CD4 T Cell-Dependent and -Independent Roles for IFN-γ in Blood-Stage Malaria.

Production of IFN-γ by CD4 T cells is widely theorized to control Plasmodium parasite burden during blood-stage malaria. Surprisingly, the specific and crucial mechanisms through which this highly pleiotropic cytokine acts to confer protection against malarial disease remain largely untested in vivo. Here we used a CD4 T cell-restricted Cre-Lox IFN-γ excision mouse model to test whether and how CD4 T cell-derived IFN-γ controls blood-stage malaria. Although complete absence of IFN-γ compromised control of the acute and the chronic, recrudescent blood-stage infections with P. c. chabaudi, we identified a specific, albeit modest, role for CD4 T cell-derived IFN-γ in limiting parasite burden only during the chronic stages of P. c. chabaudi malaria. CD4 T cell IFN-γ promoted IgG Ab class switching to the IgG2c isotype during P. c. chabaudi malaria in C57BL/6 mice. Unexpectedly, our data do not support gross defects in phagocytic activity in IFN-γ-deficient hosts infected with blood-stage malaria. Together, our data confirm CD4 T cell-dependent roles for IFN-γ but suggest CD4 T cell-independent roles for IFN-γ in immune responses to blood-stage malaria.

Cutting Edge: Subunit Booster Vaccination Confers Sterilizing Immunity against Liver-Stage Malaria in Mice Initially Primed with a Weight-Normalized Dose of Radiation-Attenuated Sporozoites.

Radiation-attenuated sporozoite (RAS) vaccination offers hope for global malaria control through induction of protective liver-stage-specific memory CD8 T cells. Effective RAS vaccination regimens exist; however, widespread implementation remains unfeasible. A key difficulty resides in the need to administer three or more doses i.v. to achieve sufficient immunity. Strategies to reduce the number of RAS doses are therefore desirable. Here we used mice to model human immune responses to a single, suboptimal weight-normalized RAS dose administered i.v. followed by subunit vaccination to amplify liver-stage-specific memory CD8 T cells. RAS+subunit prime-boost regimens increased the numbers of liver-stage-specific memory CD8 T cells to a level greater than is present after one RAS vaccination. Both i.v. and i.m. subunit vaccine delivery induced immunity in mice, and many vaccinated mice completely cleared liver infection. These findings are particularly relevant to human vaccine development because RAS+subunit prime-boost vaccination would reduce the logistical challenges of multiple RAS-only immunizations.

RPL-6: An Achilles Needle in the Malaria Haystack?

Malaria is a global health scourge for which a highly effective vaccine remains frustratingly elusive. Recent identification of an endogenous malaria antigen that stimulates robust TRM-mediated immunity in mice by Valencia-Hernandez et al. strengthens the case for prime-and-trap malaria vaccines and will greatly aid further investigations of cellular antimalarial immunity.

Balancing in a black box: Potential immunomodulatory roles for TGF-ß signaling during blood-stage malaria.

Malarial disease caused by Plasmodium parasites challenges the mammalian immune system with a delicate balancing act. Robust inflammatory responses are required to control parasite replication within red blood cells, which if unchecked, can lead to severe anemia and fatality. However, the same inflammatory response that controls parasite replication is also associated with immunopathology and severe disease, as is exemplified by cerebral malaria. A robust literature has identified critical roles for innate, cellular, and humoral immune responses orchestrated by IFN-γ and TH1 type responses in controlling blood stage malarial disease. In contrast, TGF-ß and IL-10 have been identified as important anti-inflammatory immunomodulators that help to limit inflammation and pathology during malaria. TGF-ß is a pleiotropic cytokine, with the ability to exert a wide variety of context-dependent immunomodulatory roles.The specific mechanisms that allow TGF-ß to protect against malarial pathology remain essentially unexplored and offer a promising avenue to dissect the most critical elements of immunomodulation in avoiding severe malaria. Here we discuss potential immunomodulatory roles for TGF-ß during malaria in light of recent advances in our understanding of the role of Tregs during blood-stage malaria.

The secreted kinase ROP17 promotes Toxoplasma gondii dissemination by hijacking monocyte tissue migration.

The protozoan parasite Toxoplasma gondii is thought to exploit monocyte trafficking to facilitate dissemination across endothelial barriers such as the blood-brain barrier. Here, we analysed the migration of parasitized monocytes in model endothelial and interstitial environments. We report that infection enhanced monocyte locomotion on the surface of endothelial cells, but profoundly inhibited monocyte transmigration across endothelial barriers. By contrast, infection robustly increased monocyte and macrophage migration through collagen-rich tissues in a Rho-ROCK-dependent manner consistent with integrin-independent interstitial migration. We further demonstrated that the secreted T. gondii protein kinase ROP17 was required for enhanced tissue migration. In vivo, ROP17-deficient parasites failed to upregulate monocyte tissue migration and exhibited an early dissemination delay, leading to prolonged mouse survival. Our findings indicate that the parasite-induced changes in monocyte motility primarily facilitate the transport of T. gondii through tissues and promote systemic dissemination, rather than shuttle parasites across the blood-brain barrier via extravasation.

The hitchhiker's guide to parasite dissemination.

Toxoplasma gondii (T. gondii) is a parasitic protist that can infect nearly all nucleated cell types and tissues of warm-blooded vertebrate hosts. T. gondii utilises a unique form of gliding motility to cross cellular barriers, enter tissues, and penetrate host cells, thus enhancing spread within an infected host. However, T. gondii also disseminates by hijacking the migratory abilities of infected leukocytes. Traditionally, this process has been viewed as a route to cross biological barriers such as the blood-brain barrier. Here, we review recent findings that challenge this view by showing that infection of monocytes downregulates the program of transendothelial migration. Instead, infection by T. gondii enhances Rho-dependent interstitial migration of monocytes and macrophages, which enhances dissemination within tissues. Collectively, the available evidence indicates that T. gondii parasites use multiple means to disseminate within the host, including enhanced motility in tissues and translocation across biological barriers.

A conserved ankyrin repeat-containing protein regulates conoid stability, motility and cell invasion in Toxoplasma gondii.

Apicomplexan parasites are typified by an apical complex that contains a unique microtubule-organizing center (MTOC) that organizes the cytoskeleton. In apicomplexan parasites such as Toxoplasma gondii, the apical complex includes a spiral cap of tubulin-rich fibers called the conoid. Although described ultrastructurally, the composition and functions of the conoid are largely unknown. Here, we localize 11 previously undescribed apical proteins in T. gondii and identify an essential component named conoid protein hub 1 (CPH1), which is conserved in apicomplexan parasites. CPH1 contains ankyrin repeats that are required for structural integrity of the conoid, parasite motility, and host cell invasion. Proximity labeling and protein interaction network analysis reveal that CPH1 functions as a hub linking key motor and structural proteins that contain intrinsically disordered regions and coiled coil domains. Our findings highlight the importance of essential protein hubs in controlling biological networks of MTOCs in early-branching protozoan parasites.

Calmodulin-like proteins localized to the conoid regulate motility and cell invasion by Toxoplasma gondii.

Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth. Phenotypic analysis revealed that all three CaMs contribute to parasite motility, invasion, and egress from host cells, and that they act downstream of microneme and rhoptry secretion. Super-resolution microscopy localized all three CaMs to the conoid where they overlap with myosin H (MyoH), a motor protein that is required for invasion. Biotinylation using BirA fusions with the CaMs labeled a number of apical proteins including MyoH and its light chain MLC7, suggesting they may interact. Consistent with this hypothesis, disruption of MyoH led to degradation of CaM3, or redistribution of CaM1 and CaM2. Collectively, our findings suggest these CaMs may interact with MyoH to control motility and cell invasion.

Molecular mechanism of mRNA repression in trans by a ProQ-dependent small RNA.

Research into post-transcriptional control of mRNAs by small noncoding RNAs (sRNAs) in the model bacteria Escherichia coli and Salmonella enterica has mainly focused on sRNAs that associate with the RNA chaperone Hfq. However, the recent discovery of the protein ProQ as a common binding partner that stabilizes a distinct large class of structured sRNAs suggests that additional RNA regulons exist in these organisms. The cellular functions and molecular mechanisms of these new ProQ-dependent sRNAs are largely unknown. Here, we report in Salmonella Typhimurium the mode-of-action of RaiZ, a ProQ-dependent sRNA that is made from the 3' end of the mRNA encoding ribosome-inactivating protein RaiA. We show that RaiZ is a base-pairing sRNA that represses in trans the mRNA of histone-like protein HU-α. RaiZ forms an RNA duplex with the ribosome-binding site of hupA mRNA, facilitated by ProQ, to prevent 30S ribosome loading and protein synthesis of HU-α. Similarities and differences between ProQ- and Hfq-mediated regulation will be discussed.

Toxoplasma Actin Is Required for Efficient Host Cell Invasion.

UNLABELLED: Apicomplexan parasites actively invade host cells using a mechanism predicted to be powered by a parasite actin-dependent myosin motor. In the model apicomplexan Toxoplasma gondii, inducible knockout of the actin gene, ACT1, was recently demonstrated to limit but not completely abolish invasion. This observation has led to the provocative suggestion that T. gondii possesses alternative, ACT1-independent invasion pathways. Here, we dissected the residual invasive ability of Δact1 parasites. Surprisingly, we were able to detect residual ACT1 protein in inducible Δact1 parasites as long as 5 days after ACT1 deletion. We further found that the longer Δact1 parasites were propagated after ACT1 deletion, the more severe an invasion defect was observed. Both findings are consistent with the quantity of residual ACT1 retained in Δact1 parasites being responsible for their invasive ability. Furthermore, invasion by the Δact1 parasites was also sensitive to the actin polymerization inhibitor cytochalasin D. Finally, there was no clear defect in attachment to host cells or moving junction formation by Δact1 parasites. However, Δact1 parasites often exhibited delayed entry into host cells, suggesting a defect specific to the penetration stage of invasion. Overall, our results support a model where residual ACT1 protein retained in inducible Δact1 parasites facilitates their limited invasive ability and confirm that parasite actin is essential for efficient penetration into host cells during invasion. IMPORTANCE: The prevailing model for apicomplexan invasion has recently been suggested to require major revision, based on studies where core components of the invasion machinery were genetically disrupted using a Cre-Lox-based inducible knockout system. For the myosin component of the motor thought to power invasion, an alternative parasite myosin was recently demonstrated to functionally compensate for loss of the primary myosin involved in invasion. Here, we highlight a second mechanism that can account for the surprising ability of parasites to invade after genetic disruption of core invasion machinery. Specifically, residual actin protein present in inducible knockout parasites appears able to support their limited invasion of host cells. Our results have important implications for the interpretation of the apicomplexan invasion model and also highlight significant considerations when analyzing the phenotypes of inducible knockout parasites generated using Cre-Lox technology.

Members of the human gut microbiota involved in recovery from Vibrio cholerae infection.

Given the global burden of diarrhoeal diseases, it is important to understand how members of the gut microbiota affect the risk for, course of, and recovery from disease in children and adults. The acute, voluminous diarrhoea caused by Vibrio cholerae represents a dramatic example of enteropathogen invasion and gut microbial community disruption. Here we conduct a detailed time-series metagenomic study of faecal microbiota collected during the acute diarrhoeal and recovery phases of cholera in a cohort of Bangladeshi adults living in an area with a high burden of disease. We find that recovery is characterized by a pattern of accumulation of bacterial taxa that shows similarities to the pattern of assembly/maturation of the gut microbiota in healthy Bangladeshi children. To define the underlying mechanisms, we introduce into gnotobiotic mice an artificial community composed of human gut bacterial species that directly correlate with recovery from cholera in adults and are indicative of normal microbiota maturation in healthy Bangladeshi children. One of the species, Ruminococcus obeum, exhibits consistent increases in its relative abundance upon V. cholerae infection of the mice. Follow-up analyses, including mono- and co-colonization studies, establish that R. obeum restricts V. cholerae colonization, that R. obeum luxS (autoinducer-2 (AI-2) synthase) expression and AI-2 production increase significantly with V. cholerae invasion, and that R. obeum AI-2 causes quorum-sensing-mediated repression of several V. cholerae colonization factors. Co-colonization with V. cholerae mutants discloses that R. obeum AI-2 reduces Vibrio colonization/pathogenicity through a novel pathway that does not depend on the V. cholerae AI-2 sensor, LuxP. The approach described can be used to mine the gut microbiota of Bangladeshi or other populations for members that use autoinducers and/or other mechanisms to limit colonization with V. cholerae, or conceivably other enteropathogens.