phages generate IL-33, an IL-1 family member that induces innate immune responses through ST2 signaling. LPS, a TLR4 ligand, induces macrophages to generate prostaglandin E 2 (PGE 2) through inducible COX-2 and microsomal PGE 2 syn-thase 1 (mPGES-1) (1). We demonstrate that IL-33 production by bone marrow-derived murine macrophages (bmMFs) requires the generation of endogenous PGE 2 and the intrinsic expression of EP 2 receptors to amplify NF-B-dependent, LPS-induced IL-33 expression via exchange protein activated by cAMP (EPAC). Compared with WT cells, bmMFs lacking either mPGES-1 or EP 2 receptors displayed reduced LPS-induced IL-33 levels. A selective EP 2 agonist and, to a lesser extent, EP 4 receptor agonist potentiated LPS-induced IL-33 generation from both mPGES-1-null and WT bmMFs, whereas EP 1 and EP 3 receptor agonists were inactive. The effects of PGE 2 depended on cAMP, were mimicked by an EPAC-selective agonist, and were attenuated by EPAC-selective antagonism and knockdown. LPS-induced p38 MAPK and NF-B activations were necessary for both IL-33 production and PGE 2 generation, and exogenous PGE 2 partly reversed the suppression of IL-33 production caused by p38 MAPK and NF-B inhibition. Mice lacking mPGES-1 showed lower IL-33 levels and attenuated lung inflammation in response to repetitive Alternaria inhalation challenges. Cumulatively, our data demonstrate that endogenous PGE 2 , EP 2 receptors, and EPAC are prerequisites for maximal LPS-induced IL-33 expression and that exogenous PGE 2 can amplify IL-33 production via EP 2 and EP 4 receptors. The ubiquitous induction of mPGES-1-dependent PGE 2 may be crucial for innate immune system activation during various IL-33 driven pathologic disorders. IL-33 is a recently discovered member of the IL-1 cytokine family that binds to TLR/IL1R 2 superfamily receptor ST2 (2– 4). IL-33 is constitutively expressed in the nuclei of epithe-lial cells at barrier sites such as the skin, lung, and intestine and is also expressed by fibroblastic reticular cells and endothelial cells within lymphoid tissues (5). It interacts with histones, promotes chromatin compaction, and retards NF-B transcrip-tional activity (5). It can also be released in response to cellular damage or activation and, after proteolytic processing to an active cytokine, can bind to a heterotrimeric receptor composed of T1/ST2 and IL-1RAcP, influencing both innate and adaptive type 2 immune responses (2). In addition to barrier cells, macrophages and dendritic cells can inducibly express IL-33 and contribute to the amplification of inflammatory responses in sepsis, cardiovascular disease, and allergy (6 –9). However, little is known about the mechanisms that control such inducible IL-33 expression. Among the resident hematopoietic cells involved in innate immunity, macrophages are noteworthy for inducible expression of IL-33 in response to LPS in vitro (10 –12) and also upon infection with H3N1 or by TLR7/8 agonist stimulation (13). These same stimuli also induce macrophages to generate pros-taglandin E 2 (PGE 2), a lipid mediator that plays critical roles in pyrexia, pain sensation, and inflammation. LPS-stimulated macrophages generate PGE 2 from endogenous arachidonic acid that is converted sequentially to prostaglandin H 2 (PGH 2) and then to PGE 2 , primarily by the inducible enzymes COX-2 and microsomal PGE 2 synthase 1 (mPGES-1), respectively (1). When released, PGE 2 acts through four types of E prostanoid receptors (EP 1-EP 4) and can function as both autocrine as well as paracrine mediators (14). In addition to generating PGE 2 , macrophages also express EP receptors and respond to endog-enous and exogenous PGE 2 ex vivo. In particular, by eliciting signaling through cAMP and PKA-dependent transcription factors, PGE 2 can amplify the production of IL-6 by macro-phages and of IL-23 by dendritic cells but can also suppress the generation of IL-23 and IL-12 by human monocytes. Although exogenous PGE 2 can potentiate IL-33 expression by dendritic
Background: The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been know... more Background: The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung.
Methodology/Principal Findings: During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage.
Conclusions/Significance: While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide’s binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.
Intercellular adhesion molecules (ICAMs) belong to the immunoglobulin superfamily and
participate... more Intercellular adhesion molecules (ICAMs) belong to the immunoglobulin superfamily and participate in diverse cellular processes including host–pathogen interactions. ICAM-1 is expressed on various cell types including macrophages, whereas ICAM-4 is restricted to red blood cells. Here we report the identification of an 11-kDa synthetic protein, M5, that binds to human ICAM-1 and ICAM-4, as shown by in vitro interaction studies, surface plasmon resonance and immunolocalization. M5 greatly inhibits the invasion of macrophages and erythrocytes by Mycobacterium tuberculosis and Plasmodium falciparum, respectively. Pharmacological and siRNA-mediated inhibition of ICAM-1 expression also results in reduced M. tuberculosis invasion of macrophages. ICAM-4 binds to P. falciparum merozoites, and the addition of recombinant ICAM-4 to parasite cultures blocks invasion of erythrocytes by newly released merozoites. Our results indicate that ICAM-1 and ICAM-4 play roles in host cell invasion by M. tuberculosis and P. falciparum, respectively, either as receptors or as crucial accessory molecules.
Tuberculosis (TB) is a huge global burden, with new and resistant strains emerging at an alarming... more Tuberculosis (TB) is a huge global burden, with new and resistant strains emerging at an alarming rate, necessitating an urgent need for a new class of drug candidates. Here, we report that SL3, a novel 33-amino acid peptide, causes debilitating effects on mycobacterial morphology. Treatment with SL3 drastically inhibits the growth of Mycobacterium tuberculosis in vitro as well as in a pre-clinical mouse model for M.tb infection. Microarray analysis of SL3-expressing strain demonstrates wide-scale transcriptional disruption in M.tb. We therefore believe that SL3 and similar peptides may herald a new approach towards discovering new molecules for TB therapy.
Background: Tuberculosis (TB) is one of the most prevalent infectious diseases affecting millions... more Background: Tuberculosis (TB) is one of the most prevalent infectious diseases affecting millions worldwide. The currently available anti-TB drugs and vaccines have proved insufficient to contain this scourge, necessitating an urgent need for identification of novel drug targets and therapeutic strategies. The disruption of crucial protein-protein interactions, especially those that are responsible for virulence in Mycobacterium tuberculosis – for example the ESAT-6: CFP10 complex – are a worthy pursuit in this direction.
Background: Protein-protein interactions play a crucial role in enabling a pathogen to survive wi... more Background: Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes.
Background: The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms... more Background: The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms facilitating the intra-macrophage survival of Mycobacterium tuberculosis are as yet not entirely understood. However, there is evidence showing the involvement of host cell cytoskeleton in every step of establishment and persistence of mycobacterial infection.
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Papers by SACHIN SAMUCHIWAL
Methodology/Principal Findings: During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage.
Conclusions/Significance: While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide’s binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this
peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.
participate in diverse cellular processes including host–pathogen interactions. ICAM-1 is
expressed on various cell types including macrophages, whereas ICAM-4 is restricted to red blood cells. Here we report the identification of an 11-kDa synthetic protein, M5, that binds to human ICAM-1 and ICAM-4, as shown by in vitro interaction studies, surface plasmon resonance and immunolocalization. M5 greatly inhibits the invasion of macrophages and erythrocytes by Mycobacterium tuberculosis and Plasmodium falciparum, respectively. Pharmacological and siRNA-mediated inhibition of ICAM-1 expression also results in reduced M. tuberculosis invasion of macrophages. ICAM-4 binds to P. falciparum merozoites, and the addition of recombinant ICAM-4 to parasite cultures blocks invasion of erythrocytes by newly released merozoites. Our results indicate that ICAM-1 and ICAM-4 play roles in host cell invasion by M. tuberculosis and P. falciparum, respectively, either as receptors or as crucial accessory molecules.
Methodology/Principal Findings: During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage.
Conclusions/Significance: While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide’s binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this
peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.
participate in diverse cellular processes including host–pathogen interactions. ICAM-1 is
expressed on various cell types including macrophages, whereas ICAM-4 is restricted to red blood cells. Here we report the identification of an 11-kDa synthetic protein, M5, that binds to human ICAM-1 and ICAM-4, as shown by in vitro interaction studies, surface plasmon resonance and immunolocalization. M5 greatly inhibits the invasion of macrophages and erythrocytes by Mycobacterium tuberculosis and Plasmodium falciparum, respectively. Pharmacological and siRNA-mediated inhibition of ICAM-1 expression also results in reduced M. tuberculosis invasion of macrophages. ICAM-4 binds to P. falciparum merozoites, and the addition of recombinant ICAM-4 to parasite cultures blocks invasion of erythrocytes by newly released merozoites. Our results indicate that ICAM-1 and ICAM-4 play roles in host cell invasion by M. tuberculosis and P. falciparum, respectively, either as receptors or as crucial accessory molecules.