Overlapping and distinct roles of CDPK family members in the pre-erythrocytic stages of the rodent malaria parasite, Plasmodium berghei.

Invasion of hepatocytes by Plasmodium sporozoites initiates the pre-erythrocytic step of a malaria infection. Subsequent development of the parasite within hepatocytes and exit from them is essential for starting the disease-causing erythrocytic cycle. Identification of signaling pathways that operate in pre-erythrocytic stages provides insight into a critical step of infection and potential targets for chemoprotection from malaria. We demonstrate that P. berghei homologs of Calcium Dependent Protein Kinase 1 (CDPK1), CDPK4 and CDPK5 play overlapping but distinct roles in sporozoite invasion and parasite egress from hepatocytes. All three kinases are expressed in sporozoites. All three are required for optimal motility of sporozoites and consequently their invasion of hepatocytes. Increased cGMP can compensate for the functional loss of CDPK1 and CDPK5 during sporozoite invasion but cannot overcome loss of CDPK4. CDPK1 and CDPK5 expression is downregulated after sporozoite invasion. CDPK5 reappears in a subset of late stage liver stages and is present in all merosomes. Chemical inhibition of CDPK4 and depletion of CDPK5 in liver stages implicate these kinases in the formation and/or release of merosomes from mature liver stages. Furthermore, depletion of CDPK5 in merosomes significantly delays initiation of the erythrocytic cycle without affecting infectivity of hepatic merozoites. These data suggest that CDPK5 may be required for the rupture of merosomes. Our work provides evidence that sporozoite invasion requires CDPK1 and CDPK5, and suggests that CDPK5 participates in the release of hepatic merozoites.

A Plasmodium homolog of ER tubule-forming proteins is required for parasite virulence.

Reticulon and REEP family of proteins stabilize the high curvature of endoplasmic reticulum (ER) tubules. Plasmodium berghei Yop1 (PbYop1) is a REEP5 homolog in Plasmodium. Here, we characterize its function using a gene-knockout (Pbyop1∆). Pbyop1∆ asexual stage parasites display abnormal ER architecture and an enlarged digestive vacuole. The erythrocytic cycle of Pbyop1∆ parasites is severely attenuated and the incidence of experimental cerebral malaria is significantly decreased in Pbyop1∆-infected mice. Pbyop1∆ sporozoites have reduced speed, are slower to invade host cells but give rise to equal numbers of infected HepG2 cells, as WT sporozoites. We propose that PbYOP1's disruption may lead to defects in trafficking and secretion of a subset of proteins required for parasite development and invasion of erythrocytes. Furthermore, the maintenance of ER morphology in different parasite stages is likely to depend on different proteins.

Antimalarial proteasome inhibitor reveals collateral sensitivity from intersubunit interactions and fitness cost of resistance.

We describe noncovalent, reversible asparagine ethylenediamine (AsnEDA) inhibitors of the Plasmodium falciparum proteasome (Pf20S) ß5 subunit that spare all active subunits of human constitutive and immuno-proteasomes. The compounds are active against erythrocytic, sexual, and liver-stage parasites, against parasites resistant to current antimalarials, and against P. falciparum strains from patients in Africa. The ß5 inhibitors synergize with a ß2 inhibitor in vitro and in mice and with artemisinin. P. falciparum selected for resistance to an AsnEDA ß5 inhibitor surprisingly harbored a point mutation in the noncatalytic ß6 subunit. The ß6 mutant was resistant to the species-selective Pf20S ß5 inhibitor but remained sensitive to the species-nonselective ß5 inhibitors bortezomib and carfilzomib. Moreover, resistance to the Pf20S ß5 inhibitor was accompanied by increased sensitivity to a Pf20S ß2 inhibitor. Finally, the ß5 inhibitor-resistant mutant had a fitness cost that was exacerbated by irradiation. Thus, used in combination, multistage-active inhibitors of the Pf20S ß5 and ß2 subunits afford synergistic antimalarial activity with a potential to delay the emergence of resistance to artemisinins and each other.

Human cytomegalovirus in cancer: the mechanism of HCMV-induced carcinogenesis and its therapeutic potential.

Cancer is one of the leading causes of death worldwide. Human cytomegalovirus (HCMV), a well-studied herpesvirus, has been implicated in malignancies derived from breast, colorectal muscle, brain, and other cancers. Intricate host-virus interactions are responsible for the cascade of events that have the potential to result in the transformed phenotype of normal cells. The HCMV genome contains oncogenes that may initiate these types of cancers, and although the primary HCMV infection is usually asymptomatic, the virus remains in the body in a latent or persistent form. Viral reactivation causes severe health issues in immune-compromised individuals, including cancer patients, organ transplants, and AIDS patients. This review focuses on the immunologic mechanisms and molecular mechanisms of HCMV-induced carcinogenesis, methods of HCMV treatment, and other studies. Studies show that HCMV DNA and virus-specific antibodies are present in many types of cancers, implicating HCMV as an important player in cancer progression. Importantly, many clinical trials have been initiated to exploit HCMV as a therapeutic target for the treatment of cancer, particularly in immunotherapy strategies in the treatment of breast cancer and glioblastoma patients. Taken together, these findings support a link between HCMV infections and cellular growth that develops into cancer. More importantly, HCMV is the leading cause of birth defects in newborns, and infection with HCMV is responsible for abortions in pregnant women.

A chemical method for generating live-attenuated, replication-defective DNA viruses for vaccine development.

The development of a chemically attenuated, replication-incompetent virus vaccine can provide protection against diseases caused by DNA viruses. In this study, we have developed a method to produce live-attenuated, replication-defective viruses using centanamycin (CM), a chemical compound that alkylates the A-T-rich minor groove of the DNA and thereby blocks DNA replication. We tested the efficacy of CM to produce live-attenuated, replication-defective human cytomegalovirus, mouse cytomegalovirus, and herpes simplex virus-2 (HSV-2), suggesting a broad application for generating live-attenuated, replication-defective DNA viruses. Mass spectrometry analysis showed that CM alkylate viral DNA at the adenine-N3 position. Moreover, mice immunization with CM-attenuated mouse cytomegalovirus (MCMV) produced a robust immune response and reduced the viral load in immunized animals against challenges with live, wild-type MCMV. Our study offers a unifying and attractive therapeutic opportunity that chemically attenuated live DNA viruses can be readily developed as new frontline vaccines.

Establishment of a stable transfection method in Babesia microti and identification of a novel bidirectional promoter of Babesia microti.

Babesia microti, an emerging human pathogen, is primarily transmitted through a bite of an infected tick and blood transfusions in human. Stable transfection technique has been reported in many protozoan parasites over the past few years. However, in vivo transient and stable transfection method has not been established for Babesia microti. Here, for the first time, we present a method of transient as well as stable transfection of the Babesia microti (B. microti) in the in vivo conditions. We have identified a novel promoter of B. microti. We also demonstrated that Plasmodium berghei DHFR promoter is recognized and functional in B. microti. We show that BM-CTQ41297 promoter control the expression of two genes, which are present on either side and thus represents a bi-functional promoter in B. microti. The predicted promoter activity values using Promoter 2.0 program is higher for BM- CTQ41297 promoter than strong promoters such as ß-actin, ef-1ß, and many other promoters. Furthermore, we discovered a non-essential locus for the genetic manipulation of the parasite, allowing us to stably integrate foreign genes; GFP, mCherry, into the B. microti. The transfection using an electroporation method and genetic manipulation of B. microti is now achievable and it is possible to obtain transfected viable parasites under in vivo growing conditions. The growth curve analysis of transfected and WT B. microti are similar indicating no defects in the transgenic parasites. This study will enable other researchers in understanding the B. microti biology, host modulation and diverse parasite developmental stages using reverse genetics and holds great potential to identify novel drug targets and vaccine development.

Improved Plasmodium berghei lines for conditional mutagenesis.

Conditional mutagenesis is a powerful tool for genetic analysis in Plasmodium berghei. It allows the study of proteins that function both during the parasite's pre-erythocytic and erythrocytic development. Currently available parasite lines used for conditional mutagenesis were constructed in the NK65 strain, and express a DNA recombinase under the control of pre-erythrocytic stage-specific promoters. However, the integration of the recombinase in these lines is unstable leading to inconsistent excision of the target gene. We describe improved lines of P. berghei with stably integrated DNA recombinase that allow efficient, stage-specific excision of target genes in the widely used ANKA strain.