Synthesis and preclinical evaluation of novel 18F-vancomycin-based tracers for the detection of bacterial infections using positron emission tomography.

INTRODUCTION: Bacterial infections are a major problem in medicine, and the rapid and accurate detection of such infections is essential for optimal patient outcome. Bacterial infections can be diagnosed by nuclear imaging, but most currently available modalities are unable to discriminate infection from sterile inflammation. Bacteria-targeted positron emission tomography (PET) tracers have the potential to overcome this hurdle. In the present study, we compared three 18F-labelled PET tracers based on the clinically applied antibiotic vancomycin for targeted imaging of Gram-positive bacteria. METHODS: [18F]FB-NHS and [18F]BODIPY-FL-NHS were conjugated to vancomycin. The resulting conjugates, together with our previously developed [18F]PQ-VE1-vancomycin, were tested for stability, lipophilicity, selective binding to Gram-positive bacteria, antimicrobial activity and biodistribution. For the first time, the pharmacokinetic properties of all three tracers were compared in healthy animals to identify potential binding sites. RESULTS: [18F]FB-vancomycin, [18F]BODIPY-FL-vancomycin, and [18F]PQ-VE1-vancomycin were successfully synthesized with radiochemical yields of 11.7%, 2.6%, and 0.8%, respectively. [18F]FB-vancomycin exhibited poor in vitro and in vivo stability and, accordingly, no bacterial binding. In contrast, [18F]BODIPY-FL-vancomycin and [18F]PQ-VE1-vancomycin showed strong and specific binding to Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), which was outcompeted by unlabeled vancomycin only at concentrations exceeding clinically relevant vancomycin blood levels. Biodistribution showed renal clearance of [18F]PQ-VE1-vancomycin and [18F]BODIPY-FL-vancomycin with low non-specific accumulation in muscles, fat and bones. CONCLUSION: Here we present the synthesis and first evaluation of the vancomycin-based PET tracers [18F]BODIPY-FL-vancomycin and [18F]PQ-VE1-vancomycin for image-guided detection of Gram-positive bacteria. Our study paves the way towards real-time bacteria-targeted diagnosis of soft tissue and implant-associated infections that are oftentimes caused by Gram-positive bacteria, even after prophylactic treatment with vancomycin.

Targeted optical fluorescence imaging: a meta-narrative review and future perspectives.

PURPOSE: The aim of this review is to give an overview of the current status of targeted optical fluorescence imaging in the field of oncology, cardiovascular, infectious and inflammatory diseases to further promote clinical translation. METHODS: A meta-narrative approach was taken to systematically describe the relevant literature. Consecutively, each field was assigned a developmental stage regarding the clinical implementation of optical fluorescence imaging. RESULTS: Optical fluorescence imaging is leaning towards clinical implementation in gastrointestinal and head and neck cancers, closely followed by pulmonary, neuro, breast and gynaecological oncology. In cardiovascular and infectious disease, optical imaging is in a less advanced/proof of concept stage. CONCLUSION: Targeted optical fluorescence imaging is rapidly evolving and expanding into the clinic, especially in the field of oncology. However, the imaging modality still has to overcome some major challenges before it can be part of the standard of care in the clinic, such as the provision of pivotal trial data. Intensive multidisciplinary (pre-)clinical joined forces are essential to overcome the delivery of such compelling phase III registration trial data and subsequent regulatory approval and reimbursement hurdles to advance clinical implementation of targeted optical fluorescence imaging as part of standard practice.

The phosphoenolpyruvate:sugar phosphotransferase system is involved in sensitivity to the glucosylated bacteriocin sublancin.

The mode of action of a group of glycosylated antimicrobial peptides known as glycocins remains to be elucidated. In the current study of one glycocin, sublancin, we identified the phosphoenolpyruvate:sugar phosphotransferase system (PTS) of Bacillus species as a key player in bacterial sensitivity. Sublancin kills several Gram-positive bacteria, such as Bacillus species and Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). Unlike other classes of bacteriocins for which the PTS is involved in their mechanism of action, we show that the addition of PTS-requiring sugars leads to increased resistance rather than increased sensitivity, suggesting that sublancin has a distinct mechanism of action. Collectively, our present mutagenesis and genomic studies demonstrate that the histidine-containing phosphocarrier protein (HPr) and domain A of enzyme II (PtsG) in particular are critical determinants for bacterial sensitivity to sublancin.

Overview of molecular typing methods for outbreak detection and epidemiological surveillance.

Typing methods for discriminating different bacterial isolates of the same species are essential epidemiological tools in infection prevention and control. Traditional typing systems based on phenotypes, such as serotype, biotype, phage-type, or antibiogram, have been used for many years. However, more recent methods that examine the relatedness of isolates at a molecular level have revolutionised our ability to differentiate among bacterial types and subtypes. Importantly, the development of molecular methods has provided new tools for enhanced surveillance and outbreak detection. This has resulted in better implementation of rational infection control programmes and efficient allocation of resources across Europe. The emergence of benchtop sequencers using next generation sequencing technology makes bacterial whole genome sequencing (WGS) feasible even in small research and clinical laboratories. WGS has already been used for the characterisation of bacterial isolates in several large outbreaks in Europe and, in the near future, is likely to replace currently used typing methodologies due to its ultimate resolution. However, WGS is still too laborious and time-consuming to obtain useful data in routine surveillance. Also, a largely unresolved question is how genome sequences must be examined for epidemiological characterisation. In the coming years, the lessons learnt from currently used molecular methods will allow us to condense the WGS data into epidemiologically useful information. On this basis, we have reviewed current and new molecular typing methods for outbreak detection and epidemiological surveillance of bacterial pathogens in clinical practice, aiming to give an overview of their specific advantages and disadvantages.

Mapping the pathways to staphylococcal pathogenesis by comparative secretomics.

The gram-positive bacterium Staphylococcus aureus is a frequent component of the human microbial flora that can turn into a dangerous pathogen. As such, this organism is capable of infecting almost every tissue and organ system in the human body. It does so by actively exporting a variety of virulence factors to the cell surface and extracellular milieu. Upon reaching their respective destinations, these virulence factors have pivotal roles in the colonization and subversion of the human host. It is therefore of major importance to obtain a clear understanding of the protein transport pathways that are active in S. aureus. The present review aims to provide a state-of-the-art roadmap of staphylococcal secretomes, which include both protein transport pathways and the extracytoplasmic proteins of these organisms. Specifically, an overview is presented of the exported virulence factors, pathways for protein transport, signals for cellular protein retention or secretion, and the exoproteomes of different S. aureus isolates. The focus is on S. aureus, but comparisons with Staphylococcus epidermidis and other gram-positive bacteria, such as Bacillus subtilis, are included where appropriate. Importantly, the results of genomic and proteomic studies on S. aureus secretomes are integrated through a comparative "secretomics" approach, resulting in the first definition of the core and variant secretomes of this bacterium. While the core secretome seems to be largely employed for general housekeeping functions which are necessary to thrive in particular niches provided by the human host, the variant secretome seems to contain the "gadgets" that S. aureus needs to conquer these well-protected niches.

Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon.

Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein assembly, but not in degradation, were suppressed by overproduction of yeast mitochondrial Lon, an ATP-dependent serine protease. Suppression by Lon was enhanced by inactivation of the proteolytic site and was prevented by mutation of the ATP-binding site. It is suggested that the mitochondrial proteases Lon, Afg3p, and Rca1p can also serve a chaperone-like function in the assembly of mitochondrial protein complexes.

ATP-dependent proteases that also chaperone protein biogenesis.

The ATP-dependent proteases Clp and FtsH from bacteria, as well as mitochondrial homologs of FtsH and Lon from yeast, may act as chaperones; they mediate not only proteolysis, but also the insertion of proteins into membranes and the disassembly or oligomerization of protein complexes. The coordination of such processes with selective proteolysis may function in the quality control of protein biogenesis.

Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome.

One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.

Conserved Citrullinating Exoenzymes in Porphyromonas Species.

Porphyromonas gingivalis is one of the major oral pathogens implicated in the widespread inflammatory disorder periodontitis. Moreover, in recent years, P. gingivalis has been associated with the autoimmune disease rheumatoid arthritis. The peptidylarginine deiminase enzyme of P. gingivalis (PPAD) is a major virulence factor that catalyzes the citrullination of both bacterial and host proteins, potentially contributing to production of anticitrullinated protein antibodies. Considering that these antibodies are very specific for rheumatoid arthritis, PPAD appears to be a link between P. gingivalis, periodontitis, and the autoimmune disorder rheumatoid arthritis. PPAD was thus far considered unique among prokaryotes, with P. gingivalis being the only bacterium known to produce and secrete it. To challenge this hypothesis, we investigated the possible secretion of PPAD by 11 previously collected Porphyromonas isolates from a dog, 2 sheep, 3 cats, 4 monkeys, and a jaguar with periodontitis. Our analyses uncovered the presence of secreted PPAD homologues in 8 isolates that were identified as Porphyromonas gulae (from a dog, monkeys, and cats) and Porphyromonas loveana (from sheep). In all 3 PPAD-producing Porphyromonas species, the dominant form of the secreted PPAD was associated with outer membrane vesicles, while a minor fraction was soluble. Our results prove for the first time that the citrullinating PPAD exoenzyme is not unique to only 1 prokaryotic species. Instead, we show that PPAD is produced by at least 2 other oral pathogens.

The population structure of Staphylococcus aureus in China and Europe assessed by multiple-locus variable number tandem repeat analysis; clues to geographical origins of emergence and dissemination.

To compare the genetic population structure of Staphylococcus aureus from China and Europe, 1294 human isolates were characterized by multiple-locus variable number tandem repeat analysis (MLVA). In total, MLVA identified 17 MLVA complexes (MCs), comprising 260 MLVA types (MTs) among the Chinese isolates and 372 MTs among the European isolates. The five most frequent MCs among the Chinese isolates belonged to MC398, MC5 subclade a, MC8, MC437 and MC7 and made up 55% of the sample. For the European isolates, the five most frequent MCs consisted of MC5 subclade a, MC45, MC8, MC30 and MC22, which accounted for 64% of the sample. Phylogeographic analysis of the major MCs shared between China and Europe points to a European origin of MC8 but cannot provide a consistent signal for MC5 subclade a, probably indicating a different origin. Diversity and frequency distributions of other lineages were also compared. Altogether, this study provides the first snapshot of two extant populations of S. aureus from Europe and China, and important clues on the emergence and dissemination of different lineages of S. aureus.

Improving protein secretion by engineering components of the bacterial translocation machinery.

The increased insight into the mechanism of bacterial protein translocation has resulted in new concepts for the production of heterologous proteins. The periplasm of gram-negative bacteria is revealed to have a role as a 'protein construction compartment', which can be used to fold complex proteins. Passage across the outer membrane, however, remains a challenge due to the high selectivity of the outer membrane translocase. In gram-positive bacteria, slow folding at the membrane-cell-wall interface can make heterologous proteins vulnerable to degradation by wall-associated proteases. The recent identification of thiol-disulfide oxidoreductases in Bacillus subtilis might open the possibility of secreting proteins containing multiple disulfide bonds from this host.

Staphylococcus aureus spa type t437: identification of the most dominant community-associated clone from Asia across Europe.

Methicillin-resistant Staphylococcus aureus (MRSA) belonging to the multilocus sequence type clonal complex 59 (MLST CC59) is the predominant community-associated MRSA clone in Asia. This clone, which is primarily linked with the spa type t437, has so far only been reported in low numbers among large epidemiological studies in Europe. Nevertheless, the overall numbers identified in some Northern European reference laboratories have increased during the past decade. To determine whether the S. aureus t437 clone is present in other European countries, and to assess its genetic diversity across Europe, we analysed 147 S. aureus t437 isolates from 11 European countries collected over a period of 11 years using multiple locus variable number tandem repeat fingerprinting/analysis (MLVF/MLVA) and MLST. Additionally 16 S. aureus t437 isolates from healthy carriers and patients from China were included. Most isolates were shown to be monophyletic with 98% of the isolates belonging to the single MLVA complex 621, to which nearly all included isolates from China also belonged. More importantly, all MLST-typed isolates belonged to CC59. Our study implies that the European S. aureus t437 population represents a genetically tight cluster, irrespective of the year, country and site of isolation. This underpins the view that S. aureus CC59 has been introduced into several European countries, not being restricted to particular geographical regions or specific host environments. The European S. aureus t437 isolates thus bear the general hallmarks of a high-risk clone.

The chemistry and enzymology of the type I signal peptidases.

The discovery that proteins exported from the cytoplasm are typically synthesized as larger precursors with cleavable signal peptides has focused interest on the peptidases that remove the signal peptides. Here, we review the membrane-bound peptidases dedicated to the processing of protein precursors that are found in the plasma membrane of prokaryotes and the endoplasmic reticulum, the mitochondrial inner membrane, and the chloroplast thylakoidal membrane of eukaryotes. These peptidases are termed type I signal (or leader) peptidases. They share the unusual feature of being resistant to the general inhibitors of the four well-characterized peptidase classes. The eukaryotic and prokaryotic signal peptidases appear to belong to a single peptidase family. This review emphasizes the evolutionary concepts, current knowledge of the catalytic mechanism, and substrate specificity requirements of the signal peptidases.

In vitro assay for the Bacillus subtilis signal peptidase SipS: systems for efficient in vitro transcription-translation and processing of precursors of secreted proteins.

The signal peptidase (SPase) SipS of Bacillus subtilis is responsible for the processing of precursors of secreted proteins. It differs from the SPases of Gram-negative bacteria in structure and specificity. To assay the activity of SipS in vitro, two efficient transcription-translation systems for the synthesis of radio-labelled precursors were developed. The systems were completely derived from B. subtilis. Post-translational in vitro processing of pre-staphylokinase by SipS was demonstrated. SipS activity was stimulated in vitro by several non-ionic detergents, whereas it was not affected by a large variety of proteinase inhibitors. SipS shares the latter property with other SPases.

Lack of specific hybridization between the lep genes of Salmonella typhimurium and Bacillus licheniformis.

This paper describes an attempt to clone the Bacillus licheniformis lep gene, encoding signal peptidase, using the Salmonella typhimurium lep gene as a hybridization probe. Although a hybridizing fragment was obtained, DNA sequence analysis indicated that it did not contain the lep gene. Instead, the protein encoded by the cloned fragment showed similarity with a variety of L-asparaginases.

Protein secretion and possible roles for multiple signal peptidases for precursor processing in bacilli.

Bacillus subtilis is one of the best known Gram-positive bacteria at both the genetic and physiological level. The entire sequence of its chromosome is known and efficient tools for the genetic modification of this bacterium are available. Moreover, B. subtilis and related Bacillus species are widely used in biotechnology, in particular for the production of secreted enzymes. Although bacilli can secrete large amounts of several native enzymes, the use of these bacteria for the production of heterologous enzymes has frequently resulted in low yields. Here we describe the identification of several components of the Bacillus protein secretion machinery. These components can now be engineered for optimal protein secretion. Special emphasis is given on type I signal peptidases, which remove signal peptides from secretory precursor proteins. Five genes specifying such enzymes (sip, for signal peptidase) are present on the B. subtilis chromosome. Although none of the sip genes is essential by itself, a specific combination of mutations in these genes is lethal. The expression pattern of some of the sip genes coincides with that of many secretory proteins, which seems to reflect an adaptation to high demands on the secretion machinery. Although the various B. subtilis type I signal peptidases have at least partially overlapping substrate specificities, clear differences in substrate preferences are also evident. These observations have implications for the engineering of the processing apparatus for improved secretion of native and heterologous proteins by Bacillus.

The Sip(Sli) gene of Streptomyces lividans TK24 specifies an unusual signal peptidase with a putative C-terminal transmembrane anchor.

Type I signal peptidases (SPases) are a widespread family of enzymes which remove signal peptides from proteins translocated across cellular membranes. Here, we report the first isolation of a gene coding for type I signal peptidase of Streptomyces, denoted Sip(Sli). The sip(sli) gene specifies a protein of 291 amino acids. Thus Sip(Sli) is much larger (approximately 100 amino acids) than other known SPases of Gram-positive bacteria and resembles SPases of Gram-negative bacteria, showing the highest degree of similarity to an SPase of the cyanobacterium Phormidium laminosum. Sip(Sli) contains conserved serine and lysine residues, which are believed to be required for the catalytic activity. Similar to other known SPases from Gram-positive bacteria, Sip(Sli) seems to have only one N-terminal transmembrane anchor. In addition, Sip(Sli) seems to contain a second transmembrane anchor at the C-terminus, which is an unusual feature for type I signal peptidases.

Identification of the potential active site of the signal peptidase SipS of Bacillus subtilis. Structural and functional similarities with LexA-like proteases.

Signal peptidases remove signal peptides from secretory proteins. By comparing the type I signal peptidase, SipS, of Bacillus subtilis with signal peptidases from prokaryotes, mitochondria, and the endoplasmic reticular membrane, patterns of conserved amino acids were discovered. The conserved residues of SipS were altered by site-directed mutagenesis. Replacement of methionine 44 by alanine yielded an enzyme with increased activity. Two residues (aspartic acid 146 and arginine 84) appeared to be conformational determinants; three other residues (serine 43, lysine 83, and aspartic acid 153) were critical for activity. Comparison of SipS with other proteases requiring serine, lysine, or aspartic acid residues in catalysis revealed sequence similarity between the region of SipS around serine 43 and lysine 83 and the active-site region of LexA-like proteases. Furthermore, self-cleavage sites of LexA-like proteases closely resembled signal peptidase cleavage sites. Together with the finding that serine and lysine residues are critical for activity of the signal peptidase of Escherichia coli (Tschantz, W.R., Sung, M., Delgado-Partin, V.M., and Dalbey, R.E. (1993) J. Biol. Chem. 268, 27349-27354), our data indicate that type I signal peptidases and LexA-like proteases are structurally and functionally related serine proteases. A model envisaging a catalytic serine-lysine dyad in prokaryotic type I signal peptidases is proposed to accommodate our observations.

Signal peptidase I overproduction results in increased efficiencies of export and maturation of hybrid secretory proteins in Escherichia coli.

The effects of 25-fold overproduction of Escherichia coli signal peptidase I (SPase I) on the processing kinetics of various (hybrid) secretory proteins, comprising fusions between signal sequence functions selected from the Bacillus subtilis chromosome and the mature part of TEM-beta-lactamase, were studied in E. coli. One precursor (pre[A2d]-beta-lactamase) showed an enhanced processing rate, and consequently, a highly improved release of the mature enzyme into the periplasm. A minor fraction of a second hybrid precursor (pre[A13i]-beta-lactamase), which was not processed under standard conditions of SPase I synthesis, was shown to be processed under conditions of SPase I overproduction. However, this did not result in efficient release of the mature beta-lactamase into the periplasm. In contrast, the processing rates of wild-type pre-beta-lactamase and pre(A2)-beta-lactamase, already high under standard conditions, were not detectably altered by SPase I overproduction. These results demonstrate that the availability of SPase I can be a limiting factor in protein export in E. coli, in particular with respect to (hybrid) precursor proteins showing low (SPase I) processing efficiencies.