Anionic Lipopolysaccharide Research Articles

Low-Molecular Weight Branched Polyethylenimine Reduces Cytokine Secretion from Human Immune System Monocytes Stimulated with Bacterial and Fungal PAMPs.

The innate immune system is an evolutionarily conserved pathogen recognition mechanism that serves as the first line of defense against tissue damage or pathogen invasion. Unlike the adaptive immunity that recruits T-cells and specific antibodies against antigens, innate immune cells express pathogen recognition receptors (PRRs) that can detect various pathogen-associated molecular patterns (PAMPs) released by invading pathogens. Microbial molecular patterns, such as lipopolysaccharide (LPS) from Gram-negative bacteria, trigger signaling cascades in the host that result in the production of pro-inflammatory cytokines. LPS stimulation produces a strong immune response and excessive LPS signaling leads to dysregulation of the immune response. However, dysregulated inflammatory response during wound healing often results in chronic non-healing wounds that are difficult to control. In this work, we present data demonstrating partial neutralization of anionic LPS molecules using cationic branched polyethylenimine (BPEI). The anionic sites on the LPS molecules from Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) are the lipid A moiety and BPEI binding create steric factors that hinder the binding of PRR signaling co-factors. This reduces the production of pro-inflammatory TNF-α cytokines. However, the anionic sites of Pseudomonas aeruginosa (P. aeruginosa) LPS are in the O-antigen region and subsequent BPEI binding slightly reduces TNF-α cytokine production. Fortunately, BPEI can reduce TNF-α cytokine expression in response to stimulation by intact P. aeruginosa bacterial cells and fungal zymosan PAMPs. Thus low-molecular weight (600 Da) BPEI may be able to counter dysregulated inflammation in chronic wounds and promote successful repair following tissue injury.

Amine Basicity of Quinoline ATP Synthase Inhibitors Drives Antibacterial Activity against Pseudomonas aeruginosa.

Pseudomonas aeruginosa (PA), a Gram-negative pathogen, is a common cause of nosocomial infections, especially in immunocompromised and cystic fibrosis patients. PA is intrinsically resistant to many currently prescribed antibiotics due to its tightly packed, anionic lipopolysaccharide outer membrane, efflux pumps, and ability to form biofilms. PA can acquire additional resistance through mutation and horizontal gene transfer. PA ATP synthase is an attractive target for antibiotic development because it is essential for cell survival even under fermentation conditions. Previously, we developed two lead quinoline compounds that were capable of selectively inhibiting PA ATP synthase and acting as antibacterial agents against multidrug-resistant PA. Herein we conduct a structure-activity relationship analysis of the lead compounds through the synthesis and evaluation of 18 quinoline derivatives. These compounds function as new antibacterial agents while providing insight into the balance of physical properties needed to promote cellular entry while maintaining PA ATP synthase inhibition.

Antibacterial Synthetic Nanocelluloses Synergizing with a Metal-Chelating Agent.

Antibacterial materials composed of biodegradable and biocompatible constituents that are produced via eco-friendly synthetic strategies will become an attractive alternative to antibiotics to combat antibiotic-resistant bacteria. In this study, we demonstrated the antibacterial properties of nanosheet-shaped crystalline assemblies of enzymatically synthesized aminated cellulose oligomers (namely, surface-aminated synthetic nanocelluloses) and their synergy with a metal-chelating antibacterial agent, ethylenediaminetetraacetic acid (EDTA). Growth curves and colony counting assays revealed that the surface-aminated cellulose assemblies had an antibacterial effect against Gram-negative Escherichia coli (E. coli). The cationic assemblies appeared to destabilize the cell wall of E. coli through electrostatic interactions with anionic lipopolysaccharide (LPS) molecules on the outer membrane. The antibacterial properties were significantly enhanced by the concurrent use of EDTA, which potentially removed metal ions from LPS molecules, resulting in synergistic bactericidal effects. No antibacterial activity of the surface-aminated cellulose assemblies was observed against Gram-positive Staphylococcus aureus even in the presence of EDTA, further supporting the contribution of electrostatic interactions between the cationic assemblies and anionic LPS to the activity against Gram-negative bacteria. Analysis using quartz crystal microbalance with dissipation monitoring revealed the attractive interaction of the surface-aminated cellulose assembly with LPS Ra monolayers artificially produced on the device substrate.

Structural insights on the selective interaction of the histidine-rich piscidin antimicrobial peptide Of-Pis1 with membranes

Of-Pis1 is a potent piscidin antimicrobial peptide (AMP), recently isolated from rock bream (Oplegnathus fasciatus). This rich in histidines and glycines 24-amino acid peptide displays high and broad antimicrobial activity and no significant hemolytic toxicity against human erythrocytes, suggesting low toxicity. To better understand the mechanism of action of Of-Pis1 and its potential selectivity, using NMR and CD spectroscopies, we studied the interaction with eukaryotic and procaryotic membranes and membrane models. Anionic sodium dodecyl sulfate (SDS) and lipopolysaccharide (LPS) micelles were used to mimic procaryotic membranes, while zwitterionic dodecyl phosphocholine (DPC) was used as eukaryotic membrane surrogate. In an aqueous environment, Of-Pis1 adopts a flexible random coil conformation. In DPC and SDS instead, the N-terminal region of Of-Pis1 forms an amphipathic α-helix with the non-polar face in close contact with the micelles. Slower solvent exchange and higher pKas of the histidine residues in SDS than in DPC suggest that Of-Pis1 interacts more tightly with SDS. Of-Pis1 also binds tightly and structurally perturbs LPS micelles. Of-Pis1 interacts with both Escherichia coli and mammalian cell membranes, but only in the presence of Escherichia coli membranes it populates the helical conformation. Furthermore, ligand-based NMR experiments support a tighter and more specific interaction with bacterial than with eukaryotic membranes. Overall, these data clearly show the selective interaction of this broadly active AMP with bacterial over eukaryotic membranes. The conformational information is discussed in terms of Of-Pis1 amino acid sequence and composition to provide insights useful to design more potent and selective AMPs.

Type B CTD Proteins Secreted by the Type IX Secretion System Associate with PorP-like Proteins for Cell Surface Anchorage

The Bacteroidetes type IX secretion system (T9SS) consists of at least 20 components that translocate proteins with type A or type B C-terminal domain (CTD) signals across the outer membrane (OM). While type A CTD proteins are anchored to the cell surface via covalent linkage to the anionic lipopolysaccharide, it is still unclear how type B CTD proteins are anchored to the cell surface. Moreover, very little is known about the PorE and PorP components of the T9SS. In this study, for the first time, we identified a complex comprising the OM β-barrel protein PorP, the OM-associated periplasmic protein PorE and the type B CTD protein PG1035. Cross-linking studies supported direct interactions between PorE-PorP and PorP-PG1035. Furthermore, we show that the formation of the PorE-PorP-PG1035 complex was independent of PorU and PorV. Additionally, the Flavobacterium johnsoniae PorP-like protein, SprF, was found bound to the major gliding motility adhesin, SprB, which is also a type B CTD protein. Together, these results suggest that type B-CTD proteins may anchor to the cell surface by binding to their respective PorP-like proteins.

Intermolecular latency regulates the essential C-terminal signal peptidase and sortase of the Porphyromonas gingivalis type-IX secretion system

Porphyromonas gingivalis is a keystone pathogen of the human dysbiotic oral microbiome that causes severe periodontitis. It employs a type-IX secretion system (T9SS) to shuttle proteins across the outer membrane (OM) for virulence. Uniquely, T9SS cargoes carry a C-terminal domain (CTD) as a secretion signal, which is cleaved and replaced with anionic lipopolysaccharide by transpeptidation for extracellular anchorage to the OM. Both reactions are carried out by PorU, the only known dual-function, C-terminal signal peptidase and sortase. PorU is itself secreted by the T9SS, but its CTD is not removed; instead, intact PorU combines with PorQ, PorV, and PorZ in the OM-inserted "attachment complex." Herein, we revealed that PorU transits between active monomers and latent dimers and solved the crystal structure of the ∼260-kDa dimer. PorU has an elongated shape ∼130 Å in length and consists of seven domains. The first three form an intertwined N-terminal cluster likely engaged in substrate binding. They are followed by a gingipain-type catalytic domain (CD), two immunoglobulin-like domains (IGL), and the CTD. In the first IGL, a long "latency β-hairpin" protrudes ∼30 Å from the surface to form an intermolecular β-barrel with β-strands from the symmetric CD, which is in a latent conformation. Homology modeling of the competent CD followed by in vivo validation through a cohort of mutant strains revealed that PorU is transported and functions as a monomer through a C690/H657 catalytic dyad. Thus, dimerization is an intermolecular mechanism for PorU regulation to prevent untimely activity until joining the attachment complex.

Articles of Significant Interest in This Issue

Porphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, a disease leading to tooth loss involving complex interactions between the oral microbiota and host. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to surface anionic lipopolysaccharide (A-LPS), which binds/stores extracted iron as a black-pigmented heme dimer. Glew et al. (e00631-20) show that P. gingivalis can exchange A-LPS between cells to complement A-LPS biosynthetic mutants and regain pigmentation and T9SS-dependent protein anchorage. The potential for P. gingivalis to exchange LPS and iron with other oral species reveals an important disease mechanism.

Complementation in trans of Porphyromonas gingivalis Lipopolysaccharide Biosynthetic Mutants Demonstrates Lipopolysaccharide Exchange.

Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host.IMPORTANCEPorphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.

Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

A novel cecropin B-derived peptide with antibacterial and potential anti-inflammatory properties.

Cecropins, originally found in insects, are a group of cationic antimicrobial peptides. Most cecropins have an amphipathic N-terminal segment and a largely hydrophobic C-terminal segment, and normally form a helix-hinge-helix structure. In this study, we developed the novel 32-residue cecropin-like peptide cecropin DH by deleting the hinge region (Alanine-Glycine-Proline) of cecropin B isolated from Chinese oak silk moth, Antheraea pernyi. Cecropin DH possesses effective antibacterial activity, particularly against Gram-negative bacteria, with very low cytotoxicity against mammalian cells. Interactions between cecropin DH and the highly anionic lipopolysaccharide (LPS) component of the Gram-negative bacterial outer membrane indicate that it is capable of dissociating LPS micelles and disrupting LPS aggregates into smaller assemblies, which may play a vital role in its antimicrobial activity. Using LPS-stimulated mouse macrophage RAW264.7 cells, we found that cecropin DH exerted higher potential anti-inflammatory activity than cecropin B, as demonstrated by the inhibition of pro-inflammatory cytokines nitric oxide production and secretion of tumor necrosis factor-α. In conclusion, cecropin DH has potential as a therapeutic agent for both antibacterial and anti-inflammatory applications.

PorV is an Outer Membrane Shuttle Protein for the Type IX Secretion System

Porphyromonas gingivalis is a keystone pathogen associated with chronic periodontitis. Major virulence factors named gingipains (cysteine proteinases, RgpA, RgpB and Kgp) are secreted via the Type IX Secretion System (T9SS). These, together with approximately 30 other proteins, are secreted to the cell surface and anchored to the outer membrane by covalent modification to anionic lipopolysaccharide (A-LPS) via the novel Gram negative sortase, PorU. PorU is localised on the cell surface and cleaves the C-terminal domain signal (CTD) of T9SS substrates and conjugates their new C-termini to A-LPS. A 440 kDa-attachment complex was identified in the wild-type (WT) comprising of PorU:PorV:PorQ:PorZ. In mutant strains, sub-complexes comprising PorU:PorV or PorQ:PorZ were also identified at smaller native sizes suggesting that PorU and PorZ are anchored to the cell surface via interaction with the PorV and PorQ outer membrane proteins, respectively. Analysis of porU mutants and a CTD cleavage mutant revealed accumulation of immature T9SS substrates in a PorV-bound form. Quantitative label-free proteomics of WT whole cell lysates estimated that the proportion of secretion channels:attachment complexes:free PorV:T9SS substrates was 1:6:110:2000 supporting a role for PorV as a shuttle protein delivering secreted proteins to the attachment complex for CTD signal cleavage and A-LPS modification.

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two β-propeller domains and a C-terminal β-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

PG1058 Is a Novel Multidomain Protein Component of the Bacterial Type IX Secretion System.

Porphyromonas gingivalis utilises the Bacteroidetes-specific type IX secretion system (T9SS) to export proteins across the outer membrane (OM), including virulence factors such as the gingipains. The secreted proteins have a conserved carboxy-terminal domain essential for type IX secretion that is cleaved upon export. In P. gingivalis the T9SS substrates undergo glycosylation with anionic lipopolysaccharide (A-LPS) and are attached to the OM. In this study, comparative analyses of 24 Bacteroidetes genomes identified ten putative novel components of the T9SS in P. gingivalis, one of which was PG1058. Computer modelling of the PG1058 structure predicted a novel N- to C-terminal architecture comprising a tetratricopeptide repeat (TPR) domain, a β-propeller domain, a carboxypeptidase regulatory domain-like fold (CRD) and an OmpA_C-like putative peptidoglycan binding domain. Inactivation of pg1058 in P. gingivalis resulted in loss of both colonial pigmentation and surface-associated proteolytic activity; a phenotype common to T9SS mutants. Immunoblot and LC-MS/MS analyses of subcellular fractions revealed T9SS substrates accumulated within the pg1058 mutant periplasm whilst whole-cell ELISA showed the Kgp gingipain was absent from the cell surface, confirming perturbed T9SS function. Immunoblot, TEM and whole-cell ELISA analyses indicated A-LPS was produced and present on the pg1058 mutant cell surface although it was not linked to T9SS substrate proteins. This indicated that PG1058 is crucial for export of T9SS substrates but not for the translocation of A-LPS. PG1058 is a predicted lipoprotein and was localised to the periplasmic side of the OM using whole-cell ELISA, immunoblot and LC-MS/MS analyses of subcellular fractions. The structural prediction and localisation of PG1058 suggests that it may have a role as an essential scaffold linking the periplasmic and OM components of the T9SS.

The Effect of Lipopolysaccharide Core Oligosaccharide Size on the Electrostatic Binding of Antimicrobial Proteins to Models of the Gram Negative Bacterial Outer Membrane.

Understanding the electrostatic interactions between bacterial membranes and exogenous proteins is crucial to designing effective antimicrobial agents against Gram-negative bacteria. Here we study, using neutron reflecometry under multiple isotopic contrast conditions, the role of the uncharged sugar groups in the outer core region of lipopolysaccharide (LPS) in protecting the phosphate-rich inner core region from electrostatic interactions with antimicrobial proteins. Models of the asymmetric Gram negative outer membrane on silicon were prepared with phopshatidylcholine (PC) in the inner leaflet (closest to the silicon), whereas rough LPS was used to form the outer leaflet (facing the bulk solution). We show how salt concentration can be used to reversibly alter the binding affinity of a protein antibiotic colicin N (ColN) to the anionic LPS confirming that the interaction is electrostatic in nature. By examining the interaction of ColN with two rough LPS types with different-sized core oligosaccharide regions we demonstrate the role of uncharged sugars in blocking short-range electrostatic interactions between the cationic antibiotics and the vulnerable anionic phosphate groups.

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal β-sandwich domain.

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel β-strands organized in two β-sheets, packed into a β-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.

Spheres of influence: Porphyromonas gingivalis outer membrane vesicles.

Outer membrane vesicles (OMVs) are asymmetrical single bilayer membranous nanostructures produced by Gram-negative bacteria important for bacterial interaction with the environment. Porphyromonas gingivalis, a keystone pathogen associated with chronic periodontitis, produces OMVs that act as a virulence factor secretion system contributing to its pathogenicity. Despite their biological importance, the mechanisms of OMV biogenesis have not been fully elucidated. The ~14 times more curvature of the OMV membrane than cell outer membrane (OM) indicates that OMV biogenesis requires energy expenditure for significant curvature of the OMV membrane. In P.gingivalis, we propose that this may be achieved by upregulating the production of certain inner or outer leaflet lipids, which causes localized outward curvature of the OM. This results in selection of anionic lipopolysaccharide (A-LPS) and associated C-terminal domain (CTD) -family proteins on the outer surface due to their ability to accommodate the curvature. Deacylation of A-LPS may further enable increased curvature leading to OMV formation. Porphyromonas gingivalis OMVs that are selectively enriched in CTD-family proteins, largely the gingipains, can support bacterial coaggregation, promote biofilm development and act as an intercessor for the transport of non-motile bacteria by motile bacteria. The P.gingivalis OMVs are also believed to contribute to host interaction and colonization, evasion of immune defense mechanisms, and destruction of periodontal tissues. They may be crucial for both micro- and macronutrient capture, especially heme and probably other assimilable compounds for its own benefit and that of the wider biofilm community.

Medium effects on minimum inhibitory concentrations of nylon-3 polymers against E. coli.

Minimum inhibitory concentrations (MICs) against E. coli were measured for three nylon-3 polymers using Luria-Bertani broth (LB), brain-heart infusion broth (BHI), and a chemically defined complete medium (EZRDM). The polymers differ in the ratio of hydrophobic to cationic subunits. The cationic homopolymer is inert against E. coli in BHI and LB, but becomes highly potent in EZRDM. A mixed hydrophobic/cationic polymer with a hydrophobic t-butylbenzoyl group at its N-terminus is effective in BHI, but becomes more effective in EZRDM. Supplementation of EZRDM with the tryptic digest of casein (often found in LB) recapitulates the LB and BHI behavior. Additional evidence suggests that polyanionic peptides present in LB and BHI may form electrostatic complexes with cationic polymers, decreasing activity by diminishing binding to the anionic lipopolysaccharide layer of E. coli. In contrast, two natural antimicrobial peptides show no medium effects. Thus, the use of a chemically defined medium helps to reveal factors that influence antimicrobial potency of cationic polymers and functional differences between these polymers and evolved antimicrobial peptides.

Analysis of a Lys-specific serine endopeptidase secreted via the type IX secretion system inPorphyromonas gingivalis

Porphyromonas gingivalis, a significant causative agent of adult periodontitis, possesses a novel secretion system called the type IX secretion system (T9SS). A number of virulence factors, such as Arg-gingipain (Rgp), are translocated onto the cell surface and into the extracellular milieu via the T9SS. In this study, we found that the PGN_1416 90- to 120-kDa diffuse protein bands were located in the outer membrane fraction and that the presence of the bands was dependent on genes involved in the T9SS and the formation of anionic lipopolysaccharide (A-LPS). These data strongly suggest that the PGN_1416 protein is secreted by the T9SS and anchored onto the cell surface by binding to A-LPS. Enzymatic analysis using outer membrane fractions suggested that the PGN_1416 protein has a Lys-specific serine endopeptidase activity and that its activation requires processing by Rgp. Homologues of the gene encoding PGN_1416, which is referred to as pepK, were found in bacteria belonging to the phyla Bacteroidetes and Proteobacteria, whereas homologues encoding the C-terminal domain, which is essential for T9SS-mediated secretion, and the catalytic domain were only observed in bacteria belonging to the Bacteroidetes phylum.

Effects of chitosan-nanoparticle-mediated tight junction opening on the oral absorption of endotoxins

Recently, we reported a pH-responsive nanoparticle (NP) system shelled with chitosan (CS), which could effectively increase the oral absorption of insulin and produce a hypoglycemic effect, presumably due to the CS-mediated tight junction (TJ) opening. It has been often questioned whether CS can also enhance the absorption of endotoxins present in the small intestine. To address this concern, we studied the effect of CS NPs on the absorption of lipopolysaccharide (LPS), the most commonly found toxin in the gastrointestinal tract. To follow their biodistribution by the single-photon emission computed tomography/computed tomography, LPS and insulin were labeled with 99mTc-pertechnetate ( 99mTc-LPS) and 123iodine ( 123I-insulin), respectively. The 99mTc-LPS was ingested 1 h prior to the administration of the 123I-insulin-loaded NPs to mimic the physiological conditions. The confocal and TEM micrographs show that the orally administered CS NPs were able to adhere and infiltrate through the mucus layer, approach the epithelial cells and mediate to open their TJs. The radioactivity associated with LPS was mainly restricted to the gastrointestinal tract, whereas 123I-insulin started to appear in the urinary bladder at 3 h post administration. This observation indicates that the insulin-loaded in CS NPs can traverse across the intestinal epithelium and enter the systemic circulation, whereas LPS was unable to do so, probably because of the charge repulsion between the anionic LPS in the form of micelles and the negatively charged mucus layer. Our in vivo toxicity study further confirms that the enhancement of paracellular permeation by CS NPs did not promote the absorption of LPS. These results suggest that CS NPs can be used as a safe carrier for oral delivery of protein drugs.

Analysis of the Networks Controlling the Antimicrobial-Peptide-Dependent Induction of Klebsiella pneumoniae Virulence Factors

Antimicrobial peptides (APs) impose a threat to the survival of pathogens, and it is reasonable to postulate that bacteria have developed strategies to counteract them. Polymyxins are becoming the last resort to treat infections caused by multidrug-resistant Gram-negative bacteria and, similar to APs, they interact with the anionic lipopolysaccharide. Given that polymyxins and APs share the initial target, it is possible that bacterial defense mechanisms against polymyxins will be also effective against host APs. We sought to determine whether exposure to polymyxin will increase Klebsiella pneumoniae resistance to host APs. Indeed, exposure of K. pneumoniae to polymyxin induces cross-resistance not only to polymyxin itself but also to APs present in the airways. Polymyxin treatment upregulates the expression of the capsule polysaccharide operon and the loci required to modify the lipid A with aminoarabinose and palmitate with a concomitant increase in capsule and lipid A species containing such modifications. Moreover, these surface changes contribute to APs resistance and also to polymyxin-induced cross-resistance to APs. Bacterial loads of lipid A mutants in trachea and lungs of intranasally infected mice were lower than those of wild-type strain. PhoPQ, PmrAB, and the Rcs system govern polymyxin-induced transcriptional changes, and there is a cross talk between PhoPQ and the Rcs system. Our findings support the notion that Klebsiella activates a defense program against APs that is controlled by three signaling systems. Therapeutic strategies directed to prevent the activation of this program could be a new approach worth exploring to facilitate the clearance of the pathogen from the airways.