An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.     

Maladjusted Host Immune Responses Induce Experimental Cerebral Malaria-Like Pathology in a Murine Borrelia and Plasmodium Co-Infection Model

In the Plasmodium infected host, a balance between pro- and anti-inflammatory responses is required to clear the parasites without inducing major host pathology. Clinical reports suggest that bacterial infection in conjunction with malaria aggravates disease and raises both mortality and morbidity in these patients. In this study, we investigated the immune responses in BALB/c mice, co-infected with Plasmodium berghei NK65 parasites and the relapsing fever bacterium Borrelia duttonii. In contrast to single infections, we identified in the co-infected mice a reduction of L-Arginine levels in the serum. It indicated diminished bioavailability of NO, which argued for a dysfunctional endothelium. Consistent with this, we observed increased sequestration of CD8+ cells in the brain as well over expression of ICAM-1 and VCAM by brain endothelial cells. Co-infected mice further showed an increased inflammatory response through IL-1β and TNF-α, as well as inability to down regulate the same through IL-10. In addition we found loss of synchronicity of pro- and anti-inflammatory signals seen in dendritic cells and macrophages, as well as increased numbers of regulatory T-cells. Our study shows that a situation mimicking experimental cerebral malaria (ECM) is induced in co-infected mice due to loss of timing and control over regulatory mechanisms in antigen presenting cells.     

Ultrastructure of an Unusual Erythrocytic Form of Plasmodium berghei*

SYNOPSIS. Unusual dense forms were discovered in ultrathin sections of Plasmodium berghei-infected rat erythrocytes. These parasites frequently occurred with one or more typical trophozoites in a single blood cell. They appeared darker than both the neighboring trophozoites and the host erythrocyte. Ribosomes were visible in clusters in their compact cytoplasm. The endoplasmic reticulum, when present, had dilated cisternae often containing a material of low density. Large food vacuoles werecommonly seen along with the small vesicles harboring pigment granules. The single large nucleus had dense nucleoplasm. Multilaminated membraned bodies and sausage-shaped vacuoles were, seen in some of the parasites. The exact identity of this form of P. berghei is not known. Its possible significance is discussed with particular reference to the differentiation of gametocytes.     

Experimental Genetics of Plasmodium berghei NFU in the Apicoplast Iron-Sulfur Cluster Biogenesis Pathway

Eukaryotic pathogens of the phylum Apicomplexa contain a non-photosynthetic plastid, termed apicoplast. Within this organelle distinct iron-sulfur [Fe-S] cluster proteins are likely central to biosynthesis pathways, including generation of isoprenoids and lipoic acid. Here, we targeted a nuclear-encoded component of the apicoplast [Fe-S] cluster biosynthesis pathway by experimental genetics in the murine malaria parasite Plasmodium berghei. We show that ablation of the gene encoding a nitrogen fixation factor U (NifU)-like domain containing protein (NFUapi) resulted in parasites that were able to complete the entire life cycle indicating redundant or non-essential functions. nfu ^– parasites displayed reduced merosome formation in vitro, suggesting that apicoplast NFUapi plays an auxiliary role in establishing a blood stage infection. NFUapi fused to a combined fluorescent protein-epitope tag delineates the Plasmodium apicoplast and was tested to revisit inhibition of liver stage development by azithromycin and fosmidomycin. We show that the branched apicoplast signal is entirely abolished by azithromycin treatment, while fosmidomycin had no effect on apicoplast morphology. In conclusion, our experimental genetics analysis supports specialized and/or redundant role(s) for NFUapi in the [Fe-S] cluster biosynthesis pathway in the apicoplast of a malarial parasite.     

Chromosome translocation in Plasmodium berghei.

We describe a chromosome translocation in a karyotype mutant of the rodent malarial parasite Plasmodium berghei. In this mutant (named EP) a small chromosome (chromosome 7), which has exhibited a size range between 0.9 and 1.4 Mb in other clones of P. berghei, is translocated to chromosome 13 or 14 with a size of about 3 Mb. By comparison of Apa-I restriction fragments of the chromosomes from mutant EP and from a reference clone (named HP) of P. berghei, we found evidence for a junction of subtelomeric chromosome 7 sequences and internal chromosome 13/14 sequences. In addition, a new chromosome of 1.4 Mb (named EP7) is present in mutant EP, which is (mainly) composed of sequences of chromosome 13/14. EP7 contains one telomeric region derived from chromosome 13/14. We found evidence that internal sequences of chromosome 13/14 are joined to telomeric sequences in the other telomeric region of EP7. The karyotype of mutant EP was stable during asexual and sexual multiplication and we found no indications for phenotypic changes.     

Long-term in vitro cultivation of Plasmodium berghei

Long-term in vitro culture of Plasmodium berghei was established using the Petri dish candle jar method of Trager and Jensen (1976). Cultures were established at 22, 27 and 37°C. As optimal growth was observed at 27°C, subsequent cultivation was carried out at this temperature. RPMI 1640 medium was modified by incorporating additional glucose (1 mg ml⁻¹) and bactopeptone (1 mg ml⁻¹) in the medium. This medium was found suitable for maintenance of mouse erythrocytes in vitro. P. berghei cultures were maintained using candle jars and this modified RPMI 1640 medium for 45 weeks.     

Functional characterization of an LCCL-lectin domain containing protein family in Plasmodium berghei

Using bioinformatic, proteomic, immunofluorescence, and genetic cross methods, we have functionally characterized a family of putative parasite ligands as potential mediators of cell-cell interactions. We name these proteins the Limulus clotting factor C, Coch-5b2, and Lgl1 (LCCL)-lectin adhesive-like protein (LAP) family. We demonstrate that this family is conserved amongst Plasmodium spp. It possesses a unique arrangement of adhesive protein domains normally associated with extracellular proteins. The proteins are expressed predominantly, though not exclusively, in the mosquito stages of the life cycle. We test the hypothesis that these proteins are surface proteins with 1 member of this gene family, lap1, and provide evidence that it is expressed on the surface of Plasmodium berghei sporozoites. Finally, through genetic crosses of wild-type Pblap1+ and transgenic Pblap1- parasites, we show that the null phenotype previously reported for sporozoite development in a Pblap1- mutant can be rescued within a heterokaryotic oocyst and that infectious Pblap1 sporozoites can be formed. The mutant is not rescued by coparasitization of mosquitoes with a mixture Pblap1+ and Pblap1- homokaryotic oocysts.