The ubiquitin ligation machinery in the defense against bacterial pathogens

The ubiquitin system is an important part of the host cellular defense program during bacterial infection. This is in particular evident for a number of bacteria including Salmonella Typhimurium and Mycobacterium tuberculosis which—inventively as part of their invasion strategy or accidentally upon rupture of seized host endomembranes—become exposed to the host cytosol. Ubiquitylation is involved in the detection and clearance of these bacteria as well as in the activation of innate immune and inflammatory signaling. Remarkably, all these defense responses seem to emanate from a dense layer of ubiquitin which coats the invading pathogens. In this review, we focus on the diverse group of host cell E3 ubiquitin ligases that help to tailor this ubiquitin coat. In particular, we address how the divergent ubiquitin conjugation mechanisms of these ligases contribute to the complexity of the anti‐bacterial coating and the recruitment of different ubiquitin‐binding effectors. We also discuss the activation and coordination of the different E3 ligases and which strategies bacteria evolved to evade the activities of the host ubiquitin system.     

Influenza A inhibits Th17-mediated host defense against bacterial pneumonia in mice

Staphylococcus aureus is a significant cause of hospital and community acquired pneumonia and causes secondary infection after influenza A. Recently, patients with hyper-IgE syndrome, who often present with S. aureus infections of the lung and skin, were found to have mutations in STAT3, required for Th17 immunity, suggesting a potential critical role for Th17 cells in S. aureus pneumonia. Indeed, IL-17R(-/-) and IL-22(-/-) mice displayed impaired bacterial clearance of S. aureus compared with that of wild-type mice. Mice challenged with influenza A PR/8/34 H1N1 and subsequently with S. aureus had increased inflammation and decreased clearance of both virus and bacteria. Coinfection resulted in greater type I and II IFN production in the lung compared with that with virus infection alone. Importantly, influenza A coinfection resulted in substantially decreased IL-17, IL-22, and IL-23 production after S. aureus infection. The decrease in S. aureus-induced IL-17, IL-22, and IL-23 was independent of type II IFN but required type I IFN production in influenza A-infected mice. Furthermore, overexpression of IL-23 in influenza A, S. aureus-coinfected mice rescued the induction of IL-17 and IL-22 and markedly improved bacterial clearance. These data indicate a novel mechanism by which influenza A-induced type I IFNs inhibit Th17 immunity and increase susceptibility to secondary bacterial pneumonia.     

Commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4

Background  

Major burn is associated with decreased gut barrier function and increased bacterial translocation (BT). This study is to investigate whether commensal microflora induce host defense and decrease BT in burn mice.     

The protective effect of endotoxin in X-irradiated mice

In mice X-irradiated with lethal dose (750 r) and mid-lethal dose (550 r) the protective effect of bacterial endotoxin isolated from strains ofSalmonella typhi has been found to be limited to a short period before irradiation and administration of endotoxin in repeated doses did not enhance protection. Application of endotoxin 4 days after X-rays resulted in increase of lethality of irradiation and earlier deaths of experimental animals were observed. Active intravenous and intraperitoneal immunization with endogenous strains ofEscherichia coli isolated from the intestinal flora of mice had a demonstrable protective effect when compared with passive immunization.     

Inducible defense mechanism against nitric oxide in Candida albicans

The yeast Candida albicans is an opportunistic pathogen that threatens patients with compromised immune systems. Immune cell defenses against C. albicans are complex but typically involve the production of reactive oxygen species and nitrogen radicals such as nitric oxide (NO) that damage the yeast or inhibit its growth. Whether Candida defends itself against NO and the molecules responsible for this defense have yet to be determined. The defense against NO in various bacteria and the yeast Saccharomyces cerevisiae involves an NO-scavenging flavohemoglobin. The C. albicans genome contains three genes encoding flavohemoglobin-related proteins, CaYHB1, CaYHB4, and CaYHB5. To assess their roles in NO metabolism, we constructed strains lacking each of these genes and demonstrated that just one, CaYHB1, is responsible for NO consumption and detoxification. In C. albicans, NO metabolic activity and CaYHB1 mRNA levels are rapidly induced by NO and NO-generating agents. Loss of CaYHB1 increases the sensitivity of C. albicans to NO-mediated growth inhibition. In mice, infections with Candida strains lacking CaYHB1 still resulted in lethality, but virulence was decreased compared to that in wild-type strains. Thus, C. albicans possesses a rapid, specific, and highly inducible NO defense mechanism involving one of three putative flavohemoglobin genes.     

On the mechanism of translocation of dihydrostreptomycin across the bacterial cytoplasmic membrane

This review examines two mechanisms, the channel and the uniport, proposed to explain the rapid, energy-dependent (EDP-II) phase of transport of dihydrostreptomycin (and streptomycin) across the bacterial cytoplasmic membrane. Bioenergetic and kinetic predictions are made from these two mechanisms and compared with available experimental data. Both the above mechanisms would be expected to lead to reversible transport kinetics, and to observable uptake of dihydrostreptomycin by respiring cytoplasmic membrane vesicles. However, transport is kinetically irreversible and is not observed in membrane vesicles (although the membrane vesicle findings need further confirmation), so the author rejects the proposed channel and uniport mechanisms. A possible mechanism of dihydrostreptomycin transport that would be consistent with the above experimental data, would be one in which a chemical reaction occurred as an obligatory part of the translocation cycle. Such a mechanism could be classified as primary translocation. The author emphasizes that this hypothesis is put forward to stimulate further experimental testing; it is not proposed to be a definitive explanation of the mechanism of energy-dependent dihydrostreptomycin transport.     

The properties of Escherichia isolated from the bodies of mice in bacterial translocation after immobilization stress

As revealed in experiments on mice, 6-hour immobilization stress initiates the process of the translocation of intestinal flora to mesenteric lymph nodes and the blood. This process is accompanied by the infection of parenchymatous organs (the liver, the spleen, the kidneys, the lungs) and the increase of the concentration of E. coli in the proximal sections of the digestive tract (the duodenum and the jejunum). As the result of the comparative analysis of the phenotypic signs of bacterial isolates obtained from intestinal and "extraintestinal" E. coli populations, the accumulation of clones with highly pronounced seroresistance and such persistence characteristics as anticomplementary and antilysozume signs, as well as resistance to the bactericidal action of leukocytic cation protein with a molecular weight of 11.0-11.5 kD, has been found to occur in the body (the blood, parenchymatous organs and the small intestine).     

Inhibition of tissue inflammation and bacterial translocation as one of the protective mechanisms of Saccharomyces boulardii against Salmonella infection in mice

Growing evidences suggest that Saccharomyces boulardii (SB) is efficacious against bacterial infections and inflammatory bowel diseases. This study investigated the effects of treatment with SB provided in a murine model of typhoid fever. Mice were divided into two groups: (1) control animals challenged with Salmonella Typhimurium (ST), and (2) animals receiving SB, and then challenged with ST. At days 0, 1, 5, 10 and 15 post-challenge, animals were euthanized and tissues collected to analyze bacterial translocation, cytokines, signaling pathways and histological analysis. Survival rate and animal weight were also evaluated. Treatment with SB increased survival rate and inhibited translocation of bacteria after ST challenge. Histological data showed that SB also protected mice against liver damage induced by ST. SB decreased levels of inflammatory cytokines and activation of mitogen-activated protein kinases (p38, JNK and ERK1/2), phospho-IκB, p65-RelA, phospho-jun and c-fos in the colon, signal pathways involved in the activation of inflammation induced by ST. Further experiments revealed that probiotic effects were due, at least in part, to the binding of ST to the yeast. Such binding diminishes ST translocation, resulting in decreased activation of signaling pathways which lead to intestinal inflammation in a murine model of typhoid fever.     

A host defense mechanism involving CFTR-mediated bicarbonate secretion in bacterial prostatitis

Background

Prostatitis is associated with a characteristic increase in prostatic fluid pH; however, the underlying mechanism and its physiological significance have not been elucidated.

Methodology/Principal Findings

In this study a primary culture of rat prostatic epithelial cells and a rat prostatitis model were used. Here we reported the involvement of CFTR, a cAMP-activated anion channel conducting both Cl⁻ and HCO₃ ⁻, in mediating prostate HCO₃ ⁻ secretion and its possible role in bacterial killing. Upon Escherichia coli (E coli)-LPS challenge, the expression of CFTR and carbonic anhydrase II (CA II), along with several pro-inflammatory cytokines was up-regulated in the primary culture of rat prostate epithelial cells. Inhibiting CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO₃ ⁻ content (>50 mM), rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO₃ ⁻ on bacterial killing was confirmed by its ability to increase cAMP production and suppress bacterial initiation factors in E coli. The relevance of the CFTR-mediated HCO₃ ⁻ secretion in humans was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues.

Conclusions/Significance

The CFTR and its mediated HCO₃ ⁻ secretion may be up-regulated in prostatitis as a host defense mechanism.     

Effect of age on bacterial translocation from the gastrointestinal tract in mice.

To clarify the effects of age on bacterial translocation from the gastrointestinal tract, mice at the age of 1, 2, 4, 6, 12, and 15 months were antibiotic-decontaminated for 4 days and then inoculated orally with streptomycin-resistant Escherichia coli C25. Mice treated with cyclophosphamide and untreated controls were tested for bacterial translocation to the mesenteric lymph nodes (MLN) 2 days later. The population levels of E. coli C25 in cyclophosphamide-treated and untreated mice were approximately 10(9.3) and 10(9.5) per gram of cecum, respectively, at each tested age. There were no significant differences in the incidence of translocation of E. coli C25 to MLN at any of the tested ages, whereas the number of E. coli C25 detected in MLN was higher in young mice than in aged mice in both the cyclophosphamide-treated and untreated groups. These findings suggest that bacterial translocation from the GI tract may be a more important problem in young animals than in aged animals.     

Molecular mechanism of proton-coupled ligand translocation by the bacterial efflux pump EmrE

The current surge in bacterial multi-drug resistance (MDR) is one of the largest challenges to public health, threatening to render ineffective many therapies we rely on for treatment of serious infections. Understanding different factors that contribute to MDR is hence crucial from the global “one health” perspective. In this contribution, we focus on the prototypical broad-selectivity proton-coupled antiporter EmrE, one of the smallest known ligand transporters that confers resistance to aromatic cations in a number of clinically relevant species. As an asymmetric homodimer undergoing an “alternating access” protomer-swap conformational change, it serves as a model for the mechanistic understanding of more complex drug transporters. Here, we present a free energy and solvent accessibility analysis that indicates the presence of two complementary ligand translocation pathways that remain operative in a broad range of conditions. Our simulations show a previously undescribed desolvated apo state and anticorrelated accessibility in the ligand-bound state, explaining on a structural level why EmrE does not disrupt the pH gradient through futile proton transfer. By comparing the behavior of a number of model charged and/or aromatic ligands, we also explain the origin of selectivity of EmrE towards a broad class of aromatic cations. Finally, we explore unbiased pathways of ligand entry and exit to identify correlated structural changes implicated in ligand binding and release, as well as characterize key intermediates of occupancy changes.     

IL-22 mediates mucosal host defense against Gram-negative bacterial pneumonia

Emerging evidence supports the concept that T helper type 17 (T(H)17) cells, in addition to mediating autoimmunity, have key roles in mucosal immunity against extracellular pathogens. Interleukin-22 (IL-22) and IL-17A are both effector cytokines produced by the T(H)17 lineage, and both were crucial for maintaining local control of the Gram-negative pulmonary pathogen, Klebsiella pneumoniae. Although both cytokines regulated CXC chemokines and granulocyte colony-stimulating factor production in the lung, only IL-22 increased lung epithelial cell proliferation and increased transepithelial resistance to injury. These data support the concept that the T(H)17 cell lineage and its effector molecules have evolved to effect host defense against extracellular pathogens at mucosal sites.     

Variances in the levels of serum lysozyme in X-irradiated mice

The level of serum lysozyme in mice varies in relation to the dose of irradiation in several phases. High doses result in a progressive decrease whereas a decrease on the first and about the ninth day is observed after irradiation with lower doses. A rise in lysozyme level occurs on the third day. Enhancement of lysozyme levels was observed after application of 20—50 r. Administration of lysozyme did not affect the deaths of mice receiving with massive doses of irradiation.     

Protein washdown as a defense mechanism against myocardial edema

Myocardial edema occurs in many pathological conditions. We hypothesized that protein washdown at the myocardial microvascular exchange barrier would change the distribution of interstitial proteins from large to small molecules and diminish the effect of washdown on the colloid osmotic pressure (COP) of interstitial fluid and lymph. Dogs were instrumented with coronary sinus balloon-tipped catheters and myocardial lymphatic cannulas to manipulate myocardial lymph flow and to collect lymph. Myocardial venous pressure was elevated by balloon inflation to increase transmicrovascular fluid flux and myocardial lymph flow. COP of lymph was measured directly and was also calculated from protein concentration. Decreases occurred in both protein concentration and COP of lymph. The proportion of lymph protein accounted for by albumin increased significantly, whereas that accounted for by beta-lipoprotein decreased significantly. The change in the calculated plasma-to-lymph COP gradient was significantly greater than the change in the measured COP gradient. We conclude that the change in the distribution of interstitial fluid protein species decreases the effect of protein washdown on interstitial fluid COP and limits its effectiveness as a defense mechanism against myocardial edema formation.     

The translocation mechanism of P-glycoprotein

Multidrug transporters are involved in mediating the failure of chemotherapy in treating several serious diseases. The archetypal multidrug transporter P-glycoprotein (P-gp) confers resistance to a large number of chemically and functionally unrelated anti-cancer drugs by mediating efflux from cancer cells. The ability to efflux such a large number of drugs remains a biological enigma and the lack of mechanistic understanding of the translocation pathway used by P-gp prevents rational design of compounds to inhibit its function. The translocation pathway is critically dependent on ATP hydrolysis and drug interaction with P-gp is possible at one of a multitude of allosterically linked binding sites. However, aspects such as coupling stoichiometry, molecular properties of binding sites and the nature of conformational changes remain unresolved or the centre of considerable controversy. The present review attempts to utilise the available data to generate a detailed sequence of events in the translocation pathway for this dexterous protein.     

Modulation of bacterial translocation in mice mediated through lactose and human milk oligosaccharides

Massive resection of the small intestine in infants is imposed to the regulation of several intestinal pathological situations, as intestinal adaptation cannot be relied upon. Many nutritional disturbances are occurring following surgery procedure. In this vein, long-term parenteral feeding is adopt to improve prognosis not always successfully. Clostridia and more specifically Clostridium perfringens, are suspected to participate in the physiopathology of the rising situation. In order to investigate the effect of lactose and human milk neutral oligosaccharides (HMNOs) on Clostridia, germfree mice were inoculated either with enterotoxigenic C.perfringens strain isolated from a patient with NEC, or with a human microbiota harboring C.clostridioforme group(HF). In this vein, different doses of lactose were administrated during 2 weeks in adult mice on an attempt to evaluate the lactase activity. Intake of lactose (70 g/L) and HMNOs (7 g/L) in C.perfringens monoassociated mice induced mortality within a week. In HF mice, no mortality was observed. An increase in Clostridia occurrence was observed in the median ileum after intake of 7 g lactose (p = 0.017). Higher clostridial numbers occurred in caecum following intake of 70 g lactose (p < 0.05) and HMNOs (p < 0.025). Bifidobacteria were found increased from distal ileum to colon following 70 g of lactose intake, whereas they decreased in the caecum of mice drinking lower lactose concentrations. Finally, bacteremia was more frequent in 70 g lactose/L mice (p < 0.02), whereas at lower doses of lactose bifidobacterial translocation was observed.As a result, human milk oligosaccharides could favor clostridial population when reaching the lower intestine. The shortness of the small intestine in infants underwent massive intestinal resection seems to be associated to an incomplete breakdown of lactose. Enteral feeds formulas deprived in lactose would be more suitable in enteral feeding of infants.