One problem, multiple potential targets: Where are we now in the development of small molecule inhibitors against Shiga toxin?

Shiga toxin-producing Escherichia coli (STEC) are a group of enteric pathogens which carry phage-encoded Shiga toxins (Stx). STEC infections begin with severe abdominal pain and non-bloody diarrhoea, which can progress to bloody diarrhoea after approximately 4-days post-infection. In high-risk groups such as children and the elderly, patients may develop haemolytic uremic syndrome (HUS). HUS is characterised by microangiopathic haemolytic anaemia, thrombocytopenia, and in severe disease acute renal failure. Traditional antibiotics have been linked with increased toxin production due to the activation of recA-mediated bacterial stress response, resulting in poorer patient outcomes. Therefore, treatment relies on supportive therapies. Antivirulence strategies have been explored as an alternative treatment for bacterial infections and blockers of virulence factors such as the Type III Secretion System. Recent improvements in the mechanistic understanding of the Stx pathway have led to the design of inhibitors to disrupt the pathway, leading to toxin-mediated ribosome damage. However, compounds have yet to progress beyond Phase III clinical trials successfully. This review explores the progress in developing small molecule inhibitors by collating lead compounds derived from in-silico and experimental approaches.

Enhancing a multi-purpose artificial urine for culture and gene expression studies of uropathogenic Escherichia coli strains.

AIMS: The main objective of this study was to modify a recently reported multi-purpose artificial urine (MP-AU) for culture and gene expression studies of uropathogenic Escherichia coli (UPEC) strains. METHODS AND RESULTS: We used liquid chromatography mass spectrometry (LC-MS) to identify and adjust the metabolic profile of MP-AU closer to that of pooled human urine (PHU). Modification in this way facilitated growth of UPEC strains with growth rates similar to those obtained in PHU. Transcriptomic analysis of UPEC strains cultured in enhanced artificial urine (enhanced AU) and PHU showed that the gene expression profiles are similar, with <7% of genes differentially expressed between the two conditions. CONCLUSIONS: Enhancing an MP-AU with metabolites identified in PHU allows the enhanced AU to be used as a substitute for the culture and in vitro gene expression studies of UPEC strains.

Steroid-free combination of 5-azacytidine and venetoclax for the treatment of multiple myeloma.

Multiple Myeloma (MM) is an incurable plasma cell malignancy, that despite an unprecedented increase in overall survival, lacks truly risk-adapted or targeted treatments. A proportion of patients with MM depend on BCL-2 for survival and recently the BCL-2 antagonist venetoclax has shown clinical efficacy and safety in t(11;14) and BCL-2 overexpressing MM. However, only a small proportion of MM patients rely on BCL-2 (~20%), there is a need to broaden the patient population outside of t(11;14) that can be treated with venetoclax. Therefore, we took an unbiased screening approach and screened epigenetic modifiers to enhance venetoclax sensitivity in two non-BCL-2 dependent MM cell lines. The demethylase inhibitor 5-azacytidine was one of the lead hits from the screen, and the enhanced cell killing of the combination was confirmed in additional MM cell lines. Using dynamic BH3 profiling and immunoprecipitations we identified the potential mechanism of synergy is due to increased NOXA expression, through the integrated stress response. Knockdown of PMAIP1 or PKR partially rescues cell death of the venetoclax and 5-azacytidine combination treatment. The addition of a steroid to the combination treatment did not enhance the cell death and interestingly we found enhanced death of the immune cells with steroid addition, suggesting that a steroid-sparing regimen may be more beneficial in MM. Lastly, we show for the first time in primary MM patient samples, that 5-azacytidine enhances the response to venetoclax ex-vivo, across diverse anti-apoptotic dependencies (BCL-2 or MCL-1) and diverse cytogenetic backgrounds. Overall, our data identifies 5-azacytidine and venetoclax as an effective treatment combination and this could be a tolerable steroid-sparing regimen, particularly for elderly MM patients.

Current understandings of colibactin regulation.

The biosynthetic machinery for the production of colibactin is encoded by 19 genes (clbA - S) within the pks pathogenicity island harboured by many E. coli of the B2-phylogroup. Colibactin is a potent genotoxic metabolite which causes DNA-damage and which has potential roles in microbial competition and fitness of pks+ bacteria. Colibactin has also been strongly implicated in the development of colorectal cancer. Given the genotoxicity of colibactin and the metabolic cost of its synthesis, the regulatory system governing the clb cluster is accordingly highly complex, and many of the mechanisms remain to be elucidated. In this review we summarise the current understanding of regulation of colibactin biosynthesis by internal molecular components and how these factors are modulated by signals from the external environment.

Bacterial outer membrane vesicles provide an alternative pathway for trafficking of Escherichia coli O157 type III secreted effectors to epithelial cells.

IMPORTANCE: Bacteria can package protein cargo into nanosized membrane blebs that are shed from the bacterial membrane and released into the environment. Here, we report that a type of pathogenic bacteria called enterohemorrhagic Escherichia coli O157 (EHEC) uses their membrane blebs (outer membrane vesicles) to package components of their type 3 secretion system and send them into host cells, where they can manipulate host signaling pathways including those involved in infection response, such as immunity. Usually, EHEC use a needle-like apparatus to inject these components into host cells, but packaging them into membrane blebs that get taken up by host cells is another way of delivery that can bypass the need for a functioning injection system.

D-Serine reduces the expression of the cytopathic genotoxin colibactin.

Some Escherichia coli strains harbour the pks island, a 54 kb genomic island encoding the biosynthesis genes for a genotoxic compound named colibactin. In eukaryotic cells, colibactin can induce DNA damage, cell cycle arrest and chromosomal instability. Production of colibactin has been implicated in the development of colorectal cancer (CRC). In this study, we demonstrate the inhibitory effect of D-Serine on the expression of the pks island in both prototypic and clinically-associated colibactin-producing strains and determine the implications for cytopathic effects on host cells. We also tested a comprehensive panel of proteinogenic L-amino acids and corresponding D-enantiomers for their ability to modulate clbB transcription. Whilst several D-amino acids exhibited the ability to inhibit expression of clbB, D-Serine exerted the strongest repressing activity (>3.8-fold) and thus, we focussed additional experiments on D-Serine. To investigate the cellular effect, we investigated if repression of colibactin by D-Serine could reduce the cytopathic responses normally observed during infection of HeLa cells with pks + strains. Levels of γ-H2AX (a marker of DNA double strand breaks) were reduced 2.75-fold in cells infected with D-Serine treatment. Moreover, exposure of pks + E. coli to D-Serine during infection caused a reduction in cellular senescence that was observable at 72 h post infection. The recent finding of an association between pks-carrying commensal E. coli and CRC, highlights the necessity for the development of colibactin targeting therapeutics. Here we show that D-Serine can reduce expression of colibactin, and inhibit downstream cellular cytopathy, illuminating its potential to prevent colibactin-associated disease.

Distinct ecological fitness factors coordinated by a conserved Escherichia coli regulator during systemic bloodstream infection.

The ability of bacterial pathogens to adapt to host niches is driven by the carriage and regulation of genes that benefit pathogenic lifestyles. Genes that encode virulence or fitness-enhancing factors must be regulated in response to changing host environments to allow rapid response to challenges presented by the host. Furthermore, this process can be controlled by preexisting transcription factors (TFs) that acquire new roles in tailoring regulatory networks, specifically in pathogens. However, the mechanisms underlying this process are poorly understood. The highly conserved Escherichia coli TF YhaJ exhibits distinct genome-binding dynamics and transcriptome control in pathotypes that occupy different host niches, such as uropathogenic E. coli (UPEC). Here, we report that this important regulator is required for UPEC systemic survival during murine bloodstream infection (BSI). This advantage is gained through the coordinated regulation of a small regulon comprised of both virulence and metabolic genes. YhaJ coordinates activation of both Type 1 and F1C fimbriae, as well as biosynthesis of the amino acid tryptophan, by both direct and indirect mechanisms. Deletion of yhaJ or the individual genes under its control leads to attenuated survival during BSI. Furthermore, all three systems are up-regulated in response to signals derived from serum or systemic host tissue, but not urine, suggesting a niche-specific regulatory trigger that enhances UPEC fitness via pleiotropic mechanisms. Collectively, our results identify YhaJ as a pathotype-specific regulatory aide, enhancing the expression of key genes that are collectively required for UPEC bloodstream pathogenesis.

Control of resistance against bacteriophage killing by a metabolic regulator in meningitis-associated Escherichia coli.

Ecologically beneficial traits in bacteria are encoded by intrinsic and horizontally acquired genes. However, such traits are not universal, and the highly mosaic nature of bacterial genomes requires control at the transcriptional level to drive these processes. It has emerged that regulatory flexibility is widespread in the Escherichia coli species, whereby preexisting transcription factors can acquire new and unrelated roles in regulating beneficial traits. DsdC is the regulator of D-serine tolerance in E. coli, is essential for D-serine catabolism, and is often encoded by two copies in neonatal meningitis-associated E. coli (NMEC). Here, we reveal that DsdC is a global regulator of transcription in NMEC and does not require D-serine for the control of novel beneficial traits. We show that DsdC binds the chromosome in an unusual manner, with many binding sites arranged in clusters spanning entire operons and within gene coding sequences, such as neuO. Importantly, we identify neuO as the most significantly down-regulated gene in a strain deleted for both dsdC copies, in both the presence and absence of D-serine. NeuO is prophage encoded in several NMEC K1 isolates and mediates capsule O-acetylation but has no effect on attachment to or invasion of human brain endothelial cells. Instead, we demonstrate that NeuO provides resistance against K1 bacteriophage attack and that this critical function is regulated by DsdC. This work highlights how a horizontally acquired enzyme that functions in cell-surface modulation can be controlled by an intrinsic regulator to provide a key ecological benefit to an E. coli pathotype.

A trans-Pt(II) hedgehog pathway inhibitor complex with cytotoxicity towards breast cancer stem cells and triple negative breast cancer cells.

The first example of a Pt complex of GANT61, a hedgehog (Hh) pathway inhibitor is reported. Reaction of cis-[Pt(II)Cl2(dmso)2] with one equivalent of 4-pyridine carboxaldehyde (4-PCA, control ligand) or one equivalent of GANT61 (Hh pathway inhibitor) in acetone at rt for 30 minutes afforded trans-[Pt(II)Cl2(dmso)(4-PCA)] (1) and trans-[Pt(II)Cl2(dmso)(GANT61)] (2) respectively, where 4-PCA and GANT61 are N-donor ligands. The structures of 1 and 2 were fully characterised by elemental analysis, 1H NMR, 13C NMR and IR spectroscopy and X-ray crystallography. 1 and 2 undergo isomerisation from trans- to cis-in solution and therefore the biological activity of 2 is also associated with the cis-configuration. The in vitro cytotoxicity data show that 2 is a potent inhibitor of the growth of breast CSC-depleted HMLER and breast CSC-enriched HMLER-shEcad cells. Furthermore 2 markedly reduced the size and viability and significantly reduced the number of CSC-enriched HMLER-shEcad mammospheres formed. 2 also induced apoptosis with low micromolar IC50 values against two triple negative breast cancer lines, MDA-MB-231 (MDA231) and BT549. 2, which possesses the Hh pathway inhibitor GANT61 as an N donor ligand exhibits far superior anti-CSC activity including in the CSC-enriched mammosphere model and activity against TNBC cells as compared to its control analogue, the trans-Pt(II) 4-PCA complex 1. The trans-Pt GANT61 complex 2 has also been shown to cause DNA damage and inhibit the Hh pathway at the level of GLI.

Genome sequence of the aurodox-producing bacterium Streptomyces goldiniensis ATCC 21386.

We report the genome sequence of Streptomyces goldiniensis ATCC 21386, a strain which produces the anti-bacterial and anti-virulence polyketide, aurodox. The genome of S. goldiniensis ATCC 21386 was sequenced using a multiplatform hybrid approach, revealing a linear genome of ~10 Mbp with a G+C content of 71%. The genome sequence revealed 36 putative biosynthetic gene clusters (BGCs), including a large region of 271 Kbp that was rich in biosynthetic capability. The genome sequence is deposited in DDBJ/EMBL/GenBank with the accession number PRJNA602141.

Crystal structures of WrbA, a spurious target of the salicylidene acylhydrazide inhibitors of type III secretion in Gram-negative pathogens, and verification of improved specificity of next-generation compounds.

The enterohemorrhagic Escherichia coli pathotype is responsible for severe and dangerous infections in humans. Establishment of the infection requires colonization of the gastro-intestinal tract, which is dependent on the Type III Secretion System. The Type III Secretion System (T3SS) allows attachment of the pathogen to the mammalian host cell and cytoskeletal rearrangements within the host cell. Blocking the functionality of the T3SS is likely to reduce colonization and therefore limit the disease. This route offers an alternative to antibiotics, and problems with the development of antibiotics resistance. Salicylidene acylhydrazides have been shown to have an inhibitory effect on the T3SS in several pathogens. However, the main target of these compounds is still unclear. Past work has identified a number of putative protein targets of these compounds, one of which being WrbA. Whilst WrbA is considered an off-target interaction, this study presents the effect of the salicylidne acylhydrazide compounds on the activity of WrbA, along with crystal structures of WrbA from Yersinia pseudotuberculosis and Salmonella serovar Typhimurium; the latter also containing parts of the compound in the structure. We also present data showing that the original compounds were unstable in acidic conditions, and that later compounds showed improved stability.

Biosynthesis of Aurodox, a Type III Secretion System Inhibitor from Streptomyces goldiniensis.

The global increase in antimicrobial-resistant infections means that there is a need to develop new antimicrobial molecules and strategies to combat the issue. Aurodox is a linear polyketide natural product that is produced by Streptomyces goldiniensis, yet little is known about aurodox biosynthesis or the nature of the biosynthetic gene cluster (BGC) that encodes its production. To gain a deeper understanding of aurodox biosynthesis by S. goldiniensis, the whole genome of the organism was sequenced, revealing the presence of an 87 kb hybrid polyketide synthase/non-ribosomal peptide synthetase (PKS/NRPS) BGC. The aurodox BGC shares significant homology with the kirromycin BGC from S. collinus Tϋ 365. However, the genetic organization of the BGC differs significantly. The candidate aurodox gene cluster was cloned and expressed in a heterologous host to demonstrate that it was responsible for aurodox biosynthesis and disruption of the primary PKS gene (aurAI) abolished aurodox production. These data supported a model whereby the initial core biosynthetic reactions involved in aurodox biosynthesis followed that of kirromycin. Cloning aurM* from S. goldiniensis and expressing this in the kirromycin producer S. collinus Tϋ 365 enabled methylation of the pyridone group, suggesting this is the last step in biosynthesis. This methylation step is also sufficient to confer the unique type III secretion system inhibitory properties to aurodox. IMPORTANCE Enterohemorrhagic Escherichia coli (EHEC) is a significant global pathogen for which traditional antibiotic treatment is not recommended. Aurodox inhibits the ability of EHEC to establish infection in the host gut through the specific targeting of the type III secretion system while circumventing the induction of toxin production associated with traditional antibiotics. These properties suggest aurodox could be a promising anti-virulence compound for EHEC, which merits further investigation. Here, we characterized the aurodox biosynthetic gene cluster from Streptomyces goldiniensis and established the key enzymatic steps of aurodox biosynthesis that give rise to the unique anti-virulence activity. These data provide the basis for future chemical and genetic approaches to produce aurodox derivatives with increased efficacy and the potential to engineer novel elfamycins.

Advances in the Design and Development of PROTAC-mediated HDAC Degradation.

Due to developments in modern chemistry, previously uundruggable substrates are now targetable thanks to selective degradation using the ubiquitin-proteasomal degradation system. PROteolysis TArgeting Chimeras (PROTACs) are heterobifunctional molecules designed specifically to degrade target proteins. They are of significant interest to industry and academia as they are highly specific and can target previously undruggable target proteins from transcription factors to enzymes. More than 15 degraders are expected to be evaluated in clinical trials by the end of 2021. Herein, we describe recent advances in the design and development of PROTAC-mediated degradation of histone deacetylases (HDACs). PROTAC-mediated degradation of HDACs can offer some significant advantages over direct inhibition, such as the use of substoichiometric doses and the potential to disrupt enzyme-independent HDAC function. We discuss the potential implication of the degradation of HDACs in comparison with HDAC knockout studies. Along with the selection of HDAC inhibitors and E3 ligase ligands for the design of PROTACs. The potential utility of HDAC PROTACs in various disease pathologies from cancer to inflammation to neurodegeneration is driving the interest in this field.

A Metabolomics approach for the diagnosis Of SecondAry InfeCtions in COVID-19 (MOSAIC): a study protocol.

BACKGROUND: Critically ill patients with COVID-19 are at an increased risk of developing secondary bacterial infections. These are both difficult to diagnose and are associated with an increased mortality. Metabolomics may aid clinicians in diagnosing secondary bacterial infections in COVID-19 through identification and quantification of disease specific biomarkers, with the aim of identifying underlying causative microorganisms and directing antimicrobial therapy. METHODS: This is a multi-centre prospective diagnostic observational study. Patients with COVID-19 will be recruited from critical care units in three Scottish hospitals. Three serial blood samples will be taken from patients, and an additional sample taken if a patient shows clinical or microbiological evidence of secondary infection. Samples will be analysed using LC-MS and subjected to bioinformatic processing and statistical analysis to explore the metabolite changes associated with bacterial infections in COVID-19 patients. Comparisons of the data sets will be made with standard microbiological and biochemical methods of diagnosing infection. DISCUSSION: Metabolomics analyses may provide additional strategies for identifying secondary infections, which might permit faster initiation of specific tailored antimicrobial therapy to critically ill patients with COVID-19.

d-Serine induces distinct transcriptomes in diverse Escherichia coli pathotypes.

Appropriate interpretation of environmental signals facilitates niche specificity in pathogenic bacteria. However, the responses of niche-specific pathogens to common host signals are poorly understood. d-Serine (d-ser) is a toxic metabolite present in highly variable concentrations at different colonization sites within the human host that we previously found is capable of inducing changes in gene expression. In this study, we made the striking observation that the global transcriptional response of three Escherichia coli pathotypes - enterohaemorrhagic E. coli (EHEC), uropathogenic E. coli (UPEC) and neonatal meningitis-associated E. coli (NMEC) - to d-ser was highly distinct. In fact, we identified no single differentially expressed gene common to all three strains. We observed the induction of ribosome-associated genes in extraintestinal pathogens UPEC and NMEC only, and the induction of purine metabolism genes in gut-restricted EHEC, and UPEC indicating distinct transcriptional responses to a common signal. UPEC and NMEC encode dsdCXA - a genetic locus required for detoxification and hence normal growth in the presence of d-ser. Specific transcriptional responses were induced in strains accumulating d-ser (WT EHEC and UPEC/NMEC mutants lacking the d-ser-responsive transcriptional activator DsdC), corroborating the notion that d-ser is an unfavourable metabolite if not metabolized. Importantly, many of the UPEC-associated transcriptome alterations correlate with published data on the urinary transcriptome, supporting the hypothesis that d-ser sensing forms a key part of urinary niche adaptation in this pathotype. Collectively, our results demonstrate distinct pleiotropic responses to a common metabolite in diverse E. coli pathotypes, with important implications for niche selectivity.

Heterogeneity in populations of enterohaemorrhagic Escherichia coli undergoing D-serine adaptation.

Phenotypic and genetic heterogeneities are conserved features of prokaryotic populations. During periods of stress, this programmed diversity increases the likelihood that variants within the population will survive the adverse conditions, allowing for proliferation. Phenotypic heterogeneity can have a mutational or indeed a non-mutational basis as observed in bet-hedging strategies adopted by antibiotic-tolerant persister cells. Genetic variants can arise by phase variation (slip-strand mispairing, promoter inversions etc.), nucleotide polymorphisms resulting from replication errors or larger rearrangements such as deletions and insertions. In the face of selective pressures, these alterations may be neutral, beneficial or deleterious.We recently described the genetic basis of tolerance to a normally toxic metabolite, D-serine (D-ser) in enterohaemorrhagic E. coli (EHEC). Here we summarize our work in the context of population dynamics, provide further discussion on the distinction between these tolerance mechanisms and the importance of heterogeneity for maximising adaptive potential.

Prokaryotic life finds a way: insights from evolutionary experimentation in bacteria.

While evolution proceeds through the generation of random variant alleles, the application of selective pressures can select for subsets of mutations that confer fitness-improving physiological benefits. This, in essence, defines the process of adaptive evolution. The rapid replication rate of bacteria has allowed for the design of experiments to study these processes over a reasonable timeframe within a laboratory setting. This has been greatly assisted by advances in tractability of diverse microorganisms, next generation sequencing technologies and bioinformatic analysis pipelines. Examining the processes by which organisms adapt their genetic code to cope with sub-optimal growth conditions has yielded a wealth of molecular insight into diverse biological processes. Here we discuss how the study of adaptive evolutionary trajectories in bacteria has allowed for improved understanding of stress responses, revealed important insight into microbial physiology, allowed for the production of highly optimised strains for use in biotechnology and increased our knowledge of the role of genomic plasticity in chronic infections.

The force awakens: The dark side of mechanosensing in bacterial pathogens.

For many bacteria, the ability to sense physical stimuli such as contact with a surface or a potential host cell is vital for survival and proliferation. This ability, and subsequent attachment, confers a wide range of benefits to bacteria and many species have evolved to take advantage of this. Despite the impressive diversity of bacterial pathogens and their virulence factors, mechanosensory mechanisms are often conserved. These include sensing impedance of flagellar rotation and resistance to type IV pili retraction. There are additional mechanisms that rely on the use of specific membrane-bound adhesins to sense either surface proximity or shear forces. This review aims to examine these mechanosensors, and how they are used by pathogenic bacteria to sense physical features in their environment. We will explore how these sensors generate and transmit signals which can trigger modulation of virulence-associated gene expression in some of the most common bacterial pathogens: Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli and Vibrio species.