A Pseudomonas aeruginosa small RNA regulates chronic and acute infection.

The ability to switch between different lifestyles allows bacterial pathogens to thrive in diverse ecological niches1,2. However, a molecular understanding of their lifestyle changes within the human host is lacking. Here, by directly examining bacterial gene expression in human-derived samples, we discover a gene that orchestrates the transition between chronic and acute infection in the opportunistic pathogen Pseudomonas aeruginosa. The expression level of this gene, here named sicX, is the highest of the P. aeruginosa genes expressed in human chronic wound and cystic fibrosis infections, but it is expressed at extremely low levels during standard laboratory growth. We show that sicX encodes a small RNA that is strongly induced by low-oxygen conditions and post-transcriptionally regulates anaerobic ubiquinone biosynthesis. Deletion of sicX causes P. aeruginosa to switch from a chronic to an acute lifestyle in multiple mammalian models of infection. Notably, sicX is also a biomarker for this chronic-to-acute transition, as it is the most downregulated gene when a chronic infection is dispersed to cause acute septicaemia. This work solves a decades-old question regarding the molecular basis underlying the chronic-to-acute switch in P. aeruginosa and suggests oxygen as a primary environmental driver of acute lethality.

Convergent evolution in toxin detection and resistance provides evidence for conserved bacterial-fungal interactions.

Microbes rarely exist in isolation and instead form complex polymicrobial communities. As a result, microbes have developed intricate offensive and defensive strategies that enhance their fitness in these complex communities. Thus, identifying and understanding the molecular mechanisms controlling polymicrobial interactions is critical for understanding the function of microbial communities. In this study, we show that the gram-negative opportunistic human pathogen Pseudomonas aeruginosa, which frequently causes infection alongside a plethora of other microbes including fungi, encodes a genetic network which can detect and defend against gliotoxin, a potent, disulfide-containing antimicrobial produced by the ubiquitous filamentous fungus Aspergillus fumigatus. We show that gliotoxin exposure disrupts P. aeruginosa zinc homeostasis, leading to transcriptional activation of a gene encoding a previously uncharacterized dithiol oxidase (herein named as DnoP), which detoxifies gliotoxin and structurally related toxins. Despite sharing little homology to the A. fumigatus gliotoxin resistance protein (GliT), the enzymatic mechanism of DnoP from P. aeruginosa appears to be identical that used by A. fumigatus. Thus, DnoP and its transcriptional induction by low zinc represent a rare example of both convergent evolution of toxin defense and environmental cue sensing across kingdoms. Collectively, these data provide compelling evidence that P. aeruginosa has evolved to survive exposure to an A. fumigatus disulfide-containing toxin in the natural environment.

The Glyoxylate Shunt, 60 Years On.

2017 marks the 60th anniversary of Krebs' seminal paper on the glyoxylate shunt (and coincidentally, also the 80th anniversary of his discovery of the citric acid cycle). Sixty years on, we have witnessed substantial developments in our understanding of how flux is partitioned between the glyoxylate shunt and the oxidative decarboxylation steps of the citric acid cycle. The last decade has shown us that the beautifully elegant textbook mechanism that regulates carbon flux through the shunt in E. coli is an oversimplification of the situation in many other bacteria. The aim of this review is to assess how this new knowledge is impacting our understanding of flux control at the TCA cycle/glyoxylate shunt branch point in a wider range of genera, and to summarize recent findings implicating a role for the glyoxylate shunt in cellular functions other than metabolism.

Structure-Based Discovery of Lipoteichoic Acid Synthase Inhibitors.

Lipoteichoic acid synthase (LtaS) is a key enzyme for the cell wall biosynthesis of Gram-positive bacteria. Gram-positive bacteria that lack lipoteichoic acid (LTA) exhibit impaired cell division and growth defects. Thus, LtaS appears to be an attractive antimicrobial target. The pharmacology around LtaS remains largely unexplored with only two small-molecule LtaS inhibitors reported, namely "compound 1771" and the Congo red dye. Structure-based drug discovery efforts against LtaS remain unattempted due to the lack of an inhibitor-bound structure of LtaS. To address this, we combined the use of a molecular docking technique with molecular dynamics (MD) simulations to model a plausible binding mode of compound 1771 to the extracellular catalytic domain of LtaS (eLtaS). The model was validated using alanine mutagenesis studies combined with isothermal titration calorimetry. Additionally, lead optimization driven by our computational model resulted in an improved version of compound 1771, namely, compound 4 which showed greater affinity for binding to eLtaS than compound 1771 in biophysical assays. Compound 4 reduced LTA production in S. aureus dose-dependently, induced aberrant morphology as seen for LTA-deficient bacteria, and significantly reduced bacteria titers in the lung of mice infected with S. aureus. Analysis of our MD simulation trajectories revealed the possible formation of a transient cryptic pocket in eLtaS. Virtual screening (VS) against the cryptic pocket led to the identification of a new class of inhibitors that could potentiate ß-lactams against methicillin-resistant S. aureus. Our overall workflow and data should encourage further drug design campaign against LtaS. Finally, our work reinforces the importance of considering protein conformational flexibility to a successful VS endeavor.

Blastomycosis in solid organ transplant recipients-A retrospective series from southeastern Wisconsin.

Blastomycosis is a fungal infection caused primarily by Blastomyces dermatitis. The fungus is endemic to the Ohio, Mississippi, and St. Lawrence River areas of the United States. Organ transplant recipients are at risk of blastomycosis due to pharmacologic immunosuppression. Over a 20-year period, 30 cases of blastomycosis post-solid organ transplantation were identified at our center. The cumulative incidence of blastomycosis among SOT recipients was 0.99%. There was a male predominance (70% male) and a median age of 59 at the time of diagnosis. Regarding transplant type, 23 patients received kidney transplants, 4 received liver transplants, 2 received pancreas transplants and 1 received a heart transplant. Median time to blastomycosis identification post-transplant was 67.8 months (range: 1-188 months). Amphotericin B was used as initiation therapy in most cases, followed by itraconazole, voriconazole, or in select cases fluconazole or posaconazole maintenance therapy. Regarding comorbid conditions, 87% of patients had diabetes, 50% had congestive heart failure, and 20% had chronic pulmonary disease. Nine patients (30%) developed blastomycosis-related acute respiratory distress syndrome, 33% of these died with a median time to death of 22 days (range 20 days to 2 months); these were the only deaths attributable to blastomycosis.

Gluconeogenic precursor availability regulates flux through the glyoxylate shunt in Pseudomonas aeruginosa.

The glyoxylate shunt bypasses the oxidative decarboxylation steps of the tricarboxylic acid (TCA) cycle, thereby conserving carbon skeletons for gluconeogenesis and biomass production. In Escherichia coli, carbon flux is redirected through the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), following phosphorylation and inactivation of the TCA cycle enzyme, isocitrate dehydrogenase (ICD), by the kinase/phosphatase, AceK. In contrast, mycobacterial species lack AceK and employ a phosphorylation-insensitive isocitrate dehydrogenase (IDH), which is allosterically activated by the product of ICL activity, glyoxylate. However, Pseudomonas aeruginosa expresses IDH, ICD, ICL, and AceK, raising the question of how these enzymes are regulated to ensure proper flux distribution between the competing pathways. Here, we present the structure, kinetics, and regulation of ICL, IDH, and ICD from P. aeruginosa We found that flux partitioning is coordinated through reciprocal regulation of these enzymes, linking distribution of carbon flux to the availability of the key gluconeogenic precursors, oxaloacetate and pyruvate. Specifically, a greater abundance of these metabolites activated IDH and inhibited ICL, leading to increased TCA cycle flux. Regulation was also exerted through AceK-dependent phosphorylation of ICD; high levels of acetyl-CoA (which would be expected to accumulate when oxaloacetate is limiting) stimulated the kinase activity of AceK, whereas high levels of oxaloacetate stimulated its phosphatase activity. In summary, the TCA cycle-glyoxylate shunt branch point in P. aeruginosa has a complex enzymology that is profoundly different from those in other species characterized to date. Presumably, this reflects its predilection for consuming fatty acids, especially during infection scenarios.

Loving the poison: the methylcitrate cycle and bacterial pathogenesis.

Propionate is an abundant catabolite in nature and represents a rich potential source of carbon for the organisms that can utilize it. However, propionate and propionate-derived catabolites are also toxic to cells, so propionate catabolism can alternatively be viewed as a detoxification mechanism. In this review, we summarize recent progress made in understanding how prokaryotes catabolize propionic acid, how these pathways are regulated and how they might be exploited to develop novel antibacterial interventions.

Aspergillus fumigatus protein phosphatase PpzA is involved in iron assimilation, secondary metabolite production, and virulence.

Metal restriction imposed by mammalian hosts during an infection is a common mechanism of defence to reduce or avoid the pathogen infection. Metals are essential for organism survival due to its involvement in several biological processes. Aspergillus fumigatus causes invasive aspergillosis, a disease that typically manifests in immunocompromised patients. A. fumigatus PpzA, the catalytic subunit of protein phosphatase Z (PPZ), has been recently identified as associated with iron assimilation. A. fumigatus has 2 high-affinity mechanisms of iron acquisition during infection: reductive iron assimilation and siderophore-mediated iron uptake. It has been shown that siderophore production is important for A. fumigatus virulence, differently to the reductive iron uptake system. Transcriptomic and proteomic comparisons between ∆ppzA and wild-type strains under iron starvation showed that PpzA has a broad influence on genes involved in secondary metabolism. Liquid chromatography-mass spectrometry under standard and iron starvation conditions confirmed that the ΔppzA mutant had reduced production of pyripyropene A, fumagillin, fumiquinazoline A, triacetyl-fusarinine C, and helvolic acid. The ΔppzA was shown to be avirulent in a neutropenic murine model of invasive pulmonary aspergillosis. PpzA plays an important role at the interface between iron starvation, regulation of SM production, and pathogenicity in A. fumigatus.

Transcriptomics technologies.

Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst noncoding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. The first attempts to study the whole transcriptome began in the early 1990s, and technological advances since the late 1990s have made transcriptomics a widespread discipline. Transcriptomics has been defined by repeated technological innovations that transform the field. There are two key contemporary techniques in the field: microarrays, which quantify a set of predetermined sequences, and RNA sequencing (RNA-Seq), which uses high-throughput sequencing to capture all sequences. Measuring the expression of an organism's genes in different tissues, conditions, or time points gives information on how genes are regulated and reveals details of an organism's biology. It can also help to infer the functions of previously unannotated genes. Transcriptomic analysis has enabled the study of how gene expression changes in different organisms and has been instrumental in the understanding of human disease. An analysis of gene expression in its entirety allows detection of broad coordinated trends which cannot be discerned by more targeted assays.

Discovery of novel fragments inhibiting O-acetylserine sulphhydrylase by combining scaffold hopping and ligand-based drug design.

Several bacteria rely on the reductive sulphur assimilation pathway, absent in mammals, to synthesise cysteine. Reduction of virulence and decrease in antibiotic resistance have already been associated with mutations on the genes that codify cysteine biosynthetic enzymes. Therefore, inhibition of cysteine biosynthesis has emerged as a promising strategy to find new potential agents for the treatment of bacterial infection. Following our previous efforts to explore OASS inhibition and to expand and diversify our library, a scaffold hopping approach was carried out, with the aim of identifying a novel fragment for further development. This novel chemical tool, endowed with favourable pharmacological characteristics, was successfully developed, and a preliminary Structure-Activity Relationship investigation was carried out.

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence.

The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways.

Interplay between Gliotoxin Resistance, Secretion, and the Methyl/Methionine Cycle in Aspergillus fumigatus.

Mechanistic studies on gliotoxin biosynthesis and self-protection in Aspergillus fumigatus, both of which require the gliotoxin oxidoreductase GliT, have revealed a rich landscape of highly novel biochemistries, yet key aspects of this complex molecular architecture remain obscure. Here we show that an A. fumigatus ΔgliA strain is completely deficient in gliotoxin secretion but still retains the ability to efflux bisdethiobis(methylthio)gliotoxin (BmGT). This correlates with a significant increase in sensitivity to exogenous gliotoxin because gliotoxin trapped inside the cell leads to (i) activation of the gli cluster, as disabling gli cluster activation, via gliZ deletion, attenuates the sensitivity of an A. fumigatus ΔgliT strain to gliotoxin, thus implicating cluster activation as a factor in gliotoxin sensitivity, and (ii) increased methylation activity due to excess substrate (dithiol gliotoxin) for the gliotoxin bis-thiomethyltransferase GtmA. Intracellular dithiol gliotoxin is oxidized by GliT and subsequently effluxed by GliA. In the absence of GliA, gliotoxin persists in the cell and is converted to BmGT, with levels significantly higher than those in the wild type. Similarly, in the ΔgliT strain, gliotoxin oxidation is impeded, and methylation occurs unchecked, leading to significant S-adenosylmethionine (SAM) depletion and S-adenosylhomocysteine (SAH) overproduction. This in turn significantly contributes to the observed hypersensitivity of gliT-deficient A. fumigatus to gliotoxin. Our observations reveal a key role for GliT in preventing dysregulation of the methyl/methionine cycle to control intracellular SAM and SAH homeostasis during gliotoxin biosynthesis and exposure. Moreover, we reveal attenuated GliT abundance in the A. fumigatus ΔgliK strain, but not the ΔgliG strain, following exposure to gliotoxin, correlating with relative sensitivities. Overall, we illuminate new systems interactions that have evolved in gliotoxin-producing, compared to gliotoxin-naive, fungi to facilitate their cellular presence.

Why should early-career scientists publish in society journals.

In this editorial, written by early-career scientists, we advocate for the invaluable role of society journals in our scientific community. By choosing to support these journals as authors, peer reviewers, and as editors, we can reinforce our academic growth and benefit from their re-investment back into the scientific ecosystem. Considering the numerous clear merits of this system for future generations of microbiologists and more broadly, society, we argue that early-career researchers should publish our high-quality research in society journals to shape the future of science and scientific publishing landscape.

The inaugural mBio Junior Editorial Board-lessons learned and the path forward toward improving the peer review process.

The inaugural Junior Editorial Board (JEB) of mBio consisted of 64 early-career researchers active from 2022 to 2023. The goal of the JEB was to train early-career researchers in the art of peer review under the guidance of experienced editors. JEB members gained hands-on experience in peer review by participating in modules detailing the publishing process through the lenses of the journal, editor, and reviewer. Ultimately, JEB members applied this new knowledge by reviewing mBio manuscripts. Here, we summarize the background, the mission, and the achievements of the first mBio JEB. We also include possible trajectories for the future editions of this important program.

The Aspergillus fumigatus protein GliK protects against oxidative stress and is essential for gliotoxin biosynthesis.

The function of a number of genes in the gliotoxin biosynthetic cluster (gli) in Aspergillus fumigatus remains unknown. Here, we demonstrate that gliK deletion from two strains of A. fumigatus completely abolished gliotoxin biosynthesis. Furthermore, exogenous H(2)O(2) (1 mM), but not gliotoxin, significantly induced A. fumigatus gliK expression (P = 0.0101). While both mutants exhibited significant sensitivity to both exogenous gliotoxin (P < 0.001) and H(2)O(2) (P < 0.01), unexpectedly, exogenous gliotoxin relieved H(2)O(2)-induced growth inhibition in a dose-dependent manner (0 to 10 µg/ml). Gliotoxin-containing organic extracts derived from A. fumigatus ATCC 26933 significantly inhibited (P < 0.05) the growth of the ΔgliK(26933) deletion mutant. The A. fumigatus ΔgliK(26933) mutant secreted metabolites, devoid of disulfide linkages or free thiols, that were detectable by reverse-phase high-performance liquid chromatography and liquid chromatography-mass spectrometry with m/z 394 to 396. These metabolites (m/z 394 to 396) were present at significantly higher levels in the culture supernatants of the A. fumigatus ΔgliK(26933) mutant than in those of the wild type (P = 0.0024 [fold difference, 24] and P = 0.0003 [fold difference, 9.6], respectively) and were absent from A. fumigatus ΔgliG. Significantly elevated levels of ergothioneine were present in aqueous mycelial extracts of the A. fumigatus ΔgliK(26933) mutant compared to the wild type (P < 0.001). Determination of the gliotoxin uptake rate revealed a significant difference (P = 0.0045) between that of A. fumigatus ATCC 46645 (9.3 pg/mg mycelium/min) and the ΔgliK(46645) mutant (31.4 pg/mg mycelium/min), strongly suggesting that gliK absence and the presence of elevated ergothioneine levels impede exogenously added gliotoxin efflux. Our results confirm a role for gliK in gliotoxin biosynthesis and reveal new insights into gliotoxin functionality in A. fumigatus.

Illuminating Siderophore Transporter Functionality with Thiopeptide Antibiotics.

The Gram-negative opportunistic pathogen Pseudomonas aeruginosa is a leading cause of infections and mortality in immunocompromised patients. This organism can overcome iron deprivation during infection via the synthesis of two iron-chelating siderophores, pyoverdine and pyochelin, which scavenge iron from host proteins. P. aeruginosa can also uptake xenosiderophores produced by other bacteria or fungi using dedicated transporter systems. The precise substrate specificity of these siderophore transporters remains to be determined. The thiopeptide antibiotic thiostrepton exploits the pyoverdine transporters FpvA and FpvB to cross the outer membrane and reach intracellular targets. Using a series of intricate biochemical experiments, a recent study by Chan and Burrows capitalized on the specificity of thiostrepton to uncover that FpvB transports the xenosiderophores ferrichrome and ferrioxamine B with higher affinity than pyoverdine. This surprising result highlights an alternative uptake pathway for these siderophores and has significant implications for our understanding of iron acquisition in this organism.

Impact of Growth Rate on the Protein-mRNA Ratio in Pseudomonas aeruginosa.

Our understanding of how bacterial pathogens colonize and persist during human infection has been hampered by the limited characterization of bacterial physiology during infection and a research bias toward in vitro, fast-growing bacteria. Recent research has begun to address these gaps in knowledge by directly quantifying bacterial mRNA levels during human infection, with the goal of assessing microbial community function at the infection site. However, mRNA levels are not always predictive of protein levels, which are the primary functional units of a cell. Here, we used carefully controlled chemostat experiments to examine the relationship between mRNA and protein levels across four growth rates in the bacterial pathogen Pseudomonas aeruginosa. We found a genome-wide positive correlation between mRNA and protein abundances across all growth rates, with genes required for P. aeruginosa viability having stronger correlations than nonessential genes. We developed a statistical method to identify genes whose mRNA abundances poorly predict protein abundances and calculated an RNA-to-protein (RTP) conversion factor to improve mRNA predictions of protein levels. The application of the RTP conversion factor to publicly available transcriptome data sets was highly robust, enabling the more accurate prediction of P. aeruginosa protein levels across strains and growth conditions. Finally, the RTP conversion factor was applied to P. aeruginosa human cystic fibrosis (CF) infection transcriptomes to provide greater insights into the functionality of this bacterium in the CF lung. This study addresses a critical problem in infection microbiology by providing a framework for enhancing the functional interpretation of bacterial human infection transcriptome data. IMPORTANCE Our understanding of bacterial physiology during human infection is limited by the difficulty in assessing bacterial function at the infection site. Recent studies have begun to address this question by quantifying bacterial mRNA levels in human-derived samples using transcriptomics. One challenge for these studies is the poor predictivity of mRNA for protein levels for some genes. Here, we addressed this challenge by measuring the transcriptomes and proteomes of P. aeruginosa grown at four growth rates. Our results revealed that the growth rate does not impact the genome-wide correlation of mRNA and protein levels. We used statistical methods to identify the genes for which mRNA and protein were poorly correlated and developed an RNA-to-protein (RTP) conversion factor that improved the predictivity of protein levels across strains and growth conditions. Our results provide new insights into mRNA-protein correlations and tools to enhance our understanding of bacterial physiology from transcriptome data.

Tools of the trade: plasmid repositories and standardized plasmid manipulation for molecular and synthetic biology.

Plasmids are extrachromosomal genetic elements capable of autonomous replication within a host cell. They play a key role in bacterial ecology and evolution, facilitating the mobilization of accessory genes by horizontal gene transfer. Crucially, plasmids also serve as valuable tools in modern molecular biology. Here, we highlight recent articles aimed at implementing standardized plasmid assembly techniques and plasmid repositories to promote open science as well as to improve experimental reproducibility across laboratories. Research focused on assisting these fundamental aims is a further step towards improving standardization in molecular and synthetic biology.

Systems-Wide Dissection of Organic Acid Assimilation in Pseudomonas aeruginosa Reveals a Novel Path To Underground Metabolism.

The human pathogen Pseudomonas aeruginosa (Pa) is one of the most frequent and severe causes of nosocomial infection. This organism is also a major cause of airway infections in people with cystic fibrosis (CF). Pa is known to have a remarkable metabolic plasticity, allowing it to thrive under diverse environmental conditions and ecological niches; yet, little is known about the central metabolic pathways that sustain its growth during infection or precisely how these pathways operate. In this work, we used a combination of 'omics approaches (transcriptomics, proteomics, metabolomics, and 13C-fluxomics) and reverse genetics to provide systems-level insight into how the infection-relevant organic acids succinate and propionate are metabolized by Pa. Moreover, through structural and kinetic analysis of the 2-methylcitrate synthase (2-MCS; PrpC) and its paralogue citrate (CIT) synthase (GltA), we show how these two crucial enzymatic steps are interconnected in Pa organic acid assimilation. We found that Pa can rapidly adapt to the loss of GltA function by acquiring mutations in a transcriptional repressor, which then derepresses prpC expression. Our findings provide a clear example of how "underground metabolism," facilitated by enzyme substrate promiscuity, "rewires" Pa metabolism, allowing it to overcome the loss of a crucial enzyme. This pathogen-specific knowledge is critical for the advancement of a model-driven framework to target bacterial central metabolism. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen that, due to its unrivalled resistance to antibiotics, ubiquity in the built environment, and aggressiveness in infection scenarios, has acquired the somewhat dubious accolade of being designated a "critical priority pathogen" by the WHO. In this work, we uncover the pathways and mechanisms used by P. aeruginosa to grow on a substrate that is abundant at many infection sites: propionate. We found that if the organism is prevented from metabolizing propionate, the substrate turns from being a convenient nutrient source into a potent poison, preventing bacterial growth. We further show that one of the enzymes involved in these reactions, 2-methylcitrate synthase (PrpC), is promiscuous and can moonlight for another essential enzyme in the cell (citrate synthase). Indeed, mutations that abolish citrate synthase activity (which would normally prevent the cell from growing) can be readily overcome if the cell acquires additional mutations that increase the expression of PrpC. This is a nice example of the evolutionary utility of so-called "underground metabolism."

Cysteamine Inhibits Glycine Utilisation and Disrupts Virulence in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a major opportunistic human pathogen which employs a myriad of virulence factors. In people with cystic fibrosis (CF) P. aeruginosa frequently colonises the lungs and becomes a chronic infection that evolves to become less virulent over time, but often adapts to favour persistence in the host with alginate-producing mucoid, slow-growing, and antibiotic resistant phenotypes emerging. Cysteamine is an endogenous aminothiol which has been shown to prevent biofilm formation, reduce phenazine production, and potentiate antibiotic activity against P. aeruginosa, and has been investigated in clinical trials as an adjunct therapy for pulmonary exacerbations of CF. Here we demonstrate (for the first time in a prokaryote) that cysteamine prevents glycine utilisation by P. aeruginosa in common with previously reported activity blocking the glycine cleavage system in human cells. Despite the clear inhibition of glycine metabolism, cysteamine also inhibits hydrogen cyanide (HCN) production by P. aeruginosa, suggesting a direct interference in the regulation of virulence factor synthesis. Cysteamine impaired chemotaxis, lowered pyocyanin, pyoverdine and exopolysaccharide production, and reduced the toxicity of P. aeruginosa secreted factors in a Galleria mellonella infection model. Thus, cysteamine has additional potent anti-virulence properties targeting P. aeruginosa, further supporting its therapeutic potential in CF and other infections.