Cryptococcus neoformans (C. neoformans) penetration into the central nervous system (CNS) requires traversal of the blood-brain barrier that is composed of a single layer of human brain microvascular endothelial cells (HBMEC), but the underlying mechanisms of C. neoformans traversal remain incompletely understood. C. neoformans transcytosis of HBMEC monolayer involves rearrangements of the host cell actin cytoskeleton and small GTP-binding Rho family proteins such as Rac1 are shown to regulate host cell actin cytoskeleton. We, therefore, examined whether C. neoformans traversal of the blood-brain barrier involves host Rac1. While the levels of activated Rac1 (GTP-Rac1) in HBMEC increased significantly upon incubation with C. neoformans strains, pharmacological inhibition and down-modulation of Rac1 significantly decreased C. neoformans transcytosis of HBMEC monolayer. Also, Rac1 inhibition was efficient in preventing C. neoformans penetration into the brain. In addition, C. neoformans phospholipase B1 (Plb1) was shown to contribute to activating host cell Rac1, and STAT3 was observed to associate with GTP-Rac1 in HBMEC that were incubated with C. neoformans strain but not with its Δplb1 mutant. These findings demonstrate for the first time that C. neoformans Plb1 aids fungal traversal across the blood-brain barrier by activating host cell Rac1 and its association with STAT3, and suggest that pharmacological intervention of host-microbial interaction contributing to traversal of the blood-brain barrier may prevent C. neoformans penetration into the brain.
Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most frequent causes of hospital- and community-associated infections. Resistance to the entire class of β-lactam antibiotics, such as methicillin and penicillin, makes MRSA infections difficult to treat. Hospital-associated MRSA strains are often multi-drug resistant, leaving only lower efficiency drugs such as vancomycin as treatments options. Like many other S. aureus strains, MRSA strains produce a series of virulence factors, such as toxins and adhesion proteins. Recent findings have shed some new light on the molecular events that underlie MRSA epidemic waves. Newly emerging MRSA clones appear to have acquired phenotypic traits that render them more virulent or able to colonize better, either via mobile genetic elements or adaptation of gene expression. Acquisition of Panton-Valentine leukocidin genes and increased expression of core genome-encoded toxins are being discussed as potentially contributing to the success of the recently emerged community-associated MRSA strains. However, the molecular factors underlying the spread of hospital- and community-associated MRSA strains are still far from being completely understood, a situation calling for enhanced research efforts in that area.
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that is the most common cause of sexually transmitted bacterial infections and is the etiological agent of trachoma, the leading cause of preventable blindness. The organism infects epithelial cells of the genital tract and eyelid resulting in a damaging inflammatory response. C. trachomatis grows within a vacuole termed the inclusion, and its growth depends on numerous host factors, including lipids. Although a variety of mechanisms are involved in the acquisition of host cell cholesterol and glycosphingolipids by C. trachomatis, none of the previously documented pathways for lipid acquisition are absolutely required for growth. Here we demonstrate that multiple components of the host high density lipoprotein (HDL) biogenesis machinery including the lipid effluxers, ABCA1 and CLA 1, and their extracellular lipid acceptor, apoA-1, are recruited to the inclusion of C. trachomatis-infected cells. Furthermore, the apoA-1 that accumulates within the inclusion co-localizes with pools of phosphatidylcholine. Knockdown of ABCA1, which mediates the cellular efflux of cholesterol and phospholipids to initiate the formation of HDL in the serum, prevents the growth of C. trachomatis in infected HeLa cells. In addition, drugs that inhibit the lipid transport activities of ABCA1 and CLA 1 also inhibit the recruitment of phospholipids to the inclusion and prevent chlamydial growth. These results strongly suggest that C. trachomatis co-opts the host cell lipid transport system involved in the formation of HDL to acquire lipids, such as phosphatidylcholine, that are necessary for growth.
Innate recognition systems, including the Toll-like receptors (TLRs), play a critical role in activating host defenses and proinflammatory pathways in response to infection. Pathogens have developed strategies to subvert TLRs in order to survive and replicate within the host. The model intracellular pathogen, Francisella novicida, modulates host defenses to promote survival and replication in macrophages. TLR2, which recognizes bacterial lipoproteins (BLPs), is critical for activating host defenses and proinflammatory cytokine production in response to Francisella infection. Here we show that the F. novicida protein FTN_0757 acts to repress BLP production, dampening TLR2 activation. The ΔFTN_0757 mutant strain induced robust TLR2-dependent cytokine production in macrophages compared to wild-type bacteria, and produced increased amounts of BLPs. The deletion of one BLP (FTN_1103) from ΔFTN_0757 decreased the total BLP concentration to near wild-type levels and correlated with a decrease in the induction of TLR2 signaling. The overproduction of BLPs also contributed to the in vivo attenuation of the ΔFTN_0757 mutant, which was significantly rescued when FTN_1103 was deleted. Taken together, these data reveal a novel mechanism of immune evasion by the downregulation of BLP expression to subvert TLR2 activation, which is likely used by numerous other intracellular bacterial pathogens.
Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.
cAMP is an ancient second messenger, and is used by many organisms to regulate a wide range of cellular functions. Mycobacterium tuberculosis-complex bacteria are exceptional in that they have genes for at least 15 biochemically distinct adenylyl cyclases, the enzymes that generate cAMP. cAMP-associated gene regulation within tubercle bacilli is required for their virulence, and secretion of cAMP produced by M. tuberculosis bacteria into host macrophages disrupts the host’s immune response to infection. In this review, we discuss recent advances in our understanding of the means by which cAMP levels are controlled within mycobacteria, the importance of cAMP to M. tuberculosis during host infection, and role of cAMP in mycobacterial gene regulation. Understanding the myriad aspects of cAMP signaling in tubercle bacilli will establish new paradigms for cAMP signaling, and may contribute to new approaches for prevention and/or treatment of tuberculosis (TB) disease.
Acinetobacter baumannii is an opportunistic pathogen that has emerged as a prevalent source of nosocomial infections, most frequently causing ventilator-associated pneumonia. The emergence of pan-drug resistant strains magnifies the problem by reducing viable treatment options and effectively increasing the mortality rate associated with Acinetobacter infections. In light of this rising threat, research on A. baumannii epidemiology, antibiotic resistance, and pathogenesis is accelerating. The recent development of both in vitro and in vivo models has enabled studies probing the host-Acinetobacter interface. Bacterial genetic screens and comparative genomic studies have led to the identification of several A. baumannii virulence factors. Additionally, investigations into host defense mechanisms using animal models or cell culture have provided insight into the innate immune response to infection. This review highlights some of the key attributes of A. baumannii virulence with an emphasis on bacterial interactions with the innate immune system.
The transcellular entry of E. coli K1 through human brain microvascular endothelial cells (HBMEC) is responsible for tight junction disruption, leading to brain edema in neonatal meningitis. Previous studies demonstrated that outer membrane protein A (OmpA) of E. coli K1 interacts with its receptor, Ecgp96 to induce PKC-α phosphorylation, adherens junction (AJ) disassembly (by dislodging β-catenin from VE-cadherin), and remodeling of actin in HBMEC. We report here that IQGAP1 mediates β-catenin dissociation from AJs to promote actin polymerization required for E. coli K1 invasion of HBMEC. Overexpression of C-terminal truncated IQGAP1 (IQΔC) that cannot bind β-catenin prevents both AJ disruption and E. coli K1 entry. Of note, phospho-PKC-α interacts with the C-terminal portion of Ecgp96 as well as with VE-cadherin after IQGAP1 mediated AJ disassembly. HBMEC overexpressing either C-terminal truncated Ecgp96 (Ecgp96Δ200) or IQΔC upon infection with E. coli showed no interaction of phospho-PKC-α with Ecgp96. These data indicate that the binding of OmpA to Ecgp96 induces PKC-α phosphorylation and association of phospho-PKC-α with Ecgp96, and then signals IQGAP1 to detach β-catenin from AJs. Subsequently, IQGAP1/β-catenin bound actin translocates to the site of E. coli K1 attachment to promote invasion.
Establishment of infection by spotted fever group rickettsial species is dependent on the ability of these bacteria to adhere to and invade the host endothelium. Recent studies have attributed these processes to a handful of rickettsial surface proteins from the surface cell antigen (sca) family of autotransporters. A rickettsial autotransporter from Rickettsia conorii, Sca2, has been shown to be sufficient to mediate both adherence and invasion of human endothelial cells and to participate in intracellular actin-based motility. Here we identify a region of Sca2 capable of interacting with the mammalian cell surface and show that this function of Sca2 is independent and separable from its actin nucleation activity. Furthermore, pre-incubation of mammalian cells with the Sca2 mammalian association region prior to R. conorii infection can competitively inhibit rickettsial invasion, suggesting that Sca2 plays an important role in the initial interaction with mammalian cells. Together our results demonstrate that the Sca2 autotransporter protein in R. conorii contains distinct functional domains that likely are involved in mediating cellular interactions at the plasma membrane and the host cytosol.
In the current investigation, we analyzed all the known small nucleolar RNAs (snoRNAs) in the deeply branching protozoan parasite Giardia lamblia for potential microRNAs (miRNAs) that might be derived from them. Two putative miRNAs have since been identified by Northern blot, primer extension, 3′-RACE and co-immunoprecipitation with Giardia Argonaute (GlAgo), and designated miR6 and miR10. Giardia Dicer (GlDcr) is capable of processing the snoRNAs into the corresponding miRNAs in vitro. Potential miR6 and miR10 binding sites in Giardia genome were predicted bioinformatically. A miR6 binding site was found at the 3′-untranslated regions (UTR) of 44 variant surface protein (vsp) genes, whereas a miR10 binding site was identified at the 3′-end of 159 vsp open-reading frames. Thirty-three of these vsp genes turned out to contain binding sites for both miR6 and miR10. A reporter mRNA tagged with the 3′ end of vsp1267, which contains the target sites for both miRNAs, was translationally repressed by both miRNAs in Giardia. Episomal expression of an N-terminal c-myc tagged VSP1267 was found significantly repressed by introducing either miR6 or miR10 into the cells and the repressive effects were additive. When the 2′-O-methyl antisense oligos (ASOs) of either miR6 or miR10 was introduced, however, there was an enhancement of tagged VSP1267 expression suggesting an inhibition of the repressive effects of endogenous miR6 or miR10 by the ASOs. Of the total 220 vsp genes in Giardia, we have now found 178 of them carrying putative binding sites for all the miRNAs that have been currently identified, suggesting that miRNAs are likely the regulators of VSP expression in Giardia.
The successful navigation of malaria parasites through their life cycle, which alternates between vertebrate hosts and mosquito vectors, requires a complex interplay of metabolite synthesis and salvage pathways. Using the rodent parasite Plasmodium berghei, we have explored the synthesis and scavenging pathways for lipoic acid, a short-chain fatty acid derivative that regulates the activity of α-ketoacid dehydrogenases including pyruvate dehydrogenase. In Plasmodium, lipoic acid is either synthesized de novo in the apicoplast or is scavenged from the host into the mitochondrion. Our data show that sporozoites lacking the apicoplast lipoic acid protein ligase LipB are markedly attenuated in their infectivity for mice, and in vitro studies document a very late liver stage arrest shortly before the final phase of intra-hepatic parasite maturation. LipB-deficient asexual blood stage parasites show unimpaired rates of growth in normal in vitro or in vivo conditions. However, these parasites showed reduced growth in lipid-restricted conditions induced by treatment with the lipoic acid analog 8-bromo-octanoate or with the lipid-reducing agent clofibrate. This finding has implications for understanding Plasmodium pathogenesis in malnourished children that bear the brunt of malarial disease. This study also highlights the potential of exploiting lipid metabolism pathways for the design of genetically attenuated sporozoite vaccines.
Plasmodium berghei; lipoic acid; apicoplast; transfection; clofibrate
Galectin-3 is expressed and secreted by immune cells and has been implicated in multiple aspects of the inflammatory response. It is a glycan binding protein which can exert its functions within cells or exogenously by binding cell surface ligands, acting as a molecular bridge or activating signaling pathways. In addition, this lectin has been shown to bind to microorganisms. In this study we investigated the interaction between galectin-3 and Neisseria meningitidis, an important extracellular human pathogen, which is a leading cause of septicaemia and meningitis. Immunohistochemical analysis indicated that galectin-3 is expressed during meningococcal disease and co-localises with bacterial colonies in infected tissues from patients. We show that galectin-3 binds to N. meningitidis and we demonstrate that this interaction requires full length, intact lipopolysaccharide molecules. We found that neither exogenous nor endogenous galectin-3 contributes to phagocytosis of N. meningitidis; instead exogenous galectin-3 increases adhesion to monocytes and macrophages but not epithelial cells. Finally we used galectin-3 deficient (Gal-3−/−) mice to evaluate the contribution of galectin-3 to meningococcal bacteraemia. We found that Gal3−/−mice manifested significantly lower levels of bacteriaemia compared with wild-type mice after challenge with live bacteria, indicating that galectin-3 confers an advantage to N.meningitidis during systemic infection.
Ileal lesions in Crohn’s disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3 positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non pathogenic, environmental, commensal, or pathogenic strains involved in gastroenteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation.
Crohn’s disease; autophagy; Adherent-Invasive E. coli; ATG16L1; IRGM
Enterotoxigenic Escherichia coli (ETEC) causes human morbidity and mortality in developing nations and is an emerging threat to food safety in developed nations. The ETEC heat-labile enterotoxin (LT) not only causes diarrheal disease by deregulating host adenylate cyclase, but also enhances ETEC adherence to intestinal epithelial cells. The mechanism governing this LT pro-adherence phenotype is unclear. Here we investigated intestinal epithelial cell signal transduction pathways activated by ETEC and quantified the relative importance of these host pathways to LT-induced ETEC adherence. We show that ETEC activates both NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways through mechanisms that are primarily dependent upon LT. LT-induced NF-κB activation depends upon the cAMP-dependent activation of the Ras-like GTPase Rap1 but is independent of protein kinase A (PKA). By using inhibitors of these pathways, we demonstrate that inhibiting the p38 MAPK prevents LT from increasing ETEC adherence. By contrast, the LT pro-adherence phenotype appears unrelated to both LT-induced Rap1 activity and to subsequent NF-κB activation. We speculate that LT may alter host signal transduction to induce the presentation of ligands for ETEC adhesins in such a way that promotes ETEC adherence. Our findings provide insight into previously unexplored functions of LT and their relative importance to ETEC virulence.
Enterotoxigenic Escherichia coli; heat-labile enterotoxin; MAPK; NF-κB; Rap1
Bacillus thuringiensis ssp. israelensis (Bti) has been used worldwide for the control of dipteran insect pests. This bacterium produces several Cry and Cyt toxins that individually show activity against mosquitoes but together show synergistic effect. Previous work demonstrated that Cyt1Aa synergizes the toxic activity of Cry11Aa by functioning as a membrane-bound receptor. In the case of Cry toxins active against lepidopteran insects, receptor interaction triggers the formation of a pre-pore oligomer that is responsible for pore formation and toxicity. In this work we report that binding of Cry11Aa to Cyt1Aa facilitates the formation of a Cry11Aa pre-pore oligomeric structure that is capable of forming pores in membrane vesicles. Cry11Aa and Cyt1A point mutants affected in binding and in synergism had a correlative effect on the formation of Cry11Aa pre-pore oligomer and on pore-formation activity of Cry11Aa. These data further support that Cyt1Aa interacts with Cry11Aa and demonstrate the molecular mechanism by which Cyt1Aa synergizes or suppresses resistance to Cry11Aa, by providing a binding site for Cry11Aa that will result in an efficient formation of Cry11Aa pre-pore that inserts into membranes and forms ionic pores.
Ehrlichia chaffeensis infects monocytes/macrophages and causes human monocytic ehrlichiosis. To determine the role of type IV secretion (T4S) system in infection, candidates for T4S effectors were identified by bacterial two-hybrid screening of E. chaffeensis hypothetical proteins with positively charged C-terminus using E. chaffeensis VirD4 as bait. Of three potential T4S effectors, ECH0825 was highly upregulated early during exponential growth in a human monocytic cell line. ECH0825 was translocated from the bacterium into the host-cell cytoplasm and localized to mitochondria. Delivery of anti-ECH0825 into infected host cells significantly reduced bacterial infection. Ectopically expressed ECH0825 also localized to mitochondria and inhibited apoptosis of transfected cells in response to etoposide treatment. In double transformed yeast, ECH0825 localized to mitochondria and inhibited human Bax-induced apoptosis. Mitochondrial manganese superoxide dismutase (MnSOD) was increased over 9-fold in E. chaffeensis-infected cells, and the amount of reactive oxygen species (ROS) in infected cells was significantly lower than that in uninfected cells. Similarly, MnSOD was upregulated and the ROS level was reduced in ECH0825-transfected cells. These data suggest that, by upregulating MnSOD, ECH0825 prevents ROS-induced cellular damage and apoptosis to allow intracellular infection. This is the first example of host ROS levels linked to a bacterial T4S effector.
Ehrlichia chaffeensis; type IV secretion effector; mitochondria; apoptosis; BAX; ROS; MnSOD
Nucleotide binding-leucine rich repeat (NB-LRR) proteins function as intracellular receptors for the detection of pathogens in both plants and animals. Despite their central role in innate immunity, the molecular mechanisms that govern NB-LRR activation are poorly understood. The Arabidopsis NB-LRR protein RPS5 detects the presence of the Pseudomonas syringae effector protein AvrPphB by monitoring the status of the Arabidopsis protein kinase PBS1. AvrPphB is a cysteine protease that targets PBS1 for cleavage at a single site within the activation loop of PBS1. Using a transient expression system in the plant Nicotiana benthamiana and stable transgenic Arabidopsis plants we found that both PBS1 cleavage products are required to activate RPS5 and can do so in the absence of AvrPphB. We also found, however, that the requirement for cleavage of PBS1 could be bypassed simply by inserting five amino acids at the PBS1 cleavage site, which is located at the apex of the activation loop of PBS1. Activation of RPS5 did not require PBS1kinase function, thus RPS5 appears to sense a subtle conformational change in PBS1, rather than cleavage. This finding suggests that NB-LRR proteins may function as fine-tuned sensors of alterations in the structures of effector targets.
Gametocyte maturation in Plasmodium falciparum is a critical step in the transmission of malaria. While the majority of parasites proliferate asexually in red blood cells, a small fraction of parasites undergo sexual conversion and mature over two weeks to become competent for transmission to a mosquito vector. Immature gametocytes sequester in deep tissues while mature stages must be able to circulate, pass the spleen and present themselves to the mosquito vector in order to complete transmission. Sequestration of asexual red blood cell stage parasites has been investigated in great detail. These studies have demonstrated that induction of cytoadherence properties through specific receptor-ligand interactions coincides with a significant increase in host cell stiffness. In contrast, the adherence and biophysical properties of gametocyte-infected red blood cells have not been studied systematically. Utilizing a transgenic line for 3D live imaging, in vitro capillary assays and 3D finite element whole cell modeling, we studied the role of cellular deformability in determining the circulatory characteristics of gametocytes. Our analysis shows that the red blood cell deformability of immature gametocytes displays an overall decrease followed by rapid restoration in mature gametocytes. Intriguingly, simulations suggest that along with deformability variations, the morphological changes of the parasite may play an important role in tissue distribution in vivo. Taken together we present a model, which suggests that mature but not immature gametocytes circulate in the peripheral blood for uptake in the mosquito blood meal and transmission to another human host thus ensuring long term survival of the parasite.
Chlamydia species are obligate intracellular pathogens that are important causes of human genital tract, ocular, and respiratory infections. The bacteria replicate within a specialized membrane-bound compartment termed the inclusion and require host-derived lipids for intracellular growth and development. Emerging evidence indicates that Chlamydia has evolved clever strategies to fulfill its lipid needs by interacting with multiple host cell compartments and redirecting trafficking pathways to its intracellular niche. In this review, we highlight recent findings that have significantly expanded our understanding of how Chlamydia exploit lipid trafficking pathways to ensure the survival of this important human pathogen.
Erythrocytes infected with malaria parasites have increased permeability to diverse organic and inorganic solutes. While these permeability changes have been known for decades, the molecular basis of transport was unknown and intensively debated. CLAG3, a parasite protein previously thought to function in cytoadherence, has recently been implicated in formation of the plasmodial surface anion channel (PSAC), an unusual small conductance ion channel that mediates uptake of most solutes. Consistent with transport studies, the clag genes are conserved in all plasmodia but are absent from other genera. The encoded protein is integral to the host membrane, as also predicted by electrophysiology. An important question is whether functional channels are formed by CLAG3 alone or through interactions with other proteins. In either case, gene identification should advance our understanding of parasite biology and may lead to new therapeutics.
The IRG protein Irgm3 preserves cell survival during coxsackievirus B3 (CVB3) infection. However, the molecular mechanisms are not clear. Here, we examined the effect of Irgm3 expression on ER stress triggered by pharmacological agents or CVB3 infection. In Tet-On/Irgm3 HeLa cells, Irgm3 expression suppressed either chemical- or CVB3-induced upregulation of glucose-regulated protein78. Further, Irgm3 strongly inhibited the activation of both the PERK and ATF6 pathways of ER stress responses, which further led to the diminished phosphorylation of eIF2α, reduced cleavage/activation of transcription factor SREBP1 and attenuated induction of proapoptotic genes CHOP and GADD34. These data were further supported by experiments using Irgm3 knockout mouse embryonic fibroblasts, in which the ER stress induced by CVB3 was not relieved due to the lack of Irgm3 expression. In addition, the tunicamycin-triggered ER stress promoted the subsequent CVB3 infection. The effect of Irgm3 on ER stress and CVB3 infection was diminished by the PI3K inhibitor, LY294002, while inhibitors of ERK, JNK and p38 had no effect. These data were further corroborated by transfection of cells with a dominant negative Akt. Taken together, these data suggest that Irgm3 relieves the ER stress response via a PI3K/Akt dependent mechanism, which contributes to host defense against CVB3 infection.
Due to its unique chemical properties, nitric oxide (NO) is a pluripotent signaling and effector molecule that is implicated in a variety of biological roles. Although NO is known to function in host innate immunity against pathogen invasion, its possible roles in microbial symbioses with animal and plant hosts remain relatively less well defined. In this review, we discuss the mechanisms by which bacteria sense and/or detoxify NO. We then focus specifically on its roles in microbial symbioses of diverse eukaryotic hosts. Using the squid-vibrio light-organ symbiosis as a well-characterized example, we discuss the ways in which NO serves as a signal, antioxidant and specificity determinant in this model symbiosis. Because beneficial microbial associations are older and much more prevalent than pathogenic ones, it seems likely that the former may be evolutionary precursors of the latter. Thus, knowledge of the roles played by NO in mutualisms will provide insights into its function in disease interactions as well.
Argonaute (Ago) plays a central role in RNA interference in metazoans, but its status in lower organisms remains ill-defined. We report on the Ago complex of the unicellular protozoan, Toxoplasma gondii (Tg), an obligatory pathogen of mammalian hosts. The PIWI-like domain of TgAgo lacked the canonical DDE/H catalytic triad, explaining its weak target RNA cleavage activity. However, TgAgo associated with a stronger RNA slicer, a Tudor staphylococcal nuclease (TSN), and with a protein Arg methyl transferase, PRMT1. Mutational analysis suggested that the N-terminal RGG-repeat domain of TgAgo was methylated by PRMT1, correlating with the recruitment of TSN. The slicer activity of TgAgo was Mg2+-dependent and required perfect complementarity between the guide RNA and the target. In contrast, the TSN activity was Ca2+-dependent and required an imperfectly paired guide RNA. Ago knockout parasites showed essentially normal growth, but in contrast, the PRMT1 knockouts grew abnormally. Chemical inhibition of Arg-methylation also had an anti-parasitic effect. These results suggest that the parasitic PRMT1 plays multiple roles, and its loss affects the recruitment of a more potent second slicer to the parasitic RNA silencing complex, the exact mechanism of which remains to be determined.
The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization.