Dynamics of parasite growth in genetically diverse Plasmodium falciparum isolates.

Multiple parasite lineages with different proliferation rates or fitness may coexist within a clinical malaria isolate, resulting in complex growth interactions and variations in phenotype. To elucidate the dynamics of parasite growth in multiclonal isolates, we measured growth rates (GRs) of three Plasmodium falciparum Cambodian isolates, including IPC_3445 (MRA-1236), IPC_5202 (MRA-1240), IPC_6403 (MRA-1285), and parasite lineages previously cloned from each of these isolates by limiting dilution. Following synchronization, in vitro cultures of each parasite line were maintained over four consecutive asexual cycles (192 h), with thin smears prepared at each 48-h cycle to estimate GR and fold change in parasitemia (FCP). Cell cycle time (CCT), the duration it takes for ring-stage parasites to develop into mature schizonts, was measured by monitoring the development of 0-3-h post-invasion rings for up to 52 h post-incubation. Laboratory lines 3D7 (MRA-102) and Dd2 (MRA-150) were used as controls. Significant differences in GR, FCP, and CCT were observed between parasite isolates and clonal lineages from each isolate. The parasite lines studied here have well-defined growth phenotypes and will facilitate basic malaria research and development of novel malaria interventions. These lines are available to malaria researchers through the MR4 collection of NIAID's BEI Resources Program.

A Simple Alcohol-based Method of Oocyst Inactivation for Use in the Development of Detection Assays for Cryptosporidium.

Cryptosporidium spp. are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against Cryptosporidium oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in Cryptosporidium assay development is limited. The aims of this study were to evaluate methods of inactivation of Cryptosporidium oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on C. parvum oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, in vitro excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells in vitro. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of Cryptosporidium in spiked water samples. In summary, we have established a practical approach to inactivate C. parvum oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.

Babesia microti Immunoreactive Rhoptry-Associated Protein-1 Paralogs Are Ancestral Members of the Piroplasmid-Confined RAP-1 Family.

Babesia, Cytauxzoon and Theileria are tick-borne apicomplexan parasites of the order Piroplasmida, responsible for diseases in humans and animals. Members of the piroplasmid rhoptry-associated protein-1 (pRAP-1) family have a signature cysteine-rich domain and are important for parasite development. We propose that the closely linked B. microti genes annotated as BMR1_03g00947 and BMR1_03g00960 encode two paralogue pRAP-1-like proteins named BmIPA48 and Bm960. The two genes are tandemly arranged head to tail, highly expressed in blood stage parasites, syntenic to rap-1 genes of other piroplasmids, and share large portions of an almost identical ~225 bp sequence located in their 5' putative regulatory regions. BmIPA48 and Bm960 proteins contain a N-terminal signal peptide, share very low sequence identity (<13%) with pRAP-1 from other species, and harbor one or more transmembrane domains. Diversification of the piroplasmid-confined prap-1 family is characterized by amplification of genes, protein domains, and a high sequence polymorphism. This suggests a functional involvement of pRAP-1 at the parasite-host interface, possibly in parasite adhesion, attachment, and/or evasion of the host immune defenses. Both BmIPA48 and Bm960 are recognized by antibodies in sera from humans infected with B. microti and might be promising candidates for developing novel serodiagnosis and vaccines.

Evaluation of pathogen specific urinary peptides in tick-borne illnesses.

Mass spectrometry enhanced by nanotechnology can achieve previously unattainable sensitivity for characterizing urinary pathogen-derived peptides. We utilized mass spectrometry enhanced by affinity hydrogel particles (analytical sensitivity = 2.5 pg/mL) to study tick pathogen-specific proteins shed in the urine of patients with (1) erythema migrans rash and acute symptoms, (2) post treatment Lyme disease syndrome (PTLDS), and (3) clinical suspicion of tick-borne illnesses (TBI). Targeted pathogens were Borrelia, Babesia, Anaplasma, Rickettsia, Ehrlichia, Bartonella, Francisella, Powassan virus, tick-borne encephalitis virus, and Colorado tick fever virus. Specificity was defined by 100% amino acid sequence identity with tick-borne pathogen proteins, evolutionary taxonomic verification for related pathogens, and no identity with human or other organisms. Using a cut off of two pathogen peptides, 9/10 acute Lyme Borreliosis patients resulted positive, while we identified zero false positive in 250 controls. Two or more pathogen peptides were identified in 40% of samples from PTLDS and TBI patients (categories 2 and 3 above, n = 59/148). Collectively, 279 distinct unique tick-borne pathogen derived peptides were identified. The number of pathogen specific peptides was directly correlated with presence or absence of symptoms reported by patients (ordinal regression pseudo-R2 = 0.392, p = 0.010). Enhanced mass spectrometry is a new tool for studying tick-borne pathogen infections.

Proteomic analysis reveals pathogen-derived biomarkers of acute babesiosis in erythrocytes, plasma, and urine of infected hamsters.

Babesiosis among humans is on the rise in North America. Current diagnostic assays for the screening of babesiosis require blood collection by venipuncture, which is an invasive method. Urine on the other hand is a desirable biospecimen for biomarker analysis of Babesia microti infections because it can be collected periodically and non-invasively. Our group uses a new class of biomarker harvesting nanocage technology, which, when combined with mass spectrometry (MS), can determine the presence of parasite proteins shed in different bodily fluids of mammalian hosts, including urine. Using the hamster model of babesiosis, our nanoparticle-MS approach identified several B. microti proteins in erythrocytes, plasma, and urine samples. Surface and secreted antigens previously shown to elicit host immune responses against the parasite were particularly abundant in erythrocytes and plasma compared to other proteins. Two of these antigens, BmSA1 and BMR1_03g00947, showed different localization patterns by immunofluorescence of infected erythrocytes. Hamster urine samples from parasitemic animals harbored lower numbers of B. microti proteins compared to erythrocytes and plasma, with glycolytic enzymes, cytoskeletal components, and chaperones being the most frequently detected proteins. By applying novel nanoparticle-MS methods, a high level of analytical sensitivity can be achieved to detect multiple B. microti proteins in blood and urine. This is generally difficult to obtain with other techniques due to the masking of parasite biomarkers by the complex biomolecular matrix of bodily fluids from the host.

Update on BEI Resources for Parasitology and Arthropod Vector Research.

BEI Resources has contributed to the advancement of parasitic diseases research for over 16 years. The accessibility of our reference strains and reagents is relevant to the development of new therapeutics and vaccines. Here we provide a resource update with emphasis on the new assets for toxoplasmosis and vector research.

Analysis of the Babesia microti proteome in infected red blood cells by a combination of nanotechnology and mass spectrometry.

Proteomics of Babesia microti has lagged behind other apicomplexans despite recent genome and transcriptome studies. Here, we used a combination of nanotechnology and mass spectrometry to provide a proteomic profile of B. microti acute infection. We identified ∼500 parasite proteins in blood with functions such as transport, carbohydrate and energy metabolism, proteolysis, DNA and RNA metabolism, signaling, translation, lipid biosynthesis, and motility and invasion. We also identified surface antigens with roles in the immune response to the parasite. This first evaluation of the B. microti proteome in erythrocytes provides information for the study of intracellular survival and development of diagnostic tools using mass spectrometry.

Development of droplet digital PCR for the detection of Babesia microti and Babesia duncani.

Babesia spp. are obligate protozoan parasites of red blood cells. Transmission to humans occurs through bites from infected ticks or blood transfusion. Infections with B. microti account for the majority of the reported cases of human babesiosis in the USA. A lower incidence is caused by the more recently described species B. duncani. The current gold standard for detection of Babesia is microscopic examination of blood smears. Recent PCR-based assays, including real-time PCR, have been developed for B. microti. On the other hand, molecular assays that detect and distinguish between B. microti and B. duncani infections are lacking. Closely related species of Babesia can be differentiated due to sequence variation within the internal transcribed spacer (ITS) regions of nuclear ribosomal RNAs. In the present study, we targeted the ITS regions of B. microti and B. duncani to develop sensitive and species-specific droplet digital PCR (ddPCR) assays. The assays were shown to discriminate B. microti from B. duncani and resulted in limits of detection of ~10 gene copies. Moreover, ddPCR for these species were useful in DNA extracted from blood of experimentally infected hamsters, detecting infections of low parasitemia that were negative by microscopic examination. In summary, we have developed sensitive and specific quantitative ddPCR assays for the detection of B. microti and B. duncani in blood. Our methods could be used as sensitive approaches to monitor the progression of parasitemia in rodent models of infection as well as serve as suitable molecular tests in blood screening.

Microbotryozyma collariae gen. nov., sp. nov., a basidiomycetous yeast isolated from a plant bug Collaria oleosa (Miridae).

Two strains of a basidiomycetous yeast were derived from an insect trypanosomatid culture isolated from the intestine of a plant bug, Collaria oleosa (Heteroptera: Miridae), collected in Costa Rica. The yeast did not form ballistoconidia but reproduced only by budding. Teliospores were not observed in individual and crossed cultures of each strain. Morphological and other taxonomic characteristics of the yeast were similar to those of the species in the polyphyletic genus Rhodotorula. However, molecular phylogeny inferred from the internal transcribed spacers and D1/D2 region of the large subunit rRNA gene showed that the strains represent a new species placed among the smut fungi in the family Ustilentylomataceae, which includes Aurantiosporium subnitens, Fulvisporium restifaciens, Ustilentyloma fluitans, and Rhodotorula hordea. Given the well distinguished phylogenetic position of this novel species within the Ustilentylomataceae, we propose Microbotryozyma collariae gen. nov., sp. nov. to accommodate the yeast isolated from C. oleosa, with strain American Type Culture Collection MYA-4666(T) (= PRA303-1S = CBS 12537) designated as the type strain.

Subulatomonas tetraspora nov. gen. nov. sp. is a member of a previously unrecognized major clade of eukaryotes.

While a large number of aerobic free-living protists have been described within the last decade, the number of new anaerobic or microaerophilic microbial eukaryotic taxa has lagged behind. Here we describe a microaerophilic genus and species of amoeboflagellate isolated from a near-shore marine site off the coast at Plymouth, Massachusetts: Subulatomonas tetraspora nov. gen. nov. sp. This taxon is closely related to Breviata anathema based on both microscopical features and phylogenetic analyses of sequences of three genes: SSU-rDNA, actin, and alpha-tubulin. However, Subulatomonas tetraspora nov. gen. nov. sp. and B. anathema are morphologically distinctive, differ by 14.9% at their SSU-rDNA locus, and were isolated from marine and 'slightly brackish' environments, respectively. Phylogenetic analyses of these two taxa plus closely related sequences from environmental surveys provide support for a novel clade of eukaryotes that is distinct from the major clades including the Opisthokonta, Excavata, Amoebozoa and 'SAR' (Stramenopile, Alveolate, Rhizaria).

Evidence for a previously unrecognized mycobacterial endosymbiont in Acanthamoeba castellanii strain Ma (ATCC ® 50370 ™).

We describe the isolation of a mycobacterium from Acanthamoeba castellanii strain Ma (ATCC(®) 50370(™)). The mycobacterium resides within vacuoles of A. castellanii, can be cultured by routine methodologies, and is a member of the Mycobacterium avium complex. Previously unrecognized mycobacterial endosymbionts are likely common among strains of Acanthamoeba housed at culture collections.

Infection with Toxoplasma gondii results in dysregulation of the host cell cycle.

Mammalian cells infected with Toxoplasma gondii are characterized by a profound reprogramming of gene expression. We examined whether such transcriptional responses were linked to changes in the cell cycle of the host. Human foreskin fibroblasts (HFFs) in the G(0)/G(1) phase of the cell cycle were infected with T. gondii and FACS analysis of DNA content was performed. Cell cycle profiles revealed a promotion into the S phase followed by an arrest towards the G(2)/M boundary with infection. This response was markedly different from that of growth factor stimulation which caused cell cycle entry and completion. Transcriptional profiles of T. gondii-infected HFF showed sustained increases in transcripts associated with a G(1)/S transition and DNA synthesis coupled to an abrogation of cell cycle regulators critical in G(2)/M transition relative to growth factor stimulation. These divergent responses correlated with a distinct temporal modulation of the critical cell cycle regulator kinase ERK by infection. While the kinetics of ERK phosphorylation by EGF showed rapid and sustained activation, infected cells displayed an oscillatory pattern of activation. Our results suggest that T. gondii infection induces and maintains a 'proliferation response' in the infected cell which may fulfill critical growth requirements of the parasite during intracellular residence.

The apicomplexan pathogen Neospora caninum inhibits host cell apoptosis in the absence of discernible NF-kappa B activation.

Neospora caninum, a causative agent of bovine abortions, is an apicomplexan parasite that is closely related to the human pathogen Toxoplasma gondii. Since a number of intracellular parasites, including T. gondii, have been shown to modulate host cell apoptosis, the present study was conducted to establish whether N. caninum is similarly capable of subverting apoptotic pathways in its host cells. Our results indicated that death receptor-mediated apoptosis is repressed during N. caninum infection, and the data further showed that the executioner caspase, caspase 3, does not become activated in the infected cells. Surprisingly, nuclear translocation of the NF-kappaB subunit p65 was not detected in N. caninum-infected cells, although this host transcription factor has been shown to upregulate prosurvival genes in cells infected with T. gondii. Consistent with these findings, the distinct accumulation of phosphorylated IkappaB that is seen at the parasitophorous vacuole membrane (PVM) of T. gondii was not apparent on the N. caninum PVM. Although a putative IkappaB kinase activity was detected in N. caninum extracts, thereby implying that this parasite is capable of modulating NF-kappaB translocation into the host cell nucleus, the data collectively suggest that a profound and sustained activation of the NF-kappaB pathway is not central to the ability of N. caninum to prevent apoptosis of their host cells.

Host and parasite-derived IKK activities direct distinct temporal phases of NF-kappaB activation and target gene expression following Toxoplasma gondii infection.

Activation of NF-kappaB by the intracellular pathogen Toxoplasma gondii is associated with the localization of phosphorylated IkappaB alpha to the parasitophorous vacuole membrane (PVM). This is mediated by a parasite-derived IkappaB kinase (TgIKK) activity and is independent of host IKK function. In the present study, we examined the roles of host IKK and parasite-derived TgIKK on the temporal modulation of NF-kappaB activation. Despite the presence of TgIKK activity at the PVM, nuclear translocation of NF-kappaB and subsequent gene expression exhibited a requirement for the host IKK complex. A detailed kinetic analysis of NF-kappaB activation revealed a biphasic, hierarchical and temporally regulated response. We propose a novel paradigm for the modulation of NF-kappaB-dependent gene expression by T. gondii that involves both the host IKK complex and TgIKK activity at different phases of infection. Thus, T. gondii effectively alters gene expression in a temporal dimension by exploiting the NF-kappaB signaling machinery and subsequently rewiring the activation circuits of the infected host cell.

Detection of a novel parasite kinase activity at the Toxoplasma gondii parasitophorous vacuole membrane capable of phosphorylating host IkappaBalpha.

Toxoplasma gondii activates the NF-kappaB pathway in the infected host cell resulting in upregulation of pro-survival genes and prevention of apoptosis. Manipulation of the NF-kappaB cascade by T. gondii correlates with the localization of phosphorylated IkappaB at the parasitophorous vacuole membrane (PVM). This suggests a parasite-mediated event, involving the recruitment and activation of the host IkappaB kinase (IKK) complex, as has been observed with the related protozoan Theileria parva. In contrast to Theileria, confocal microscopy studies showed no apparent hijacking of IKKalpha, IKKbeta, or their activated phosphorylated forms at the T. gondii PVM. Remarkably, phosphorylation of IkappaBalpha at Ser 32/36 was observed at the PVM of T. gondii-infected IKKalpha-/-, IKKbeta-/- and IKKalpha/beta double-knockout (IKKalpha/beta-/-) fibroblasts, suggesting the involvement of a parasite kinase activity independent of host IKK. The presence of a putative T. gondii IkappaB kinase was examined by in vitro kinase assays using GST-IkappaBalpha constructs and protein extracts from both extracellular parasites and PVM fractions. Interestingly, an activity capable of phosphorylating IkappaBalpha at the critical Ser 32/36 sites was identified in parasite extracts, a property restricted to the IKK signalosome. Taken together, our data support the role for a T. gondii kinase involved in phosphorylation of host cell IkappaBalpha and suggest an unusual mechanism utilized by an intracellular pathogen capable of manipulating the NF-kappaB pathway.

Effects of fluoroquinolones on the migration of human phagocytes through Chlamydia pneumoniae-infected and tumor necrosis factor alpha-stimulated endothelial cells.

The anti-inflammatory activities of three quinolones, levofloxacin, moxifloxacin, and gatifloxacin, were investigated with an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVEC) were seeded in Transwell inserts, treated with serial dilutions of antibiotics, infected with Chlamydia pneumoniae, or stimulated with tumor necrosis factor alpha (TNF-alpha). Neutrophils or monocytes were also preincubated with serial dilutions of each antibiotic. TEM was assessed by light microscopic examination of the underside of the polycarbonate membrane, and levels of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) were measured by enzyme-linked immunosorbent assay. In HUVEC infected with C. pneumoniae or stimulated with TNF-alpha, all fluoroquinolones significantly decreased neutrophil and monocyte TEM, compared to antibiotic-free controls. Moxifloxacin and gatifloxacin produced a significant decrease in IL-8 in C. pneumoniae-infected and TNF-alpha-stimulated HUVEC; however, moxifloxacin was the only fluoroquinolone that produced a significant decrease in MCP-1 levels under both conditions. Results from this study indicate similarities in the anti-inflammatory activities of these fluoroquinolones, although no statistically significant decrease in chemokine secretion was observed when levofloxacin was used. Mechanisms of neutrophil and monocyte TEM inhibition by fluoroquinolone antibiotics are unknown but may be partially due to inhibition of IL-8 and MCP-1 production, respectively.

Activation of NF-kappaB by Toxoplasma gondii correlates with increased expression of antiapoptotic genes and localization of phosphorylated IkappaB to the parasitophorous vacuole membrane.

Mammalian cells infected with Toxoplasma gondii are resistant to apoptosis induced by a variety of stimuli. We have demonstrated that the host transcription factor NF-kappaB plays a pivotal role in the T.-gondii-mediated blockade of apoptosis because inhibition is lost in cells lacking the p65 (RelA) subunit of NF-kappaB (p65-/-). In the present study, we examined the effects of T. gondii infection on NF-kappaB activation and the expression of genes involved in the apoptotic cascade. Infection of wild-type mouse embryonic fibroblasts (MEFs) with T.-gondii-induced nuclear translocation of the p50 and p65 subunits of NF-kappaB as examined by immunoblotting of nuclear extracts, immunofluorescence and electrophoretic mobility shift assays. A comparison of apoptotic gene expression profiles from wild-type and p65-/- MEFs revealed distinct patterns of induction in response to T. gondii infection. In particular, the differences seen in the Bcl-2 and IAP families are consistent with the antiapoptotic responses observed in the resistant wild-type cells compared with the sensitive p65-/- fibroblasts. Consistent with NF-kappaB activation, T. gondii infection promoted phosphorylation of the inhibitor IkappaB. Interestingly, phosphorylated IkappaB was concentrated on the parasitophorous vacuole membrane (PVM), suggesting a parasite-directed event. Results from this study suggest that activation of NF-kappaB plays an important role in stimulation of antiapoptotic gene expression by T. gondii. Furthermore, recruitment of phosphorylated IkappaB to the PVM implies the presence of intrinsic factor(s) in T. gondii that might be used to manipulate the NF-kappaB signaling pathway in the host to elicit a survival response during infection.

Inhibition of caspase activation and a requirement for NF-kappaB function in the Toxoplasma gondii-mediated blockade of host apoptosis.

Mammalian cells infected with the protozoan parasite Toxoplasma gondii are resistant to many apoptotic stimuli transmitted along both the mitochondrial and death receptor pathways. Apoptosis, and its inhibition in infected cells, was examined using multiple morphological, molecular and biochemical approaches. The data strongly indicate manipulation of the host apoptotic machinery at multiple levels, focusing on the inhibition of host caspases. Activation of the pro-apoptotic caspase family of proteases is a biochemical hallmark of apoptosis. Caspase activation occurs in a highly ordered cascade triggered by the initiator caspases 8 and 9, which activate the executioner caspase, caspase 3. Our findings indicate a profound blockade of caspase activation and activity as the molecular basis for the inhibition of apoptosis in T.-gondii-infected cells. Caspase inhibition was demonstrated using multiple intrinsic and synthetic substrates. Although the specific inhibitory molecule remains to be identified, data indicate an absolute requirement for the host transcription factor NF-kappaB and, by extension, genes regulated by it. We propose that T. gondii activates the host survival response, thereby increasing the overall resistance of infected cells to apoptotic stimuli.

Effect of macrolide antibiotics on human endothelial cells activated by Chlamydia pneumoniae infection and tumor necrosis factor-alpha.

This study investigated the potential anti-inflammatory activity of 3 macrolide antibiotics, clarithromycin, roxithromycin, and azithromycin, in an in vitro model of transendothelial migration (TEM). Human umbilical vein endothelial cells (HUVECs) were seeded in Transwell inserts, treated with serial dilutions of the antibiotics, and infected with Chlamydia pneumoniae or stimulated with tumor necrosis factor (TNF)-alpha. In HUVECs infected with C. pneumoniae or stimulated with TNF-alpha, both azithromycin and roxithromycin caused significant decreases in neutrophil and monocyte TEM, compared with antibiotic-free controls. Clarithromycin had no detectable effect in either group. Azithromycin caused significant decreases in interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1, whereas roxithromycin significantly decreased IL-8. This study indicates heterogeneity in the anti-inflammatory activity of these antibiotics. Mechanisms of monocyte and neutrophil TEM inhibition by azithromycin and roxithromycin are unclear but may be partially due to inhibition of IL-8 and MCP-1 production.