Designer Liposomic Nanocarriers Are Effective Biofilm Eradicators.

Drug delivery via nanovehicles is successfully employed in several clinical settings, yet bacterial infections, forming microbial communities in the form of biofilms, present a strong challenge to therapeutic treatment due to resistance to conventional antimicrobial therapies. Liposomes can provide a versatile drug-vector strategy for biofilm treatment, but are limited by the need to balance colloidal stability with biofilm penetration. We have discovered a liposomic functionalization strategy, using membrane-embedded moieties of poly[2-(methacryloyloxy)ethyl phosphorylcholine], pMPC, that overcomes this limitation. Such pMPCylation results in liposomic stability equivalent to current functionalization strategies (mostly PEGylation, the present gold-standard), but with strikingly improved cellular uptake and cargo conveyance. Fluorimetry, cryo-electron, and fluorescence microscopies reveal a far-enhanced antibiotic delivery to model Pseudomonas aeruginosa biofilms by pMPC-liposomes, followed by faster cytosolic cargo release, resulting in significantly greater biofilm eradication than either PEGylation or free drug. Moreover, this combination of techniques uncovers the molecular mechanism underlying the enhanced interaction with bacteria, indicating it arises from bridging by divalent ions of the zwitterionic groups on the pMPC moieties to the negatively charged lipopolysaccharide chains emanating from the bacterial membranes. Our results point to pMPCylation as a transformative strategy for liposomal functionalization, leading to next-generation delivery systems for biofilm treatment.

A dynamic rhizosphere interplay between tree roots and soil bacteria under drought stress.

Root exudates are thought to play an important role in plant-microbial interactions. In return for nutrition, soil bacteria can increase the bioavailability of soil nutrients. However, root exudates typically decrease in situations such as drought, calling into question the efficacy of solvation and bacteria-dependent mineral uptake in such stress. Here, we tested the hypothesis of exudate-driven microbial priming on Cupressus saplings grown in forest soil in custom-made rhizotron boxes. A 1-month imposed drought and concomitant inoculations with a mix of Bacillus subtilis and Pseudomonas stutzeri, bacteria species isolated from the forest soil, were applied using factorial design. Direct bacteria counts and visualization by confocal microscopy showed that both bacteria associated with Cupressus roots. Interestingly, root exudation rates increased 2.3-fold with bacteria under drought, as well as irrigation. Forty-four metabolites in exudates were significantly different in concentration between irrigated and drought trees, including phenolic acid compounds and quinate. When adding these metabolites as carbon and nitrogen sources to bacterial cultures of both bacterial species, eight of nine metabolites stimulated bacterial growth. Importantly, soil phosphorous bioavailability was maintained only in inoculated trees, mitigating drought-induced decrease in leaf phosphorus and iron. Our observations of increased root exudation rate when drought and inoculation regimes were combined support the idea of root recruitment of beneficial bacteria, especially under water stress.

The soil surrounding the roots of trees, termed the rhizosphere, is full of bacteria and other communities of microorganisms. Trees secrete organic compounds in to the soil which are thought to influence the behavior of bacteria in the rhizosphere. Specifically, these root secretions, or 'exudates', attract and feed soil bacteria, which, in return, release nutrients that benefit the tree. In 2020, a group of researchers found that some trees in the Mediterranean forest produce more exudates during the long dry season. This suggests that the compounds secreted by roots may help trees to tolerate stress conditions, such as drought. To test this hypothesis, Oppenheimer-Shaanan et al. ­ including some of the researchers involved in the 2020 study ­ grew young Cupressus sempervirens conifer trees in drought conditions that starved them of the nutrients phosphorous and iron. Each tree was planted in a custom-built box which allowed easy access to roots growing in the soil. Two species of bacteria from the forest soil C. sempervirens trees naturally live in were then added to the soil in each box. Microscopy revealed that both species of bacteria, which had been tagged with fluorescent markers, were attracted to the roots of the trees, boosting the bacterial community in the rhizosphere. Oppenheimer-Shaanan et al. found that the recruitment of the two bacterial species caused the rate at which exudates were secreted from the roots to increase. Compounds in the exudate stimulated the bacteria to grow. Ultimately, levels of phosphorous and iron in the leaves of the starved trees increased when in the presence of these soil bacteria. This suggests that bacteria in the rhizosphere helps trees to survive when they are under stress and have low levels of water. These findings provide further evidence that plants and bacteria can live together in symbiosis and benefit one another. This could have important implications for forest ecology and potentially how trees are grown in orchards and gardens. For example, specific bacteria and organic compounds in the rhizosphere may be able to improve tree health. However, further work is needed to investigate whether the exudate compounds identified in this study are found more widely in nature.

Bacterial gasdermins reveal an ancient mechanism of cell death.

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.

Cyclic CMP and cyclic UMP mediate bacterial immunity against phages.

The cyclic pyrimidines 3',5'-cyclic cytidine monophosphate (cCMP) and 3',5'-cyclic uridine monophosphate (cUMP) have been reported in multiple organisms and cell types. As opposed to the cyclic nucleotides 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP), which are second messenger molecules with well-established regulatory roles across all domains of life, the biological role of cyclic pyrimidines has remained unclear. Here we report that cCMP and cUMP are second messengers functioning in bacterial immunity against viruses. We discovered a family of bacterial pyrimidine cyclase enzymes that specifically synthesize cCMP and cUMP following phage infection and demonstrate that these molecules activate immune effectors that execute an antiviral response. A crystal structure of a uridylate cyclase enzyme from this family explains the molecular mechanism of selectivity for pyrimidines as cyclization substrates. Defense systems encoding pyrimidine cyclases, denoted here Pycsar (pyrimidine cyclase system for antiphage resistance), are widespread in prokaryotes. Our results assign clear biological function to cCMP and cUMP as immunity signaling molecules in bacteria.

Bacterial Retrons Function In Anti-Phage Defense.

Retrons are bacterial genetic elements comprised of a reverse transcriptase (RT) and a non-coding RNA (ncRNA). The RT uses the ncRNA as template, generating a chimeric RNA/DNA molecule in which the RNA and DNA components are covalently linked. Although retrons were discovered three decades ago, their function remained unknown. We report that retrons function as anti-phage defense systems. The defensive unit is composed of three components: the RT, the ncRNA, and an effector protein. We examined multiple retron systems and show that they confer defense against a broad range of phages via abortive infection. Focusing on retron Ec48, we show evidence that it "guards" RecBCD, a complex with central anti-phage functions in bacteria. Inhibition of RecBCD by phage proteins activates the retron, leading to abortive infection and cell death. Thus, the Ec48 retron forms a second line of defense that is triggered if the first lines of defense have collapsed.

Cyclic GMP-AMP signalling protects bacteria against viral infection.

The cyclic GMP-AMP synthase (cGAS)-STING pathway is a central component of the cell-autonomous innate immune system in animals1,2. The cGAS protein is a sensor of cytosolic viral DNA and, upon sensing DNA, it produces a cyclic GMP-AMP (cGAMP) signalling molecule that binds to the STING protein and activates the immune response3-5. The production of cGAMP has also been detected in bacteria6, and has been shown, in Vibrio cholerae, to activate a phospholipase that degrades the inner bacterial membrane7. However, the biological role of cGAMP signalling in bacteria remains unknown. Here we show that cGAMP signalling is part of an antiphage defence system that is common in bacteria. This system is composed of a four-gene operon that encodes the bacterial cGAS and the associated phospholipase, as well as two enzymes with the eukaryotic-like domains E1, E2 and JAB. We show that this operon confers resistance against a wide variety of phages. Phage infection triggers the production of cGAMP, which-in turn-activates the phospholipase, leading to a loss of membrane integrity and to cell death before completion of phage reproduction. Diverged versions of this system appear in more than 10% of prokaryotic genomes, and we show that variants with effectors other than phospholipase also protect against phage infection. Our results suggest that the eukaryotic cGAS-STING antiviral pathway has ancient evolutionary roots that stem from microbial defences against phages.

Micro-CT X-ray imaging exposes structured diffusion barriers within biofilms.

In nature, bacteria predominantly exist as highly structured biofilms, which are held together by extracellular polymeric substance and protect their residents from environmental insults, such as antibiotics. The mechanisms supporting this phenotypic resistance are poorly understood. Recently, we identified a new mechanism maintaining biofilms - an active production of calcite minerals. In this work, a high-resolution and robust µCT technique is used to study the mineralized areas within intact bacterial biofilms. µCT is a vital tool for visualizing bacterial communities that can provide insights into the relationship between bacterial biofilm structure and function. Our results imply that dense and structured calcium carbonate lamina forms a diffusion barrier sheltering the inner cell mass of the biofilm colony. Therefore, µCT can be employed in clinical settings to predict the permeability of the biofilms. It is demonstrated that chemical interference with urease, a key enzyme in biomineralization, inhibits the assembly of complex bacterial structures, prevents the formation of mineral diffusion barriers and increases biofilm permeability. Therefore, biomineralization enzymes emerge as novel therapeutic targets for highly resistant infections.

Toxin-Antitoxin systems eliminate defective cells and preserve symmetry in Bacillus subtilis biofilms.

Toxin-antitoxin modules are gene pairs encoding a toxin and its antitoxin, and are found on the chromosomes of many bacteria, including pathogens. Here, we characterize the specific contribution of the TxpA and YqcG toxins in elimination of defective cells from developing Bacillus subtilis biofilms. On nutrient limitation, defective cells accumulated in the biofilm breaking its symmetry. Deletion of the toxins resulted in accumulation of morphologically abnormal cells, and interfered with the proper development of the multicellular community. Dual physiological responses are of significance for TxpA and YqcG activation: nitrogen deprivation enhances the transcription of both TxpA and YqcG toxins, and simultaneously sensitizes the biofilm cells to their activity. Furthermore, we demonstrate that while both toxins when overexpressed affect the morphology of the developing biofilm, the toxin TxpA can act to lyse and dissolve pre-established B. subtilis biofilms.

Not so simple, not so subtle: the interspecies competition between Bacillus simplex and Bacillus subtilis and its impact on the evolution of biofilms.

Bacillus subtilis biofilms have a fundamental role in shaping the soil ecosystem. During this process, they unavoidably interact with neighbour bacterial species. We studied the interspecies interactions between biofilms of the soil-residing bacteria B. subtilis and related Bacillus species. We found that proximity between the biofilms triggered recruitment of motile B. subtilis cells, which engulfed the competing Bacillus simplex colony. Upon interaction, B. subtilis secreted surfactin and cannibalism toxins, at concentrations that were inert to B. subtilis itself, which eliminated the B. simplex colony, as well as colonies of Bacillus toyonensis. Surfactin toxicity was correlated with the presence of short carbon-tail length isomers, and synergistic with the cannibalism toxins. Importantly, during biofilm development and interspecies interactions a subpopulation in B. subtilis biofilm lost its native plasmid, leading to increased virulence against the competing Bacillus species. Overall, these findings indicate that genetic programs and traits that have little effect on biofilm development when each species is grown in isolation have a dramatic impact when different bacterial species interact.

Spatio-temporal assembly of functional mineral scaffolds within microbial biofilms.

Historically, multicellular bacterial communities, known as biofilms, have been thought to be held together solely by a self-produced extracellular matrix. Our study identified a novel mechanism maintaining Bacillus subtilis and Mycobacterium smegmatis biofilms-active production of calcite minerals. We studied, for the first time, the effects of mutants defective in biomineralization and calcite formation on biofilm development, resilience and morphology. We demonstrated that an intrinsic rise in carbon dioxide levels within the biofilm is a strong trigger for the initiation of calcite-dependent patterning. The calcite-dependent patterns provide resistance to environmental insults and increase the overall fitness of the microbial community. Our results suggest that it is highly feasible that the formation of mineral scaffolds plays a cardinal and conserved role in bacterial multicellularity.

Disturbance of the bacterial cell wall specifically interferes with biofilm formation.

In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment.

Small molecules are natural triggers for the disassembly of biofilms.

Gigantic bacterial communities, termed biofilms, thrive in a variety of situations. Held together by a protective matrix, a biofilm is a bacterial fortress whose inhabitants are much better protected against environmental insults than free-living bacteria. However, knowing how single bacteria can break away from the community could be harnessed to break up biofilms that form on prosthetic devices implanted into the human body. This review demonstrates how small secreted molecules can elegantly mediate the disassembly of biofilms. Four different mechanisms for natural triggers of disassembly are highlighted: signals and cues, cell envelope-modifying molecules, anti-matrix molecules, and molecules that promote cell death.

Relacin, a novel antibacterial agent targeting the Stringent Response.

Finding bacterial cellular targets for developing novel antibiotics has become a major challenge in fighting resistant pathogenic bacteria. We present a novel compound, Relacin, designed to inhibit (p)ppGpp production by the ubiquitous bacterial enzyme RelA that triggers the Stringent Response. Relacin inhibits RelA in vitro and reduces (p)ppGpp production in vivo. Moreover, Relacin affects entry into stationary phase in Gram positive bacteria, leading to a dramatic reduction in cell viability. When Relacin is added to sporulating Bacillus subtilis cells, it strongly perturbs spore formation regardless of the time of addition. Spore formation is also impeded in the pathogenic bacterium Bacillus anthracis that causes the acute anthrax disease. Finally, the formation of multicellular biofilms is markedly disrupted by Relacin. Thus, we establish that Relacin, a novel ppGpp analogue, interferes with bacterial long term survival strategies, placing it as an attractive new antibacterial agent.

c-di-AMP reports DNA integrity during sporulation in Bacillus subtilis.

The bacterium Bacillus subtilis produces the DNA integrity scanning protein (DisA), a checkpoint protein that delays sporulation in response to DNA damage. DisA scans the chromosome and pauses at sites of DNA lesions. Structural analysis showed that DisA synthesizes the small molecule cyclic diadenosine monophosphate (c-di-AMP). Here, we demonstrate that the intracellular concentration of c-di-AMP rises markedly at the onset of sporulation in a DisA-dependent manner. Furthermore, exposing sporulating cells to DNA-damaging agents leads to a global decrease in the level of this molecule. This drop was associated with stalled DisA complexes that halt c-di-AMP production and with increased levels of the c-di-AMP-degrading enzyme YybT. Reduced c-di-AMP levels cause a delay in sporulation that can be reversed by external supplementation of the molecule. Thus, c-di-AMP acts as a secondary messenger, coupling DNA integrity with progression of sporulation.

A checkpoint protein that scans the chromosome for damage at the start of sporulation in Bacillus subtilis.

In response to DNA damage, cells activate checkpoint signaling cascades to control cell-cycle progression and elicit DNA repair in order to maintain genomic integrity. The sensing and repair of lesions is critical for Bacillus subtilis cells entering the developmental process of sporulation as damaged DNA may prevent the cells from completing spore morphogenesis. We report the identification of the protein DisA (DNA integrity scanning protein, annotated YacK), which is required to delay the initiation of sporulation in response to chromosomal damage. DisA is a nonspecific DNA binding protein that forms a single focus, which moves rapidly within the bacterial cell, pausing at sites of DNA damage. We propose that the DisA focus scans along the chromosomes searching for lesions. Upon encountering a lesion, DisA delays entry into sporulation until the damage is repaired.