1887

Abstract

Several inducers of chlamydial persistence have been described, including interferon- (IFN-), IFN-, IFN-, and tumour necrosis factor- (TNF-) exposure, and iron, amino acid or glucose deprivation. A tissue-culture model of /herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. This study was designed to ascertain whether co-infection-induced persistence is mediated by a previously characterized mechanism. Luminex assays indicate that IFN-, IFN-, and TNF- are not released from co-infected cells. Semiquantitative RT-PCR studies demonstrate that IFN-, IFN-, indoleamine 2,3-dioxygenase, lymphotoxin- and inducible nitric oxide synthase are not expressed during co-infection. These data indicate that viral-induced persistence is not stimulated by any persistence-associated cytokine. Supplementation of co-infected cells with excess amino acids, iron-saturated holotransferrin, glucose or a combination of amino acids and iron does not restore chlamydial infectivity, demonstrating that HSV-2-induced persistence is not mediated by depletion of these nutrients. Finally, inclusions within co-infected cells continue to enlarge and incorporate C-NBD-ceramide, indicating that HSV-2 co-infection does not inhibit vesicular transport to the developing inclusion. Collectively these data demonstrate that co-infection-induced persistence is not mediated by any currently characterized persistence inducer or anti-chlamydial pathway. Previous studies indicate that HSV-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting that viral attachment and/or entry may trigger a novel host pathway which restricts chlamydial development.

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2008-03-01
2019-10-17
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Semi-quantitative RT-PCR primer and synthetic DNA target sequences. [ PDF] (57 kb) /HSV co-infected cells do not produce nitric oxide synthase. Total cellular RNA from mock-, and HSV-2 singly- and co-infected HeLa cells was used for semiquantitative RT-PCR with primers specific to human (a) and 18S rRNA (b). A dilution series of synthetic DNA targets or host genomic DNA served as an amplification control for each gene. =8. [ PDF] (405 kb) HSV-2 co-infection does not stimulate or expression. (a) -infected HeLa cultures were either exposed to diluent (-IFN-γ) or 50 U ml of IFN-γ (+IFN-γ) beginning immediately after infection. Total RNA was isolated at 48 h post-infection as described and subjected to RT-PCR using primers specific for human . (b, c)Total cellular RNA was isolated from mock-, and HSV-2 singly- and co-infected HeLa cultures for semiquantitative RT-PCR using primers specific to human (b) and chlamydial (c). A dilution series of synthetic DNA targets ( ) or chlamydial genomic DNA ( ) served as an amplification control for each gene. Chlamydial and human amplimers were quantified as described and normalized to chlamydial ( ) or host ( ) genome copy number (data not shown). =8. [ PDF] (581 kb) HSV-2 induction of chlamydial persistence is not mediated by nutrient deprivation. (a) Cultures of HeLa cells were mock-, singly-infected +/- Desferal or co-infected. Protein concentrations were standardized using the MicroBCA kit (Pierce) prior to analysis by ELISA. Ferritin concentration is expressed as ng per ml sample±SEM; =3. Asterisks (*) indicate ferritin concentrations that are significantly different (by -test) compared to mock-infected cells ( <0.05). (b, c) Cultures of mock-, and HSV-2 singly- and co-infected HeLa cells were refed with either MEM (controls) or MEM+6 mg hTF ml , 5X (1.4 mM) essential and non-essential amino acids or 450 mg ml glucose following HSV-2 adsorption and harvested for EB titration at 20 h post HSV-2 infection. EB titres are expressed as IFU per ml sample±SEM; =3. Asterisks (*) indicate titres that are significantly different (by -test) compared to those from singly-infected cells ( <0.05). The data shown are representative of three independent experiments. [ PDF] (727 kb)

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Semi-quantitative RT-PCR primer and synthetic DNA target sequences. [ PDF] (57 kb) /HSV co-infected cells do not produce nitric oxide synthase. Total cellular RNA from mock-, and HSV-2 singly- and co-infected HeLa cells was used for semiquantitative RT-PCR with primers specific to human (a) and 18S rRNA (b). A dilution series of synthetic DNA targets or host genomic DNA served as an amplification control for each gene. =8. [ PDF] (405 kb) HSV-2 co-infection does not stimulate or expression. (a) -infected HeLa cultures were either exposed to diluent (-IFN-γ) or 50 U ml of IFN-γ (+IFN-γ) beginning immediately after infection. Total RNA was isolated at 48 h post-infection as described and subjected to RT-PCR using primers specific for human . (b, c)Total cellular RNA was isolated from mock-, and HSV-2 singly- and co-infected HeLa cultures for semiquantitative RT-PCR using primers specific to human (b) and chlamydial (c). A dilution series of synthetic DNA targets ( ) or chlamydial genomic DNA ( ) served as an amplification control for each gene. Chlamydial and human amplimers were quantified as described and normalized to chlamydial ( ) or host ( ) genome copy number (data not shown). =8. [ PDF] (581 kb) HSV-2 induction of chlamydial persistence is not mediated by nutrient deprivation. (a) Cultures of HeLa cells were mock-, singly-infected +/- Desferal or co-infected. Protein concentrations were standardized using the MicroBCA kit (Pierce) prior to analysis by ELISA. Ferritin concentration is expressed as ng per ml sample±SEM; =3. Asterisks (*) indicate ferritin concentrations that are significantly different (by -test) compared to mock-infected cells ( <0.05). (b, c) Cultures of mock-, and HSV-2 singly- and co-infected HeLa cells were refed with either MEM (controls) or MEM+6 mg hTF ml , 5X (1.4 mM) essential and non-essential amino acids or 450 mg ml glucose following HSV-2 adsorption and harvested for EB titration at 20 h post HSV-2 infection. EB titres are expressed as IFU per ml sample±SEM; =3. Asterisks (*) indicate titres that are significantly different (by -test) compared to those from singly-infected cells ( <0.05). The data shown are representative of three independent experiments. [ PDF] (727 kb)

PDF

Semi-quantitative RT-PCR primer and synthetic DNA target sequences. [ PDF] (57 kb) /HSV co-infected cells do not produce nitric oxide synthase. Total cellular RNA from mock-, and HSV-2 singly- and co-infected HeLa cells was used for semiquantitative RT-PCR with primers specific to human (a) and 18S rRNA (b). A dilution series of synthetic DNA targets or host genomic DNA served as an amplification control for each gene. =8. [ PDF] (405 kb) HSV-2 co-infection does not stimulate or expression. (a) -infected HeLa cultures were either exposed to diluent (-IFN-γ) or 50 U ml of IFN-γ (+IFN-γ) beginning immediately after infection. Total RNA was isolated at 48 h post-infection as described and subjected to RT-PCR using primers specific for human . (b, c)Total cellular RNA was isolated from mock-, and HSV-2 singly- and co-infected HeLa cultures for semiquantitative RT-PCR using primers specific to human (b) and chlamydial (c). A dilution series of synthetic DNA targets ( ) or chlamydial genomic DNA ( ) served as an amplification control for each gene. Chlamydial and human amplimers were quantified as described and normalized to chlamydial ( ) or host ( ) genome copy number (data not shown). =8. [ PDF] (581 kb) HSV-2 induction of chlamydial persistence is not mediated by nutrient deprivation. (a) Cultures of HeLa cells were mock-, singly-infected +/- Desferal or co-infected. Protein concentrations were standardized using the MicroBCA kit (Pierce) prior to analysis by ELISA. Ferritin concentration is expressed as ng per ml sample±SEM; =3. Asterisks (*) indicate ferritin concentrations that are significantly different (by -test) compared to mock-infected cells ( <0.05). (b, c) Cultures of mock-, and HSV-2 singly- and co-infected HeLa cells were refed with either MEM (controls) or MEM+6 mg hTF ml , 5X (1.4 mM) essential and non-essential amino acids or 450 mg ml glucose following HSV-2 adsorption and harvested for EB titration at 20 h post HSV-2 infection. EB titres are expressed as IFU per ml sample±SEM; =3. Asterisks (*) indicate titres that are significantly different (by -test) compared to those from singly-infected cells ( <0.05). The data shown are representative of three independent experiments. [ PDF] (727 kb)

PDF

Semi-quantitative RT-PCR primer and synthetic DNA target sequences. [ PDF] (57 kb) /HSV co-infected cells do not produce nitric oxide synthase. Total cellular RNA from mock-, and HSV-2 singly- and co-infected HeLa cells was used for semiquantitative RT-PCR with primers specific to human (a) and 18S rRNA (b). A dilution series of synthetic DNA targets or host genomic DNA served as an amplification control for each gene. =8. [ PDF] (405 kb) HSV-2 co-infection does not stimulate or expression. (a) -infected HeLa cultures were either exposed to diluent (-IFN-γ) or 50 U ml of IFN-γ (+IFN-γ) beginning immediately after infection. Total RNA was isolated at 48 h post-infection as described and subjected to RT-PCR using primers specific for human . (b, c)Total cellular RNA was isolated from mock-, and HSV-2 singly- and co-infected HeLa cultures for semiquantitative RT-PCR using primers specific to human (b) and chlamydial (c). A dilution series of synthetic DNA targets ( ) or chlamydial genomic DNA ( ) served as an amplification control for each gene. Chlamydial and human amplimers were quantified as described and normalized to chlamydial ( ) or host ( ) genome copy number (data not shown). =8. [ PDF] (581 kb) HSV-2 induction of chlamydial persistence is not mediated by nutrient deprivation. (a) Cultures of HeLa cells were mock-, singly-infected +/- Desferal or co-infected. Protein concentrations were standardized using the MicroBCA kit (Pierce) prior to analysis by ELISA. Ferritin concentration is expressed as ng per ml sample±SEM; =3. Asterisks (*) indicate ferritin concentrations that are significantly different (by -test) compared to mock-infected cells ( <0.05). (b, c) Cultures of mock-, and HSV-2 singly- and co-infected HeLa cells were refed with either MEM (controls) or MEM+6 mg hTF ml , 5X (1.4 mM) essential and non-essential amino acids or 450 mg ml glucose following HSV-2 adsorption and harvested for EB titration at 20 h post HSV-2 infection. EB titres are expressed as IFU per ml sample±SEM; =3. Asterisks (*) indicate titres that are significantly different (by -test) compared to those from singly-infected cells ( <0.05). The data shown are representative of three independent experiments. [ PDF] (727 kb)

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