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Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans -translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans -translation by these molecules. These results show that trans -translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways. Antibiotic-resistant bacterial pathogens pose a substantial threat to human health. Here, aided by structural analyses, the authors describe the molecular mechanism behind the activity of a series of compounds that inhibit trans-translation and are effective in eradicating N. gonorrhoeae infection in mice.

Artemisinin resistance has emerged in Southeast Asia, endangering the substantial progress in malaria elimination worldwide. It is associated with mutations in the PfK13 protein, but how PfK13 mediates artemisinin resistance is not completely understood. Here we used a new antibody against PfK13 to show that the PfK13 protein is expressed in all stages of the asexual intraerythrocytic cycle as well as in gametocytes and is partially localized in the endoplasmic reticulum. By introducing four PfK13 mutations into the 3D7 strain and reverting these mutations in field parasite isolates, we determined the impacts of these mutations identified in the parasite populations from northern Myanmar on the ring stage using the in vitro ring survival assay. The introduction of the N458Y mutation into the 3D7 background significantly increased the survival rates of the ring-stage parasites but at the cost of the reduced fitness of the parasites. Introduction of the F446I mutation, the most prevalent PfK13 mutation in northern Myanmar, did not result in a significant increase in ring-stage survival after exposure to dihydroartemisinin (DHA), but these parasites showed extended ring-stage development. Further, parasites with the F446I mutation showed only a marginal loss of fitness, partially explaining its high frequency in northern Myanmar. Conversely, reverting all these mutations, except for the C469Y mutation, back to their respective wild types reduced the ring-stage survival of these isolates in response to in vitro DHA treatment. Mutations in the Plasmodium falciparum Kelch 13 (PfK13) protein are associated with artemisinin resistance. PfK13 is essential for asexual erythrocytic development, but its function is not known. We tagged the PfK13 protein with green fluorescent protein in P. falciparum to study its expression and localization in asexual and sexual stages. We used a new antibody against PfK13 to show that the PfK13 protein is expressed ubiquitously in both asexual erythrocytic stages and gametocytes and is localized in punctate structures, partially overlapping an endoplasmic reticulum marker. We introduced into the 3D7 strain four PfK13 mutations (F446I, N458Y, C469Y, and F495L) identified in parasites from the China-Myanmar border area and characterized the in vitro artemisinin response phenotypes of the mutants. We found that all the parasites with the introduced PfK13 mutations showed higher survival rates in the ring-stage survival assay (RSA) than the wild-type (WT) control, but only parasites with N458Y displayed a significantly higher RSA value (26.3%) than the WT control. After these PfK13 mutations were reverted back to the WT in field parasite isolates, all revertant parasites except those with the C469Y mutation showed significantly lower RSA values than their respective parental isolates. Although the 3D7 parasites with introduced F446I, the predominant PfK13 mutation in northern Myanmar, did not show significantly higher RSA values than the WT, they had prolonged ring-stage development and showed very little fitness cost in in vitro culture competition assays. In comparison, parasites with the N458Y mutations also had a prolonged ring stage and showed upregulated resistance pathways in response to artemisinin, but this mutation produced a significant fitness cost, potentially leading to their lower prevalence in the Greater Mekong subregion. IMPORTANCE Artemisinin resistance has emerged in Southeast Asia, endangering the substantial progress in malaria elimination worldwide. It is associated with mutations in the PfK13 protein, but how PfK13 mediates artemisinin resistance is not completely understood. Here we used a new antibody against PfK13 to show that the PfK13 protein is expressed in all stages of the asexual intraerythrocytic cycle as well as in gametocytes and is partially localized in the endoplasmic reticulum. By introducing four PfK13 mutations into the 3D7 strain and reverting these mutations in field parasite isolates, we determined the impacts of these mutations identified in the parasite populations from northern Myanmar on the ring stage using the in vitro ring survival assay. The introduction of the N458Y mutation into the 3D7 background significantly increased the survival rates of the ring-stage parasites but at the cost of the reduced fitness of the parasites. Introduction of the F446I mutation, the most prevalent PfK13 mutation in northern Myanmar, did not result in a significant increase in ring-stage survival after exposure to dihydroartemisinin (DHA), but these parasites showed extended ring-stage development. Further, parasites with the F446I mutation showed only a marginal loss of fitness, partially explaining its high frequency in northern Myanmar. Conversely, reverting all these mutations, except for the C469Y mutation, back to their respective wild types reduced the ring-stage survival of these isolates in response to in vitro DHA treatment.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1(GFP/+) mice. CX₃CR1-GFP⁺ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX₃CR1-GFP⁺ Ly6C(low) monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX₃CR1-GFP⁺ cells in the blood and the tissue showed CD4⁺ T cell dependent accumulation of PD-L2⁺ CX₃CR1-GFP⁺ AAM in the tissues as granulomas form. By adoptive transfer of Ly6C(high) and Ly6C(low) monocytes into infected mice, we found that AAM originate primarily from transferred Ly6C(high) monocytes, but that these cells may transition through a Ly6C(low) state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6C(high) monocytes via help from CD4⁺ T cells.

Background The recent emergence and spread of artemisinin resistance in the Greater Mekong Subregion poses a great threat to malaria control and elimination. A K13-propeller gene ( K13 ), PF3D7_1343700 , has been associated lately with artemisinin resistance both in vitro and in vivo . This study aimed to investigate the K13 polymorphisms in Plasmodium falciparum parasites from the China-Myanmar border area where artemisinin use has the longest history. Methods A total of 180 archived P. falciparum isolates containing 191 parasite clones, mainly collected in 2007–2012 from the China-Myanmar area, were used to obtain the full-length K13 gene sequences. Results Seventeen point mutations were identified in 46.1% (88/191) parasite clones, of which seven were new. The F446I mutation predominated in 27.2% of the parasite clones. The C580Y mutation that is correlated with artemisinin resistance was detected at a low frequency of 1.6%. Collectively, 43.1% of the parasite clones contained point mutations in the kelch domain of the K13 gene. Moreover, there was a trend of increase in the frequency of parasites carrying kelch domain mutations through the years of sample collection. In addition, a microsatellite variation in the N-terminus of the K13 protein was found to have reached a high frequency (69.1%). Conclusions This study documented the presence of mutations in the K13 gene in parasite populations from the China-Myanmar border. Mutations present in the kelch domain have become prevalent (>40%). A predominant mutation F446I and a prevalent microsatellite variation in the N-terminus were identified, but their importance in artemisinin resistance remains to be elucidated.

Plasmodium falciparum malaria remains one of the most serious health problems globally and a protective malaria vaccine is desperately needed. Vaccination with attenuated parasites elicits multiple cellular effector mechanisms that lead to Plasmodium liver stage elimination. While granule-mediated cytotoxicity requires contact between CD8+ effector T cells and infected hepatocytes, cytokine secretion should allow parasite killing over longer distances. To better understand the mechanism of parasite elimination in vivo, we monitored the dynamics of CD8+ T cells in the livers of naïve, immunized and sporozoite-infected mice by intravital microscopy. We found that immunization of BALB/c mice with attenuated P. yoelii 17XNL sporozoites significantly increases the velocity of CD8+ T cells patrolling the hepatic microvasculature from 2.69±0.34 μm/min in naïve mice to 5.74±0.66 μm/min, 9.26±0.92 μm/min, and 7.11±0.73 μm/min in mice immunized with irradiated, early genetically attenuated (Pyuis4-deficient), and late genetically attenuated (Pyfabb/f-deficient) parasites, respectively. Sporozoite infection of immunized mice revealed a 97% and 63% reduction in liver stage density and volume, respectively, compared to naïve controls. To examine cellular mechanisms of immunity in situ, naïve mice were passively immunized with hepatic or splenic CD8+ T cells. Unexpectedly, adoptive transfer rendered the motile CD8+ T cells from immunized mice immotile in the liver of P. yoelii infected mice. Similarly, when mice were simultaneously inoculated with viable sporozoites and CD8+ T cells, velocities 18 h later were also significantly reduced to 0.68±0.10 μm/min, 1.53±0.22 μm/min, and 1.06±0.26 μm/min for CD8+ T cells from mice immunized with irradiated wild type sporozoites, Pyfabb/f-deficient parasites, and P. yoelii CS280-288 peptide, respectively. Because immobilized CD8+ T cells are unable to make contact with infected hepatocytes, soluble mediators could potentially play a key role in parasite elimination under these experimental conditions.

Drug resistance has emerged as one of the greatest challenges facing malaria control. The recent emergence of resistance to artemisinin (ART) and its partner drugs in ART-based combination therapies (ACT) is threatening the efficacy of this front-line regimen for treating Plasmodium falciparum parasites. Thus, an understanding of the molecular mechanisms that underlie the resistance to ART and the partner drugs has become a high priority for resistance containment and malaria management. Using genome-wide association studies, we investigated the associations of genome-wide single nucleotide polymorphisms with in vitro sensitivities to 10 commonly used antimalarial drugs in 94 P. falciparum isolates from the China-Myanmar border area, a region with the longest history of ART usage. We identified several loci associated with various drugs, including those containing pfcrt and pfdhfr . Of particular interest is a locus on chromosome 10 containing the autophagy-related protein 18 (ATG18 ) associated with decreased sensitivities to dihydroartemisinin, artemether and piperaquine – an ACT partner drug in this area. ATG18 is a phosphatidylinositol-3-phosphate binding protein essential for autophagy and recently identified as a potential ART target. Further investigations on the ATG18 and genes at the chromosome 10 locus may provide an important lead for a connection between ART resistance and autophagy.

Falcipain-2a ([FP2a] PF3D7_1115700) is a Plasmodium falciparum cysteine protease and hemoglobinase. Functional FP2a is required for potent activity of artemisinin, and in vitro selection for artemisinin resistance selected for an FP2a nonsense mutation. To investigate associations between FP2a polymorphisms and artemisinin resistance and to characterize the diversity of the enzyme in parasites from the China-Myanmar border, we sequenced the full-length FP2a gene in 140 P falciparum isolates collected during 2004-2011. The isolates were grouped into 8 different haplotype groups. Haplotype group I appeared in samples obtained after 2008, coinciding with implementation of artemisinin-based combination therapy in this region. In functional studies, compared with wild-type parasites, the FP2a haplotypes demonstrated increased ring survival, and all haplotype groups exhibited significantly reduced FP2a activity, with group I showing the slowest protease kinetics and reduced parasite fitness. These results suggest that altered hemoglobin digestion due to FP2a mutations may contribute to artemisinin resistance.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1GFP/+ mice. CX3CR1-GFP+ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX3CR1-GFP+ Ly6Clow monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX3CR1-GFP+ cells in the blood and the tissue showed CD4+ T cell dependent accumulation of PD-L2+ CX3CR1-GFP+ AAM in the tissues as granulomas form. By adoptive transfer of Ly6Chigh and Ly6Clow monocytes into infected mice, we found that AAM originate primarily from transferred Ly6Chigh monocytes, but that these cells may transition through a Ly6Clow state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6Chigh monocytes via help from CD4+ T cells.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected CX3CR1GFP/+ mice. CX3CR1-GFP+ monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of CX3CR1-GFP+ Ly6Clow monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of CX3CR1-GFP+ cells in the blood and the tissue showed CD4+ T cell dependent accumulation of PD-L2+ CX3CR1-GFP+ AAM in the tissues as granulomas form. By adoptive transfer of Ly6Chigh and Ly6Clow monocytes into infected mice, we found that AAM originate primarily from transferred Ly6Chigh monocytes, but that these cells may transition through a Ly6Clow state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited Ly6Chigh monocytes via help from CD4+ T cells. Macrophages will adopt different characteristics based on different types of inflammatory responses. During infection by parasitic helminths such as Schistosoma mansoni, macrophages adopt an “alternatively activated” or M2 phenotype (AAM). These AAM are important for protecting liver hepatocytes from damage caused by the parasite eggs. Here, we examine the cellular source of AAM in the liver of mice infected with S. mansoni. We find that AAM during S. mansoni infection come from monocytes and not from tissue resident macrophages. Monocytes can be separated into Ly6Chigh and Ly6Clow monocyte subsets. We demonstrate that it is the Ly6Chigh monocytes that are the precursors of AAM in the liver granulomas, but they might adopt the behavior of Ly6Clow monocytes in response to schistosome eggs. Additionally, these Ly6CHigh monocytes require help from CD4+ T cells in order to differentiate into AAM or to maintain this phenotype.

Alternatively activated macrophages (AAM) that accumulate during chronic T helper 2 inflammatory conditions may arise through proliferation of resident macrophages or recruitment of monocyte-derived cells. Liver granulomas that form around eggs of the helminth parasite Schistosoma mansoni require AAM to limit tissue damage. Here, we characterized monocyte and macrophage dynamics in the livers of infected [CX3CR1.sup.GFP/+] mice. [CX.sub.3][CR1-GFP.sup.+] monocytes and macrophages accumulated around eggs and in granulomas during infection and upregulated PD-L2 expression, indicating differentiation into AAM. Intravital imaging of [CX.sub.3][CR1-GFP.sup.+] [Ly6C.sup.low] monocytes revealed alterations in patrolling behavior including arrest around eggs that were not encased in granulomas. Differential labeling of [CX.sub.3][CR1-GFP.sup.+] cells in the blood and the tissue showed [CD4.sup.+] T cell dependent accumulation of [PD-L2.sup.+] [CX.sub.3][CR1-GFP.sup.+] AAM in the tissues as granulomas form. By adoptive transfer of [Ly6C.sup.high] and [Ly6C.sup.low] monocytes into infected mice, we found that AAM originate primarily from transferred [Ly6C.sup.high] monocytes, but that these cells may transition through a [Ly6C.sup.low] state and adopt patrolling behavior in the vasculature. Thus, during chronic helminth infection AAM can arise from recruited [Ly6C.sup.high] monocytes via help from [CD4.sup.+] T cells.