Ana Rodriguez

The silent path to thousands of merozoites: the Plasmodium liver stage

Plasmodium sporozoites are deposited in the skin of their vertebrate hosts through the bite of an infected female Anopheles mosquito. Most of these parasites find a blood vessel and travel in the peripheral blood circulation until they reach the liver sinusoids. Once there, the sporozoites cross the sinusoidal wall and migrate through several hepatocytes before they infect a final hepatocyte, with the formation of a parasitophorous vacuole, in which the intrahepatic form of the parasite grows and multiplies. During this period, each sporozoite generates thousands of merozoites. As the development of Plasmodium sporozoites inside hepatocytes is an obligatory step before the onset of disease, understanding the parasites requirements during this period is crucial for the development of any form of early intervention. This Review summarizes our current knowledge on this stage of the Plasmodium life cycle.

Malaria Blood Stage Suppression of Liver Stage Immunity by Dendritic Cells

Malaria starts with Plasmodium sporozoites infection of the hosts liver, where development into blood stage parasites occurs. It is not clear why natural infections do not induce protection against the initial liver stage and generate low CD8+ T cell responses. Using a rodent malaria model, we show that Plasmodium blood stage infection suppresses CD8+ T cell immune responses that were induced against the initial liver stage. Blood stage Plasmodium affects dendritic cell (DC) functions, inhibiting maturation and the capacity to initiate immune responses and inverting the interleukin (IL)-12/IL-10 secretion pattern. The interaction of blood stage parasites with DCs induces the secretion of soluble factors that inhibit the activation of CD8+ T cells in vitro and the suppression of protective CD8+ T cell responses against the liver stage in vivo. We propose that blood stage infection induces DCs to suppress CD8+ T cell responses in natural malaria infections. This evasion mechanism leaves the host unprotected against reinfection by inhibiting the immune response against the initial liver stage of the disease.

In Vitro Antifungal Evaluation and Structure-Activity Relationships of a New Series of Chalcone Derivatives and Synthetic Analogues, with Inhibitory Properties Against Polymers of the Fungal Cell Wall

Here we report the synthesis, in vitro antifungal evaluation and SAR study of 41 chalcones and analogues. In addition, all active structures were tested for their capacity of inhibiting Saccharomyces cerevisiae beta(1,3)-glucan synthase and chitin synthase, enzymes that catalyze the synthesis of the major polymers of the fungal cell wall.

The myriad roles of cyclic AMP in microbial pathogens: from signal to sword

All organisms must sense and respond to their external environments, and this signal transduction often involves second messengers such as cyclic nucleotides. One such nucleotide is cyclic AMP, a universal second messenger that is used by diverse forms of life, including mammals, fungi, protozoa and bacteria. In this review, we discuss the many roles of cAMP in bacterial, fungal and protozoan pathogens and its contributions to microbial pathogenesis. These roles include the coordination of intracellular processes, such as virulence gene expression, with extracellular signals from the environment, and the manipulation of host immunity by increasing cAMP levels in host cells during infection.

Migration through host cells activates Plasmodium sporozoites for infection

Plasmodium sporozoites, the infective stage of the malaria parasite transmitted by mosquitoes, migrate through several hepatocytes before infecting a final one. Migration through hepatocytes occurs by breaching their plasma membranes, and final infection takes place with the formation of a vacuole around the sporozoite. Once in the liver, sporozoites have already reached their target cells, making migration through hepatocytes prior to infection seem unnecessary. Here we show that this migration is required for infection of hepatocytes. Migration through host cells, but not passive contact with hepatocytes, induces the exocytosis of sporozoite apical organelles, a prerequisite for infection with formation of a vacuole. Sporozoite activation induced by migration through host cells is an essential step of Plasmodium life cycle.

Hepatocyte growth factor and its receptor are required for malaria infection

Plasmodium, the causative agent of malaria, must first infect hepatocytes to initiate a mammalian infection. Sporozoites migrate through several hepatocytes, by breaching their plasma membranes, before infection is finally established in one of them. Here we show that wounding of hepatocytes by sporozoite migration induces the secretion of hepatocyte growth factor (HGF), which renders hepatocytes susceptible to infection. Infection depends on activation of the HGF receptor, MET, by secreted HGF. The malaria parasite exploits MET not as a primary binding site, but as a mediator of signals that make the host cell susceptible to infection. HGF/MET signaling induces rearrangements of the host-cell actin cytoskeleton that are required for the early development of the parasites within hepatocytes. Our findings identify HGF and MET as potential targets for new approaches to malaria prevention.

Growth Hormone: Metabolic Clearance Rates, Integrated Concentrations, and Production Rates in Normal Adults and the Effect of Prednisone

A constant withdrawal pump was used to determine the integrated concentration of growth hormone (ICGH) which was used in conjunction with the metabolic clearance rate (MCR) of growth hormone (GH) to calculate the GH production rates (GHPR) in normal adults, acromegalics, and normal controls receiving prednisone. The mean ICGH for 22 premenopausal females on no medication was 3.0+/-1.6 ng/ml (sd) which is significantly lower (P < 0.005) than the mean of 6.6+/-2.9 for 10 women receiving oral contraceptives and significantly higher than the means of 1.5+/-0.75 for 5 postmenopausal females (P < 0.05) and 1.8+/-1.0 for 16 adult males (P < 0.01) which are comparable. The mean GHPRs in mg/24 hr per m(2) for the four groups are: normal females = 0.52+/-0.24 (sd), females receiving contraceptive pills = 1.65+/-0.58 (P < 0.005), postmenopausal females = 0.26+/-0.12 (P < 0.025), and adult males 0.35+/-0.23 (P < 0.025).Three untreated acromegalic patients had ICGHs of 59, 82, and 93 ng/ml and GHPRs ranging from 14.5 to 17.9 mg/24 hr. Prednisone in a dose of 20 mg t.i.d. for 8 days significantly decreased both the ICGH and GHPR. Alternate day prednisone (60 mg in a single q.o.d. dose) resulted in less consistent inhibition of GH release which may play a role in the more normal growth seen in children receiving q.o.d. prednisone.

HGF/MET signalling protects Plasmodium-infected host cells from apoptosis.

Plasmodium, the causative agent of malaria, migrates through several hepatocytes before initiating a malaria infection. We have previously shown that this process induces the secretion of hepatocyte growth factor (HGF) by traversed cells, which renders neighbour hepatocytes susceptible to infection. The signalling initiated by HGF through its receptor MET has multifunctional effects on various cell types. Our results reveal a major role for apoptosis protection of host cells by HGF/MET signalling on the host susceptibility to infection. Inhibition of HGF/MET signalling induces a specific increase in apoptosis of infected cells leading to a great reduction on infection. Since HGF/MET signalling is capable of protecting cells from apoptosis by using both PI3‐kinase/Akt and, to a lesser extent, MAPK pathways, we determined the impact of these pathways on Plasmodium sporozoite infection. Although inhibition of either of these pathways leads to a reduction in infection, inhibition of PI3‐kinase/Akt pathway caused a stronger effect, which correlated with a higher level of apoptosis in infected host cells. Altogether, the results show that the HGF/MET signalling requirement for infection is mediated by its anti‐apoptotic signal effects. These results demonstrate for the first time that active inhibition of apoptosis in host cell during infection by Plasmodium is required for a successful infection.

Adenylyl Cyclase α and cAMP Signaling Mediate Plasmodium Sporozoite Apical Regulated Exocytosis and Hepatocyte Infection

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase α (ACα), a gene containing regions with high homology to adenylyl cyclases. PbACα-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACα, as re-introduction of ACα in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACα and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes.

New Compound Sets Identified from High Throughput Phenotypic Screening Against Three Kinetoplastid Parasites: An Open Resource

Using whole-cell phenotypic assays, the GlaxoSmithKline high-throughput screening (HTS) diversity set of 1.8 million compounds was screened against the three kinetoplastids most relevant to human disease, i.e. Leishmania donovani, Trypanosoma cruzi and Trypanosoma brucei. Secondary confirmatory and orthogonal intracellular anti-parasiticidal assays were conducted, and the potential for non-specific cytotoxicity determined. Hit compounds were chemically clustered and triaged for desirable physicochemical properties. The hypothetical biological target space covered by these diversity sets was investigated through bioinformatics methodologies. Consequently, three anti-kinetoplastid chemical boxes of ~200 compounds each were assembled. Functional analyses of these compounds suggest a wide array of potential modes of action against kinetoplastid kinases, proteases and cytochromes as well as potential host–pathogen targets. This is the first published parallel high throughput screening of a pharma compound collection against kinetoplastids. The compound sets are provided as an open resource for future lead discovery programs, and to address important research questions.