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Dr Willa Huston

Biography

I am a Molecular Microbiologist (PhD awarded 2004) with an interest in Chlamydia and chlamydial diseases. My work has mainly focussed on understanding the chlamydial mechanisms of disease, persistence, and how the disease causes infertility in women. I am also interested in the role proteases have in pathogenesis and biology. I have strong expertise in intracellular infection models and human disease models. My team has research projects on-going in chlamydial biology, human disease factors, diagnosis and treatment and we welcome enquiries from interested students or collaborators.

Professional

I am a member of

The Australian Society for Microbiology

The International Proteolysis Society

Franklin Women

I am an active participant in school based activities and Scientist in Schools

Image of Willa Huston
Senior Lecturer, School of Life Sciences
Associate Member, ithree - Institute of Infection, Immunity and Innovation
Higher Education and Academic Practice, Microbiology
 
Phone
+61 2 9514 3449

Research Interests

My group is focussed on understanding how chlamydial causes infertility in women and how this intracellular bacterial pathogen interacts with human cells.

We are focussed on understanding the bacterial pathogenic factors in order to develop future improved diagnostics and therapeutics for humans.

We are also interested in chlamydia and Koalas and the development of new drugs to treat this devastating disease.

Can supervise: Yes
I am experienced in teaching microbiology, molecular microbiology, chemistry and genetics.

Books

Cole, L.J., Huston, W.M. & Moir, J.W. 2008, Delivery of nitric oxide for analysis of the function of cytochrome c'..
On delivery of nitric oxide (NO) to protein samples (e.g., cytochrome c'), for spectroscopic experiments it is important to avoid exposure to oxygen and to remove contaminants from the NO gas. We describe a number of techniques for steady-state UV/Vis spectrophotometry and pre-steady-state stopped-flow spectrophotometry analysis of cytochrome c'.

Chapters

Marsh, J.W., Gloeckl, S., Tyndall, J.D.A. & Huston, W.M. 2012, 'The role of HtrA as a chaperone and protease in bacterial pathogenesis' in Boulanger, A. & Blanc, M. (eds), Bacterial Pathogens: Virulence Mechanisms, Diagnosis, and Management, Nova Science Publishers, Inc, Happauge, New York, pp. 117-164.
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Marsh, J.W., Gloeckl, S., Tyndall, J.D.A. & Huston, W.M. 2011, 'The role of HtrA as a chaperone and protease in bacterial pathogenesis' in Berhardt, L.V. (ed), Advances in Medicine and Biology, Nova Biomedical, Hauppage, New York, pp. 87-121.
This continuing series gathers and presents original research results on the leading edge of medicine and biology.

Journal articles

Paul, B., Kim, H.S., Kerr, M.C., Huston, W.M., Teasdale, R.D. & Collins, B.M. 2017, 'Structural basis for the hijacking of endosomal sorting nexin proteins by Chlamydia trachomatis.', Elife, vol. 6.
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During infection chlamydial pathogens form an intracellular membrane-bound replicative niche termed the inclusion, which is enriched with bacterial transmembrane proteins called Incs. Incs bind and manipulate host cell proteins to promote inclusion expansion and provide camouflage against innate immune responses. Sorting nexin (SNX) proteins that normally function in endosomal membrane trafficking are a major class of inclusion-associated host proteins, and are recruited by IncE/CT116. Crystal structures of the SNX5 phox-homology (PX) domain in complex with IncE define the precise molecular basis for these interactions. The binding site is unique to SNX5 and related family members SNX6 and SNX32. Intriguingly the site is also conserved in SNX5 homologues throughout evolution, suggesting that IncE captures SNX5-related proteins by mimicking a native host protein interaction. These findings thus provide the first mechanistic insights both into how chlamydial Incs hijack host proteins, and how SNX5-related PX domains function as scaffolds in protein complex assembly.
Kerr, M.C., Gomez, G.A., Ferguson, C., Tanzer, M.C., Murphy, J.M., Yap, A.S., Parton, R.G., Huston, W.M. & Teasdale, R.D. 2017, 'Laser-mediated rupture of chlamydial inclusions triggers pathogen egress and host cell necrosis.', Nat Commun, vol. 8, p. 14729.
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Remarkably little is known about how intracellular pathogens exit the host cell in order to infect new hosts. Pathogenic chlamydiae egress by first rupturing their replicative niche (the inclusion) before rapidly lysing the host cell. Here we apply a laser ablation strategy to specifically disrupt the chlamydial inclusion, thereby uncoupling inclusion rupture from the subsequent cell lysis and allowing us to dissect the molecular events involved in each step. Pharmacological inhibition of host cell calpains inhibits inclusion rupture, but not subsequent cell lysis. Further, we demonstrate that inclusion rupture triggers a rapid necrotic cell death pathway independent of BAK, BAX, RIP1 and caspases. Both processes work sequentially to efficiently liberate the pathogen from the host cytoplasm, promoting secondary infection. These results reconcile the pathogen's known capacity to promote host cell survival and induce cell death.
Lawrence, A., Eglezos, S. & Huston, W. 2016, 'Environmental Legionella spp. collected in urban test sites of South East Queensland, Australia, are virulent to human macrophages in vitro.', Research in microbiology, vol. 167, no. 2, pp. 149-153.
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Legionellae are frequent contaminants of potable water supplies, resulting in sporadic infections and occasional outbreaks. Isolates of Legionella were collected from urban test sites within South East Queensland and evaluated for their virulence potential in vitro. Two strains (from the species Legionella londiniensis and Legionella quinlivanii) were demonstrated to have the ability to infect human macrophages, while a strain from the species Legionella anisa did not maintain an infection over the same time course. This suggests that the spectrum of urban environmentally associated Legionella with potential to cause human disease might be greater than currently considered.
Zhu, Y., Pham, T.H., Nhiep, T.H., Vu, N.M., Marcellin, E., Chakrabortti, A., Wang, Y., Waanders, J., Lo, R., Huston, W.M., Bansal, N., Nielsen, L.K., Liang, Z.X. & Turner, M.S. 2016, 'Cyclic-di-AMP synthesis by the diadenylate cyclase CdaA is modulated by the peptidoglycan biosynthesis enzyme GlmM in Lactococcus lactis.', Molecular microbiology, vol. 99, no. 6, pp. 1015-1027.
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The second messenger cyclic-di-AMP plays important roles in growth, virulence, cell wall homeostasis, potassium transport and affects resistance to antibiotics, heat and osmotic stress. Most Firmicutes contain only one c-di-AMP synthesizing diadenylate cyclase (CdaA) however little is known about signals and effectors controlling CdaA activity and c-di-AMP levels. In this study, a genetic screen was employed to identify components which affect the c-di-AMP level in Lactococcus. We characterised suppressor mutations that restored osmoresistance to spontaneous c-di-AMP phosphodiesterase gdpP mutants which contain high c-di-AMP levels. Loss-of-function and gain-of-function mutations were identified in the cdaA and gdpP genes, respectively, which led to lower c-di-AMP levels. A mutation was also identified in the phosphoglucosamine mutase gene glmM which is commonly located within the cdaA operon in bacteria. The glmM I154F mutation resulted in a lowering of the c-di-AMP level and a reduction in the key peptidoglycan precursor UDP-N-acetylglucosamine in L. lactis. C-di-AMP synthesis by CdaA was shown to be inhibited by GlmM(I154F) more than GlmM and GlmM(I154F) was found to bind more strongly to CdaA than GlmM. These findings identify GlmM as a c-di-AMP level modulating protein and provide a direct connection between c-di-AMP synthesis and peptidoglycan biosynthesis.
Menon, S., Alexander, K., Timms, P., Allan, J.A. & Huston, W.M. 2016, 'CXCL10, CXCL11, HLA-A and IL-1 beta are induced in peripheral blood mononuclear cells from women with Chlamydia trachomatis related infertility', PATHOGENS AND DISEASE, vol. 74, no. 1.
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Teo, W.X., Kerr, M.C., Huston, W.M. & Teasdale, R.D. 2016, 'Sortilin is associated with the chlamydial inclusion and is modulated during infection.', Biology open, vol. 5, no. 4, pp. 429-435.
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Chlamydia species are obligate intracellular pathogens that have a major impact on human health. The pathogen replicates within an intracellular niche called an inclusion and is thought to rely heavily on host-derived proteins and lipids, including ceramide. Sortilin is a transmembrane receptor implicated in the trafficking of acid sphingomyelinase, which is responsible for catalysing the breakdown of sphingomyelin to ceramide. In this study, we examined the role of sortilin in Chlamydia trachomatis L2 development. Western immunoblotting and immunocytochemistry analysis revealed that endogenous sortilin is not only associated with the inclusion, but that protein levels increase in infected cells. RNAi-mediated depletion of sortilin, however, had no detectable impact on ceramide delivery to the inclusion or the production of infectious progeny. This study demonstrates that whilst Chlamydia redirects sortilin trafficking to the chlamydial inclusion, RNAi knockdown of sortilin expression is insufficient to determine if this pathway is requisite for the development of the pathogen.
Menon, S., Stansfield, S.H., Walsh, M., Hope, E., Isaia, L., Righarts, A.A., Niupulusu, T., Temese, S.V.A., Iosefa-Siitia, L., Auvaa, L., Tapelu, S.A., Motu, M.F., Suaalii-Sauni, T., Timms, P., Hill, P.C. & Huston, W.M. 2016, 'Sero-epidemiological assessment of Chlamydia trachomatis infection and sub-fertility in Samoan women.', BMC infectious diseases, vol. 16, p. 175.
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In our recent village-based cross-sectional study, the prevalence of nucleic acid amplification technique (NAAT) diagnosed Chlamydia trachomatis (CT) in sexually active Samoan women was very high (36 %), and test positivity was associated with sub-fertility. We conducted a serological and epidemiological analysis in these participants to identify if serological data can provide further insight into the potential contribution of CT to sub-fertility in this population.Serological prediction of CT associated sub-fertility was conducted using a series of commercial tests. The correlation between fertility or sub-fertility, behavioral factors, and serologically predicted CT associated sub-fertility was determined.A positive antibody reaction against the Chlamydia Major Outer Membrane Protein (MOMP) was significantly associated with sub-fertility, with 50 % of infertile women being positive. Serum IgG and IgA antibodies against MOMP correlated with current infection measured by urine NAAT, suggesting longer term infections are common in this population. Chlamydia pneumoniae antibodies were frequently detected in this population (84 %), and unexpectedly, were significantly associated with sub-fertility.The high prevalence of chlamydial infection and of positive chlamydial sub-fertility results suggests that CT is an important and frequent contributory factor to sub-fertility in this population.
Menon, S., Stansfield, S.H., Logan, B., Hocking, J.S., Timms, P., Rombauts, L., Allan, J. & Huston, W.M. 2016, 'Development and evaluation of a multi-antigen peptide ELISA for the diagnosis of Chlamydia trachomatis related infertility in women.', Journal of medical microbiology.
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Chlamydia trachomatis results in tubal factor infertility in some women. Diagnosis of this tubal infertility is difficult and typically involves laparoscopy or hysterosalpingography to detect the tubal blockages. Numerous serological tests have been developed however are presently not used for diagnosis without subsequent surgical during the infertility investigation. This study aimed to develop a highly specific serological assay for chlamydial tubal factor infertility in women that could be used to recommend direct progression to IVF treatment for women who are positive. Women were recruited from a variety of settings including; women seeking fertility treatment, sexual health and GP consults, or antenatal care (n=259). The serological assay was developed using sera from a large group of women by using infertile MIF positive women with tubal damage as the positives compared to infertile, or acute infection, and/or fertile controls (negatives). The new multi-peptide ELISA was highly specific for the detection of tubal factor infertility (p=0.011) compared to another ELISA (p=0.022) and MIF (p=0.099). The sensitivity of the assay should be improved before clinical utility. Potentially a two-step testing protocol could be used during the initial infertility investigation where MIF followed by a highly specific ELISA could be used to recommend direct progression to IVF for women who are positive.
Lawrence, A., Fraser, T., Gillett, A., Tyndall, J.D., Timms, P., Polkinghorne, A. & Huston, W.M. 2016, 'Chlamydia Serine Protease Inhibitor, targeting HtrA, as a New Treatment for Koala Chlamydia infection.', Scientific reports, vol. 6, p. 31466.
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The koala, an iconic marsupial native to Australia, is a threatened species in many parts of the country. One major factor in the decline is disease caused by infection with Chlamydia. Current therapeutic strategies to treat chlamydiosis in the koala are limited. This study examines the effectiveness of an inhibitor, JO146, which targets the HtrA serine protease for treatment of C. pecorum and C. pneumoniae in vitro and ex vivo with the aim of developing a novel therapeutic for koala Chlamydia infections. Clinical isolates from koalas were examined for their susceptibility to JO146. In vitro studies demonstrated that treatment with JO146 during the mid-replicative phase of C. pecorum or C. pneumoniae infections resulted in a significant loss of infectious progeny. Ex vivo primary koala tissue cultures were used to demonstrate the efficacy of JO146 and the non-toxic nature of this compound on peripheral blood mononuclear cells and primary cell lines established from koala tissues collected at necropsy. Our results suggest that inhibition of the serine protease HtrA could be a novel treatment strategy for chlamydiosis in koalas.
Ziklo, N., Huston, W.M., Hocking, J.S. & Timms, P. 2016, 'Chlamydia trachomatis Genital Tract Infections: When Host Immune Response and the Microbiome Collide', Trends in Microbiology, vol. 24, no. 9, pp. 750-765.
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© 2016Genital infections with Chlamydia trachomatis continue to be a major health problem worldwide. While some individuals clear their infection (presumed to be the result of an effective Th1/interferon- response), others develop chronic infections and some are prone to repeat infections. In females in particular, chronic asymptomatic infections are common and can lead to pelvic inflammatory disease and infertility. Recent studies suggest that the genital tract microbiota could be a significant factor and explain person-to-person variation in C. trachomatis infections. One hypothesis suggests that C. trachomatis can use its trpBA genes to rescue tryptophan from indole, which is a product of anaerobic members of the genital tract microbiota. Women with particular microbiota types, such as seen in bacterial vaginosis, have increased numbers of anaerobes, and this would enable the chlamydia in these individuals to overcome the host's interferon- attempts to eliminate it, resulting in more repeat and/or chronic infections.
Ziklo, N., Huston, W.M., Taing, K., Katouli, M. & Timms, P. 2016, 'In vitro rescue of genital strains of Chlamydia trachomatis from interferon- and tryptophan depletion with indole-positive, but not indole-negative Prevotella spp.', BMC Microbiol, vol. 16, no. 1, p. 286.
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BACKGROUND: The natural course of sexually transmitted infections caused by Chlamydia trachomatis varies between individuals. In addition to parasite and host effects, the vaginal microbiota might play a key role in the outcome of C. trachomatis infections. Interferon-gamma (IFN-), known for its anti-chlamydial properties, activates the expression of indoleamine 2,3-dioxygenase (IDO1) in epithelial cells, an enzyme that catabolizes the amino acid L- tryptophan into N-formylkynurenine, depleting the host cell's pool of tryptophan. Although C. trachomatis is a tryptophan auxotroph, urogenital strains (but not ocular strains) have been shown in vitro to have the ability to produce tryptophan from indole using the tryptophan synthase (trpBA) gene. It has been suggested that indole producing bacteria from the vaginal microbiota could influence the outcome of Chlamydia infection. RESULTS: We used two in vitro models (treatment with IFN- or direct limitation of tryptophan), to study the effects of direct rescue by the addition of exogenous indole, or by the addition of culture supernatant from indole-positive versus indole-negative Prevotella strains, on the growth and infectivity of C. trachomatis. We found that only supernatants from the indole-positive strains, P. intermedia and P. nigrescens, were able to rescue tryptophan-starved C. trachomatis. In addition, we analyzed vaginal secretion samples to determine physiological indole concentrations. In spite of the complexity of vaginal secretions, we demonstrated that for some vaginal specimens with higher indole levels, there was a link to higher recovery of the Chlamydia under tryptophan-starved conditions, lending preliminary support to the critical role of the IFN--tryptophan-indole axis in vivo. CONCLUSIONS: Our data provide evidence for the ability of both exogenous indole as well as supernatant from indole producing bacteria such as Prevotella, to rescue genital C. trachomatis from tryptophan starvation. This...
Christensen, S., Grøftehauge, M.K., Byriel, K., Huston, W.M., Furlong, E., Heras, B., Martin, J.L. & McMahon, R.M. 2016, 'Structural and Biochemical Characterization of Chlamydia trachomatis DsbA Reveals a Cysteine-Rich and Weakly Oxidising Oxidoreductase.', PLoS One, vol. 11, no. 12, p. e0168485.
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The Gram negative bacteria Chlamydia trachomatis is an obligate intracellular human pathogen that can cause pelvic inflammatory disease, infertility and blinding trachoma. C. trachomatis encodes a homolog of the dithiol oxidoreductase DsbA. Bacterial DsbA proteins introduce disulfide bonds to folding proteins providing structural bracing for secreted virulence factors, consequently these proteins are potential targets for antimicrobial drugs. Despite sharing functional and structural characteristics, the DsbA enzymes studied to date vary widely in their redox character. In this study we show that the truncated soluble form of the predicted membrane anchored protein C. trachomatis DsbA (CtDsbA) has oxidase activity and redox properties broadly similar to other characterized DsbA proteins. However CtDsbA is distinguished from other DsbAs by having six cysteines, including a second disulfide bond, and an unusual dipeptide sequence in its catalytic motif (Cys-Ser-Ala-Cys). We report the 2.7 Å crystal structure of CtDsbA revealing a typical DsbA fold, which is most similar to that of DsbA-II type proteins. Consistent with this, the catalytic surface of CtDsbA is negatively charged and lacks the hydrophobic groove found in EcDsbA and DsbAs from other enterobacteriaceae. Biochemical characterization of CtDsbA reveals it to be weakly oxidizing compared to other DsbAs and with only a mildly destabilizing active site disulfide bond. Analysis of the crystal structure suggests that this redox character is consistent with a lack of contributing factors to stabilize the active site nucleophilic thiolate relative to more oxidizing DsbA proteins.
Walsh, M.S., Hope, E., Isaia, L., Righarts, A., Niupulusu, T., Temese, S.V., Iosefa-Siitia, L., Auvaa, L., Tapelu, S.A., Motu, M.F., Edwards, C., Wernick, M., Huston, W.M., Suaalii-Sauni, T. & Hill, P.C. 2015, 'Prevalence of Chlamydia trachomatis infection in Samoan women aged 18 to 29 and assessment of possible risk factors: a community-based study.', Transactions of the Royal Society of Tropical Medicine and Hygiene, vol. 109, no. 4, pp. 245-251.
Knowledge about genital Chlamydia trachomatis (CT) infections in the Pacific is limited. In this study we investigated CT infection in Samoan women.We recruited women having unprotected sex aged 18 to 29 years from 41 Samoan villages. They completed a questionnaire and provided a urine sample for CT testing by PCR. Associations between CT infection and possible risk factors were explored using logistic regression.Altogether, 239 women were recruited; 86 (36.0%; weighted estimate of prevalence: 41.9%; 95% CI: 33.4-50.5%) were positive for CT infection. A higher proportion of women aged 18 to 24 were positive (54/145; 37.2%) than those aged 25 to 29 (32/94; 34.0%; p=0.20). Being single (OR 1.92; 95% CI: 1.02-3.63) and having two or more lifetime sexual partners (OR 3.02; 95% CI: 1.19-7.67) were associated with CT infection; 27.6% of those with one lifetime partner were positive. Participants who had a previous pregnancy were less likely to be positive (OR 0.49; 95% CI: 0.27-0.87). Primiparous and multiparous women were less likely to be positive than nulliparous women (OR 0.54; 95% CI: 0.30-0.99 and OR 0.46; 95% CI: 0.24-0.89, respectively).The prevalence of CT infection in these Samoan women is very high. Further studies, including investigating the prevalence of CT infection in men, and strategies for sustainable control are needed.
Chacko, A., Beagley, K.W., Timms, P. & Huston, W.M. 2015, 'Human Chlamydia pneumoniae isolates demonstrate ability to recover infectivity following penicillin treatment whereas animal isolates do not.', FEMS microbiology letters, vol. 362, no. 6.
Chlamydia pneumoniae strains have recently been demonstrated to have substantially different capacities to enter and recover from IFN--induced persistence, depending on whether they are from human or animal host sources. Here, we examined the ability of two human and two animal strains to enter and be rescued from penicillin-induced persistence. The ability to form inclusions after the addition of penicillin was much reduced in the two animal isolates (koala LPCoLN, bandicoot B21) compared to the two human isolates (respiratory AR39 and heart A03). The penicillin treatment resulted in a dose-dependent loss of infectious progeny for all isolates, with the human strains failing to produce infectious progeny at lower doses of penicillin than the animal strains. The most remarkable finding however was the contrasting ability of the isolates to recover infectious progeny production after rescue by removal of the penicillin (at 72 h) and continued culture. The animal isolates both showed virtually no recovery from the penicillin treatment conditions. In contrast, the human isolates showed a significant ability to recovery infectivity, with the heart isolate (A03) showing the most marked recovery. Combined, these data further support the hypothesis that the ability to establish and recover from persistence appears to be enhanced in human C. pneumoniae strains compared to animal strains.
Kong, F.Y., Tabrizi, S.N., Fairley, C.K., Vodstrcil, L.A., Huston, W.M., Chen, M., Bradshaw, C. & Hocking, J.S. 2015, 'The efficacy of azithromycin and doxycycline for the treatment of rectal chlamydia infection: a systematic review and meta-analysis.', The Journal of antimicrobial chemotherapy, vol. 70, no. 5, pp. 1290-1297.
There are increasing concerns about treatment failure following treatment for rectal chlamydia with 1 g of azithromycin. A systematic review and meta-analysis was conducted to investigate the efficacy of 1 g of azithromycin as a single dose or 100 mg of doxycycline twice daily for 7 days for the treatment of rectal chlamydia.Medline, Embase, PubMed, Cochrane Controlled Trials Register, Australia New Zealand Clinical Trial Register and ClinicalTrials.gov were searched to the end of April 2014. Studies using 1 g of azithromycin or 7 days of doxycycline for the treatment of rectal chlamydia were eligible. Gender, diagnostic test, serovar, symptomatic status, other sexually transmitted infections, follow-up time, attrition and microbial cure were extracted. Meta-analysis was used to calculate pooled (i) azithromycin and doxycycline efficacy and (ii) efficacy difference.All eight included studies were observational. The random-effects pooled efficacy for azithromycin (based on eight studies) was 82.9% (95% CI 76.0%-89.8%; I(2)=71.0%; P<0.01) and for doxycycline (based on five studies) was 99.6% (95% CI 98.6%-100%; I(2)=0%; P=0.571), resulting in a random-effects pooled efficacy difference (based on five studies) of 19.9% (95% CI 11.4%-28.3%; I(2)=48.5%; P=0.101) in favour of doxycycline.The efficacy of single-dose azithromycin may be considerably lower than 1 week of doxycycline for treating rectal chlamydia. However, the available evidence is very poor. Robust randomized controlled trials are urgently required.
Vodstrcil, L.A., McIver, R., Huston, W.M., Tabrizi, S.N., Timms, P. & Hocking, J.S. 2015, 'The Epidemiology of Chlamydia trachomatis Organism Load During Genital Infection: A Systematic Review.', The Journal of infectious diseases, vol. 211, no. 10, pp. 1628-1645.
BACKGROUND: The role of organism load in Chlamydia trachomatis infection is not well understood. We conducted a systematic review to investigate the epidemiology of C. trachomatis organism load in human genital chlamydia infection. METHODS: Embase, PubMed, and Medline databases were searched for literature published through August 2014. English-language publications that quantified load in humans were eligible. Participant characteristics and laboratory data were extracted. RESULTS: A total of 737 records were identified, and 29 publications involving 40 883 participants were included. In women, load was highest for cervical swabs and lowest for urine specimens. In men, load was highest for rectal swabs and similar for urethral swabs and urine specimens. Evidence of any association between load and age, serovar, risk of transmission, hormone levels, and concurrent sexually transmitted infections was inconsistent. Eight of 9 culture-based studies found an association between load and signs and symptoms, in contrast with only 3 of 8 nucleic acid amplification test (NAAT)-based studies (P = .03). CONCLUSION: Chlamydia organism load varies by specimen type and site of sampling, and viable chlamydia organism load may be a more important indicator of severity of infection than total load measured by NAAT.
Hocking, J.S., Kong, F.Y., Timms, P., Huston, W.M. & Tabrizi, S.N. 2015, 'Treatment of rectal chlamydia infection may be more complicated than we originally thought.', The Journal of antimicrobial chemotherapy, vol. 70, no. 4, pp. 961-964.
Rectal chlamydia diagnoses have been increasing among MSM and may also rise among women as anal sex rates increase among heterosexuals. However, there is growing concern about treatment for rectal chlamydia with treatment failures of up to 22% being reported. This article addresses factors that may be contributing to treatment failure for rectal chlamydia, including the pharmacokinetic properties of azithromycin and doxycycline in rectal tissue, the ability of chlamydia to transform into a persistent state that is less responsive to antimicrobial therapy, the impact of the rectal microbiome on chlamydia, heterotypic resistance, failure to detect cases of lymphogranuloma venereum and the performance of screening tests. If we are to reduce the burden of genital chlamydia, treatment for rectal chlamydia must be efficacious. This highlights the need for randomized controlled trial evidence comparing azithromycin with doxycycline for the treatment of rectal chlamydia.
Menon, S., Timms, P., Allan, J.A., Alexander, K., Rombauts, L., Horner, P., Keltz, M., Hocking, J. & Huston, W.M. 2015, 'Human and Pathogen Factors Associated with Chlamydia trachomatis-Related Infertility in Women', CLINICAL MICROBIOLOGY REVIEWS, vol. 28, no. 4, pp. 969-985.
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Marsh, J.W., Wee, B.A., Tyndall, J.D.A., Lott, W.B., Bastidas, R.J., Caldwell, H.D., Valdivia, R.H., Kari, L. & Huston, W.M. 2015, 'A Chlamydia trachomatis strain with a chemically generated amino acid substitution (P370L) in the cthtrA gene shows reduced elementary body production', BMC Microbiology, vol. 15, no. 194, pp. 1-13.
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Roulis, E., Bachmann, N.L., Myers, G.S.A., Huston, W., Summersgill, J., Hudson, A., Dreses-Werringloer, U., Polkinghorne, A. & Timms, P. 2015, 'Comparative genomic analysis of human Chlamydia pneumoniae isolates from respiratory, brain and cardiac tissues.', Genomics, vol. 106, no. 6, pp. 373-383.
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Chlamydia pneumoniae is an obligate intracellular bacterium implicated in a wide range of human diseases including atherosclerosis and Alzheimer's disease. Efforts to understand the relationships between C. pneumoniae detected in these diseases have been hindered by the availability of sequence data for non-respiratory strains. In this study, we sequenced the whole genomes for C. pneumoniae isolates from atherosclerosis and Alzheimer's disease, and compared these to previously published C. pneumoniae genomes. Phylogenetic analyses of these new C. pneumoniae strains indicate two sub-groups within human C. pneumoniae, and suggest that both recombination and mutation events have driven the evolution of human C. pneumoniae. Further fine-detailed analyses of these new C. pneumoniae sequences show several genetically variable loci. This suggests that similar strains of C. pneumoniae are found in the brain, lungs and cardiovascular system and that only minor genetic differences may contribute to the adaptation of particular strains in human disease.
Roulis, E., Bachmann, N., Humphrys, M., Myers, G., Huston, W., Polkinghorne, A. & Timms, P. 2015, 'Phylogenetic analysis of human Chlamydia pneumoniae strains reveals a distinct Australian indigenous clade that predates European exploration of the continent.', BMC genomics, vol. 16, p. 1094.
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The obligate intracellular bacterium Chlamydia pneumoniae is a common respiratory pathogen, which has been found in a range of hosts including humans, marsupials and amphibians. Whole genome comparisons of human C. pneumoniae have previously highlighted a highly conserved nucleotide sequence, with minor but key polymorphisms and additional coding capacity when human and animal strains are compared.In this study, we sequenced three Australian human C. pneumoniae strains, two of which were isolated from patients in remote indigenous communities, and compared them to all available C. pneumoniae genomes. Our study demonstrated a phylogenetically distinct human C. pneumoniae clade containing the two indigenous Australian strains, with estimates that the most recent common ancestor of these strains predates the arrival of European settlers to Australia. We describe several polymorphisms characteristic to these strains, some of which are similar in sequence to animal C. pneumoniae strains, as well as evidence to suggest that several recombination events have shaped these distinct strains.Our study reveals a greater sequence diversity amongst both human and animal C. pneumoniae strains, and suggests that a wider range of strains may be circulating in the human population than current sampling indicates.
Deeudom, M., Huston, W. & Moir, J.W.B. 2015, 'Erratum to: Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR [Antonie van Leeuwenhoek, 10.1007/s10482-015-0400-z]', Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, vol. 107, no. 4, p. 1117.
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Deeudom, M., Huston, W. & Moir, J.W.B. 2015, 'Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR', Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, vol. 107, no. 4, pp. 1107-1116.
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&copy; 2015, Springer International Publishing Switzerland.The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626&nbsp;nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the Laz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.
Ong, V.A., Lawrence, A., Timms, P., Vodstrcil, L.A., Tabrizi, S.N., Beagley, K.W., Allan, J.A., Hocking, J.S. & Huston, W.M. 2015, 'In vitro susceptibility of recent Chlamydia trachomatis clinical isolates to the CtHtrA inhibitor JO146', MICROBES AND INFECTION, vol. 17, no. 11-12, pp. 738-744.
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Patel, P., De Boer, L., Timms, P. & Huston, W.M. 2014, 'Evidence of a conserved role for Chlamydia HtrA in the replication phase of the chlamydial developmental cycle.', Microbes and infection / Institut Pasteur, vol. 16, no. 8, pp. 690-694.
Identification of the HtrA inhibitor JO146 previously enabled us to demonstrate an essential function for HtrA during the mid-replicative phase of the Chlamydia trachomatis developmental cycle. Here we extend our investigations to other members of the Chlamydia genus. C. trachomatis isolates with distinct replicative phase growth kinetics showed significant loss of viable infectious progeny after HtrA was inhibited during the replicative phase. Mid-replicative phase addition of JO146 was also significantly detrimental to Chlamydia pecorum, Chlamydia suis and Chlamydia cavie. These data combined indicate that HtrA has a conserved critical role during the replicative phase of the chlamydial developmental cycle.
Chacko, A., Barker, C.J., Beagley, K.W., Hodson, M.P., Plan, M.R., Timms, P. & Huston, W.M. 2014, 'Increased sensitivity to tryptophan bioavailability is a positive adaptation by the human strains of Chlamydia pneumoniae.', Molecular microbiology, vol. 93, no. 4, pp. 797-813.
One of the most significant activities induced by interferon-gamma against intracellular pathogens is the induction of IDO (indoleamine 2,3-dioxygenase) expression, which subsequently results in the depletion of tryptophan. We tested the hypothesis that human strains of Chlamydia pneumoniae are more sensitive to tryptophan limitation than animal C. pneumoniae strains. The human strains were significantly more sensitive to IFN- than the animal strains in a lung epithelia cell model (BEAS-2B), with exposure to 1Uml(-1) IFN- resulting in complete loss of infectious yield of human strains, compared to the animal strains where reductions in infectious progeny were around 3.5-4.0 log. Strikingly, the IFN- induced loss of ability to form infectious progeny production was completely rescued by removal of the IFN- and addition of exogenous tryptophan for the human strains, but not the animal strains. In fact, a human heart strain was more capable of entering a non-infectious, viable persistent stage when exposed to IFN- and was also more effectively rescued, compared to a human respiratory strain. Exquisite susceptibility to IFN-, specifically due to tryptophan availability appears to be a core adaptation of the human C. pneumoniae strains, which may reflect the chronic nature of their infections in this host.
Huston, W.M., Barker, C.J., Chacko, A. & Timms, P. 2014, 'Evolution to a chronic disease niche correlates with increased sensitivity to tryptophan availability for the obligate intracellular bacterium Chlamydia pneumoniae.', Journal of bacteriology, vol. 196, no. 11, pp. 1915-1924.
The chlamydiae are obligate intracellular parasites that have evolved specific interactions with their various hosts and host cell types to ensure their successful survival and consequential pathogenesis. The species Chlamydia pneumoniae is ubiquitous, with serological studies showing that most humans are infected at some stage in their lifetime. While most human infections are asymptomatic, C. pneumoniae can cause more-severe respiratory disease and pneumonia and has been linked to chronic diseases such as asthma, atherosclerosis, and even Alzheimer's disease. The widely dispersed animal-adapted C. pneumoniae strains cause an equally wide range of diseases in their hosts. It is emerging that the ability of C. pneumoniae to survive inside its target cells, including evasion of the host's immune attack mechanisms, is linked to the acquisition of key metabolites. Tryptophan and arginine are key checkpoint compounds in this host-parasite battle. Interestingly, the animal strains of C. pneumoniae have a slightly larger genome, enabling them to cope better with metabolite restrictions. It therefore appears that as the evolutionarily more ancient animal strains have evolved to infect humans, they have selectively become more "susceptible" to the levels of key metabolites, such as tryptophan. While this might initially appear to be a weakness, it allows these human C. pneumoniae strains to exquisitely sense host immune attack and respond by rapidly reverting to a persistent phase. During persistence, they reduce their metabolic levels, halting progression of their developmental cycle, waiting until the hostile external conditions have passed before they reemerge.
Bengtson Nash, S., Dawson, A., Burkhard, M., Waugh, C. & Huston, W. 2014, 'Detoxification enzyme activities (CYP1A1 and GST) in the skin of humpback whales as a function of organochlorine burdens and migration status.', Aquatic toxicology (Amsterdam, Netherlands), vol. 155, pp. 207-212.
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The activities of glutathione-s-transferase (GST) and cytochrome P-450 1A1 (CYP1A1) enzymes were measured in freshly extracted epidermis of live-biopsied, migrating, southern hemisphere humpback whales (Megaptera novaeangliae). The two quantified enzyme activities did not correlate strongly with each other. Similarly, neither correlated strongly with any of the organochlorine compound groups previously measured in the superficial blubber of the sample biopsy core, likely reflecting the anticipated low levels of typical aryl-hydrocarbon receptor ligands. GST activity did not differ significantly between genders or between northward (early migration) or southward (late migration) migrating cohorts. Indeed, the inter-individual variability in GST measurements was relatively low. This observation raises the possibility that measured activities were basal activities and that GST function was inherently impacted by the fasting state of the sampled animals, as seen in other species. These results do not support the implementation of CYP1A1 or GST as effective biomarkers of organochlorine contaminant burdens in southern hemisphere populations of humpback whales as advocated for other cetacean species. Further investigation of GST activity in feeding versus fasting cohorts may, however, provide some insight into the fasting metabolism of these behaviourally adapted populations.
Lawrence, A., K Nicholls, S., H Stansfield, S. & M Huston, W. 2014, 'Characterization of the tail-specific protease (Tsp) from Legionella.', The Journal of general and applied microbiology, vol. 60, no. 3, pp. 95-100.
Bacterial tail-specific proteases (Tsps) have been attributed a wide variety of functions including intracellular virulence, cell wall morphology, proteolytic signal cascades and stress response. This study tested the hypothesis that Tsp has a key function for the transmissive form of Legionella pneumophila. A tsp mutant was generated in Legionella pneumophila 130b and the characteristics of this strain and the isogenic wild-type were examined using a range of growth and proteomic analyses. Recombinant Tsp protein was also produced and analyzed. The L. pneumophila tsp mutant showed no defect in growth on rich media or during thermo-osmotic stress conditions. In addition, no defects in cellular morphology were observed when the cells were examined using transmission electron microscopy. Purified recombinant Tsp was found to be an active protease with a narrow substrate range. Proteome analysis using iTRAQ (5% coverage of the proteome) found that, of those proteins detected, only 5 had different levels in the tsp mutant compared to the wild type. ACP (Acyl Carrier Protein), which has a key role for Legionella differentiation to the infectious form, was reduced in the tsp mutant; however, tsp(-) was able to infect and replicate inside macrophages to the same extent as the wild type. Combined, these data demonstrate that Tsp is a protease but is not essential for Legionella growth or cell infection. Thus, Tsp may have functional redundancy in Legionella.
Gloeckl, S., Ong, V., Patel, P., Tyndall, J., Timms, P., Beagley, K., Allan, J., Armitage, C., Turnbull, L., Whitchurch, C.B., Merdanovic, M., Ehrmann, M., Powers, J., Oleksyszyn, J., Verdoes, M., Bogyo, M. & Huston, W. 2013, 'Identification Of A Serine Protease Inhibitor Which Causes Inclusion Vacuole Reduction And Is Lethal To Chlamydia Trachomatis', Molecular Microbiology, vol. 89, no. 4, pp. 676-689.
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The mechanistic details of the pathogenesis of Chlamydia, an obligate intracellular pathogen of global importance, have eluded scientists due to the scarcity of traditional molecular genetic tools to investigate this organism. Here we report a chemical b
Ong, V.A., Marsh, J.W., Lawrence, A., Allan, J.A., Timms, P. & Huston, W.M. 2013, 'The protease inhibitor JO146 demonstrates a critical role for CtHtrA for Chlamydia trachomatis reversion from penicillin persistence', FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, vol. 3.
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Cunningham, K., Stansfield, S.H., Patel, P., Menon, S., Kienzle, V., Allan, J.A. & Huston, W.M. 2013, 'The IL-6 response to Chlamydia from primary reproductive epithelial cells is highly variable and may be involved in differential susceptibility to the immunopathological consequences of chlamydial infection', BMC IMMUNOLOGY, vol. 14.
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Carey, A.J., Huston, W.M., Cunningham, K.A., Hafner, L.M., Timms, P. & Beagley, K.W. 2013, 'Characterization of in vitro Chlamydia muridarum persistence and utilization in an in vivo mouse model of Chlamydia vaccine.', American journal of reproductive immunology (New York, N.Y. : 1989), vol. 69, no. 5, pp. 475-485.
PROBLEM: Chlamydia trachomatis genital tract infections are easily treated with antibiotics; however, the majority of infections are asymptomatic and therefore untreated, highlighting the need for a vaccine. Because most infections are asymptomatic, vaccination could potentially be administered to individuals who may have an acute infection at that time. In such individuals, the effect of vaccination on the existing infection is unknown; however, one potential outcome could be the development of a persistent infection. In vitro chlamydial persistence has been well characterized in various strains; however, there have been no reported studies in C.&nbsp;muridarum. METHOD OF STUDY: We performed ultrastructural characterization and transcriptome analysis of selected genes. We then used the transcriptional profiles of the selected genes to examine whether intranasal immunization of mice during an active genital infection would induce persistence in the upper reproductive tract of female mice. RESULTS AND CONCLUSIONS: We found that persistence developed in the oviducts of mice as a result of immunization. This is a significant finding, not only because it is the first time that C.&nbsp;muridarum persistence has been characterized in vitro, but also due to the fact that there is a minimal characterization of in vivo persistence of any chlamydial species. This highlights the importance of the timing of vaccination in individuals.
Stansfield, S.H., Patel, P., Debattista, J., Armitage, C.W., Cunningham, K., Timms, P., Allan, J., Mittal, A. & Huston, W.M. 2013, 'Proof of concept: A bioinformatic and serological screening method for identifying new peptide antigens for Chlamydia trachomatis related sequelae in women.', Results in immunology, vol. 3, pp. 33-39.
This study aimed to identify new peptide antigens from Chlamydia (C.) trachomatis in a proof of concept approach which could be used to develop an epitope-based serological diagnostic for C. trachomatis related infertility in women. A bioinformatics analysis was conducted examining several immunodominant proteins from C. trachomatis to identify predicted immunoglobulin epitopes unique to C. trachomatis. A peptide array of these epitopes was screened against participant sera. The participants (all female) were categorized into the following cohorts based on their infection and gynecological history; acute (single treated infection with C. trachomatis), multiple (more than one C. trachomatis infection, all treated), sequelae (PID or tubal infertility with a history of C. trachomatis infection), and infertile (no history of C. trachomatis infection and no detected tubal damage). The bioinformatics strategy identified several promising epitopes. Participants who reacted positively in the peptide 11 ELISA were found to have an increased likelihood of being in the sequelae cohort compared to the infertile cohort with an odds ratio of 16.3 (95% c.i. 1.65-160), with 95% specificity and 46% sensitivity (0.19-0.74). The peptide 11 ELISA has the potential to be further developed as a screening tool for use during the early IVF work up and provides proof of concept that there may be further peptide antigens which could be identified using bioinformatics and screening approaches.
Marsh, J.W., Lott, W.B., Tyndall, J.D. & Huston, W.W. 2013, 'Proteolytic activation of Chlamydia trachomatis HTRA is mediated by PDZ1 domain interactions with protease domain loops L3 and LC and beta strand 5.', Cellular & molecular biology letters, vol. 18, no. 4, pp. 522-537.
Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand 5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand 5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.
Hocking, J.S., Vodstrcil, L.A., Huston, W.M., Timms, P., Chen, M.Y., Worthington, K., McIver, R. & Tabrizi, S.N. 2013, 'A cohort study of Chlamydia trachomatis treatment failure in women: a study protocol.', BMC infectious diseases, vol. 13, p. 379.
BACKGROUND: Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection in the developed world and diagnosis rates have increased dramatically over the last decade. Repeat infections of chlamydia are very common and may represent re-infection from an untreated partner or treatment failure. The aim of this cohort study is to estimate the proportion of women infected with chlamydia who experience treatment failure after treatment with 1 gram azithromycin. METHODS/DESIGN: This cohort study will follow women diagnosed with chlamydia for up to 56 days post treatment. Women will provide weekly genital specimens for further assay. The primary outcome is the proportion of women who are classified as having treatment failure 28, 42 or 56 days after recruitment. Comprehensive sexual behavior data collection and the detection of Y chromosome DNA and high discriminatory chlamydial genotyping will be used to differentiate between chlamydia re-infection and treatment failure. Azithromycin levels in high-vaginal specimens will be measured using a validated liquid chromatography-tandem mass spectrometry method to assess whether poor azithromycin absorption could be a cause of treatment failure. Chlamydia culture and minimal inhibitory concentrations will be performed to further characterize the chlamydia infections. DISCUSSION: Distinguishing between treatment failure and re-infection is important in order to refine treatment recommendations and focus infection control mechanisms. If a large proportion of repeat chlamydia infections are due to antibiotic treatment failure, then international recommendations on chlamydia treatment may need to be re-evaluated. If most are re-infections, then strategies to expedite partner treatment are necessary.
Gloeckl, S., Tyndall, J.D., Stansfield, S.H., Timms, P. & Huston, W.M. 2012, 'the active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions.', Journal of molecular microbiology and biotechnology, vol. 22, no. 1, pp. 10-16.
HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.
Huston, W.M., Harvie, M., Mittal, A., Timms, P. & Beagley, K.W. 2012, 'Vaccination to protect against infection of the female reproductive tract.', Expert review of clinical immunology, vol. 8, no. 1, pp. 81-94.
Infection of the female genital tract can result in serious morbidities and mortalities from reproductive disability, pelvic inflammatory disease and cancer, to impacts on the fetus, such as infant blindness. While therapeutic agents are available, frequent testing and treatment is required to prevent the occurrence of the severe disease sequelae. Hence, sexually transmitted infections remain a major public health burden with ongoing social and economic barriers to prevention and treatment. Unfortunately, while there are two success stories in the development of vaccines to protect against HPV infection of the female reproductive tract, many serious infectious agents impacting on the female reproductive tract still have no vaccines available. Vaccination to prevent infection of the female reproductive tract is an inherently difficult target, with many impacting factors, such as appropriate vaccination strategies/mechanisms to induce a suitable protective response locally in the genital tract, variation in the local immune responses due to the hormonal cycle, selection of vaccine antigen(s) that confers effective protection against multiple variants of a single pathogen (e.g., the different serovars of Chlamydia trachomatis) and timing of the vaccine administration prior to infection exposure. Despite these difficulties, there are numerous ongoing efforts to develop effective vaccines against these infectious agents and it is likely that this important human health field will see further major developments in the next 5 years.
Huston, W.M., Tyndall, J.D., Lott, W.B., Stansfield, S.H. & Timms, P. 2011, 'Unique residues involved in activation of the multitasking protease/chaperone HtrA from Chlamydia trachomatis.', PloS one, vol. 6, no. 9, p. e24547.
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DegP, a member of the HtrA family of proteins, conducts critical bacterial protein quality control by both chaperone and proteolysis activities. The regulatory mechanisms controlling these two distinct activities, however, are unknown. DegP activation is known to involve a unique mechanism of allosteric binding, conformational changes and oligomer formation. We have uncovered a novel role for the residues at the PDZ1:protease interface in oligomer formation specifically for chaperone substrates of Chlamydia trachomatis HtrA (DegP homolog). We have demonstrated that CtHtrA proteolysis could be activated by allosteric binding and oligomer formation. The PDZ1 activator cleft was required for the activation and oligomer formation. However, unique to CtHtrA was the critical role for residues at the PDZ1:protease interface in oligomer formation when the activator was an in vitro chaperone substrate. Furthermore, a potential in vivo chaperone substrate, the major outer membrane protein (MOMP) from Chlamydia, was able to activate CtHtrA and induce oligomer formation. Therefore, we have revealed novel residues involved in the activation of CtHtrA which are likely to have important in vivo implications for outer membrane protein assembly.
Huston, W.M., Gloeckl, S., de Boer, L., Beagley, K.W. & Timms, P. 2011, 'Apoptosis is induced in Chlamydia trachomatis-infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone.', American journal of reproductive immunology (New York, N.Y. : 1989), vol. 65, no. 5, pp. 460-465.
PROBLEM: Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. METHOD OF STUDY: Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. RESULTS: The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. CONCLUSION: This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone.
Waugh, C.A., Huston, W.M., Noad, M.J. & Bengtson Nash, S. 2011, 'Cytochrome P450 isozyme protein verified in the skin of southern hemisphere humpback whales (Megaptera novaeangliae): implications for biochemical biomarker assessment.', Marine pollution bulletin, vol. 62, no. 4, pp. 758-761.
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Large mysticete whales represent a unique challenge for chemical risk assessment. Few epidemiological investigations are possible due to the low incidence of adult stranding events. Similarly their often extreme life-history adaptations of prolonged migration and fasting challenge exposure assumptions. Molecular biomarkers offer the potential to complement information yielded through tissue chemical analysis, as well as providing evidence of a molecular response to chemical exposure. In this study we confirm the presence of cytochrome P450 reductase (CPR) and cytochrome P450 isoenzyme 1A1 (CYP1A1) in epidermal tissue of southern hemisphere humpback whales (Megaptera novaeangliae). The detection of CYP1A1 in the integument of the humpback whale affords the opportunity for further quantitative non-destructive investigations of enzyme activity as a function of chemical stress.
Huston, W.M., Armitage, C.W., Lawrence, A., Gloeckl, S., Bell, S.J., Debattista, J., Allan, J.A., Timms, P. & Res, Q.C.C. 2010, 'HtrA, RseP, and Tsp proteins do not elicit a pathology-related serum IgG response during sexually transmitted infection with Chlamydia trachomatis', JOURNAL OF REPRODUCTIVE IMMUNOLOGY, vol. 85, no. 2, pp. 168-171.
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Huston, W.M. 2010, 'Bacterial proteases from the intracellular vacuole niche; protease conservation and adaptation for pathogenic advantage.', FEMS immunology and medical microbiology, vol. 59, no. 1, pp. 1-10.
Proteases with important roles for bacterial pathogens that specifically reside within intracellular vacuoles are frequently homologous to those that have important virulence functions for other bacteria. Research has identified that some of these conserved proteases have evolved specialized functions for intracellular vacuole-residing bacteria. Unique proteases with pathogenic functions have also been described from Chlamydia, Mycobacteria, and Legionella. These findings suggest that there are further novel functions for proteases from these bacteria that remain to be described. This review summarizes the recent findings of novel protease functions from the intracellular human pathogenic bacteria that reside exclusively in vacuoles.
Beagley, K.W., Huston, W.M., Hansbro, P.M. & Timms, P. 2009, 'Chlamydial infection of immune cells: altered function and implications for disease.', Critical reviews in immunology, vol. 29, no. 4, pp. 275-305.
Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects the genital and ocular mucosa of humans, causing infections that can lead to pelvic inflammatory disease, infertility, and blinding trachoma. C. pneumoniae is a respiratory pathogen that is the cause of 12-15% of community-acquired pneumonia. Both chlamydial species were believed to be restricted to the epithelia of the genital, ocular, and respiratory mucosa; however, increasing evidence suggests that both these pathogens can be isolated from peripheral blood of both healthy individuals and patients with inflammatory conditions such as coronary artery disease and asthma. Chlamydia can also be isolated from brain tissues of patients with degenerative neurological disorders such as Alzheimer's disease and multiple sclerosis, and also from certain lymphomas. An increasing number of in vitro studies suggest that some chlamydial species can infect immune cells, at least at low levels. These infections may alter immune cell function in a way that promotes chlamydial persistence in the host and contributes to the progression of several chronic inflammatory diseases. In this paper, we review the evidence for the growth of Chlamydia in immune cells, particularly monocytes/macrophages and dendritic cells, and describe how infection may affect the function of these cells.
Huston, W.M., Theodoropoulos, C., Mathews, S.A. & Timms, P. 2008, 'Chlamydia trachomatis responds to heat shock, penicillin induced persistence, and IFN-gamma persistence by altering levels of the extracytoplasmic stress response protease HtrA.', BMC microbiology, vol. 8, p. 190.
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BACKGROUND: Chlamydia trachomatis, an obligate intracellular human pathogen, is the most prevalent bacterial sexually transmitted infection worldwide and a leading cause of preventable blindness. HtrA is a virulence and stress response periplasmic serine protease and molecular chaperone found in many bacteria. Recombinant purified C. trachomatis HtrA has been previously shown to have both activities. This investigation examined the physiological role of Chlamydia trachomatis HtrA. RESULTS: The Chlamydia trachomatis htrA gene complemented the lethal high temperature phenotype of Escherichia coli htrA- (>42 degrees C). HtrA levels were detected to increase by western blot and immunofluorescence during Chlamydia heat shock experiments. Confocal laser scanning microscopy revealed a likely periplasmic localisation of HtrA. During penicillin induced persistence of Chlamydia trachomatis, HtrA levels (as a ratio of LPS) were initially less than control acute cultures (20 h post infection) but increased to more than acute cultures at 44 h post infection. This was unlike IFN-gamma persistence where lower levels of HtrA were observed, suggesting Chlamydia trachomatis IFN-gamma persistence does not involve a broad stress response. CONCLUSION: The heterologous heat shock protection for Escherichia coli, and increased HtrA during cell wall disruption via penicillin and heat shock, indicates an important role for HtrA during high protein stress conditions for Chlamydia trachomatis.
Tan, Y.P., Giffard, P.M., Barry, D.G., Huston, W.M. & Turner, M.S. 2008, 'Random mutagenesis identifies novel genes involved in the secretion of antimicrobial, cell wall-lytic enzymes by Lactococcus lactis.', Applied and environmental microbiology, vol. 74, no. 24, pp. 7490-7496.
Lactococcus lactis is a gram-positive bacterium that is widely used in the food industry and is therefore desirable as a candidate for the production and secretion of recombinant proteins. Previously, we generated a L. lactis strain that expressed and secreted the antimicrobial cell wall-lytic enzyme lysostaphin. To identify lactococcal gene products that affect the production of lysostaphin, we isolated and characterized mutants generated by random transposon mutagenesis that had altered lysostaphin activity. Out of 35,000 mutants screened, only one with no lysostaphin activity was identified, and it was found to contain an insertion in the lysostaphin expression cassette. Ten mutants with higher lysostaphin activity contained insertions in only four different genes, which encode an uncharacterized putative transmembrane protein (llmg_0609) (three mutants), an enzyme catalyzing the first step in peptidoglycan biosynthesis (murA2) (five mutants), a putative regulator of peptidoglycan modification (trmA) (one mutant), and an uncharacterized enzyme possibly involved in ubiquinone biosynthesis (llmg_2148) (one mutant). These mutants were found to secrete larger amounts of lysostaphin than the control strain (MG1363[lss]), and the greatest increase in secretion was 9.8- to 16.1-fold, for the llmg_0609 mutants. The lysostaphin-oversecreting llmg_0609, murA2, and trmA mutants were also found to secrete larger amounts of another cell wall-lytic enzyme (the Listeria monocytogenes bacteriophage endolysin Ply511) than the control strain, indicating that the phenotype is not limited to lysostaphin.
Huston, W.M., Naylor, J., Cianciotto, N.P., Jennings, M.P. & McEwan, A.G. 2008, 'Functional analysis of the multi-copper oxidase from Legionella pneumophila.', Microbes and infection / Institut Pasteur, vol. 10, no. 5, pp. 497-503.
Multicopper oxidases have been described to have functions in copper tolerance, manganese oxidation, and iron oxidation in a range of bacteria. The putative cytoplasmic membrane multicopper oxidase from Legionella pneumophila was investigated. The mcoL gene was found to be critical for aerobic extracellular growth under either iron-limiting conditions or in the presence of ferrous Fe(II) iron, as a sole source of this essential metal. The mcoL mutants showed minor growth defects when grown in the presence of Fe(III) as the iron source. In contrast, intracellular growth and survival was not affected by the absence of the mcoL gene regardless of available iron concentration. The evidence presented here could indicate a possible role for mcoL in prevention of the toxic effects of ferrous iron during aerobic conditions. However, a function in high-affinity acquisition of iron could also be possible given the inability of the McoL mutants to grow aerobically under iron-limiting conditions.
Huston, W.M., Swedberg, J.E., Harris, J.M., Walsh, T.P., Mathews, S.A. & Timms, P. 2007, 'The temperature activated HtrA protease from pathogen Chlamydia trachomatis acts as both a chaperone and protease at 37 degrees C.', FEBS letters, vol. 581, no. 18, pp. 3382-3386.
Characterization of the protease, HtrA, from pathogen Chlamydia trachomatis is presented. The purified recombinant protein was a serine endoprotease, specific for unfolded proteins, and temperature activated above 34 degrees C. Chaperone activity was observed, although this appeared target-dependent. Inactive protease (S247A) was able to chaperone insulin B-chain, irrespective of temperature, but at 30 degrees C only HtrA and not S247A displayed significant chaperone activity for alpha-lactalbumin. These data demonstrate that chaperone activity may involve functional protease domain and that C. trachomatis HtrA functions as both a chaperone and protease at 37 degrees C. These properties are consistent with the developmental cycle of this obligate intracellular bacterium.
Huston, W.M., Harhangi, H.R., Leech, A.P., Butler, C.S., Jetten, M.S., Op den Camp, H.J. & Moir, J.W. 2007, 'Expression and characterisation of a major c-type cytochrome encoded by gene kustc0563 from Kuenenia stuttgartiensis as a recombinant protein in Escherichia coli.', Protein expression and purification, vol. 51, no. 1, pp. 28-33.
The purification of small quantities of a major small c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis has recently been reported. In order to characterise this protein further we have expressed the gene encoding this cytochrome in Escherichia coli and have purified the protein to homogeneity. The protein is directed to the E. coli periplasm using its native signal sequence suggesting that it may be translocated via a Sec-type system in K. stuttgartiensis. The cytochrome has the visible spectroscopic properties typical of a low-spin c-type cytochrome, but these spectroscopic features broaden in high salt solutions. The oxidised cytochrome was able to bind the ligands NO and cyanide. A redox potential of +230 mV suggests that the protein is suitable to act as an electron carrier protein that may be involved in the respiratory chain between hydrazine oxidation and the reduction of nitrite. The predicted protein sequence for the cytochrome suggests it to be a predominantly alpha-helical protein, and this is supported by circular dichroism.
Huston, W.M., Andrew, C.R., Servid, A.E., McKay, A.L., Leech, A.P., Butler, C.S. & Moir, J.W. 2006, 'Heterologous overexpression and purification of cytochrome c' from Rhodobacter capsulatus and a mutant (K42E) in the dimerization region. Mutation does not alter oligomerization but impacts the heme iron spin state and nitric oxide binding properties.', Biochemistry, vol. 45, no. 14, pp. 4388-4395.
Rhodobacter capsulatus cytochrome c' (RCCP) has been overexpressed in Escherichia coli, and its spectroscopic and ligand-binding properties have been investigated. It is concluded that the heterologously expressed protein is assembled correctly, as judged by UV-vis absorption, EPR, and resonance Raman (RR) spectroscopy of the unligated protein as well as forms in which the heme is ligated by CO or NO. To probe the oligomerization state of RCCP and its potential influence on heme reactivity, we have compared the properties of wild-type RCCP with a mutant (K42E) that lacks a salt bridge at the subunit interface. Analytical ultracentrifugation indicates that wild-type and K42E proteins are both monomeric in solution, contrary to the homodimeric structure of the crystalline state. Surprisingly, the K42E mutation produces a number of changes at the heme center (nearly 20 A distant), including perturbation of the ferric spin-state equilibrium and a change in the ferrous heme-nitrosyl complex from a six-coordinate/five-coordinate mixture to a predominantly five-coordinate heme-NO species. RR spectra indicate that ferrous K42E and wild-type RCCP both have relatively high Fe-His stretching frequencies, suggesting that the more favored five-coordinate heme-nitrosyl formation in K42E is not caused by a weaker Fe2+-His bond. Nevertheless, the altered reactivity of ferrous K42E with NO, together with its modified ferric spin state, shows that structural changes originating at the dimer interface can affect the properties of the heme center, raising the exciting possibility that intermolecular encounters at the protein surface might modulate the reactivity of cytochrome c' in vivo.
Huston, W.M., Lowe, E.C., Butler, C.S. & Moir, J.W. 2005, 'Purification and characterization of cytochrome c' from Neisseria meningitidis.', Biochemical Society transactions, vol. 33, no. Pt 1, pp. 187-189.
Cytochrome c', a c-type cytochrome with unique spectroscopic and magnetic properties, has been characterized in a variety of denitrifying and photosynthetic bacteria. Cytochrome c' has a role in defence and/or removal of NO but the mechanism of action is not clear. To examine the function of cytochrome c' from Neisseria meningitidis, the protein was purified after heterologous overexpression in Escherichia coli. The electronic spectra of the oxidized c' demonstrated a pH-dependent transition (over the pH range of 6-10) typical of known c'-type cytochromes. Interestingly, the form in which NO is supplied determines the redox state of the resultant haem-nitrosyl complex. Fe(III)-NO complexes were formed when Fe(II) or Fe(III) cytochrome c' was sparged with NO gas, whereas an Fe(II)-NO complex was generated when NO was supplied using DEA NONOate (diazeniumdiolate).
Andrew, C.R., Kemper, L.J., Busche, T.L., Tiwari, A.M., Kecskes, M.C., Stafford, J.M., Croft, L.C., Lu, S., Moënne-Loccoz, P., Huston, W., Moir, J.W. & Eady, R.R. 2005, 'Accessibility of the distal heme face, rather than Fe-His bond strength, determines the heme-nitrosyl coordination number of cytochromes c': evidence from spectroscopic studies.', Biochemistry, vol. 44, no. 24, pp. 8664-8672.
The heme coordination chemistry and spectroscopic properties of Rhodobacter capsulatus cytochrome c' (RCCP) have been compared to data from Alcaligenes xylosoxidans (AXCP), with the aim of understanding the basis for their different reactivities with nitric oxide (NO). Whereas ferrous AXCP reacts with NO to form a predominantly five-coordinate heme-nitrosyl complex via a six-coordinate intermediate, RCCP forms an equilibrium mixture of six-coordinate and five-coordinate heme-nitrosyl species in approximately equal proportions. Ferrous RCCP and AXCP both exhibit high Fe-His stretching frequencies (227 and 231 cm(-)(1), respectively), suggesting that factors other than the Fe-His bond strength account for their differences in heme-nitrosyl coordination number. Resonance Raman spectra of ferrous-nitrosyl RCCP confirm the presence of both five-coordinate and six-coordinate heme-NO complexes. The six-coordinate heme-nitrosyl of RCCP exhibits a fairly typical Fe-NO stretching frequency (569 cm(-)(1)), in contrast to the relatively high value (579 cm(-)(1)) of the AXCP six-coordinate heme-nitrosyl intermediate. It is proposed that NO experiences greater steric hindrance in binding to the distal face of AXCP, as compared to RCCP, leading to a more distorted Fe-N-O geometry and an elevated Fe-NO stretching frequency. Evidence that RCCP has a more accessible distal coordination site than in AXCP stems from the fact that ferric RCCP readily forms a heme complex with exogenous imidazole, whereas AXCP does not. A model is proposed in which distal heme-face accessibility, rather than the proximal Fe-His bond strength, determines the heme-nitrosyl coordination number in cytochromes c'.
Huston, W.M., Potter, A.J., Jennings, M.P., Rello, J., Hauser, A.R. & McEwan, A.G. 2004, 'Survey of ferroxidase expression and siderophore production in clinical isolates of Pseudomonas aeruginosa.', Journal of clinical microbiology, vol. 42, no. 6, pp. 2806-2809.
Ferroxidase (encoded by the mco gene), a component of a ferrous iron uptake pathway in Pseudomonas aeruginosa, was detected in all of the 35 respiratory clinical isolates surveyed; in contrast, considerable variation in siderophore expression was observed. The ubiquitous expression of this periplasmic ferroxidase suggests that it plays a key role in iron uptake in this opportunistic pathogen.
Huston, W.M., Jennings, M.P. & McEwan, A.G. 2002, 'The multicopper oxidase of Pseudomonas aeruginosa is a ferroxidase with a central role in iron acquisition.', Molecular microbiology, vol. 45, no. 6, pp. 1741-1750.
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Recently it has been observed that multicopper oxidases are present in a number of microbial genomes, raising the question of their function in prokaryotes. Here we describe the analysis of an mco mutant from the opportunistic pathogen Pseudomonas aeruginosa. Unlike wild-type Pseudomonas aeruginosa, the mco mutant was unable to grow aerobically on minimal media with Fe(II) as sole iron source. In contrast, both the wild-type and mutant strain were able to grow either anaerobically via denitrification with Fe(II) or aerobically with Fe(III). Analysis of iron uptake showed that the mco mutant was impaired in Fe(II) uptake but unaffected in Fe(III) uptake. Purification and analysis of the MCO protein confirmed ferroxidase activity. Taken together, these data show that the mco gene encodes a multicopper oxidase that is involved in the oxidation of Fe(II) to Fe(III) subsequent to its acquisition by the cell. In view of the widespread distribution of the mco gene in bacteria, it is suggested that an iron acquisition mechanism involving multicopper oxidases may be an important and hitherto unrecognized feature of bacterial pathogenicity.
Kappler, U., Huston, W.M. & McEwan, A.G. 2002, 'Control of dimethylsulfoxide reductase expression in Rhodobacter capsulatus: the role of carbon metabolites and the response regulators DorR and RegA.', Microbiology (Reading, England), vol. 148, no. Pt 2, pp. 605-614.
Regulation of the expression of dimethylsulfoxide (DMSO) reductase was investigated in the purple phototrophic bacterium Rhodobacter capsulatus. Under phototrophic, anaerobic conditions with malate as carbon source, DMSO caused an approximately 150-fold induction of DMSO reductase activity. The response regulator DorR was required for DMSO-dependent induction and also appeared to slightly repress DMSO reductase expression in the absence of substrate. Likewise, when pyruvate replaced malate as carbon source there was an induction of DMSO reductase activity in cells grown at low light intensity (16 W m(-2)) and again this induction was dependent on DorR. The level of DMSO reductase activity in aerobically grown cells was elevated when pyruvate replaced malate as carbon source. One possible explanation for this is that acetyl phosphate, produced from pyruvate, may activate expression of DMSO reductase by direct phosphorylation of DorR, leading to low levels of induction of dor gene expression in the absence of DMSO. A mutant lacking the global response regulator of photosynthesis gene expression, RegA, exhibited high levels of DMSO reductase in the absence of DMSO, when grown phototrophically with malate as carbon source. This suggests that phosphorylated RegA acts as a repressor of dor operon expression under these conditions. It has been proposed elsewhere that RegA-dependent expression is negatively regulated by the cytochrome cbb3 oxidase. A cco mutant lacking cytochrome cbb3 exhibited significantly higher levels of phi[dorA::lacZ] activity in the presence of DMSO compared to wild-type cells and this is consistent with the above model. Pyruvate restored DMSO reductase expression in the regA mutant to the same pattern as found in wild-type cells. These data suggest that R. capsulatus contains a regulator of DMSO respiration that is distinct from DorR and RegA, is activated in the presence of pyruvate, and acts as a negative regulator of DMSO reductase expression.
Kappler, U., Huston, W.M. & McEwan, A.G. 2002, 'Control of dimethylsulfoxide reductase expression in Rhodobacter capsulatus: The role of carbon metabolites and the response regulators DorR and RegA', Microbiology, vol. 148, no. 2, pp. 605-614.
Regulation of the expression of dimethylsulfoxide (DMSO) reductase was investigated in the purple phototrophic bacterium Rhodobacter capsulatus. Under phototrophic, anaerobic conditions with malate as carbon source, DMSO caused an approximately 150-fold induction of DMSO reductase activity. The response regulator DorR was required for DMSO-dependent induction and also appeared to slightly repress DMSO reductase expression in the absence of substrate. Likewise, when pyruvate replaced malate as carbon source there was an induction of DMSO reductase activity in cells grown at low light intensity (16 W m-2) and again this induction was dependent on DorR. The level of DMSO reductase activity in aerobically grown cells was elevated when pyruvate replaced malate as carbon source. One possible explanation for this is that acetyl phosphate, produced from pyruvate, may activate expression of DMSO reductase by direct phosphorylation of DorR, leading to low levels of induction of dor gene expression in the absence of DMSO. A mutant lacking the global response regulator of photosynthesis gene expression, RegA, exhibited high levels of DMSO reductase in the absence of DMSO, when grown phototrophically with malate as carbon source. This suggests that phosphorylated RegA acts as a repressor of dor operon expression under these conditions. It has been proposed elsewhere that RegA-dependent expression is negatively regulated by the cytochrome cbb3 oxidase. A cco mutant lacking cytochrome cbb3 exhibited significantly higher levels of [dorA::lacZ] activity in the presence of DMSO compared to wild-type cells and this is consistent with the above model. Pyruvate restored DMSO reductase expression in the regA mutant to the same pattern as found in wild-type cells. These data suggest that R. capsulatus contains a regulator of DMSO respiration that is distinct from DorR and RegA, is activated in the presence of pyruvate, and acts as a negative regulator of DMSO reductase expression.