Mycobacterium tuberculosis MycP1 Protease Plays a Dual Role in Regulation of ESX-1 Secretion and Virulence

SummaryMycobacterium tuberculosis uses the ESX-1 secretion system to deliver virulence proteins during infection of host cells. Here we report a mechanism of posttranscriptional control of ESX-1 mediated by MycP1, a M. tuberculosis serine protease. We show that MycP1 is required for ESX-1 secretion but that, unexpectedly, genetic inactivation of MycP1 protease activity increases secretion of ESX-1 substrates. We demonstrate that EspB, an ESX-1 substrate required for secretion, is a target of MycP1 in vitro and in vivo. During macrophage infection, an inactive MycP1 protease mutant causes hyperactivation of ESX-1-stimulated innate signaling pathways. MycP1 is required for growth in mice during acute infection, while loss of its protease activity leads to attenuated virulence during chronic infection. As the key ESX-1 substrates ESAT-6 and CFP-10 are highly immunogenic, fine-tuning of their secretion by MycP1 may balance virulence and immune detection and be essential for successful maintenance of long-term M. tuberculosis infection

Patellin1, a Novel Sec14-Like Protein, Localizes to the Cell Plate and Binds Phosphoinositides

Membrane trafficking is central to construction of the cell plate during plant cytokinesis. Consequently, a detailed understanding of the process depends on the characterization of molecules that function in the formation, transport, targeting, and fusion of membrane vesicles to the developing plate, as well as those that participate in its consolidation and maturation into a fully functional partition. Here we report the initial biochemical and functional characterization of patellin1 (PATL1), a novel cell-plate-associated protein that is related in sequence to proteins involved in membrane trafficking in other eukaryotes. Analysis of the Arabidopsis genome indicated that PATL1 is one of a small family of Arabidopsis proteins, characterized by a variable N-terminal domain followed by two domains found in other membrane-trafficking proteins (Sec14 and Golgi dynamics domains). Results from immunolocalization and biochemical fractionation studies suggested that PATL1 is recruited from the cytoplasm to the expanding and maturing cell plate. In vesicle-binding assays, PATL1 bound to specific phosphoinositides, important regulators of membrane trafficking, with a preference for phosphatidylinositol(5)P, phosphatidylinositol(4,5)P(2), and phosphatidylinositol(3)P. Taken together, these findings suggest a role for PATL1 in membrane-trafficking events associated with cell-plate expansion or maturation and point to the involvement of phosphoinositides in cell-plate biogenesis

Direct Inhibition of Cellular Fatty Acid Synthase Impairs Replication of Respiratory Syncytial Virus and Other Respiratory Viruses

<div><p>Fatty acid synthase (FASN) catalyzes the <i>de novo</i> synthesis of palmitate, a fatty acid utilized for synthesis of more complex fatty acids, plasma membrane structure, and post-translational palmitoylation of host and viral proteins. We have developed a potent inhibitor of FASN (TVB-3166) that reduces the production of respiratory syncytial virus (RSV) progeny <i>in vitro</i> from infected human lung epithelial cells (A549) and <i>in vivo</i> from mice challenged intranasally with RSV. Addition of TVB-3166 to the culture medium of RSV-infected A549 cells reduces viral spread without inducing cytopathic effects. The antiviral effect of the FASN inhibitor is a direct consequence of reducing <i>de novo</i> palmitate synthesis; similar doses are required for both antiviral activity and inhibition of palmitate production, and the addition of exogenous palmitate to TVB-3166-treated cells restores RSV production. TVB-3166 has minimal effect on RSV entry but significantly reduces viral RNA replication, protein levels, viral particle formation and infectivity of released viral particles. TVB-3166 substantially impacts viral replication, reducing production of infectious progeny 250-fold. <i>In vivo</i>, oral administration of TVB-3166 to RSV-A (Long)-infected BALB/c mice on normal chow, starting either on the day of infection or one day post-infection, reduces RSV lung titers 21-fold and 9-fold respectively. Further, TVB-3166 also inhibits the production of RSV B, human parainfluenza 3 (PIV3), and human rhinovirus 16 (HRV16) progeny from A549, HEp2 and HeLa cells respectively. Thus, inhibition of FASN and palmitate synthesis by TVB-3166 significantly reduces RSV progeny both <i>in vitro</i> and <i>in vivo</i> and has broad-spectrum activity against other respiratory viruses. FASN inhibition may alter the composition of regions of the host cell membrane where RSV assembly or replication occurs, or change the membrane composition of RSV progeny particles, decreasing their infectivity.</p></div

FASN inhibition reduces yield of RSV A, PIV3 and HRV infectious progeny.

<p>Experiments were performed as shown in the diagram in (<b>A</b>). (<b>B</b>) A549 cells were infected with RSV A2 GFP (MOI 0.1). (<b>C</b>) HEp2 cells were infected with PIV3 (MOI 0.001). (<b>D</b>) HeLa cells were infected with HRV16 (MOI 0.0002). Progeny virus harvested from cells infected for 72 hours in the presence or absence of the FASN inhibitors TVB-3166 or TVB-2722 was used to infect Vero cells. Progeny was titered by automated microscopy (<b>B</b>) or plaque assay (<b>C, D</b>). (<b>E</b>) Infected or mock-infected cells were treated as indicated and cell viability was assessed 72 hours post treatment by Cell Titer Glo assay. Graphs represent the mean ± standard deviation of results from duplicate samples (* = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001).</p

TVB-3166 reduces infectivity of progeny virions.

<p>A549 cells were infected with RSV A2 GFP at MOI 0.1 and treated with DMSO or TVB-3166 as indicated. (<b>A</b>) Cells were fixed at 72 hpi and percent infection was calculated using automated imaging. (<b>B-D</b>) Infected cell media were collected at 72 hpi and treated with RNase A. (<b>B</b>) RNase-protected RNA was extracted from cell media and reverse-transcribed with genomic primers, and RSV N RNA levels were determined by qPCR. (<b>C</b>) Progeny virus harvested from cell media was used to infect Vero cells and progeny was titered by automated microscopy. (<b>D</b>) Infectivity of progeny virions is expressed as PFU per million RNA copies. Graphs represent the mean ± standard deviation of results from duplicate samples (* = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001).</p

FASN inhibitors reduce infectious progeny of multiple RSV strains.

<p>A549 cells were infected at MOI 0.1 with RSV strains A Long (<b>A</b>), ATCC 2012–10 (<b>B</b>), B-WV/14617/85 (<b>C</b>), or B 18537 (<b>D</b>). Progeny virus harvested from cells infected for 72 hours in the presence or absence of the FASN inhibitor TVB-3166 was used to infect Vero cells. Progeny was titered by plaque assay. Graphs represent the mean ± standard deviation of results from duplicate samples (* = <i>p</i><0.05, ** = <i>p</i><0.01).</p

Inhibition of FASN by TVB-3166 reduces RSV spread and intracellular levels of RSV protein and RNA.

<p>A549 cells were infected with RSV A2 GFP at MOI 0.1 (<b>A, B, D</b>) or MOI 2 (<b>C</b>), then treated with DMSO or TVB-3166, and harvested at the indicated times post infection. (<b>A</b>) Cells were fixed and imaged at 10X magnification by fluorescent microscopy. (<b>B</b>) Percent infection was calculated using automated imaging. (<b>C</b>) RSV F and G protein levels were detected by Western blot. (<b>D</b>) RSV F RNA levels were determined by qPCR after reverse transcription with oligo-dT or genomic primers and normalized to -actin. qPCR was performed in duplicate from duplicate samples and all C(t) values were normalized to the lowest of the four DMSO-72h C(t) values. Graphs represent the mean ± standard deviation of results from four samples (** = <i>p</i><0.01, *** = <i>p</i><0.001, **** = <i>p</i><0.0001).</p

TVB-3166 exhibits both prophylactic and therapeutic anti-RSV effects in BALB/c mice.

<p>(<b>A</b>) Twice daily treatment with TVB-3166 (p.o.), Ribavirin (i.p.) or drug vehicle (p.o.) was begun 2 hours after intranasal inoculation of mice with 5.5 x 10⁵ PFU of RSV Long. One group received vehicle on day 0 and TVB-3166 on subsequent days. Lungs and broncheoalveolar lavage (BAL) fluid were harvested five days post infection. (<b>B</b>) Lung titers were determined by plaque assay on HEp2 cells. (<b>C</b>) Percent change in initial body weight was measured daily. (<b>D</b>) Leukocyte populations in BAL were quantified by differential count. Graphs represent the mean ± standard deviation of results from groups of 5 or 10 animals (* = <i>p</i><0.05, ** = <i>p</i><0.01, *** = <i>p</i><0.001, **** = <i>p</i><0.0001).</p

Progeny virions from TVB-3166-treated cells fail to replicate in naïve cells.

<p>A549 cells were infected with RSV A2 GFP at MOI 0.3 and treated with DMSO or 0.2 μM TVB-3166 as shown in (<b>A</b>). Infected cell media were collected at 72 hpi and treated with RNase A. RNase-protected RNA was extracted from cell media and reverse-transcribed with genomic primers, and RSV progeny particle numbers (RSV N RNA levels) were determined by qPCR. Equal particle numbers of DMSO or TVB-3166 progeny virus were allowed to attach to Vero cells at 4°C, following which cells were shifted to 37°C to allow viral entry, and treated with trypsin to destroy un-internalized virus. Internalized virus was quantitated by qPCR. Viral attachment is shown in (<b>B</b>); entry and replication in (<b>C</b>). Graphs represent the mean ± standard deviation of results from duplicate samples (* = <i>p</i><0.05, **** = <i>p</i><0.0001).</p