Co-transfection of murine NXPE2 and murine glycophorin A confers reactivity with Ter-119.

Not available.

New Genera and Species of Caulobacter and Brevundimonas Bacteriophages Provide Insights into Phage Genome Evolution.

Previous studies have identified diverse bacteriophages that infect Caulobacter vibrioides strain CB15 ranging from small RNA phages to four genera of jumbo phages. In this study, we focus on 20 bacteriophages whose genomes range from 40 to 60 kb in length. Genome comparisons indicated that these diverse phages represent six Caulobacter phage genera and one additional genus that includes both Caulobacter and Brevundimonas phages. Within species, comparisons revealed that both single base changes and inserted or deleted genetic material cause the genomes of closely related phages to diverge. Among genera, the basic gene order and the orientation of key genes were retained with most of the observed variation occurring at ends of the genomes. We hypothesize that the nucleotide sequences of the ends of these phage genomes are less important than the need to maintain the size of the genome and the stability of the corresponding mRNAs.

iNOS is necessary for GBP-mediated T. gondii clearance in murine macrophages via vacuole nitration and intravacuolar network collapse.

Toxoplasma gondii is an obligate intracellular parasite of rodents and humans. Interferon-inducible guanylate binding proteins (GBPs) are mediators of T. gondii clearance, however, this mechanism is incomplete. Here, using automated spatially targeted optical micro proteomics we demonstrate that inducible nitric oxide synthetase (iNOS) is highly enriched at GBP2+ parasitophorous vacuoles (PV) in murine macrophages. iNOS expression in macrophages is necessary to limit T. gondii load in vivo and in vitro. Although iNOS activity is dispensable for GBP2 recruitment and PV membrane ruffling; parasites can replicate, egress and shed GBP2 when iNOS is inhibited. T. gondii clearance by iNOS requires nitric oxide, leading to nitration of the PV and collapse of the intravacuolar network of membranes in a chromosome 3 GBP-dependent manner. We conclude that reactive nitrogen species generated by iNOS cooperate with GBPs to target distinct structures in the PV that are necessary for optimal parasite clearance in macrophages.

Inducible nitric oxide synthase (iNOS) is necessary for GBP-mediated T. gondii restriction in murine macrophages via vacuole nitration and intravacuolar network collapse.

Toxoplasma gondii is an obligate intracellular, protozoan pathogen of rodents and humans. T. gondii's ability to grow within cells and evade cell-autonomous immunity depends on the integrity of the parasitophorous vacuole (PV). Interferon-inducible guanylate binding proteins (GBPs) are central mediators of T. gondii clearance, however, the precise mechanism linking GBP recruitment to the PV and T. gondii restriction is not clear. This knowledge gap is linked to heterogenous GBP-targeting across a population of vacuoles and the lack of tools to selectively purify the intact PV. To identify mediators of parasite clearance associated with GBP2-positive vacuoles, we employed a novel protein discovery tool automated spatially targeted optical micro proteomics (autoSTOMP). This approach identified inducible nitric oxide synthetase (iNOS) enriched at levels similar to the GBPs in infected bone marrow-derived myeloid cells. iNOS expression on myeloid cells was necessary for mice to control T. gondii growth in vivo and survive acute infection. T. gondii infection of IFNγ-primed macrophage was sufficient to robustly induce iNOS expression. iNOS restricted T. gondii infection through nitric oxide synthesis rather than arginine depletion, leading to robust and selective nitration of the PV. Optimal parasite restriction by iNOS and vacuole nitration depended on the chromosome 3 GBPs. Notably, GBP2 recruitment and ruffling of the PV membrane occurred in iNOS knockouts, however, these vacuoles contained dividing parasites. iNOS activity was necessary for the collapse of the intravacuolar network of nanotubular membranes which connects parasites to each other and the host cytosol. Based on these data we conclude reactive nitrogen species generated by iNOS cooperate with the chromosome 3 GBPs to target distinct biology of the PV that are necessary for optimal parasite clearance in murine myeloid cells.