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The glucose transporter PfHT is essential to the survival of the malaria parasite Plasmodium falciparum and has been shown to be a druggable target with high potential for pharmacological intervention. Identification of compounds against novel drug targets is crucial to combating resistance against current therapeutics. Here, we describe the development of a cell-based assay system readily adaptable to high-throughput screening that directly measures compound effects on PfHT-mediated glucose transport. Intracellular glucose concentrations are detected using a genetically encoded fluorescence resonance energy transfer (FRET)-based glucose sensor. This allows assessment of the ability of small molecules to inhibit glucose uptake with high accuracy (Z' factor of >0.8), thereby eliminating the need for radiolabeled substrates. Furthermore, we have adapted this assay to counterscreen PfHT hits against the human orthologues GLUT1, -2, -3, and -4. We report the identification of several hits after screening the Medicines for Malaria Venture (MMV) Malaria Box, a library of 400 compounds known to inhibit erythrocytic development of P. falciparum Hit compounds were characterized by determining the half-maximal inhibitory concentration (IC 50 ) for the uptake of radiolabeled glucose into isolated P. falciparum parasites. One of our hits, compound MMV009085, shows high potency and orthologue selectivity, thereby successfully validating our assay for antimalarial screening., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Borrelia burgdorferi produces extracellular vesicles containing various borrelial protein antigens when propagated in vitro in culture media. Commonly observed components of borrelial vesicle preparations are borrelial surface antigens, bovine serum albumin, and the heavy chains of rabbit immunoglobulin G and immunoglobulin M. This study employed ultracentrifugation to harvest borrelial vesicles and analyzed these preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting. We demonstrated that the rabbit mu heavy-chain band observed was devoid of OspA or at most levels below those detectable by immunoblot. We also demonstrated the recovery of borrelial vesicles at relative centrifugal forces as low as 25,000 x g, compared with the force of > 200,000 x g normally employed. Further, the mu heavy-chain band was recovered from uninoculated growth media processed at 25,000 x g, suggesting that it behaves as a particle rather than as a soluble molecule under these conditions. Lastly, vesicles were demonstrated to be present in preparations harvested from growth media supplemented with fetal calf serum, suggesting that vesicle production by B. burgdorferi can occur in the absence of immunoglobulins.

All Borrelia burgdorferi sensu lato isolates characterized to date have one or a combination of several major outer surface proteins (Osps). Mutants of B. burgdorferi lacking Osps were selected with polyclonal or monoclonal antibodies at a frequency of 10(-6) to 10(-5). One mutant that lacked OspA, -B, -C, and -D was further characterized. It was distinguished from the OspA+B+ cells by its (i) autoaggregation and slower growth rate, (ii) decreased plating efficiency on solid medium, (iii) serum and complement sensitivity, and (iv) diminished capacity to adhere to human umbilical vein endothelial cells. The Osp-less mutant was unable to evoke a detectable immune response after intradermal live cell immunization even though mutant survived in mouse skin for the same duration as wild-type cells. Polyclonal mouse serum raised against Osp-less cells inhibited growth of the mutant but not of wild-type cells, an indication that other antigens are present on the surface of the Osp-less mutant. Two types of monoclonal antibodies (MAbs) with growth-inhibiting properties for mutant cells were identified. The first type bound to a 13-kDa surface protein of B. burgdorferi sensu stricto and of B. afzelii. The MIC of the Fab fragment of one MAb of this type was 0.2 micrograms/ml. The second type of MAb to the Osp-less mutant did not bind to B. burgdorferi components by Western blotting (immunoblotting) but did not bind to unfixed, viable cells in immunofluorescence and growth inhibition assays. These studies revealed possible functions Osp proteins in borrelias, specifically serum resistance, and indicated that in the absence of Osp proteins, other antigens are expressed or become accessible at the cell surface.

The outer surface lipoprotein B (OspB) of Borrelia burgdorferi is a major component of the borrelial protein profile and has been shown to be highly immunogenic in experimentally immunized and infected mammals. However, the ospB loci of different strains show considerable heterology at the nucleic acid sequence level, and the progeny of a clonal strain of B. burgdorferi exhibited OspB polymorphisms with respect to apparent molecular weights and reactivities with monoclonal antibodies. These data suggest that OspB is not a good candidate for vaccination or diagnostic purposes. The present study describes a monoclonal antibody, designated 84C, directed against a very highly conserved domain of the OspB lipoprotein. Western immunoblot analysis with 84C demonstrated reactivity in 84.2% of human, tick, and other vertebrate isolate strains examined from widely diverse geographic regions, including strains of B. burgdorferi sensu stricto and two closely related species, B. garinii and B. afzelii. The 84C-binding region was delimited to a highly conserved 11-amino-acid region in the carboxyl terminus of OspB as demonstrated by (i) DNA sequence analysis of wild-type and 84C-resistant mutant ospB alleles and (ii) deletion mutagenesis of a recombinant ospB gene in Escherichia coli. Finally, the 84C epitope was demonstrated to be exposed on the borrelial surface in situ as (i) the monoclonal antibody 84C was able to agglutinate borrelias in culture and (ii) 84C-resistant escape variants were isolated. These data suggest that the potential value of OspB as a vaccine candidate or diagnostic tool be examined more closely, in the context of the 84C-reactive domain.

Most Borrelia burgdorferi strains have two major surface proteins, OspA and OspB. In the present study, we selected from a clonal population of infectious B. burgdorferi an OspB escape mutant, identified the genetic basis for this phenotype, and evaluated its functional activities. Selection with the anti-OspB antibody H614 was performed in vitro in medium and extended in vivo in scid mice. Mutants with a truncated OspB protein were selected at a frequency of 1 x 10(-5) to 3 x 10(-5). After no major rearrangements in DNA were detected, sequence analysis of the mutant's ospAB locus revealed a single base change in the consensus ribosomal binding sequence for ospB and a single nucleotide deletion in the ospB gene itself. The effect of these mutations was reduced expression of a truncated OspB protein. When functional abilities of the wild type and mutant were compared, the mutant had a threefold-lower capacity to penetrate a human endothelium umbilical vein cell monolayer. Infectivity of wild-type and mutant cells for scid mice was evaluated by culturing different organs, and the median infectious dose was calculated. The inoculum of mutant cells for infecting the mice was 30- to 300-fold higher than that of wild-type cells. This study shows that reduced size and expression of OspB are associated with lowered virulence of B. burgdorferi. Selection of mutants that to some degree remain infectious is one approach to defining the role of different surface proteins in the pathogenesis of Lyme disease.

Borrelia burgdorferi produces extracellular vesicles which contain some of the outer surface proteins of the bacterium (e.g., OspA and OspB). Borrelial vesicles, isolated by differential centrifugation and filtration, were tested for the ability to bind to cultured human umbilical vein endothelial (HUVE) cells in culture. The recently described lipoprotein OspD was expressed on vesicles. Vesicles exhibited differential expression of OspB and OspD in a relationship with passage number and medium serum supplement type, respectively. Qualitative immunoblotting analyses demonstrated dose-dependent, passage number-dependent adsorption of vesicles by HUVE cells. This adsorption was demonstrated to be dependent upon a borrelial component of the vesicle and not due to the presence of minor contamination with intact spirochetes. Quantitative experiments examining inhibition of B. burgdorferi-HUVE association as a function of prior vesicle-HUVE association demonstrated dependence upon (i) a borrelial component(s) in the vesicle, (ii) low passage number, and (iii) vesicle protein concentration. However, vesicle pretreatment of the HUVE cell monolayer was not requisite for this inhibition. Vesicles from highly passaged borrelias were noninhibitory for B. burgdorferi-HUVE cell association, regardless of the serum used to supplement the medium. The use of vesicles as a tool for studying B. burgdorferi pathogenesis and/or physiology is proposed.

Previously, it has been shown that polyclonal antibodies to Borrelia burgdorferi and some monoclonal antibodies (MAbs) to borrelia major surface proteins caused inhibition of adherence of the bacteria to cultured human umbilical vein endothelial (HUVE) cells. In this study, fragment antigen binding (Fab) molecules generated from the immunoglobulin G fraction of rabbit anti-recombinant OspA serum were found to inhibit the adherence of B. burgdorferi to HUVE cells by 73%. Subsequently, MAbs were generated for use in determining whether or how B. burgdorferi outer surface proteins (Osps) A and/or B are involved in mediating attachment to, and/or invasion of, HUVE cells by B. burgdorferi. Twenty-two MAbs were generated to borrelial proteins with apparent molecular masses (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 19, 31 (OspA), 34 (OspB), and 35 kDa. Fab molecules from one anti-OspA MAb, 9B3D, demonstrated an inhibitory effect on bacterial association with HUVE cells. None of the other MAbs, including the other anti-OspA MAbs, showed an inhibitory effect on cell association of greater than 5%. This effect of Fab 9B3D was concentration dependent and plateaued at approximately 6 micrograms of Fab per ml (nearly 80% inhibition of the bacterial association with the monolayer). Penetration assays and cell association experiments performed by using immunofluorescence also suggested that the inhibitory action of 9B3D occurs at the level of adherence. MAb 9B3D recognized the OspA of every North American strain tested (n = 19) but only 3 [corrected] of 20 strains from western Europe, Russia, and Japan, suggesting that the North American strains and strains from other parts of the world may use different molecules and/or different OspA epitopes to interact with endothelial cells. Immunoblots of Escherichia coli expressing different OspA fusion peptides suggested that the 9B3D epitope resides in the carboxy-terminal half of OspA. MAb 9B3D promises to be a valuable tool for elucidating the domain or domains of OspA involved in the endothelial cell cytadherence of North American strains of B. burgdorferi.

Spirochetes that share pathogen-restricted antigens with Treponema pallidum subsp. pallidum have been identified in dental plaque and diseased gingival tissues, but it is not known whether these spirochetes possess virulence characteristics. In this study, plaque spirochetes were able to transmigrate a tissue barrier in vitro and were identified on the other side by using monoclonal antibodies specific for pathogen-restricted determinants from T. pallidum subsp. pallidum. This invasive capability is shared with T. pallidum subsp. pallidum, but cultured oral and intestinal treponemes did not perforate the tissue barrier. Cocultures indicated that invasive treponemes do not create opportunities for cultivable oral treponemes to cross the barrier. These findings indicate that gingival tissues may be a port of entry for previously unrecognized invasive spirochetes in humans.

The genomic structures of spirochete species are not well characterized, and genetic studies on these organisms have been hampered by lack of a genetic exchange mechanism in these bacteria. In view of these observations, pulsed-field gel electrophoresis was used to examine the genomes of Leptospira species. Live cells, prepared in agarose plugs, were lysed in situ, and the DNA was analyzed under different electrophoretic conditions. Pulsed-field gel electrophoresis of DNA digested with infrequently cutting restriction enzymes showed that the genome of Leptospira interrogans serovar canicola is approximately 3.1 Mb, while that of the saprophytic L. biflexa serovar patoc I is 3.5 Mb. DNA forms of approximately 2,000 and 350 kb which were present in samples from L. interrogans serovars were not readily detected in nonpathogenic serovars. Three distinct populations, designated type alpha, beta, and gamma, of L. interrogans DNA molecules were further analyzed with two-dimensional gel electrophoresis. Evidence suggested that two of these DNA forms, type alpha and gamma, were linear structures. Pulsed-field gel electrophoresis has proven to be a valuable tool with which to size bacterial genomes and to take the first steps toward characterization of a form of leptospiral DNA which behaves as a linear molecule and which may be related to the virulence of L. interrogans.

Interactions of Leptospira interrogans with cultured endothelial and kidney epithelial cells were assayed by examining (i) cytoadherence of intrinsically radiolabeled leptospires to eucaryotic cell monolayers and (ii) penetration of leptospires through cell monolayers grown on polycarbonate filters in chemotaxis chambers. L. interrogans serovars attached to cultured cells in a dose- and time-dependent manner. Adherence was diminished following pretreatment of organisms with proteases, rabbit immune serum, or heat. When observed by scanning electron microscopy, most leptospires attached by both ends, rather than just one tip like Treponema pallidum. In penetration assays, 9.7% of added L. interrogans migrated through the monolayer-filter barrier, while only 0.3% of L. biflexa penetrated in the same time interval. Transmission electron microscopy revealed that organisms entered the host cell cytoplasm. These in vitro results indicate that leptospires have an invasive capacity that may be related to pathogenicity in vivo and suggest that further investigation of interactions with host cells may enhance knowledge of leptospiral virulence.

A new drug resistance plasmid-dependent RNA containing phage resembling coliphage f2 in its particle size and density is described. The phage, PRR1, will only productively infect some R(+) hosts containing the Pseudomonas drug resistance plasmid R1822. The membrane filter-salt elution patterns, RNase sensitivity, inactivation in low ionic strength solutions, and host range serve to distinguish PRR1 from coliphage f2 and two other Pseudomonas RNA phages, 7s and PP7.

The effect of Escherichia coli heat-stable (ST) enterotoxin on calcium and cyclic nucleotide metabolism in rat basophilic leukemia cell cultures was investigated. Addition of ST enterotoxin to rat basophilic leukemia cell cultures resulted in dose- and time-dependent stimulation of calcium uptake and elevation of the intracellular cyclic GMP (cGMP) concentration. The effect of ST enterotoxin on calcium uptake (P less than 0.02) and cGMP synthesis (P less than 0.02) was demonstrated after 5 and 30 min of incubation at 37 degrees C, respectively. In further studies ST enterotoxin did not enhance calcium release or the intracellular concentration of cyclic AMP. The stimulation of calcium uptake and cGMP synthesis by ST enterotoxin was inhibited by pharmacological and chemical agents which block cellular calcium entry and prostaglandin synthesis. These results demonstrate that ST enterotoxin induces calcium uptake and cGMP synthesis in rat basophilic leukemia cell cultures.

The purpose of this investigation was to develop an in vitro model with which invasion of tissues by pathogenic Treponema pallidum could be studied. Double-sided culture chambers were created by mounting abdominal walls excised from mice between two halves of small dialysis cells. The integrity of tissue barriers was confirmed by dye exclusion. T. pallidum subsp. pallidum, including intrinsically radiolabeled organisms, was introduced into one side of each chamber, and fractions from the other side were evaluated over time by dark-field microscopy and scintillation counting. Tissues were evaluated by scanning electron microscopy and immunologic staining. Motile T. pallidum, but not nonpathogenic, host-indigenous Treponema phagedenis biotype Reiter, was able to pass from one side of the chamber to the other side within 10 h. Up to 12% of the inoculum crossed the chamber within 24 h. Spirochetes were found within tissue in the greatest numbers between 6 and 8 h postinoculation. The murine abdominal wall has epithelium only on the peritoneum side, and results showed that T. pallidum required an epithelial surface on the entry side of the double-chambered cell in order to traverse the tissue barrier. This new in vitro technique may be of value in studying spirochete virulence and host resistance.

The ability of Borrelia burgdorferi, the agent of Lyme disease, to penetrate cultured human umbilical vein endothelial cell monolayers was investigated. After 4 h of coincubation, approximately 7.7% of added bacteria passed through the host cell monolayers. Electron microscopy revealed that the borreliae entered the endothelial cells and suggested that the organisms penetrated the host monolayers primarily by passing through them.

The effect of lodoxamide tromethamine, a calcium antagonist, on intestinal fluid accumulation induced by Escherichia coli heat-stable (ST) and Vibrio cholerae (CT) enterotoxins in infant mice was investigated. The simultaneous administration of lodoxamide with ST or CT enterotoxin resulted in a significant (P less than 0.01) inhibition of the intestinal fluid response. A minimum concentration of 10(-7) or 10(-8)M lodoxamide caused an inhibition (P less than 0.01) of the ST- or CT-mediated fluid response, respectively. Treatment of infant mice with buffer or drug alone did not result in fluid accumulation. A significant inhibition of ST and CT enterotoxic activities was also observed when lodoxamide was administered 30 min before (P less than 0.02) or 30 min after (P less than 0.01) toxin challenge. These data suggest that calcium may be important in the ST- or CT-mediated induction of fluid accumulation. Further studies on the potential use of lodoxamide tromethamine in both the prophylaxis and treatment of diarrheal disease appear warranted.

Three putative Treponema pallidum ligands (P1, P2, and P3) that bind host fibronectin were characterized by peptide mapping. Papain digestion of each protein yielded a comigrating peptide of approximately 12,000 molecular weight. An antibody to this protein fragment inhibited T. pallidum host cytadherence, indicating that this peptide may be the functional domain of these treponemal adhesins.

The association of the Lyme disease spirochete, Borrelia burgdorferi, with cultured human endothelial cells was investigated. Attachment was time and temperature dependent, with optimal adherence occurring after 4 h of incubation at 37 degrees C. Pretreatment of borreliae with heat, immune human serum, or monoclonal antibodies directed against outer surface protein B (OspB) reduced the attachment of organisms to host cell monolayers. These results suggest that the adherence of B. burgdorferi may be mediated, at least in part, by borrelial surface proteins.

Freshly extracted Treponema pallidum organisms treated with exogenous human fibronectin (Fn) (Fn-primed treponemes) showed a 6- to 15-fold increased level of attachment to Fn-coated cover slips and to extracellular matrix (ECM) when compared with unprimed treponemes. Treponemes primed with collagen or laminin showed no similar enhanced binding to immobilized Fn or ECM. Preexposure of immobilized Fn and ECM to anti-Fn serum but not to anti-collagen or anti-laminin serum prevented treponemal adherence. Also, the presence of proteoglycanlike molecules such as dextran sulfate or heparan sulfate inhibited Fn-primed treponemal attachment to Fn or ECM. In contrast Fn-primed treponemes did not exhibit elevated levels of attachment to eucaryotic cell monolayers. To understand the increased tropism of Fn-primed T. pallidum organisms for Fn and ECM-like surfaces, we radiolabeled freshly extracted treponemes with [35S]methionine and examined them for the presence of surface immunoreactive Fn. Magnetic protAspheres and glass beads coated with monospecific anti-Fn serum bound only 20 to 30% of radiolabeled treponemes. Nonadherent treponemes failed to bind to gelatin-agarose, further confirming the absence of surface Fn or Fn-like material. Fn-free organisms, however, did attach to Fn-coated cover slips and to cell monolayers like treponemes of the original population. Incubation of Fn-free treponemes with human Fn resulted in almost total binding of organisms to anti-Fn antibody on glass beads and also produced increased attachment to Fn-coated cover slips and ECM. These results suggest that enhanced interactions between T. pallidum and the host are dependent on the presence of Fn on syphilis spirochetes and the specific location and orientation of Fn in vivo.