Host-pathogen interactions

Nisha Singh, Agustin Rolandelli, Anya J O’Neal, Rainer L. Butler, Sourabh Samaddar, Hanna J Laukaitis-Yousey, Matthew Butnaru, Stephanie E Mohr, Norbert Perrimon, and Joao HF Pedra. 9/8/2023. “Genetic manipulation of an Ixodes scapularis cell line.” bioRxiv, Pp. 9/8/2023. 09.08.556855. Publisher's VersionAbstract
Although genetic manipulation is one of the hallmarks in model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the blacklegged tick Ixodes scapularis, an arthropod that serves as a vector of several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology dependent recombination. Targeting the E3 ubiquitin ligase X-linked inhibitor of apoptosis (xiap) and its substrate p47 led to alteration in molecular signaling within the immune deficiency (IMD) network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for genetic engineering of ticks in vivo and aid in circumventing the long-life cycle of I. scapularis, of which limits efficient and scalable molecular genetic screens.Importance Genetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of I. scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent A. phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the blacklegged tick Ixodes scapularis.Competing Interest StatementThe authors have declared no competing interest.
Agustin Rolandelli, Hanna J Laukaitis-Yousey, Haikel N Bogale, Nisha Singh, Sourabh Samaddar, Anya J O’Neal, Camila R Ferraz, Matthew Butnaru, Enzo Mameli, Baolong Xia, Tays M. Mendes, Rainer L. Butler, Liron Marnin, Francy ECabrera Paz, Luisa M Valencia, Vipin S Rana, Ciaran Skerry, Utpal Pal, Stephanie E Mohr, Norbert Perrimon, David Serre, and Joao HF Pedra. 2023. “Tick hemocytes have pleiotropic roles in microbial infection and arthropod fitness.” bioRxiv, Pp. 2023.08.31.555785. Publisher's VersionAbstract
Uncovering the complexity of systems in non-model organisms is critical for understanding arthropod immunology. Prior efforts have mostly focused on Dipteran insects, which only account for a subset of existing arthropod species in nature. Here, we describe immune cells or hemocytes from the clinically relevant tick Ixodes scapularis using bulk and single cell RNA sequencing combined with depletion via clodronate liposomes, RNA interference, Clustered Regularly Interspaced Short Palindromic Repeats activation (CRISPRa) and RNA-fluorescence in situ hybridization (FISH). We observe molecular alterations in hemocytes upon tick infestation of mammals and infection with either the Lyme disease spirochete Borrelia burgdorferi or the rickettsial agent Anaplasma phagocytophilum. We predict distinct hemocyte lineages and reveal clusters exhibiting defined signatures for immunity, metabolism, and proliferation during hematophagy. Furthermore, we perform a mechanistic characterization of two I. scapularis hemocyte markers: hemocytin and astakine. Depletion of phagocytic hemocytes affects hemocytin and astakine levels, which impacts blood feeding and molting behavior of ticks. Hemocytin specifically affects the c-Jun N-terminal kinase (JNK) signaling pathway, whereas astakine alters hemocyte proliferation in I. scapularis. Altogether, we uncover the heterogeneity and pleiotropic roles of hemocytes in ticks and provide a valuable resource for comparative biology in arthropods.Competing Interest StatementThe authors have declared no competing interest.
Ying Xu, Raghuvir Viswanatha, Oleg Sitsel, Daniel Roderer, Haifang Zhao, Christopher Ashwood, Cecilia Voelcker, Songhai Tian, Stefan Raunser, Norbert Perrimon, and Min Dong. 2022. “CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.” Nature, 610, 7931, Pp. 349-355.Abstract
Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.
2020 Sep 21

DRSC-BTRR at the GSA Molecular Parasitology Meeting XXXI

Mon Sep 21 (All day) to Thu Sep 24 (All day)

Location: 

online
The Drosophila Research & Screening Center-Biomedical Technology Research Resource (DRSC-BTRR) will be represented at the GSA's Molecular Parasitology Meeting XXXI (a virtual event). Look for a presentation by Viswanatha et al. on our work establishing CRISPR knockout screening in mosquito cell lines. Read more about DRSC-BTRR at the GSA Molecular Parasitology Meeting XXXI
Cartoon of fly host cells with virus or endosymbiotic bacteria

Cell-based RNAi screening helps reveal host-microbe interactions--two new screen reports

November 19, 2018

Laboratories at the Skirball Institute at New York University and the Boyce Thompson Institute at Cornell University reported results of two different cell-based Drosophila RNAi screens in papers published this week. The screens have in common that they looked at interactions between the host insect cells and a microbe -- the endosymbiont Wolbachia in one study and baculovirus in the other. For more, check out the newly published studies. For both these screens, the DRSC provided libraries for screens that were then performed at the host institution.

 

... Read more about Cell-based RNAi screening helps reveal host-microbe interactions--two new screen reports
Jennifer A Philips, Maura C Porto, Hui Wang, Eric J Rubin, and Norbert Perrimon. 2008. “ESCRT factors restrict mycobacterial growth.” Proc Natl Acad Sci U S A, 105, 8, Pp. 3070-5.Abstract

Nearly 1.7 billion people are infected with Mycobacterium tuberculosis. Its ability to survive intracellularly is thought to be central to its success as a pathogen, but how it does this is poorly understood. Using a Drosophila model of infection, we identify three host cell activities, Rab7, CG8743, and the ESCRT machinery, that modulate the mycobacterial phagosome. In the absence of these factors the cell no longer restricts growth of the non-pathogen Mycobacterium smegmatis. Hence, we identify factors that represent unique vulnerabilities of the host cell, because manipulation of any one of them alone is sufficient to allow a nonpathogenic mycobacterial species to proliferate. Furthermore, we demonstrate that, in mammalian cells, the ESCRT machinery plays a conserved role in restricting bacterial growth.

Christine Akimana, Souhaila Al-Khodor, and Yousef Abu Kwaik. 2010. “Host factors required for modulation of phagosome biogenesis and proliferation of Francisella tularensis within the cytosol.” PLoS One, 5, 6, Pp. e11025.Abstract

Francisella tularensis is a highly infectious facultative intracellular bacterium that can be transmitted between mammals by arthropod vectors. Similar to many other intracellular bacteria that replicate within the cytosol, such as Listeria, Shigella, Burkholderia, and Rickettsia, the virulence of F. tularensis depends on its ability to modulate biogenesis of its phagosome and to escape into the host cell cytosol where it proliferates. Recent studies have identified the F. tularensis genes required for modulation of phagosome biogenesis and escape into the host cell cytosol within human and arthropod-derived cells. However, the arthropod and mammalian host factors required for intracellular proliferation of F. tularensis are not known. We have utilized a forward genetic approach employing genome-wide RNAi screen in Drosophila melanogaster-derived cells. Screening a library of approximately 21,300 RNAi, we have identified at least 186 host factors required for intracellular bacterial proliferation. We silenced twelve mammalian homologues by RNAi in HEK293T cells and identified three conserved factors, the PI4 kinase PI4KCA, the ubiquitin hydrolase USP22, and the ubiquitin ligase CDC27, which are also required for replication in human cells. The PI4KCA and USP22 mammalian factors are not required for modulation of phagosome biogenesis or phagosomal escape but are required for proliferation within the cytosol. In contrast, the CDC27 ubiquitin ligase is required for evading lysosomal fusion and for phagosomal escape into the cytosol. Although F. tularensis interacts with the autophagy pathway during late stages of proliferation in mouse macrophages, this does not occur in human cells. Our data suggest that F. tularensis utilizes host ubiquitin turnover in distinct mechanisms during the phagosomal and cytosolic phases and phosphoinositide metabolism is essential for cytosolic proliferation of F. tularensis. Our data will facilitate deciphering molecular ecology, patho-adaptation of F. tularensis to the arthropod vector and its role in bacterial ecology and patho-evolution to infect mammals.

Sara Cherry, Tammy Doukas, Susan Armknecht, Sean Whelan, Hui Wang, Peter Sarnow, and Norbert Perrimon. 2005. “Genome-wide RNAi screen reveals a specific sensitivity of IRES-containing RNA viruses to host translation inhibition.” Genes Dev, 19, 4, Pp. 445-52.Abstract

The widespread class of RNA viruses that utilize internal ribosome entry sites (IRESs) for translation include poliovirus and Hepatitis C virus. To identify host factors required for IRES-dependent translation and viral replication, we performed a genome-wide RNAi screen in Drosophila cells infected with Drosophila C virus (DCV). We identified 66 ribosomal proteins that, when depleted, specifically inhibit DCV growth, but not a non-IRES-containing RNA virus. Moreover, treatment of flies with a translation inhibitor is protective in vivo. Finally, this increased sensitivity to ribosome levels also holds true for poliovirus infection of human cells, demonstrating the generality of these findings.

Joshua D Stender, Gabriel Pascual, Wen Liu, Minna U Kaikkonen, Kevin Do, Nathanael J Spann, Michael Boutros, Norbert Perrimon, Michael G Rosenfeld, and Christopher K Glass. 2012. “Control of proinflammatory gene programs by regulated trimethylation and demethylation of histone H4K20.” Mol Cell, 48, 1, Pp. 28-38.Abstract

Regulation of genes that initiate and amplify inflammatory programs of gene expression is achieved by signal-dependent exchange of coregulator complexes that function to read, write, and erase specific histone modifications linked to transcriptional activation or repression. Here, we provide evidence for the role of trimethylated histone H4 lysine 20 (H4K20me3) as a repression checkpoint that restricts expression of toll-like receptor 4 (TLR4) target genes in macrophages. H4K20me3 is deposited at the promoters of a subset of these genes by the SMYD5 histone methyltransferase through its association with NCoR corepressor complexes. Signal-dependent erasure of H4K20me3 is required for effective gene activation and is achieved by NF-κB-dependent delivery of the histone demethylase PHF2. Liver X receptors antagonize TLR4-dependent gene activation by maintaining NCoR/SMYD5-mediated repression. These findings reveal a histone H4K20 trimethylation/demethylation strategy that integrates positive and negative signaling inputs that control immunity and homeostasis.

Jennifer A Philips, Eric J Rubin, and Norbert Perrimon. 2005. “Drosophila RNAi screen reveals CD36 family member required for mycobacterial infection.” Science, 309, 5738, Pp. 1251-3.Abstract

Certain pathogens, such as Mycobacterium tuberculosis, survive within the hostile intracellular environment of a macrophage. To identify host factors required for mycobacterial entry and survival within macrophages, we performed a genomewide RNA interference screen in Drosophila macrophage-like cells, using Mycobacterium fortuitum. We identified factors required for general phagocytosis, as well as those needed specifically for mycobacterial infection. One specific factor, Peste (Pes), is a CD36 family member required for uptake of mycobacteria, but not Escherichia coli or Staphylococcus aureus. Moreover, mammalian class B scavenger receptors (SRs) conferred uptake of bacteria into nonphagocytic cells, with SR-BI and SR-BII uniquely mediating uptake of M. fortuitum, which suggests a conserved role for class B SRs in pattern recognition and innate immunity.

Isabelle Derré, Marc Pypaert, Alice Dautry-Varsat, and Hervé Agaisse. 2007. “RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection.” PLoS Pathog, 3, 10, Pp. 1446-58.Abstract

Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate's knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific.

October M Sessions, Nicholas J Barrows, Jayme A Souza-Neto, Timothy J Robinson, Christine L Hershey, Mary A Rodgers, Jose L Ramirez, George Dimopoulos, Priscilla L Yang, James L Pearson, and Mariano A Garcia-Blanco. 2009. “Discovery of insect and human dengue virus host factors.” Nature, 458, 7241, Pp. 1047-50.Abstract

Dengue fever is the most frequent arthropod-borne viral disease of humans, with almost half of the world's population at risk of infection. The high prevalence, lack of an effective vaccine, and absence of specific treatment conspire to make dengue fever a global public health threat. Given their compact genomes, dengue viruses (DENV-1-4) and other flaviviruses probably require an extensive number of host factors; however, only a limited number of human, and an even smaller number of insect host factors, have been identified. Here we identify insect host factors required for DENV-2 propagation, by carrying out a genome-wide RNA interference screen in Drosophila melanogaster cells using a well-established 22,632 double-stranded RNA library. This screen identified 116 candidate dengue virus host factors (DVHFs). Although some were previously associated with flaviviruses (for example, V-ATPases and alpha-glucosidases), most of the DVHFs were newly implicated in dengue virus propagation. The dipteran DVHFs had 82 readily recognizable human homologues and, using a targeted short-interfering-RNA screen, we showed that 42 of these are human DVHFs. This indicates notable conservation of required factors between dipteran and human hosts. This work suggests new approaches to control infection in the insect vector and the mammalian host.

Theresa S Moser, Russell G Jones, Craig B Thompson, Carolyn B Coyne, and Sara Cherry. 2010. “A kinome RNAi screen identified AMPK as promoting poxvirus entry through the control of actin dynamics.” PLoS Pathog, 6, 6, Pp. e1000954.Abstract

Poxviruses include medically important human pathogens, yet little is known about the specific cellular factors essential for their replication. To identify genes essential for poxvirus infection, we used high-throughput RNA interference to screen the Drosophila kinome for factors required for vaccinia infection. We identified seven genes including the three subunits of AMPK as promoting vaccinia infection. AMPK not only facilitated infection in insect cells, but also in mammalian cells. Moreover, we found that AMPK is required for macropinocytosis, a major endocytic entry pathway for vaccinia. Furthermore, we show that AMPK contributes to other virus-independent actin-dependent processes including lamellipodia formation and wound healing, independent of the known AMPK activators LKB1 and CaMKK. Therefore, AMPK plays a highly conserved role in poxvirus infection and actin dynamics independent of its role as an energy regulator.

Hervé Agaisse, Laura S Burrack, Jennifer A Philips, Eric J Rubin, Norbert Perrimon, and Darren E Higgins. 2005. “Genome-wide RNAi screen for host factors required for intracellular bacterial infection.” Science, 309, 5738, Pp. 1248-51.Abstract

Most studies of host-pathogen interactions have focused on pathogen-specific virulence determinants. Here, we report a genome-wide RNA interference screen to identify host factors required for intracellular bacterial pathogenesis. Using Drosophila cells and the cytosolic pathogen Listeria monocytogenes, we identified 305 double-stranded RNAs targeting a wide range of cellular functions that altered L. monocytogenes infection. Comparison to a similar screen with Mycobacterium fortuitum, a vacuolar pathogen, identified host factors that may play a general role in intracellular pathogenesis and factors that specifically affect access to the cytosol by L. monocytogenes.

Sara Cherry, Amit Kunte, Hui Wang, Carolyn Coyne, Robert B Rawson, and Norbert Perrimon. 2006. “COPI activity coupled with fatty acid biosynthesis is required for viral replication.” PLoS Pathog, 2, 10, Pp. e102.Abstract

During infection by diverse viral families, RNA replication occurs on the surface of virally induced cytoplasmic membranes of cellular origin. How this process is regulated, and which cellular factors are required, has been unclear. Moreover, the host-pathogen interactions that facilitate the formation of this new compartment might represent critical determinants of viral pathogenesis, and their elucidation may lead to novel insights into the coordination of vesicular trafficking events during infection. Here we show that in Drosophila cells, Drosophila C virus remodels the Golgi apparatus and forms a novel vesicular compartment, on the surface of which viral RNA replication takes place. Using genome-wide RNA interference screening, we found that this step in the viral lifecycle requires at least two host encoded pathways: the coat protein complex I (COPI) coatamer and fatty acid biosynthesis. Our results integrate, clarify, and extend numerous observations concerning the cell biology of viral replication, allowing us to conclude that the coupling of new cellular membrane formation with the budding of these vesicles from the Golgi apparatus allows for the regulated generation of this new virogenic organelle, which is essential for viral replication. Additionally, because these pathways are also limiting in flies and in human cells infected with the related RNA virus poliovirus, they may represent novel targets for antiviral therapies.