Synthesis of trypanosome hsp70 mRNA is resistant to disruption of trans-splicing by heat shock

Synthesis of mRNA in trypanosomes involves an apparent trans-splicing reaction whereby a common 39-nucleotide mini-exon sequence is joined to the protein-coding exon of a mRNA precursor. We have previously shown (Muhich, M. L., and Boothroyd, J. C. (1988) Mol. Cell. Biol. 8, 3837-3846) that the trans-splicing pathway of Trypanosoma brucei is sensitive to disruption by severe heat shock. Here we demonstrate that the synthesis of heat shock protein 70 (hsp 70) mRNA in T. brucei is apparently resistant to the heat-induced disruption of splicing. The 5'-ends of hsp 70 mRNAs are shown to be identical for molecules synthesized at either normal or heat shock temperatures, and in both cases, the 5'-terminal mini-exon sequence is present. These results suggest that T. brucei has evolved a mechanism which directly compensates for the deleterious effects of heat shock on trans-splicing, one which allows for the continued mini-exon-dependent trans-splicing of selected pre-mRNAs.     

Post‐release dispersal and spawning movements of a translocated lake sturgeon (Acipenser fulvescens,Rafinesque 1817) population in the Mattagami River,Ontario

Species translocations are increasingly being used as a management tool to mitigate population losses due to such factors as habitat degradation and fragmentation, but post‐introduction follow‐up is relatively sparse. Post‐translocation telemetry can assess success by identifying activity, emigrations, survival, habitat usage, and reproductive events, aiding in the continued management of translocated populations and informing future efforts. This study assessed movement of translocated adult lake sturgeon (Acipenser fulvescens) immediately post‐release and a decade later, and tested for associations between environmental variables and spawning movements. Prior to their translocation in 2002, 13 of 51 adult lake sturgeon were surgically implanted with radio telemetry tags and tracked for 1 year. In 2011 and 2013, eight additional adults were captured within the reintroduction site and implanted with radio‐tags. Six of the 13 sturgeon tagged in 2002 dispersed downstream over a dam during the early post‐release period. In spring 2014, tagged adults were tracked to the spillway at the release area's inflow, and spawning was confirmed by larval captures. Movement data for tagged adults differed between the two tracking periods, showing marked differences in behaviour over time. Water velocity was correlated with upstream and downstream spawning movements, with water temperature also correlated with downstream movement. Research regarding post‐translocation movement and dispersal provides insight on behavioural responses following translocation, and may improve outcomes by informing future efforts.     

Identification of methylation markers for the prediction of nodal metastasis in oral and oropharyngeal squamous cell carcinoma

Hypermethylation is an important mechanism for the dynamic regulation of gene expression, necessary for metastasizing tumour cells. Our aim is to identify methylation tumour markers that have a predictive value for the presence of regional lymph node metastases in patients with oral and oropharyngeal squamous cell carcinoma (OOSCC). Significantly differentially expressed genes were retrieved from four reported microarray expression profiles comparing pN0 and pN+ head-neck tumours, and one expression array identifying functionally hypermethylated genes. Additional metastasis-associated genes were included from the literature. Thus genes were selected that influence the development of nodal metastases and might be regulated by methylation. Methylation-specific PCR (MSP) primers were designed and tested on 8 head-neck squamous cell carcinoma cell lines and technically validated on 10 formalin-fixed paraffin-embedded (FFPE) OOSCC cases. Predictive value was assessed in a clinical series of 70 FFPE OOSCC with pathologically determined nodal status. Five out of 28 methylation markers (OCLN, CDKN2A, MGMT, MLH1 and DAPK1) were frequently differentially methylated in OOSCC. Of these, MGMT methylation was associated with pN0 status (P = 0.02) and with lower immunoexpression (P = 0.02). DAPK1 methylation was associated with pN+ status (P = 0.008) but did not associate with protein expression. In conclusion, out of 28 candidate genes, two (7%) showed a predictive value for the pN status. Both genes, DAPK1 and MGMT, have predictive value for nodal metastasis in a clinical group of OOSCC. Therefore DNA methylation markers are capable of contributing to diagnosis and treatment selection in OOSCC. To efficiently identify additional new methylation markers, genome-wide methods are needed.     

Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication

Apicomplexan parasites exhibit an unusual mechanism of host cell penetration. A central player in this process is the protein apical membrane antigen 1 (AMA1). Although essential for invasion, the precise functional roles AMA1 plays have been unclear. Several recent studies have provided important functional insight into its role within the multiprotein complex that comprises the moving junction (MJ). Initially formed at the apical tip of the invading parasite, the MJ represents a ring-like region of contact between the surfaces of the invading parasite and the host cell as the invaginated host plasma membrane is forced inward by the penetrating parasite. This review discusses these and other recent insights into AMA1 with particular emphasis on studies conducted in Plasmodium and Toxoplasma.     

Toxoplasma polymorphic effectors determine macrophage polarization and intestinal inflammation

European and North American strains of the parasite Toxoplasma gondii belong to three distinct clonal lineages, type I, type II, and type III, which differ in virulence. Understanding the basis of Toxoplasma strain differences and how secreted effectors work to achieve chronic infection is a major goal of current research. Here we show that type I and III infected macrophages, a cell type required for host immunity to Toxoplasma, are alternatively activated, while type II infected macrophages are classically activated. The Toxoplasma rhoptry kinase ROP16, which activates STAT6, is responsible for alternative activation. The Toxoplasma dense granule protein GRA15, which activates NF-κB, promotes classical activation by type II parasites. These effectors antagonistically regulate many of the same genes, and mice infected with type II parasites expressing type I ROP16 are?protected against Toxoplasma-induced ileitis. Thus, polymorphisms in determinants that modulate?macrophage activation influence the ability of Toxoplasma to establish a chronic infection.     

Toxoplasma Co-opts Host Cells It Does Not Invade

Like many intracellular microbes, the protozoan parasite Toxoplasma gondii injects effector proteins into cells it invades. One group of these effector proteins is injected from specialized organelles called the rhoptries, which have previously been described to discharge their contents only during successful invasion of a host cell. In this report, using several reporter systems, we show that in vitro the parasite injects rhoptry proteins into cells it does not productively invade and that the rhoptry effector proteins can manipulate the uninfected cell in a similar manner to infected cells. In addition, as one of the reporter systems uses a rhoptry:Cre recombinase fusion protein, we show that in Cre-reporter mice infected with an encysting Toxoplasma-Cre strain, uninfected-injected cells, which could be derived from aborted invasion or cell-intrinsic killing after invasion, are actually more common than infected-injected cells, especially in the mouse brain, where Toxoplasma encysts and persists. This phenomenon has important implications for how Toxoplasma globally affects its host and opens a new avenue for how other intracellular microbes may similarly manipulate the host environment at large.     

A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins

The ability of mice to resist infection with the protozoan parasite, Toxoplasma gondii, depends in large part on the function of members of a complex family of atypical large GTPases, the interferon-gamma-inducible immunity-related GTPases (IRG proteins). Nevertheless, some strains of T. gondii are highly virulent for mice because, as recently shown, they secrete a polymorphic protein kinase, ROP18, from the rhoptries into the host cell cytosol at the moment of cell invasion. Depending on the allele, ROP18 can act as a virulence factor for T. gondii by phosphorylating and thereby inactivating mouse IRG proteins. In this article we show that IRG proteins interact not only with ROP18, but also strongly with the products of another polymorphic locus, ROP5, already implicated as a major virulence factor from genetic crosses, but whose function has previously been a complete mystery. ROP5 proteins are members of the same protein family as ROP18 kinases but are pseudokinases by sequence, structure, and function. We show by a combination of genetic and biochemical approaches that ROP5 proteins act as essential co-factors for ROP18 and present evidence that they work by enforcing an inactive GDP-dependent conformation on the IRG target protein. By doing so they prevent GTP-dependent activation and simultaneously expose the target threonines on the switch I loop for phosphorylation by ROP18, resulting in permanent inactivation of the protein. This represents a novel mechanism in which a pseudokinase facilitates the phosphorylation of a target by a partner kinase by preparing the substrate for phosphorylation, rather than by upregulation of the activity of the kinase itself.