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.

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.

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.

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.

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.

The Bacillus secretion stress response is an indicator for alpha-amylase production levels.

AIMS: Overproduced alpha-amylases in Bacillus subtilis provoke a specific stress response involving the CssRS two-component system, which controls expression of the HtrA and HtrB proteases. Previously, the B. subtilis TepA protein was implicated in high-level alpha-amylase secretion. Our present studies were aimed at investigating a possible role of TepA in secretion stress management, and characterizing the intensity of the secretion stress response in relation to alpha-amylase production. METHODS AND RESULTS: The expression of a transcriptional htrB-lacZ gene fusion, and the levels of alpha-amylase production were monitored simultaneously using tepA mutant B. subtilis strains. TepA was shown to be dispensable for secretion stress management. Importantly, however, the levels of htrB-lacZ expression can be correlated with the levels of alpha-amylase production. CONCLUSION: Our observations show that the secretion stress response can serve as an indicator for alpha-amylase production levels. SIGNIFICANCE AND IMPACT OF STUDY: Conceivably, this stress response can be employed to monitor the biotechnological production of various secretory proteins by the Bacillus cell factory.

A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress.

The Gram-positive eubacterium Bacillus subtilis is well known for its high capacity to secrete proteins into the environment. Even though high-level secretion of proteins is an efficient process, it imposes stress on the cell. The present studies were aimed at the identification of systems required to combat this so-called secretion stress. A two-component regulatory system, named CssR-CssS, was identified, which bears resemblance to the CpxR-CpxA system of Escherichia coli. The results show that the CssR/S system is required for the cell to survive the severe secretion stress caused by a combination of high-level production of the alpha-amylase AmyQ and reduced levels of the extracytoplasmic folding factor PrsA. As shown with a prsA3 mutation, the Css system is required to degrade misfolded exported proteins at the membrane-cell wall interface. This view is supported by the observation that transcription of the htrA gene, encoding a predicted membrane-bound protease of B. subtilis, is strictly controlled by CssS. Notably, CssS represents the first identified sensor for extracytoplasmic protein misfolding in a Gram-positive eubacterium. In conclusion, the results show that quality control systems for extracytoplasmic protein folding are not exclusively present in the periplasm of Gram-negative eubacteria, but also in the Gram-positive cell envelope.

A proteomic view on genome-based signal peptide predictions.

The availability of complete genome sequences has allowed the prediction of all exported proteins of the corresponding organisms with dedicated algorithms. Even though numerous studies report on genome-based predictions of signal peptides and cell retention signals, they lack a proteomic verification. For example, 180 secretory and 114 lipoprotein signal peptides were predicted recently for the Gram-positive eubacterium Bacillus subtilis. In the present studies, proteomic approaches were used to define the extracellular complement of the B. subtilis secretome. Using different growth conditions and a hyper-secreting mutant, approximately 200 extracellular proteins were visualized by two-dimensional (2D) gel electrophoresis, of which 82 were identified by mass spectrometry. These include 41 proteins that have a potential signal peptide with a type I signal peptidase (SPase) cleavage site, and lack a retention signal. Strikingly, the remaining 41 proteins were predicted previously to be cell associated because of the apparent absence of a signal peptide (22), or the presence of specific cell retention signals in addition to an export signal (19). To test the importance of the five type I SPases and the unique lipoprotein-specific SPase of B. subtilis, the extracellular proteome of (multiple) SPase mutants was analyzed. Surprisingly, only the processing of the polytopic membrane protein YfnI was strongly inhibited in Spase I mutants, showing for the first time that a native eubacterial membrane protein is a genuine Spase I substrate. Furthermore, a mutation affecting lipoprotein modification and processing resulted in the shedding of at least 23 (lipo-)proteins into the medium. In conclusion, our observations show that genome-based predictions reflect the actual composition of the extracellular proteome for approximately 50%. Major problems are currently encountered with the prediction of extracellular proteins lacking signal peptides (including cytoplasmic proteins) and lipoproteins.

Distinction between major and minor Bacillus signal peptidases based on phylogenetic and structural criteria.

The processing of secretory preproteins by signal peptidases (SPases) is essential for cell viability. As previously shown for Bacillus subtilis, only certain SPases of organisms containing multiple paralogous SPases are essential. This allows a distinction between SPases that are of major and minor importance for cell viability. Notably, the functional difference between major and minor SPases is not reflected clearly in sequence alignments. Here, we have successfully used molecular phylogeny to predict major and minor SPases. The results were verified with SPases from various bacilli. As predicted, the latter enzymes behaved as major or minor SPases when expressed in B. subtilis. Strikingly, molecular modeling indicated that the active site geometry is not a critical parameter for the classification of major and minor Bacillus SPases. Even though the substrate binding site of the minor SPase SipV is smaller than that of other known SPases, SipV could be converted into a major SPase without changing this site. Instead, replacement of amino-terminal residues of SipV with corresponding residues of the major SPase SipS was sufficient for conversion of SipV into a major SPase. This suggests that differences between major and minor SPases are based on activities other than substrate cleavage site selection.

A truncated soluble Bacillus signal peptidase produced in Escherichia coli is subject to self-cleavage at its active site.

Soluble forms of Bacillus signal peptidases which lack their unique amino-terminal membrane anchor are prone to degradation, which precludes their high-level production in the cytoplasm of Escherichia coli. Here, we show that the degradation of soluble forms of the Bacillus signal peptidase SipS is largely due to self-cleavage. First, catalytically inactive soluble forms of this signal peptidase were not prone to degradation; in fact, these mutant proteins were produced at very high levels in E. coli. Second, the purified active soluble form of SipS displayed self-cleavage in vitro. Third, as determined by N-terminal sequencing, at least one of the sites of self-cleavage (between Ser15 and Met16 of the truncated enzyme) strongly resembles a typical signal peptidase cleavage site. Self-cleavage at the latter position results in complete inactivation of the enzyme, as Ser15 forms a catalytic dyad with Lys55. Ironically, self-cleavage between Ser15 and Met16 cannot be prevented by mutagenesis of Gly13 and Ser15, which conform to the -1, -3 rule for signal peptidase recognition, because these residues are critical for signal peptidase activity.

TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway.

The recent discovery of a ubiquitous translocation pathway, specifically required for proteins with a twin-arginine motif in their signal peptide, has focused interest on its membrane-bound components, one of which is known as TatC. Unlike most organisms of which the genome has been sequenced completely, the Gram-positive eubacterium Bacillus subtilis contains two tatC-like genes denoted tatCd and tatCy. The corresponding TatCd and TatCy proteins have the potential to be involved in the translocation of 27 proteins with putative twin-arginine signal peptides of which approximately 6-14 are likely to be secreted into the growth medium. Using a proteomic approach, we show that PhoD of B. subtilis, a phosphodiesterase belonging to a novel protein family of which all known members are synthesized with typical twin-arginine signal peptides, is secreted via the twin-arginine translocation pathway. Strikingly, TatCd is of major importance for the secretion of PhoD, whereas TatCy is not required for this process. Thus, TatC appears to be a specificity determinant for protein secretion via the Tat pathway. Based on our observations, we hypothesize that the TatC-determined pathway specificity is based on specific interactions between TatC-like proteins and other pathway components, such as TatA, of which three paralogues are present in B. subtilis.

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 serine and histidine residues are critical for activity of the ER-type signal peptidase SipW of Bacillus subtilis.

Type I signal peptidases (SPases) are required for the removal of signal peptides from translocated proteins and, subsequently, release of the mature protein from the trans side of the membrane. Interestingly, prokaryotic (P-type) and endoplasmic reticular (ER-type) SPases are functionally equivalent, but structurally quite different, forming two distinct SPase families that share only few conserved residues. P-type SPases were, so far, exclusively identified in eubacteria and organelles, whereas ER-type SPases were found in the three kingdoms of life. Strikingly, the presence of ER-type SPases appears to be limited to sporulating Gram-positive eubacteria. The present studies were aimed at the identification of potential active site residues of the ER-type SPase SipW of Bacillus subtilis, which is required for processing of the spore-associated protein TasA. Conserved serine, histidine, and aspartic acid residues are critical for SipW activity, suggesting that the ER-type SPases employ a Ser-His-Asp catalytic triad or, alternatively, a Ser-His catalytic dyad. In contrast, the P-type SPases employ a Ser-Lys catalytic dyad (Paetzel, M., Dalbey, R. E., and Strynadka, N. C. J. (1998) Nature 396, 186-190). Notably, catalytic activity of SipW was not only essential for pre-TasA processing, but also for the incorporation of mature TasA into spores.

The potential active site of the lipoprotein-specific (type II) signal peptidase of Bacillus subtilis.

Type II signal peptidases (SPase II) remove signal peptides from lipid-modified preproteins of eubacteria. As the catalytic mechanism employed by type II SPases was not known, the present studies were aimed at the identification of their potential active site residues. Comparison of the deduced amino acid sequences of 19 known type II SPases revealed the presence of five conserved domains. The importance of the 15 best conserved residues in these domains was investigated using the type II SPase of Bacillus subtilis, which, unlike SPase II of Escherichia coli, is not essential for viability. The results showed that only six residues are important for SPase II activity. These are Asp-14, Asn-99, Asp-102, Asn-126, Ala-128, and Asp-129. Only Asp-14 was required for stability of SPase II, indicating that the other five residues are required for catalysis, the active site geometry, or the specific recognition of lipid-modified preproteins. As Asp-102 and Asp-129 are the only residues invoked in the known catalytic mechanisms of proteases, we hypothesize that these two residues are directly involved in SPase II-mediated catalysis. This implies that type II SPases belong to a novel family of aspartic proteases.

Functional analysis of paralogous thiol-disulfide oxidoreductases in Bacillus subtilis.

The in vivo formation of disulfide bonds, which is critical for the stability and/or activity of many proteins, is catalyzed by thiol-disulfide oxidoreductases. In the present studies, we show that the Gram-positive eubacterium Bacillus subtilis contains three genes, denoted bdbA, bdbB, and bdbC, for thiol-disulfide oxidoreductases. Escherichia coli alkaline phosphatase, containing two disulfide bonds, was unstable when secreted by B. subtilis cells lacking BdbB or BdbC, and notably, the expression levels of bdbB and bdbC appeared to set a limit for the secretion of active alkaline phosphatase. Cells lacking BdbC also showed decreased stability of cell-associated forms of E. coli TEM-beta-lactamase, containing one disulfide bond. In contrast, BdbA was not required for the stability of alkaline phosphatase or beta-lactamase. Because BdbB and BdbC are typical membrane proteins, our findings suggest that they promote protein folding at the membrane-cell wall interface. Interestingly, pre-beta-lactamase processing to its mature form was stimulated in cells lacking BdbC, suggesting that the unfolded form of this precursor is a preferred substrate for signal peptidase. Surprisingly, cells lacking BdbC did not develop competence for DNA uptake, indicating the involvement of disulfide bond-containing proteins in this process. Unlike E. coli and yeast, none of the thiol-disulfide oxidoreductases of B. subtilis was required for growth in the presence of reducing agents. In conclusion, our observations indicate that BdbB and BdbC have a general role in disulfide bond formation, whereas BdbA may be dedicated to a specific process.

Signal peptide peptidase- and ClpP-like proteins of Bacillus subtilis required for efficient translocation and processing of secretory proteins.

Signal peptides direct the export of secretory proteins from the cytoplasm. After processing by signal peptidase, they are degraded in the membrane and cytoplasm. The resulting fragments can have signaling functions. These observations suggest important roles for signal peptide peptidases. The present studies show that the Gram-positive eubacterium Bacillus subtilis contains two genes for proteins, denoted SppA and TepA, with similarity to the signal peptide peptidase A of Escherichia coli. Notably, TepA also shows similarity to ClpP proteases. SppA of B. subtilis was only required for efficient processing of pre-proteins under conditions of hyper-secretion. In contrast, TepA depletion had a strong effect on pre-protein translocation across the membrane and subsequent processing, not only under conditions of hyper-secretion. Unlike SppA, which is a typical membrane protein, TepA appears to have a cytosolic localization, which is consistent with the observation that TepA is involved in early stages of the secretion process. Our observations demonstrate that SppA and TepA have a role in protein secretion in B. subtilis. Based on their similarity to known proteases, it seems likely that SppA and TepA are specifically required for the degradation of proteins or (signal) peptides that are inhibitory to protein translocation.