A Murine Skin Infection Model Capable of Differentiating the Dermatopathology of Community-Associated MRSA Strain USA300 from Other MRSA Strains.

USA300 is a predominant and highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strain that is a leading cause of skin and soft tissue infections. We established a murine intradermal infection model capable of demonstrating dermatopathological differences between USA300 and other MRSA strains. In this model, USA300 induced dermonecrosis, uniformly presenting as extensive open lesions with a histologically documented profound inflammatory cell infiltrate extending below the subcutis. In contrast, USA400 and a colonizing control strain M92 caused only localized non-ulcerated skin infections associated with a mild focal inflammatory infiltrate. It was also determined that the dermonecrosis induced by USA300 was associated with significantly increased neutrophil recruitment, inhibition of an antibacterial response, and increased production of cytokines/chemokines associated with disease severity. These results suggest that induction of severe skin lesions by USA300 is related to over-activation of neutrophils, inhibition of host antibacterial responses, and selective alteration of host cytokine/chemokine profiles.

Mass spectrometry-based Shiga toxin identification: A clinical validation.

After we published our preliminary study on the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) and curated E. coli toxin databases on the identification of E. coli Shiga toxins (Stxs) in the Journal of Proteomics in year 2018, we were encouraged to further refine the method and test clinical isolates. In this study, different concentrations of mitomycin C (MMC) and ciprofloxacin (CF), two common antibiotic/chemotherapy agents capable of stimulating Stx production, were first tested and compared on three reference strains and eight clinical isolates to observe the toxin induction and subsequent identification. Notably, no differences were observed between the two agents other than the concentrations applied. Seventeen more clinical isolates were then tested using fixed MMC and CF concentrations and sample amount. This study confirms that the majority of stx2-positive E. coli strains can be stimulated to produce sufficient toxin for confident identification. This does not occur with stx1-positive E. coli isolates, however, despite the fact that both Stxs can be identified for several isolates without MMC or CF stimulation. BIOLOGICAL SIGNIFICANCE: Stxs, especially Stx2, are very important causes of severe food-borne disease, even death. This study confirms that receptor analogue-based affinity enrichment of Stxs, after MMC or CF treatment of E. coli, is useful for fast and accurate Stx2 identification through LC-MS/MS.

Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome.

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1ß secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1ß production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficient THP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.

Mass spectrometry-based Shiga toxin identification: An optimized approach.

Toxin expression is a key factor in Shiga toxin (Stx)-producing E. coli, a common pathogen involved in foodborne disease outbreaks. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based approach has been used in this study to identify commonly reported E. coli toxins, with a focus on Shiga toxins (Stxs). Different sample preparation methods using variable culture conditions and concentrations of mitomycin C (MMC), a common antibiotic/chemotherapy agent capable of stimulating Stx production, were first tested on reference strains EDL933 and 90-2380 by LC-MS/MS detection of tryptic digests of receptor-analogue affinity binding enriched Stx preparations from culture supernatants and lysates. A curated E. coli protein toxin database was also used for faster and more straightforward toxin identification. With eight more genetically confirmed E. coli strains examined to verify the method, this preliminary study indicates that receptor-analogue based affinity enrichment on cell lysate or supernatant is a sensitive and accurate method for Stx identification. BIOLOGICAL SIGNIFICANCE: The existence of Stx is very important for identifying Stx-producing E. coli and implementing a clinical treatment regime. This study demonstrates for the first time that using a curated E. coli toxin database, together with receptor-analogue-based affinity enrichment of Stxs after MMC treatment of E. coli, is an easy and appropriate approach for fast and accurate Stx identification through LC-MS/MS.

BfpI, BfpJ, and BfpK Minor Pilins Are Important for the Function and Biogenesis of Bundle-Forming Pili Expressed by Enteropathogenic Escherichia coli.

UNLABELLED: Enteropathogenic Escherichia coli (EPEC) remains a significant cause of infant diarrheal illness and associated morbidity and mortality in developing countries. EPEC strains are characterized by their ability to colonize the small intestines of their hosts by a multistep program involving initial loose attachment to intestinal epithelial cells followed by an intimate adhesion phase. The initial loose interaction of typical EPEC with host intestinal cells is mediated by bundle-forming pili (BFP). BFP are type 4b pili (T4bP) based on structural and functional properties shared with T4bP expressed by other bacteria. The major structural subunit of BFP is called bundlin, a T4b pilin expressed from the bfpA gene in the BFP operon, which contains three additional genes that encode the pilin-like proteins BfpI, BfpJ, and BfpK. In this study, we show that, in the absence of the BFP retraction ATPase (BfpF), BfpI, BfpJ, and BfpK are dispensable for BFP biogenesis. We also demonstrate that these three minor pilins are incorporated along with bundlin into the BFP filament and contribute to its structural integrity and host cell adhesive properties. The results confirm that previous findings in T4aP systems can be extended to a model T4bP such as BFP. IMPORTANCE: Bundle-forming pili contribute to the host colonization strategy of enteropathogenic Escherichia coli. The studies described here investigate the role for three minor pilin subunits in the structure and function of BFP in EPEC. The studies also suggest that these subunits could be antigens for vaccine development.

The Effects of Shiga Toxin 1, 2 and Their Subunits on Cytokine and Chemokine Expression by Human Macrophage-Like THP-1 Cells.

Infection by Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC) results in severe diarrhea, hemorrhagic colitis, and, occasionally, hemolytic-uremic syndrome (HUS). HUS is associated with an increase in pro-inflammatory cytokines and chemokines, many of which are produced by macrophages in the kidneys, indicating that localized host innate immunity likely plays a role in renal pathogenesis. EHEC serotypes may express one or two classes of serologically defined but structurally and functionally-related Shiga toxins called Stx1 and Stx2. Of these, Stx2 appears to be linked to higher rates of HUS than Stx1. To investigate a possible reason for this, we exposed human macrophage-like THP-1 cells to Stx1 or Stx2 and then used the Luminex multiplex system to assess cytokine/chemokine concentrations in culture supernatant solutions. This analysis revealed that, relative to Stx1, Stx2 significantly caused increased expression of GRO, G-CSF, IL-1ß, IL-8 and TNFα in macrophage-like THP-1 cells. This was determined to not be due to a difference in cytotoxicity since both Stx1 and Stx2 displayed similar cytotoxic activities on macrophage-like THP-1 cells. These observations indicate that, in vitro, Stx2 can provoke a greater pro-inflammatory response than Stx1 in macrophages and provides a possible partial explanation for higher rates of HUS in patients infected with EHEC strains expressing Stx2. To begin to determine a mechanism for Shiga toxin-mediated cytokine production, we exposed macrophage-like THP-1 cells to Stx1 or Stx2 A and B subunits. Luminex analysis of cytokines in cell culture supernatant solutions demonstrated that neither subunit alone induced a cytokine response in THP-1 cells.

Structural and evolutionary analyses show unique stabilization strategies in the type IV pili of Clostridium difficile.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, biofilm formation, cellular adhesion, and horizontal gene transfer. However, many Gram-positive species, including Clostridium difficile, also produce type IV pili. Here, we identify the major subunit of the type IV pili of C. difficile, PilA1, and describe multiple 3D structures of PilA1, demonstrating the diversity found in three strains of C. difficile. We also model the incorporation of both PilA1 and a minor pilin, PilJ, into the pilus fiber. Although PilA1 contains no cysteine residues, and therefore cannot form the disulfide bonds found in all Gram-negative type IV pilins, it adopts unique strategies to achieve a typical pilin fold. The structures of PilA1 and PilJ exhibit similarities with the type IVb pilins from Gram-negative bacteria that suggest that the type IV pili of C. difficile are involved in microcolony formation.

Synthesis and structure-activity relationship studies of N-terminal analogues of the antimicrobial peptide tridecaptin A(1).

Chemical synthesis was used to increase the potency of the antimicrobial lipopeptide tridecaptin A1. Lipid tail modification proved to be an ideal platform for synthesizing structurally simpler analogues that are not readily accessible by isolation. The stereochemical elements of the tridecaptin A1 lipid tail are not essential for antimicrobial activity and could be replaced with hydrophobic aliphatic or aromatic groups. Some simpler analogues displayed potent antimicrobial activity against Gram-negative bacteria, including Campylobacter jejuni, Escherichia coli O157:H7, and multidrug resistant Klebsiella pneumoniae.

Structure of Clostridium difficile PilJ exhibits unprecedented divergence from known type IV pilins.

Type IV pili are produced by many pathogenic Gram-negative bacteria and are important for processes as diverse as twitching motility, cellular adhesion, and colonization. Recently, there has been an increased appreciation of the ability of Gram-positive species, including Clostridium difficile, to produce Type IV pili. Here we report the first three-dimensional structure of a Gram-positive Type IV pilin, PilJ, demonstrate its incorporation into Type IV pili, and offer insights into how the Type IV pili of C. difficile may assemble and function. PilJ has several unique structural features, including a dual-pilin fold and the incorporation of a structural zinc ion. We show that PilJ is incorporated into Type IV pili in C. difficile and present a model in which the incorporation of PilJ into pili exposes the C-terminal domain of PilJ to create a novel interaction surface.

The crystal structure of shiga toxin type 2 with bound disaccharide guides the design of a heterobifunctional toxin inhibitor.

Shiga toxin type 2 (Stx2a) is clinically most closely associated with enterohemorrhagic E. coli O157:H7-mediated hemorrhagic colitis that sometimes progresses to hemolytic-uremic syndrome. The ability to express the toxin has been acquired by other Escherichia coli strains, and outbreaks of food poisoning have caused significant mortality rates as, for example, in the 2011 outbreak in northern Germany. Stx2a, an AB5 toxin, gains entry into human cells via the glycosphingolipid receptor Gb3. We have determined the first crystal structure of a disaccharide analog of Gb3 bound to the B5 pentamer of Stx2a holotoxin. In this Gb3 analog,-GalNAc replaces the terminal-Gal residue. This co-crystal structure confirms previous inferences that two of the primary binding sites identified in theB5 pentamer of Stx1 are also functional in Stx2a. This knowledge provides a rationale for the synthesis and evaluation of heterobifunctional antagonists for E. coli toxins that target Stx2a. Incorporation of GalNAc Gb3 trisaccharide in a heterobifunctional ligand with an attached pyruvate acetal, a ligand for human amyloid P component, and conjugation to poly[acrylamide-co-(3-azidopropylmethacrylamide)] produced a polymer that neutralized Stx2a in a mouse model of Shigatoxemia.

Effects of nitric oxide and reactive oxygen species on HIF-1α stabilization following clostridium difficile toxin exposure of the Caco-2 epithelial cell line.

BACKGROUND/AIMS: Stabilization of the hypoxia-inducible factor (HIF-1α) is proposed to provide a protective host-response to C. difficile intoxication. Here, we aimed to elucidate whether nitric oxide and/or reactive oxygen species produced during C. difficile toxin exposure could influence HIF-1α stability and initiate protection against epithelial cell damage. METHODS/RESULTS: HIF-1α and inducible nitric oxide synthase (iNOS) proteins were up-regulated whereas factor-inhibiting HIF-1 (FIH-1) protein was down-regulated in Caco-2 epithelial cell monolayers with in vitro toxin exposure. We demonstrate using the biotin-switch assay that the stabilization of HIF-1α protein occurred via iNOS-dependent nitrosylation. Inhibition of iNOS activity by selective inhibitor (1400W) attenuated HIF-1α stabilization and exacerbated toxin-dependent disruptions in Caco-2 monolayer morphology and tight junctional integrity in vitro. Treatment of Caco-2 cell monolayers with N-actylcysteine (NAC), a scavenger of reactive oxygen species (ROS), attenuated toxin-dependent increases in iNOS and HIF-1α protein levels but had no effect on FIH-1 responses. In addition, mice that were exposed to C. difficile toxin in vivo also demonstrated a significant increase in HIF-1α protein and nitrosylation levels. CONCLUSION: Taken together, these data suggest that important synergistic actions exist between nitric oxide and ROS to stabilize HIF-1α and its innate, protective actions in the context of C. difficile toxin-mediated epithelial injury.

Sustained yield forestry in Sweden and Russia: how does it correspond to sustainable forest management policy?

This paper analyzes how sustained yield (SY) forestry is defined and implemented in Sweden and Russia, two countries with different forest-industrial regimes. We first compare definitions of SY forestry in national legislation and policies. Then we study forest management planning in two large forest management units with respect to: delivered forest products and values, how the harvest level of timber is defined, where the harvest takes place, and what treatments are used to sustain desired forest products and values. In Sweden SY forestry is maximum yield based on high-input forest management, and in Russia it is forestry based on natural regeneration with minimum investments in silviculture. We conclude that how SY forestry contributes to SFM depends on the context. Finally, we discuss the consequences of SY forestry as performed in Sweden and Russia related to its ability to support diverse forest functions, as envisioned in sustainable forest management policy.

Virulence potential and adherence properties of Escherichia coli that produce CTX-M and NDM ß-lactamases.

The success of certain sequence types such as ST131 that produce CTX-M or NDM ß-lactamases, and ST405 that produce CTX-M ß-lactamases, among extraintestinal Escherichia coli (ExPEC) had previously been linked to a combination of antimicrobial resistance and certain virulence factors. The adherence properties of these sequence types to gastro-intestinal epithelial cells had not been investigated. A study was therefore designed to investigate the phylogenetic groups, virulence factors and adherence properties of E. coli sequence types ST101, ST131 and ST405 that produce CTX-M-15 and NDM-1. Our results show that ST131 was positive for phylogenetic group B2, ST101 for B1 and ST404 for D. ST131 had more virulence factors than ST101 or ST405. Interestingly, ST101 adhered more avidly to HEp-2 and Caco-2 cells than did ST131 and ST405. Our study showed that adherence to gastro-intestinal cells did not seem to play an important role in the worldwide epidemiological success of ST131 and ST405. The exact role of ExPEC-associated virulence genes is unknown and it is unlikely that one set of factors determines the virulence properties and epidemiological success of certain sequence types. Future investigations should be undertaken to study the microbiological and ecological factors that make certain sequence types among ExPEC such successful pathogens.

Intrarectal instillation of Clostridium difficile toxin A triggers colonic inflammation and tissue damage: development of a novel and efficient mouse model of Clostridium difficile toxin exposure.

Clostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated "ileal loop." This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ of C. difficile infection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1ß. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.

Impact of the nature and size of the polymeric backbone on the ability of heterobifunctional ligands to mediate shiga toxin and serum amyloid p component ternary complex formation.

Inhibition of AB(5)-type bacterial toxins can be achieved by heterobifunctional ligands (BAITs) that mediate assembly of supramolecular complexes involving the toxin's pentameric cell membrane-binding subunit and an endogenous protein, serum amyloid P component, of the innate immune system. Effective in vivo protection from Shiga toxin Type 1 (Stx1) is achieved by polymer-bound, heterobifunctional inhibitors-adaptors (PolyBAITs), which exhibit prolonged half-life in circulation and by mediating formation of face-to-face SAP-AB(5) complexes, block receptor recognition sites and redirect toxins to the spleen and liver for degradation. Direct correlation between solid-phase activity and protective dose of PolyBAITs both in the cytotoxicity assay and in vivo indicate that the mechanism of protection from intoxication is inhibition of toxin binding to the host cell membrane. The polymeric scaffold influences the activity not only by clustering active binding fragments but also by sterically interfering with the supramolecular complex assembly. Thus, inhibitors based on N-(2-hydroxypropyl) methacrylamide (HPMA) show significantly lower activity than polyacrylamide-based analogs. The detrimental steric effect can partially be alleviated by extending the length of the spacer, which separates pendant ligand from the backbone, as well as extending the spacer, which spans the distance between binding moieties within each heterobifunctional ligand. Herein we report that polymer size and payload of the active ligand had moderate effects on the inhibitor's activity.

Lipopolysaccharide renders transgenic mice expressing human serum amyloid P component sensitive to Shiga toxin 2.

Transgenic C57BL/6 mice expressing human serum amyloid P component (HuSAP) are resistant to Shiga toxin 2 (Stx2) at dosages that are lethal in HuSAP-negative wild-type mice. However, it is well established that Stx2 initiates extra-intestinal complications such as the haemolytic-uremic syndrome despite the presence of HuSAP in human sera. We now demonstrate that co-administering purified Escherichia coli O55 lipopolysaccharide (LPS), at a dosage of 300 ng/g body weight, to HuSAP-transgenic mice increases their susceptibility to the lethal effects of Stx2. The enhanced susceptibility to Stx2 correlated with an increased expression of genes encoding the pro-inflammatory cytokine TNFα and chemokines of the CXC and CC families in the kidneys of LPS-treated mice, 48 hours after the Stx2/LPS challenge. Co-administering the glucocorticoid dexamethasone, but not the LPS neutralizing cationic peptide LL-37, protected LPS-sensitized HuSAP-transgenic mice from lethal doses of Stx2. Dexamethasone protection was specifically associated with decreased expression of the same inflammatory mediators (CXC and CC-type chemokines and TNFα) linked to enhanced susceptibility caused by LPS. The studies reveal further details about the complex cascade of host-related events that are initiated by Stx2 as well as establish a new animal model system in which to investigate strategies for diminishing serious Stx2-mediated complications in humans infected with enterohemorrhagic E. coli strains.

Mutagenic analysis of the Clostridium difficile flagellar proteins, FliC and FliD, and their contribution to virulence in hamsters.

Although toxins A and B are known to be important contributors to the acute phase of Clostridium difficile infection, the role of colonization and adherence to host tissues in the overall pathogenesis of these organisms remains unclear. Consequently, we used the recently introduced intron-based ClosTron gene interruption system to eliminate the expression of two reported C. difficile colonization factors, the major flagellar structural subunit (FliC) and the flagellar cap protein (FliD), to gain greater insight into how flagella and motility contribute to C. difficile's pathogenic strategy. The results demonstrate that interrupting either the fliC or the fliD gene results in a complete loss of flagella, as well as motility, in C. difficile. However, both the fliC and fliD mutant strains adhered better than the wild-type 630Δerm strain to human intestine-derived Caco-2 cells, suggesting that flagella and motility do not contribute to, or may even interfere with, C. difficile adherence to epithelial cell surfaces in vitro. Moreover, we found that the mutant strains were more virulent in hamsters, indicating either that flagella are unnecessary for virulence or that repression of motility may be a pathogenic strategy employed by C. difficile in hamsters.

Binding of Clostridium difficile toxins to human milk oligosaccharides.

The binding of recombinant fragments of the C-terminal cell-binding domains of the two large exotoxins, toxin A (TcdA) and toxin B (TcdB), expressed by Clostridium difficile and a library consisting of the most abundant neutral and acidic human milk oligosaccharides (HMOs) was examined quantitatively at 25°C and pH 7 using the direct electrospray ionization mass spectrometry (ES-MS) assay. The results of the ES-MS measurements indicate that both toxin fragments investigated, TcdB-B1 and TcdA-A2, which possess one and two carbohydrate binding sites, respectively, bind specifically to HMOs ranging in size from tri- to heptasaccharides. Notably, five of the HMOs tested bind to both toxins: Fuc(α1-2)Gal(ß1-4)Glc, Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc, Fuc(α1-2)Gal(ß1-3)GlcNAc(ß1-3)Gal(ß1-4)Glc, Gal(ß1-3)[Fuc(α1-4)]GlcNAc(ß1-3)Gal(ß1-4)Glc and Gal(ß1-4)[Fuc(α1-3)]GlcNAc(ß1-3)Gal(ß1-4)Glc. However, the binding of the HMOs is uniformly weak, with apparent affinities ≤10(3 )M(-1). The results of molecular docking simulations, taken together with the experimental binding data, suggest that a disaccharide moiety (lactose or lactosamine) represents the core HMO recognition element for both toxin fragments. The results of a Verocytotoxicity neutralization assay reveal that HMOs do not significantly inhibit the cytotoxic effects of TcdA or TcdB. The absence of protection is attributed to the very weak intrinsic affinities that the toxins exhibit towards the HMOs.