Optimized expression of Plasmodium falciparum erythrocyte membrane protein 1 domains in Escherichia coli

BACKGROUND: The expression of recombinant proteins in Escherichia coli is an important and frequently used tool within malaria research, however, this method remains problematic. High A/T versus C/G content and frequent lysine and arginine repeats in the Plasmodium falciparum genome are thought to be the main reason for early termination in the mRNA translation process. Therefore, the majority of P. falciparum derived recombinant proteins is expressed only as truncated forms or appears as insoluble inclusion bodies within the bacterial cells. METHODS: Several domains of PfEMP1 genes obtained from different P. falciparum strains were expressed in E. coli as GST-fusion proteins. Expression was carried out under various culture conditions with a main focus on the time point of induction in relation to the bacterial growth stage. RESULTS AND CONCLUSIONS: When expressed in E. coli recombinant proteins derived from P. falciparum sequences are often truncated and tend to aggregate what in turn leads to the formation of insoluble inclusion bodies. The analysis of various factors influencing the expression revealed that the time point of induction plays a key role in successful expression of A/T rich sequences into their native conformation. Contrary to recommended procedures, initiation of expression at post-log instead of mid-log growth phase generated significantly increased amounts of soluble protein of a high quality. Furthermore, these proteins were shown to be functionally active. Other factors such as temperature, pH, bacterial proteases or the codon optimization for E. coli had little or no effect on the quality of the recombinant protein, nevertheless, optimizing these factors might be beneficial for each individual construct. In conclusion, changing the timepoint of induction and conducting expression at the post-log stage where the bacteria have entered a decelerated growth phase, greatly facilitates and improves the expression of sequences containing rare codons

A Novel DBL-Domain of the P. falciparum 332 Molecule Possibly Involved in Erythrocyte Adhesion

Plasmodium falciparum malaria is brought about by the asexual stages of the parasite residing in human red blood cells (RBC). Contact between the erythrocyte surface and the merozoite is the first step for successful invasion and proliferation of the parasite. A number of different pathways utilised by the parasite to adhere and invade the host RBC have been characterized, but the complete biology of this process remains elusive. We here report the identification of an open reading frame (ORF) representing a hitherto unknown second exon of the Pf332 gene that encodes a cysteine-rich polypeptide with a high degree of similarity to the Duffy-binding-like (DBL) domain of the erythrocyte-binding-ligand (EBL) family. The sequence of this DBL-domain is conserved and expressed in all parasite clones/strains investigated. In addition, the expression level of Pf332 correlates with proliferation efficiency of the parasites in vitro. Antibodies raised against the DBL-domain are able to reduce the invasion efficiency of different parasite clones/strains. Analysis of the DBL-domain revealed its ability to bind to uninfected human RBC, and moreover demonstrated association with the iRBC surface. Thus, Pf332 is a molecule with a potential role to support merozoite invasion. Due to the high level of conservation in sequence, the novel DBL-domain of Pf332 is of possible importance for development of novel anti-malaria drugs and vaccines

Rapid Communication Rescue of Hantaan virus minigenomes

Hantavirus infections are a major public health concern worldwide. Their widespread geographical distribution and their ability to produce serious, often fatal, human disease underline the need for a system that allows manipulation of these viruses. We describe here the first successful establishment of a reverse genetics technology for Hantaan virus, the prototype of the genus Hantavirus. The system offers a unique opportunity to study the biology of hantaviruses, the pathogenesis of the diseases, and the efficacy of antiviral and prophylactic measures against hantavirus infections

Reverse Genetics for Crimean-Congo Hemorrhagic Fever Virus

The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus Nairovirus, family Bunyaviridae. The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family Bunyaviridae. This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates

Functional Analysis of the Noncoding Regions of the Uukuniemi Virus (Bunyaviridae) RNA Segments

The role of the variable portion of the noncoding regions (NCRs) of the three Bunyaviridae RNA segments (L, M, S) in transcription, replication, and packaging was studied using the recently developed plasmid-driven RNA polymerase I minigenome system for Uukuniemi (UUK) virus, genus Phlebovirus (11), as a model. Comparison of the different segments showed that all NCRs were sufficient to mediate transcription/replication of a minigenome but demonstrated decreased promoter strength in the order M > L > S. Chimeric minigenomes with flanking NCRs from different genome segments revealed that the number of total base pairs within the inverted, partially complementary ends was important for transcription and replication. Point mutations increasing the base-pairing potential produced increased reporter expression, indicating that complementarity between the 5′ and 3′ ends is crucial for promoter activity. The role of the intergenic region (IGR) located between the two open reading frames of the ambisense UUK virus S segment was analyzed by inserting this sequence element downstream of the reporter genes. The presence of the IGR was found to enhance reporter expression, demonstrating that efficient transcription termination, regulated by the IGR, is important for optimal minigenome mRNA translation. Finally, genome packaging efficacy varied for different NCRs and was strongest for L followed by M and S. Strong reporter gene activity was still observed after seven consecutive cell culture passages, indicating a selective rather than random genome-packaging mechanism. In summary, our results demonstrate that the NCRs from all three segments contain the necessary signals to initiate transcription and replication as well as packaging. Based on promoter strength, M-segment NCRs may be the preferred choice for the development of reverse genetics and minigenome rescue systems for bunyaviruses

Identification of a Polyclonal B-Cell Activator in Plasmodium falciparum

Polyclonal B-cell activation and hypergammaglobulinemia are prominent features of human malaria. We report here that Plasmodium falciparum-infected erythrocytes directly adhere to and activate peripheral blood B cells from nonimmune donors. The infected erythrocytes employ the cysteine-rich interdomain region 1α (CIDR1α) of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to interact with the B cells. Stimulation with recombinant CIDR1α induces proliferation, an increase in B-cell size, expression of activation molecules, and secretion of immunoglobulins (immunoglobulin M) and cytokines (tumor necrosis factor alpha and interleukin-6). Furthermore, CIDR1α binds to Fab and Fc fragments of human immunoglobulins and to immunoglobulins purified from the sera of different animal species. This binding pattern is similar to that of the polyclonal B-cell activator Staphylococcus aureus protein A. Our findings shed light on the understanding of the molecular basis of polyclonal B-cell activation during malaria infections. The results suggest that the var gene family encoding PfEMP1 has evolved not only to mediate the sequestration of infected erythrocytes but also to manipulate the immune system to enhance the survival of the parasite

A retrospective TBNET assessment of linezolid safety, tolerability and efficacy in multidrug-resistant tuberculosis

Linezolid is used to treat patients with multidrug-resistant (MDR)/extensively drug-resistant (XDR)-tuberculosis (TB) cases, although clinical data on its safety, tolerability and efficacy are lacking. We performed a retrospective, nonrandomised, unblinded observational study evaluating the safety and tolerability of linezolid at 600 mg q.d. or b.i.d. in MDR/XDR-TB treatment in four European countries. Efficacy evaluation compared end-points of 45 linezolid-treated against 110 linezolid-nontreated cases. Out of 195 MDR/XDR-TB patients, 85 were treated with linezolid for a mean of 221 days. Of these, 35 (41.2%) out of 85 experienced major side-effects attributed to linezolid (anaemia, thrombocytopenia and/or polyneuropathy), requiring discontinuation in 27 (77%) cases. Most side-effects occurred after 60 days of treatment. Twice-daily administration produced more major side-effects than once-daily dosing (p = 0.0004), with no difference in efficacy found. Outcomes were similar in patients treated with/without linezolid (p = 0.8), although linezolid-treated cases had more first-line (p = 0.002) and second-line (p = 0.02) drug resistance and a higher number of previous treatment regimens (4.5 versus 2.3; p = 0.07). Linezolid 600 mg q.d. added to an individualised multidrug regimen may improve the chance of bacteriological conversion, providing a better chance of treatment success in only the most complicated MDR/XDR-TB cases. Its safety profile does not warrant use in cases for which there are other, safer, alternatives