Bacterial Type I Signal Peptidases as Antibiotic Targets

Smitha Rao CV; Jozef Anné

Disclosures

Future Microbiol. 2011;6(11):1279-1296. 

In This Article

Abstract and Introduction

Abstract

Despite an alarming increase in morbidity and mortality caused by multidrug-resistant bacteria, the number of antibiotics available to efficiently combat them is dwindling. Consequently, there is a pressing need for new drugs, preferably with novel modes of action to avert the problem of cross-resistance. Several new targets have been proposed, including proteins essential in the protein secretion pathway such as the type I signal peptidase (SPase), indispensable for the release of the signal peptide during secretion of Sec- and Tat-dependent proteins. The type I SPase is considered to be an attractive target because it is essential, substantially different from the eukaryotic counterpart, and its active site is located at the outer leaflet of the cytoplasmic membrane, permitting relatively easy access to potential inhibitors. A few SPase inhibitors have already been identified, but their suitability as drugs is yet to be confirmed. An overview is given on the currently known SPase inhibitors, how they can give valuable information on the structural, biochemical and target validation aspects of the SPases, the approaches to identify them, and their future potential as drugs.

Introduction

Since their introduction into the market more than 70 years ago, antibiotics have continued to contribute immensely to human health in combating infections. Antibiotics have been losing efficiency as a consequence of the remarkable adaptability of bacteria[1,2] as well as their uncontrolled and inappropriate use, that has resulted in a dramatic increase in resistant bacteria. In the period between the 1930s and 1960s, arsenals of new antibiotic classes were introduced (Figure 1), a majority of which are still employed in current clinical practice. Nearly four decades elapsed before two new classes of antibiotics (oxazolidinone and lipopeptides) entered into the market in 2000 and 2003, drugs efficient against methicillin and/or vancomycin-resistant Gram-positive bacteria.[3,4] The reasons behind the innovation gap are a decreasing attention to the antibacterial research within the pharmaceutical industry and the increased time, cost and complications involved in the drug development and approval process, resulting in decreased return on investment.[5] Not surprisingly, most of the antibiotics have come from a small set of molecular scaffolds whose functional lifetimes have been prolonged by cycles of synthetic adaptations.

Figure 1.

The dwindling antibiotic development pipeline.

Until about two decades ago, if one antibiotic encountered resistance, there was always another to take its place, but with the emergence of multidrug- (MDR), extreme drug- and pan-drug resistant (PDR) bacteria, this is no longer the case. These strains have limited treatment options, causing tens of thousands of deaths each year, worldwide.[201] The interested reader is directed to[201] for the facts and figures, the factors driving antimicrobial resistance and why it is a global problem.

Of major concern are methicillin-resistant (MRSA)/vancomycin-intermediate (VISA)/vancomycin-resistant Staphylococcus aureus (VRSA), MDR and PDR Gram-negative bacilli, such as Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae, and MDR and extensively drug-resistant Mycobacterium tuberculosis (MDR-TB and XDR-TB, respectively). In addition, an increase in the use of indwelling and prosthetic devices has added biofilm-associated bacteria (e.g., Staphylococcus epidermidis), which are less sensitive to antibiotics, to the treatment challenge. Novel antibiotics are urgently needed[6] as the drug-development pipeline is almost empty.[7]

The availability of complete bacterial genome sequences (with the first, Haemophilus influenza, genome in 1995) spurred a renewed interest in novel antibiotic research. Genomic information fostered the target-based approach of finding new classes of drugs with a novel mode of action. Several unconventional targets have been selected based on genomics, isolated, characterized and screened in pursuit of novel drug candidates.[8] However, the success rate has been rather disappointing. Genomic information is now being used to find genes that encode the production of natural products. Since these are usually clustered in the genome, analysis is facilitated and with the help of bioinformatics tools, prediction of biosynthetic pathways and structures has become possible. This has recently resulted in the discovery of many novel natural compounds.[9] Another approach to find new antibiotics is to target previously unexploited essential enzymes amongst others, those involved in the fatty acid metabolic pathway, cell division, DNA replication and others. For a recent review see.[9] Other proteins that are considered interesting targets are those that are essential in protein secretion, such as the type I signal peptidases (SPases)[10] and SecA.[11] Since in bacteria, approximately 25–30% of the proteins synthesized are destined to function at the cell envelope or outside, hindering protein secretion will finally result in cell death as proteins to be secreted will accumulate in the membrane and disturb its function.

The majority of proteins to be secreted are synthesized as preproteins with an amino-terminal extension called the signal peptide (SP) which, apart from other functions, serves primarily as a postal code for transmembrane transport.[12] Preproteins are targeted and transported across the membrane via the general secretion pathway (Sec pathway), which is essential and universal in bacteria, or in some cases, via the twin-arginine translocation (Tat) pathway that also exists in several bacteria (for a review see[13]). A major difference between the two pathways is that while the former translocates unfolded proteins, the latter transports folded proteins across the membrane. The Tat-pathway is shown to be important for transport of several virulence factors.[14] Both pathways consist of separate targeting components and, in each case, a membrane-embedded pore (the translocon) is present through which the preprotein is translocated (Figure 2). In both pathways, during or shortly after translocation of the preproteins across the membrane, the hydrophobic SP is cleaved off by enzymes called the signal peptidases, ensuring the release of the mature protein from the membrane.[15] In a major role, the type I signal peptidase, also known as leader peptidase (Lep), a membrane-bound endopeptidase, processes nonlipoproteins, thereby enabling them to reach their final destination. In this article, the term SPase(s) refers to the bacterial type I SPase(s) and not to the other types of SPases (SPase II or SPase IV) that co-exist in bacteria.

Figure 2.

Protein transport across the membrane in Escherichia coli.
Preproteins are routed from the site of synthesis (cytoplasm) to the membrane by the secretion (Sec) or the twin-arginine translocation (Tat) pathway. The Sec system consists of a membrane-embedded, protein-conducting channel (PCC) comprising of three proteins (SecY, SecE and SecG), which form the Sec translocase and a peripherally associated, ATP-driven motor protein, SecA. SecD, SecF and YajC form the translocon-associated complex and YidC is involved in membrane protein integration and folding. In the Sec pathway, the transport of unfolded proteins occurs either post- or co-translationally. In case of the former, the fully synthesized preprotein detaches from the ribosome and is directed to the Sec translocase, with the help of a chaperone, SecB. In co-translational targeting, the SRP binds to the signal sequence of the secretory protein while it emerges from the ribosome and the entire ternary complex of SRP/ribosome/nascent secretory protein chain is targeted to the Sec translocase with the aid of SRP receptor (SR or FtsY). SecA accepts the unfolded proteins and threads them through the transmembrane channel (PCC).75 The Tat system, dedicated for transport of folded proteins, consists of three membrane-integrated subunits, namely TatA, TatB and TatC, that together form receptor and a protein-conducting machinery for Tat substrates. TatC and TatB form a complex that is involved in recognition of Tat signal sequences and their insertion into the membrane, while TatA mediates the actual translocation event.76 During or shortly after translocation of the proteins, the hydrophobic signal peptide of most nonlipoproteins is cleaved off by type I signal peptidase, resulting in the release of the mature protein.
SRP: Signal recognition particle.

This article aims at summarizing the recent developments in the area of SPases, with regard to their use as drug targets. As with most novel targets, SPases did not achieve the quick success that was probably expected of the target-based approach. Nevertheless, enormous efforts from industry as well as academia have kept SPases in focus for over a decade.

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