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Importance: Toxoplasma gondii (Tg) is a ubiquitous parasitic pathogen, infecting about one-third of the global population. Tg is controlled in immunocompetent people by mechanisms that are not fully understood. Tg infection drives the production of the inflammatory cytokine interferon gamma (IFNγ), which upregulates intracellular anti-pathogen defense pathways. In this study, we describe host proteins p97/VCP, UBXD1, and ANKRD13A that control Tg at the parasitophorous vacuole (PV) in IFNγ-stimulated endothelial cells. p97/VCP is an ATPase that interacts with a network of cofactors and is active in a wide range of ubiquitin-dependent cellular processes. We demonstrate that PV ubiquitination is a pre-requisite for recruitment of these host defense proteins, and their deposition directs Tg PVs to acidification in endothelial cells. We show that p97/VCP universally targets PVs in human cells and restricts Tg in different human cell types. Overall, these findings reveal new players of intracellular host defense of a vacuolated pathogen., Competing Interests: The authors declare no conflict of interest.

Understanding how immune challenges elicit different responses is critical for diagnosing and deciphering immune regulation. Using a modular strategy to interpret the complex transcriptional host response in mouse models of infection and inflammation, we show a breadth of immune responses in the lung. Lung immune signatures are dominated by either IFN-γ and IFN-inducible, IL-17-induced neutrophil- or allergy-associated gene expression. Type I IFN and IFN-γ-inducible, but not IL-17- or allergy-associated signatures, are preserved in the blood. While IL-17-associated genes identified in lung are detected in blood, the allergy signature is only detectable in blood CD4 + effector cells. Type I IFN-inducible genes are abrogated in the absence of IFN-γ signaling and decrease in the absence of IFNAR signaling, both independently contributing to the regulation of granulocyte responses and pathology during Toxoplasma gondii infection. Our framework provides an ideal tool for comparative analyses of transcriptional signatures contributing to protection or pathogenesis in disease.

For image-based infection biology, accurate unbiased quantification of host-pathogen interactions is essential, yet often performed manually or using limited enumeration employing simple image analysis algorithms based on image segmentation. Host protein recruitment to pathogens is often refractory to accurate automated assessment due to its heterogeneous nature. An intuitive intelligent image analysis program to assess host protein recruitment within general cellular pathogen defense is lacking. We present HRMAn (Host Response to Microbe Analysis), an open-source image analysis platform based on machine learning algorithms and deep learning. We show that HRMAn has the capacity to learn phenotypes from the data, without relying on researcher-based assumptions. Using Toxoplasma gondii and Salmonella enterica Typhimurium we demonstrate HRMAn's capacity to recognize, classify and quantify pathogen killing, replication and cellular defense responses. HRMAn thus presents the only intelligent solution operating at human capacity suitable for both single image and high content image analysis., Editorial Note: This article has been through an editorial process in which the authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., Competing Interests: DF, AY, BC, JW, MB, MH, JM, EF No competing interests declared, (© 2019, Fisch et al.)

The tertiary structure of the monomeric yeast glyoxalase I has been modeled based on the crystal structure of the dimeric human glyoxalase I and a sequence alignment of the two enzymes. The model suggests that yeast glyoxalase I has two active sites contained in a single polypeptide. To investigate this, a recombinant expression clone of yeast glyoxalase I was constructed for overproduction of the enzyme in Escherichia coli. Each putative active site was inactivated by site-directed mutagenesis. According to the alignment, glutamate 163 and glutamate 318 in yeast glyoxalase I correspond to glutamate 172 in human glyoxalase I, a Zn(II) ligand and proposed general base in the catalytic mechanism. The residues were each replaced by glutamine and a double mutant containing both mutations was also constructed. Steady-state kinetics and metal analyses of the recombinant enzymes corroborate that yeast glyoxalase I has two functional active sites. The activities of the catalytic sites seem to be somewhat different. The metal ions bound in the active sites are probably one Fe(II) and one Zn(II), but Mn(II) may replace Zn(II). Yeast glyoxalase I appears to be one of the few enzymes that are present as a single polypeptide with two active sites that catalyze the same reaction.