Truncated Rv2820c enhances mycobacterial virulence ex vivo and in vivo.

Three hypervirulent strains of Mycobacterium tuberculosis isolated from patients suffering from tuberculous meningitis were shown to grow more rapidly inside human macrophages in our previous study. In the current investigation, genomic polymorphisms in these hypervirulent strains were examined using microarray-based comparative genomic hybridization. Among the five genomic polymorphisms identified, two are in-frame deletion (Rv0071/4 and Rv0613c/6c), two are frameshift deletion (Rv1758' and Rv2820c'), and one is gene replacement (Mb3159). The five genomic polymorphisms were transformed into Mycobacterium smegmatis strain mc(2)155 and the survivability of recombinants inside the human monocytic cell line THP-1 was measured. Interestingly, only the recombinant possessing the Rv2820c' survived significantly better than the vector control after 6 h of ex vivo infection (P < 0.001, one-way ANOVA). The Rv2820c' was later transformed into Mycobacterium marinum strain M and the recombinant was used to infect zebrafish. The in vivo infection also showed that the zebrafish infected with the recombinant possessing the Rv2820c' died significantly faster than the vector control (P = 0.006, log-rank test). The 3' truncation in the Rv2820c' was caused by the Beijing/W-defining deletion RD207 and is commonly found in the Beijing/W strains. The current study demonstrated that the truncated Rv2820c of Beijing/W strains could enhance mycobacterial virulence ex vivo and in vivo. This enhancement, however, was not observed for the intact Rv2820c of the non-Beijing/W strains. The presence of the 3' truncated portion of Rv2820c may interfere with overall protein folding and render the Rv2820c of the non-Beijing/W strains non-functional.

Molecular characterization of clinical isolates of Mycobacterium tuberculosis and their association with phenotypic virulence in human macrophages.

Among 125 clinical isolates of Mycobacterium tuberculosis collected in Hong Kong and Shanghai, China, between 2002 and 2004, IS6110 typing revealed that 71 strains (57%) belonged to the Beijing family. The intracellular growth of the strains in human peripheral blood monocyte-derived macrophages was measured ex vivo on days 0, 3, 6, and 10. Among all tested strains, three hypervirulent strains showed significant increases in intracellular growth after 10 days of incubation. With an initial bacterial load of 10(4) CFU, most of the clinical isolates and H37Ra (an avirulent strain) exhibited no intracellular survival on day 10, while the three hypervirulent strains together with H37Rv (a virulent strain) showed on average a two- to fourfold rise in CFU count. These three hypervirulent strains belonging to a non-Beijing family were isolated from patients suffering from tuberculosis meningitis. Cytokines secreted by gamma interferon-activated macrophages were measured daily after challenge with selected strains of M. tuberculosis. The levels of tumor necrosis factor alpha were elevated after 24 h of infection among all strains, but the levels were significantly lower among the three hypervirulent strains, whereas interleukin 10 (IL-10) and IL-12 were not detected. Results were concordant with the differential expression of the corresponding cytokine genes in activated macrophages, as monitored by real-time PCR. Our findings highlighted that these three hypervirulent strains may possess an innate mechanism for escaping host immunity, which accounts for their characteristic virulence in patients presenting with a more severe form of disease.

A three-dimensional assay for measurement of viral-induced oncolysis.

Oncolytic viruses represent a novel cancer treatment strategy. Despite their promising preclinical data, however, corresponding clinical trials have disappointed. To aid preclinical analyses, we hypothesized that three-dimensional tumor cell clusters or spheroids might provide an assay system superior to conventional monolayer cell cultures. Spheroids show viral infection, replication and oncolytic patterns distinct from conventional monolayer assays. Therefore, viral tumor penetration and oncolysis measurements may be improved with such three-dimensional models. Also, preclinical analyses of oncolytic viruses frequently measure mitochondrial activity, but more accurate measures of oncolysis might involve quantitation of intracellular protein release. Therefore, we measured luciferase released from luciferase-expressing spheroids and found unique patterns that maintained consistency with various viruses and doses. The relative variations between viruses and doses may represent temporal differences in oncolysis dynamics. Analysis of five recombinant replicative adenoviruses with promise for clinical application showed that Ad5/3-Delta24 produced the most luciferase release 1 week after infection and achieved the earliest and highest peak luciferase release level. Ad5/3-Delta24 also effected the earliest subtotal spheroid cell death. These findings closely parallel monolayer oncolysis assays with these agents. Therefore, the luciferase-expressing tumor spheroid assay represents a promising three-dimensional model for preclinical analysis of replicative oncolytic agents.

A cyclooxygenase-2 promoter-based conditionally replicating adenovirus with enhanced infectivity for treatment of ovarian adenocarcinoma.

Conditionally replicating adenoviruses (CRADs) take advantage of tumor-specific characteristics for preferential replication and subsequent oncolysis of cancer cells. The antitumor effect is determined by the capability to infect tumor cells. Here, we used RGDCRADcox-2R, which features the cyclooxygenase-2 promoter for replication control and an integrin binding RGD-4C motif for enhanced infectivity of ovarian cancer cells. RGDCRADcox-2R replicated in and killed human ovarian cancer cells effectively, while the replication in nonmalignant cells was low. Importantly, the therapeutic efficacy, as evaluated in an orthotopic model of peritoneally disseminated ovarian cancer, was significantly improved and toxicity was lower than with a wild-type virus. Thus, this CRAD could be tested for treatment of ovarian cancer in humans.

Preclinical evaluation of a class of infectivity-enhanced adenoviral vectors in ovarian cancer gene therapy.

Ovarian carcinoma cells are often infected inefficiently by adenoviruses (Ad) due to low expression of coxsackie-adenovirus receptors (CAR), hindering the application of adenovirus-mediated gene therapy in ovarian cancer. In this study, we explored a class of infectivity-enhanced Ad vectors, which contain CAR-independent targeting motifs RGD (Ad5.RGD), polylysine (Ad5.pK7), or both (Ad5.RGD.pK7), for their utility in ovarian cancer gene therapy using in vitro and in vivo model systems. We found that these vectors infected established ovarian carcinoma cell lines and primary ovarian cancer cells with significantly enhanced infectivity. Among them, Ad5.RGD.pK7 appeared to be most efficient. Further, we evaluated their gene delivery efficiency using two different ovarian cancer mouse models--subcutaneous and intraperitoneal human ovarian cancer xenografts. All of the modified vectors appeared to be more efficient than the unmodified Ad5 vector in both models, although some of the differences are not statistically significant. Of these, Ad5.RGD.pK7 exhibited the highest efficacy in the subcutaneous tumor model, while Ad5.pK7 worked most efficiently in the intraperitoneal tumor model. These preclinical results suggest that Ad5.RGD.pK7 and Ad5.pK7 may be very useful in ovarian cancer gene therapy.

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters.

The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.

Regulation of the human Na(+)-glucose cotransporter gene, SGLT1, by HNF-1 and Sp1.

The Na(+)-glucose cotransporter (SGLT1) is expressed primarily by small intestinal epithelial cells and transports the monosaccharides glucose and galactose across the apical membrane. Here we describe the isolation and characterization of 5.3 kb of the 5'-flanking region of the SGLT1 gene by transiently transfecting reporter constructs into a variety of epithelial cell lines. A fragment (nt -235 to +22) of the promoter showed strong activity in the intestinal cell line Caco-2 but was inactive in a nonintestinal epithelial cell line (Chinese hamster ovary). Within this region, three cis-elements, a hepatocyte nuclear factor-1 (HNF-1) and two GC box sites are critical for maintaining the gene's basal level of expression. The two GC boxes bind to several members of the Sp1 family of transcription factors and, in the presence of HNF-1, synergistically upregulate transactivation of the promoter. A novel 16-bp element just downstream of one GC box was also shown to influence the interaction of Sp1 to its binding site. In summary, we report the identification and characterization of the human SGLT1 minimal promoter and the critical role that HNF-1 and Sp1-multigene members have in enhancing the basal level of its transcription in Caco-2 cells.

Transcriptional control of the murine polymeric IgA receptor promoter by glucocorticoids.

Glucocorticoids have been implicated as an important regulator of intestinal epithelial cell ontogeny. The polymeric IgA receptor (pIgR) is expressed in the intestinal epithelial layer and is regulated by several mediators, including glucocorticoids. The mechanism of how corticosteroids alter the transcriptional regulation of pIgR expression has not been defined. In this study, we demonstrated that glucocorticoids upregulate steady-state pIgR mRNA levels in the proximal intestine of suckling rats and in the IEC-6 intestinal cell line. We performed functional analysis of the 5'-flanking region in the presence of glucocorticoids and its receptor using the intestinal cell line Caco-2. We screened 4.7 kb of the upstream region of the murine gene and identified the most potent steroid response element to reside between nt -215 and -163 relative to the start of transcription. Substitution mutation analysis of this region identified the location of the putative steroid response element to be between nt -195 and -176. In vitro DNase I footprint analysis using the recombinant glucocorticoid receptor DNA binding domain confirmed a single area of protection that spans the nt identified by mutagenesis analysis. Electrophoretic mobility shift assays of the putative element confirmed the binding of both recombinant and cell synthesized glucocorticoid receptor in a specific manner. In summary, we report the identification and characterization of the glucocorticoid-DNA response element located in the immediate 5'-upstream region of the murine pIgR gene.

Missense mutations in SGLT1 cause glucose-galactose malabsorption by trafficking defects.

Glucose-galactose malabsorption (GGM) is an autosomal recessive disorder caused by defects in the Na+/glucose cotransporter (SGLT1). Neonates present with severe diarrhea while on any diet containing glucose and/or galactose [1]. This study focuses on a patient of Swiss and Dominican descent. All 15 exons of SGLT1 were screened using single stranded conformational polymorphism analyses, and aberrant PCR products were sequenced. Two missense mutations, Gly318Arg and Ala468Val, were identified. SGLT1 mutants were expressed in Xenopus laevis oocytes for radiotracer uptake, electrophysiological experiments, and Western blotting. Uptakes of [14C]alpha-methyl-d-glucoside by the mutants were 5% or less than that of wild-type. Two-electrode voltage-clamp experiments confirmed the transport defects, as no noticeable sugar-induced current could be elicited from either mutant [2]. Western blots of cell protein showed levels of each SGLT1 mutant protein comparable to that of wild-type, and that both were core-glycosylated. Presteady-state current measurements indicated an absence of SGLT1 in the plasma membrane. We suggest that the compound heterozygote missense mutations G318R and A468V lead to GGM in this patient by defective trafficking of mutant proteins from the endoplasmic reticulum to the plasma membrane.

Structure and function of the Na+/glucose cotransporter.

Cotransporters are a major class of membrane transport proteins that are responsible for the accumulation of nutrients, neurotransmitters, osmolytes and ions in cells from bacteria to man. The energy for solute accumulation comes from the proton and/or sodium electrochemical gradients that exist across cell membranes. A major problem in biology is how transport is coupled to these electrochemical potential gradients. The primary example of this class of membrane proteins is the intestinal brush border Na+/glucose cotransporter (SGLT1), first described by Bob Crane in 1960. Over 35 members of the SGLT1 gene family have been identified in animal cells, yeast and bacteria, and all share a common core structure of 13 transmembrane (TM) helices. Electrophysiological techniques have been used to examine the function of several family members, chimeras and mutants expressed in heterologous systems such as Xenopus laevis oocytes. These have revealed that cotransporters are multi-functional proteins: they are responsible for 1). uncoupled passive Na+ transport (Na+ uniport); 2). down-hill water transport in the absence of substrate; 3). Na+/substrate cotransport; and 4). Na+/substrate/water cotransport. The sugar binding and translocation pathway is formed by 4 TM helices near the C-terminal of the protein, helices 10-13. We propose that the N-terminal domains of SGLT1 are responsible for Na+ binding and/or translocation, and that Na+/glucose cotransport results from interactions between the N- and C-terminal domains of the protein.

Characterization of the 5'-flanking region of the murine polymeric IgA receptor gene.

The regulatory elements that control basal and activated transcriptional expression of the polymeric IgA receptor gene (pIgR) have not been defined. In this study, we performed functional analysis of the murine pIgR 5'-upstream region. Transient transfection studies identified the gene's minimal promoter to reside within 110 nucleotides upstream from the start of transcription. Substitution mutations of this region identified both a putative activator (-78 to -70) and a repressor (-66 to -52) element. DNase I footprint analysis confirmed an area of protection that spans from nucleotides -85 to -62. Mobility shift assays of the putative region confirmed binding of upstream stimulatory factor 1 (USF1) to an E box element at positions -75 and -70, representing the putative enhancer. Overexpression studies using various forms of USF suggest that both USF1 and USF2 enhance activity of the pIgR minimal promoter. We report the identification and characterization of the murine pIgR minimal promoter, as well as the critical role of USF in enhancing its basal level of transcription in Caco-2 cells.

Neutralization of conservative charged transmembrane residues in the Na+/glucose cotransporter SGLT1.

Our goal was to identify pairs of charged residues in the membrane domains of the Na+/glucose cotransporter (SGLT1) that form salt bridges, to obtain information about packing of the transmembrane helices. The strategy was to neutralize Glu225, Asp273, Asp294, and Lys321 in helices 6-8, express the mutants in oocytes, measure [14C]-alphaMDG uptake, and then attempt to find second-site mutations of opposite charge that restored function. alphaMDG uptake by E225A was identical to that by SGLT1, whereas transport was reduced by over 90% for D273A, D294A, and K321A and was not restored in the double mutants D273A/K321A or D294A/K321A. This suggested that K321 did not form salt bridges with D273 or D294 and that E225 was not involved in salt-bridging. Neutralization of K321 dramatically changed the Na+ uniport and Na+/glucose cotransport kinetics. The maximum rate of uniport in K321A increased 3-5-fold with a decrease in the apparent affinity for Na+ (70 vs 3 mM) and no change in apparent H+ affinity (0.5 microM). The change in Na+ affinity caused a +50 mV shift in the charge/voltage (Q/V) and relaxation time constant (tau)/voltage curves in the presteady-state kinetics. The presteady-state kinetics in H+ remained unchanged. The lower Na+ affinity resulted also in a 200-fold increase in the apparent K0.5 for alphaMDG and phlorizin. Replacements of K321 with alanine, valine, glutamine, arginine, or glutamic acid residues changed the steady-state kinetics in a similar way. Therefore, we suggest that K321 determines, directly or indirectly, (i) the rate and selectivity of SGLT1 uniport activity and (ii) the apparent affinities of SGLT1 for Na+, and indirectly sugar in the cotransport mode.

Conformational changes couple Na+ and glucose transport.

The mechanism by which cotransport proteins couple their substrates across cell membranes is not known. A commonly proposed model is that cotransport results from ligand-induced conformational transitions that change the accessibility of ligand-binding sites from one side of the membrane to the other. To test this model, we have measured the accessibility of covalent probes to a cysteine residue (Q457C) placed in the putative sugar-translocation domain of the Na+/glucose cotransporter (SGLT1). The mutant protein Q457C was able to transport sugar, but transport was abolished after alkylation by methanethiosulfonate reagents. Alkylation blocked sugar translocation but not sugar binding. Accessibility of Q457C to alkylating reagents required external Na+ and was blocked by external sugar and phlorizin. The voltage dependence of accessibility was directly correlated with the presteady-state charge movement of SGLT1. Voltage-jump experiments with rhodamine-6-maleimide-labeled Q457C showed that the time course and level of changes in fluorescence closely followed the presteady-state charge movement. We conclude that conformational changes are responsible for the coupling of Na+ and sugar transport and that Q457 plays a critical role in sugar translocation by SGLT1.

Genomic cloning and structural analysis of the murine polymeric receptor (pIgR) gene and promoter region.

The role of the polymeric receptor (pIgR) is to transport polymeric IgA across various mucosal epithelial layers. Although several mammalian pIgR cDNAs, including mouse, have been cloned, genomic structure has only been partially analyzed in the human, and neither its 5'-upstream region nor its transcriptional start site is known. We report the isolation and characterization of the murine pIgR gene that spans 32 kb and contains 11 exons. The general organization of the murine gene, including its intron/exon boundaries was similar to its human homolog; however, the second intron was 7.2 kb in the mouse vs. only 0.8 kb in humans. Primer extension and 5'-RACE independently identified the identical transcriptional initiation site. Sequence analysis of 350 base pairs in the 5'-flanking region revealed several motifs, including a TATA box, and putative interferon-gamma, HNF-3beta and AP1 sites. In summary, we have isolated the murine pIgR gene and described its structure and organization.