Serine deamination by human serine racemase synergizes with antibiotics to curtail the replication of Chlamydia trachomatis.

The obligate intracellular bacterium, Chlamydia trachomatis, has evolved to depend on its human host for many metabolites, including most amino acids and three of the four nucleotides. Given this, it is not surprising that depletion of a single amino acid in the host cell growth medium blocks chlamydial replication. Paradoxically, supra-normal levels of some amino acids also block productive replication of Chlamydia. Here, we have determined how elevated serine levels, generated by exogenous supplementation, impede chlamydial inclusion development and reduce the generation of infectious progeny. Our findings reveal that human serine racemase, which is broadly expressed in multiple tissues, potentiates the anti-chlamydial effect of elevated serine concentrations. In addition to reversibly converting l-serine to d-serine, serine racemase also deaminates serine via ß-elimination. We have determined that d-serine does not directly impact Chlamydia; rather, ammonia generated by serine deamination limits the productive chlamydial replication. Our findings imply that ammonia produced within host cells can traverse the chlamydial inclusion membrane. Further, this property of serine deaminase can be exploited to sensitize Chlamydia to concentrations of doxycycline that are otherwise not bactericidal. Because exogenously elevated levels of serine can be tolerated over extended periods, the broad expression pattern of serine racemase indicates it to be a host enzyme whose activity can be directed against multiple intracellular bacterial pathogens. From a therapeutic perspective, demonstrating host metabolism can be skewed to generate an anti-bacterial metabolite that synergizes with antibiotics, we believe our results provide a new approach to target intracellular pathogens.

Ammonia generation by tryptophan synthase drives a key genetic difference between genital and ocular Chlamydia trachomatis isolates.

A striking difference between genital and ocular clinical isolates of Chlamydia trachomatis is that only the former express a functional tryptophan synthase and therefore can synthesize tryptophan by indole salvage. Ocular isolates uniformly cannot use indole due to inactivating mutations within tryptophan synthase, indicating a selection against maintaining this enzyme in the ocular environment. Here, we demonstrate that this selection occurs in two steps. First, specific indole derivatives, produced by the human gut microbiome and present in serum, rapidly induce expression of C. trachomatis tryptophan synthase, even under conditions of tryptophan sufficiency. We demonstrate that these indole derivatives function by acting as de-repressors of C. trachomatis TrpR. Second, trp operon de-repression is profoundly deleterious when infected cells are in an indole-deficient environment, because in the absence of indole, tryptophan synthase deaminates serine to pyruvate and ammonia. We have used biochemical and genetic approaches to demonstrate that expression of wild-type tryptophan synthase is required for the bactericidal production of ammonia. Pertinently, although these indole derivatives de-repress the trpRBA operon of C. trachomatis strains with trpA or trpB mutations, no ammonia is produced, and no deleterious effects are observed. Our studies demonstrate that tryptophan synthase can catalyze the ammonia-generating ß-elimination reaction within any live bacterium. Our results also likely explain previous observations demonstrating that the same indole derivatives inhibit the growth of other pathogenic bacterial species, and why high serum levels of these indole derivatives are favorable for the prognosis of diseased conditions associated with bacterial dysbiosis.

LACK, a RACK1 ortholog, facilitates cytochrome c oxidase subunit expression to promote Leishmania major fitness.

Leishmania are kinetoplastid parasites that cause the sandfly-transmitted disease leishmaniasis. To maintain fitness throughout their infectious life cycle, Leishmania must undergo rapid metabolic adaptations to the dramatically distinct environments encountered during transition between sandfly and vertebrate hosts. We performed proteomic and immunoblot analyses of attenuated L. major strains deficient for LACK, the Leishmania ortholog of the mammalian receptor for activated c kinase (RACK1), that is important for parasite thermotolerance and virulence. This approach identified cytochrome c oxidase (LmCOX) subunit IV as a LACK-dependent fitness protein. Consistent with decreased levels of LmCOX subunit IV at mammalian temperature, and in amastigotes, LmCOX activity and mitochondrial function were also impaired in LACK-deficient L. major under these conditions. Importantly, overexpression of LmCOX subunit IV in LACK-deficient L. major restored thermotolerance and macrophage infectivity. Interestingly, overexpression of LmCOX subunit IV enhanced LmCOX subunit VI expression at mammalian temperature. Collectively, our data suggest LACK promotes Leishmania adaptation to the mammalian host environment by sustaining LmCOX subunit IV expression and hence energy metabolism in response to stress stimuli such as heat. These findings extend the repertoire of RACK1 protein utility to include a role in mitochondrial function.

RNA-dependent recruitment of the origin recognition complex.

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.

Inhibition of indoleamine 2,3-dioxygenase activity by levo-1-methyl tryptophan blocks gamma interferon-induced Chlamydia trachomatis persistence in human epithelial cells.

Gamma interferon (IFN-γ) induces expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) in human epithelial cells, the permissive cells for the obligate intracellular bacterium Chlamydia trachomatis. IDO1 depletes tryptophan by catabolizing it to kynurenine with consequences for C. trachomatis, which is a tryptophan auxotroph. In vitro studies reveal that tryptophan depletion can result in the formation of persistent (viable but noncultivable) chlamydial forms. Here, we tested the effects of the IDO1 inhibitor, levo-1-methyl-tryptophan (L-1MT), on IFN-γ-induced C. trachomatis persistence. We found that addition of 0.2 mM L-1MT to IFN-γ-exposed infected HeLa cell cultures restricted IDO1 activity at the mid-stage (20 h postinfection [hpi]) of the chlamydial developmental cycle. This delayed tryptophan depletion until the late stage (38 hpi) of the cycle. Parallel morphological and gene expression studies indicated a consequence of the delay was a block in the induction of C. trachomatis persistence by IFN-γ. Furthermore, L-1MT addition allowed C. trachomatis to undergo secondary differentiation, albeit with limited productive multiplication of the bacterium. IFN-γ-induced persistent infections in epithelial cells have been previously reported to be more resistant to doxycycline than normal productive infections in vitro. Pertinent to this observation, we found that L-1MT significantly improved the efficacy of doxycycline in clearing persistent C. trachomatis forms. It has been postulated that persistent forms of C. trachomatis may contribute to chronic chlamydial disease. Our findings suggest that IDO1 inhibitors such as L-1MT might provide a novel means to investigate, and potentially target, persistent chlamydial forms, particularly in conjunction with conventional therapeutics.

Zinc coordination is required for and regulates transcription activation by Epstein-Barr nuclear antigen 1.

Epstein-Barr Nuclear Antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B-cells. Upon binding a cluster of 20 cognate binding-sites termed the family of repeats, EBNA1 transactivates promoters for EBV genes that are required for immortalization. A small domain, termed UR1, that is 25 amino-acids in length, has been identified previously as essential for EBNA1 to activate transcription. In this study, we have elucidated how UR1 contributes to EBNA1's ability to transactivate. We show that zinc is necessary for EBNA1 to activate transcription, and that UR1 coordinates zinc through a pair of essential cysteines contained within it. UR1 dimerizes upon coordinating zinc, indicating that EBNA1 contains a second dimerization interface in its amino-terminus. There is a strong correlation between UR1-mediated dimerization and EBNA1's ability to transactivate cooperatively. Point mutants of EBNA1 that disrupt zinc coordination also prevent self-association, and do not activate transcription cooperatively. Further, we demonstrate that UR1 acts as a molecular sensor that regulates the ability of EBNA1 to activate transcription in response to changes in redox and oxygen partial pressure (pO(2)). Mild oxidative stress mimicking such environmental changes decreases EBNA1-dependent transcription in a lymphoblastoid cell-line. Coincident with a reduction in EBNA1-dependent transcription, reductions are observed in EBNA2 and LMP1 protein levels. Although these changes do not affect LCL survival, treated cells accumulate in G0/G1. These findings are discussed in the context of EBV latency in body compartments that differ strikingly in their pO(2) and redox potential.

Optimal transactivation by Epstein-Barr nuclear antigen 1 requires the UR1 and ATH1 domains.

Epstein-Barr nuclear antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B cells. EBNA1 transactivates viral promoters for genes that are necessary for immortalization when it is bound to a cluster of 20 cognate binding sites, termed the family of repeats. A region of EBNA1 from amino acids (aa) 40 to 89, termed linking region 1 (LR1), has been identified previously as being sufficient for transactivation. LR1 contains two domains that are conserved in the EBNA1 orthologs of other gamma herpesviruses. The first of these, termed unique region 1 (UR1), corresponds to aa 65 to 89 of EBNA1. UR1 is necessary for transactivation and contains a conserved recognition site for cyclic AMP-dependent protein kinase (PKA), corresponding to serine 78 of EBNA1. We have pharmacologically modulated PKA activity to determine if PKA controls EBNA1's ability to transactivate. Our results indicate that PKA activators and inhibitors do not affect transactivation by EBNA1. In addition, site-directed mutagenesis demonstrates that transactivation is not influenced by the phosphorylation status of serine 78 in the UR1 domain. The second conserved domain within LR1 is a glycine-arginine repeat, corresponding to aa 40 to 54 of EBNA1. This domain, termed ATH1, functions as an AT-hook, a DNA-binding motif found in architectural transcription factors such as HMGA1a. We demonstrate that deletion of the ATH1 domain decreases EBNA1 transactivation ability, which is consistent with a transcriptional role for ATH1. Furthermore, transactivation is restored when ATH1 is replaced by equivalent AT-hook motifs from HMGA1a. Our data strongly indicate a role for AT-hooks in EBNA1's ability to transactivate, a function necessary for EBV to immortalize naïve B-cells.

Diametrically opposed effects of hypoxia and oxidative stress on two viral transactivators.

BACKGROUND: Many pathogens exist in multiple physiological niches within the host. Differences between aerobic and anaerobic conditions are known to alter the expression of bacterial virulence factors, typically through the conditional activity of transactivators that modulate their expression. More recently, changes in physiological niches have been shown to affect the expression of viral genes. For many viruses, differences in oxygen tension between hypoxia and normoxia alter gene expression or function. Oxygen tension also affects many mammalian transactivators including AP-1, NFkB, and p53 by affecting the reduced state of critical cysteines in these proteins. We have recently determined that an essential cys-x-x-cys motif in the EBNA1 transactivator of Epstein-Barr virus is redox-regulated, such that transactivation is favoured under reducing conditions. The crucial Tat transactivator of human immunodeficiency virus (HIV) has an essential cysteine-rich region, and is also regulated by redox. Contrary to EBNA1, it is reported that Tat's activity is increased by oxidative stress. Here we have compared the effects of hypoxia, oxidative stress, and cellular redox modulators on EBNA1 and Tat. RESULTS: Our results indicate that unlike EBNA1, Tat is less active during hypoxia. Agents that generate hydroxyl and superoxide radicals reduce EBNA1's activity but increase transactivation by Tat. The cellular redox modulator, APE1/Ref-1, increases EBNA1's activity, without any effect on Tat. Conversely, thioredoxin reductase 1 (TRR1) reduces Tat's function without any effect on EBNA1. CONCLUSIONS: We conclude that oxygen partial pressure and oxidative stress affects the functions of EBNA1 and Tat in a dramatically opposed fashion. Tat is more active during oxidative stress, whereas EBNA1's activity is compromised under these conditions. The two proteins respond to differing cellular redox modulators, suggesting that the oxidized cysteine adduct is a disulfide bond(s) in Tat, but sulfenic acid in EBNA1. The effect of oxygen partial pressure on transactivator function suggests that changes in redox may underlie differences in virus-infected cells dependent upon the physiological niches they traffic to.

Epstein-Barr Nuclear Antigen 1 modulates replication of oriP-plasmids by impeding replication and transcription fork migration through the family of repeats.

BACKGROUND: Epstein-Barr virus is replicated once per cell-cycle, and partitioned equally in latently infected cells. Both these processes require a single viral cis-element, termed oriP, and a single viral protein, EBNA1. EBNA1 binds two clusters of binding sites in oriP, termed the dyad symmetry element (DS) and the family of repeats (FR), which function as a replication element and partitioning element respectively. Wild-type FR contains 20 binding sites for EBNA1. RESULTS: We, and others, have determined previously that decreasing the number of EBNA1-binding sites in FR increases the efficiency with which oriP-plasmids are replicated. Here we demonstrate that the wild-type number of binding sites in FR impedes the migration of replication and transcription forks. Further, splitting FR into two widely separated sets of ten binding sites causes a ten-fold increase in the efficiency with which oriP-plasmids are established in cells expressing EBNA1. We have also determined that EBNA1 bound to FR impairs the migration of transcription forks in a manner dependent on the number of EBNA1-binding sites in FR. CONCLUSION: We conclude that EBNA1 bound to FR regulates the replication of oriP-plasmids by impeding the migration of replication forks. Upon binding FR, EBNA1 also blocks the migration of transcription forks. Thus, in addition to regulating oriP replication, EBNA1 bound to FR also decreases the probability of detrimental collisions between two opposing replication forks, or between a transcription fork and a replication fork.

Cholesterol Supplementation During Production Increases the Infectivity of Retroviral and Lentiviral Vectors Pseudotyped with the Vesicular Stomatitis Virus Glycoprotein (VSV-G).

Cholesterol, a major component of plasma membrane lipid rafts, is important for assembly and budding of enveloped viruses, including influenza and HIV-1. Cholesterol depletion impairs virus assembly and infectivity. This study examined the effects of exogenous cholesterol addition (delivered as a complex with methyl beta cyclodextrin) on the production of Molony murine leukemia virus retroviral vector and HIV-1-based lentiviral vector pseudotyped with the vesicular stomatitis virus glycoprotein (VSV-G). Cholesterol supplementation before and during vector production enhanced the infectivity of retroviral and lentiviral vectors up to 4-fold and 6-fold, respectively. In contrast, the amount of retroviral vector produced was unchanged, and that of lentiviral vector was increased less than two-fold. Both free cholesterol and cholesterol ester content in 293-gag-pol producer cells increased with cholesterol addition. In contrast, the phospholipids headgroup composition was essentially unchanged by cholesterol supplementation in 293-gag-pol packaging cells. Based on these results, it is proposed that cholesterol supplementation increases the infectivity of VSV-G-pseudotyped retroviral and lentiviral vectors, possibly by altering the composition of the producer cell membrane where the viral vectors are assembled and bud, and/or by changing the lipid composition of the viral vectors.

Molecular character of influenza A/H1N1 2009: Implications for spread and control.

The world is experiencing a pandemic of influenza that emerged in March 2009, due to a novel strain designated influenza A/H1N1 2009. This strain is closest in molecular sequence to swine influenza viruses, but differs from all previously known influenza by a minimum of 6.1%, and from prior "seasonal" H1N1 by 27.2%, giving it great potential for widespread human infection. While spread into India was delayed for two months by an aggressive interdiction program, since 1 August 2009 most cases in India have been indigenous. H1N1 2009 has differentially struck younger patients who are naïve susceptibles to its antigenic subtype, while sparing those >60 who have crossreactive antibody from prior experience with influenza decades ago and the 1977 "swine flu" vaccine distributed in the United States. It also appears to more severely affect pregnant women. It emanated from a single source in central Mexico, but its precise geographical and circumstantial origins, from either Eurasia or the Americas, remain uncertain. While currently a mild pandemic by the standard of past pandemics, the seriousness of H1N1 2009 especially among children should not be underestimated. There is potential for the virus, which continues to adapt to humans, to change over time into a more severe etiologic agent by any of several foreseeable mutations. Mass acceptance of the novel H1N1 2009 vaccine worldwide will be essential to its control. Having spread globally in a few months, affecting millions of people, it is likely to remain circulating in the human population for a decade or more.

Chlamydia trachomatis-infected cells and uninfected-bystander cells exhibit diametrically opposed responses to interferon gamma.

The intracellular bacterial pathogen, Chlamydia trachomatis, is a tryptophan auxotroph. Therefore, induction of the host tryptophan catabolizing enzyme, indoleamine-2,3-dioxgenase-1 (IDO1), by interferon gamma (IFNγ) is one of the primary protective responses against chlamydial infection. However, despite the presence of a robust IFNγ response, active and replicating C. trachomatis can be detected in cervical secretions of women. We hypothesized that a primary C. trachomatis infection may evade the IFNγ response, and that the protective effect of this cytokine results from its activation of tryptophan catabolism in bystander cells. To test this hypothesis, we developed a novel method to separate a pool of cells exposed to C. trachomatis into pure populations of live infected and bystander cells and applied this technique to distinguish between the effects of IFNγ on infected and bystander cells. Our findings revealed that the protective induction of IDO1 is suppressed specifically within primary infected cells because Chlamydia attenuates the nuclear import of activated STAT1 following IFNγ exposure, without affecting STAT1 levels or phosphorylation. Critically, the IFNγ-mediated induction of IDO1 activity is unhindered in bystander cells. Therefore, the IDO1-mediated tryptophan catabolism is functional in these cells, transforming these bystander cells into inhospitable hosts for a secondary C. trachomatis infection.

Cell Intrinsic Factors Modulate the Effects of IFNγ on the Development of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular bacterial pathogen that cannot synthesize several amino acids, including tryptophan. Rather, C. trachomatis acquires these essential metabolites from its human host cell. Chlamydial dependence on host-provided tryptophan underlies a major host defense mechanism against the bacterium; namely, the induction of the host tryptophan-catabolizing enzyme, indoleamine 2,3- dioxygenase (IDO1) by interferon gamma (IFNγ), which leads to eradication of C. trachomatis by tryptophan starvation. For this reason, IFNγ is proposed to be the major host protective cytokine against genital C. trachomatis infections. The protective effect of IFNγ against C. trachomatis can be recapitulated in vitro using epithelial cell-lines such as the cervical carcinoma derived cell-line Hela, the Hela subclone HEp-2, and the cervical carcinoma derived cell-line ME180. Addition of IFNγ to these cells infected with C. trachomatis results in a strong bactericidal or bacteriostatic effect dependent on the concentration of IFNγ administered. Unlike Hela, HEp-2, and ME180, there are other human epithelial, or epithelial-like cell-lines where administration of IFNγ does not affect chlamydial replication, although they express the IFNγ receptor (IFNGR). In this report, we have characterized the mechanisms that underlie this dichotomy using the cell-lines C33A and 293. Akin to Hela, C33A is derived from a human cervical carcinoma, while 293 cells were produced by transfection of adenovirus type 5 DNA into embryonic kidney cells. We demonstrate that although IFNGR is expressed at high levels in C33A cells, its ligation by IFNγ does not result in STAT1 phosphorylation, an essential step for activation of the IDO1 promoter. Our results indicate that although the IFNγ-dependent signaling cascade is intact in 293 cells; the IDO1 promoter is not activated in these cells because it is epigenetically silenced, most likely by DNA methylation. Because polymorphisms in IFNγ, IFNGR, and the IDO1 promoter are known to affect other human infections or diseased states, our results indicate that the effect of allelic differences in these genes and the pathways they activate should be evaluated for their effect on C. trachomatis pathology.

The High-Risk Human Papillomavirus E6 Oncogene Exacerbates the Negative Effect of Tryptophan Starvation on the Development of Chlamydia trachomatis.

Chlamydia trachomatis is an obligate intracellular pathogen that requires specific essential nutrients from the host cell, one of which is the amino acid tryptophan. In this context interferon gamma (IFNγ) is the major host protective cytokine against chlamydial infections because it induces the expression of the host enzyme, indoleamine 2,3-dioxygenase 1, that degrades tryptophan, thereby restricting bacterial replication. The mechanism by which IFNγ acts has been dissected in vitro using epithelial cell-lines such as HeLa, HEp-2, or the primary-like endocervical cell-line A2EN. All these cell-lines express the high-risk human papillomavirus oncogenes E6 & E7. While screening cell-lines to identify those suitable for C. trachomatis co-infections with other genital pathogens, we unexpectedly found that tryptophan starvation did not completely block chlamydial development in cell-lines that were HR-HPV negative, such as C33A and 293. Therefore, we tested the hypothesis that HR-HPV oncogenes modulate the effect of tryptophan starvation on chlamydial development by comparing chlamydial development in HeLa and C33A cell-lines that were both derived from cervical carcinomas. Our results indicate that during tryptophan depletion, unlike HeLa, C33A cells generate sufficient intracellular tryptophan via proteasomal activity to permit C. trachomatis replication. By generating stable derivatives of C33A that expressed HPV16 E6, E7 or E6 & E7, we found that E6 expression alone was sufficient to convert C33A cells to behave like HeLa during tryptophan starvation. The reduced tryptophan levels in HeLa cells have a biological consequence; akin to the previously described effect of IFNγ, tryptophan starvation protects C. trachomatis from clearance by doxycycline in HeLa but not C33A cells. Curiously, when compared to the known Homo sapiens proteome, the representation of tryptophan in the HR-HPV E6 & E6AP degradome is substantially lower, possibly providing a mechanism that underlies the lowered intracellular free tryptophan levels in E6-expressing cells during starvation.

Transduction efficiency of pantropic retroviral vectors is controlled by the envelope plasmid to vector plasmid ratio.

Pantropic retroviral vectors pseudotyped with vesicular stomatitis virus envelope G protein (VSV-G) are typically produced by transient transfection of the VSV-G expression plasmid because constitutive expression of VSV-G is cytotoxic. To produce pantropic vectors, the VSV-G expression plasmid and the vector plasmid are cotransfected into a packaging cell line, such as 293-gag-pol. Typically, the ratio of VSV-G plasmid to the vector plasmid ranges from 0.33 to 1.0. However, it is not clear that this range is optimal for vector production. In this study we have systematically examined the effect of the ratio of VSV-G plasmid (pVSV-G) to vector plasmid on vector production. For this, 293-gag-pol stable packaging cells were cotransfected with pVSV-G and an enhanced green fluorescent protein- (EGFP-) expressing retroviral vector plasmid (pLTR-EGFP) by use of lipofectamine. Vector was collected following transfection and used to transduce three target cell lines, namely, 3T3 fibroblasts, telomerase-immortalized human diploid fibroblasts (HDF), and the human hepatoma cell line HuH7. Transduction efficiency was evaluated for vectors produced at different pVSV-G:pLTR-EGFP ratios such that the total amount of plasmid transfected into 293-gag-pol cells was kept constant. Our results indicate that transduction efficiency is greatest when the pVSV-G:pLTR-EGFP ratio is substantially below 1.0. For 3T3 and HDF cells, the maximum transduction efficiency was obtained when a ratio of pVSV-G:pLTR-EGFP ranging from 0.053 to 0.2 was used for transfection. The relative magnitude of this effect was greater for lower transduction efficiencies in control cultures. For HuH7 cells, the beneficial effects were smaller than those observed when HDF or 3T3 cells were used. The difference in transduction efficiency for vector produced under various pVSV-G:pLTR-EGFP ratios was not due to differences in the proliferation of packaging cells or target cells. Further characterization showed that the amount of vector RNA relative to p30gag decreased as the ratio of pVSV-G:pLTR-EGFP increased. These results indicate that transduction efficiency increases with increasing levels of vector RNA as long as a minimally sufficient level of pantropic envelope protein is expressed.

Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis.

RPE65 is a membrane-associated retinoid isomerase involved in the visual cycle responsible for sustaining vision. Many mutations in the human RPE65 gene are associated with distinct forms of retinal degenerative diseases. The pathogenic mechanisms for most of these mutations remain poorly understood. Here, we show that three Leber congenital amaurosis -associated RPE65 mutants (R91W, Y249C and R515W) undergo rapid proteasomal degradation mediated by the 26 S proteasome non-ATPase regulatory subunit 13 (PSMD13) in cultured human retinal pigment epithelium (RPE) cells. These mutant proteins formed cytosolic inclusion bodies or high molecular weight complexes via disulfide bonds. The mutations are mapped on non-active sites but severely reduced isomerase activity of RPE65. At 30°C, however, the enzymatic function and membrane-association of the mutant RPE65s are significantly rescued possibly due to proper folding. In addition, PSMD13 displayed a drastically decreased effect on degradation of the mutant proteins in the cells grown at 30°C. These results suggest that PSMD13 plays a critical role in regulating pathogenicity of the mutations and the molecular basis for the PSMD13-mediated rapid degradation and loss of function of the mutants is misfolding of RPE65.

Dual effect of interferon (IFNγ)-induced nitric oxide on tumorigenesis and intracellular bacteria.

Nitric oxide (NO) is a key messenger involved in numerous physiological functions including inflammatory and immune responses. The functions of NO and their underlying mechanisms have been elucidated by extensive studies over the past 10 years. However, the complexity of the interactions between different levels of NO and multiple aspects of tumor development/progression as well as bacterial pathogenesis has led to apparently conflicting findings. The precise role of NO in bacterial and tumor pathogenesis involves a multitude of inter- and intracellular signaling pathways in which interferon gamma signaling and L-arginine metabolism are the major pathways involved in NO synthesis and regulation. The availability of the amino acid L-Arg can be a key factor to control the expression of inducible nitric oxide synthase (NOS2) and cellular NO levels. The role played by the NOS2/NO system both in bacterial pathogenesis and in tumor development is complex due to the dual role these molecules can play promoting or inhibiting infections and cancer. This duality brings to the table a double challenge to determine the net impact of NO on cancer or bacterial behavior and to define the therapeutic role of NO-centered anticancer or antibacterial strategies. We believe that a comprehensive and dynamic understanding of the cascade of molecular and cellular events underlying tumor biology and bacterial pathogenesis that are affected by NO will allow researchers to exploit the potential antitumor and antibacterial properties of drugs interfering with NO metabolism. The contrasting roles of NO/NOS2 in these processes are clarified in this chapter.

Influence of the tryptophan-indole-IFNγ axis on human genital Chlamydia trachomatis infection: role of vaginal co-infections.

The natural history of genital Chlamydia trachomatis infections can vary widely; infections can spontaneously resolve but can also last from months to years, potentially progressing to cause significant pathology. The host and bacterial factors underlying this wide variation are not completely understood, but emphasize the bacterium's capacity to evade/adapt to the genital immune response, and/or exploit local environmental conditions to survive this immune response. IFNγ is considered to be a primary host protective cytokine against endocervical C. trachomatis infections. IFNγ acts by inducing the host enzyme indoleamine 2,3-dioxgenase, which catabolizes tryptophan, thereby depriving the bacterium of this essential amino acid. In vitro studies have revealed that tryptophan deprivation causes Chlamydia to enter a viable but non-infectious growth pattern that is termed a persistent growth form, characterized by a unique morphology and gene expression pattern. Provision of tryptophan can reactivate the bacterium to the normal developmental cycle. There is a significant difference in the capacity of ocular and genital C. trachomatis serovars to counter tryptophan deprivation. The latter uniquely encode a functional tryptophan synthase to synthesize tryptophan via indole salvage, should indole be available in the infection microenvironment. In vitro studies have confirmed the capacity of indole to mitigate the effects of IFNγ; it has been suggested that a perturbed vaginal microbiome may provide a source of indole in vivo. Consistent with this hypothesis, the microbiome associated with bacterial vaginosis includes species that encode a tryptophanase to produce indole. In this review, we discuss the natural history of genital chlamydial infections, morphological and molecular changes imposed by IFNγ on Chlamydia, and finally, the microenvironmental conditions associated with vaginal co-infections that can ameliorate the effects of IFNγ on C. trachomatis.

Morphologic and molecular evaluation of Chlamydia trachomatis growth in human endocervix reveals distinct growth patterns.

In vitro models of Chlamydia trachomatis growth have long been studied to predict growth in vivo. Alternative or persistent growth modes in vitro have been shown to occur under the influence of numerous stressors but have not been studied in vivo. Here, we report the development of methods for sampling human infections from the endocervix in a manner that permits a multifaceted analysis of the bacteria, host and the endocervical environment. Our approach permits evaluating total bacterial load, transcriptional patterns, morphology by immunofluorescence and electron microscopy, and levels of cytokines and nutrients in the infection microenvironment. By applying this approach to two pilot patients with disparate infections, we have determined that their contrasting growth patterns correlate with strikingly distinct transcriptional biomarkers, and are associated with differences in local levels of IFNγ. Our multifaceted approach will be useful to dissect infections in the human host and be useful in identifying patients at risk for chronic disease. Importantly, the molecular and morphological analyses described here indicate that persistent growth forms can be isolated from the human endocervix when the infection microenvironment resembles the in vitro model of IFNγ-induced persistence.

Modulation of MICA on the surface of Chlamydia trachomatis-infected endocervical epithelial cells promotes NK cell-mediated killing.

Chlamydia trachomatis serovars D-K are obligate intracellular bacteria that have tropism for the columnar epithelial cells of the genital tract. Chlamydia trachomatis infection has been reported to induce modifications in immune cell ligand expression on epithelial host cells. In this study, we used an in vitro infection model that resulted in a partial infection of C. trachomatis-exposed primary-like immortalized endocervical epithelial cells (A2EN). Using this model, we demonstrated that expression of the natural killer (NK) cell activating ligand, MHC class I-related protein A (MICA), was upregulated on C. trachomatis-infected, but not on noninfected bystander cells. MICA upregulation was concomitant with MHC class I downregulation and impacted the susceptibility of C. trachomatis-infected cells to NK cell activity. The specificity of MICA upregulation was reflected by a higher cytolytic activity of an NK cell line (NK92MI) against C. trachomatis-infected cells compared with uninfected control cells. Significantly, data also indicated that NK cells exerted a partial, but incomplete sterilizing effect on C. trachomatis as shown by the reduction in recoverable inclusion forming units (IFU) when cocultured with C. trachomatis-infected cells. Taken together, our data suggest that NK cells may play a significant role in the ability of the host to counter C. trachomatis infection.