Monica Prado

Features of autophagic cell death in Plasmodium liver-stage parasites.

Analyzing molecular determinants of Plasmodium parasite cell death is a promising approach for exploring new avenues in the fight against malaria. Three major forms of cell death (apoptosis, necrosis and autophagic cell death) have been described in multicellular organisms but which cell death processes exist in protozoa is still a matter of debate. Here we suggest that all three types of cell death occur in Plasmodium liver-stage parasites. Whereas typical molecular markers for apoptosis and necrosis have not been found in the genome of Plasmodium parasites, we identified genes coding for putative autophagy-marker proteins and thus concentrated on autophagic cell death. We characterized the Plasmodium berghei homolog of the prominent autophagy marker protein Atg8/LC3 and found that it localized to the apicoplast. A relocalization of PbAtg8 to autophagosome-like vesicles or vacuoles that appear in dying parasites was not, however, observed. This strongly suggests that the function of this protein in liver-stage parasites is restricted to apicoplast biology.

Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms

Plasmodium parasites are transmitted by Anopheles mosquitoes to the mammalian host and actively infect hepatocytes after passive transport in the bloodstream to the liver. In their target host hepatocyte, parasites reside within a parasitophorous vacuole (PV). In the present study it was shown that the parasitophorous vacuole membrane (PVM) can be targeted by autophagy marker proteins LC3, ubiquitin, and SQSTM1/p62 as well as by lysosomes in a process resembling selective autophagy. The dynamics of autophagy marker proteins in individual Plasmodium berghei-infected hepatocytes were followed by live imaging throughout the entire development of the parasite in the liver. Although the host cell very efficiently recognized the invading parasite in its vacuole, the majority of parasites survived this initial attack. Successful parasite development correlated with the gradual loss of all analyzed autophagy marker proteins and associated lysosomes from the PVM. However, other autophagic events like nonselective canonical autophagy in the host cell continued. This was indicated as LC3, although not labeling the PVM anymore, still localized to autophagosomes in the infected host cell. It appears that growing parasites even benefit from this form of nonselective host cell autophagy as an additional source of nutrients, as in host cells deficient for autophagy, parasite growth was retarded and could partly be rescued by the supply of additional amino acid in the medium. Importantly, mouse infections with P. berghei sporozoites confirmed LC3 dynamics, the positive effect of autophagy activation on parasite growth, and negative effects upon autophagy inhibition.

Acute physiopathological effects of honeybee (Apis mellifera) envenoming by subcutaneous route in a mouse model

Bee stings are a health concern in the Americas, where fatal envenomings due to massive attacks by Africanized honeybees have been documented in the last decades. Most studies on the toxic effects of honeybee venom in experimental animals have been performed using the intravenous or intraperitoneal injection routes. The aim of this study was to develop a mouse model that would better resemble a massive honeybee attack by using the subcutaneous (s.c.) route to induce a severe, sublethal systemic envenoming. An array of acute venom effects were characterized, including biochemical, hematological, histological, and inflammatory alterations, after the s.c. injection of 0.5 median lethal dose of venom. Rapid increases in serum alanine (ALT) and aspartate (AST) transaminases, creatinine, urea nitrogen, uric acid, sodium and chloride electrolytes, and creatine kinase (CK) were recorded, indicating damage to liver, kidneys, and skeletal muscle. Also, coagulation disturbances (fibrinogen decrease, and moderate delay in prothrombin and partial thromboplastin times) were demonstrated. Circulating platelet and leukocyte numbers remained unaltered, but a hemoconcentration effect (hematocrit and hemoglobin increase) was observed. This effect might be related to the marked edema induced by the venom. In addition, this inflammatory response included a systemic increase in cytokines (IL-1 beta, IL-6, TNF-alpha), together with an elevation of serum malondialdehyde and nitric oxide. The myotoxic effects of venom, melittin, and phospholipase A(2) were demonstrated after injection by s.c. route. No synergistic myotoxicity between melittin and PLA(2) was observed. Moreover, these two components, when injected at equivalent concentrations to those present in venom, induced a lower increase in serum CK than venom, suggesting that other components also contribute to its strong systemic toxicity towards skeletal muscle. The model here presented may be useful in preclinical studies to assess therapeutic antivenoms developed to cope with the problem of massive bee attacks.

Percepción del riesgo y estrategias de comunicación social sobre el dengue en las Américas

Dengue is clearly a very serious public health problem. In the Americas the number of dengue cases has been increasing since the 1960s, and outbreaks of the disease have been occurring more frequently. Furthermore, the density of infestation with the disease vector, the Aedes aegypti mosquito, is high in the Americas. The general strategy for preventing and controlling dengue and dengue hemorrhagic fever is based on promoting behavior changes that lead to incorporating the community in controlling the disease, particularly the vector. In order to achieve this, mass communication programs on dengue should have two primary aims: converting information into practice and encouraging the community to take over prevention and control measures. The new generation of programs should be designed based on the local sanitation structure (water distribution and waste disposal) as well as information on community organizations and the roles of different family members. Furthermore, the new programs should incorporate all the following ten components: epidemiological surveillance, intersectoral actions, community participation, managing the environment and basic services, patient care, case reporting, education, using insecticides and vector control, training, and preparing for emergencies. Communication should be aimed at modifying the behavior of individuals and the community by empowering them to carry out prevention and control measures.

The autophagic machinery ensures nonlytic transmission of mycobacteria

Significance Pathogenic mycobacteria can be transmitted by direct ejection from one host cell to another. However, the mechanism of ejection, and how lysing the host cell is prevented are unknown. This study explains how the host cell remains intact and alive while Mycobacterium marinum breaks through its plasma membrane during ejection. We show that a membraneous cup is specifically recruited to the distal pole of ejecting M. marinum. We demonstrate that these membranes are formed by the canonical autophagic pathway, though they do not mature to autophagolysosomes. Disruption of autophagy causes the host cells to become leaky and die during ejection. This dramatically reduces cell-to-cell transmission of the infection, demonstrating an important and unexpected role for autophagy in maintaining plasma membrane integrity during mycobacterial infection. In contrast to mechanisms mediating uptake of intracellular bacterial pathogens, bacterial egress and cell-to-cell transmission are poorly understood. Previously, we showed that the transmission of pathogenic mycobacteria between phagocytic cells also depends on nonlytic ejection through an F-actin based structure, called the ejectosome. How the host cell maintains integrity of its plasma membrane during the ejection process was unknown. Here, we reveal an unexpected function for the autophagic machinery in nonlytic spreading of bacteria. We show that ejecting mycobacteria are escorted by a distinct polar autophagocytic vacuole. If autophagy is impaired, cell-to-cell transmission is inhibited, the host plasma membrane becomes compromised and the host cells die. These findings highlight a previously unidentified, highly ordered interaction between bacteria and the autophagic pathway and might represent the ancient way to ensure nonlytic egress of bacteria.

The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites

Sequestration of red blood cells infected with the human malaria parasite Plasmodium falciparum in organs such as the brain is considered important for pathogenicity. A similar phenomenon has been observed in mouse models of malaria, using the rodent parasite Plasmodium berghei, but it is unclear whether the P. falciparum proteins known to be involved in this process are conserved in the rodent parasite. Here we identify the P. berghei orthologues of two such key factors of P. falciparum, SBP1 and MAHRP1. Red blood cells infected with P. berghei parasites lacking SBP1 or MAHRP1a fail to bind the endothelial receptor CD36 and show reduced sequestration and virulence in mice. Complementation of the mutant P. berghei parasites with the respective P. falciparum SBP1 and MAHRP1 orthologues restores sequestration and virulence. These findings reveal evolutionary conservation of the machinery underlying sequestration of divergent malaria parasites and support the notion that the P. berghei rodent model is an adequate tool for research on malaria virulence.

A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine

Recently it has been shown in rodent malaria models that immunisation with genetically attenuated Plasmodium parasites can confer sterile protection against challenge with virulent parasites. For the mass production of live attenuated Plasmodium parasites for vaccination, safety is a prerequisite. Knockout of a single gene is not sufficient for such a strategy since the parasite can likely compensate for such a genetic modification and a single surviving parasite is sufficient to kill an immunised individual. Parasites must therefore be at least double-attenuated when generating a safe vaccine strain. Genetic double-attenuation can be achieved by knocking out two essential genes or by combining a single gene knockout with the expression of a protein toxic for the parasite. We generated a double-attenuated Plasmodium berghei strain that is deficient in fatty acid synthesis by the knockout of the pdh-e1α gene, introducing a second attenuation by the liver stage-specific expression of the pore-forming bacterial toxin perfringolysin O. With this double genetically attenuated parasite strain, a superior attenuation was indeed achieved compared with single-attenuated strains that were either deficient in pyruvate dehydrogenase (PDH)-E1 or expressed perfringolysin O. In vivo, both single-attenuated strains resulted in breakthrough infections even if low to moderate doses of sporozoites (2,000-5,000) were administered. In contrast, the double genetically attenuated parasite strain, given at moderate doses of 5,000 sporozoites, did not result in blood stage infection and even when administered at 5- to 20-fold higher doses, only single and delayed breakthrough infections were observed. Prime booster immunisation with the double genetically attenuated parasite strain completely protected a susceptible mouse strain from malaria and even a single immunisation conferred protection in some cases and lead to a markedly delayed onset of blood stage infection in others. Importantly, premature rupture of the parasitophorous vacuole membrane by liver stage-specific perfringolysin O expression did not induce host cell death and soluble parasite proteins, which are released into the host cell cytoplasm, have the potential to be processed and presented via MHC class I molecules. This, in turn, might support immunological responses against Plasmodium-infected hepatocytes.

Mortality due to Hymenoptera stings in Costa Rica, 1985-2006

OBJECTIVE To analyze mortality due to Hymenoptera stings in Costa Rica during 1985-2006. METHODS Records of deaths due to Hymenoptera stings in 1985-2006 were retrieved from Instituto Nacional de Estadística y Censos (National Statistics and Census Institute). Mortality rates were calculated on the basis of national population reports, as of 1 July of each year. Information for each case included age, gender, and the province in which the death occurred. In addition, reports of Hymenoptera sting accidents received by the Centro Nacional de Intoxicaciones (National Poison Center, CNI) in 1995-2006 were obtained to assess exposure to these insects. RESULTS Over the 22-year period analyzed, 52 fatalities due to Hymenoptera stings were recorded. Annual mortality rates varied from 0-1.73 per 1 million inhabitants, with a mean of 0.74 (95% confidence interval: 0.46-0.93). The majority of deaths occurred in males (88.5%), representing a male to female ratio of 7.7:1. A predominance of fatalities was observed in the elderly (50 years of age and older), as well as in children less than 10 years of age. The province with the highest mortality rate was Guanacaste. The CNI documented 1,591 reports of Hymenoptera stings (mostly by bees) in 1995-2006, resulting in an annual average of 133 cases, with only a slight predominance of males over females (1.4:1). CONCLUSIONS Stings by Hymenoptera, mostly by bees, constitute a frequent occurrence in Costa Rica that can be life-threatening in a small proportion of cases, most often in males and the elderly. The annual number of fatalities fluctuated from 0-6, averaging 2.4 deaths per year. Awareness should be raised not only among the general population, but also among health care personnel that should consider this risk in the clinical management of patients stung by Hymenoptera.

Shedding of host autophagic proteins from the parasitophorous vacuolar membrane of Plasmodium berghei.

The hepatic stage of the malaria parasite Plasmodium is accompanied by an autophagy-mediated host response directly targeting the parasitophorous vacuolar membrane (PVM) harbouring the parasite. Removal of the PVM-associated autophagic proteins such as ubiquitin, p62, and LC3 correlates with parasite survival. Yet, it is unclear how Plasmodium avoids the deleterious effects of selective autophagy. Here we show that parasites trap host autophagic factors in the tubovesicular network (TVN), an expansion of the PVM into the host cytoplasm. In proliferating parasites, PVM-associated LC3 becomes immediately redirected into the TVN, where it accumulates distally from the parasite’s replicative centre. Finally, the host factors are shed as vesicles into the host cytoplasm. This strategy may enable the parasite to balance the benefits of the enhanced host catabolic activity with the risk of being eliminated by the cell’s cytosolic immune defence.

A Cysteine Protease Inhibitor of Plasmodium berghei Is Essential for Exo-erythrocytic Development

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.