Explosive Cell Lysis Research Articles

Genotoxic stress stimulates eDNA release via explosive cell lysis and thereby promotes streamer formation of Burkholderia cenocepacia H111 cultured in a microfluidic device

DNA is a component of biofilms, but the triggers of DNA release during biofilm formation and how DNA contributes to biofilm development are poorly investigated. One key mechanism involved in DNA release is explosive cell lysis, which is a consequence of prophage induction. In this article, the role of explosive cell lysis in biofilm formation was investigated in the opportunistic human pathogen Burkholderia cenocepacia H111 (H111). Biofilm streamers, flow-suspended biofilm filaments, were used as a biofilm model in this study, as DNA is an essential component of their matrix. H111 contains three prophages on chromosome 1 of its genome, and the involvement of each prophage in causing explosive cell lysis of the host and subsequent DNA and membrane vesicle (MV) release, as well as their contribution to streamer formation, were studied in the presence and absence of genotoxic stress. The results show that two of the three prophages of H111 encode functional lytic prophages that can be induced by genotoxic stress and their activation causes DNA and MVs release by explosive cell lysis. Furthermore, it is shown that the released DNA enables the strain to develop biofilm streamers, and streamer formation can be enhanced by genotoxic stress. Overall, this study demonstrates the involvement of prophages in streamer formation and uncovers an often-overlooked problem with the use of antibiotics that trigger the bacterial SOS response for the treatment of bacterial infections.

Droplet duos on water display pairing, autonomous motion, and periodic eruption

Under non-equilibrium conditions, liquid droplets dynamically couple with their milieu through the continuous flux of matter and energy, forming active systems capable of self-organizing functions reminiscent of those of living organisms. Among the various dynamic behaviors demonstrated by cells, the pairing of heterogeneous cell units is necessary to enable collective activity and cell fusion (to reprogram somatic cells). Furthermore, the cyclic occurrence of eruptive events such as necroptosis or explosive cell lysis is necessary to maintain cell functions. However, unlike the self-propulsion behavior of cells, cyclic cellular behavior involving pairing and eruption has not been successfully modeled using artificial systems. Here, we show that a simple droplet system based on quasi-immiscible hydrophobic oils (perfluorodecalin and decane) deposited on water, mimics such complex cellular dynamics. Perfluorodecalin and decane droplet duos form autonomously moving Janus or coaxial structures, depending on their volumes. Notably, the system with a coaxial structure demonstrates cyclic behavior, alternating between autonomous motion and eruption. Despite their complexity, the dynamic behaviors of the system are consistently explained in terms of the spreading properties of perfluorodecalin/decane duplex interfacial films.

Murine Norovirus Interaction with Enterobacter cloacae Leads to Changes in Membrane Stability and Packaging of Lipid and Metabolite Vesicle Content.

Outer membrane vesicles (OMVs) are a primary means of communication for Gram-negative bacteria. The specific role of vesicle components in cellular communication and how components are packaged are still under investigation, but a correlation exists between OMV biogenesis and content. The two primary mechanisms of OMV biogenesis are membrane blebbing and explosive cell lysis, and vesicle content is based on the biogenesis mechanism. Hypervesiculation, which can be induced by stress conditions, also influences OMV content. Norovirus interaction with Enterobacter cloacae induces stress responses leading to increased OMV production and changes in DNA content, protein content, and vesicle size. The presence of genomic DNA and cytoplasmic proteins in these OMVs suggests some of the vesicles are formed by explosive cell lysis, so reduction or loss of these components indicates a shift away from this mechanism of biogenesis. Based on this, further investigation into bacterial stability and OMV content was conducted. Results showed that norovirus induced a dramatic shift in OMV lipid content. Specifically, the increased accumulation of phospholipids is associated with increased blebbing, thereby supporting previous observations that noroviruses shift the mechanism of OMV biogenesis. Slight differences in OMV metabolite content were also observed. While norovirus induced changes in OMV content, it did not change the lipid content of the bacterial outer membrane or the metabolite content of the bacterial cell. Overall, these results indicate that norovirus induces significant changes to OMV lipid architecture and cargo, which may be linked to a change in the mechanism of vesicle biogenesis. IMPORTANCE Extracellular vesicles from commensal bacteria are recognized for their importance in modulating host immune responses, and vesicle content is related to their impact on the host. Therefore, understanding how vesicles are formed and how their content shifts in response to stress conditions is necessary for elucidating their downstream functions. Our recent work has demonstrated that interactions between noroviruses and Enterobacter cloacae induce bacterial stress responses leading to hypervesiculation. In this article, we characterize and compare the lipid and metabolomic cargo of E. cloacae vesicles generated in the presence and absence of norovirus and show that viral interactions induce significant changes in vesicle content. Furthermore, we probe how these changes and changes to the bacterial cell may be indicative of a shift in the mechanism of vesicle biogenesis. Importantly, we find that noroviruses induce significant changes in vesicle lipid architecture and cargo that may be responsible for the immunogenic activity of these vesicles.

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition.

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P.aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.

Composition and functions of bacterial membrane vesicles.

Extracellular vesicles are produced by species across all domains of life, suggesting that vesiculation represents a fundamental principle of living matter. In Gram-negative bacteria, membrane vesicles (MVs) can originate either from blebs of the outer membrane or from endolysin-triggered explosive cell lysis, which is often induced by genotoxic stress. Although less is known about the mechanisms of vesiculation in Gram-positive and Gram-neutral bacteria, recent research has shown that both lysis and blebbing mechanisms also exist in these organisms. Evidence has accumulated over the past years that different biogenesis routes lead to distinct types of MV with varied structure and composition. In this Review, we discuss the different types of MV and their potential cargo packaging mechanisms. We summarize current knowledge regarding how MV composition determines their various functions including support of bacterial growth via the disposal of waste material, nutrient scavenging, export of bioactive molecules, DNA transfer, neutralization of phages, antibiotics and bactericidal functions, delivery of virulence factors and toxins to host cells and inflammatory and immunomodulatory effects. We also discuss the advantages of MV-mediated secretion compared with classic bacterial secretion systems and we introduce the concept of quantal secretion.

L-form conversion in Gram-positive bacteria enables escape from phage infection

At the end of a lytic bacteriophage replication cycle in Gram-positive bacteria, peptidoglycan-degrading endolysins that cause explosive cell lysis of the host can also attack non-infected bystander cells. Here we show that in osmotically stabilized environments, Listeria monocytogenes can evade phage predation by transient conversion to a cell wall-deficient L-form state. This L-form escape is triggered by endolysins disintegrating the cell wall from without, leading to turgor-driven extrusion of wall-deficient, yet viable L-form cells. Remarkably, in the absence of phage predation, we show that L-forms can quickly revert to the walled state. These findings suggest that L-form conversion represents a population-level persistence mechanism to evade complete eradication by phage attack. Importantly, we also demonstrate phage-mediated L-form switching of the urinary tract pathogen Enterococcus faecalis in human urine, which underscores that this escape route may be widespread and has important implications for phage- and endolysin-based therapeutic interventions.

Visualizing extracellular vesicle biogenesis in gram-positive bacteria using super-resolution microscopy

BackgroundRecently, bacterial extracellular vesicles (EVs) have been considered to play crucial roles in various biological processes and have great potential for developing cancer therapeutics and biomedicine. However, studies on bacterial EVs have mainly focused on outer membrane vesicles released from gram-negative bacteria since the outermost peptidoglycan layer in gram-positive bacteria is thought to preclude the release of EVs as a physical barrier.ResultsHere, we examined the ultrastructural organization of the EV produced by gram-positive bacteria using super-resolution stochastic optical reconstruction microscopy (STORM) at the nanoscale, which has not been resolved using conventional microscopy. Based on the super-resolution images of EVs, we propose three major mechanisms of EV biogenesis, i.e., membrane blebbing (mechanisms 1 and 2) or explosive cell lysis (mechanism 3), which are different from the mechanisms in gram-negative bacteria, despite some similarities.ConclusionsThese findings highlight the significant role of cell wall degradation in regulating various mechanisms of EV biogenesis and call for a reassessment of previously unresolved EV biogenesis in gram-positive bacteria.

The role of peptidoglycan hydrolases in the formation and toxicity of Pseudomonas aeruginosa membrane vesicles.

Bacterial membrane vesicles (MVs) have been reported to kill other bacteria. In the case of Pseudomonas aeruginosa the bactericidal activity has been attributed to an unidentified 26kDa peptidoglycan (PG) hydrolase that is associated with MVs and gives rise to a lytic band on zymograms using murein sacculi as substrate. In this study, we employed a proteomics approach to show that this PG hydrolase is the AmphD3 amidase. The analysis of an amphD3 mutant as well as of an AmphD3 overexpression derivative revealed that this enzyme is not required for the bactericidal activity of P. aeruginosa MVs but is involved in cell wall recycling and thus protects the cell against PG damage. Another 23kDa PG hydrolase, which we observed on zymograms of SOS-induced MVs, was identified as the endolysin Lys, which triggers explosive cell lysis but is shown to be dispensable for MV-mediated killing. We conclude that the lytic activities observed on zymograms do not correlate with the bactericidal potential of MVs. We demonstrate that P. aeruginosa MVs are enriched for several autolysins, suggesting that the predatory activity of MVs depends on the combined action of different murein hydrolases.

Phage-Mediated Explosive Cell Lysis Induces the Formation of a Different Type of O-IMV in Shewanella vesiculosa M7T.

Shewanella vesiculosa M7T is a cold-adapted Antarctic bacterium that has a great capacity to secrete membrane vesicles (MVs), making it a potentially excellent model for studying the vesiculation process. S. vesiculosa M7T undergoes a blebbing mechanism to produce different types of MVs, including outer membrane vesicles and outer-inner membrane vesicles (O-IMVs). More recently, other mechanisms have been considered that could lead to the formation of O-IMVs derived from prophage-mediated explosive cell lysis in other bacteria, but it is not clear if they are of the same type. The bacterial growth phase could also have a great impact on the type of MVs, although there are few studies on the subject. In this study, we used high-resolution flow cytometry, transmission electron microscopy, and cryo-electron microscopy (Cryo-EM) analysis to determine the amount and types of MVs S. vesiculosa M7T secreted during different growth phases. We show that MV secretion increases during the transition from the late exponential to the stationary phase. Moreover, prophage-mediated explosive cell lysis is activated in S. vesiculosa M7T, increasing the heterogeneity of both single- and double-layer MVs. The sequenced DNA fragments from the MVs covered the entire genome, confirming this explosive cell lysis mechanism. A different structure and biogenesis mechanisms for the explosive cell lysis-derived double-layered MVs was observed, and we propose to name them explosive O-IMVs, distinguishing them from the blebbing O-IMVs; their separation is a first step to elucidate their different functions. In our study, we used for the first time sorting by flow cytometry and Cryo-EM analyses to isolate bacterial MVs based on their nucleic acid content. Further improvements and implementation of bacterial MV separation techniques is essential to develop more in-depth knowledge of MVs.

Bacteriophage infection of Escherichia coli leads to the formation of membrane vesicles via both explosive cell lysis and membrane blebbing.

Membrane vesicles (MVs) are membrane-bound spherical nanostructures that prevail in all three domains of life. In Gram-negative bacteria, MVs are thought to be produced through blebbing of the outer membrane and are often referred to as outer membrane vesicles (OMVs). We have recently described another mechanism of MV formation in Pseudomonas aeruginosa that involves explosive cell-lysis events, which shatters cellular membranes into fragments that rapidly anneal into MVs. Interestingly, MVs are often observed within preparations of lytic bacteriophage, however the source of these MVs and their association with bacteriophage infection has not been explored. In this study we aimed to determine if MV formation is associated with lytic bacteriophage infection. Live super-resolution microscopy demonstrated that explosive cell lysis of Escherichia coli cells infected with either bacteriophage T4 or T7, resulted in the formation of MVs derived from shattered membrane fragments. Infection by either bacteriophage was also associated with the formation of membrane blebs on intact bacteria. TEM revealed multiple classes of MVs within phage lysates, consistent with multiple mechanisms of MV formation. These findings suggest that bacteriophage infection may be a major contributor to the abundance of bacterial MVs in nature.

Live cell dynamics of production, explosive release and killing activity of phage tail-like weapons for Pseudomonas kin exclusion

Interference competition among bacteria requires a highly specialized, narrow-spectrum weaponry when targeting closely-related competitors while sparing individuals from the same clonal population. Here we investigated mechanisms by which environmentally important Pseudomonas bacteria with plant-beneficial activity perform kin interference competition. We show that killing between phylogenetically closely-related strains involves contractile phage tail-like devices called R-tailocins that puncture target cell membranes. Using live-cell imaging, we evidence that R-tailocins are produced at the cell center, transported to the cell poles and ejected by explosive cell lysis. This enables their dispersal over several tens of micrometers to reach targeted cells. We visualize R-tailocin-mediated competition dynamics between closely-related Pseudomonas strains at the single-cell level, both in non-induced condition and upon artificial induction. We document the fatal impact of cellular self-sacrifice coupled to deployment of phage tail-like weaponry in the microenvironment of kin bacterial competitors, emphasizing the necessity for microscale assessment of microbial competitions.

Multiple holins contribute to extracellular DNA release in Pseudomonas aeruginosa biofilms

Bacterial biofilms are composed of aggregates of cells encased within a matrix of extracellular polymeric substances (EPS). One key EPS component is extracellular DNA (eDNA), which acts as a ‘glue’, facilitating cell–cell and cell–substratum interactions. We have previously demonstrated that eDNA is produced in Pseudomonas aeruginosa biofilms via explosive cell lysis. This phenomenon involves a subset of the bacterial population explosively lysing, due to peptidoglycan degradation by the endolysin Lys. Here we demonstrate that in P. aeruginosa three holins, AlpB, CidA and Hol, are involved in Lys-mediated eDNA release within both submerged (hydrated) and interstitial (actively expanding) biofilms, albeit to different extents, depending upon the type of biofilm and the stage of biofilm development. We also demonstrate that eDNA release events determine the sites at which cells begin to cluster to initiate microcolony formation during the early stages of submerged biofilm development. Furthermore, our results show that sustained release of eDNA is required for cell cluster consolidation and subsequent microcolony development in submerged biofilms. Overall, this study adds to our understanding of how eDNA release is controlled temporally and spatially within P. aeruginosa biofilms.

Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa is an opportunistic human pathogen causing devastating acute and chronic infections in individuals with compromised immune systems. Its highly notorious persistence in clinical settings is attributed to its ability to form antibiotic-resistant biofilms. Biofilm is an architecture built mostly by autogenic extracellular polymeric substances which function as a scaffold to encase the bacteria together on surfaces, and to protect them from environmental stresses, impedes phagocytosis and thereby conferring the capacity for colonization and long-term persistence. Here we review the current knowledge on P. aeruginosa biofilms, its development stages, and molecular mechanisms of invasion and persistence conferred by biofilms. Explosive cell lysis within bacterial biofilm to produce essential communal materials, and interspecies biofilms of P. aeruginosa and commensal Streptococcus which impedes P. aeruginosa virulence and possibly improves disease conditions will also be discussed. Recent research on diagnostics of P. aeruginosa infections will be investigated. Finally, therapeutic strategies for the treatment of P. aeruginosa biofilms along with their advantages and limitations will be compiled.

Characterization and proteomic analysis of outer membrane vesicles from a commensal microbe, Enterobacter cloacae

Outer membrane vesicles (OMVs) are membrane-enclosed spherical entities released by gram-negative bacteria and are important for bacterial survival under stress conditions. There have been numerous studies on OMVs released by gram-negative pathogenic bacteria, but an understanding of the functions and characteristics of the OMVs produced by commensal microbes is still lacking. Enterobacter cloacae is a gram-negative commensal bacterium present in the human gut microbiome, but this organism can also function as an opportunistic pathogen. Understanding the OMV-mediated communication route between bacteria-bacteria or bacteria-host is essential for the determination of the biological functions of the commensal bacterium in the gut and delineating between benign and virulent characteristics. In this study, we have described a proteome of E. cloacae OMVs, which are membrane vesicles in a size range of 20–300 nm. Proteomic analysis showed the presence of membrane-bound proteins, including transporters, receptors, signaling molecules, and protein channels. The physical and proteomic analyses also indicate this bacterium uses two mechanisms for OMV production. This study is one of the few existing descriptions of the proteomic profile of OMVs generated by a commensal Proteobacteria, and the first report of OMVs produced by E. cloacae. SignificanceThis study prioritizes the importance of understanding the vesicular proteome of the human commensal bacterium, Enterobacter cloacae. We demonstrate for the first time that the gram-negative bacterium E. cloacae ATCC 13047 produces outer membrane vesicles (OMVs). The proteomic analysis showed enrichment of membrane-bound proteins in these vesicles. Understanding the cargo proteins of OMVs will help in exploring the physiological and functional role of these vesicles in the human microbiome and how they assist in the conversion of a bacterium from commensal to pathogen under certain conditions. While EM images reveal vesicles budding from the bacterial surface, the presence of cytoplasmic proteins and genomic DNA within the OMVs indicate that explosive cell lysis is an additional mechanism of biogenesis for these OMVs along with outer membrane blebbing. This research encourages future work on characterizing membrane vesicles produced by commensal bacterial and investigating their role in cell to cell communication.

Bacterial communication through membrane vesicles.

Bacteria can communicate through diffusible signaling molecules that are perceived by cognate receptors. It is now well established that bacterial communication regulates hundreds of genes. Hydrophobic molecules which do not diffuse in aqueous environments alone have been identified in bacterial communication, that raised the question on how these molecules are transported between cells and trigger gene expressions. Recent studies show that these hydrophobic signaling molecules, including a long-chain N-acyl homoserine lactone signal produced in Paracoccus denitrificans, are carried by membrane vesicles (MVs). MVs were thought to be formed only through the blebbing of the cell membrane, but new findings in Pseudomonas aeruginosa and Bacillus subtilis revealed that different types of MVs can be formed through explosive cell lysis or bubbling cell death, which findings have certain implications on our view of bacterial interactions.

Nontypeable Haemophilus influenzae releases DNA and DNABII proteins via a T4SS-like complex and ComE of the type IV pilus machinery

Biofilms formed by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and resistance to treatment of multiple human respiratory tract diseases including otitis media, chronic rhinosinusitis, and exacerbations of both cystic fibrosis and chronic obstructive pulmonary disease. Extracellular DNA (eDNA) and associated DNABII proteins are essential to the overall architecture and structural integrity of biofilms formed by NTHI and all other bacterial pathogens tested to date. Although cell lysis and outer-membrane vesicle extrusion are possible means by which these canonically intracellular components might be released into the extracellular environment for incorporation into the biofilm matrix, we hypothesized that NTHI additionally used a mechanism of active DNA release. Herein, we describe a mechanism whereby DNA and associated DNABII proteins transit from the bacterial cytoplasm to the periplasm via an inner-membrane pore complex (TraC and TraG) with homology to type IV secretion-like systems. These components exit the bacterial cell through the ComE pore through which the NTHI type IV pilus is expressed. The described mechanism is independent of explosive cell lysis or cell death, and the release of DNA is confined to a discrete subpolar location, which suggests a novel form of DNA release from viable NTHI. Identification of the mechanisms and determination of the kinetics by which critical biofilm matrix-stabilizing components are released will aid in the design of novel biofilm-targeted therapeutic and preventative strategies for diseases caused by NTHI and many other human pathogens known to integrate eDNA and DNABII proteins into their biofilm matrix.

Biofilms: Have exploding cells blown up MV dogma?

Membrane vesicles in Pseudomonas aeruginosa biofilms are not produced by blebbing of the outer membrane but instead by explosive cell lysis, which also releases extracellular DNA into the biofilm matrix.

Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms

Many bacteria produce extracellular and surface-associated components such as membrane vesicles (MVs), extracellular DNA and moonlighting cytosolic proteins for which the biogenesis and export pathways are not fully understood. Here we show that the explosive cell lysis of a sub-population of cells accounts for the liberation of cytosolic content in Pseudomonas aeruginosa biofilms. Super-resolution microscopy reveals that explosive cell lysis also produces shattered membrane fragments that rapidly form MVs. A prophage endolysin encoded within the R- and F-pyocin gene cluster is essential for explosive cell lysis. Endolysin-deficient mutants are defective in MV production and biofilm development, consistent with a crucial role in the biogenesis of MVs and liberation of extracellular DNA and other biofilm matrix components. Our findings reveal that explosive cell lysis, mediated through the activity of a cryptic prophage endolysin, acts as a mechanism for the production of bacterial MVs.