Heidi Wood

Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates

We previously reported that laboratory reference strains of Chlamydia trachomatis differing in infection organotropism correlated with inactivating mutations in the pathogens tryptophan synthase (trpBA) genes. Here, we have applied functional genomics to extend this work and find that the paradigm established for reference serovars also applies to clinical isolates - specifically, all ocular trachoma isolates tested have inactivating mutations in the synthase, whereas all genital isolates encode a functional enzyme. Moreover, functional enzyme activity was directly correlated to IFN-gamma resistance through an indole rescue mechanism. Hence, a strong selective pressure exists for genital strains to maintain a functional synthase capable of using indole for tryptophan biosynthesis. The fact that ocular serovars (serovar B) isolated from the genital tract were found to possess a functional synthase provided further persuasive evidence of this association. These results argue that there is an important host-parasite relationship between chlamydial genital strains and the human host that determines organotropism of infection and the pathophysiology of disease. We speculate that this relationship involves the production of indole by components of the vaginal microbial flora, allowing chlamydiae to escape IFN-gamma-mediated eradication and thus establish persistent infection.

Comparison of Gamma Interferon-Mediated Antichlamydial Defense Mechanisms in Human and Mouse Cells

ABSTRACT Gamma interferon (IFN-γ)-induced effector mechanisms have potent antichlamydial activities that are critical to host defense. The most prominent and well-studied effectors are indoleamine dioxygenase (IDO) and nitric oxide (NO) synthase. The relative contributions of these mechanisms as inhibitors of chlamydial in vitro growth have been extensively studied using different host cells, induction mechanisms, and chlamydial strains with conflicting results. Here, we have undertaken a comparative analysis of cytokine- and lipopolysaccharide (LPS)-induced IDO and NO using an extensive assortment of human and murine host cells infected with human and murine chlamydial strains. Following cytokine (IFN-γ or tumor necrosis factor alpha) and/or LPS treatment, the majority of human cell lines induced IDO but failed to produce NO. Conversely, the majority of mouse cell lines studied produced NO, not IDO. Induction of IDO in human cell lines inhibited growth of L2 and mouse pneumonitis agent, now referred to as Chlamydia muridarum MoPn equally in all but two lines, and inhibition was completely reversible by the addition of tryptophan. IFN-γ treatment of mouse cell lines resulted in substantially greater reduction of L2 than MoPn growth. However, despite elevated NO production by murine cells, blockage of NO synthesis with the l-arginine analogue N-monomethyl-l-arginine only partially rescued chlamydial growth, suggesting the presence of another IFN-γ-inducible antichlamydial mechanism unique to murine cells. Moreover, NO generated from the chemical nitric oxide donor sodium nitroprusside showed little direct effect on chlamydial infectivity or growth, indicating a natural resistance to NO. Finally, IFN-γ-inducible IDO expression in human HeLa cells was inhibited following exogenous NO treatment, resulting in a permissive environment for chlamydial growth. In summary, cytokine- and LPS-inducible effectors produced by human and mouse cells differ and, importantly, these host-specific effector responses result in chlamydial strain-specific antimicrobial activities.

Regulation of tryptophan synthase gene expression in Chlamydia trachomatis.

We previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase (trpA and trpB). The results presented here indicate that C. trachomatis also expresses the tryptophan repressor gene (trpR). The complement of genes regulated by tryptophan levels in C. trachomatis is limited to trpBA and trpR. trp gene expression was repressed if chlamydiae‐infected HeLa cells were cultured the presence of tryptophan and induced if grown in tryptophan‐depleted medium or in the presence of IFN‐γ. Furthermore, expression of the trp genes in strains which encode a functional tryptophan synthase is repressed when infected cells are cultured in the presence of the tryptophan precursor indole. Results from experiments with cycloheximide, an inhibitor of eukaryotic protein synthesis, indicate that in addition to the absolute size of the intracellular tryptophan pool, host competition for available tryptophan plays a key role in regulating expression of the trp genes. The tryptophan analogue, 5‐fluorotryptophan, repressed trp gene expression and induced the formation of aberrant organisms of C. trachomatis. The growth‐inhibitory properties of 5‐fluorotryptophan could be reversed with exogenous tryptophan but not indole. In total, our results indicate that the ability to regulate trp gene expression in response to tryptophan availability is advantageous for the intracellular survival of this organism. Furthermore, the fact that C. trachomatis has retained the capacity to respond to tryptophan limitation supports the view that the in vivo antichlamydial effect of IFN‐γ is via the induction of the tryptophan‐degrading enzyme, indoleamine 2,3‐dioxygenase.

Tryptophan recycling is responsible for the interferon‐γ resistance of Chlamydia psittaci GPIC in indoleamine dioxygenase‐expressing host cells

Comparative genomics indicates that vast differences in Chlamydia sp. host range and disease characteristics can be traced back to subtle variations in gene content within a region of the chromosome termed the plasticity zone. Genes required for tryptophan biosynthesis are located in the plasticity zone; however, the complement of genes encoded varies depending on the chlamydial species examined. Of the sequenced chlamydia genomes, Chlamydia psittaci GPIC contains the most complete tryptophan biosynthesis operon, encoding trpRDCFBA. Immediately downstream of the trp operon are genes encoding kynureninase and ribose phosphate pyrophosphokinase. Here, we show that, in GPIC, these genes are transcribed as a single transcript, the expression of which is regulated by tryptophan. Complementation analyses, using various mutant Escherichia coli isolates, indicate that the tryptophan biosynthesis, kynureninase and ribose phosphate pyrophosphokinase gene products are functional. Furthermore, growth of C. psittaci GPIC in HeLa cells, cultured in tryptophan‐free medium, could be rescued by the addition of anthranilate, kynurenine or indole. In total, our results indicate that this complement of genes enables GPIC to recycle tryptophan and thus accounts for the interferon‐γ resistant phenotype displayed in indoleamine‐2,3‐dioxygenase‐expressing host cells.

Chlamydial Heat-Shock Protein–60 Antibody and Correlation with Chlamydia pneumoniae in Atherosclerotic Plaques

A study was performed to determine whether serum antibody to Chlamydial heat-shock protein-60 (CHSP-60) and C-reactive protein (CRP) were associated with the presence of Chlamydia pneumoniae in atheromatous plaques in 75 patients. The mean (+/-SD) ELISA optical density (OD) of anti-CHSP-60 was 0.19+/-0.15 in 54 patients with detectable C. pneumoniae antigen, versus an OD of 0.11+/-0.08 in 21 patients without detectable C. pneumoniae I antigen (P=.008). Higher anti-CHSP-60 at an OD > or =0.12 was present in 38 (70.4%) of patients with detectable C. pneumoniae in atheromas, compared with 5 (23.8%) of patients without C. pneumoniae antigen (P<.001; 2-tailed test). The mean CRP concentration was 7.4+/-10.3 mg/L in patients with detectable C. pneumoniae antigen, versus 5.7+/-6.1 mg/L in those without (P=.556). Immune response to CHSP-60 may play a role in atherogenesis, but CRP serum levels does not appear to be related to C. pneumoniae infection.

Rabies virus phosphoprotein interacts with mitochondrial Complex I and induces mitochondrial dysfunction and oxidative stress

Our previous studies in an experimental model of rabies showed neuronal process degeneration in association with severe clinical disease. Cultured adult rodent dorsal root ganglion neurons infected with challenge virus standard (CVS)-11 strain of rabies virus (RABV) showed axonal swellings and reduced axonal growth with evidence of oxidative stress. We have shown that CVS infection alters a variety of mitochondrial parameters and increases reactive oxygen species (ROS) production and mitochondrial Complex I activity vs. mock infection. We have hypothesized that a RABV protein targets mitochondria and triggers dysfunction. Mitochondrial extracts of mouse neuroblastoma cells were analyzed with a proteomics approach. We have identified peptides belonging to the RABV nucleocapsid protein (N), phosphoprotein (P), and glycoprotein (G), and our data indicate that the extract was most highly enriched with P. P was also detected by immunoblotting in RABV-infected purified mitochondrial extracts and also in Complex I immunoprecipitates from the extracts but not in mock-infected extracts. A plasmid expressing P in cells increased Complex I activity and increased ROS generation, whereas expression of other RABV proteins did not. We have analyzed recombinant plasmids encoding various P gene segments. Expression of a peptide from amino acid 139–172 increased Complex I activity and ROS generation similar to expression of the entire P protein, whereas peptides that did not contain this region did not increase Complex I activity or induce ROS generation. These results indicate that a region of the RABV P interacts with Complex I in mitochondria causing mitochondrial dysfunction, increased generation of ROS, and oxidative stress.

Is there a reservoir of sub-clinical lymphogranuloma venereum and non-LGV Chlamydia trachomatis infection in men who have sex with men?

The aim of this study was to determine if a reservoir of sub-clinical LGV infection exists in men who have sex with men (MSM), as this finding might account for the recent rise in lymphogranuloma venereum (LGV) Chlamydia trachomatis infections among MSM in Canada. MSM without proctitis were enrolled between January and August 2006 in a cross-sectional study. Rectal, urine, serology and pharyngeal specimens were tested for specific C. trachomatis serovars. The median age of the 253 participants was 43 years; 53% were HIV+. We found no active cases of LGV infection; but 20 (8%) participants had positive serology. Thirteen participants (5%) had non-LGV C. trachomatis infections. Unprotected anopenetrative intercourse, rectal enema and drug use were associated with non-LGV C. trachomatis infection. Sub-clinical rectal non-LGV C. trachomatis infection was relatively common but LGV was not identified in our sample. Further studies of screening for non-LGV chlamydia infection in MSM are needed.

In vivo and in vitro studies of Chlamydia trachomatis TrpR:DNA interactions

We previously reported that Chlamydia trachomatis expresses the genes encoding tryptophan synthase (trpBA) and the tryptophan repressor (trpR). Here we employ primer extension analysis to identify the transcriptional origins of both trpR and trpBA, allowing for the identification of the putative operator sequences for both trpR and trpBA. Moreover we demonstrate that native recombinant chlamydial TrpR binds to the predicted operator sequence upstream of trpR. A restriction endonuclease protection assay was designed and used to demonstrate that 5‐fluorotryptophan was the only tryptophan analogue capable of activating binding of native recombinant chlamydial TrpR to its operator. Additionally, 5‐fluorotryptophan was the only analogue that repressed expression of trpBA at a level analogous to l‐tryptophan itself. Based on these findings, a mutant selection protocol was designed and a C. trachomatis isolate containing a frameshift mutation in trpR was isolated. This chlamydial mutant synthesizes a truncated TrpR protein that cannot regulate expression of trpBA and trpR in response to changes in tryptophan levels. These findings provide the first genetic proof that TrpR acts as a negative regulator of transcription in C. trachomatis.

Role of nuclear factor-κB in oxidative stress associated with rabies virus infection of adult rat dorsal root ganglion neurons

ABSTRACT Recent studies in an experimental model of rabies showed major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult rat dorsal root ganglion (DRG) neurons infected with the challenge virus standard-11 strain of rabies virus (CVS) showed axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress and reduced axonal growth compared to that of mock-infected DRG neurons. We have evaluated whether nuclear factor (NF)-κB might act as a critical bridge linking CVS infection and oxidative stress. On Western immunoblotting, CVS infection induced expression of the NF-κB p50 subunit compared to that of mock infection. Ciliary neurotrophic factor, a potent activator of NF-κB, had no effect on mock-infected rat DRG neurons and reduced the number of 4-HNE-labeled puncta. SN50, a peptide inhibitor of NF-κB, and CVS infection had an additive effect in producing axonal swellings, indicating that NF-κB is neuroprotective. The fluorescent signal for subunit p50 was quantitatively evaluated in the nucleus and cytoplasm of mock- and CVS-infected rat DRG neurons. At 24 h postinfection (p.i.), there was a significant increase in the nucleus/cytoplasm ratio, indicating increased transcriptional activity of NF-κB, perhaps as a response to stress. At both 48 and 72 h p.i., there was significantly reduced nuclear localization of NF-κB. CVS infection may induce oxidative stress by inhibiting nuclear activation of NF-κB. A rabies virus protein may directly inhibit NF-κB activity. Further investigations are needed to gain a better understanding of the basic mechanisms involved in the oxidative damage associated with rabies virus infection.

Seroprevalence of Seven Zoonotic Pathogens in Pregnant Women from the Caribbean

Studies examining the prevalence of zoonotic agents in the Caribbean are very limited. The objective of this study was to examine the seroprevalence of seven zoonotic agents among individuals residing on 10 English-speaking Caribbean countries. Sera from healthy, pregnant women were collected from Antigua-Barbuda, Belize, Bermuda, Dominica, Grenada, Jamaica, Montserrat, St. Kitts-Nevis, St. Lucia, and St. Vincent-Grenadines and tested for the presence of IgG antibodies to dengue virus, hepatitis E virus, hantaviruses, leptospiral agents, spotted fever group rickettsiae (SFGR), typhus group rickettsiae (TGR), and Coxiella burnetii (Q fever). The highest seroprevalence values were observed for dengue virus, SFGR, and leptospirosis, although the lowest seroprevalence values were observed for hepatitis E virus, C. burnetii, and TGR. Antibodies to hantaviruses were not detected in any individuals.