A Salmonella Toxin Promotes Persister Formation through Acetylation of tRNA.

The recalcitrance of many bacterial infections to antibiotic treatment is thought to be due to the presence of persisters that are non-growing, antibiotic-insensitive cells. Eventually, persisters resume growth, accounting for relapses of infection. Salmonella is an important pathogen that causes disease through its ability to survive inside macrophages. After macrophage phagocytosis, a significant proportion of the Salmonella population forms non-growing persisters through the action of toxin-antitoxin modules. Here we reveal that one such toxin, TacT, is an acetyltransferase that blocks the primary amine group of amino acids on charged tRNA molecules, thereby inhibiting translation and promoting persister formation. Furthermore, we report the crystal structure of TacT and note unique structural features, including two positively charged surface patches that are essential for toxicity. Finally, we identify a detoxifying mechanism in Salmonella wherein peptidyl-tRNA hydrolase counteracts TacT-dependent growth arrest, explaining how bacterial persisters can resume growth.

Auxiliary interfaces support the evolution of specific toxin-antitoxin pairing.

Toxin-antitoxin (TA) systems are a large family of genes implicated in the regulation of bacterial growth and its arrest in response to attacks. These systems encode nonsecreted toxins and antitoxins that specifically pair, even when present in several paralogous copies per genome. Salmonella enterica serovar Typhimurium contains three paralogous TacAT systems that block bacterial translation. We determined the crystal structures of the three TacAT complexes to understand the structural basis of specific TA neutralization and the evolution of such specific pairing. In the present study, we show that alteration of a discrete structural add-on element on the toxin drives specific recognition by their cognate antitoxin underpinning insulation of the three pairs. Similar to other TA families, the region supporting TA-specific pairing is key to neutralization. Our work reveals that additional TA interfaces beside the main neutralization interface increase the safe space for evolution of pairing specificity.

The Salmonella Effector SpvD Is a Cysteine Hydrolase with a Serovar-specific Polymorphism Influencing Catalytic Activity, Suppression of Immune Responses, and Bacterial Virulence.

Many bacterial pathogens secrete virulence (effector) proteins that interfere with immune signaling in their host. SpvD is a Salmonella enterica effector protein that we previously demonstrated to negatively regulate the NF-κB signaling pathway and promote virulence of S. enterica serovar Typhimurium in mice. To shed light on the mechanistic basis for these observations, we determined the crystal structure of SpvD and show that it adopts a papain-like fold with a characteristic cysteine-histidine-aspartate catalytic triad comprising Cys-73, His-162, and Asp-182. SpvD possessed an in vitro deconjugative activity on aminoluciferin-linked peptide and protein substrates in vitro A C73A mutation abolished SpvD activity, demonstrating that an intact catalytic triad is required for its function. Taken together, these results strongly suggest that SpvD is a cysteine protease. The amino acid sequence of SpvD is highly conserved across different S. enterica serovars, but residue 161, located close to the catalytic triad, is variable, with serovar Typhimurium SpvD having an arginine and serovar Enteritidis a glycine at this position. This variation affected hydrolytic activity of the enzyme on artificial substrates and can be explained by substrate accessibility to the active site. Interestingly, the SpvDG161 variant more potently inhibited NF-κB-mediated immune responses in cells in vitro and increased virulence of serovar Typhimurium in mice. In summary, our results explain the biochemical basis for the effect of virulence protein SpvD and demonstrate that a single amino acid polymorphism can affect the overall virulence of a bacterial pathogen in its host.

Diverse structural approaches to haem appropriation by pathogenic bacteria.

The critical need for iron presents a challenge for pathogenic bacteria that must survive in an environment bereft of accessible iron due to a natural low bioavailability and their host's nutritional immunity. Appropriating haem, either direct from host haemoproteins or by secreting haem-scavenging haemophores, is one way pathogenic bacteria can overcome this challenge. After capturing their target, haem appropriation systems must remove haem from a high-affinity binding site (on the host haemoprotein or bacterial haemophore) and transfer it to a binding site of lower affinity on a bacterial receptor. Structural information is now available to show how, using a combination of induced structural changes and steric clashes, bacteria are able to extract haem from haemophores, haemopexin and haemoglobin. This review focuses on structural descriptions of these bacterial haem acquisition systems, summarising how they bind haem and their target haemoproteins with particularly emphasis on the mechanism of haem extraction.

Molecular stripping underpins derepression of a toxin-antitoxin system.

Transcription factors control gene expression; among these, transcriptional repressors must liberate the promoter for derepression to occur. Toxin-antitoxin (TA) modules are bacterial elements that autoregulate their transcription by binding the promoter in a T:A ratio-dependent manner, known as conditional cooperativity. The molecular basis of how excess toxin triggers derepression has remained elusive, largely because monitoring the rearrangement of promoter-repressor complexes, which underpin derepression, is challenging. Here, we dissect the autoregulation of the Salmonella enterica tacAT3 module. Using a combination of assays targeting DNA binding and promoter activity, as well as structural characterization, we determine the essential TA and DNA elements required to control transcription, and we reconstitute a repression-to-derepression path. We demonstrate that excess toxin triggers molecular stripping of the repressor complex off the DNA through multiple allosteric changes causing DNA distortion and ultimately leading to derepression. Thus, our work provides important insight into the mechanisms underlying conditional cooperativity.

Pirates of the haemoglobin.

Not all treasure is silver and gold; for pathogenic bacteria, iron is the most precious and the most pillaged of metallic elements. Iron is essential for the survival and growth of all life; however free iron is scarce for bacteria inside human hosts. As a mechanism of defence, humans have evolved ways to store iron so as to render it inaccessible for invading pathogens, such as keeping the metal bound to iron-carrying proteins. For bacteria to survive within humans, they must therefore evolve counters to this defence to compete with these proteins for iron binding, or directly steal iron from them. The most populous form of iron in humans is haem: a functionally significant coordination complex that is central to oxygen transport and predominantly bound by haemoglobin. Haemoglobin is therefore the largest source of iron in humans and, as a result, bacterial pathogens in critical need of iron have evolved complex and creative ways to acquire haem from haemoglobin. Bacteria of all cell wall types have the ability to bind haemoglobin at their cell surface, to accept the haem from it and transport this to the cytoplasm for downstream uses. This review describes the systems employed by various pathogenic bacteria to utilise haemoglobin as an iron source within human hosts and discusses their contribution to virulence.

Activity of acetyltransferase toxins involved in Salmonella persister formation during macrophage infection.

Non-typhoidal Salmonella strains are responsible for invasive infections associated with high mortality and recurrence in sub-Saharan Africa, and there is strong evidence for clonal relapse following antibiotic treatment. Persisters are non-growing bacteria that are thought to be responsible for the recalcitrance of many infections to antibiotics. Toxin-antitoxin systems are stress-responsive elements that are important for Salmonella persister formation, specifically during infection. Here, we report the analysis of persister formation of clinical invasive strains of Salmonella Typhimurium and Enteritidis in human primary macrophages. We show that all the invasive clinical isolates of both serovars that we tested produce high levels of persisters following internalization by human macrophages. Our genome comparison reveals that S. Enteritidis and S. Typhimurium strains contain three acetyltransferase toxins that we characterize structurally and functionally. We show that all induce the persister state by inhibiting translation through acetylation of aminoacyl-tRNAs. However, they differ in their potency and target partially different subsets of aminoacyl-tRNAs, potentially accounting for their non-redundant effect.

The development of novel LTA4H modulators to selectively target LTB4 generation.

The pro-inflammatory mediator leukotriene B4 (LTB4) is implicated in the pathologies of an array of diseases and thus represents an attractive therapeutic target. The enzyme leukotriene A4 hydrolase (LTA4H) catalyses the distal step in LTB4 synthesis and hence inhibitors of this enzyme have been actively pursued. Despite potent LTA4H inhibitors entering clinical trials all have failed to show efficacy. We recently identified a secondary anti-inflammatory role for LTA4H in degrading the neutrophil chemoattractant Pro-Gly-Pro (PGP) and rationalized that the failure of conventional LTA4H inhibitors may be that they inadvertently prevented PGP degradation. We demonstrate that these inhibitors do indeed fail to discriminate between the dual activities of LTA4H, and enable PGP accumulation in mice. Accordingly, we have developed novel compounds that potently inhibit LTB4 generation whilst leaving PGP degradation unperturbed. These novel compounds could represent a safer and superior class of LTA4H inhibitors for translation into the clinic.

Structures of the DfsB Protein Family Suggest a Cationic, Helical Sibling Lethal Factor Peptide.

Bacteria have developed a variety of mechanisms for surviving harsh environmental conditions, nutrient stress and overpopulation. Paenibacillus dendritiformis produces a lethal protein (Slf) that is able to induce cell death in neighbouring colonies and a phenotypic switch in more distant ones. Slf is derived from the secreted precursor protein, DfsB, after proteolytic processing. Here, we present new crystal structures of DfsB homologues from a variety of bacterial species and a surprising version present in the yeast Saccharomyces cerevisiae. Adopting a four-helix bundle decorated with a further three short helices within intervening loops, DfsB belongs to a non-enzymatic class of the DinB fold. The structure suggests that the biologically active Slf fragment may possess a C-terminal helix rich in basic and aromatic residues that suggest a functional mechanism akin to that for cationic antimicrobial peptides.

Purification, crystallization and characterization of the Pseudomonas outer membrane protein FapF, a functional amyloid transporter.

Bacteria often produce extracellular amyloid fibres via a multi-component secretion system. Aggregation-prone, unstructured subunits cross the periplasm and are secreted through the outer membrane, after which they self-assemble. Here, significant progress is presented towards solving the high-resolution crystal structure of the novel amyloid transporter FapF from Pseudomonas, which facilitates the secretion of the amyloid-forming polypeptide FapC across the bacterial outer membrane. This represents the first step towards obtaining structural insight into the products of the Pseudomonas fap operon. Initial attempts at crystallizing full-length and N-terminally truncated constructs by refolding techniques were not successful; however, after preparing FapF106-430 from the membrane fraction, reproducible crystals were obtained using the sitting-drop method of vapour diffusion. Diffraction data have been processed to 2.5 Šresolution. These crystals belonged to the monoclinic space group C121, with unit-cell parameters a = 143.4, b = 124.6, c = 80.4 Å, α = γ = 90, ß = 96.32° and three monomers in the asymmetric unit. It was found that the switch to complete detergent exchange into C8E4 was crucial for forming well diffracting crystals, and it is suggested that this combined with limited proteolysis is a potentially useful protocol for membrane ß-barrel protein crystallography. The three-dimensional structure of FapF will provide invaluable information on the mechanistic differences of biogenesis between the curli and Fap functional amyloid systems.

Structural analysis of haemoglobin binding by HpuA from the Neisseriaceae family.

The Neisseriaceae family of bacteria causes a range of diseases including meningitis, septicaemia, gonorrhoea and endocarditis, and extracts haem from haemoglobin as an important iron source within the iron-limited environment of its human host. Herein we report crystal structures of apo- and haemoglobin-bound HpuA, an essential component of this haem import system. The interface involves long loops on the bacterial receptor that present hydrophobic side chains for packing against the surface of haemoglobin. Interestingly, our structural and biochemical analyses of Kingella denitrificans and Neisseria gonorrhoeae HpuA mutants, although validating the interactions observed in the crystal structure, show how Neisseriaceae have the fascinating ability to diversify functional sequences and yet retain the haemoglobin binding function. Our results present the first description of HpuA's role in direct binding of haemoglobin.