ParA superfamily ͉ polymerization ͉ plasmid partition ͉ ATPase T he precise distribution of newly replicated genomes to progeny cells is imperative for stable transmission of genetic information. In bacteria, the most well characterized segregation mechanisms are specified by low-copy-number plasmids. These systems most frequently comprise two plasmid-encoded proteins, often termed ParA and ParB, that assemble on a cis-acting centromeric site. ParB directly binds the centromere, whereas ParA is recruited by interactions with ParB. The resulting segrosome complex is a positioning apparatus that localizes the attached plasmids to specific subcellular addresses (1, 2).The segregation locus of multidrug-resistance plasmid TP228 in Escherichia coli consists of the parF and parG genes and nearby parH centromere (3). ParG (8.6 kDa) is the prototype of a class of small proteins involved in accurate segregation that are unrelated phylogenetically to ParB, but that fulfil analogous functions as centromere-binding factors (1,4,5). ParG is dimeric, with symmetric C-terminal domains that interleave into a ribbon-helixhelix fold that is crucial for DNA binding, and unstructured N-terminal tails (4, 6). Additional to its role as a centromerebinding protein, ParG is a transcriptional repressor of the parFG genes: transient associations between the flexible and folded domains in complex with target DNA modulate organization of a higher-order complex critical for transcriptional repression (7).The ParA superfamily of ATPases, widely encoded by both chromosomes and plasmids, is characterized by a variant Walkertype ATP-binding motif (8). ParF (22.0 kDa) epitomizes one clade of the superfamily (3). In common with other ParA proteins, ParF is a weak ATPase whose nucleotide hydrolysis is enhanced Ϸ30-fold by ParG (9). ATP binding and/or hydrolysis by ParA proteins has long been recognized as a crucial facet of the segregation process, although its mechanistic purpose was uncertain (10-12). We have recently shown that ATP binding stimulates the polymerization of ParF into extensive multistranded filaments, whereas ADP antagonizes filamentation. ParG is another key modulator of polymerization (9). Mutagenesis of the ATP-binding site in Parf perturbed DNA segregation in vivo, ATP hydrolysis, and polymerization. We envisage that segrosome formation is initiated by site-specific binding of ParG to parH, generating paired complexes of specific topology. ParF is then recruited. ParF polymerization within the complex is controlled by nucleotide binding, by ParG-mediated stimulation of ATP hydrolysis, by remodeling effects of ParG, and, more speculatively, by cell cycle signals. Polymerization, or depolymerization, invokes separation of paired plasmids and their segregation in opposite poleward directions (1, 9).Arginine fingers stimulate nucleotide hydrolysis by NTPases through the action of an arginine side chain inserted into the catalytic niche (13,14). The arginine stabilizes the transition state through neutralization of negative charge...

ParG is the prototype of a group of small (<10 kDa) proteins involved in accurate plasmid segregation. The protein is a dimeric DNA binding factor, which consists of symmetric paired C-terminal domains that interleave into a ribbon-helix-helix fold that is crucial for the inter-

SUMMARYThe high variability among strains and isolates of Trypanosoma cruzi and the existence of shared antigenic determinants with other pathogens, particularly with members of the Leishmania genus make difficult the specific diagnosis of Chagas' disease. The data reported in this paper show that the T. cruzi KMP11 protein is an immunodominant antigen highly recognized by the sera from chagasic and leishmaniasis patients. By the use of amino-and carboxyl-terminal truncated KMP11 recombinant proteins and synthetic peptides, evidence is provided that while the sera from chagasic patients recognize linear peptides the sera from patients with visceral leishmaniasis must be predominantly directed against conformational epitopes. We found that a particular linear determinant, located in the carboxyl-terminal region of the protein, is recognized with high specificity and sensitivity only by sera from Chagas' disease patients, suggesting it could be a good candidate for differential serodiagnosis of Chagas' disease.

The humoral immune response against Leishmania braziliensis histone H1 by patients with cutaneous leishmaniasis is described. For this purpose, the protein was purified as a recombinant protein in a prokaryotic expression system and was assayed by enzyme-linked immunosorbent assay (ELISA) with a collection of sera from patients with cutaneous leishmaniasis and Chagas' disease. The assays showed that L. braziliensis histone H1 was recognized by 66% of the serum samples from patients with leishmaniasis and by 40% of the serum samples from patients with Chagas' disease, indicating that it acts as an immunogen during cutaneous leishmaniasis. In order to locate the linear antigenic determinants of this protein, a collection of synthetic peptides covering the L. braziliensis histone H1sequence was tested by ELISA. The experiments showed that the main antigenic determinant is located in the central region of this protein. Our results show that the recombinant L. braziliensis histone H1 is recognized by a significant percentage of serum samples from patients with cutaneous leishmaniasis, but use of this protein as a tool for the diagnosis of cutaneous leishmaniasis is hampered by the cross-reaction with sera from patients with Chagas' disease.Histones are evolutionarily conserved proteins which associate with DNA to form the chromatin structural unit in eukaryotes, the nucleosome. The name histone H1 is applied to a family of small basic proteins which take part in the stabilization of the nucleosomes and facilitate the assembly of chromatin into higher-order structures. Histone H1 proteins have been described in different trypanosomatids like Crithidia fasciculata (7), Trypanosoma cruzi (1), Trypanosoma brucei (4), Leishmania major (9), and Leishmania braziliensis (13). All of these H1 proteins are smaller than their counterparts from higher eukaryotes due to their lack of a central globular domain. This fact has been related to the imperfect condensation of chromatin in trypanosomatid chromosomes during cell division (8).The first report of the elicitation of a humoral immune response against parasite histones during infection was made in 1995, in which a response against Leishmania infantum H2A during canine visceral leishmaniasis (CVL) was described (16). Similar responses against histone H3, histone H2B, and a fragment of histone H4 from L. infantum were described thereafter (17, 18). The investigators mapped the linear epitopes of histones using synthetic peptides. Their findings led to the conclusion that the humoral response against the L. infantum histones during CVL was triggered by the less conserved regions of the molecule, which correspond to the amino-and carboxy-terminal ends of the protein (15).The term leishmaniasis is applied to a spectrum of diseases caused by different species of the genus Leishmania. This parasite is endemic in 88 countries worldwide, and 350 million people are considered to be at risk of leishmaniasis. Of the 2 million new cases discovered every year, about 1.5 million are cutaneou...

Leishmania spp. is a protozoan parasite that affects millions of people around the world. At present, there is no effective vaccine to prevent leishmaniases in humans. A major limitation in vaccine development is the lack of precise understanding of the particular immunological mechanisms that allow parasite survival in the host. The parasite-host cell interaction induces dramatic changes in transcriptome patterns in both organisms, therefore, a detailed analysis of gene expression in infected tissues will contribute to the evaluation of drug and vaccine candidates, the identification of potential biomarkers, and the understanding of the immunological pathways that lead to protection or progression of disease. In this large-scale analysis, differential expression of 112 immune-related genes has been analyzed using high-throughput qPCR in spleens of infected and naïve Balb/c mice at four different time points. This analysis revealed that early response against Leishmania infection is characterized by the upregulation of Th1 markers and M1-macrophage activation molecules such as Ifng, Stat1, Cxcl9, Cxcl10, Ccr5, Cxcr3, Xcl1, and Ccl3. This activation doesn't protect spleen from infection, since parasitic burden rises along time. This marked difference in gene expression between infected and control mice disappears during intermediate stages of infection, probably related to the strong anti-inflammatory and immunosuppresory signals that are activated early upon infection (Ctla4) or remain activated throughout the experiment (Il18bp). The overexpression of these Th1/M1 markers is restored later in the chronic phase (8 wpi), suggesting the generation of a classical “protective response” against leishmaniasis. Nonetheless, the parasitic burden rockets at this timepoint. This apparent contradiction can be explained by the generation of a regulatory immune response characterized by overexpression of Ifng, Tnfa, Il10, and downregulation Il4 that counteracts the Th1/M1 response. This large pool of data was also used to identify potential biomarkers of infection and parasitic burden in spleen, on the bases of two different regression models. Given the results, gene expression signature analysis appears as a useful tool to identify mechanisms involved in disease outcome and to establish a rational approach for the identification of potential biomarkers useful for monitoring disease progression, new therapies or vaccine development.