Host Cell Growth Research Articles

Human CAP cells represent a novel source for functional, miRNA-loaded exosome production.

Exosomes represent a promising delivery tool for nucleic acid-based pharmaceuticals. They are highly suitable for transporting therapeutic miRNAs to tumor cells, due to their natural membrane components. Further, exosomes are capable of effectively protecting nucleic acids against ribonucleases and enable the delivery of their content through cell membranes. However, no suitable production host for miRNA containing exosomes of non-tumorigenic origin has yet been identified. In this study we engineered an immortalised human amniocyte cell line (CAP® cells), whose exosomes were enriched and characterised. The cell line modifications not only enabled the production of GFP-labelled but also pro-apoptotic miRNA containing exosomes without negative influence on host cell growth. Furthermore, we demonstrated that pro-apoptotic miRNA containing CAP exosomes are taken up by ovarian cancer cells. Strikingly, delivery of functional exosomal miRNA led to downregulation of several reported target genes in the treated tumor cells. In summary, we revealed CAP cells of non-tumorigenic origin as a novel and efficient exosome production host with the potential to produce functional miRNA-loaded exosomes.

Uneven host cell growth causes lysogenic virus induction in the Baltic Sea.

In the Baltic Sea redoxcline, lysogenic viruses infecting prokaryotes have rarely been detected using the commonly used inducing agent mitomycin C. However, it is well known that not all viruses are induceable by mitomycin C and growing evidence suggests that changes in trophic conditions may trigger the induction of lysogenic viruses. We hypothesized that using antibiotics to simulate a strong change in trophic conditions for antibiotica-resistant cells due to reduced competition for resources might lead to the induction of lysogenic viruses into the lytic cycle within these cells. This hypothesis was tested by incubating prokaryotes obtained throughout the Baltic Sea redoxcline in seawater with substantially reduced numbers of viruses. We used a mixture of the protein synthesis-inhibiting antibiotics streptomycin and erythromycin to induce the desired changes in trophic conditions for resistant cells and at the same time ensuring that no progeny viruses were formed in sensitive cells. No inducible lysogenic viruses could be detected in incubations amended with mitomycin C. Yet, the presence of streptomycin and erythromycin increased virus-induced mortality of prokaryotes by 56–930% compared to controls, resulting in the induction of lysogenic viruses equivalent to 2–14% of in situ prokaryotic abundance. The results indicate the existence of a previously unrecognized induction mechanism for lysogenic viruses in the Baltic Sea redoxcline, as the mode of action distinctly differs between the used antibiotics (no virus production within affected cells) and mitomycin C (lysogenic viruses are produced within affected cells). Obtaining accurate experimental data on levels of lysogeny in prokaryotic host cells remains challenging, as relying on mitomycin C alone may severely underestimate lysogeny.

Advances in researches on mechanism of macrophage migration inhibitory factor regulating parasite-host immune interaction

Macrophage migration inhibitory factor (MIF), a type of pleiotropic immunoregulatory cytokine with a specific structure, participates in the regulation of host cell growth and migration and immune responses. Following parasitic infections, hosts may produce MIF and then participate in the parasite-host interactions. In addition, parasites may secrete parasite-derived MIF, and they jointly participate in parasite-host interactions. This paper reviews the regulation of MIF gene expression following parasitic infections, the role of MIF in parasite-host immune system interactions, and important signaling pathways of MIF-mediated immune responses.

Bacterial quorum-sensing signal IQS induces host cell apoptosis by targeting POT1-p53 signalling pathway.

Pseudomonas aeruginosa, an opportunistic life-threatening human bacterial pathogen, employs quorum-sensing (QS) signal molecules to modulate virulence gene expression. 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde (IQS) is a recently identified QS signal that integrates the canonical lasR-type QS of P. aeruginosa and host phosphate stress response to fine-tune its virulence production for a successful infection. To address the role of IQS in pathogen-host interaction, we here present that IQS inhibits host cell growth and stimulates apoptosis in a dosage-dependent manner. By downregulating the telomere-protecting protein POT1 in host cells, IQS activates CHK1, CHK2, and p53 in an Ataxia telangiectasia mutated (ATM)/ATM and RAD3-related (ATR)-dependent manner and induces DNA damage response. Overexpression of POT1 in host cells presents a resistance to IQS treatment. These results suggest a pivotal role of IQS in host apoptosis, highlighting the complexity of pathogenesis mechanisms developed by P. aeruginosa during infection.

Mutation of a Staphylococcus aureus temperate bacteriophage to a virulent one and evaluation of its application

Bacteriophages have been suggested as alternative antimicrobial agents based on their host specificity and lytic activity. Therefore, it is necessary to obtain a virulent phage from a temperate one using molecular techniques to control Staphylococcus aureus efficiently. SA13, a novel temperate phage infecting S. aureus, was isolated and characterized. From this phage, mutant phages were generated by random deletion mutations, and a virulent mutant phage SA13m was selected. Comparative genome analysis revealed that the SA13m genome contains various nucleotide deletions in six genes encoding three hypothetical proteins and three lysogeny-associated proteins, including putative integrase, putative CI, and putative anti-repressor proteins. Mitomycin C induction of SA13m-resistant strains revealed that this mutant phage does not form lysogen, suggesting that SA13m is a virulent phage. In addition, SA13m showed rapid and long-lasting host cell growth inhibition activity. Furthermore, application of SA13m in sterilized milk showed that S. aureus was reduced to non-detectable levels both at refrigerator temperature (4 °C) and room temperature (25 °C), suggesting that SA13m can efficiently control the growth of S. aureus in foods. The virulent mutant phage SA13m could be used as a promising biocontrol agent against S. aureus without lysogen formation.

Dipenyleneiodonium Induces Growth Inhibition of Toxoplasma gondii through ROS Induction in ARPE-19 Cells.

Based on the reactive oxygen species (ROS) regulatory properties of diphenyleneiodonium (DPI), we investigated the effects of DPI on host-infected T. gondii proliferation and determined specific concentration that inhibit the intracellular parasite growth but without severe toxic effect on human retinal pigment epithelial (ARPE-19) cells. As a result, it is observed that host superoxide, mitochondria superoxide and H2O2 levels can be increased by DPI, significantly, followed by suppression of T. gondii infection and proliferation. The involvement of ROS in anti-parasitic effect of DPI was confirmed by finding that DPI effect on T. gondii can be reversed by ROS scavengers, N-acetyl-L-cysteine and ascorbic acid. These results suggest that, in ARPE-19 cell, DPI can enhance host ROS generation to prevent T. gondii growth. Our study showed DPI is capable of suppressing T. gondii growth in host cells while minimizing the un-favorite side-effect to host cell. These results imply that DPI as a promising candidate material for novel drug development that can ameliorate toxoplasmosis based on ROS regulation.

A possible alternative for chromosome dimer resolution in E. coli

Dimer formation is a serious threat to the stable maintenance of ColE1-like plasmids. Dimers form infrequently by homologous recombination but accumulate rapidly by having two origins of replication. This results in elevated plasmid loss and a reduction in host cell growth rate. Plasmid dimers are resolved to monomers by the XerCD recombinase plus accessory proteins ArgR and PepA, acting at the cer recombination site. The circular chromosome of E. coli also forms dimers infrequently, and consequent failure of chromosome partition results in filamentation, SOS induction, and failure of cell division. Site-specific recombination is required for dimer resolution during cell division in a process facilitated by XerCD acting at the dif (deletion induced filamentation) site near the E. coli chromosome terminus. ArgR and PepA accessory proteins are nonessential, but the septum-associated protein FtsK is necessary for dimer segregation, suggesting the XerCD/difcomplex interacts with division septums. Our preliminary work had revealed homology between cer and a 170 bp chromosomal site (tcs, terminal region cer-like site) 1.2 min from dif. The tcs site and surrounding region was cloned into plasmids, dimer plasmids were purified and then assayed for tcs-mediated recombination. Our results demonstrate that a construct with a 500 bp tcs insert supports XerCD-mediated recombination, whereas smaller constructs were recombination-deficient. The absence of plasmid monomerization in mutant strains indicates that ArgR and PepA are required for recombination. Additionally, the tcs knockout strain displayed moderate filamentation of cells. Given the similarities between cer, tcs, and dif, these results suggest that tcs could facilitate chromosome dimer resolution.

Avian Intestinal Mucus Modulates Campylobacter jejuni Gene Expression in a Host-Specific Manner.

Campylobacter jejuni is a leading cause of bacterial foodborne illness in humans worldwide. However, C. jejuni naturally colonizes poultry without causing pathology where it resides deep within mucus of the cecal crypts. Mucus may modulate the pathogenicity of C. jejuni in a species-specific manner, where it is pathogenic in humans and asymptomatic in poultry. Little is known about how intestinal mucus from different host species affects C. jejuni gene expression. In this study we characterized the growth and transcriptome of C. jejuni NCTC11168 cultured in defined media supplemented with or without mucus isolated from avian (chicken or turkey) or mammalian (cow, pig, or sheep) sources. C. jejuni showed substantially improved growth over defined media, with mucus from all species, showing that intestinal mucus was an energy source for C. jejuni. Seventy-three genes were differentially expressed when C. jejuni was cultured in avian vs. mammalian mucus. Genes associated with iron acquisition and resistance to oxidative stress were significantly increased in avian mucus. Many of the differentially expressed genes were flanked by differentially expressed antisense RNA asRNA, suggesting a role in gene regulation. This study highlights the interactions between C. jejuni and host mucus and the impact on gene expression, growth and invasion of host cells, suggesting important responses to environmental cues that facilitate intestinal colonization.IMPORTANCE Campylobacter jejuni infection of humans is an important health problem world-wide and is the leading bacterial cause of foodborne illnesses in U.S. The main route for exposure for humans is consumption of poultry meat contaminated during processing. C. jejuni is frequently found in poultry, residing within the mucus of the intestinal tract without causing disease. It is not clear why C. jejuni causes disease in some animals and humans, while leaving birds without symptoms. To understand its activity in birds, we characterized C. jejuni responses to poultry mucus to identify genes turned on in the intestinal tract of birds. We identified genes important for colonization and persistence within the poultry gut, turned on when C. jejuni was exposed to poultry mucus. Our findings are an important step in understanding how C. jejuni responds and interacts in the poultry gut, and may identify ways to reduce C. jejuni in birds.

The Multifaceted Roles of Autophagy in Flavivirus-Host Interactions.

Autophagy is an evolutionarily conserved cellular process in which intracellular components are eliminated via lysosomal degradation to supply nutrients for organelle biogenesis and metabolic homeostasis. Flavivirus infections underlie multiple human diseases and thus exert an immense burden on public health worldwide. Mounting evidence indicates that host autophagy is subverted to modulate the life cycles of flaviviruses, such as hepatitis C virus, dengue virus, Japanese encephalitis virus, West Nile virus and Zika virus. The diverse interplay between autophagy and flavivirus infection not only regulates viral growth in host cells but also counteracts host stress responses induced by viral infection. In this review, we summarize the current knowledge on the role of autophagy in the flavivirus life cycle. We also discuss the impacts of virus-induced autophagy on the pathogeneses of flavivirus-associated diseases and the potential use of autophagy as a therapeutic target for curing flavivirus infections and related human diseases.

Proto-oncogenes in a eukaryotic unicellular organism play essential roles in plasmodial growth in host cells

BackgroundThe eukaryotic unicellular protist Plasmodiophora brassicae is an endocellular parasite of cruciferous plants. In host cortical cells, this protist develops a unicellular structure that is termed the plasmodium. The plasmodium is actually a multinucleated cell, which subsequently splits and forms resting spores. The mechanism for the growth of this endocellular parasite in host cell is unclear.ResultsHere, combining de novo genome sequence and transcriptome analysis of strain ZJ-1, we identified top five significant enriched KEGG pathways of differentially expressed genes (DEGs), namely translation, cell growth and death, cell communication, cell motility and cancers. We detected 171 proto-oncogenes from the genome of P. brassicae that were implicated in cancer-related pathways, of which 46 were differential expression genes. Three predicted proto-oncogenes (Pb-Raf1, Pb-Raf2, and Pb-MYB), which showed homology to the human proto-oncogenes Raf and MYB, were specifically activated during the plasmodial growth in host cortical cells, demonstrating their involvement in the multinucleate development stage of the unicellular protist organism. Gene networks involved in the tumorigenic-related signaling transduction pathways and the activation of 12 core genes were identified. Inhibition of phosphoinositol-3-kinase relieved the clubroot symptom and significantly suppressed the development process of plasmodia.ConclusionsProto-oncogene-related regulatory mechanisms play an important role in the plasmodial growth of P. brassicae.

TOR-autophagy branch signaling via Imp1 dictates plant-microbe biotrophic interface longevity.

Like other intracellular eukaryotic phytopathogens, the devastating rice blast fungus Magnaporthe (Pyricularia) oryzae first infects living host cells by elaborating invasive hyphae (IH) surrounded by a plant-derived membrane. This forms an extended biotrophic interface enclosing an apoplastic compartment into which fungal effectors can be deployed to evade host detection. M. oryzae also forms a focal, plant membrane-rich structure, the biotrophic interfacial complex (BIC), that accumulates cytoplasmic effectors for translocation into host cells. Molecular decision-making processes integrating fungal growth and metabolism in host cells with interface function and dynamics are unknown. Here, we report unanticipated roles for the M. oryzae Target-of-Rapamycin (TOR) nutrient-signaling pathway in mediating plant-fungal biotrophic interface membrane integrity. Through a forward genetics screen for M. oryzae mutant strains resistant to the specific TOR kinase inhibitor rapamycin, we discovered IMP1 encoding a novel vacuolar protein required for membrane trafficking, V-ATPase assembly, organelle acidification and autophagy induction. During infection, Δimp1 deletants developed intracellular IH in the first infected rice cell following cuticle penetration. However, fluorescently labeled effector probes revealed that interface membrane integrity became compromised as biotrophy progressed, abolishing the BIC and releasing apoplastic effectors into host cytoplasm. Growth between rice cells was restricted. TOR-independent autophagy activation in Δimp1 deletants (following infection) remediated interface function and cell-to-cell growth. Autophagy inhibition in wild type (following infection) recapitulated Δimp1. In addition to vacuoles, Imp1GFP localized to IH membranes in an autophagy-dependent manner. Collectively, our results suggest TOR-Imp1-autophagy branch signaling mediates membrane homeostasis to prevent catastrophic erosion of the biotrophic interface, thus facilitating fungal growth in living rice cells. The significance of this work lays in elaborating a novel molecular mechanism of infection stressing the dominance of fungal metabolism and metabolic control in sustaining long-term plant-microbe interactions. This work also has implications for understanding the enigmatic biotrophy to necrotrophy transition.

Interstrain transfer of the prophage ϕNM2 in staphylococcal strains

Staphylococcus aureus is a successful pathogen in part because the bacterium can adapt rapidly to selective pressures imparted by the external environment. Horizontal gene transfer (HGT) plays an integral role in the evolution of bacterial genomes, and phage transduction is likely to be the most common and important HGT mechanism for S. aureus. Phage can transfer not only its own genome DNA but also host bacterial DNA with or without pathogenicity islands to other bacteria. Here, we demonstrate that the staphylococcal prophage ϕNM2 could transfer between strains Newman and NCTC8325/NCTC8325-4 by simulating a natural situation in laboratory without mitomycin C or ultra-violet light treatment. This transference may be caused by direct contact between Newman and NCTC8325/NCTC8325-4 instead of phage particles released in Newman culture’s supernatant. The rates of successful horizontal genetic transfer in recipients NCTC8325 and NCTC8325-4 were 2.1% and 1.8%, respectively. Prophage ϕNM2 was integrated with one direction at an intergenic region between rpmF and isdB in all 17 lysogenic isolates. Phage particles were spontaneously released from lysogenic strains again and had no noticeable influence on the growth of host cells. The results reported herein provide insight into how mobile genetic elements such as prophages can lead to the emergence of genetic diversity among S. aureus strains.

Metabolic constraints on Magnaporthe biotrophy: loss of de novo asparagine biosynthesis aborts invasive hyphal growth in the first infected rice cell.

The blast fungus Magnaporthe oryzae devastates global rice yields and is an emerging threat to wheat. Determining the metabolic strategies underlying M. oryzae growth in host cells could lead to the development of new plant protection approaches against blast. Here, we targeted asparagine synthetase (encoded by ASN1), which is required for the terminal step in asparagine production from aspartate and glutamine, the sole pathway to de novo asparagine biosynthesis in M. oryzae. Consequently, the Δasn1 mutant strains could not grow on minimal media without asparagine supplementation. Spores harvested from supplemented plates could form appressoria and penetrate rice leaf surfaces, but biotrophic growth was aborted and the Δasn1 strains were nonpathogenic. This work provides strong genetic evidence that de novo asparagine biosynthesis, and not acquisition from the host, is a critical and potentially exploitable metabolic strategy employed by M. oryzae in order to successfully colonize rice cells.

Leishmania infection activates host mTOR for its survival by M2macrophage polarization.

Mammalian target of rapamycin (mTOR) is a central regulator of growth and immunity of host cells. It's involvement in cancer and tuberculosis is well documented but least explored in Leishmania donovani invasion of host cells. Therefore, in the present study, we aimed to investigate the role of mTOR in M2 macrophage polarization for Leishmania survival. We observed that Leishmania infection activated host mTOR pathway characterized by phosphorylation of mTOR, 70S6K and 4-EBP1. Inhibition of mTOR resulted in decreased parasite load and percent infectivity. Moreover, Leishmania infection triggered cell proliferation as was evidenced by increased expression of cyclin A and p-RPS6. mTOR activation during Leishmania infection resulted in reduced expression of M1 macrophage markers (eg, ROS, NO, iNOS, NOX-1, IL-12, IL-1β and TNF-α), and increased expression of M2 macrophage markers (eg, arginase-1, IL-10, TGF-β, CD206 and CD163). Furthermore, we observed that in case of Leishmania infection, mTOR inhibition increased the translocation of NF-κB to nucleus and deactivation of STAT-3. Eventually, we observed that inhibition of M2 macrophage polarization reduced Leishmania survival inside macrophages. Therefore, our findings suggest that mTOR plays a crucial role in regulation of M2 macrophage polarization and direct the innate immune homeostasis towards parasite survival inside host.

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method

We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth

Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins.

Tissue engineering in pediatric urology - a critical appraisal.

Tissue engineering is defined as the combination of biomaterials and bioengineering principles together with cell transplantation or directed growth of host cells to develop a biological replacement tissue or organ that can be a substitute for normal tissue both in structure and function. Despite early promising preclinical studies, clinical translation of tissue engineering in pediatric urology into humans has been unsuccessful both for cell-seeded and acellular scaffolds. This can be ascribed to various factors, including the use of only non-diseased models that inaccurately describe the structural and functional modifications of diseased tissue. The paper addresses potential future strategies to overcome the limitations experienced in clinical applications so far. This includes the use of stem cells of various origins (mesenchymal stem cells, hematopoietic stem/progenitor cells, urine-derived stem cells, and progenitor cells of the urothelium) as well as the need for a deeper understanding of signaling pathways and directing tissue ingrowth and differentiation through the concept of dynamic reciprocity. The development of smart scaffolds that release trophic factors in a set and timely manner will probably improve regeneration. Modulation of innate immune response as a major contributor to tissue regeneration outcome is also addressed. It is unlikely that only one of these strategies alone will lead to clinically applicable tissue engineering strategies in pediatric urology. In the meanwhile, the fundamental new insights into regenerative processes already obtained in the attempts of tissue engineering of the lower urogenital tract remain our greatest gain.

Introgression and gene family contraction drive the evolution of lifestyle and host shifts of hypocrealean fungi

ABSTRACTHypocrealean fungi (Ascomycota) are known for their diversity of lifestyles. Their vital influences on agricultural and natural ecosystems have resulted in a number of sequenced genomes, which provide essential data for genomic analysis. Totally, 45 hypocrealean fungal genomes constructed a phylogeny. The phylogeny showed that plant pathogens in Nectriaceae diverged earliest, followed by animal pathogens in Cordycipitaceae, Ophiocordycipitaceae and Clavicipitaceae with mycoparasites in Hypocreaceae. Insect/nematode pathogens and grass endophytes in Clavicipitaceae diverged at last. Gene families associated with host-derived nutrients are significantly contracted in diverged lineages compared with the ancestral species. Introgression was detected in certain lineages of hypocrealean fungi, and the main functions of the genes located in the introgressed regions are involved in host recognition, transcriptional regulation, stress response and cell growth regulation. These results indicate that contraction of gene families and introgression might be main mechanisms to drive lifestyle differentiation and evolution and host shift of hypocrealean fungi.

Metal TiO2 Nanotube Layers for the Treatment of Dental Implant Infections.

Titanium oxide nanotube layers with silver and zinc nanoparticles are attracting increasing attention in the design of bone and dental implants due to their antimicrobial potential and their ability to control host cell adhesion, growth, and differentiation. However, recent reports indicate that the etiology of dental infections is more complex than has been previously considered. Therefore, the antimicrobial potential of dental implants should be evaluated against at least several different microorganisms cooperating in human mouth colonization. In this study, Ag and Zn nanoparticles incorporated into titanium oxide nanotubular layers were studied with regard to how they affect Candida albicans, Candida parapsilosis, and Streptococcus mutans. Layers of titanium oxide nanotubes with an average diameter of 110 nm were fabricated by electrochemical anodization, annealed at 650 °C, and modified with approx. 5 wt % Ag or Zn nanoparticles. The surfaces were examined with the scanning electron microscopy-energy dispersive X-ray analysis, scanning transmission electron microscopy, and X-ray photoelectron spectroscopy techniques and subjected to evaluation of microbial-killing and microbial adhesion-inhibiting potency. In a 1.5 h long adhesion test, the samples were found more effective toward yeast strains than toward S. mutans. In a release-killing test, the microorganisms were almost completely eliminated by the samples, either within 3 h of contact (for S. mutans) or 24 h of contact (for both yeast strains). Although further improvement is advisable, it seems that Ag and Zn nanoparticles incorporated into TiO2 nanotubular surfaces provide a powerful tool for reducing the incidence of bone implant infections. Their high bidirectional activity (against both Candida species and S. mutans) makes the layers tested particularly promising for the design of dental implants.

T91. DEVELOPMENT OF NOVEL BIS-AMIDINES FOR THE TREATMENT OF TOXOPLASMOSIS

BackgroundToxoplasma infections constitute a worldwide public health problem responsible for significant morbidity. Symptoms of acute infection by the agent, the apicomplexan parasite Toxoplasma gondii, can range from mild to life-threatening depending upon the immune status of the host. Toxoplasma infections are often unrecognized in immune competent hosts but infection can lead to the long-term establishment of cysts in the brain and thus a persistent infection. Such tissue cysts are associated with altered behavior and cognition in mouse models. Furthermore, serological evidence of Toxoplasma infection has been associated with an increased risk of recent onset psychosis, schizophrenia, and other psychiatric disorders in humans. Adjunct treatment of Toxoplasma-seropositive individuals afflicted with schizophrenia with effective anti-parasitic medications could be beneficial by eliminating the parasite cysts, thereby possibly alleviating psychiatric symptoms. However, currently available medications are ineffective against the cyst form of the organism responsible for persistent infection. Others have published the anti-Toxoplasma potential of derivatives of the bis-amidine pentamidine. We designed and synthesized a panel of novel bis-amidines and explored their anti-Toxoplasma efficacy.MethodsWe designed and synthesized an array of twelve novel bis-amidines. These compounds were examined for in vitro activity against T. gondii tachyzoites first using a colorimetric assay employing the β-gal producing strain RH-2F to examine the effect of the compounds over 5 days of parasite growth in human fibroblast host cells. This was followed by several immunofluorescence-based assays to determine if compounds can directly act on tachyzoites (red/green invasion assay), on an established infection of host cells (replication assay), and whether the compounds are parasiticidal, ie. can rid the host cells of tachyzoite infection after one dose (recovery assay). A rodent model of acute toxoplasmosis was established for examining in vivo efficacy of lead compounds.ResultsFour of these compounds proved highly efficacious in vitro with 50% inhibitory (IC50) values ranging from 200 nM to 1.3 µM. Therapeutic indices based on the ratio between the median cell cytotoxic dose and the IC50 ranged from 4 to 84. These four compounds all inhibited tachyzoite invasion and significantly inhibited in vitro replication over a 24-hour period at nanomolar concentrations. Additionally, two of the compounds evaluated thus far appear to be parasiticidal in vitro at 1 - 2 µM.DiscussionOur results suggest that bis-amidines can be designed to be effective against experimental Toxoplasma infections. The parasiticidal activity of some of the compounds make them serious candidates for further drug development. Further experiments to determine 1) in vivo efficacy in mouse models of persistent infection, and 2) synergy with antipsychotics and mood stabilizers will investigate the possibility that these compounds could be used as adjunct treatment in Toxoplasma-positive individuals suffering with schizophrenia and other psychiatric disorders.