A combination of nirmatrelvir and ombitasvir boosts inhibition of SARS-CoV-2 replication.

Antiviral therapeutics are highly effective countermeasures for the treatment of coronavirus disease 2019 (COVID-19). However, development of resistance to antivirals undermines their effectiveness. Combining multiple antivirals during patient treatment has the potential to overcome the evolutionary selective pressure towards antiviral resistance, as well as provide a more robust and efficacious treatment option. The current evidence for effective antiviral combinations to inhibit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication is limited. Here, we demonstrate a combination of nirmatrelvir with ombitasvir, to jointly bring about potent inhibition of SARS-CoV-2 replication. We developed an in vitro 384- well plate cytopathic effect assay for the evaluation of antiviral combinations against Calu-3 cells infected with SARS-CoV-2 and found, that a combination of ombitasvir and nirmatrelvir was synergistic; thereby decreasing the nirmatrelvir IC50 by approx. 16-fold. The increased potency of the nirmatrelvir-ombitasvir combination, over nirmatrelvir alone afforded a greater than 3 log10 reduction in viral titre, which is sufficient to fully prevent the detection of progeny SARS-CoV-2 viral particles at 48 h post infection. The mechanism of this potentiated effect was shown to be, in-part, due to joint inhibition of the 3-chymotrypsin-like protease via a positive allosteric modulation mechanism.

Azetidines Kill Multidrug-Resistant Mycobacterium tuberculosis without Detectable Resistance by Blocking Mycolate Assembly.

Tuberculosis (TB) is the leading cause of global morbidity and mortality resulting from infectious disease, with over 10.6 million new cases and 1.4 million deaths in 2021. This global emergency is exacerbated by the emergence of multidrug-resistant MDR-TB and extensively drug-resistant XDR-TB; therefore, new drugs and new drug targets are urgently required. From a whole cell phenotypic screen, a series of azetidines derivatives termed BGAz, which elicit potent bactericidal activity with MIC99 values <10 µM against drug-sensitive Mycobacterium tuberculosis and MDR-TB, were identified. These compounds demonstrate no detectable drug resistance. The mode of action and target deconvolution studies suggest that these compounds inhibit mycobacterial growth by interfering with cell envelope biogenesis, specifically late-stage mycolic acid biosynthesis. Transcriptomic analysis demonstrates that the BGAz compounds tested display a mode of action distinct from the existing mycobacterial cell wall inhibitors. In addition, the compounds tested exhibit toxicological and PK/PD profiles that pave the way for their development as antitubercular chemotherapies.

Biofilms in tuberculosis: What have we learnt in the past decade and what is still unexplored?

Elucidating how Mycobacterium tuberculosis produces biofilms, and its impact for tuberculosis (TB) pathogenesis is gaining momentum. Here, we discuss recent findings reported over the last decade, which help us gain insights into the association between biofilm formation and TB pathogenesis. A new appreciation of extracellular TB phenotypes found in lung lesions will drive drug and vaccine discovery forward to new possibilities.

Mycobacterium tuberculosis modifies cell wall carbohydrates during biofilm growth with a concomitant reduction in complement activation.

The development of new vaccines for TB needs to be underpinned by an understanding of both the molecular and cellular mechanisms of host-pathogen interactions and how the immune response can be modulated to achieve protection from disease. Complement orchestrates many aspects of the innate and adaptive immune responses. However, little is known about the contribution of the complement pathways during TB disease, particularly with respect to mycobacterial phenotype. Extracellular communities (biofilms) of M. tuberculosis are found in the acellular rim of granulomas, during disease, and these are likely to be present in post-primary TB episodes, in necrotic lesions. Our study aimed to determine which mycobacterial cell wall components were altered during biofilm growth and how these cell wall alterations modified the complement response. We have shown that M. tuberculosis biofilms modified their cell wall carbohydrates and elicited reduced classical and lectin pathway activation. Consistent with this finding was the reduction of C3b/iC3b deposition on biofilm cell wall carbohydrate extracts. Here, we have highlighted the role of cell wall carbohydrate alterations during biofilm growth of M. tuberculosis and subsequent modulation of complement activation.

Time series analysis and mechanistic modelling of heterogeneity and sero-reversion in antibody responses to mild SARS­CoV-2 infection.

BACKGROUND: SARS-CoV-2 serology is used to identify prior infection at individual and at population level. Extended longitudinal studies with multi-timepoint sampling to evaluate dynamic changes in antibody levels are required to identify the time horizon in which these applications of serology are valid, and to explore the longevity of protective humoral immunity. METHODS: Healthcare workers were recruited to a prospective cohort study from the first SARS-CoV-2 epidemic peak in London, undergoing weekly symptom screen, viral PCR and blood sampling over 16-21 weeks. Serological analysis (n =12,990) was performed using semi-quantitative Euroimmun IgG to viral spike S1 domain and Roche total antibody to viral nucleocapsid protein (NP) assays. Comparisons were made to pseudovirus neutralizing antibody measurements. FINDINGS: A total of 157/729 (21.5%) participants developed positive SARS-CoV-2 serology by one or other assay, of whom 31.0% were asymptomatic and there were no deaths. Peak Euroimmun anti-S1 and Roche anti-NP measurements correlated (r = 0.57, p<0.0001) but only anti-S1 measurements correlated with near-contemporary pseudovirus neutralising antibody titres (measured at 16-18 weeks, r = 0.57, p<0.0001). By 21 weeks' follow-up, 31/143 (21.7%) anti-S1 and 6/150 (4.0%) anti-NP measurements reverted to negative. Mathematical modelling revealed faster clearance of anti-S1 compared to anti-NP (median half-life of 2.5 weeks versus 4.0 weeks), earlier transition to lower levels of antibody production (median of 8 versus 13 weeks), and greater reductions in relative antibody production rate after the transition (median of 35% versus 50%). INTERPRETATION: Mild SARS-CoV-2 infection is associated with heterogeneous serological responses in Euroimmun anti-S1 and Roche anti-NP assays. Anti-S1 responses showed faster rates of clearance, more rapid transition from high to low level production rate and greater reduction in production rate after this transition. In mild infection, anti-S1 serology alone may underestimate incident infections. The mechanisms that underpin faster clearance and lower rates of sustained anti-S1 production may impact on the longevity of humoral immunity. FUNDING: Charitable donations via Barts Charity, Wellcome Trust, NIHR.

Optimisation of Mycobacterium bovis BCG Fermentation and Storage Survival.

Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) was generated over a century ago for protection against Mycobacterium tuberculosis (Mtb) and is one the oldest vaccines still in use. The BCG vaccine is currently produced using a pellicle growth method, which is a complex and lengthy process that has been challenging to standardise. Fermentation for BCG vaccine production would reduce the complexity associated with pellicle growth and increase batch to batch reproducibility. This more standardised growth lends itself to quantification of the total number of bacilli in the BCG vaccine by alternative approaches, such as flow cytometry, which can also provide information about the metabolic status of the bacterial population. The aim of the work reported here was to determine which batch fermentation conditions and storage conditions give the most favourable outcomes in terms of the yield and stability of live M. bovis BCG Danish bacilli. We compared different media and assessed growth over time in culture, using total viable counts, total bacterial counts, and turbidity throughout culture. We applied fluorescent viability dyes and flow cytometry to measure real-time within-culture viability. Culture samples were stored in different cryoprotectants at different temperatures to assess the effect of these combined conditions on bacterial titres. Roisin's minimal medium and Middlebrook 7H9 medium gave comparable, high titres in fermenters. Flow cytometry proved to be a useful tool for enumeration of total bacterial counts and in the assessment of within-culture cell viability and cell death. Of the cryoprotectants evaluated, 5% (v/v) DMSO showed the most significant positive effect on survival and reduced the negative effects of low temperature storage on M. bovis BCG Danish viability. In conclusion, we have shown a reproducible, more standardised approach for the production, evaluation, and storage of high titre, viable, BCG vaccine.

Bioreactor-Grown Bacillus of Calmette and Guérin (BCG) Vaccine Protects Badgers against Virulent Mycobacterium bovis When Administered Orally: Identifying Limitations in Baited Vaccine Delivery.

Bovine tuberculosis (TB) in Great Britain adversely affects animal health and welfare and is a cause of considerable economic loss. The situation is exacerbated by European badgers (Meles meles) acting as a wildlife source of recurrent Mycobacterium bovis infection to cattle. Vaccination of badgers against TB is a possible means to reduce and control bovine TB. The delivery of vaccine in oral bait holds the best prospect for vaccinating badgers over a wide geographical area. There are practical limitations over the volume and concentration of Bacillus of Calmette and Guérin (BCG) that can be prepared for inclusion in bait. The production of BCG in a bioreactor may overcome these issues. We evaluated the efficacy of oral, bioreactor-grown BCG against experimental TB in badgers. We demonstrated repeatable protection through the direct administration of at least 2.0 × 108 colony forming units of BCG to the oral cavity, whereas vaccination via voluntary consumption of bait containing the same preparation of BCG did not result in demonstrable protection at the group-level, although a minority of badgers consuming bait showed immunological responses and protection after challenge equivalent to badgers receiving oral vaccine by direct administration. The need to deliver oral BCG in the context of a palatable and environmentally robust bait appears to introduce such variation in BCG delivery to sites of immune induction in the badger as to render experimental studies variable and inconsistent.

Noncytotoxic Pyrrolobenzodiazepine-Ciprofloxacin Conjugate with Activity against Mycobacterium tuberculosis.

The development of new antitubercular agents for the treatment of infections caused by multidrug-resistant (MDR) Mycobacterium tuberculosis is an urgent priority. Pyrrolobenzodiazepines (PBDs) are a promising class of antibacterial agents that were initially discovered and isolated from a range of Streptomyces species. Recently, C8-linked PBD monomers have been shown to work by inhibiting DNA gyrase and have demonstrated activity against M. tuberculosis. However, both PBD monomers and dimers are toxic to eukaryotic cells, limiting their development as antibacterial agents. To eliminate the toxicity associated with PBDs and explore the effect of C8-modification with a known antibacterial agent with the same mechanism of action (i.e., ciprofloxacin, a gyrase inhibitor), we synthesized a C8-linked PBD-ciprofloxacin (PBD-CIP, 3) hybrid. The hybrid compound displayed minimum inhibitory concentration values of 0.4 or 2.1 µg/mL against drug-sensitive and drug-resistant M. tuberculosis strains, respectively. A molecular modeling study showed good interaction of compound 3 with wild-type M. tuberculosis DNA gyrase, suggesting gyrase inhibition as a possible mechanism of action. Compound 3 is a nontoxic combination hybrid that can be utilized as a new scaffold and further optimized to develop new antitubercular agents.

Re-growth of Mycobacterium tuberculosis populations exposed to antibiotic combinations is due to the presence of isoniazid and not bacterial growth rate.

Modulation of growth rate in Mycobacterium tuberculosis is key to its survival in the host; particularly with regard to its adaptation during chronic infection when the growth rate is very slow. The resulting physiological changes will influence the way this pathogen interacts with the host and responds to antibiotics. Therefore, it is important that we understand how growth rate impacts antibiotic efficacy, particularly with respect to recovery/relapse. This is the first study that has asked how growth rates influence the mycobacterial responses to combinations of frontline antimycobacterials, isoniazid (INH), rifampicin (RIF), and pyrazinamide (PZA), using continuous cultures. Time-course profiles of log-transformed total viable counts for cultures, controlled at either a fast growth rate (23.1. mean generation time (MGT)) or slow growth rate (69.3h MGT), were analysed with the fitting of a mathematical model by nonlinear regression that accounted for the dilution rate in the chemostat, and profiled kill rates and recovery in culture. Using this approach, we show that populations growing more slowly were generally less susceptible to all treatments. We observed a higher kill rate associated with INH (compared to RIF or PZA) and the appearance of re-growth. In line with this observation, re-growth was not observed with RIF-exposure, which provided a slower bactericidal response. The sequential additions of RIF and PZA did not eliminate re-growth. We consider here that faster, early bactericidal activity is not what is required for successful sterilisation of M. tuberculosis, but instead slower elimination of bacilli followed by reduced recovery of the bacterial population.

Utilisation of the Prestwick Chemical Library to identify drugs that inhibit the growth of mycobacteria.

Tuberculosis (TB) is an infectious bacterial disease that kills approximately 1.3 million people every year. Despite global efforts to reduce both the incidence and mortality associated with TB, the emergence of drug resistant strains has slowed any progress made towards combating the spread of this deadly disease. The current TB drug regimen is inadequate, takes months to complete and poses significant challenges when administering to patients suffering from drug resistant TB. New treatments that are faster, simpler and more affordable are urgently required. Arguably, a good strategy to discover new drugs is to start with an old drug. Here, we have screened a library of 1200 FDA approved drugs from the Prestwick Chemical library using a GFP microplate assay. Drugs were screened against GFP expressing strains of Mycobacterium smegmatis and Mycobacterium bovis BCG as surrogates for Mycobacterium tuberculosis, the causative agent of TB in humans. We identified several classes of drugs that displayed antimycobacterial activity against both M. smegmatis and BCG, however each organism also displayed some selectivity towards certain drug classes. Variant analysis of whole genomes sequenced for resistant mutants raised to florfenicol, vanoxerine and pentamidine highlight new pathways that could be exploited in drug repurposing programmes.

Mode of Action of Kanglemycin A, an Ansamycin Natural Product that Is Active against Rifampicin-Resistant Mycobacterium tuberculosis.

Antibiotic-resistant bacterial pathogens pose an urgent healthcare threat, prompting a demand for new medicines. We report the mode of action of the natural ansamycin antibiotic kanglemycin A (KglA). KglA binds bacterial RNA polymerase at the rifampicin-binding pocket but maintains potency against RNA polymerases containing rifampicin-resistant mutations. KglA has antibiotic activity against rifampicin-resistant Gram-positive bacteria and multidrug-resistant Mycobacterium tuberculosis (MDR-M. tuberculosis). The X-ray crystal structures of KglA with the Escherichia coli RNA polymerase holoenzyme and Thermus thermophilus RNA polymerase-promoter complex reveal an altered-compared with rifampicin-conformation of KglA within the rifampicin-binding pocket. Unique deoxysugar and succinate ansa bridge substituents make additional contacts with a separate, hydrophobic pocket of RNA polymerase and preclude the formation of initial dinucleotides, respectively. Previous ansa-chain modifications in the rifamycin series have proven unsuccessful. Thus, KglA represents a key starting point for the development of a new class of ansa-chain derivatized ansamycins to tackle rifampicin resistance.

A Flow Cytometry Method for Assessing M. tuberculosis Responses to Antibiotics.

Traditional drug susceptibility methods can take several days or weeks of incubation between drug exposure and confirmation of sensitivity or resistance. In addition, these methods do not capture information about viable organisms that are not immediately culturable after drug exposure. Here, we present a rapid fluorescence detection method for assessing the susceptibility of M. tuberculosis to antibiotics. Fluorescent markers Calcein violet-AM and SYTOX-green are used for measuring cell viability or cell death and to capture information about the susceptibility of the whole population and not just those bacteria that can grow in media postexposure. Postexposure to the antibiotic, the method gives a rapid readout of drug susceptibility, as well as insights into the concentration and time-dependent drug activity following antibiotic exposure.

Application of Continuous Culture for Assessing Antibiotic Activity Against Mycobacterium tuberculosis.

There is a proportion of the M. tuberculosis population that is refractory to the bactericidal action of antituberculosis antibiotics due to phenotypic tolerance. This tolerance can be impacted by environmental stimuli and the subsequent physiological state of the organism. It may be the result of preexisting populations of slow growing/non replicating bacteria that are protected from antibiotic action. It still remains unclear how the slow growth of M. tuberculosis contributes to antibiotic resistance and antibiotic tolerance. Here, we present a method for assessing the activity of antibiotics against M. tuberculosis using continuous culture, which is the only system that can be used to control bacterial growth rate and study the impact of slow or fast growth on the organism's response to antibiotic exposure.

Diverse drug-resistant subpopulations of Mycobacterium tuberculosis are sustained in continuous culture.

Drug resistance to tuberculosis (TB) has become more widespread over the past decade. As such, understanding the emergence and fitness of antibiotic-resistant subpopulations is crucial for the development of new interventions. Here we use a simple mathematical model to explain the differences in the response to isoniazid (INH) of Mycobacterium tuberculosis cells cultured under two growth rates in a chemostat. We obtain posterior distributions of model parameters consistent with data using a Markov chain Monte Carlo (MCMC) method. We explore the dynamics of diverse INH-resistant subpopulations consistent with these data in a multi-population model. We find that the simple model captures the qualitative behaviour of the cultures under both dilution rates and also present testable predictions about how diversity is maintained in such cultures.

The effect of growth rate on pyrazinamide activity in Mycobacterium tuberculosis - insights for early bactericidal activity?

BACKGROUND: Pyrazinamide (PZA) plays an essential part in the shortened six-month tuberculosis (TB) treatment course due to its activity against slow-growing and non-replicating organisms. We tested whether PZA preferentially targets slow growing cells of Mycobacterium tuberculosis that could be representative of bacteria that remain after the initial kill with isoniazid (INH), by observing the response of either slow growing or fast growing bacilli to differing concentrations of PZA. METHODS: M. tuberculosis H37Rv was grown in continuous culture at either a constant fast growth rate (Mean Generation Time (MGT) of 23.1 h) or slow growth rate (69.3 h MGT) at a controlled dissolved oxygen tension of 10 % and a controlled acidity at pH 6.3 ± 0.1. Cultures were exposed to step-wise increases in the concentration of PZA (25 to 500 µgml(-1)) every two MGTs, and bacterial survival was measured. PZA-induced global gene expression was explored for each increase in PZA-concentration, using DNA microarray. RESULTS: At a constant pH 6.3, actively dividing mycobacteria were susceptible to PZA, with similar responses to increasing concentrations of PZA at both growth rates. Three distinct phases of drug response could be distingished for both slow growing (69.3 h MGT) and fast growing (23.1 h MGT) bacilli. A bacteriostatic phase at a low concentration of PZA was followed by a recovery period in which the culture adapted to the presence of PZA and bacteria were actively dividing in steady-state. In contrast, there was a rapid loss of viability at bactericidal concentrations. There was a notable delay in the onset of the recovery period in quickly dividing cells compared with those dividing more slowly. Fast growers and slow growers adapted to PZA-exposure via very similar mechanisms; through reduced gene expression of tRNA, 50S, and 30S ribosomal proteins. CONCLUSIONS: PZA had an equivalent level of activity against fast growing and slow growing M. tuberculosis. At both growth rates drug-tolerance to sub-lethal concentrations may have been due to reduced expression of tRNA, 50S, and 30S ribosomal proteins. The findings from this study show that PZA has utility against more than one phenotypic sub-population of bacilli and could be re-assessed for its early bactericidal activity, in combination with other drugs, during TB treatment.

A Flow Cytometry Method for Rapidly Assessing Mycobacterium tuberculosis Responses to Antibiotics with Different Modes of Action.

Current methods for assessing the drug susceptibility of Mycobacterium tuberculosis are lengthy and do not capture information about viable organisms that are not immediately culturable under standard laboratory conditions as a result of antibiotic exposure. We have developed a rapid dual-fluorescence flow cytometry method using markers for cell viability and death. We show that the fluorescent marker calcein violet with an acetoxy-methyl ester group (CV-AM) can differentiate between populations of M. tuberculosis growing at different rates, while Sytox green (SG) can differentiate between live and dead mycobacteria. M. tuberculosis was exposed to isoniazid or rifampin at different concentrations over time and either dual stained with CV-AM and SG and analyzed by flow cytometry or plated to determine the viability of the cells. Although similar trends in the loss of viability were observed when the results of flow cytometry and the plate counting methods were compared, there was a lack of correlation between these two approaches, as the flow cytometry analysis potentially captured information about cell populations that were unable to grow under standard conditions. The flow cytometry approach had an additional advantage in that it could provide insights into the mode of action of the drug: antibiotics targeting the cell wall gave a flow cytometry profile distinct from those inhibiting intracellular processes. This rapid drug susceptibility testing method could identify more effective antimycobacterials, provide information about their potential mode of action, and accelerate their progress to the clinic.

Mycobacterium tuberculosis Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in katG Codon Ser315.

An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any M. tuberculosis population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for M. tuberculosis. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in katG codon Ser315 which are present in approximately 50-90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with katG mutations distributed throughout the gene. Mutations in katG codon Ser315 appear to have a fitness cost in vitro and particularly in fast growing cultures. Our results suggest a requirement for functional katG-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why katG codon Ser315 mutations are favoured in the slow growing cultures.

Non-replicating Mycobacterium tuberculosis elicits a reduced infectivity profile with corresponding modifications to the cell wall and extracellular matrix.

A key feature of Mycobacterium tuberculosis is its ability to become dormant in the host. Little is known of the mechanisms by which these bacilli are able to persist in this state. Therefore, the focus of this study was to emulate environmental conditions encountered by M. tuberculosis in the granuloma, and determine the effect of such conditions on the physiology and infectivity of the organism. Non-replicating persistent (NRP) M. tuberculosis was established by the gradual depletion of nutrients in an oxygen-replete and controlled environment. In contrast to rapidly dividing bacilli, NRP bacteria exhibited a distinct phenotype by accumulating an extracellular matrix rich in free mycolate and lipoglycans, with increased arabinosylation. Microarray studies demonstrated a substantial down-regulation of genes involved in energy metabolism in NRP bacteria. Despite this reduction in metabolic activity, cells were still able to infect guinea pigs, but with a delay in the development of disease when compared to exponential phase bacilli. Using these approaches to investigate the interplay between the changing environment of the host and altered physiology of NRP bacteria, this study sheds new light on the conditions that are pertinent to M. tuberculosis dormancy and how this organism could be establishing latent disease.

Application of continuous culture for measuring the effect of environmental stress on mutation frequency in Mycobacterium tuberculosis.

The ability of all pathogens to survive within the host is key to their success in establishing disease. Environmental conditions that affect the growth of a pathogen in the host include nutrient status, environmental pH, oxygen availability, and host defences. Studying the response of Mycobacterium tuberculosis (M. tuberculosis) exposed to these relevant host conditions in vitro will further increase our understanding of how these environments have an impact on the molecular mechanisms M. tuberculosis adopts to combat the effects of external influences such as antimycobacterials. The methods presented here are used to investigate the effect of environmental factors on the development of drug-resistant M. tuberculosis. Cultures grown under controlled conditions in continuous culture are sampled and the frequency with which resistant mutants develop are determined. These studies provide data that aid our understanding of the complex interaction between the host environment and invading bacterium that allow resistant strains to develop and continue to cause disease.

An integrated machine learning approach for predicting DosR-regulated genes in Mycobacterium tuberculosis.

BACKGROUND: DosR is an important regulator of the response to stress such as limited oxygen availability in Mycobacterium tuberculosis. Time course gene expression data enable us to dissect this response on the gene regulatory level. The mRNA expression profile of a regulator, however, is not necessarily a direct reflection of its activity. Knowing the transcription factor activity (TFA) can be exploited to predict novel target genes regulated by the same transcription factor. Various approaches have been proposed to reconstruct TFAs from gene expression data. Most of them capture only a first-order approximation to the complex transcriptional processes by assuming linear gene responses and linear dynamics in TFA, or ignore the temporal information in data from such systems. RESULTS: In this paper, we approach the problem of inferring dynamic hidden TFAs using Gaussian processes (GP). We are able to model dynamic TFAs and to account for both linear and nonlinear gene responses. To test the validity of the proposed approach, we reconstruct the hidden TFA of p53, a tumour suppressor activated by DNA damage, using published time course gene expression data. Our reconstructed TFA is closer to the experimentally determined profile of p53 concentration than that from the original study. We then apply the model to time course gene expression data obtained from chemostat cultures of M. tuberculosis under reduced oxygen availability. After estimation of the TFA of DosR based on a number of known target genes using the GP model, we predict novel DosR-regulated genes: the parameters of the model are interpreted as relevance parameters indicating an existing functional relationship between TFA and gene expression. We further improve the prediction by integrating promoter sequence information in a logistic regression model. Apart from the documented DosR-regulated genes, our prediction yields ten novel genes under direct control of DosR. CONCLUSIONS: Chemostat cultures are an ideal experimental system for controlling noise and variability when monitoring the response of bacterial organisms such as M. tuberculosis to finely controlled changes in culture conditions and available metabolites. Nonlinear hidden TFA dynamics of regulators can be reconstructed remarkably well with Gaussian processes from such data. Moreover, estimated parameters of the GP can be used to assess whether a gene is controlled by the reconstructed TFA or not. It is straightforward to combine these parameters with further information, such as the presence of binding motifs, to increase prediction accuracy.