A Three-Hybrid System to Probe In Vivo Protein-Protein Interactions: Application to the Essential Proteins of the RD1 Complex of M. tuberculosis

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. METHODOLOGY/PRINCIPAL FINDINGS: The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3. CONCLUSIONS/SIGNIFICANCE: The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes

Expression of the ARPC4 subunit of human Arp2/3 severely affects mycobacterium tuberculosis growth and suppresses immunogenic response in murine macrophages

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. Methodology/Principal Findings: Here we show that expression of ARPC4, a subunit of the Actin related protein 2/3 (Arp2/3) protein complex, severely affects the pathogen’s growth. TEM studies display shedding of the mycobacterial outer-coat. Furthermore, in infected macrophages, mycobacteria expressing ARPC4 were cleared off at a much faster rate, and were unable to mount a pro-inflammatory cytokine response. The translocation of ARPC4-expressing mycobacteria to the lysosome of the infected macrophage was also impaired. Additionally, the ARPC4 subunit was shown to interact with Rv1626, an essential secretory mycobacterial protein. Real-time PCR analysis showed that upon expression of ARPC4 in mycobacteria, Rv1626 expression is downregulated as much as six-fold. Rv1626 was found to also interact with mammalian cytoskeleton protein, Arp2/3, and enhance the rate of actin polymerization. Conclusions/Significance: With crystal structures for Rv1626 and ARPC4 subunit already known, our finding lays out the effect of a novel molecule on mycobacteria, and represents a viable starting point for developing potent peptidomimetics

A peptide fragment from the human COX3 protein disrupts association of Mycobacterium tuberculosisvirulence proteins ESAT-6 and CFP10, inhibits mycobacterial growth and mounts protective immune response

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. METHODS: We therefore sought to improvise a method to attenuate M. tuberculosis while retaining the latter’s antigenic properties. We screened peptide libraries for potent ESAT-6 binders capable of dissociating CFP10 from ESAT-6. We assessed the disruption by a peptide named HCL2, of the ESAT-6:CFP10 complex and studied its effects on mycobacterial survival and virulence. RESULTS: We found that HCL2, derived from the human cytochrome c oxidase subunit 3 (COX3) protein, disrupts ESAT-6:CFP10 complex, binds ESAT-6 potently, disintegrates bacterial cell wall and inhibits extracellular as well as intracellular mycobacterial growth. In addition, an HCL2 expressing M. tuberculosis strain induces both Th1 and Th17 host protective responses. CONCLUSIONS: Disruption of ESAT-6:CFP10 association could, therefore, be an alternate method for attenuating M. tuberculosis, and a possible route towards future vaccine generation

Host ICAMs play a role in cell invasion by Mycobacterium tuberculosis and Plasmodium falciparum

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Eicosanoid control over antigen presenting cells in asthma

Asthma is a common lung disease affecting 300 million people worldwide. Allergic asthma is recognized as a prototypical Th2 disorder, orchestrated by an aberrant adaptive CD4+ T helper (Th2/Th17) cell immune response against airborne allergens, that leads to eosinophilic inflammation, reversible bronchoconstriction, and mucus overproduction. Other forms of asthma are controlled by an eosinophil-rich innate ILC2 response driven by epithelial damage, whereas in some patients with more neutrophilia, the disease is driven by Th17 cells. Dendritic cells (DCs) and macrophages are crucial regulators of type 2 immunity in asthma. Numerous lipid mediators including the eicosanoids prostaglandins and leukotrienes influence key functions of these cells, leading to either pro- or anti-inflammatory effects on disease outcome. In this review, we will discuss how eicosanoids affect the functions of DCs and macrophages in the asthmatic lung and how this leads to aberrant T cell differentiation that causes disease

Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis, and Concurrently Affect the Pathogen's Growth

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

ILC2s—Trailblazers in the Host Response Against Intestinal Helminths

Group 2 innate lymphoid cells (ILC2s) were first discovered in experimental studies of intestinal helminth infection—and much of our current knowledge of ILC2 activation and function is based on the use of these models. It is perhaps not surprising therefore that these cells have also been found to play a key role in mediating protection against these large multicellular parasites. ILC2s have been intensively studied over the last decade, and are known to respond quickly and robustly to the presence of helminths—both by increasing in number and producing type 2 cytokines. These mediators function to activate and repair epithelial barriers, to recruit other innate cells such as eosinophils, and to help activate T helper 2 cells. More recent investigations have focused on the mechanisms by which the host senses helminth parasites to activate ILC2s. Such studies have identified novel stromal cell types as being involved in this process—including intestinal tuft cells and enteric neurons, which respond to the presence of helminths and activate ILC2s by producing IL-25 and Neuromedin, respectively. In the current review, we will outline the latest insights into ILC2 activation and discuss the requirement for—or redundancy of—ILC2s in providing protective immunity against intestinal helminth parasites

Endogenous prostaglandin E2 amplifies IL-33 production by macrophages through an E prostanoid (EP)2/EP4-cAMP-EPACdependent pathway

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 9.5px Helvetica} span.s1 {font: 6.0px Helvetica} <p>When activated through toll-like receptors (TLRs), macrophages</p> <p>generate IL-33, an IL-1 family member that induces</p> <p>innate immune responses through ST2 signaling. LPS, a TLR4</p> <p>ligand, induces macrophages to generate prostaglandin E2</p> <p>(PGE2) through inducible COX-2 and microsomal PGE2 synthase</p> <p>1 (mPGES-1) (1). We demonstrate that IL-33 production</p> <p>by bone marrow-derived murine macrophages (bmMFs) requires</p> <p>the generation of endogenous PGE2 and the intrinsic</p> <p>expression of EP2 receptors to amplify NF-B-dependent, LPS-induced</p> <p>IL-33 expression via exchange protein activated by cAMP</p> <p>(EPAC). Compared with WT cells, bmMFs lacking either</p> <p>mPGES-1 or EP2 receptors displayed reduced LPS-induced IL-33</p> <p>levels. A selective EP2 agonist and, to a lesser extent, EP4 receptor</p> <p>agonist potentiated LPS-induced IL-33 generation from both</p> <p>mPGES-1-null and WT bmMFs, whereas EP1 and EP3 receptor</p> <p>agonists were inactive. The effects of PGE2 depended on cAMP,</p> <p>were mimicked by an EPAC-selective agonist, and were attenuated</p> <p>by EPAC-selective antagonism and knockdown. LPS-induced p38</p> <p>MAPK and NF-B activations were necessary for both IL-33 production</p> <p>andPGE2 generation, and exogenousPGE2 partly reversed</p> <p>the suppression of IL-33 production caused by p38 MAPK and</p> <p>NF-B inhibition. Mice lacking mPGES-1 showed lower IL-33 levels</p> <p>and attenuated lung inflammation in response to repetitive</p> <p>Alternaria inhalation challenges. Cumulatively, our data demonstrate</p> <p>that endogenousPGE2, EP2 receptors, andEPACare prerequisites</p> <p>for maximal LPS-induced IL-33 expression and that</p> <p>exogenous PGE2 can amplify IL-33 production via EP2 and EP4</p> <p>receptors. The ubiquitous induction of mPGES-1-dependent</p> <p>PGE2 may be crucial for innate immune system activation during</p> <p>various IL-33 driven pathologic disorders.</p

A Three-Hybrid System to Probe In Vivo Protein-Protein Interactions: Application to the Essential Proteins of the RD1 Complex of M. tuberculosis

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 9.0px Helvetica} span.s1 {font: 9.5px Helvetica} <p>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</p> <p>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.</p><p><br></p><p>The protein-protein interactions involved in M. tuberculosis pathogenesis have been used<br></p> <p>as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide</p> <p>candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and</p> <p>another peptide SL3.</p><p><br></p> <p>The validation of the three-hybrid system paves the way for finding new peptides that are</p> <p>stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.</p

Expression of the ARPC4 Subunit of Human Arp2/3 Severely Affects Mycobacterium tuberculosis Growth and Suppresses Immunogenic Response in Murine Macrophages

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 9.0px Helvetica} span.s1 {font: 9.5px Helvetica} <p>Background: The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms facilitating the intramacrophage</p> <p>survival of Mycobacterium tuberculosis are as yet not entirely understood. However, there is evidence showing</p> <p>the involvement of host cell cytoskeleton in every step of establishment and persistence of mycobacterial infection.</p> <p>Methodology/Principal Findings: Here we show that expression of ARPC4, a subunit of the Actin related protein 2/3 (Arp2/</p> <p>3) protein complex, severely affects the pathogen’s growth. TEM studies display shedding of the mycobacterial outer-coat.</p> <p>Furthermore, in infected macrophages, mycobacteria expressing ARPC4 were cleared off at a much faster rate, and were</p> <p>unable to mount a pro-inflammatory cytokine response. The translocation of ARPC4-expressing mycobacteria to the</p> <p>lysosome of the infected macrophage was also impaired. Additionally, the ARPC4 subunit was shown to interact with</p> <p>Rv1626, an essential secretory mycobacterial protein. Real-time PCR analysis showed that upon expression of ARPC4 in</p> <p>mycobacteria, Rv1626 expression is downregulated as much as six-fold. Rv1626 was found to also interact with mammalian</p> <p>cytoskeleton protein, Arp2/3, and enhance the rate of actin polymerization.</p> <p>Conclusions/Significance: With crystal structures for Rv1626 and ARPC4 subunit already known, our finding lays out the</p> <p>effect of a novel molecule on mycobacteria, and represents a viable starting point for developing potent peptidomimetics.</p