Proceedings of the National Academy of Sciences, 1999
An important effector of Ca 2+ signaling in animals and yeast is the Ca 2+ /calmodulin-dependent ... more An important effector of Ca 2+ signaling in animals and yeast is the Ca 2+ /calmodulin-dependent protein phosphatase calcineurin. However, the biochemical identity of plant calcineurin remained elusive. Here we report the molecular characterization of AtCBL ( A rabidopsis t haliana c alcineurin B - l ike protein) from Arabidopsis . The protein is most similar to mammalian calcineurin B, the regulatory subunit of the phosphatase. AtCBL also shows significant similarity with another Ca 2+ -binding protein, the neuronal calcium sensor in animals. It contains typical EF-hand motifs with Ca 2+ -binding capability, as confirmed by in vitro Ca 2+ -binding assays, and it interacts in vivo with rat calcineurin A in the yeast two-hybrid system. Interaction of AtCBL1 and rat calcineurin A complemented the salt-sensitive phenotype in a yeast calcineurin B mutant. Cloning of cDNAs revealed that AtCBL proteins are encoded by a family of at least six genes in Arabidopsis . Genes for three isoforms...
Women are at significant risk of heterosexually transmitted human immunodeficiency virus (HIV) in... more Women are at significant risk of heterosexually transmitted human immunodeficiency virus (HIV) infection, with the mucosal epithelium of the cervix and vagina serving as a major portal of entry. The cervicovaginal mucosa naturally harbors dynamic microflora composed predominantly of lactobacilli, which may be genetically modified to serve as a more efficient protective barrier against the heterosexual transmission of HIV. We selected a vaginal strain of Lactobacillus , L. jensenii 1153, for genetic modification to display surface-anchored anti-HIV proteins. Genomic sequencing analyses revealed that L. jensenii 1153 encodes several unique high-molecular-weight cell wall-anchored proteins with a C-terminal cell wall sorting LPQTG motif. In this report, we employed these proteins to express a surface-anchored two-domain CD4 (2D CD4) molecule in L. jensenii 1153. Our studies indicated that the C-terminal cell wall sorting signal LPQTG motif alone is insufficient to drive the surface exp...
Background-We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization ... more Background-We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates. Methods-Vaginal and rectal microflora in Chinese rhesus macaques (Macaca mulatta) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization. Results-Chinese rhesus macaques harbored abundant vaginal Lactobacillus, with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals. Conclusions-The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus-based topical microbicides.
The optimized CV-N expression cassette was stably integrated in single copy into the lactobacillu... more The optimized CV-N expression cassette was stably integrated in single copy into the lactobacillus bacterial chromosome, and resolved from extraneous plasmid DNA and antibiotic resistance determinants. The L. jensenii-expressed CV-N dramatically decreased CCR5-trophic HIV ...
Women are at significant risk of human immunodeficiency virus (HIV) infection, with the cervicova... more Women are at significant risk of human immunodeficiency virus (HIV) infection, with the cervicovaginal mucosa serving as a major portal for virus entry. Female-initiated preventatives, including topical microbicides, are urgently needed to help curtail the HIV/AIDS pandemic. Here we report on the development of a novel, live microbicide that employs a natural vaginal strain of Lactobacillus jensenii engineered to deliver the potent HIV inhibitor cyanovirin-N (CV-N). To facilitate efficient expression of CV-N by this bacterium, the L. jensenii 1153 genome was sequenced, allowing identification of native regulatory elements and sites for the chromosomal integration of heterologous genes. A CV-N expression cassette was optimized and shown to produce high levels of structurally intact CV-N when expressed in L. jensenii. Lactobacillus-derived CV-N was capable of inhibiting CCR5-tropic HIV BaL infectivity in vitro with a 50% inhibitory concentration of 0.3 nM. The CV-N expression cassette was stably integrated as a single copy into the bacterial chromosome and resolved from extraneous plasmid DNA without adversely affecting the bacterial phenotype. This bacterial strain was capable of colonizing the vagina and producing full-length CV-N when administered intravaginally to mice during estrus phase. The CV-N-producing Lactobacillus was genetically stable when propagated in vitro and in vivo. This work represents a major step towards the development of an inexpensive yet durable protein-based microbicide to block the heterosexual transmission of HIV in women.
Proceedings of the National Academy of Sciences, 1999
An important effector of Ca 2+ signaling in animals and yeast is the Ca 2+ /calmodulin-dependent ... more An important effector of Ca 2+ signaling in animals and yeast is the Ca 2+ /calmodulin-dependent protein phosphatase calcineurin. However, the biochemical identity of plant calcineurin remained elusive. Here we report the molecular characterization of AtCBL ( A rabidopsis t haliana c alcineurin B - l ike protein) from Arabidopsis . The protein is most similar to mammalian calcineurin B, the regulatory subunit of the phosphatase. AtCBL also shows significant similarity with another Ca 2+ -binding protein, the neuronal calcium sensor in animals. It contains typical EF-hand motifs with Ca 2+ -binding capability, as confirmed by in vitro Ca 2+ -binding assays, and it interacts in vivo with rat calcineurin A in the yeast two-hybrid system. Interaction of AtCBL1 and rat calcineurin A complemented the salt-sensitive phenotype in a yeast calcineurin B mutant. Cloning of cDNAs revealed that AtCBL proteins are encoded by a family of at least six genes in Arabidopsis . Genes for three isoforms...
Women are at significant risk of heterosexually transmitted human immunodeficiency virus (HIV) in... more Women are at significant risk of heterosexually transmitted human immunodeficiency virus (HIV) infection, with the mucosal epithelium of the cervix and vagina serving as a major portal of entry. The cervicovaginal mucosa naturally harbors dynamic microflora composed predominantly of lactobacilli, which may be genetically modified to serve as a more efficient protective barrier against the heterosexual transmission of HIV. We selected a vaginal strain of Lactobacillus , L. jensenii 1153, for genetic modification to display surface-anchored anti-HIV proteins. Genomic sequencing analyses revealed that L. jensenii 1153 encodes several unique high-molecular-weight cell wall-anchored proteins with a C-terminal cell wall sorting LPQTG motif. In this report, we employed these proteins to express a surface-anchored two-domain CD4 (2D CD4) molecule in L. jensenii 1153. Our studies indicated that the C-terminal cell wall sorting signal LPQTG motif alone is insufficient to drive the surface exp...
Background-We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization ... more Background-We sought to establish a nonhuman primate model of vaginal Lactobacillus colonization suitable for evaluating live microbial microbicide candidates. Methods-Vaginal and rectal microflora in Chinese rhesus macaques (Macaca mulatta) were analyzed, with cultivable bacteria identified by 16S rRNA gene sequencing. Live lactobacilli were intravaginally administered to evaluate bacterial colonization. Results-Chinese rhesus macaques harbored abundant vaginal Lactobacillus, with Lactobacillus johnsonii as the predominant species. Like humans, most examined macaques harbored only one vaginal Lactobacillus species. Vaginal and rectal Lactobacillus isolates from the same animal exhibited different genetic and biochemical profiles. Vaginal Lactobacillus was cleared by a vaginal suppository of azithromycin, and endogenous L. johnsonii was subsequently restored by intravaginal inoculation. Importantly, prolonged colonization of a human vaginal Lactobacillus jensenii was established in these animals. Conclusions-The Chinese rhesus macaque harbors vaginal Lactobacillus and is a potentially useful model to support the pre-clinical evaluation of Lactobacillus-based topical microbicides.
The optimized CV-N expression cassette was stably integrated in single copy into the lactobacillu... more The optimized CV-N expression cassette was stably integrated in single copy into the lactobacillus bacterial chromosome, and resolved from extraneous plasmid DNA and antibiotic resistance determinants. The L. jensenii-expressed CV-N dramatically decreased CCR5-trophic HIV ...
Women are at significant risk of human immunodeficiency virus (HIV) infection, with the cervicova... more Women are at significant risk of human immunodeficiency virus (HIV) infection, with the cervicovaginal mucosa serving as a major portal for virus entry. Female-initiated preventatives, including topical microbicides, are urgently needed to help curtail the HIV/AIDS pandemic. Here we report on the development of a novel, live microbicide that employs a natural vaginal strain of Lactobacillus jensenii engineered to deliver the potent HIV inhibitor cyanovirin-N (CV-N). To facilitate efficient expression of CV-N by this bacterium, the L. jensenii 1153 genome was sequenced, allowing identification of native regulatory elements and sites for the chromosomal integration of heterologous genes. A CV-N expression cassette was optimized and shown to produce high levels of structurally intact CV-N when expressed in L. jensenii. Lactobacillus-derived CV-N was capable of inhibiting CCR5-tropic HIV BaL infectivity in vitro with a 50% inhibitory concentration of 0.3 nM. The CV-N expression cassette was stably integrated as a single copy into the bacterial chromosome and resolved from extraneous plasmid DNA without adversely affecting the bacterial phenotype. This bacterial strain was capable of colonizing the vagina and producing full-length CV-N when administered intravaginally to mice during estrus phase. The CV-N-producing Lactobacillus was genetically stable when propagated in vitro and in vivo. This work represents a major step towards the development of an inexpensive yet durable protein-based microbicide to block the heterosexual transmission of HIV in women.
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Papers by Qiang Xu