16 research outputs found
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
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
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
Host ICAMs play a role in cell invasion by Mycobacterium tuberculosis and Plasmodium falciparum
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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
A novel peptide interferes with Mycobacterium tuberculosis virulence and survival
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
Identification of novel single nucleotide polymorphisms in the DGAT1 gene of buffaloes by PCR-SSCP
Diacylglycerol O-acyltransferase 1 (DGAT1) is a microsomal enzyme that catalyzes the final step of triglyceride synthesis. The DGAT1 gene is a strong functional candidate for determining milk fat content in cattle. In this work, we used PCR-SSCP (polymerase chain reaction-single-strand conformation polymorphism) and DNA sequencing to examine polymorphism in the region spanning exon 7 to exon 9 of the DGAT1 gene in Murrah and Pandharpuri buffaloes. Three alleles (A, B and C) and four novel single-nucleotide polymorphisms were identified in the buffalo DGAT1 gene. The frequencies of the alleles differed between the two buffalo breeds, with allele C being present in Murrah but not in Pandharpuri buffalo. The allele variation detected in this work may influence DGAT1 expression and function. The results described here could be useful in examining the association between the DGAT1 gene and milk traits in buffalo
Rv1626-ARPC4 protein-protein interaction <i>in vitro</i>.
<p><b>A.</b> Coomassie-stained purification gel of ARPC4-His<sub>6X</sub>. ARPC4-His<sub>6X</sub> protein was purified under denaturing conditions on a Ni-NTA column. 15% SDS polyacrylamide gel showing purified C-terminal His-tagged ARPC4 protein, stained with coomassie blue. Lane 1: uninduced cell lysate, Lane 2: induced cell lysate, Lane 3: supernatant fraction of cell lysate (load), Lane 4: final wash, Lane 5: low molecular weight protein marker (the sizes, in kDa, are indicated on the left of the panel), Lane 6–7: purified protein elution fractions. <b>B.</b> Coomassie-stained purification gel of GST-Rv1626 proteins GST-Rv1626 protein was purified under native conditions on a Glutathione Sepharose-4B resin column. 15% SDS polyacrylamide gel showing the purified GST-Rv1626 protein, stained with coomassie blue. Lane 1: uninduced cell lysate, Lane 2: induced cell lysate, Lane 3–7 (except 4) are fractions collected from the column; Lane 3: wash fraction, Lane 4: low molecular weight protein marker (the sizes, in kDa, are indicated on the left of the panel), Lane 5–7: purified protein elution fractions. <b>C.</b> Dot Far-Western Blots showing <i>in vitro</i> protein-protein interactions. 1 µg each of purified ARPC4-His<sub>6X</sub>, GST, GST-Rv1626 or BSA proteins were immobilized on nitrocellulose strips that were incubated overnight in 1 µg/mL solution of the either GST-Rv1626 or GST protein. The strips were then developed using the antibody indicated in the parenthesis.</p
Rv1626-Arp2/3 protein-protein interaction.
<p><b>A.</b> Dot Far-Western Blots showing <i>in vitro</i> protein-protein interaction between Arp2/3 and Rv1626. 1 µg each of Arp2/3 and BSA proteins were immobilized and the strips incubated overnight in 1 µg/mL solution of the either GST-Rv1626 or GST protein followed by development using anti-GST antibody. <b>B.</b> Rv1626 interaction with Arp2/3 enhances Actin Polymerization. Polymerization of G-actin to F-actin leads to an increase in the fluorescence intensity of pyrene-labelled actin, plotted here as a function of time. The figure shows the effect of Rv1626 protein on the rate of actin polymerization. Rv1626 (500 nM alone, black); Arp2/3 (10 nM) in the presence of Rv1626 (500 nM, red), VCA domain (400 nM, blue), and an unrelated protein (500 nM, green).</p