Heat shock potentiates aminoglycosides against gram-negative bacteria by enhancing antibiotic uptake, protein aggregation, and ROS.

The potentiation of antibiotics is a promising strategy for combatting antibiotic-resistant/tolerant bacteria. Herein, we report that a 5-min sublethal heat shock enhances the bactericidal actions of aminoglycoside antibiotics by six orders of magnitude against both exponential- and stationary-phase Escherichia coli. This combined treatment also effectively kills various E. coli persisters, E. coli clinical isolates, and numerous gram-negative but not gram-positive bacteria and enables aminoglycosides at 5% of minimum inhibitory concentrations to eradicate multidrug-resistant pathogens Acinetobacter baumannii and Klebsiella pneumoniae. Mechanistically, the potentiation is achieved comprehensively by heat shock-enhanced proton motive force that thus promotes the bacterial uptake of aminoglycosides, as well as by increasing irreversible protein aggregation and reactive oxygen species that further augment the downstream lethality of aminoglycosides. Consistently, protonophores, chemical chaperones, antioxidants, and anaerobic culturing abolish heat shock-enhanced aminoglycoside lethality. We also demonstrate as a proof of concept that infrared irradiation- or photothermal nanosphere-induced thermal treatments potentiate aminoglycoside killing of Pseudomonas aeruginosa in a mouse acute skin wound model. Our study advances the understanding of the mechanism of actions of aminoglycosides and demonstrates a high potential for thermal ablation in curing bacterial infections when combined with aminoglycosides.

Conformational change as a mechanism for toxin activation in bacterial toxin-antitoxin systems.

Toxin/antitoxin (TA) systems are present in nearly every prokaryotic genome and play the important physiological roles of phage inhibition by reducing metabolism (this includes persistence for the extreme case of complete cessation of metabolism), genetic element stabilization, and biofilm formation. TA systems have also been incorporated into other cell systems, such as CRISPR-Cas and phage quorum sensing. For the simplest and best-studied case, proteinaceous toxins and antitoxins (i.e., type II), toxin activity is masked by direct binding of the antitoxin. A long-standing, unresolved question in the TA field is how toxins are activated when bound to antitoxins at nanomolar affinity. The current paradigm envisions preferential degradation of the antitoxin by a protease, but this is highly unlikely in that a protease cannot discriminate between bound toxin and bound antitoxin because both are highly structured. Strikingly, recent results from several studies show one likely mechanism for toxin activation is conformational changes in the TA complex that result in the release or activation of the toxin as a result of a protein trigger, such as that from phages, and as a result of thermally-driven refolding dynamics.

Conjugative plasmid-encoded toxin-antitoxin system PrpT/PrpA directly controls plasmid copy number.

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Tryptophan-metabolizing gut microbes regulate adult neurogenesis via the aryl hydrocarbon receptor.

While modulatory effects of gut microbes on neurological phenotypes have been reported, the mechanisms remain largely unknown. Here, we demonstrate that indole, a tryptophan metabolite produced by tryptophanase-expressing gut microbes, elicits neurogenic effects in the adult mouse hippocampus. Neurogenesis is reduced in germ-free (GF) mice and in GF mice monocolonized with a single-gene tnaA knockout (KO) mutant Escherichia coli unable to produce indole. External administration of systemic indole increases adult neurogenesis in the dentate gyrus in these mouse models and in specific pathogen-free (SPF) control mice. Indole-treated mice display elevated synaptic markers postsynaptic density protein 95 and synaptophysin, suggesting synaptic maturation effects in vivo. By contrast, neurogenesis is not induced by indole in aryl hydrocarbon receptor KO (AhR-/-) mice or in ex vivo neurospheres derived from them. Neural progenitor cells exposed to indole exit the cell cycle, terminally differentiate, and mature into neurons that display longer and more branched neurites. These effects are not observed with kynurenine, another AhR ligand. The indole-AhR-mediated signaling pathway elevated the expression of ß-catenin, Neurog2, and VEGF-α genes, thus identifying a molecular pathway connecting gut microbiota composition and their metabolic function to neurogenesis in the adult hippocampus. Our data have implications for the understanding of mechanisms of brain aging and for potential next-generation therapeutic opportunities.

Emerging applications of bacteria as antitumor agents.

Bacteria are associated with the human body and colonize the gut, skin, and mucous membranes. These associations can be either symbiotic or pathogenic. In either case, bacteria derive more benefit from their host. The ability of bacteria to enter and survive within the human body can be exploited for human benefit. They can be used as a vehicle for delivering or producing bioactive molecules, such as toxins and lytic enzymes, and eventually for killing tumor cells. Clostridium and Salmonella have been shown to infect and survive within the human body, including in tumors. There is a need to develop genetic circuits, which enable bacterial cells to carry out the following activities: (i) escape the human immune system, (ii) invade tumors, (iii) multiply within the tumorous cells, (iv) produce toxins via quorum sensing at low cell densities, and (v) express suicide genes to undergo cell death or cell lysis after the tumor has been lysed. Thus, bacteria have the potential to be exploited as anticancer agents.

Novel polyadenylylation-dependent neutralization mechanism of the HEPN/MNT toxin/antitoxin system.

The two-gene module HEPN/MNT is predicted to be the most abundant toxin/antitoxin (TA) system in prokaryotes. However, its physiological function and neutralization mechanism remains obscure. Here, we discovered that the MntA antitoxin (MNT-domain protein) acts as an adenylyltransferase and chemically modifies the HepT toxin (HEPN-domain protein) to block its toxicity as an RNase. Biochemical and structural studies revealed that MntA mediates the transfer of three AMPs to a tyrosine residue next to the RNase domain of HepT in Shewanella oneidensis. Furthermore, in vitro enzymatic assays showed that the three AMPs are transferred to HepT by MntA consecutively with ATP serving as the substrate, and this polyadenylylation is crucial for reducing HepT toxicity. Additionally, the GSX10DXD motif, which is conserved among MntA proteins, is the key active motif for polyadenylylating and neutralizing HepT. Thus, HepT/MntA represents a new type of TA system, and the polyadenylylation-dependent TA neutralization mechanism is prevalent in bacteria and archaea.

CRISPR-Cas Controls Cryptic Prophages.

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12. We show here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 E. coli spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including rzoD), Rac, Qin (including ydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.

Adaptation without Specialization Early in a Host Shift.

AbstractStudents of speciation debate the role of performance trade-offs across different environments early in speciation. We tested for early performance trade-offs with a host shift experiment using a member of the Enchenopa binotata species complex of treehoppers (Hemiptera: Membracidae). In this clade of plant-feeding insects, different species live on different host plants and exhibit strong behavioral and physiological host specialization. After five generations, the experimental host shifts resulted either in no adaptation or in adaptation without specialization. The latter result was more likely in sympatry; in allopatry, populations on novel host plants were more likely to become extinct. We conclude that in the early stages of speciation, adaptation to novel host plants does not necessarily bring about performance trade-offs on ancestral environments. Adaptation may be facilitated rather than hindered by gene flow, which prevents extinction. Additional causes of specialization and assortative mating may be required if colonization of novel environments is to result in speciation.

'Viable but non-culturable cells' are dead.

Most bacteria lead lives of quiet desperation, so they sleep. By sleeping, bacteria survive ubiquitous stress, such as antibiotics, and can resuscitate to reconstitute infections. As for other nearly universal and highly regulated processes such as biofilm formation, in persistence, a small population of cells have an elegantly-regulated pathway to become dormant. By inactivating their ribosomes, persister cells sleep through stress and resuscitate once (i) the stress is removed, (ii) nutrients are presented and (iii) ribosome content reaches a threshold. During stress, cells often become spheroid and die, becoming hollow, membrane-enclosed vessels. How cellular content is lost is unclear, but it is obvious that these 'cell shells' are dead; i.e., 'There's no there there'. Critically, due to their intact membranes, the shells appear with membrane-impenetrant stains as 'viable' particles. Unfortunately, the microbiology field of 'viable but non-culturable cells' (VBNCs), though important for demonstrating the existence of dormant bacteria as a result of myriad stress states, has often mistaken these non-viable shells as viable particles that mysteriously may be reborn, when an appropriate incantation is made. We argue here, based on experimental data, that if resuscitation occurs, it is the persister (always-viable) cell population that revives, rather than the cell husks, which are dead.

Vibrio splendidus persister cells induced by host coelomic fluids show a similar phenotype to antibiotic-induced counterparts.

Persister cells are dormant variants of regular cells that are multidrug tolerant and have heterogeneous phenotypes; these cells are a potential threat to hosts because they can escape the immune system or antibiotic treatments and reconstitute infectious. Skin ulcer syndrome (SUS) frequently occurs in the sea cucumber (Apostichopus japonicus), and Vibrio splendidus is one of the main bacterial pathogens of SUS. This study found that the active cells of V. splendidus became persister cells more readily in the presence of A. japonicus coelomic fluids. We showed that the A. japonicus coelomic fluids plus antibiotics induce 100-fold more persister cells in V. splendidus compared with antibiotics alone via nine sets of experiments including assays for antibiotic resistance, metabolic activity, and single-cell phenotypes. Furthermore, the coelomic fluids-induced persister cells showed similar phenotypes as the antibiotic-induced persister cells. Further investigation showed that guanosine pentaphosphate/tetraphosphate (henceforth ppGpp) and SOS response pathway involved in the formation of persister cells as determined using real-time RT-PCR. In addition, single-cell observations showed that, similar to the antibiotic-induced V. splendidus persister cells, the coelomic fluids-induced persister cells have five resuscitation phenotypes: no growth, expansion, elongation, elongation and then division, and elongation followed by death/disappearance. In addition, dark foci formed in the majority of persister cells for both the antibiotic-induced and coelomic fluids-induced persister cells. Our results highlight that the pathogen V. splendidus might escape from the host immune system by entering the persister state during the process of infection due to exposure to coelomic fluids.

Escherichia coli cryptic prophages sense nutrients to influence persister cell resuscitation.

Cryptic prophages are not genomic junk but instead enable cells to combat myriad stresses as an active stress response. How these phage fossils affect persister cell resuscitation has, however, not been explored. Persister cells form as a result of stresses such as starvation, antibiotics and oxidative conditions, and resuscitation of these persister cells likely causes recurring infections such as those associated with tuberculosis, cystic fibrosis and Lyme disease. Deletion of each of the nine Escherichia coli cryptic prophages has no effect on persister cell formation. Strikingly, elimination of each cryptic prophage results in an increase in persister cell resuscitation with a dramatic increase in resuscitation upon deleting all nine prophages. This increased resuscitation includes eliminating the need for a carbon source and is due to activation of the phosphate import system resulting from inactivating the transcriptional regulator AlpA of the CP4-57 cryptic prophage. Deletion of alpA increases persister resuscitation, and AlpA represses phosphate regulator PhoR. Both phosphate regulators PhoP and PhoB stimulate resuscitation. This suggests a novel cellular stress mechanism controlled by cryptic prophages: regulation of phosphate uptake which controls the exit of the cell from dormancy and prevents premature resuscitation in the absence of nutrients.

(p)ppGpp and Its Role in Bacterial Persistence: New Challenges.

Antibiotic failure not only is due to the development of resistance by pathogens but can also often be explained by persistence and tolerance. Persistence and tolerance can be included in the "persistent phenotype," with high relevance for clinics. Two of the most important molecular mechanisms involved in tolerance and persistence are toxin-antitoxin (TA) modules and signaling via guanosine pentaphosphate/tetraphosphate [(p)ppGpp], also known as "magic spot." (p)ppGpp is a very important stress alarmone which orchestrates the stringent response in bacteria; hence, (p)ppGpp is produced during amino acid or fatty acid starvation by proteins belonging to the RelA/SpoT homolog family (RSH). However, (p)ppGpp levels can also accumulate in response to a wide range of signals, including oxygen variation, pH downshift, osmotic shock, temperature shift, or even exposure to darkness. Furthermore, the stringent response is not only involved in responses to environmental stresses (starvation for carbon sources, fatty acids, and phosphates or heat shock), but it is also used in bacterial pathogenesis, host invasion, and antibiotic tolerance and persistence. Given the exhaustive and contradictory literature surrounding the role of (p)ppGpp in bacterial persistence, and with the aim of summarizing what is known so far about the magic spot in this bacterial stage, this review provides new insights into the link between the stringent response and persistence. Moreover, we review some of the innovative treatments that have (p)ppGpp as a target, which are in the spotlight of the scientific community as candidates for effective antipersistence agents.

Persister cells resuscitate via ribosome modification by 23S rRNA pseudouridine synthase RluD.

Upon a wide range of stress conditions (e.g. nutrient, antibiotic, oxidative), a subpopulation of bacterial cells known as persisters survives by halting metabolism. These cells resuscitate rapidly to reconstitute infections once the stress is removed and nutrients are provided. However, how these dormant cells resuscitate is not understood well but involves reactivating ribosomes. By screening 10,000 compounds directly for stimulating Escherichia coli persister cell resuscitation, we identified that 2-{[2-(4-bromophenyl)-2-oxoethyl]thio}-3-ethyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (BPOET) stimulates resuscitation. Critically, by screening 4267 E. coli proteins, we determined that BPOET activates hibernating ribosomes via 23S rRNA pseudouridine synthase RluD, which increases ribosome activity. Corroborating the increased waking with RluD, production of RluD increased the number of active ribosomes in persister cells. Also, inactivating the small RNA RybB which represses rluD led to faster persister resuscitation. Hence, persister cells resuscitate via activation of RluD.

ppGpp ribosome dimerization model for bacterial persister formation and resuscitation.

Stress is ubiquitous for bacteria and can convert a subpopulation of cells into a dormant state known as persistence, in which cells are tolerant to antimicrobials. These cells revive rapidly when the stress is removed and are likely the cause of many recurring infections such as those associated with tuberculosis, cystic fibrosis, and Lyme disease. However, how persister cells are formed is not understood well. Here we propose the ppGpp ribosome dimerization persister (PRDP) model in which the alarmone guanosine pentaphosphate/tetraphosphate (henceforth ppGpp) generates persister cells directly by inactivating ribosomes via the ribosome modulation factor (RMF), the hibernation promoting factor (Hpf), and the ribosome-associated inhibitor (RaiA). We demonstrate that persister cells contain a large fraction of 100S ribosomes, that inactivation of RMF, HpF, and RaiA reduces persistence and increases single-cell persister resuscitation and that ppGpp has no effect on single-cell persister resuscitation. Hence, a direct connection between ppGpp and persistence is shown along with evidence of the importance of ribosome dimerization in persistence and for active ribosomes during resuscitation.

σ54 -Dependent regulator DVU2956 switches Desulfovibrio vulgaris from biofilm formation to planktonic growth and regulates hydrogen sulfide production.

Microbiologically influenced corrosion causes $100 billion in damage per year, and biofilms formed by sulfate-reducing bacteria (SRB) are the major culprit. However, little is known about the regulation of SRB biofilm formation. Using Desulfovibrio vulgaris as a model SRB organism, we compared the transcriptomes of biofilm and planktonic cells and identified that the gene for σ54 -dependent regulator DVU2956 is repressed in biofilms. Utilizing a novel promoter that is primarily transcribed in biofilms (Pdvu0304 ), we found production of DVU2956 inhibits biofilm formation by 70%. Corroborating this result, deleting dvu2956 increased biofilm formation, and this biofilm phenotype could be complemented. By producing proteins in biofilms from genes controlled by DVU2956 (dvu2960 and dvu2962), biofilm formation was inhibited almost completely. A second round of RNA-seq for the production of DVU2956 revealed DVU2956 influences electron transport via an Hmc complex (high-molecular-weight cytochrome c encoded by dvu0531-dvu0536) and the Fe-only hydrogenase (encoded by dvu1769, hydA and dvu1770, hydB) to control H2 S production. Corroborating these results, producing DVU2956 in biofilms decreased H2 S production by half, deleting dvu2956 increased H2 S production by 131 ± 5%, and producing DVU2956 in the dvu2956 strain reduced H2 S production. Therefore, DVU2956 maintains SRB in the planktonic state and reduces H2 S formation.

Symbiosis of a P2-family phage and deep-sea Shewanella putrefaciens.

Almost all bacterial genomes harbour prophages, yet it remains unknown why prophages integrate into tRNA-related genes. Approximately 1/3 of Shewanella isolates harbour a prophage at the tmRNA (ssrA) gene. Here, we discovered a P2-family prophage integrated at the 3'-end of ssrA in the deep-sea bacterium S. putrefaciens. We found that ~0.1% of host cells are lysed to release P2 constitutively during host growth. P2 phage production is induced by a prophage-encoded Rep protein and its excision is induced by the Cox protein. We also found that P2 genome excision leads to the disruption of wobble base pairing of SsrA due to site-specific recombination, thus disrupting the trans-translation function of SsrA. We further demonstrated that P2 excision greatly hinders growth in seawater medium and inhibits biofilm formation. Complementation with a functional SsrA in the P2-excised strain completely restores the growth defects in seawater medium and partially restores biofilm formation. Additionally, we found that products of the P2 genes also increase biofilm formation. Taken together, this study illustrates a symbiotic relationship between P2 and its marine host, thus providing multiple benefits for both sides when a phage is integrated but suffers from reduced fitness when the prophage is excised.

Identification of a potent indigoid persister antimicrobial by screening dormant cells.

The subpopulation of bacterial cells that survive myriad stress conditions (e.g., nutrient deprivation and antimicrobials) by ceasing metabolism, revive by activating ribosomes. These resuscitated cells can reconstitute infections; hence, it is imperative to discover compounds which eradicate persister cells. By screening 10,000 compounds directly for persister cell killing, we identified 5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride (NPIMA) kills Escherichia coli persister cells more effectively than the best indigoid found to date, 5-iodoindole, and better than the DNA-crosslinker cisplatin. In addition, NPIMA eradicated Pseudomonas aeruginosa persister cells in a manner comparable to cisplatin. NPIMA also eradicated Staphylococcus aureus persister cells but was less effective than cisplatin. Critically, NPIMA kills Gram-positive and Gram-negative bacteria by damaging membranes and causing lysis as demonstrated by microscopy and release of extracellular DNA and protein. Furthermore, NPIMA was effective in reducing P. aeruginosa and S. aureus cell numbers in a wound model, and no resistance was found after 1 week. Hence, we identified a potent indigoid that kills persister cells by damaging their membranes.

Quorum sensing between Gram-negative bacteria responsible for methane production in a complex waste sewage sludge consortium.

Quorum sensing (QS) plays a key role in activating bacterial functions through small molecules called autoinducers. In this study, the QS of Gram-negative bacteria in waste sewage sludge (WSS) was downregulated by adding the quorum quenching enzyme, AiiM lactonase, which cleaved the acyl-homoserine lactone (AHL) autoinducer signals from Gram-negative bacteria, and subsequently methane production was inhibited by over 400%. The pH was lowered after 2 days in the anaerobic fermentation whereas protease activity at the hydrolysis step was almost the same with or without AiiM. The production of acetic acid significantly increased during the fermentation in the presence of AiiM. The bacterial community at day 2 indicated that the population of Gram-positive bacteria increased in the presence of AiiM, and the percentage of Gram-negative bacteria decreased in the WSS containing AiiM. The change in the bacterial community in the presence of AiiM may be due to the different antimicrobial agents produced in the WSS because some of the Gram-positive bacteria were killed by adding the solid-phase extraction (SPE) fraction from the WSS without AiiM. In contrast, the SPE fraction with AiiM had reduced bactericidal activity against Gram-negative bacteria. Thus, bacterial signaling between Gram-negative bacteria is critical for methane production by the microbial consortia.

Indole: An evolutionarily conserved influencer of behavior across kingdoms.

Indole is a key environmental cue that is used by many organisms. Based on its biochemistry, we suggest indole is used so universally, and by such different organisms, because it derives from the metabolism of tryptophan, a resource essential for many species yet rare in nature. These properties make it a valuable, environmental cue for resources almost universally important for promoting fitness. We then describe how indole is used to coordinate actions within organisms, to influence the behavior of conspecifics and can even be used to change the behavior of species that belong to other kingdoms. Drawing on the evolutionary framework that has been developed for understanding animal communication, we show how this is diversely achieved by indole acting as a cue, a manipulative signal, and an honest signal, as well as how indole can be used synergistically to amplify information conveyed by other molecules. Clarifying these distinct functions of indole identifies patterns that transcend different kingdoms of organisms.

Living biofouling-resistant membranes as a model for the beneficial use of engineered biofilms.

Membrane systems are used increasingly for water treatment, recycling water from wastewater, during food processing, and energy production. They thus are a key technology to ensure water, energy, and food sustainability. However, biofouling, the build-up of microbes and their polymeric matrix, clogs these systems and reduces their efficiency. Realizing that a microbial film is inevitable, we engineered a beneficial biofilm that prevents membrane biofouling, limiting its own thickness by sensing the number of its cells that are present via a quorum-sensing circuit. The beneficial biofilm also prevents biofilm formation by deleterious bacteria by secreting nitric oxide, a general biofilm dispersal agent, as demonstrated by both short-term dead-end filtration and long-term cross-flow filtration tests. In addition, the beneficial biofilm was engineered to produce an epoxide hydrolase so that it efficiently removes the environmental pollutant epichlorohydrin. Thus, we have created a living biofouling-resistant membrane system that simultaneously reduces biofouling and provides a platform for biodegradation of persistent organic pollutants.