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Meta J Kuehn - One of the best experts on this subject based on the ideXlab platform.
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protective plant immune responses are elicited by Bacterial Outer Membrane vesicles
Cell Reports, 2021Co-Authors: Hannah M Mcmillan, Sophia G Zebell, Jean B. Ristaino, Xinnian Dong, Meta J KuehnAbstract:Summary Bacterial Outer Membrane vesicles (OMVs) perform a variety of functions in Bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and are exploited as vaccines. However, little work has focused on the interactions of OMVs with plant hosts. Here, we report that OMVs from Pseudomonas syringae and P. fluorescens activate plant immune responses that protect against Bacterial and oomycete pathogens. OMV-mediated immunomodulatory activity from these species displayed different sensitivity to biochemical stressors, reflecting differences in OMV content. Importantly, OMV-mediated plant responses are distinct from those triggered by conserved Bacterial epitopes or effector molecules alone. Our study shows that OMV-induced protective immune responses are independent of the T3SS and protein, but that OMV-mediated seedling growth inhibition largely depends on proteinaceous components. OMVs provide a unique opportunity to understand the interplay between virulence and host response strategies and add a new dimension to consider in host-microbe interactions.
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Protective Plant Immune Responses are Elicited by Bacterial Outer Membrane Vesicles
2020Co-Authors: Hannah M Mcmillan, Sophia G Zebell, Jean B. Ristaino, Xinnian Dong, Meta J KuehnAbstract:Bacterial Outer Membrane vesicles (OMVs) perform a variety of functions in Bacterial survival and virulence. In mammalian systems, OMVs activate immune responses and have been exploited as vaccines. However, little work has focused on the role that OMVs play during interactions with plant hosts. Here we report that OMVs from the pathogenic Pseudomonas syringae and the beneficial Pseudomonas fluorescens activate plant immune responses that protect against Bacterial and oomycete pathogens. OMVs from these two species display different sensitivity to biochemical stressors, which could indicate differences in OMV cargo packaging. Furthermore, our study shows that OMV-induced protective immune responses are T3SS- and protein-independent, while OMV-mediated seedling growth inhibition largely depends on protein cargo. Importantly, OMV-mediated plant responses are distinct from those triggered by PAMP/MAMPs or effector molecules alone. OMVs provide a unique opportunity to study virulence factors in combination and add a new layer of interaction and complexity to host-microbe interactions.
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inflammasome activation by Bacterial Outer Membrane vesicles requires guanylate binding proteins
Mbio, 2017Co-Authors: Ryan Finethy, Meta J Kuehn, Sarah Luoma, Nichole Orenchrivera, Eric M Feeley, Arun K Haldar, Masahiro Yamamoto, Thirumaladevi Kanneganti, Jorn CoersAbstract:The Gram-negative Bacterial cell wall component lipopolysaccharide (LPS) is recognized by the noncanonical inflammasome protein caspase-11 in the cytosol of infected host cells and thereby prompts an inflammatory immune response linked to sepsis. Host guanylate binding proteins (GBPs) promote infection-induced caspase-11 activation in tissue culture models, and yet their in vivo role in LPS-mediated sepsis has remained unexplored. LPS can be released from lysed bacteria as "free" LPS aggregates or actively secreted by live bacteria as a component of Outer Membrane vesicles (OMVs). Here, we report that GBPs control inflammation and sepsis in mice injected with either free LPS or purified OMVs derived from Gram-negative Escherichia coli In agreement with our observations from in vivo experiments, we demonstrate that macrophages lacking GBP2 expression fail to induce pyroptotic cell death and proinflammatory interleukin-1β (IL-1β) and IL-18 secretion when exposed to OMVs. We propose that in order to activate caspase-11 in vivo, GBPs control the processing of bacterium-derived OMVs by macrophages as well as the processing of circulating free LPS by as-yet-undetermined cell types.IMPORTANCE The Bacterial cell wall component LPS is a strong inducer of inflammation and is responsible for much of the toxicity of Gram-negative bacteria. Bacteria shed some of their cell wall and its associated LPS in the form of Outer Membrane vesicles (OMVs). Recent work demonstrated that secreted OMVs deliver LPS into the host cell cytosol by an unknown mechanism, resulting in the activation of the proinflammatory LPS sensor caspase-11. Here, we show that activation of cytosolic caspase-11 by OMVs requires additional host factors, the so-called guanylate binding proteins (GBPs). The discovery of GBPs as regulators of OMV-mediated inflammation paves the way toward a mechanistic understanding of the host response toward Bacterial OMVs and may lead to effective strategies to ameliorate inflammation induced by Bacterial infections.
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modulation of Bacterial Outer Membrane vesicle production by envelope structure and content
BMC Microbiology, 2014Co-Authors: Carmen Schwechheimer, Meta J KuehnAbstract:Background Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where Outer Membrane vesicles (OMVs) are small spherical particles on the order of 50 to 250 nm composed of Outer Membrane (OM) and lumenal periplasmic content. Vesicle functions have been elucidated in some detail, showing their importance in virulence factor secretion, Bacterial survival, and biofilm formation in pathogenesis. Furthermore, OMVs serve as an envelope stress response, protecting the secreting bacteria from internal protein misfolding stress, as well as external envelope stressors. Despite their important functional roles very little is known about the regulation and mechanism of vesicle production. Based on the envelope architecture and prior characterization of the hypervesiculation phenotypes for mutants lacking the lipoprotein, Lpp, which is involved in the covalent OM-peptidoglycan (PG) crosslinks, it is expected that an inverse relationship exists between OMV production and PG-crosslinked Lpp.
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modulation of Bacterial Outer Membrane vesicle production by envelope structure and content
BMC Microbiology, 2014Co-Authors: Carmen Schwechheimer, Adam Kulp, Meta J KuehnAbstract:Vesiculation is a ubiquitous secretion process of Gram-negative bacteria, where Outer Membrane vesicles (OMVs) are small spherical particles on the order of 50 to 250 nm composed of Outer Membrane (OM) and lumenal periplasmic content. Vesicle functions have been elucidated in some detail, showing their importance in virulence factor secretion, Bacterial survival, and biofilm formation in pathogenesis. Furthermore, OMVs serve as an envelope stress response, protecting the secreting bacteria from internal protein misfolding stress, as well as external envelope stressors. Despite their important functional roles very little is known about the regulation and mechanism of vesicle production. Based on the envelope architecture and prior characterization of the hypervesiculation phenotypes for mutants lacking the lipoprotein, Lpp, which is involved in the covalent OM-peptidoglycan (PG) crosslinks, it is expected that an inverse relationship exists between OMV production and PG-crosslinked Lpp. In this study, we found that subtle modifications of PG remodeling and crosslinking modulate OMV production, inversely correlating with bound Lpp levels. However, this inverse relationship was not found in strains in which OMV production is driven by an increase in “periplasmic pressure” resulting from the accumulation of protein, PG fragments, or lipopolysaccharide. In addition, the characterization of an nlpA deletion in backgrounds lacking either Lpp- or OmpA-mediated envelope crosslinks demonstrated a novel role for NlpA in envelope architecture. From this work, we conclude that OMV production can be driven by distinct Lpp concentration-dependent and Lpp concentration-independent pathways.
Harris D Bernstein - One of the best experts on this subject based on the ideXlab platform.
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cryo em structures reveal multiple stages of Bacterial Outer Membrane protein folding
bioRxiv, 2021Co-Authors: Matthew Thomas Doyle, John R Jimah, Jenny E Hinshaw, Harris D BernsteinAbstract:TransMembrane β barrel proteins are folded into the Outer Membrane (OM) of Gram-negative bacteria by the β barrel assembly machine (BAM) via an unexplained process that occurs without known external energy sources. Here we used single-particle cryo-EM to visualize the folding dynamics of a model β barrel protein (EspP) by BAM. We found that BAM binds the highly conserved β signal motif of EspP to correctly orient β strands in the OM during folding. We also found that the folding of EspP proceeds via remarkable hybrid-barrel intermediates in which Membrane integrated β sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the Membrane surrounding the EspP intermediates and suggest that β sheets progressively fold towards BamA to form a β barrel. Along with in vivo experiments that tracked β barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate β barrel folding.
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Bam complex-mediated assembly of Bacterial Outer Membrane proteins synthesized in an in vitro translation system.
Scientific Reports, 2020Co-Authors: Sunyia Hussain, Janine H. Peterson, Harris D BernsteinAbstract:Bacterial Outer Membrane proteins (OMPs) contain a unique "β barrel" segment that is inserted into the Membrane by the barrel assembly machinery (Bam) complex by an unknown mechanism. OMP assembly has been reconstituted in vitro, but assembly reactions have involved the use of urea-denatured protein purified from inclusion bodies. Here we show that the E. coli Bam complex catalyzes the efficient assembly of OMPs synthesized de novo in a coupled in vitro transcription/translation system. Interestingly, the in vitro translated forms of the OMPs we analyzed were assembled more rapidly and were effectively engaged by fewer periplasmic chaperones than their urea-denatured counterparts. Taken together, our results strongly suggest that the mode of production influences the conformational states sampled by OMPs and thereby affects their recognition by both chaperones and the Bam complex. Besides providing insights into OMP biogenesis, our work describes a novel, streamlined method to reconstitute OMP assembly in vitro.
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Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway.
mBio, 2019Co-Authors: Rakesh Sikdar, Harris D BernsteinAbstract:ABSTRACT Trimeric autotransporter adhesins (TAAs) are a family of Bacterial Outer Membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular (“passenger”) domain and a short C-terminal segment that contributes four β strands to a single 12-stranded β barrel. The mechanism by which the passenger domains are translocated across the OM and the energetics of the translocation reaction are poorly understood. To address these issues, we examined the secretion of modified versions of the passenger domain of UpaG, a TAA produced by Escherichia coli CFT073. Using the SpyTag-SpyCatcher system to probe passenger domain localization, we found that both intrinsically disordered polypeptides fused to the UpaG passenger domain and artificially disulfide-bonded polypeptides were secreted effectively but relatively slowly. Surprisingly, we also found that in some cases, the three nonnative passenger domain segments associated with a single trimer were secreted sequentially. Photo-cross-linking experiments indicated that incompletely assembled UpaG derivatives remained bound to the barrel assembly machinery (Bam) complex until all three passenger domains were fully secreted. Taken together, our results strongly suggest that the secretion of polypeptides through the TAA pathway is coordinated with the assembly of the β barrel domain and that the folding of passenger domains in the extracellular space maximizes the rate of secretion. Furthermore, our work provides evidence for an unprecedented sequential mode of protein translocation, at least under specific experimental conditions. IMPORTANCE Trimeric autotransporter adhesins (TAAs) are specialized Bacterial Outer Membrane proteins consisting of three identical subunits. TAAs contain large extracellular domains that trimerize and promote virulence, but the mechanism by which they are secreted is poorly understood. We found that the extracellular domains of a native TAA were secreted rapidly but that disordered and artificially folded polypeptides fused to native passenger domains were secreted in a slow, sequential fashion. Our results strongly suggest that the efficient secretion of native extracellular domains is driven by their trimerization following export but that alternative energy sources can be harnessed to secrete nonnative polypeptides. Furthermore, we obtained evidence that TAA extracellular domains are secreted before the assembly of the linked Membrane spanning domain is completed.
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Bacterial Outer Membrane proteins assemble via asymmetric interactions with the bama β barrel
Nature Communications, 2019Co-Authors: Matthew T Doyle, Harris D BernsteinAbstract:The integration of β-barrel proteins into the Bacterial Outer Membrane (OM) is catalysed by the β-barrel assembly machinery (BAM). The central BAM subunit (BamA) itself contains a β-barrel domain that is essential for OM protein biogenesis, but its mechanism of action is unknown. To elucidate its function, here we develop a method to trap a native Escherichia coli β-barrel protein bound stably to BamA at a late stage of assembly in vivo. Using disulfide-bond crosslinking, we find that the first β-strand of a laterally ‘open’ form of the BamA β-barrel forms a rigid interface with the C-terminal β-strand of the substrate. In contrast, the lipid-facing surface of the last two BamA β-strands forms weaker, conformationally heterogeneous interactions with the first β-strand of the substrate that likely represent intermediate assembly states. Based on our results, we propose that BamA promotes the Membrane integration of partially folded β-barrels by a ‘swing’ mechanism. The integration of β-barrel proteins into the Bacterial Outer Membrane (OM) is catalysed by the β-barrel assembly machinery (BAM). Here authors develop a method to trap an E. coli β-barrel protein bound stably to BamA at a late stage of assembly in vivo which provides insights BamA mediated Membrane integration.
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Bacterial Outer Membrane proteins assemble via asymmetric interactions with the BamA β-barrel.
Nature Communications, 2019Co-Authors: Matthew T Doyle, Harris D BernsteinAbstract:The integration of β-barrel proteins into the Bacterial Outer Membrane (OM) is catalysed by the β-barrel assembly machinery (BAM). The central BAM subunit (BamA) itself contains a β-barrel domain that is essential for OM protein biogenesis, but its mechanism of action is unknown. To elucidate its function, here we develop a method to trap a native Escherichia coli β-barrel protein bound stably to BamA at a late stage of assembly in vivo. Using disulfide-bond crosslinking, we find that the first β-strand of a laterally 'open' form of the BamA β-barrel forms a rigid interface with the C-terminal β-strand of the substrate. In contrast, the lipid-facing surface of the last two BamA β-strands forms weaker, conformationally heterogeneous interactions with the first β-strand of the substrate that likely represent intermediate assembly states. Based on our results, we propose that BamA promotes the Membrane integration of partially folded β-barrels by a 'swing' mechanism.
Jan Tommassen - One of the best experts on this subject based on the ideXlab platform.
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Biogenesis of the gram-negative Bacterial Outer Membrane
Annual Review of Microbiology, 2007Co-Authors: Martine P. Bos, Viviane Robert, Jan TommassenAbstract:The cell envelope of gram-negative bacteria consists of two Membranes, the inner and the Outer Membrane, that are separated by the periplasm. The Outer Membrane consists of phospholipids, lipopolysaccharides, integral Membrane proteins, and lipoproteins. These components are synthesized in the cytoplasm or at the inner leaflet of the inner Membrane and have to be transported across the inner Membrane and through the periplasm to assemble eventually in the correct Membrane. Recent studies in Neisseria meningitidis and Escherichia coli have led to the identification of several machineries implicated in these transport and assembly processes.
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Lipopolysaccharide Transport to the Bacterial Outer Membrane in Spheroplasts
Journal of Biological Chemistry, 2004Co-Authors: Boris Tefsen, Jan Tommassen, Jeroen Geurtsen, Frank Beckers, Hans De CockAbstract:The mechanism of lipopolysaccharide (LPS) transport in Gram-negative bacteria from the inner Membrane to the Outer Membrane is largely unknown. Here, we investigated the possibility that LPS transport proceeds via a soluble intermediate associated with a periplasmic chaperone analogous to the Lol-dependent transport mechanism of lipoproteins. Whereas newly synthesized lipoproteins could be released from spheroplasts of Escherichia coli upon addition of a periplasmic extract containing LolA, de novo synthesized LPS was not released. We demonstrate that LPS synthesized de novo in spheroplasts co-fractionated with the Outer Membranes and that this co-fractionation was dependent on the presence in the spheroplasts of a functional MsbA protein, the protein responsible for the flip-flop of LPS across the inner Membrane. The Outer Membrane localization of the LPS was confirmed by its modification by the Outer Membrane enzyme CrcA (PagP). We conclude that a substantial amount of LPS was translocated to the Outer Membrane in spheroplasts, suggesting that transport proceeds via contact sites between the two Membranes. In contrast to LPS, de novo synthesized phospholipids were not transported to the Outer Membrane in spheroplasts. Apparently, LPS and phospholipids have different requirements for their transport to the Outer Membrane.
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Biogenesis of the Gram-Negative Bacterial Outer Membrane
Current Opinion in Microbiology, 2004Co-Authors: Martine P. Bos, Jan TommassenAbstract:Gram-negative bacteria are bounded by two Membranes. The Outer Membrane consists of phospholipids, lipopolysaccharides, lipoproteins and integral Outer Membrane proteins, all of which are synthesized in the cytoplasm. Recently, much progress has been made in the elucidation of the mechanisms of transport of these molecules over the inner Membrane, through the periplasm and into the Outer Membrane, in part by exploiting the extraordinary capacity of Neisseria meningitidis to survive without lipopolysaccharide.
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Folding of a Bacterial Outer Membrane protein during passage through the periplasm
The EMBO Journal, 1997Co-Authors: Elaine F Eppens, Nico Nouwen, Jan TommassenAbstract:The transport of Bacterial Outer Membrane proteins to their destination might be either a one-step process via the contact zones between the inner and Outer Membrane or a two-step process, implicating a periplasmic intermediate that inserts into the Membrane. Furthermore, folding might precede insertion or vice versa. To address these questions, we have made use of the known 3D-structure of the trimeric porin PhoE of Escherichia coli to engineer intramolecular disulfide bridges into this protein at positions that are not exposed to the periplasm once the protein is correctly assembled. The mutations did not interfere with the biogenesis of the protein, and disulfide bond formation appeared to be dependent on the periplasmic enzyme DsbA, which catalyzes disulfide bond formation in the periplasm. This proves that the protein passes through the periplasm on its way to the Outer Membrane. Furthermore, since the disulfide bonds create elements of tertiary structure within the mutant proteins, it appears that these proteins are at least partially folded before they insert into the Outer Membrane.
Nicolas Bayan - One of the best experts on this subject based on the ideXlab platform.
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in vitro multimerization and Membrane insertion of Bacterial Outer Membrane secretin puld
Journal of Molecular Biology, 2008Co-Authors: Ingrid Guilvout, Mohamed Chami, Andreas Engel, Anthony P. Pugsley, Nicolas Bayan, Catherine Berrier, Alexandre GhaziAbstract:Synthesis of the Klebsiella oxytoca Outer Membrane secretin PulD, or its Membrane-associated core domain, in a liposome-supplemented Escherichia coli in vitro transcription-translation system resulted in the formation of multimers that appeared as typical dodecameric secretin rings when examined by negative-stain electron microscopy. Cryo-electron microscopy of unstained liposomes and differential extraction by urea indicated that the secretin particles were inserted into the liposome Membranes. When made in the presence of the detergent Brij-35, PulD and the core domain were synthesized as monomers. Both proteins caused almost immediate growth cessation when synthesized in E. coli without a signal peptide. The small amounts of PulD synthesized before cell death appeared as multimers with characteristics similar to those of the normal Outer Membrane secretin dodecamers. It was concluded that multimerization and Membrane insertion are intrinsic properties of secretin PulD that are independent of a specific Membrane environment or Membrane-associated factors. The closely related Erwinia chrysanthemi secretin OutD behaved similarly to PulD in all assays, but the more distantly related Neisseria meningitidis secretin PilQ did not form multimers either when made in vitro in the presence of liposomes or when made in E. coli without its signal peptide. This is the first report of the apparently spontaneous in vitro assembly and Membrane insertion of a large Outer Membrane protein complex. Spontaneous multimerization and insertion appear to be restricted to Outer Membrane proteins closely related to PulD.
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Bacterial Outer Membrane secretin PulD assembles and inserts into the inner Membrane in the absence of its pilotin
The EMBO Journal, 2006Co-Authors: Ingrid Guilvout, Mohamed Chami, Andreas Engel, Anthony P. Pugsley, Nicolas BayanAbstract:Dodecamerization and insertion of the Outer Membrane secretin PulD is entirely determined by the C-terminal half of the polypeptide (PulD-CS). In the absence of its cognate chaperone PulS, PulD-CS and PulD mislocalize to the inner Membrane, from which they are extractable with detergents but not urea. Electron microscopy of PulD-CS purified from the inner Membrane revealed apparently normal dodecameric complexes. Electron microscopy of PulD-CS and PulD in inner Membrane vesicles revealed inserted secretin complexes. Mislocalization of PulD or PulD-CS to this Membrane induces the phage shock response, probably as a result of a decreased Membrane electrochemical potential. Production of PulD in the absence of the phage shock response protein PspA and PulS caused a substantial drop in Membrane potential and was lethal. Thus, PulD-CS and PulD assemble in the inner Membrane if they do not associate with PulS. We propose that PulS prevents premature multimerization of PulD and accompanies it through the periplasm to the Outer Membrane. PulD is the first Bacterial Outer Membrane protein with demonstrated ability to insert efficiently into the inner Membrane.
Yong Song Gho - One of the best experts on this subject based on the ideXlab platform.
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Bacterial Outer Membrane vesicles suppress tumor by interferon-γ-mediated antitumor response.
Nature Communications, 2017Co-Authors: Oh Youn Kim, Hyun Taek Park, Nhung Thi Hong Dinh, Seng Jin Choi, Jaewook Lee, Ji Hyun Kim, Seung-woo Lee, Yong Song GhoAbstract:Gram-negative bacteria actively secrete Outer Membrane vesicles, spherical nano-meter-sized proteolipids enriched with Outer Membrane proteins, to the surroundings. Outer Membrane vesicles have gained wide interests as non-living complex vaccines or delivery vehicles. However, no study has used Outer Membrane vesicles in treating cancer thus far. Here we investigate the potential of Bacterial Outer Membrane vesicles as therapeutic agents to treat cancer via immunotherapy. Our results show remarkable capability of Bacterial Outer Membrane vesicles to effectively induce long-term antitumor immune responses that can fully eradicate established tumors without notable adverse effects. Moreover, systematically administered Bacterial Outer Membrane vesicles specifically target and accumulate in the tumor tissue, and subsequently induce the production of antitumor cytokines CXCL10 and interferon-γ. This antitumor effect is interferon-γ dependent, as interferon-γ-deficient mice could not induce such Outer Membrane vesicle-mediated immune response. Together, our results herein demonstrate the potential of Bacterial Outer Membrane vesicles as effective immunotherapeutic agent that can treat various cancers without apparent adverse effects. Bacterial Outer Membrane vesicles (OMVs) contain immunogens but no study has yet examined their potential in treating cancer. Here, the authors demonstrate that OMVs can suppress established tumours and prevent tumour metastasis by an interferon-γ mediated antitumor response.
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proteomic profiling of gram negative Bacterial Outer Membrane vesicles current perspectives
Proteomics Clinical Applications, 2016Co-Authors: Jaewook Lee, Oh Youn Kim, Yong Song GhoAbstract:Outer Membrane vesicles (OMVs) are extracellular vesicles derived from Gram-negative bacteria. Recent progress in the studies of Gram-negative Bacterial extracellular vesicles implies that OMVs may function as intercellular communicasomes in bacteria-bacteria and bacteria-host interactions. Current MS-based high-throughput proteomic analyses of Gram-negative Bacterial OMVs have identified thousands of vesicular proteins and provided clues to reveal the biogenesis and pathophysiological functions of Gram-negative Bacterial OMVs. The future directions of proteomics of Gram-negative Bacterial OMVs may include the isolation strategy of Gram-negative Bacterial OMVs to thoroughly exclude nonvesicular contaminants and proteomics of Gram-negative Bacterial OMVs derived from diverse conditions as well as body fluids of bacterium-infected hosts. We hope this review will shed light on future research in this emerging field of proteomics of extracellular vesicles derived from Gram-negative bacteria and contribute to the development of OMV-based diagnostic tools and effective vaccines.
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proteomics in gram negative Bacterial Outer Membrane vesicles
Mass Spectrometry Reviews, 2008Co-Authors: Eunyoung Lee, Dongsic Choi, Kwang Pyo Kim, Yong Song GhoAbstract:Gram-negative bacteria constitutively secrete Outer Membrane vesicles (OMVs) into the extracellular milieu. Recent research in this area has revealed that OMVs may act as intercellular communicasomes in polyspecies communities by enhancing Bacterial survival and pathogenesis in hosts. However, the mechanisms of vesicle formation and the pathophysiological roles of OMVs have not been clearly defined. While it is obvious that mass spectrometry-based proteomics offers great opportunities for improving our knowledge of Bacterial OMVs, limited proteomic data are available for OMVs. The present review aims to give an overview of the previous biochemical, biological, and proteomic studies in the emerging field of Bacterial OMVs, and to give future directions for high-throughput and comparative proteomic studies of OMVs that originate from diverse Gram-negative bacteria under various environmental conditions. This article will hopefully stimulate further efforts to construct a comprehensive proteome database of Bacterial OMVs that will help us not only to elucidate the biogenesis and functions of OMVs but also to develop diagnostic tools, vaccines, and antibiotics effective against pathogenic bacteria.