Cyril Badaut

Expression of the endogenous type II secretion pathway in Escherichia coli leads to chitinase secretion

Escherichia coli K‐12, the most widely used laboratory bacterium, does not secrete proteins into the extracellular medium under standard growth conditions, despite possessing chromosomal genes encoding a putative type II secretion machinery (secreton). We show that in wild‐type E.coli K‐12, divergent transcription of the two operons in the main chromosomal gsp locus, encoding the majority of the secreton components, is silenced by the nucleoid‐structuring protein H‐NS. In mutants lacking H‐NS, the secreton genes cloned on a moderate‐copy‐number plasmid are expressed and promote efficient secretion of the endogenous, co‐regulated endochitinase ChiA. This is the first time that secretion of an endogenous extracellular protein has been demonstrated in E.coli K‐12.

An In Vivo and In Vitro Model of Plasmodium falciparum Rosetting and Autoagglutination Mediated by varO, a Group A var Gene Encoding a Frequent Serotype

ABSTRACT In the Saimiri sciureus monkey, erythrocytes infected with the varO antigenic variant of the Plasmodium falciparum Palo Alto 89F5 clone bind uninfected red blood cells (rosetting), form autoagglutinates, and have a high multiplication rate, three phenotypic characteristics that are associated with severe malaria in human patients. We report here that varO parasites express a var gene having the characteristics of group A var genes, and we show that the varO Duffy binding-like 1α1 (DBL1α1) domain is implicated in the rosetting of both S. sciureus and human erythrocytes. The soluble varO N-terminal sequence (NTS)-DBL1α1 recombinant domain, produced in a baculovirus-insect cell system, induced high titers of antibodies that reacted with varO-infected red blood cells and disrupted varO rosettes. varO parasites were culture adapted in vitro using human erythrocytes. They formed rosettes and autoagglutinates, and they had the same surface serotype and expressed the same varO gene as the monkey-propagated parasites. To develop an in vitro model with highly homogeneous varO parasites, rosette purification was combined with positive selection by panning with a varO NTS-DBL1α1-specific mouse monoclonal antibody. The single-variant, clonal parasites were used to analyze seroprevalence for varO at the village level in a setting where malaria is holoendemic (Dielmo, Senegal). We found 93.6% (95% confidence interval, 89.7 to 96.4%) seroprevalence for varO surface-reacting antibodies and 86.7% (95% confidence interval, 82.8 to 91.6%) seroprevalence for the recombinant NTS-DBL1α1 domain, and virtually all permanent residents had seroconverted by the age of 5 years. These data imply that the varO model is a relevant in vivo and in vitro model for rosetting and autoagglutination that can be used for rational development of vaccine candidates and therapeutic strategies aimed at preventing malaria pathology.

The ChiA (YheB) protein of Escherichia coli K-12 is an endochitinase whose gene is negatively controlled by the nucleoid-structuring protein H-NS

The chromosome of Escherichia coli K‐12 contains a putative gene, yheB (chiA), at centisome 74.7, whose product shows sequence similarity with chitinases of bacterial and viral origin. We cloned the chiA (yheB) gene and demonstrated that it codes for a 94.5 kDa periplasmic protein with endochitinase/lysozyme activity. Under standard laboratory growth conditions, chiA expression is very low, as shown by the Lac− phenotype of a chiA transcriptional fusion to a promoterless lacZ reporter. To identify factors that control chitinase gene expression, we generated random Tn10 insertions in the chromosome of the fusion‐containing strain, selecting for a Lac+ phenotype. The majority of the mutations that caused a Lac+ phenotype mapped to the hns gene, encoding the nucleoid‐structuring protein H‐NS. Transcription of chiA in vivo is driven by a single σ70 promoter and is derepressed in an hns mutant. Using a competitive gel retardation assay, we demonstrated that H‐NS binds directly and with high affinity to the chiA promoter region. In addition to hns, other E. coli mutations causing defects in global regulatory proteins, such as fis, crp or stpA in combination with hns, increased chiA expression to different extents, as did decreasing the growth temperature from 37°C to 30°C. A possible physiological function of ChiA (YheB) endochitinase in E. coli K‐12 is discussed.

Specific stimulation of HIV-1 replication in human placental trophoblasts by an antigen of Plasmodium falciparum.

Epidemiological data point to an increased risk of HIV-1 mother-to-child transmission in pregnant women with malaria, by unknown mechanisms. We show here that surface binding of a recombinant Plasmodium falciparum adhesin to chondroitin sulphate A proteoglycans increases HIV-1 replication in the human placental cell line BeWo, probably by a P. falciparum adhesin-induced long-terminal repeat-driven TNF-α stimulation. This suggests that placental malaria could increase the risk of HIV-1 transmission in utero.

Functional and Immunological Characterization of a Duffy Binding–Like–γ Domain from Plasmodium falciparum Erythrocyte Membrane Protein–1 Expressed by a Placental Isolate

A recombinant Duffy binding-like (DBL)- gamma domain from a previously identified placental isolate, 732, was expressed by use of the baculovirus/insect cell system and was purified in milligram quantities. The recombinant protein binds specifically to chondroitin sulfate A (CSA) and inhibits CSA binding by placental infected erythrocytes (IEs). Polyclonal antibodies raised against the domain recognized the surfaces of live IEs from CSA-adherent clinical placental isolates. These antibodies also abrogated the in vitro binding of IEs to CSA. The 732 DBL-3 gamma domain was specifically recognized by plasma from pregnant women but not by plasma from control subjects. In addition, the protein was, comparatively, significantly more reactive with plasma from women with infected placentas, strongly suggesting that the 732 DBL-3 gamma domain carries preferentially IE-expressed immunogenic epitopes. High levels of plasma antibodies to the recombinant domain were associated with reduced placental parasite density. This is the first report of a recombinant DBL- gamma domain derived from a placental isolate that shows CSA-binding properties.

Structural and immunological correlations between the variable blocks of the VAR2CSA domain DBL6ε from two Plasmodium falciparum parasite lines.

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), a family of adhesins of the falciparum species of the malaria parasite, is exposed on the surface of the infected erythrocyte. In general, only one PfEMP1 variant is expressed at a time but switching between variants occurs, changing both host-cell receptor specificity and serotype. The PfEMP1 variant VAR2CSA causes sequestration of infected erythrocytes in the intervillous spaces of the placenta via the glycosaminoglycan chondroitin sulfate A. This leads to pregnancy-associated malaria, which has severe consequences for the fetus and mother. The extracellular region of VAR2CSA comprises six DBL (Duffy-binding-like) domains and a single CIDR (cysteine-rich inter-domain region) domain. The C-terminal domain DBL6ε, the most polymorphic domain of VAR2CSA, has seven regions of high variability termed variable blocks (VBs). Here we have determined the crystal structure of DBL6ε from the FCR3 parasite line and have compared it with the previously determined structure of that from the 3D7 line. We found significant differences particularly in the N-terminal region, which contains the first VB (VB1). Although DBL6ε is the most variable VAR2CSA domain, DBL6ε-FCR3 and DBL6ε-3D7 react with IgG purified from immune sera of pregnant women. Furthermore, IgG purified on one domain cross-reacts with the other, confirming the presence of cross-reactive epitopes. We also examined reactivity of immune sera to the four least variable VB (VB1, VB2, VB4 and VB5) using peptides with the consensus sequence closest, in turn, to the FCR3 or 3D7 domain. These results provide new molecular insights into immune escape by parasites expressing the VAR2CSA variant.

A Plasmodium falciparum antigen increases HIV-1 replication in a human placenta-derived cell line

Material and methods The placenta-derived choriocarcinoma cell line BeWo and monocyte-derived macrophages (MDM) were infected with varying doses of luciferase reporter HIV-1 pseudotyped with the vesicular stomatitis virus protein G. A recombinant DBL3γ domain, derived from a placental Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) adhesin domain (DBL3γ-732), that binds to chondroitin sulfate A (CSA) and a non-CSA-binding PfEMP1 adhesin domain, DBL1α-varO domain were used to stimulate infected cells. In some experiments, DBL3γ732 was preincubated with the Fab fragment of a specific or an irrelevant monoclonal antibodies (mAb) that inhibits, or not, binding to CSA. TNF-α was measured in the culture supernatants and luciferase activity was quantified at different time points in cell lysates to evaluate viral replication. Results Addition of DBL3γ-732 to BeWo cells, led to a dosedependent increase of HIV-1 replication of up to 400 times the control level in the absence of DBL3γ-732. This enhancement was specific since it was inhibited by Fab fragment of an antiDBL3γ-732 monoclonal antibody but not by the Fab of an irrelevant mAb. In contrast, the addition of DBL1α-varO domain does not increase viral replication. In MDM which presents surface CSA, both DBL domains strongly inhibit viral replication. The effect of DBL3γ-732 on HIV-1 replication is most likely mediated by TNF-α as this cytokine is significantly increased by DBL3γ-732 binding to BeWo cells and MDM.