Macrophages are the major reservoir of latent murine gammaherpesvirus 68 in peritoneal cells

B cells have previously been identified as the major hematopoietic cell type harboring latent gammaherpesvirus 68 (gammaHV68) (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). However, we have shown that gammaHV68 efficiently establishes latency in B-cell-deficient mice (K. E. Weck, M. L. Barkon, L. I. Yoo, S. H. Speck, and H. W. Virgin, J. Virol. 70:6775-6780, 1996), demonstrating that B cells are not required for gammaHV68 latency. To understand this dichotomy, we determined whether hematopoietic cell types, in addition to B cells, carry latent gammaHV68. We observed a high frequency of cells that reactivate latent gammaHV68 in peritoneal exudate cells (PECs) derived from both B-cell-deficient and normal C57BL/6 mice. PECs were composed primarily of macrophages in B-cell-deficient mice and of macrophages plus B cells in normal C57BL/6 mice. To determine which cells in PECs from C57BL/6 mice carry latent gammaHV68, we developed a limiting-dilution PCR assay to quantitate the frequency of cells carrying the gammaHV68 genome in fluorescence-activated cell sorter-purified cell populations. We also quantitated the contribution of individual cell populations to the total frequency of cells carrying latent gammaHV68. At early times after infection, the frequency of PECs that reactivated gammaHV68 correlated very closely with the frequency of PECs carrying the gammaHV68 genome, validating measurement of the frequency of viral-genome-positive cells as a measure of latency in this cell population. F4/80-positive macrophage-enriched, lymphocyte-depleted PECs harbored most of the gammaHV68 genome and efficiently reactivated gammaHV68, while CD19-positive, B-cell-enriched PECs harbored about a 10-fold lower frequency of gammaHV68 genome-positive cells. CD4-positive, T-cell-enriched PECs contained only a very low frequency of gammaHV68 genome-positive cells, consistent with previous analyses indicating that T cells are not a reservoir for gammaHV68 latency (N. P. Sunil-Chandra, S. Efstathiou, and A. A. Nash, J. Gen. Virol. 73:3275-3279, 1992). Since macrophages are bone marrow derived, we determined whether elicitation of a large inflammatory response in the peritoneum would recruit additional latent cells into the peritoneum. Thioglycolate inoculation increased the total number of PECs by about 20-fold but did not affect the frequency of cells that reactivate gammaHV68, consistent with a bone marrow reservoir for latent gammaHV68. These experiments demonstrate gammaHV68 latency in two different hematopoietic cell types, F4/80-positive macrophages and CD19-positive B cells, and argue for a bone marrow reservoir for latent gammaHV68.     

Antiplatelet and anticoagulant therapy in elective percutaneous coronary intervention

Thrombosis plays a major role in acute vessel closure both after coronary balloon angioplasty and after stenting. This review will address the role of antiplatelet and anticoagulant therapy in preventing early thrombotic complications after percutaneous coronary intervention. The focus will be on agents that are routinely available and commonly used.     

Complete genome sequence of the extremely acidophilic methanotroph isolate V4, Methylacidiphilum infernorum, a representative of the bacterial phylum Verrucomicrobia

Background

The phylum Verrucomicrobia is a widespread but poorly characterized bacterial clade. Although cultivation-independent approaches detect representatives of this phylum in a wide range of environments, including soils, seawater, hot springs and human gastrointestinal tract, only few have been isolated in pure culture. We have recently reported cultivation and initial characterization of an extremely acidophilic methanotrophic member of the Verrucomicrobia, strain V4, isolated from the Hell's Gate geothermal area in New Zealand. Similar organisms were independently isolated from geothermal systems in Italy and Russia.

Results

We report the complete genome sequence of strain V4, the first one from a representative of the Verrucomicrobia. Isolate V4, initially named "Methylokorus infernorum" (and recently renamed Methylacidiphilum infernorum) is an autotrophic bacterium with a streamlined genome of ~2.3 Mbp that encodes simple signal transduction pathways and has a limited potential for regulation of gene expression. Central metabolism of M. infernorum was reconstructed almost completely and revealed highly interconnected pathways of autotrophic central metabolism and modifications of C₁-utilization pathways compared to other known methylotrophs. The M. infernorum genome does not encode tubulin, which was previously discovered in bacteria of the genus Prosthecobacter, or close homologs of any other signature eukaryotic proteins. Phylogenetic analysis of ribosomal proteins and RNA polymerase subunits unequivocally supports grouping Planctomycetes, Verrucomicrobia and Chlamydiae into a single clade, the PVC superphylum, despite dramatically different gene content in members of these three groups. Comparative-genomic analysis suggests that evolution of the M. infernorum lineage involved extensive horizontal gene exchange with a variety of bacteria. The genome of M. infernorum shows apparent adaptations for existence under extremely acidic conditions including a major upward shift in the isoelectric points of proteins.

Conclusion

The results of genome analysis of M. infernorum support the monophyly of the PVC superphylum. M. infernorum possesses a streamlined genome but seems to have acquired numerous genes including those for enzymes of methylotrophic pathways via horizontal gene transfer, in particular, from Proteobacteria.

Reviewers

This article was reviewed by John A. Fuerst, Ludmila Chistoserdova, and Radhey S. Gupta.     

Occurrence of nonmevalonate and mevalonate pathways for isoprenoid biosynthesis in bacteria of different taxonomic groups

The effect of fosmidomycin and mevinoline, inhibitors of the nonmevalonate and the mevalonate pathways of isoprenoid biosynthesis, respectively, on the growth of 34 anaerobic and 10 aerobic prokaryotic strains was studied. Fosmidomycin at the concentrations used was shown to inhibit the growth of 9 (of 10) representatives of the family Microbacteriaceae, 4 (of 5) strains of Thermoanaerobacter, and 11 (of 12) strains of Clostridium, whereas mevinoline inhibited the growth of lactobacilli (Carnobacterium), methanogenic and sulfate-reducing bacteria insensitive to fosmidomycin. During the late growth phase, four strains of actinobacteria (of nine) accumulated the compound, which, upon oxidation, generates a long-lived free radical; three strains synthesized 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC). It was concluded that the difference in the sensitivity of the organisms to fosmidomycin and mevinoline might serve as a test to differentiate several representatives of the family Microbacteriaceae. The use of mevinoline for inhibiting methanogens in ecological investigations seems to be promising.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 185–190.Original Russian Text Copyright © 2005 by Trutko, Dorofeeva, Shcherbakova, Chuvilskaya, Laurinavichus, Binyukov, Ostrovskii, Hintz, Wiesner, Jomaa, Akimenko.     

Prevalence of nonmevalonate and mevalonate pathways for isoprenoid biosynthesis among bacteria of different systematic groups

The effect of fosmidomycin and mevinoline, inhibitors of the nonmevalonate and the mevalonate pathway of isoprenoid biosynthesis, respectively, on the growth of 34 anaerobic and 10 aerobic prokaryotic strains was studied. Fosmidomycin at the concentrations used was shown to inhibit the growth of 9 (of 10) representatives of the family Microbacteriaceae, 4 (of 5) strains of Thermoanaerobacter, and 11 (of 12) strains of Clostridium, whereas mevinoline inhibited the growth of lactobacilli (Carnobacterium), methanogenic and sulfate-reducing bacteria insensitive to fosmidomycin. During the late growth phase, four strains of actinobacteria (of nine) accumulate the compound, which, upon oxidation, generates a long-lived free radical; three strains synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEC). It was concluded that the difference in the sensitivity of the organisms to fosmidomycin and mevinoline might serve as a test to differentiate several representatives of the family Microbacteriaceae. The use of mevinoline for inhibiting methanogens in ecological investigations seems to be promising.     

Differentiation of human gamma-delta T cells towards distinct memory phenotypes

Vgamma9/Vdelta2 T cells comprise a small population of peripheral T cells responding towards the low molecular weight antigen, (E)-4-hydroxy-3-methyl-but-2-enyl-pyrophosphate (HMB-PP). HMB-PP-stimulated Vgamma9/Vdelta2 T cells proliferated, expressed CCL5/RANTES, and upregulated markers like CD16, CD25, CD69, and CD94, in the presence of either IL-15 or IL-21. Vgamma9/Vdelta2 T cells grown in the presence of IL-15 differentiated into an effector/memory population characterized by production of TNF-alpha, expression of CD45RO and CCR5, and lack of CD62L, CD81, and CCR7. In contrast, Vgamma9/Vdelta2 T cells grown with IL-21 differentiated into putative central memory CD45RO(+) T cells that did not produce TNF-alpha, IFN-gamma, or IL-4, and maintained expression of CD62L, CD81, and CCR7.     

Structure-activity relationships of novel anti-malarial agents. Part 4: N-(3-benzoyl-4-tolylacetylaminophenyl)-3-(5-aryl-2-furyl)acrylic acid amides

In a previous report, we have described novel anti-malarial compounds based on a 2,5-diaminobenzophenone scaffold. Here, we have invesigated acryloyl derivatives carrying a biaryl structure consisting of a terminal aryl residue and a central 2-furyl ring. Several compounds were obtained in the series of para-substituted phenylfurylacryloyl derivatives that displayed improved anti-malarial activity in comparison to earlier described derivatives. From the structure-activity relationships it can be deduced that there has to be a lipophilic moiety in the para-position of the terminal phenyl residue. Furthermore, there are indications that, alternatively, activity may benefit from the presence of a polar moiety with hydrogen bond acceptor properties.     

Structure-activity relationships of novel anti-malarial agents. Part 6: N-(4-arylpropionylamino-3-benzoylphenyl)-[5-(4-nitrophenyl)-2-furyl]acrylic acid amides

We have demonstrated that the p-trifluoromethylphenylpropionylamino residue at the 2-position of the core structure leads to an active benzophenone-type anti-malarial agent. The attempt to improve water solubility by introduction of an amino group into the alpha-position of the arylpropionyl residue resulted in decreased activity.     

LytB protein catalyzes the terminal step of the 2-C-methyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis

Recombinant LytB protein from the thermophilic eubacterium Aquifex aeolicus produced in Escherichia coli was purified to apparent homogeneity. The purified LytB protein catalyzed the reduction of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) in a defined in vitro system. The reaction products were identified as isopentenyl diphosphate and dimethylallyl diphosphate. A spectrophotometric assay was established to determine the steady-state kinetic parameters of LytB protein. The maximal specific activity of 6.6+/-0.3 micromol x min(-1) x mg(-1) protein was determined at pH 7.5 and 60 degrees C. The k(cat) value of the LytB protein was 3.7+/-0.2 s(-1) and the K(m) value for HMBPP was 590+/-60 microM.     

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP(+) reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP(+) reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.