Identification of PhIL1, a novel cytoskeletal protein of the Toxoplasma gondii pellicle, through photosensitized labeling with 5-[125I]iodonaphthalene-1-azide

The pellicle of the protozoan parasite Toxoplasma gondii is a unique triple bilayer structure, consisting of the plasma membrane and two tightly apposed membranes of the underlying inner membrane complex. Integral membrane proteins of the pellicle are likely to play critical roles in host cell recognition, attachment, and invasion, but few such proteins have been identified. This is in large part because the parasite surface is dominated by a family of abundant and highly immunogenic glycosylphosphatidylinositol (GPI)-anchored proteins, which has made the identification of non-GPI-linked proteins difficult. To identify such proteins, we have developed a radiolabeling approach using the hydrophobic, photoactivatable compound 5-[(125)I]iodonaphthalene-1-azide (INA). INA can be activated by photosensitizing fluorochromes; by restricting these fluorochromes to the pellicle, [(125)I]INA labeling will selectively target non-GPI-anchored membrane-embedded proteins of the pellicle. We demonstrate here that three known membrane proteins of the pellicle can indeed be labeled by photosensitization with INA. In addition, this approach has identified a novel 22-kDa protein, named PhIL1 (photosensitized INA-labeled protein 1), with unexpected properties. While the INA labeling of PhIL1 is consistent with an integral membrane protein, the protein has neither a transmembrane domain nor predicted sites of lipid modification. PhIL1 is conserved in apicomplexan parasites and localizes to the parasite periphery, concentrated at the apical end just basal to the conoid. Detergent extraction and immunolocalization data suggest that PhIL1 associates with the parasite cytoskeleton.     

Protein kinase and phosphatase signaling in Mycobacterium tuberculosis physiology and pathogenesis

Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB), evades the antimicrobial defenses of the host and survives within the infected individual through a complex set of strategies. These include active prevention of host cellular killing processes as well as overwhelming adaptive gene expression. In the past decade, we have gained an increased understanding of how mycobacteria not only have the ability to adapt to a changing host environment but also actively interfere with the signaling machinery within the host cell to counteract or inhibit parts of the killing apparatus employed by the macrophage. Mtb is able to sense its environment via a set of phospho-signaling proteins which mediate its response and interaction with the host in a coordinated manner. In this review, we summarize the current knowledge about selected Mtb serine, threonine, and tyrosine kinase and phosphatase signaling proteins, focusing on the protein kinases, PknG and PtkA, and the protein phosphatase, PtpA.     

A protein kinase inhibitor as an antimycobacterial agent

The protein kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7) was found to inhibit the growth of two different mycobacterial strains, the slow-growing Mycobacterium bovis Bacille Calmette Guerin (BCG) and the fast-growing saprophyte Mycobacterium smegmatis mc2 155, in a dose-dependent manner. While screening for the effect of kinase inhibitors on mycobacterial growth, millimolar concentrations of H7 induced a 40% decrease in the growth of M. bovis BCG when measured as a function of oxidative phosphorylation. This H7-induced decrease in growth was shown to involve a 2-log fold decrease in the viable counts of M. smegmatis within a 48-h period and a 50% reduction in the number of BCG viable counts within a 10-day period. Micromolar concentrations of H7 compound induced a significant decrease in the activity of the Mycobacterium tuberculosis protein serine/threonine kinase (PSTK) PknB. The inhibition of mycobacterial growth as well as the inhibition of a representative M. tuberculosis protein serine/threonine kinase PknB suggests that conventional PSTK inhibitors can be used to study the role that the mycobacterial PSTK family plays in controlling bacterial growth.     

Mycobacterium smegmatis biofilm formation and sliding motility are affected by the serine/threonine protein kinase PknF

Eighteen 'eukaryotic-like' serine/threonine kinases are present in the Mycobacterium smegmatis genome. One of them encoded by the ORF 3677 demonstrates high similarity to the Mycobacterium tuberculosis protein kinase PknF. A merodiploid strain was generated, which showed reduced growth associated with irregular cell structure. The merodiploid strain displayed altered colony morphology, defective sliding motility and biofilm formation. These data indicate a role for PknF in biofilm formation, possibly associated with alterations in glycopeptidolipid composition.     

Hydrophobic inactivation of influenza viruses confers preservation of viral structure with enhanced immunogenicity

The use of inactivated influenza virus for the development of vaccines with broad heterosubtypic protection requires selective inactivation techniques that eliminate viral infectivity while preserving structural integrity. Here we tested if a hydrophobic inactivation approach reported for retroviruses could be applied to the influenza virus. By this approach, the transmembrane domains of viral envelope proteins are selectively targeted by the hydrophobic photoactivatable compound 1,5-iodonaphthyl-azide (INA). This probe partitions into the lipid bilayer of the viral envelope and upon far UV irradiation reacts selectively with membrane-embedded domains of proteins and lipids while the protein domains that localize outside the bilayer remain unaffected. INA treatment of influenza virus blocked infection in a dose-dependent manner without disrupting the virion or affecting neuraminidase activity. Moreover, the virus maintained the full activity in inducing pH-dependent lipid mixing, but pH-dependent redistribution of viral envelope proteins into the target cell membrane was completely blocked. These results indicate that INA selectively blocks fusion of the virus with the target cell membrane at the pore formation and expansion step. Using a murine model of influenza virus infection, INA-inactivated influenza virus induced potent anti-influenza virus serum antibody and T-cell responses, similar to live virus immunization, and protected against heterosubtypic challenge. INA treatment of influenza A virus produced a virus that is noninfectious, intact, and fully maintains the functional activity associated with the ectodomains of its two major envelope proteins, neuraminidase and hemagglutinin. When used as a vaccine given intranasally (i.n.), INA-inactivated influenza virus induced immune responses similar to live virus infection.     

Cholesterol is accumulated by mycobacteria but its degradation is limited to non-pathogenic fast-growing mycobacteria

In this report we show that fast-growing non-pathogenic mycobacteria degrade cholesterol from liquid media, and are able to grow on cholesterol as a sole carbon source. In contrast, slow-growing mycobacteria, including pathogenic Mycobacterium tuberculosis and bacillus Calmette-Guérin (BCG), do not degrade and use cholesterol as a carbon source. Nevertheless, pathogenic mycobacteria are able to uptake, modify, and accumulate cholesterol from liquid growth media, and form a zone of clearance around a colony when plated on solid media containing cholesterol. These data suggest that cholesterol may have a role in mycobacterial infection other than its use as carbon source.     

The crystal structure of 1-D-myo-inosityl 2-acetamido-2-deoxy-alpha-D-glucopyranoside deacetylase (MshB) from Mycobacterium tuberculosis reveals a zinc hydrolase with a lactate dehydrogenase fold

Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two beta-sheets and two alpha-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.     

Identification and characterization of a diamide sensitive mutant of Mycobacterium smegmatis

A mutant, T7, highly sensitive to oxidative stress as caused by diamide was isolated from a Mycobacterium smegmatis mc(2)155 transposon mutant library. While wild-type M. smegmatis is able to grow well on solid media supplemented with 10 mM diamide, T7 is only able to grow on solid media containing up to 1 mM diamide. This mutant is also sensitive to other thiol modifying agents such as iodoacetamide and chlorodinitrobenzene. By sequencing the genomic DNA flanking the transposon, T7 was found to be mutated in the region upstream of the homolog of M. tuberculosis Rv0274 open reading frame. Sequence analysis revealed that Rv0274 is a member of a superfamily of metalloenzymes comprising enzymes such as extradiol dioxygenases, glyoxalases, and fosfomycin resistant glutathione transferases. Cloning and epichromosomal expression of M. tuberculosis Rv0274 in the mutant resulted in complementation of the sensitivity to diamide.