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

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

Activation of infected macrophages and inter-compartmental localization of mycobacteria within A.

<p>The plots depict reduced activation of H37RV/ARPC4 infected macrophages. <b>a)</b> Percentage of cells expressing PD1 among CD11B+ cells is shown in the dot plots with mean±STDEV. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B and PD1 antibodies and samples were acquired by flow cytometry. Data shown here are representative of three independent experiments. <b>b)</b> Cell death in H37Rv and H37Rv/ARPC4 Mtb infected macrophages. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains in 1∶10 ratio, were surface-stained with anti-CD11B, CD11C antibodies followed by PI staining for 20 minutes prior to acquisition by flow cytometry to assess cell death in infected macrophages. The percentage of cells expressing PI among CD11B±cells is shown with mean±STDEV. Data shown here are representative of three independent experiments. <b>c)</b> Expression of macrophage activation markers. Intraperitoneal macrophages isolated from mice were cultured and, 48 hours post-infection with H37Rv and H37Rv/ARPC4 strains at 10 MOI, were surface-stained with anti-CD11B, CD11C, MHCII, CD14, CD54, CD80, CD86 and CD69 antibodies and samples were acquired by flow cytometry. CD11B+ cells were gated for expression of MHCII, CD14, CD54, CD80 and CD86 and the percentage of cells expressing these markers are shown in the histogram overlay plots with mean±STDEV. Data shown here are representative of three independent experiments. <b>B.</b> Confocal Microscopy images showing that H37Rv/ARPC4 mycobacterial strain is less infectious and persistent in macrophage. FITC-labelled mycobacterial cells (green) were used for infecting macrophages (red) at an MOI of 5. At specific time-points, the cells were fixed, permeabilized and stained with anti-LAMP1 antibodies, followed by Alexa Fluor 594 goat anti-rat antibodies. (a)- (d) shows the infected macrophage cells at 0, 24, 48 and 72 hours' time-points post-infection, respectively. (e) and (f) are the graphical representation of the percentage of macrophage cells infected with FITC-labeled mycobacteria at different time-points and the percentage of bacteria localized in lysosomes in the infected cells, respectively. Experiments were repeated thrice and similar results were obtained.</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

Rv1626-ARPC4 protein-protein interaction by bacterial two-hybrid.

<p><b>A.</b> Bacterial two-hybrid plate showing Rv1626 and ARPC4 interaction where blue colored colonies show interaction between the bait and target proteins. BacterioMatch two-hybrid <i>E. coli</i> reporter strain was co-transformed with Rv1626-pBTnn+ARPC4-pTRGnn; pBTnn+ARPC4-pTRGnn (negative control); LGF2-pBT+Gal11p-pTRG (positive control); and Rv1626-pBTnn+Gal11p-pTRG (negative control) plasmids. Two individual colonies from each co-transformant were patched on X-gal indicator plate. <b>B.</b> Histogram of liquid β-galactosidase assay for quantitative estimation of interaction strength of Rv1626 and ARPC4 compared to that with positive and negative controls. Co-transformants ARPC4-pTRGnn+pBTnn and Gal11p-pTRG+Rv1626-pBTnn were taken as the negative controls; CFP10-pTRGnn+ESAT6-pBTnn and Gal11p-pTRG+LGF2-pBT were taken as positive controls; and these were used to compare the β-galactosidase activity of co-transformant ARPC4-pTRGnn+Rv1626-pBTnn. Enzyme activity is a direct indication of the strength of interaction of the bait and target proteins and, is expressed in terms of Miller Units. All interactions were found to be statistically significant by Student’s t-test (*, P<0.05).</p

Sequences of the DNA primers used for PCR amplification of various genes described in the present study.

<p>Sequences of the DNA primers used for PCR amplification of various genes described in the present study.</p

Rv1626-ARPC4 protein-protein interaction <i>in vitro</i>.

<p><b>A.</b> Coomassie-stained purification gel of ARPC4-His_6X. ARPC4-His_6X 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_6X, 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

Effect of ARPC4 on the growth of <i>M. tuberculosis</i>.

<p>A. An individual colony each from mycobacteria harbouring either ARPC4pVV16 (H37Rv/ARPC4) or pVV16 empty vector (H37Rv/pVV16) was picked for growth curve analysis. Optical density of both cultures was measured at 600 nm. Mean±s.d values are plotted against time (in Days). A similar growth curve pattern was observed each time the experiment was repeated. Triangular data points represent OD₆₀₀ values of H37Rv/pVV16 samples (control); circles represent OD₆₀₀ values of H37Rv/ARPC4 samples. <b>B.</b> Cultures were grown in 7H9 medium at 37°C with shaking under axenic conditions. The figure shows result of CFU counts of H37Rv/ARPC4 and H37Rv/pVV16 at days 0, 7 and 14. A significant decline in the survival of mycobacteria carrying ARPC4pVV16 was observed in comparison to the vector control. <b>C.</b> Effect of ARPC4 expression on cell morphology growth of <i>M. tuberculosis</i>. Transmission electron micrographs of H37Rv (panel a) and mycobacterial cells harboring ARPC4pVV16 (H37Rv/ARPC4) (panels b and c). <b>D.</b> Relative fold change in the transcript levels of <i>rv1626</i> and <i>bfrB</i> genes. The <i>rv1626</i> gene (dark grey) was found to be significantly down-regulated (6-fold) in H37Rv/ARPC4 cells when compared with the H37Rv mycobacterial cells, whereas the unrelated <i>bfrB</i> gene (light grey) expression was not much perturbed. The 16S rRNA gene was used as the normalization internal control. Experiment was performed in triplicates.</p