PLOS Pathogens

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposis sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NF{kappa}B signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection. Author SummaryChemical modifications on mRNA, such as m6A, are functionally linked to all stages of mRNA metabolism and regulate a variety of biological processes. As such, m6A modification offers unique possibilities for viruses to modulate both viral and host gene expression. m6A has been identified on transcripts encoded by a wide range of viruses and studies to investigate m6A function have highlighted distinct roles in virus life cycles. In addition, cellular transcripts undergoing differential m6A status during infection may also be important for virus replication. In this study we investigate the impact of differential m6A modification in host transcripts during KSHV lytic replication, by identifying transcripts with altered methylation profiles between latent and lytic replication programmes. We show that increased m6A content in one of these cellular mRNAs, GPRC5A, enhances its stability and correlates with increased abundance during KSHV lytic replication. Moreover, the importance of GPRC5A is demonstrated by depletion studies, showing that GPRC5A enhances KSHV lytic replication by inhibiting cell signalling pathways.

The bacterial pathogen Legionella pneumophila delivers more than 330 effector proteins into host cells through its Dot/Icm type IV secretion system (T4SS) to facilitate its intracellular replication. A number of these effectors modulate organelle trafficking pathways to create a membrane-bound niche called the Legionella containing vacuole (LCV). In this study, we found that L. pneumophila induces F-actin accumulation in host cell cortex by its Dot/Icm substrate RavJ (Lpg0944). RavJ harbors an C101H138D170 motif associated with human tissue transglutaminases (TGs). We showed that RavJ catalyzes a covalent linkage between actin and the Motin family proteins Angiomotin (AMOT) and Angiomotin-like 1 (AMOTL1), proteins known to regulate tube formation and cell migration. Further study revealed that RavJ-induced crosslink between actin and AMOT occurs on its Gln354 residue. Crosslink between actin and AMOT significantly reduces the binding between actin and its binding partner cofilin, suggesting that RavJ inhibits actin depolymerization. We also demonstrated that the metaeffector LegL1 directly interacts with RavJ to antagonize its transglutaminase activity, leading to reduced crosslink between actin and Motin proteins. Our results reveal a novel mechanism of modulating the host actin cytoskeleton by L. pneumophila.

Malaria is caused by apicomplexan parasites of the genus Plasmodium, which are transmitted through the bite of Anopheles mosquitoes that inject sporozoites (SPZs) into the skin. SPZs migrate to and infect the liver for an initial round of replication. SPZs and liver stages have long been considered as ideal targets for malaria vaccines. The main SPZ surface protein, the circumsporozoite protein (CSP), is the target of currently approved malaria vaccines and prophylactic antibody therapies. Studies in rodent malaria models have shown that anti-CSP antibodies exert their protective effect mainly in the skin, but some of the most potent anti-CSP monoclonal antibodies show additional protective effects in the vasculature and liver. Other SPZ proteins involved at different steps of the infection process may thus represent additional targets for antibody-mediated neutralization. Three 6-cysteine (6-Cys) domain proteins (P36, P52 and B9) play an essential role during SPZ invasion of hepatocytes, yet their molecular function and whether they can be targeted by neutralizing antibodies remains unknown. Here, to fill this gap, we combined an integrative structural biology approach with functional experiments in the P. berghei rodent malaria model. AlphaFold-based structural modeling followed by experimental validation via electron microscopy and small-angle X-ray scattering indicated that the P36-P52 heterodimer displays a head-to-tail architecture with an interaction interface that is largely conserved among Plasmodium species. The structural models supported the rational design of an epitope tagging approach, which, combined with neutralizing assays, revealed that antibodies against P36 and P52 can efficiently block invasion of hepatocytes by SPZs in culture conditions. The data show that the inhibitory activity of antibodies heavily depends on epitope position and revealed that antibody-exposed vulnerable sites lie on the membrane-distal side of the P36-P52 heterodimer. In contrast, antibodies targeting B9 had no inhibitory effect on SPZ invasion, irrespective of epitope positioning. These data show that the invasion step could be targeted by antibodies and indicate that the P36-P52 complex may be considered as a potential target for the development of next generation pre-erythrocytic malaria vaccines or therapeutic antibodies.

Glucocorticoids are a major class of therapeutic anti-inflammatory and immunosuppressive drugs prescribed to patients with inflammatory diseases, to avoid transplant rejection, and as part of cancer chemotherapy. However, exposure to these drugs increases the risk of opportunistic infections such as with the fungus Aspergillus fumigatus. Prolonged glucocorticoid therapy is one of the main risks for invasive aspergillosis, which causes mortality in >50% of infected patients. The mechanisms by which glucocorticoids increase susceptibility to A. fumigatus are poorly understood. Here, we used a zebrafish larva-Aspergillus infection model to identify innate immune mechanisms altered by glucocorticoid treatment. Infected larvae exposed to dexamethasone succumb to the infection at a significantly higher rate than control larvae. However, both macrophages and neutrophils are still recruited to the site of infection and dexamethasone treatment does not significantly affect fungal spore killing. Instead, the primary effect of dexamethasone manifests later in infection with treated larvae exhibiting increased invasive hyphal growth. In line with this, dexamethasone predominantly inhibits neutrophil function, rather than macrophage function. Dexamethasone-induced mortality also depends on the glucocorticoid receptor. One pathway that glucocorticoids can inhibit is NF-{kappa}B activation and we report that dexamethasone partially suppresses NF-{kappa}B activation at the infection site by inducing the transcription of I{kappa}B via the glucocorticoid receptor. Independent CRISPR/Cas9 targeting of IKK{gamma} to prevent NF-{kappa}B activation also increases invasive A. fumigatus growth and larval mortality. However, dexamethasone treatment of IKK{gamma} crispant larvae further increases invasive hyphal growth, suggesting that dexamethasone may suppress other pathways in addition to NF-{kappa}B to promote host susceptibility. Collectively, we find that dexamethasone acts through the glucocorticoid receptor to suppress NF-{kappa}B-mediated neutrophil control of A. fumigatus hyphae in zebrafish larvae. Author SummaryGlucocorticoids are drugs that stop inflammation and suppress the immune system. Glucocorticoids are effective in treating inflammatory diseases such as asthma and arthritis, preventing organ rejection after transplant surgery, and in ameliorating the side effects of cancer chemotherapy. However, as these drugs suppress the immune system, patients taking glucocorticoids are more prone to infections such as with the environmental fungus Aspergillus fumigatus. The specific mechanisms that glucocorticoids inhibit to increase susceptibility to infection are largely unknown. Here, we used a larval zebrafish model of A. fumigatus infection to determine that glucocorticoids mainly suppress the ability of neutrophils to control the fungal hyphal growth that causes tissue damage. Our study provides insight into future strategies to treat A. fumigatus infection in patients undergoing glucocorticoid therapy.

Upon sensing Yersinia pseudotuberculosis (Yptb), receptor-mediated pathways are stimulated to trigger polymorphonuclear (PMN) antimicrobial responses. Yptb injects multiple Type 3 secreted effector proteins, Yops (Yersinia outer proteins), that possess distinct biochemical functions, into PMNs to inhibit PMN responses. Here, we show that several Yops, YopE, YopH, and YopO, each partially interfered with CD63 mobilization to the plasma membrane, a marker for primary degranulation. The host pathways involved in CD63 mobilization are complex and it is not completely understood how Yops collaborate to inactivate this process. Here, CRISPR/Cas9 technology was used in an immortalized system of myeloid progenitor cells (Cas9-ER-HoxB8) to generate a panel of knockout PMN cell lines. To probe the impact of different Yops on the neutrophil pathways activated upon encountering Yptb, we interrogated the panel of genetically modified neutrophils with genetically modified bacteria. This approach of targeted gene deletion to inactivate specific pathways/proteins uncovered host pathways that synergize to induce CD63 mobilization that are distinctly targeted by YopE and YopH. YopE specifically inhibited CD63 mobilization in the absence of SKAP2, a YopH target, whereas YopH inhibited CD63 mobilization in the absence of RhoG, a YopE target, indicating that these Yops inactivate distinct signaling pathways contributing to CD63 mobilization. Furthermore, the SKAP2-independent pathway inactivated by YopE is involved in primary granule release and ROS production. Overall, this work highlights the diverse Yop-mediated mechanisms that WT-Yptb employs to effectively disarm PMN responses and provides an avenue to untangle neutrophil signaling pathways targeted by pathogens using Cas9-ER-HoxB8 cells. Author SummaryWhen sensing invading bacteria, neutrophils become activated through multiple receptors that trigger signal-transduction cascades resulting in the generation antimicrobial responses. The enteric pathogen, Yersinia pseudotuberculosis (Yptb), is equipped to effectively inhibit these responses using its collection of effector proteins (Yops). Here, we developed a system to overcome the limitations of performing genetic manipulations in neutrophils by implementing CRISPR/Cas9 technology in an engineered system of myeloid progenitor cells (Cas9-ER-HoxB8) that can be induced to differentiate into neutrophils. By infecting genetically modified neutrophils with Yptb strains expressing individual Yops, we identified distinct host signaling pathways that synergize to induce neutrophil antimicrobial responses. Our findings provide insight into several signaling events triggered by Yptb infection and show how YopE and YopH target distinct pathways to block vesicle trafficking and extracellular ROS production. This powerful genetic system can be applied to other pathogens to dissect the intricacies of neutrophil-pathogen interactions.

Human immune deficiency virus (HIV) infection of microglial cells in the brain leads to chronic neuroinflammation, which is antecedent to the development of HIV-associated neurocognitive disorders (HAND) in the majority of patients. Productively HIV infected microglia release multiple neurotoxins including proinflammatory cytokines and HIV proteins such as envelope glycoprotein (gp120) and transactivator of transcription (Tat). However, powerful counteracting silencing mechanisms in microglial cells result in the rapid shutdown of HIV expression to limit neuronal damage. Here we investigated whether the Nerve Growth Factor IB-like nuclear receptor Nurr1 (NR4A2), which is a repressor of inflammation in the brain, acts to directly restrict HIV expression. HIV silencing was substantially enhanced by Nurr1 agonists in both immortalized human microglial cells (h{micro}glia) and induced pluripotent stem cells (iPSC)-derived human microglial cells (iMG). Overexpression of Nurr1 led to viral suppression, whereas by contrast, knock down (KD) of endogenous Nurr1 blocked HIV silencing. Chromatin immunoprecipitation (ChIP) assays showed that Nurr1 mediates recruitment of the CoREST/HDAC1/G9a/EZH2 transcription repressor complex to HIV promoter resulting in epigenetic silencing of active HIV. Transcriptomic studies demonstrated that in addition to repressing HIV transcription, Nurr1 also downregulated numerous cellular genes involved in inflammation, cell cycle, and metabolism, thus promoting HIV latency and microglial homoeostasis. Thus, Nurr1 plays a pivotal role in modulating the cycles of proviral reactivation by cytokines and potentiating the proviral transcriptional shutdown. These data highlight the therapeutic potential of Nurr1 agonists for inducing HIV silencing and microglial homeostasis and amelioration of the neuroinflammation associated with HAND. AUTHOR SUMMARYHIV enters the brain almost immediately after infection where it infects perivascular macrophages, microglia and, to a less extent, astrocytes. In previous work using an immortalized human microglial cell model, we observed that integrated HIV constantly underwent cycles of reactivation and subsequent silencing. In the present study, we found that the Nurr1 nuclear receptor is a key mediator of HIV silencing. The functional activation of Nurr1 by specific agonists, or the over expression of Nurr1, resulted in rapid silencing of activated HIV in microglial cells. Global gene expression analysis confirmed that Nurr1 not only repressed HIV expression but also regulated numerous genes involved in microglial homeostasis and inflammation. Thus, Nurr1 is pivotal for HIV silencing and repression of inflammation in the brain and is a promising therapeutic target for treatment of HAND.

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages requires substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct nuclear sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNA seq data show that TgFBXL2 conditional depletion induces the expression of genes necessary for sexual commitment. We suggest that TgFBXL2 is a latent guardian of sexual stage development in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of sexual development. AUTHOR SUMMARYToxoplasma gondii is a protozoan parasite that replicates sexually in felids and asexually in nearly all other mammals with each life stage having a specific transcriptional profile. When life stage specific transcription is not properly controlled, the parasite dies and therefore its important to understand what inhibits expression of sexual stage genes during asexual growth and vice versa. Here we identify a ubiquitin E3 ligase complex that inhibits sexual stage gene expression during asexual growth.

Factors influencing Kaposis sarcoma-associated herpesvirus (KSHV) transmission and the early stages of KSHV infection in the human immune system remain poorly characterized. KSHV is known to extensively manipulate the host immune system and the cytokine milieu, and cytokines are known to influence the progression of KSHV-associated diseases. Here, using our unique model of KSHV infection in tonsil lymphocytes, we investigate the influence of host cytokines on the establishment of KSHV infection in human B cells. Our data demonstrate that KSHV manipulates the host cytokine microenvironment during early infection and susceptibility is generally associated with downregulation of multiple cytokines. However, we show that IL-21 signaling promotes KSHV infection by promoting both plasma cell numbers and increasing KSHV infection in plasma cells as early as 3 days post-infection. Our data reveal that this phenotype is dependent upon a specific milieu of T cells, that includes IL-21 producing Th17, Tc17 and CD8+ central memory T cells. These results suggest that IL-21 plays a significant role in the early stages of KSHV infection in the human immune system and that specific immunological states favor the initial establishment of KSHV infection by increasing infection in plasma cells.

NOT1 is the scaffold of the CCR4-NOT complex, a highly conserved multi-protein complex that regulates gene expression in eukaryotes. As opposed to most eukaryotes in which NO1 is encoded by a single gene, malaria parasites, Plasmodium falciparum, carry two NOT1 paralogues, PfNOT1.1 and PfNOT1.2. Here we showed that the two PfNOT1 proteins function as mutually exclusive scaffolds within the PfCCR4-NOT protein complexes that are abundantly located in the parasite cytoplasm. Intriguingly, the two PfNOT1 paralogues appear to have directly opposing functions in regulation of mRNA abundance across the P. falciparum IDC, in which PfNTO1.1 and PfNOT1.2 induces and suppresses transcript abundance during their active transcription, respectively. Targeted disruption of either of the PfNOT1 gene causes defective growth and lower invasion rates presumably due to the deregulation the P. falciparum IDC transcriptional cascade. We also demonstrate that the regulatory function of both PfNOT1.1 and PfNOT1.2 are related to another PfCCR4-NOT subunit, PfCaf1, which indicates their activity during post-transcriptional regulation. Indeed RNA decay studies suggest the active role of both PfNOT1 proteins in regulation of mRNA stability in a directly opposing manner. Author summaryCCR4-NOT complex is a highly conserved multi-protein complex that regulates gene expression in eukaryotes. NOT1 serves as the scaffold of the complex and plays important roles in gene regulation both transcriptionally and post-transcriptionally. As opposed to other eukaryotes, P. falciparum encodes two paralogues of PfNOT1, raising the question as to the significance to possess an additional copy of PfNOT1 in the parasite. Here we described antagonistic regulatory functions of two PfNOT1 paralogues in gene expression during the 48-hour intraerythrocytic developmental cycle. We also reported that their regulatory functions are predominantly post-transcriptional and proposed a model in which distinct PfCCR4-NOT complexes defined by mutually exclusive PfNOT1 scaffolds differentially regulate PfCAF1 function in mRNA decay. This study highlights the importance of post-transcriptional regulation in P. falciparum and provides novel insights into mechanisms of gene regulation in this organism. The unique presence of two PfNOT1 paralogues may also open avenues for the development of new drug targets for anti-malarial control.

Kaposis sarcoma-associated herpesvirus (KSHV) causes several human diseases including Kaposis sarcoma (KS), a leading cause of cancer in Africa and in patients with AIDS. KS tumor cells harbor KSHV predominantly in a latent form, while typically <5% contain lytic replicating virus. Because both latent and lytic stages likely contribute to cancer initiation and progression, continued dissection of host regulators of this biological switch will provide insights into fundamental pathways controlling the KSHV life cycle and related disease pathogenesis. Several cellular protein kinases have been reported to promote or restrict KSHV reactivation, but our knowledge of these signaling mediators and pathways is incomplete. We employed a polypharmacology-based kinome screen to identifiy specific kinases that regulate KSHV reactivation. Those identified by the screen and validated by knockdown experiments included several kinases that enhance lytic reactivation: ERBB2 (HER2 or neu), ERBB3 (HER3), ERBB4 (HER4), MKNK2 (MNK2), ITK, TEC, and DSTYK (RIPK5). Conversely, ERBB1 (EGFR1 or HER1), MKNK1 (MNK1) and FRK (PTK5) were found to promote the maintenance of latency. Mechanistic characterization of ERBB2 pro-lytic functions revealed a signaling connection between ERBB2 and the activation of CREB1, a transcription factor that drives KSHV lytic gene expression. These studies provided a proof-of-principle application of a polypharmacology-based kinome screen for the study of KSHV reactivation and enabled the discovery of both kinase inhibitors and specific kinases that regulate the KSHV latent-to-lytic replication switch. Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) causes Kaposis sarcoma, a cancer particularly prevalent in Africa. In cancer cells, the virus persists in a quiescent form called latency, in which only a few viral genes are made. Periodically, the virus switches into an active replicative cycle in which most of the viral genes are made and new virus is produced. What controls the switch from latency to active replication is not well understood, but cellular kinases, enzymes that control many cellular processes, have been implicated. Using a cell culture model of KSHV reactivation along with an innovative screening method that probes the effects of many cellular kinases simultaneously, we identified drugs that significantly limit KSHV reactivation, as well as specific kinases that either enhance or restrict KSHV replicative cycle. Among these were the ERBB kinases which are known to regulate growth of cancer cells. Understanding how these and other kinases contribute to the switch leading to production of more infectious virus helps us understand the mediators and mechanisms of KSHV diseases. Additionally, because kinase inhibitors are proving to be effective for treating other diseases including some cancers, identifying ones that restrict KSHV replicative cycle may lead to new approaches to treating KSHV-related diseases.

The Interferon-Stimulated Gene 15 (ISG15) is a ubiquitin-like protein induced by viral and bacterial infection. ISG15 covalently modifies host and pathogenic proteins in a process called ISGylation. Yet, the consequences of ISGylation on protein fate and function remain to be determined. Here we sought to assess whether ISGylation would be protective following bacterial pneumonia caused by Francisella novicida. We found that infection with F. novicida induces ISGylation both in vitro in macrophages and in vivo in the lung, liver, and spleen of mice infected intranasally. Surprisingly, ISG15 and ISGylation do not affect bacterial burden in the lung in vivo, but in a model of enhanced ISGylation (usp18C61A/C61A) mice have decreased respiratory distress relative to Isg15-/- animals. In order to understand the mechanism which underlies this phenotype, we mapped the ISGylome of F. novicida-infected mouse lungs using label-free quantitative mass spectrometry and identified enrichment in ISGylation of proteins involved in the innate immune response and cytosolic nucleotide signaling. We validated ISGylation of the sterile alpha motif and HD-containing protein 1 (SAMHD1) via immunoprecipitation. SAMHD1 depletes cytosolic dinucleotide stores critical for retroviral replication but it is unknown how its activity could affect bacterial infection. Structure-function analysis indicates that ISG15 modification sites in usp18C61A/C61A mice could prevent SAMHD1 dimerization and therefore abrogate function. Accordingly, deletion of SAMHD1 in fibroblasts with enhanced ISGylation reduces bacterial load. Taken together, unchecked ISGylation plays a protective role in F. novicida infection in vivo through improved respiratory function. Thus, inhibiting USP18 may be a promising therapeutic strategy for both viral and bacterial pneumonia. Author summaryFrancisella tularensis is a bacterial pathogen responsible for the disease tularemia, which can result in severe respiratory infection if as few as ten bacteria are inhaled. Our cells have many ways of managing infections, including the production of proteins designed to fight off foreign pathogens. One protein produced following infection is the interferon-stimulated gene 15 (ISG15). ISG15 is a ubiquitin-like molecule, meaning that it can be chemically attached to other proteins. When bound ISG15 changes the stability, interacting partners, or function of its target in a process termed ISGylation. Here we show that ISG15 is produced following infection with Francisella. We found that enhanced ISGylation led to less severe respiratory symptoms. To better understand the mechanism by which ISGylation protects from infection we identified the ISG15-modified proteins in the lung using mass-spectrometry-based proteomics. We found protein targets that are involved in the control of immune signaling pathways including sterile alpha motif and HD-containing protein 1 (SAMHD1) which, when deleted in cells with enhanced ISGylation, leads to better bacterial clearance. Together, we show that enhanced ISGylation plays a protective role following bacterial pneumonia, indicating that targeting this pathway could prove a beneficial therapeutic in both bacterial and viral respiratory diseases.

Detection of viral infection leads to both cell-intrinsic and cell-extrinsic responses. In mammals, cell-intrinsic detection of viral infection leads to cell-extrinsic activation of STAT (Signal Transduction and Activators of Transcription) proteins, a family of transcription factors that promote anti-viral defense. In the nematode C. elegans, STA-1/STAT is a negative regulator of anti-viral defense, but it is not known if it acts cell-intrinsically or cell-extrinsically and whether it has functional domains conserved with mammalian STATs. Here we show that C. elegans STA-1 protein disappears from nuclei of cells infected with the natural viral pathogen, Orsay virus, but remains nuclear in uninfected cells, indicating a cell-intrinsic site of action. During viral infection, STA-1 forms cytoplasmic puncta that interact with the RNA viral sensor DRH-1, suggesting that DRH-1 helps restrain this immune-repressive factor. STA-1 overexpression causes increased susceptibility to viral infection, in a manner dependent on conserved residues important for DNA binding, nuclear localization and phosphorylation. Structural predictions indicate that STA-1 is most similar to STAT5 proteins in mammals, which have known immune-repressive roles. Our transcriptomic analysis demonstrates that C. elegans STA-1 regulates a general anti-pathogen program, including genes upregulated later during viral infection. Altogether, our findings provide insight into conserved and distinct features of STA-1 in C. elegans, indicating an ancient role for cell-intrinsic, immune-repressive STATs. Author SummaryAll living organisms must detect viral infections and mount a defense to survive. One major antiviral defense pathway in mammals is the interferon response, which involves sensing viral infection in one cell, and delivering an interferon message to neighboring cells. These neighboring cells then turn on anti-viral defense gene expression using proteins called STAT transcription factors. We study anti-viral defense in the roundworm C. elegans, and in this study show that viral infected cells themselves use a STAT protein called STA-1, with perhaps a lesser role for STA-1 in neighboring cells, in contrast to mammals. We also extend on previous findings that STA-1 turns off anti-viral gene expression, and we analyze regions in the protein to demonstrate that STA-1 is bona fide transcription factor with an immune-repressive role. Structural prediction analysis of STA-1 indicates it is most similar to STAT5 in mammals, suggesting an ancient role for this protein as an immune-repressive factor acting directly in virally infected cells.

The Toxoplasma gondii cytoskeleton contains an intermediate filament network, supporting a quilt of alveolar sheets forming the inner membrane complex (IMC), undergirded by 22 subpellicular microtubules (SPMTs). Embedded within the IMC are the apical annuli: 5-6 ring-shaped pores facilitating dense granule exocytosis. Here we describe a novel apical annuli protein, AAP7. AAP7 depletion causes a severe fitness defect. In stable AAP7-depleted (ATc-resistant) parasites, LMBD3 no longer traffics to the annuli, but accumulates among the secretory organelles. This suggests AAP7 is required to traffic LMDB3 to the plasma membrane through a novel route. Moreover, it indicated that AAP7 connects plasma membrane embedded LMDB3 to the AAP proteins embedded in the IMC sutures. Functionally, AAP7 depletion results in reduced secretion of dense granule proteins. Specifically, parasitophorous vacuole membrane pore forming GRA17 secretion is reduced, causing bubble vacuoles. GRA17 overexpression overcomes AAP7 depletion and reduces bubble vacuoles revealing the critical defect. An additional AAP7 depletion phenotype is the accumulation of polyglutamylated SPMTs at the basal end, indicating slow turnover. Lastly, from a comparative angle, we investigated annuli in Sarcocystis neurona, revealing 6 apical annuli. This is surprising considering S. neuronas 11 alveolar vesicles and expected 11 annuli. Ergo, annuli architecture does not take cues from IMC suture positioning. In summary, our analysis of AAP7 led to equally versatile and novel insights in apical annuli architecture, their assembly (uncovering a potentially novel trafficking process), how they interface with the IMC and impact the SPMTs, and their critical function in facilitating GRA17 secretion required for the pore across the parasitophorous vacuole membrane

Plasmodium spp., the parasites that are the causative agents of malaria, encode a repertoire of divergent protein kinases that coordinate essential processes including cell division and host cell invasion, yet the functions of many kinases are poorly defined. Plasmodium Protein Kinase 2 (PK2) is essential for asexual blood-stage proliferation and has been implicated in P. falciparum merozoite invasion of red blood cells. However, its role in the sexual stages of the Plasmodium life cycle responsible for transmission is unknown. Here, using live cell imaging, functional analyses, ultrastructure microscopy and phosphoproteomics, we demonstrate that PK2 has a significant role in the Plasmodium berghei life cycle in the mosquito. We show that PK2 is expressed in merozoites, ookinetes and sporozoites - the invasive stages of the parasite life cycle. A conditional knockdown approach revealed that PK2 is required for the ookinete to oocyst transition in the mosquito midgut, potentially associated with altered microneme positioning. Using haemocoel injection to bypass the midgut barrier revealed that PK2 is also required for sporozoite development after midgut invasion. Following PK2 knockdown, global proteome abundance was largely unaffected at 24 h post activation, whereas phosphoproteomics identified changes in phosphorylation of proteins linked to midgut traversal, parasite architecture, and gene regulation. These studies provide insight into the importance of PK2 function in Plasmodium sexual stages and parasite transmission through the mosquito, highlighting its essential function during the three invasive stages of the parasites life cycle.

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo, and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue, and measured variation between infected cells. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneity of the infected hepatocyte in situ and provide insights into how the parasite may alter the local microenvironment to influence its survival and modulate immunity.

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary co-factor for host immunity. Heme oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of heme. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish heme oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from mycobacterial infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against mycobacterial infection by reducing infection-induced iron accumulation and ferroptosis.

Pathogenesis of Pseudomonas aeruginosa infections can include bacterial survival inside epithelial cells. Previously, we showed this involves multiple roles played by the type three-secretion system (T3SS), and specifically the effector ExoS. This includes ExoS-dependent inhibition of a lytic host cell response that subsequently enables intracellular replication. Here, we studied the underlying cell death response to intracellular P. aeruginosa, comparing wild-type to T3SS mutants varying in capacity to induce cell death and that localize to different intracellular compartments. Results showed that corneal epithelial cell death induced by intracellular P. aeruginosa lacking the T3SS, which remains in vacuoles, correlated with activation of NF-{kappa}B as measured by p65 relocalization and TNF transcription and secretion. Deletion of caspase-4 through CRISPR-Cas9 mutagenesis delayed cell death caused by these intracellular T3SS mutants. Caspase-4 deletion also countered more rapid cell death caused by T3SS effector-null mutants still expressing the TSSS apparatus that traffic to the host cell cytoplasm, and in doing so rescued intracellular replication normally dependent on ExoS. While HeLa cells lacked a lytic death response to T3SS mutants, it was found to be enabled by interferon gamma treatment. Together, these results show that epithelial cells can activate the noncanonical inflammasome pathway to limit proliferation of intracellular P. aeruginosa, not fully dependent on bacterially-driven vacuole escape. Since ExoS inhibits the lytic response, the data implicate targeting of caspase-4, an intracellular pattern recognition receptor, as another contributor to the role of ExoS in the intracellular lifestyle of P. aeruginosa. ImportancePseudomonas aeruginosa can exhibit an intracellular lifestyle within epithelial cells in vivo and in vitro. The type three secretion system (T3SS) effector ExoS contributes via multiple mechanisms, including extending the life of invaded host cells. Here, we aimed to understand the underlying cell death inhibited by ExoS when P. aeruginosa is intracellular. Results showed that intracellular P. aeruginosa lacking T3SS effectors could elicit rapid cell lysis via the non-canonical inflammasome pathway. Caspase-4 contributed to cell lysis even when the intracellular bacteria lacked the entire T33S and were consequently unable to escape vacuoles, representing a naturally occurring subpopulation during wildtype infection. Together, the data show the caspase-4 inflammasome as an epithelial cell defense against intracellular P. aeruginosa, and implicate its targeting as another mechanism by which ExoS preserves the host cell replicative niche.

Apicomplexan parasites possess several specialized structures to invade their host cells and replicate successfully. One of these is the inner membrane complex (IMC), a peripheral membrane-cytoskeletal system underneath the plasma membrane. It is composed of a series of flattened, membrane-bound vesicles and a cytoskeletal subpellicular network (SPN) comprised of intermediate filament-like proteins called alveolins. While the alveolin proteins are conserved throughout the Apicomplexa and the broader Alveolata, their precise functions and interactions remain poorly understood. Here, we describe the function of one of these alveolin proteins, TgIMC6. Disruption of IMC6 resulted in striking morphological defects that led to aberrant motility, invasion, and replication. Deletion analyses revealed that the alveolin domain alone is largely sufficient to restore localization and partially sufficient for function. As this highlights the importance of the IMC6 alveolin domain, we implemented unnatural amino acid photoreactive crosslinking to the alveolin domain and identified multiple binding interfaces between IMC6 and two other cytoskeletal proteins - IMC3 and ILP1. To our knowledge, this provides the first direct evidence of protein-protein interactions in the alveolin domain and supports the long-held hypothesis that the alveolin domain is responsible for filament formation. Collectively, our study features the conserved alveolin proteins as critical components that maintain the parasites structural integrity and highlights the alveolin domain as a key mediator of SPN architecture.

The human malaria parasite Plasmodium falciparum (Pf) expresses ten different thrombospondin type 1 repeat (TSR) domain-bearing proteins at different stages throughout its life cycle. TSRs can be modified by two types of glycosylation: O-fucosylation at conserved serine (S) or threonine (T) residues and C-mannosylation at conserved tryptophan (W) residues. PfTRAP, which is expressed in mosquito-stage sporozoites, has one TSR domain that is O-fucosylated at Thr256 and C-mannosylated at Trp250. We employed site-directed mutagenesis by CRISPR/Cas9 gene editing to generate two PfTRAP glyco-null mutant parasites, PfTRAP_T256A and PfTRAP_W250F, and assessed the fitness of these mutant parasites across the life cycle compared to the wild-type NF54 line as well as a PfTRAP knockout line. The PfTRAP glyco-null parasites exhibited major fitness defects comparable to knockout: sporozoites were unable to productively colonize the salivary glands and were highly impaired in gliding motility and the ability to invade cultured human hepatocytes. PfTRAP abundance in these mutants was significantly decreased despite normal transcript levels. Biophysical assays with recombinant proteins confirmed that glycosylation of the PfTRAP TSR stabilizes the domain and is likely required for its folding and secretion. These findings demonstrate that glycosylation of PfTRAPs TSR is critical for its proper expression and function, and underscore the importance of TSR glycosylation in the mosquito stage of the life cycle. IMPORTANCEMalaria is a mosquito-borne disease caused by Plasmodium parasites, of which P. falciparum is the deadliest. Plasmodium has ten proteins bearing thrombospondin type 1 repeats (TSRs), protein folds that aid cell-cell recognition and binding. Each of Plasmodiums ten TSR-bearing proteins is important for invading tissues in the mosquito vector and human host. TSRs are decorated with sugar molecules, a modification termed glycosylation. To better understand the importance of TSR glycosylation in Plasmodium, we investigated the P. falciparum protein TRAP, which is only expressed in mosquito-stage parasite forms called sporozoites. When PfTRAP was mutated to prevent glycosylation, abundance of the protein significantly decreased and parasites were unable to colonize the mosquito salivary glands. Furthermore, these mutant sporozoites were unable to move or to invade human liver cells. Our study reveals how TSR glycosylation can support the function of proteins that are required for parasite virulence.

1.Orthoflaviviruses (OFVs) are the primary cause of arboviral disease worldwide, leading to large numbers of hospitalisations, sequelae and deaths associated. Viral genome replication is catalysed by non-structural protein 5 (NS5), the largest and most conserved protein in these viruses. Here, we used quantitative proteomics to identify host cell interactors of Zika virus (ZIKV), Usutu virus (USUV), and West Nile virus (WNV) NS5 proteins. A total of 141 proteins were detected in the analysis, being 26 of them associated with all three NS5s. Downregulation of myoferlin and LGALS3BP led to decreases in the virus yields, indicating that their presence is needed for efficient virus replication. Conversely, silencing of mitophagy-related prohibitin 2 (PHB2) resulted in significant increases in ZIKV and USUV titres and genomic RNA. Supporting a connection between this antiviral behaviour and mitophagy, an inhibitor of this pathway also led to larger virus titres and viral RNA copies. During infection, PHB2 protein levels gradually decreased, becoming transiently undetectable, and suggesting that it is specifically targeted by the virus for degradation. Ectopic expression of NS5 alone did not affect PHB2 intracellular abundance, however a significant decrease was observed in cells expressing the viral protease NS2B/NS3. Its elimination, was more pronounced in cells where both viral enzymes were co-expressed, supporting that NS5 could be assisting PHB2 recognition by NS2B/NS3. Overall, our results support that PHB2 is an orthoflaviviral restriction factor which is targeted for degradation to favour viral replication. 2. IMPORTANCEOrthoflaviviruses (OFVs) are one of the main causes of disease transmitted by mosquitoes, with millions of infections occurring annually, and large numbers of deaths associated. Some important members include viruses causing dengue, yellow fever, and Zika-associated disease (e.g. microcephaly). OFVs contain a genome which is copied by a highly conserved viral protein termed NS5. Prohibitin 2 (PHB2) is a host cellular protein involved in different cellular processes including mitophagy, a mechanism to remove damaged mitochondria. Here, we show that human PHB2 interacts with orthoflaviviral NS5. Depletion of PHB2 results in increased viral yields, suggesting that this protein restricts orthoflaviviral infection. We found that PHB2 is targeted for degradation by the orthoflaviviral protease in a process aided by NS5, supporting that several viral proteins act concertedly to eliminate PHB2, counteracting its antiviral activity, and thus favouring infection.