Plasmodium falciparum cysteine protease Falcipain 3: A potential enzyme for proteolytic processing of histone acetyltransferase PfGCN5.

In spite of 150 years of studying malaria, the unique features of the malarial parasite, Plasmodium, still perplex researchers. One of the methods by which the parasite manages its gene expression is epigenetic regulation, the champion of which is PfGCN5, an essential enzyme responsible for acetylating histone proteins. PfGCN5 is a ∼170 kDa chromatin-remodeling enzyme that harbors the conserved bromodomain and acetyltransferase domain situated in its C-terminus domain. Although the PfGCN5 proteolytic processing is essential for its activity, the specific protease involved in this process still remains elusive. Identification of PfGCN5 interacting proteins through immunoprecipitation (IP) followed by LC-tandem mass spectrometry analysis revealed the presence of food vacuolar proteins, such as the cysteine protease Falcipain 3 (FP3), in addition to the typical members of the PfGCN5 complex. The direct interaction between FP3 and PfGCN5 was further validated by in vitro pull-down assay as well as IP assay. Subsequently, use of cysteine protease inhibitor E64d led to the inhibition of protease-specific processing of PfGCN5 with concomitant enrichment and co-localization of PfGCN5 and FP3 around the food vacuole as evidenced by confocal microscopy as well as electron microscopy. Remarkably, the proteolytic cleavage of the nuclear protein PfGCN5 by food vacuolar protease FP3 is exceptional and atypical in eukaryotic organisms. Targeting the proteolytic processing of GCN5 and the associated protease FP3 could provide a novel approach for drug development aimed at addressing the growing resistance of parasites to current antimalarial drugs.

Unusual weak and delayed GTPase activity of FtsZ from human pathogenic bacteria Helicobacter pylori.

Actively treadmilling FtsZ acts as the pivotal scaffold for bacterial cell divisome components providing them with a circumferential ride along the site of future division. FtsZ from slow growing Helicobacter pylori (HpFtsZ), a class I carcinogen which thrives abundantly in the acidic environment is poorly understood. We studied HpFtsZ as a function of pH, cations and time and compared it with well-studied E. coli FtsZ (EcFtsZ). HpFtsZ shows pH dependent GTPase activity which is inhibited under acidic conditions. Mg+2 ions play an indispensable role in its GTPase activity, however, higher Mg+2 levels negatively affect its activity. As compared to EcFtsZ, HpFtsZ exhibits lower and slower nucleotide hydrolyzing activity. Molecular Dynamics Simulation studies of FtsZ reveal that GTP binding induces a rewiring of the hydrogen bond network which results in reduction of the binding cleft volume leading to the spontaneous release of GTP. The GTPase activity is linked to the extent of reduction in the binding cleft volume, which is also supported by the binding free energy analysis. Evidently, HpFtsZ is a pH sensitive GTPase with low efficiency that may reflect on the overall slow growth rate of H. pylori.

Genome-wide binding sites of Plasmodium falciparum mini chromosome maintenance protein MCM6 show new insights into parasite DNA replication.

Multiple rounds of DNA replication take place in various stages of the life cycle in the human malaria parasite Plasmodium falciparum. Previous bioinformatics analysis has shown the presence of putative Autonomously Replicating Sequence (ARS) like sequences in the Plasmodium genome. However, the actual sites and frequency of replication origins in the P. falciparum genome based on experimental data still remain elusive. Minichromosome maintenance (MCM) proteins are recruited by the Origin recognition complex (ORC) to the origins of replication in eukaryotes including P. falciparum. We used PfMCM6 for chromatin immunoprecipitation followed by sequencing (ChIP-seq) in the quest for identification of putative replication origins in the parasite. PfMCM6 DNA binding sites annotation revealed high enrichment at exon regions. This is contrary to higher eukaryotes that show an inclination of origin sites towards transcriptional start sites. ChIP-seq results were further validated by ChIP-qPCR results as well as nascent strand abundance assay at the selected PfMCM6 enriched sites that also showed preferential binding of PfORC1 suggesting potential of these sites as origin sites. Further, PfMCM6 ChIP-seq data showed a positive correlation with previously published histone H4K8Ac genome-wide binding sites but not with H3K9Ac sites suggesting epigenetic control of replication initiation sites in the parasites. Overall, our data show the genome-wide distribution of PfMCM6 binding sites with their potential as replication origins in this deadly human pathogen that not only broadens our knowledge of parasite DNA replication and its unique biology, it may help to find new avenues for intervention processes.

Role of PfMYST in DNA replication in Plasmodium falciparum.

Chromatin modification through histone acetylation/deacetylation is important for the regulation of transcription as well as DNA replication in eukaryotes. PfGCN5 and PfMYST are two well-studied histone acetyltransferases in Plasmodium. PfMYST containing the MYST domain, zinc finger domain, and the chromodomain primarily acetylates histone 4. Here, we show that PfMYST is expressed in two isoforms, a long version (∼72 kDa) and a short version (∼45 kDa) of the protein, while the shorter version is predominantly present in the nucleus. Further, the association of PfMYST with the putative Plasmodium autonomously replicating sequences (PfARS) was found to be much stronger than the binding of PfGCN5 in these regions with concomitant enrichment of the H4 acetylation level. The binding of PfMYST at these sites was also correlated with another replication protein PfORC1 as well as with the replicating stage (trophozoite) of the parasite. Collectively these results show for the first time the potential role of PfMYST in parasite DNA replication through chromatin modification that may be found useful for the intervention of parasite growth.

The tetrameric structure of Plasmodium falciparum phosphoglycerate mutase is critical for optimal enzymatic activity.

The glycolytic enzyme phosphoglycerate mutase (PGM) is of utmost importance for overall cellular metabolism and has emerged as a novel therapeutic target in cancer cells. This enzyme is also conserved in the rapidly proliferating malarial parasite Plasmodium falciparum, which have a similar metabolic framework as cancer cells and rely on glycolysis as the sole energy-yielding process during intraerythrocytic development. There is no redundancy among the annotated PGM enzymes in Plasmodium, and PfPGM1 is absolutely required for the parasite survival as evidenced by conditional knockdown in our study. A detailed comparison of PfPGM1 with its counterparts followed by in-depth structure-function analysis revealed unique attributes of this parasitic protein. Here, we report for the first time the importance of oligomerization for the optimal functioning of the enzyme in vivo, as earlier studies in eukaryotes only focused on the effects in vitro. We show that single point mutation of the amino acid residue W68 led to complete loss of tetramerization and diminished catalytic activity in vitro. Additionally, ectopic expression of the WT PfPGM1 protein enhanced parasite growth, whereas the monomeric form of PfPGM1 failed to provide growth advantage. Furthermore, mutation of the evolutionarily conserved residue K100 led to a drastic reduction in enzymatic activity. The indispensable nature of this parasite enzyme highlights the potential of PfPGM1 as a therapeutic target against malaria, and targeting the interfacial residues critical for oligomerization can serve as a focal point for promising drug development strategies that may not be restricted to malaria only.

Role of unique loops in oligomerization and ATPase function of Plasmodium falciparum gyrase B.

DNA gyrase is an ATP dependent Type IIA topoisomerase that is unique to prokaryotes. Interestingly DNA gyrase has also been found in the apicoplasts of apicomplexan parasites like Plasmodium falciparum (Pf) the causative agent of Malaria. Gyrase B (GyrB), a subunit of gyrase A2 B2 complex has an N-terminal domain (GyrBN) which is endowed with ATPase activity. We reported earlier that PfGyrB exhibits ATP-independent dimerization unlike its bacterial counterparts. Here we report the role of two unique regions (L1 and L2) identified in PfGyrBN. Deletions of L1 alone (PfGyrBNΔL1), or L1 and L2 together (PfGyrBNΔL1ΔL2) have indicated that these regions may play an important role in ATPase activity and the oligomeric state of PfGyrBN. Our experiments show that the deletion of L1 region disrupts the dimer interface of PfGyrBN and reduces its ATPase activity. Further through ITC experiments we show that the binding affinity of ATP to PfGyrBN is reduced upon the deletion of L1 region. We have observed a reduction in ATPase activity for of all three proteins PfGyrBN, PfGyrBNΔL1, and PfGyrBNΔL1ΔL2 in presence of coumermycin. Our results suggests that L1 region of PfGyrBN is likely to be functionally important and may provide a unique dimer interface that affects its enzymatic activity. Since deletion of L1 region decreases the affinity of ATP to the protein, this region can be targeted toward designing novel inhibitors of ATP hydrolysis.

Antibacterial action of acriflavine hydrochloride for eradication of the gastric pathogen Helicobacter pylori.

Helicobacter pylori, a type 1 carcinogen, accounts for numerous gastric cancer-related deaths worldwide. Repurposing existing drugs or developing new ones for a combinatorial approach against increasing antimicrobial resistance is the need of the hour. This study highlights the efficacy of acriflavine hydrochloride (ACF-HCl) in inhibiting the growth of H. pylori reference strain and antibiotic-resistant clinical isolates at low concentrations. ACF-HCl inhibits H. pylori growth at MIC value 10 times less than that in Escherichia coli, another Gram-negative bacteria. Furthermore, ACF-HCl demonstrates synergistic effect with clarithromycin, a commonly used antibiotic against H. pylori. ACF-HCl treatment also eradicates H. pylori infection in the mice model efficiently. Our in vitro data indicate that bacterial membrane is the prime target. The novel action of ACF-HCl against antibiotic-resistant clinical isolates, synergistic effect with the conventional antibiotic clarithromycin and eradication of H. pylori from infected mice highlight the potential of ACF-HCl as a promising therapeutic agent against H. pylori by itself as well as for combinatorial therapy.

Synthesis of Novel Ciprofloxacin-Based Hybrid Molecules toward Potent Antimalarial Activity.

Antimalarial drug resistance is a serious obstacle in the persistent quest to eradicate malaria. There is a need for potent chemical agents that are able to act on drug-resistant Plasmodium falciparum populations at reasonable concentrations without any related toxicity to the host. By rational drug design, we envisaged to address this issue by generating a novel hybrid drug possessing two pharmacophores that can act on two unique and independent targets within the cell. We synthesized a new class of ciprofloxacin-based hybrid molecules, which have been integrated with acridine, quinolone, sulphonamide, and cinnamoyl pharmacophores (1-4). We realized a potent chloroquinolone-ciprofloxacin-based antimalarial hybrid (2, CQ-CFX) whose mechanism of action is unlike that of its parent molecules indicating a unique biological target. CQ-CFX is not only potent against CQ-resistant and susceptible strains of Plasmodium falciparum at low nanomolar concentrations (IC50 values are 63.17 ± 1.2 nM and 25.52 ± 4.45 nM, respectively) but is also not toxic to mammalian and bacterial systems up to 20 µM and 1 µM, respectively.

Potent in vivo antimalarial activity of water-soluble artemisinin nano-preparations.

Artemisinin is a remarkable compound whose derivatives and combinations with multiple drugs have been utilized at the forefront of malaria treatment. However, the inherent issues of the parent compound such as poor bioavailability, short serum half-life, and high first-pass metabolism partially limit further applications of this drug. In this study, we enhanced the aqueous phase solubility of artemisinin by encapsulating it in two nanocarriers based on the polymer polycaprolactone (ART-PCL) and lipid-based Large Unilamellar Vesicles (ART-LIPO) respectively. Both nanoformulations exhibit in vitro parasite killing activity against Plasmodium falciparum with the ART-LIPO performing at comparable efficacy to the control drug solubilized in ethanol. These water-soluble formulations showed potent in vivo antimalarial activity as well in the mouse model of malaria at equivalent doses of the parent drug. Additionally, the artemisinin-PCL nanoformulation used in combination with either pyrimethamine or chloroquine increased the survival of the Plasmodium berghei infected mice for more than 34 days and effectively cured the mice of the infection. We highlight the potential for polymer and liposome-based nanocarriers in improving not only the aqueous phase solubility of artemisinin but also concomitantly retaining its therapeutic efficacy in vivo as well.

Plasmodium falciparum GCN5 acetyltransferase follows a novel proteolytic processing pathway that is essential for its function.

The pathogenesis of human malarial parasite Plasmodium falciparum is interlinked with its timely control of gene expression during its complex life cycle. In this organism, gene expression is partially controlled through epigenetic mechanisms, the regulation of which is, hence, of paramount importance to the parasite. The P. falciparum (Pf)-GCN5 histone acetyltransferase (HAT), an essential enzyme, acetylates histone 3 and regulates global gene expression in the parasite. Here, we show the existence of a novel proteolytic processing for PfGCN5 that is crucial for its activity in vivo We find that a cysteine protease-like enzyme is required for the processing of PfGCN5 protein. Immunofluorescence and immuno-electron microscopy analysis suggest that the processing event occurs in the vicinity of the digestive vacuole of the parasite following its trafficking through the classical ER-Golgi secretory pathway, before it subsequently reaches the nucleus. Furthermore, blocking of PfGCN5 processing leads to the concomitant reduction of its occupancy at the gene promoters and a reduced H3K9 acetylation level at these promoters, highlighting the important correlation between the processing event and PfGCN5 activity. Altogether, our study reveals a unique processing event for a nuclear protein PfGCN5 with unforeseen role of a food vacuolar cysteine protease. This leads to a possibility of the development of new antimalarials against these targets.This article has an associated First Person interview with the first author of the paper.

Helicobacter pylori helicase loader protein Hp0897 shows unique functions of N- and C-terminal regions.

Helicase loaders are required for the loading of helicases at the vicinity of replication origins. In Helicobacter pylori, Hp0897 has been shown to be a potential helicase loader for replicative helicase (HpDnaB) although it does not show any sequence homology with conventional DnaC like helicase loader proteins. Therefore, it is important to investigate the in vivo role of Hp0897 and structure-function analysis with respect to domain mapping of Hp0897 and HpDnaB. Although HporiC is divided into oriC1 and oriC2, the latter has been assigned as functional origin based on loading of initiator protein HpDnaA. Using chromatin immunoprecipitation (ChIP) experiment, we show preferential binding of Hp0897 at oriC2 over oriC1 like HpDnaA highlighting its helicase loader function in vivo. Furthermore, we generated series of deletion mutants for HpDnaB and Hp0897 that enabled us to map the domains of interaction between these two proteins. Interestingly, the C-terminal domain of Hp0897 (Hp0897CTD) shows stronger interaction with HpDnaB over the N-terminal region of Hp0897 (Hp0897NTD). Similar to the full-length protein, Hp0897CTD also stimulates the DNA binding activity of HpDnaB. Furthermore, overexpression of Hp0897 full-length protein in H. pylori leads to an elongated cell phenotype. While the overexpression of Hp0897CTD does not show a phenotype of cell elongation, overexpression of Hp0897NTD shows extensive cell elongation. These results highlight the possible role of Hp0897CTD in helicase loading and Hp0897NTD's unique function linked to cell division that make Hp0897 as a potential drug target against H. pylori.

In silico identification of inhibitors against Plasmodium falciparum histone deacetylase 1 (PfHDAC-1).

In erythrocytes, actively multiplying Plasmodium falciparum parasites exhibit a unique signature of virulence associated histone modifications, thereby epigenetically regulating the expression of the majority of genes. Histone acetylation is one such modification, effectuated and maintained by the dynamic interplay of two functionally antagonist enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs). Their inhibition leads to hypo/hyperacetylation and is known to be deleterious for P. falciparum, and hence they have become attractive molecular targets to design novel antimalarials. Many compounds, including four Food and Drug Administration (FDA) approved drugs, have been developed so far to inhibit HDAC activity but are not suitable to treat malaria as they lack selectivity and cause cytotoxicity in mammalian cells. In this study, we used comparative modeling and molecular docking to establish different binding modes of nonselective and selective compounds in the PfHDAC-1 (a class I HDAC protein in P. falciparum) active site and identified the involvement of active site nonidentical residues in binding of selective compounds. Further, we have applied virtual screening with precise selection criteria and molecular dynamics simulation to identify novel potential inhibitors against PfHDAC-1. We report 20 compounds (10 from ChEMBL and 10 from analogues compound library) bearing seven scaffolds having better affinity toward PfHDAC-1. Sixteen of these compounds are known antimalarials with 14 having activity in the nanomolar range against various drug resistant and sensitive strains of P. falciparum. The cytotoxicity of these compounds against various human cell lines are reported at relatively higher concentration and hence can be used as potential PfHDAC-1 inhibitors in P. falciparum. These findings indeed show great potential for using the above molecules as prospective antimalarials.

Role of tyrosine residue (Y213) in nuclear retention of PCNA1 in human malaria parasite Plasmodium falciparum.

Proliferating Cell Nuclear Antigen (PCNA) undergoes several post-translational modifications including phosphorylation leading to its regulation in mammalian and yeast systems. Plasmodium falciparum possesses two PCNAs (PCNA1 & PCNA2) with an edge of PfPCNA1 over PfPCNA2 for DNA replication. Recent phospho-proteome data report phosphorylation of S191 residue without its functional implication. In mammalian cells, phosphorylation of HsPCNA at Y211 stabilizes chromatin bound PCNA. We find tyrosine (but not S191) to be conserved in PfPCNAs and it is important for its nuclear localization and foci formation of PfPCNA1. Further, a Y213F mutation in PfPCNA1 leads to its functional loss both in yeast and parasite. We highlight the importance of evolutionarily conserved tyrosine in PCNA from parasite to mammal linked with DNA replication and cell proliferation.

Identification of a novel trafficking pathway exporting a replication protein, Orc2 to nucleus via classical secretory pathway in Plasmodium falciparum.

Malaria parasites use an extensive secretory pathway to traffic a number of proteins within itself and beyond. In higher eukaryotes, Endoplasmic Reticulum (ER) membrane bound transcription factors such as SREBP are reported to get processed en route and migrate to nucleus under the influence of specific cues. However, a protein constitutively trafficked to the nucleus via classical secretory pathway has not been reported. Herein, we report the presence of a novel trafficking pathway in an apicomplexan, Plasmodium falciparum where a homologue of an Origin Recognition Complex 2 (Orc2) goes to the nucleus following its association with the ER. Our work highlights the unconventional role of ER in protein trafficking and reports for the first time an ORC homologue getting trafficked through such a pathway to the nucleus where it may be involved in DNA replication and other ancillary functions. Such trafficking pathways may have a profound impact on the cell biology of a malaria parasite and have significant implications in strategizing new antimalarials.

Screening of E. coli ß-clamp Inhibitors Revealed that Few Inhibit Helicobacter pylori More Effectively: Structural and Functional Characterization.

The characteristic of interaction with various enzymes and processivity-promoting nature during DNA replication makes ß-clamp an important drug target. Helicobacter pylori (H. pylori) have several unique features in DNA replication machinery that makes it different from other microorganisms. To find out whether difference in DNA replication proteins behavior accounts for any difference in drug response when compared to E. coli, in the present study, we have tested E. coli ß-clamp inhibitor molecules against H. pylori ß-clamp. Various approaches were used to test the binding of inhibitors to H. pylori ß-clamp including docking, surface competition assay, complex structure determination, as well as antimicrobial assay. Out of five shortlisted inhibitor molecules on the basis of docking score, three molecules, 5-chloroisatin, carprofen, and 3,4-difluorobenzamide were co-crystallized with H. pylori ß-clamp and the structures show that they bind at the protein-protein interaction site as expected. In vivo studies showed only two molecules, 5-chloroisatin, and 3,4-difluorobenzamide inhibited the growth of the pylori with MIC values in micro molar range, which is better than the inhibitory effect of the same drugs on E. coli. Therefore, the evaluation of such drugs against H. pylori may explore the possibility to use to generate species-specific pharmacophore for development of new drugs against H. pylori.

Role of Chromatin assembly factor 1 in DNA replication of Plasmodium falciparum.

Nucleosome assembly in P. falciparum could be the key process in maintaining its genomic integrity as DNA replicates more than once per cell cycle during several stages of its life cycle. Here, we report the functional characterization of P. falciparum chromatin assembly factor 1 (CAF1), which interacts with several proteins namely PfCAF2, Histones, PfHP1 and others. Consistent with the above findings, we demonstrate the presence of PfCAF1 at the telomeric repeat regions, central and subtelomeric var genes of multiple var gene family along with PfHP1. Further, we report the upregulation of PfCAF1 after treatment with genotoxic agents like MMS and HU. Together, these findings establish role of PfCAF1 in heterochromatin maintenance and as histone chaperone in nucleosome assembly and DNA damage repair.

Targeting the ß-clamp in Helicobacter pylori with FDA-approved drugs reveals micromolar inhibition by diflunisal.

The ß-clamp is the processivity-promoting factor for most of the enzymes in prokaryotic DNA replication; hence, it is a crucial drug target. In the present study, we investigated the ß-clamp from Helicobacter pylori, aiming to seek potential drug molecules against this gastric-cancer-causing bacterium. An in silico screening of Food and Drug Administration (FDA) approved drugs against the H. pylori ß-clamp, followed by its in vitro inhibition using a surface competition approach, yielded the drug diflunisal as a positive initial hit. Diflunisal inhibits the growth of H. pylori in the micromolar range. We determined the structure of diflunisal in complex with the ß-clamp to show that the drug binds at subsite I, which is a protein-protein interaction site. Successful identification of FDA-approved molecules against H. pylori may lead to better and faster drug development.

Identification and characterization of ARS-like sequences as putative origin(s) of replication in human malaria parasite Plasmodium falciparum.

DNA replication is a fundamental process in genome maintenance, and initiates from several genomic sites (origins) in eukaryotes. In Saccharomyces cerevisiae, conserved sequences known as autonomously replicating sequences (ARSs) provide a landing pad for the origin recognition complex (ORC), leading to replication initiation. Although origins from higher eukaryotes share some common sequence features, the definitive genomic organization of these sites remains elusive. The human malaria parasite Plasmodium falciparum undergoes multiple rounds of DNA replication; therefore, control of initiation events is crucial to ensure proper replication. However, the sites of DNA replication initiation and the mechanism by which replication is initiated are poorly understood. Here, we have identified and characterized putative origins in P. falciparum by bioinformatics analyses and experimental approaches. An autocorrelation measure method was initially used to search for regions with marked fluctuation (dips) in the chromosome, which we hypothesized might contain potential origins. Indeed, S. cerevisiae ARS consensus sequences were found in dip regions. Several of these P. falciparum sequences were validated with chromatin immunoprecipitation-quantitative PCR, nascent strand abundance and a plasmid stability assay. Subsequently, the same sequences were used in yeast to confirm their potential as origins in vivo. Our results identify the presence of functional ARSs in P. falciparum and provide meaningful insights into replication origins in these deadly parasites. These data could be useful in designing transgenic vectors with improved stability for transfection in P. falciparum.

Designing novel inhibitors against histone acetyltransferase (HAT: GCN5) of Plasmodium falciparum.

During active proliferation phase of intra-erythrocytic cycle, the genome of P. falciparum is regulated epigenetically and evolutionary conserved parasite-specific histone proteins are extensively acetylated. The reversible process of lysine acetylation, causing transcriptional activation and its deacetylation, causing transcriptional repression is regulated by balanced activities of HATs and HDACs. They are also known to regulate antigenic variations and gametocytic conversion in P. falciparum. These histone modifying enzymes have been identified as potential targets for development of anitmalarials in literature. PfGCN5, a HAT family member of P. falciparum is predominantly involved in H3K9 acetylation. In this study, through comparative structure and sequence analysis, we elucidate differences in the catalytic pocket of PfGCN5 which can be exploited to design selective inhibitors. Through virtual screening of known antimalarials from ChEMBL bioassay database, we mapped 10 compounds with better affinity towards PfGCN5. Further, we identified 10 more novel compounds which showed remarkably better affinity towards the Plasmodium target from analogues of mapped inhibitors from ZINC database of commercially available compounds. Comparative molecular dynamics simulation study of one of the compounds (C14) complex with PfGCN5 and HsGCN5 suggested the possible reason for its selectivity. In vitro parasite growth assay in the presence of C14 showed IC50 value at lower nanomolar range (∼ 225 nM). However, no effect in mammalian fibroblast cells was observed for C14 (up to 20 µM). Further, reduced level of HAT activity of recombinant GCN5 and H3K9Ac was observed in the parasites treated with C14. Overall, this study reports 20 potential inhibitors of PfGCN5 and experimental validation of one molecule (C14) with antimalarial activity at low nanomolar range.

'Modulation of the enzymatic activities of replicative helicase (DnaB) by interaction with Hp0897: a possible mechanism for helicase loading in Helicobacter pylori'.

DNA replication in Helicobacter pylori is initiated from a unique site (oriC) on its chromosome where several proteins assemble to form a functional replisome. The assembly of H. pylori replication machinery is similar to that of the model gram negative bacterium Escherichia coli except for the absence of DnaC needed to recruit the hexameric DnaB helicase at the replisome assembly site. In the absence of an obvious DnaC homologue inH. pylori, the question arises as to whether HpDnaB helicase is loaded at theHp-replication origin by itself or is assisted by other unidentified protein(s). A high-throughput yeast two-hybrid study has revealed two proteins of unknown functions (Hp0897 and Hp0340) that interact with HpDnaB. Here we demonstrate that Hp0897 interacts with HpDnaB helicase in vitro as well as in vivo Furthermore, the interaction stimulates the DNA binding activity of HpDnaB and modulates its adenosine triphosphate hydrolysis and helicase activities significantly. Prior complex formation of Hp0897 and HpDnaB enhances the binding/loading of DnaB onto DNA. Hp0897, along with HpDnaB, colocalizes with replication complex at initiation but does not move with the replisome during elongation. Together, these results suggest a possible role of Hp0897 in loading of HpDnaB at oriC.