Imaging Infection by Vector-Borne Protozoan Parasites Using Whole-Mouse Bioluminescence.

Vector-borne protozoan parasites such as Plasmodium spp. Leishmania spp. and Trypanosoma brucei are responsible for several serious diseases. Significant advances in parasitology have been made using rodent models combined with live imaging techniques, including whole-mouse bioluminescence imaging (BLI). This technique has been applied to investigate parasite dissemination, infectivity, and growth. It has also been used in drug and vaccine testing. This chapter focuses on the methods that utilize whole-mouse BLI to (i) evaluate the homing and infectivity of Plasmodium berghei sporozoites; (ii) conduct in vivo testing of promising chemical entities against Leishmania infantum infection; and (iii) study molecular mechanisms of host susceptibility to Trypanosoma brucei brucei infection.

MAEBL Contributes to Plasmodium Sporozoite Adhesiveness.

The sole currently approved malaria vaccine targets the circumsporozoite protein-the protein that densely coats the surface of sporozoites, the parasite stage deposited in the skin of the mammalian host by infected mosquitoes. However, this vaccine only confers moderate protection against clinical diseases in children, impelling a continuous search for novel candidates. In this work, we studied the importance of the membrane-associated erythrocyte binding-like protein (MAEBL) for infection by Plasmodium sporozoites. Using transgenic parasites and live imaging in mice, we show that the absence of MAEBL reduces Plasmodium berghei hemolymph sporozoite infectivity to mice. Moreover, we found that maebl knockout (maebl-) sporozoites display reduced adhesion, including to cultured hepatocytes, which could contribute to the defects in multiple biological processes, such as in gliding motility, hepatocyte wounding, and invasion. The maebl- defective phenotypes in mosquito salivary gland and liver infection were reverted by genetic complementation. Using a parasite line expressing a C-terminal myc-tagged MAEBL, we found that MAEBL levels peak in midgut and hemolymph parasites but drop after sporozoite entry into the salivary glands, where the labeling was found to be heterogeneous among sporozoites. MAEBL was found associated, not only with micronemes, but also with the surface of mature sporozoites. Overall, our data provide further insight into the role of MAEBL in sporozoite infectivity and may contribute to the design of future immune interventions.

TRSP is dispensable for the Plasmodium pre-erythrocytic phase.

Plasmodium sporozoites deposited in the skin following a mosquito bite must migrate and invade blood vessels to complete their development in the liver. Once in the bloodstream, sporozoites arrest in the liver sinusoids, but the molecular determinants that mediate this specific homing are not yet genetically defined. Here we investigate the involvement of the thrombospondin-related sporozoite protein (TRSP) in this process using knockout Plasmodium berghei parasites and in vivo bioluminescence imaging in mice. Resorting to a homing assay, trsp knockout sporozoites were found to arrest in the liver similar to control parasites. Moreover, we found no defects in the establishment of infection in mice following inoculation of trsp knockout sporozoites via intravenous and cutaneous injection or mosquito bite. Accordingly, mutant sporozoites were also able to successfully invade hepatocytes in vitro. Altogether, these results suggest TRSP may have a redundant role in the completion of the pre-erythrocytic phase of the malaria parasite. Nonetheless, identifying molecules with paramount roles in this phase could aid in the search for new antigens needed for the design of a protective vaccine against malaria.

The crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1: Implications to protein function and drug design.

The de novo crystal structure of the Leishmania infantum Silent Information Regulator 2 related protein 1 (LiSir2rp1) has been solved at 1.99Å in complex with an acetyl-lysine peptide substrate. The structure is broadly commensurate with Hst2/SIRT2 proteins of yeast and human origin, reproducing many of the structural features common to these sirtuin deacetylases, including the characteristic small zinc-binding domain, and the larger Rossmann-fold domain involved in NAD+-binding interactions. The two domains are linked via a cofactor binding loop ordered in open conformation. The peptide substrate binds to the LiSir2rp1 protein via a cleft formed between the small and large domains, with the acetyl-lysine side chain inserting further into the resultant hydrophobic tunnel. Crystals were obtained only with recombinant LiSir2rp1 possessing an extensive internal deletion of a proteolytically-sensitive region unique to the sirtuins of kinetoplastid origin. Deletion of 51 internal amino acids (P253-E303) from LiSir2rp1 did not appear to alter peptide substrate interactions in deacetylation assays, but was indispensable to obtain crystals. Removal of this potentially flexible region, that otherwise extends from the classical structural elements of the Rossmann-fold, specifically the ß8-ß9 connector, appears to result in lower accumulation of the protein when expressed from episomal vectors in L. infantum SIR2rp1 single knockout promastigotes. The biological function of the large serine-rich insertion in kinetoplastid/trypanosomatid sirtuins, highlighted as a disordered region with strong potential for post-translational modification, remains unknown but may confer additional cellular functions that are distinct from their human counterparts. These unique molecular features, along with the resolution of the first kinetoplastid sirtuin deacetylase structure, present novel opportunities for drug design against a protein target previously established as essential to parasite survival and proliferation.

In vivo imaging of pathogen homing to the host tissues.

Hematogenous dissemination followed by tissue tropism is a characteristic of the infectious process of many pathogens including those transmitted by blood-feeding vectors. After entering into the blood circulation, these pathogens must arrest in the target organ before they infect a specific tissue. Here, we describe a non-invasive method to visualize and quantify the homing of pathogens to the host tissues. By using in vivo bioluminescence imaging we quantify the accumulation of luciferase-expressing parasites in the host organs during the first minutes following their intravascular inoculation in mice. Using this technique we show that in the malarial infection, once in the blood circulation, most of bioluminescent Plasmodium berghei sporozoites, the parasite stage transmitted to the host skin by a mosquito bite, rapidly home to the liver where they invade and develop inside hepatocytes. This homing is specific to this developmental stage since blood stage parasites do not accumulate in the liver, as well as extracellular Trypanosoma brucei bloodstream forms and liver-infecting Leishmania infantum amastigotes. Finally, this method can be used to study the dynamics of tissue tropism of parasites, dissect the molecular and cellular basis of their increased arrest in organs and to evaluate immune interventions designed to block this targeted interaction.