IS free-range pork better and safer to eat than conventional pork? Many consumers think so. The well-publicized horrors of intensive pig farming have fostered the widespread assumption that, as one purveyor of free-range meats put it, “the health benefits are indisputable.” However, as yet another reminder that culinary wisdom is never conventional, scientists have found that free-range pork can be more likely than caged pork to carry dangerous bacteria and parasites. It’s not only pistachios and 50-pound tubs of peanut paste that have been infected with salmonella but also 500-pound pigs allowed to root and to roam pastures happily before butting heads with a bolt gun. The study published in the journal Foodborne Pathogens and Disease that brought these findings to light last year sampled more than 600 pigs in North Carolina, Ohio and Wisconsin. It discovered not only higher rates of salmonella in free-range pigs (54 percent versus 39 percent) but also greater levels of the pathogen toxoplasma (6.8 percent versus 1.1 percent) and, most alarming, two free-range pigs that carried the parasite trichina (as opposed to zero for confined pigs). For many years, the pork industry has been assuring cooks that a little pink in the pork is fine. Trichinosis, which can be deadly, was assumed to be history.

Apicomplexan parasites, including Plasmodium falciparum and Toxoplasma gondii (the causative agents of malaria and toxoplasmosis, respectively), are responsible for considerable morbidity and mortality worldwide. These pathogenic protozoa replicate within an intracellular vacuole inside of infected host cells, from which they must escape to initiate a new lytic cycle. By integrating cell biological, pharmacological, and genetic approaches, we provide evidence that both Plasmodium and Toxoplasma hijack host cell calpain proteases to facilitate parasite egress. Immunodepletion or inhibition of calpain-1 in hypotonically lysed and resealed erythrocytes prevented the escape of P. falciparum parasites, which was restored by adding purified calpain-1. Similarly, efficient egress of T. gondii from mammalian fibroblasts was blocked by either small interfering RNA–mediated suppression or genetic deletion of calpain activity and could be restored by genetic complementation.

Researchers at the University of Pennsylvania have discovered that parasites hijack host-cell proteins to ensure their survival and proliferation, suggesting new ways to control the diseases they cause. The study, appearing this week online in Science, was led by Doron Greenbaum, PhD, Assistant Professor of Pharmacology in the Penn School of Medicine. “Researchers can now develop ways to kill parasites by placing roadblocks in the path they use to destroy their victims,” says Greenbaum. The team discovered that malaria parasites depend upon an enzyme stolen from the host cell for successful infection. Historically, many researchers have focused on developing ways to keep parasites from entering host cells, but Greenbaum’s group was curious about an alternative route of attack: locking the parasites inside the host cell. These studies began with Plasmodium falciparum, which causes the most deadly form of human malaria. Each year, the Centers for Disease Control and Prevention report 350–500 million cases of malaria occur worldwide, killing more than a million people. In collaboration with the laboratory of Penn biologist David Roos, PhD, the work was broadened to include Toxoplasma gondii, which causes a parasitic disease called toxoplasmosis, the leading cause of birth defects worldwide and harmful to people with compromised immune systems. The CDC estimates more than 60 million people living in the U.S. carry T. gondii. “We always suspected that enzymes called proteases might be required to help parasites escape from the infected cell, but had assumed that these enzymes were produced by the parasites themselves. We had never considered that parasites might instead hijack host cell proteases. It's an ingenious system,” says Greenbaum. “Our findings open up whole new window for drug discovery.” “This work is a triumph of integrative science, combining modern techniques in chemistry, biology, genetics, pharmacology, and genomics," says Roos, the E. Otis Kendal Professor of Biology and Ellison Medical Foundation Senior Scholar of Global Infectious Diseases. Collaborations between the Greenbaum and Roos laboratories have been facilitated by proximity, as these researchers are housed in adjacent space, under the auspices of the Penn Genome Frontiers Institute. Because Plasmodium and Toxoplasma kill infected cells, they must constantly hop from cell to cell to survive. When parasites burst out of an infected cell, they leave a mess behind, shredding the dense meshwork of proteins comprising the host cell cytoskeleton and breaking the cell apart, causing cell death. But researchers were unsure what proteins the parasites were using as tools to help them break through the walls of the cell. To observe the behavior of P. falciparum parasites, the team infected human red blood cells, using pharmacological and biochemical evidence to discover that parasites activate the host protease calpain-1. Blocking or removing calpain-1, a calcium regulated protease, left parasites trapped inside the host cell. By adding calpain-1 back into the cell, parasites were able to once again blast free.

Macrophages infected with the opportunistic protozoan Toxoplasma gondii are unable to up-regulate many proinflammatory cytokine genes, including TNF (TNF-alpha), upon stimulation with LPS and other TLR ligands. In this study, we examined the influence of T. gondii on transcription factors associated with TNF-alpha transcription, as well as phosphorylation and acetylation of histone H3 at distal and proximal regions of the TNF-alpha promoter. During LPS stimulation, we found that Toxoplasma blocks nuclear accumulation of transcription factor c-Jun, but not that of cAMP response element-binding protein or NF-kappaB. However, chromatin immunoprecipitation studies revealed that binding of all of these transcription factors to the TNF promoter was decreased by T. gondii infection. Furthermore, the parasite blocked LPS-induced Ser(10) phosphorylation and Lys(9)/Lys(14) acetylation of histone H3 molecules associated with distal and proximal regions of the TNF-alpha promoter. Our results show that Toxoplasma inhibits TNF-alpha transcription by interfering with chromatin remodeling events required for transcriptional activation at the TNF promoter, revealing a new mechanism by which a eukaryotic pathogen incapacitates proinflammatory cytokine production during infection.

Toll-like receptor (TLR) signaling in macrophages is required for antipathogen responses, including the biosynthesis of nitric oxide from arginine, and is essential for immunity to Mycobacterium tuberculosis, Toxoplasma gondii and other intracellular pathogens. Here we report a 'loophole' in the TLR pathway that is advantageous to these pathogens. Intracellular pathogens induced expression of the arginine hydrolytic enzyme arginase 1 (Arg1) in mouse macrophages through the TLR pathway. In contrast to diseases dominated by T helper type 2 responses in which Arg1 expression is greatly increased by interleukin 4 and 13 signaling through the transcription factor STAT6, TLR-mediated Arg1 induction was independent of the STAT6 pathway. Specific elimination of Arg1 in macrophages favored host survival during T. gondii infection and decreased lung bacterial load during tuberculosis infection.

Cardiolipin is a mitochondria-specific phospholipid known to be intimately involved with apoptosis. However, the lack of appropriate cellular models to date restricted analysis of its role in cell death. The maturation of cardiolipin requires the transacylase tafazzin, which is mutated in the human disorder Barth syndrome. Using Barth syndrome patient-derived cells and HeLa cells in which tafazzin was knocked down, we show that cardiolipin is required for apoptosis in the type II mitochondria-dependent response to Fas stimulation. Cardiolipin provides an anchor and activating platform for caspase-8 translocation to, and embedding in, the mitochondrial membrane, where it oligomerizes and is further activated, steps that are necessary for an efficient type II apoptotic response.

It has been 100 years since Toxoplasma gondii was initially described in Tunis by Nicolle and Manceaux (1908) in the tissues of the gundi (Ctenodoactylus gundi) and in Brazil by Splendore (1908) in the tissues of a rabbit. T. gondii is a ubiquitous, Apicomplexan parasite of warm-blooded animals that can cause several clinical syndromes including encephalitis, chorioretinitis and congenital infection. Due to the extensive repertoire of applicable experimental techniques available for this pathogen it has become a model organism for the study of intracellular pathogens. Data obtained from genome-wide expression studies, including ChIP on chip and proteomics surveys, are refining our understanding of the genetic networks involved in the developmental biology of this pathogen as well as the interactions of the parasite with its host. This review addresses recent advances in our understanding of the developmental biology and host–pathogen relationships of T. gondii.

Although the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning-laser microscopy to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We found that neutrophils formed both small, transient and large, persistent swarms via a coordinated migration pattern. We provided evidence that cooperative action of neutrophils and parasite egress from host cells could trigger swarm formation. Neutrophil swarm formation coincided in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses.

The parasite Toxoplasma gondii replicates in a specialized intracellular vacuole and causes disease in many species. Protection from toxoplasmosis is mediated by CD8+ T cells, but the T. gondii antigens and host genes required for eliciting protective immunity are poorly defined. Here we identified GRA6, a polymorphic protein secreted in the parasitophorous vacuole, as the source of the immunodominant and protective decapeptide HF10 presented by the H-2Ld major histocompatibility complex class I molecule. Presentation of the HF10–H-2Ld ligand required proteolysis by ERAAP, the endoplasmic reticulum aminopeptidase associated with antigen processing. Consequently, expansion of protective CD8+ T cell populations was impaired in T. gondii–infected ERAAP-deficient mice, which were more susceptible to toxoplasmosis. Thus, endoplasmic reticulum proteolysis is critical for eliciting protective immunity to a vacuolar parasite.