The impact of transcriptome and proteome analyses on antibiotic drug discovery

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Recent scientific publications demonstrate the increasing interest in measurement of genome-wide gene expression on transcript and protein level in response to treatment with antibacterial agents. Nevertheless, the number of large bacterial transcriptome and proteome datasets available so far is limited, although a high number and diversity of antibiotic-triggered expression profiles aid to optimally exploit these technologies. The first published examples substantiate the need to establish these so-called reference compendia of bacterial expression profiles, to discover the molecular mechanism-of-action of uncharacterized bioactive substances. In addition, such compendia open up ways for novel cell-based drug screening approaches.

Introduction

Infectious diseases remain a serious threat to public health. Rapid resistance development among major pathogens makes today's antibiotics more and more ineffective raising the need to find novel broad-spectrum antibiotics [1••]. Discovery efforts, which were traditionally based on random cell-based screening or the modification of marketed antibiotics, hardly keep up with the emergence of resistance. Therefore, novel technologies and research strategies have been introduced into the antibacterial drug discovery process particularly benefiting from the availability of genomic data. During the past nine years, after the first bacterial genome was completely deciphered (Haemophilus influenzae) [2], there has been a tremendous development of genetic tools and molecular technologies, which has enabled a more diverse range of bacterial species to be studied. This development goes parallel with the elucidation of the complete genomic information of the majority of clinically relevant bacterial species. Thus, questions about bacterial physiology can be addressed more holistically than ever before.

Advanced molecular techniques based on genomic information support various antibacterial research strategies. Many diagnostic, prophylactic, and therapeutic approaches are currently pursued including vaccine design [3], probiotic strategies 4., 5. and discovery of novel natural product-derived or chemically synthesized antibiotics 6., 7.••. Not only the elucidation of the numerous mechanisms of virulence and resistance of individual bacterial pathogens [8], but also the discovery and characterization of antibiotics acting against a wide variety of bacterial species are facilitated by these techniques.

Genome-based expression profiling may represent a valuable tool for three applications in antibiotic drug discovery: (i) target identification including functionality studies of genes of unknown function, (ii) mechanism-of-action (MOA) studies with antibiotics, and (iii) development of novel types of whole-cell assays for drug screening purposes. Indeed, information about previously uncharacterized genes can be obtained from expression profiles. Co-regulation of such genes with others, of which the functional roles are known, might give hints for their importance in bacterial physiology. Thus, such data might open up avenues to identify novel bacterial targets. Here, we emphasize the impact of transcriptome and proteome analyses on discovering the MOA of uncharacterized bioactive substances because these compounds represent a valuable source of novel lead structures for antibiotic drug development. An essential prerequisite for such studies is the availability of reference compendia of antibiotic-triggered expression profiles for known antibiotics. The potential of such compendium-based approaches for MOA predictions and for the design of novel cellular drug screening assays is described below.

In this review, we focus on the application of transcriptome- and proteome-based expression profiling technologies for the discovery of novel broad-spectrum antibiotics.

Section snippets

Transcriptome and proteome – two levels of expression analysis

The genome-wide expression of genes is reflected on two levels: the mRNA level (transcriptome) and protein level (proteome). There are some essential technical prerequisites for global expression profiling studies despite the availability of genomic data. The transcriptome measurement is generally based on hybridization of RNA or cDNA with DNA probes spotted on microarrays. For reviewing technical details of different array technologies, please refer to recently published reviews 9.•, 10..

Mechanism-of-action characterization by antibiotic-triggered expression profiles

The bacterial transcriptome and proteome are dynamic entities rapidly responding to changes in environmental conditions within the order of minutes. Numerous external and internal signal molecules and signal transduction processes are responsible for adaptation of the mRNA and protein abundance. Several studies have already been reported describing the changes in the bacterial transcriptomes and proteomes towards changes of growth conditions (temperature, pH, osmolarity, nutrient availability),

Reference compendia approaches

Various efforts have been or are still being undertaken in pharma and biotech companies to generate expression data, which reflect the treatment of logarithmically growing model bacteria with antibiotics acting via different MOAs (Basilea, Bayer, GeneSoft, GPC Biotech, Eli Lilly, Pfizer, Wyeth-Ayers, among others). Such data collections serve as reference compendia for comparison with expression profiles of not yet characterized antibiotics. However, while such approaches to characterize the

Prediction of novel mechanisms-of-action based on compendia of expression profiles

The reference compendium of B. subtilis proteome profiles represents a first example for illustrating its value for MOA predictions [19••]. By visual inspection of the two-dimensional gels with subsequent spot determination, proteomic profiles for 30 different antibiotics were deduced based on a limited number of gene products. The authors were nevertheless able to identify hints of novel MOAs that are not employed by already existing reference antibiotics. For instance, they could demonstrate

Expansion of reference compendia by conditional mutant profiles

The expansion of a reference compendium with expression profiles of bacterial mutants underexpressing potential target proteins could be a helpful means to overcome the hurdle in identifying novel antibacterial MOAs more distantly related to the ones represented by reference antibiotics. Experimental handling of numerous conditional bacterial mutants underexpressing essential genes might not be trivial for compendium approaches. It is known that essential genes often need to be repressed to

Novel cell-based screening systems

Large-scale expression profiling techniques are not only tools to characterize the MOAs of unexplored natural products and synthetic compounds but they also can be used to validate the cellular mechanism of hits derived from high-throughput screening. Collections of diverse expression profiles even open up avenues to the development of novel cell-based assay systems suitable for high-throughput screening. Such datasets aid in identification and characterization of antibiotic-responsive

Conclusions

Expression profiling analyses are on the way to becoming a well-recognized tool for mechanistic studies with antibiotic agents. In most cases including the examples described in this review, expression profiling experiments have been used to validate hypotheses originated from other technologies. Nevertheless, the published expression profiling experiments document, that valuable MOA and target hypotheses can even initially be developed on the basis of reference compendia. Clearly however, only

Update

It was recently published, that the antibiotic effect of the pyrrolidine dione antibiotic moiramide B was caused by the inhibition of the bacterial acetyl-CoA carboxylase, a target of which no inhibitor with antibiotic properties was previously known [46]. The authors of this study reported that they indeed predicted this novel MOA based on expression profiling experiments ([46]; Freiberg et al., unpublished).

In addition, a panel of novel B. subtilis reporter strains has recently been

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

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