Biomedical subjects

Robert K Ernst

Publications and source records attributed to Robert K Ernst.

At least 19 recordsLinked to original sources

PubMed2007

Immunostimulatory properties of the emerging pathogen Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a multiple-antibiotic-resistant opportunistic pathogen that is being isolated with increasing frequency from patients with health-care-associated infections and especially from patients with cystic fibrosis (CF). While clinicians feel compelled to treat infections involving this organism, its potential for virulence is not well established. We evaluated the immunostimulatory properties and overall virulence of clinical isolates of S. maltophilia using the well-characterized opportunistic pathogen Pseudomonas aeruginosa PAO1 as a control. The properties of CF isolates were examined specifically to see if they have a common phenotype. The immunostimulatory properties of S. maltophilia were studied in vitro by stimulating airway epithelial and macrophage cell lines. A neonatal mouse model of pneumonia was used to determine the rates of pneumonia, bacteremia, and mortality, as well as the inflammatory response elicited by S. maltophilia infection. Respiratory and nonrespiratory S. maltophilia isolates were highly immunostimulatory and elicited significant interleukin-8 expression by airway epithelial cells, as well as tumor necrosis factor alpha (TNF-alpha) expression by macrophages. TNF-alpha signaling appears to be important in the pathogenesis of S. maltophilia infection as less than 20% of TNFR1 null mice (compared with 100% of wild-type mice) developed pneumonia and bacteremia following intranasal inoculation. The S. maltophilia isolates were weakly invasive, and low-level bacteremia with no mortality was observed. Despite the lack of invasiveness of S. maltophilia, the immunostimulatory properties of this organism and its induction of TNF-alpha expression specifically indicate that it is likely to contribute significantly to airway inflammation.

Animals↗

PubMed2006

Differentiation and distribution of colistin- and sodium dodecyl sulfate-tolerant cells in Pseudomonas aeruginosa biofilms.

During Pseudomonas aeruginosa flow cell biofilm development, the cell population differentiates into a nonmotile subpopulation which forms microcolonies and a migrating subpopulation which eventually colonizes the top of the microcolonies, resulting in the development of mushroom-shaped multicellular structures. The cap-forming subpopulation was found to develop tolerance to membrane-targeting antimicrobial agents, such as the cyclic cationic peptide colistin and the detergent sodium dodecyl sulfate. The stalk-forming subpopulation, on the other hand, was sensitive to the membrane-targeting antibacterial agents. All biofilm-associated cells were sensitive to the antibacterial agents when tested in standard plate assays. A mutation eliminating the production of type IV pili, and hence surface-associated motility, prevented the formation of regular mushroom-shaped structures in the flow cell biofilms, and the development of tolerance to the antimicrobial agents was found to be affected as well. Mutations in genes interfering with lipopolysaccharide modification (pmr) eliminated the biofilm-associated colistin tolerance phenotype. Experiments with a PAO1 strain harboring a pmr-gfp fusion showed that only the cap-forming subpopulation in biofilms treated with colistin expresses the pmr operon. These results suggest that increased antibiotic tolerance in biofilms may be a consequence of differentiation into distinct subpopulations with different phenotypic properties.

Anti-Bacterial Agents↗

PubMed2006

Lack of in vitro and in vivo recognition of Francisella tularensis subspecies lipopolysaccharide by Toll-like receptors.

Francisella tularensis is an intracellular gram-negative bacterium that is highly infectious and potentially lethal. Several subspecies exist of varying pathogenicity. Infection by only a few organisms is sufficient to cause disease depending on the model system. Lipopolysaccharide (LPS) of gram-negative bacteria is generally recognized by Toll-like receptor 4 (TLR4)/MD-2 and induces a strong proinflammatory response. Examination of human clinical F. tularensis isolates revealed that human virulent type A and type B strains produced lipid A of similar structure to the nonhuman model pathogen of mice, Francisella novicida. F. novicida LPS or lipid A is neither stimulatory nor an antagonist for human and murine cells through TLR4 or TLR2. It does not appear to interact with TLR4 or MD-2, as it is not an antagonist to other stimulatory LPS. Consistent with these observations, aerosolization of F. novicida LPS or whole bacteria induced no inflammatory response in mice. These results suggest that poor innate recognition of F. tularensis allows the bacterium to evade early recognition by the host innate immune system to promote its pathogenesis for mammals.

Animals↗

PubMed2006

The Pseudomonas aeruginosa lipid A deacylase: selection for expression and loss within the cystic fibrosis airway.

Lipopolysaccharide (LPS) is the major surface component of gram-negative bacteria, and a component of LPS, lipid A, is recognized by the innate immune system through the Toll-like receptor 4/MD-2 complex. Pseudomonas aeruginosa, an environmental gram-negative bacterium that opportunistically infects the respiratory tracts of patients with cystic fibrosis (CF), can synthesize various structures of lipid A. Lipid A from P. aeruginosa strains isolated from infants with CF has a specific structure that includes the removal of the 3 position 3-OH C10 fatty acid. Here we demonstrate increased expression of the P. aeruginosa lipid A 3-O-deacylase (PagL) in isolates from CF infants compared to that in environmental isolates. PagL activity was increased in environmental isolates by growth in medium limited for magnesium and decreased by growth at low temperature in laboratory-adapted strains of P. aeruginosa. P. aeruginosa PagL was shown to be an outer membrane protein by isopycnic density gradient centrifugation. Heterologous expression of P. aeruginosa pagL in Salmonella enterica serovar Typhimurium and Escherichia coli resulted in removal of the 3-OH C14 fatty acid from lipid A, indicating that P. aeruginosa PagL recognizes either 3-OH C10 or 3-OH C14. Finally, deacylated lipid A species were not observed in some clinical P. aeruginosa isolates from patients with severe pulmonary disease, suggesting that loss of PagL function can occur during long-term adaptation to the CF airway.

Acylation↗

PubMed2006

Characterization of late acyltransferase genes of Yersinia pestis and their role in temperature-dependent lipid A variation.

Yersinia pestis is an important human pathogen that is maintained in flea-rodent enzootic cycles in many parts of the world. During its life cycle, Y. pestis senses host-specific environmental cues such as temperature and regulates gene expression appropriately to adapt to the insect or mammalian host. For example, Y. pestis synthesizes different forms of lipid A when grown at temperatures corresponding to the in vivo environments of the mammalian host and the flea vector. At 37 degrees C, tetra-acylated lipid A is the major form; but at 26 degrees C or below, hexa-acylated lipid A predominates. In this study, we show that the Y. pestis msbB (lpxM) and lpxP homologs encode the acyltransferases that add C12 and C(16:1) groups, respectively, to lipid IV(A) to generate the hexa-acylated form, and that their expression is upregulated at 21 degrees C in vitro and in the flea midgut. A Y. pestis deltamsbB deltalpxP double mutant that did not produce hexa-acylated lipid A was more sensitive to cecropin A, but not to polymyxin B. This mutant was able to infect and block fleas as well as the parental wild-type strain, indicating that the low-temperature-dependent change to hexa-acylated lipid A synthesis is not required for survival in the flea gut.

Acyltransferases↗

PubMed2005

Exopolysaccharides from Burkholderia cenocepacia inhibit neutrophil chemotaxis and scavenge reactive oxygen species.

Bacteria belonging to the Burkholderia cepacia complex are important opportunistic pathogens in compromised hosts, particularly patients with cystic fibrosis or chronic granulomatous disease. Isolates of B. cepacia complex may produce large amounts of exopolysaccharides (EPS) that endow the bacteria with a mucoid phenotype and appear to facilitate bacterial persistence during infection. We showed that EPS from a clinical B. cenocepacia isolate interfered with the function of human neutrophils in vitro; it inhibited chemotaxis and production of reactive oxygen species (ROS), both essential components of innate neutrophil-mediated host defenses. These inhibitory effects were not due to cytotoxicity or interference with intracellular calcium signaling. EPS also inhibited enzymatic generation of ROS in cell-free systems, indicating that it scavenges these bactericidal products. B. cenocepacia EPS is structurally distinct from Pseudomonas aeruginosa alginate, yet they share the capacity to scavenge ROS and inhibit chemotaxis. These properties could explain why the two bacterial species resist clearance from the infected cystic fibrosis lung.

Burkholderia↗

PubMed2005

LPS, TLR4 and infectious disease diversity.

Innate immune receptors recognize microorganism-specific motifs. One such receptor-ligand complex is formed between the mammalian Toll-like receptor 4 (TLR4)-MD2-CD14 complex and bacterial lipopolysaccharide (LPS). Recent research indicates that there is significant phylogenetic and individual diversity in TLR4-mediated responses. In addition, the diversity of LPS structures and the differential recognition of these structures by TLR4 have been associated with several bacterial diseases. This review will examine the hypothesis that the variability of bacterial ligands such as LPS and their innate immune receptors is an important factor in determining the outcome of infectious disease.

Animals↗

PubMed2005

Mouse paneth cell secretory responses to cell surface glycolipids of virulent and attenuated pathogenic bacteria.

Mouse Paneth cells respond to bacteria and bacterial cell surface antigens by discharging secretory granules into the lumen of small intestinal crypts (T. Ayabe et al., Nat. Immunol. 1:113-118, 2000). To investigate mechanisms regulating these responses, purified surface glycolipid molecules with known acyl chain modifications and attenuated properties were tested for the ability to stimulate Paneth cell secretion. The antigens included lipopolysaccharide (LPS) from wild-type and msbB-null Escherichia coli and phoP-null and phoP-constitutive Salmonella enterica serovar Typhimurium strains, as well as LPS, lipid A, and lipoteichoic acid from Pseudomonas aeruginosa and Listeria monocytogenes grown in Mg2+-limited media. Measurements of total secreted protein, secreted lysozyme, and the bactericidal peptide activities of collected secretions showed that the purified antigens elicited similar secretory responses from Paneth cells in mouse crypts ex vivo, regardless of glycolipid acyl chain modification. Despite their impaired Tlr4 pathway, Paneth cells in ex vivo C3H/HeJ mouse crypts released equivalent amounts of bactericidal peptide activity in response to purified bacterial antigens, including lipid A. Thus, mouse Paneth cells respond equivalently to purified bacterial cell envelope glycolipids, regardless of functional Tlr4, the structural properties of glycolipid acyl chains, or their association with virulence in humans.

Animals↗

PubMed2005

Inhibition of Salmonella enterica serovar Typhimurium lipopolysaccharide deacylation by aminoarabinose membrane modification.

Salmonella enterica serovar Typhimurium remodels the lipid A component of lipopolysaccharide, a major component of the outer membrane, to survive within animals. The activation of the sensor kinase PhoQ in host environments increases the synthesis of enzymes that deacylate, palmitoylate, hydroxylate, and attach aminoarabinose to lipid A, also known as endotoxin. These modifications promote bacterial resistance to antimicrobial peptides and reduce the host recognition of lipid A by Toll-like receptor 4. The Salmonella lipid A 3-O-deacylase, PagL, is an outer membrane protein whose expression is regulated by PhoQ. In S. enterica serovar Typhimurium strains that had the ability to add aminoarabinose to lipid A, 3-O-deacylated lipid A species were not detected, despite the PhoQ induction of PagL protein expression. In contrast, strains defective for the aminoarabinose modification of lipid A demonstrated in vivo PagL activity, indicating that this membrane modification inhibited PagL's enzymatic activity. Since not all lipid A molecules are modified with aminoarabinose upon PhoQ activation, these results cannot be ascribed to the substrate specificity of PagL. PagL-dependent deacylation was detected in sonically disrupted membranes and membranes treated with the nonionic detergent n-octyl-beta-d-glucopyranoside, suggesting that perturbation of the intact outer membrane releases PagL from posttranslational inhibition by aminoarabinose-containing membranes. Taken together, these results suggest that PagL enzymatic deacylation is posttranslationally inhibited by membrane environments, which either sequester PagL from its substrate or alter its conformation.

Arabinose↗

PubMed2005

Purification and characterization of deacylated and/or palmitoylated lipid A species unique to Salmonella enterica serovar Typhimurium.

The Salmonella enterica serovar Typhimurium virulence gene products PhoP/PhoQ sense host microenvironments to regulate the expression of a lipid A 3-O-deacylase, PagL, and a lipid A palmitoyltransferase, PagP. Therefore, deacylation and/or palmitoylation of lipid A could occur in Salmonellae adapted to host environments. The PhoP/PhoQ-regulated modification of lipid A alters host recognition and signaling, and may play an important role in host defense against Salmonellae infection. Here we report the purification and characterization of modified lipid A species. Deacylated lipid A, deacylated and palmitoylated lipid A, and palmitoylated lipid A species were generated in Escherichia coli cells heterologously expressing salmonellae PagL and/or PagP, and then purified by sequential thin-layer chromatography. The purified lipid A species showed m/z values that correspond to single lipid A species on mass spectrometry analysis. The modified lipid A species showed reduced ability to induce cellular signaling through Toll-like receptor 4, suggesting a specific function of the lipid A modifications in the pathogenesis of salmonellae infection.

Acylation↗

PubMed2004

Respiratory epithelial cells regulate lung inflammation in response to inhaled endotoxin.

To determine the role of respiratory epithelial cells in the inflammatory response to inhaled endotoxin, we selectively inhibited NF-kappa B activation in the respiratory epithelium using a mutant I kappa B-alpha construct that functioned as a dominant negative inhibitor of NF-kappa B translocation (dnI kappa B-alpha). We developed two lines of transgenic mice in which expression of dnI kappa B-alpha was targeted to the distal airway epithelium using the human surfactant apoprotein C promoter. Transgene expression was localized to the epithelium of the terminal bronchioles and alveoli. After inhalation of LPS, nuclear translocation of NF-kappa B was evident in bronchiolar epithelium of nontransgenic but not of transgenic mice. This defect was associated with impaired neutrophilic lung inflammation 4 h after LPS challenge and diminished levels of TNF-alpha, IL-1 beta, macrophage inflammatory protein-2, and KC in lung homogenates. Expression of TNF-alpha within bronchiolar epithelial cells and of VCAM-1 within peribronchiolar endothelial cells was reduced in transgenic animals. Thus targeted inhibition of NF-kappa B activation in distal airway epithelial cells impaired the inflammatory response to inhaled LPS. These data provide causal evidence that distal airway epithelial cells and the signals they transduce play a physiological role in lung inflammation in vivo.

Administration, Inhalation↗

PubMed2004

3-O-deacylation of lipid A by PagL, a PhoP/PhoQ-regulated deacylase of Salmonella typhimurium, modulates signaling through Toll-like receptor 4.

Toll-like receptor 4 (TLR4)-mediated responses, which are induced by the lipid A portion of lipopolysaccharide, are important for host defense against Salmonellae infection. A variety of different data indicate that the acylation state of lipid A can alter TLR4-mediated responses. The S. typhimurium virulence gene product PhoP/PhoQ signals the presence of host microenvironments to regulate the expression of a lipid A 3-O-deacylase, PagL, and a lipid A palmitoyltransferase, PagP. We now demonstrate that 3-O-deacylation and palmitoylation of lipid A decreases its ability to induce TLR4-mediated signaling. Deacylated lipid A, deacylated and palmitoylated lipid A, palmitoylated lipid A, and unmodified lipid A species were purified from Escherichia coli heterologously expressing PagL and/or PagP. The purified lipid A preparations showed spectra of a single lipid A species on mass spectrometry and gave a single band on thin layer chromatography. The activity of purified lipid A species was examined using human and mouse cell lines that express recombinant human TLR4. Compared with unmodified lipid A, the modified lipid A species are 30-100-fold less active in the ability to induce NF-kappaB-dependent reporter activation. These results suggest that the lipid A modifications reduce TLR4-signaling as part of Salmonellae adaptation to host environments.

Acyltransferases↗

PubMed2004

Factors affecting the escape of Francisella tularensis from the phagolysosome.

The highly virulent bacterium Francisella tularensis is well adapted to the intracellular habitat but the mechanisms behind its intracellular survival have been elusive. Recently, it was shown that the bacterium is capable of escaping from the phagosome of human and mouse monocytic cells. Here it is shown that this escape is affected by gamma interferon (IFN-gamma) treatment of mouse peritoneal exudate cells since in treated cells the proportion that escaped was significantly lower (80%) than in untreated cells (97%) as determined by transmission electron microscopy. By contrast, < 1% of mutant bacteria lacking expression of a 23 kDa protein denoted IglC were able to escape from the phagosome. Infection with the DeltaiglC strain complemented with the iglC gene resulted in 60% of the bacteria escaping from the phagosome. Whereas IFN-gamma treatment conferred a static effect on intracellular wild-type bacteria, the treatment had a bactericidal effect on the DeltaiglC strain. The results show that the activation status of infected cells affects the escape of F. tularensis from the phagosome. An even more profound effect on this escape is related to expression of IglC by F. tularensis. Its absence rendered the mutant bacteria incapable of escaping from the phagosome and of multiplying intracellularly.

Animals↗

PubMed2004

Variation in lipid A structure in the pathogenic yersiniae.

Important pathogens in the genus Yersinia include the plague bacillus Yersinia pestis and two enteropathogenic species, Yersinia pseudotuberculosis and Yersinia enterocolitica. A shift in growth temperature induced changes in the number and type of acyl groups on the lipid A of all three species. After growth at 37 degrees C, Y. pestis lipopolysaccharide (LPS) contained the tetra-acylated lipid IV(A) and smaller amounts of lipid IV(A) modified with C10 or C12 acyl groups, Y. pseudotuberculosis contained the same forms as part of a more heterogeneous population in which lipid IV(A) modified with C16:0 predominated, and Y. enterocolitica produced a unique tetra-acylated lipid A. When grown at 21 degrees C, however, the three yersiniae synthesized LPS containing predominantly hexa-acylated lipid A. This more complex lipid A stimulated human monocytes to secrete tumour necrosis factor-alpha, whereas the lipid A synthesized by the three species at 37 degrees C did not. The Y. pestis phoP gene was required for aminoarabinose modification of lipid A, but not for the temperature-dependent acylation changes. The results suggest that the production of a less immunostimulatory form of LPS upon entry into the mammalian host is a conserved pathogenesis mechanism in the genus Yersinia, and that species-specific lipid A forms may be important for life cycle and pathogenicity differences.

Animals↗

PubMed2004

PmrAB, a two-component regulatory system of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial peptides and addition of aminoarabinose to lipid A.

Spontaneous polymyxin-resistant mutants of Pseudomonas aeruginosa were isolated. The mutations responsible for this phenotype were mapped to a two-component signal transduction system similar to PmrAB of Salmonella enterica serovar Typhimurium. Lipid A of these mutants contained aminoarabinose, an inducible modification that is associated with polymyxin resistance. Thus, P. aeruginosa possesses a mechanism that induces resistance to cationic antimicrobial peptides in response to environmental conditions.

Antimicrobial Cationic Peptides↗

PubMed2004

Deacylation and palmitoylation of lipid A by Salmonellae outer membrane enzymes modulate host signaling through Toll-like receptor 4.

The Salmonella typhimurium virulence gene products, PhoP/PhoQ sense host micro-environments to regulate the expression of a lipid A 3-O-deacylase, PagL, and a lipid A palmitoyltransferase, PagP. Therefore, deacylation and/or palmitoylation of lipid A could occur in Salmonellae adapted to host environments. The acylation state of lipid A can alter host recognition and signaling by Toll-like receptor (TLR) 4, and may play an important role in host defenses against Salmonellae infection. Deacylated lipid A, deacylated and palmitoylated lipid A, palmitoylated lipid A, and unmodified lipid A species were purified, and the activity was examined using cell lines expressing recombinant human or mouse TLR4. Compared with unmodified lipid A, the modified lipid A species are 10-100-fold less active. These results suggest that PagL and PagP modify lipid A to reduce TLR4-signaling as part of Salmonellae adaptation to the host environment.

Acyltransferases↗

PubMed2003

Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation.

In this study, large-scale qualitative and quantitative proteomic technology was applied to the analysis of the opportunistic bacterial pathogen Pseudomonas aeruginosa grown under magnesium limitation, an environmental condition previously shown to induce expression of various virulence factors. For quantitative analysis, whole cell and membrane proteins were differentially labeled with isotope-coded affinity tag (ICAT) reagents and ICAT reagent-labeled peptides were separated by two-dimensional chromatography prior to analysis by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) in an ion trap mass spectrometer (ITMS). To increase the number of protein identifications, gas-phase fractionation (GPF) in the m/z dimension was employed for analysis of ICAT peptides derived from whole cell extracts. The experiments confirmed expression of 1331 P. aeruginosa proteins of which 145 were differentially expressed upon limitation of magnesium. A number of conserved Gram-negative magnesium stress-response proteins involved in bacterial virulence were among the most abundant proteins induced in low magnesium. Comparative ICAT analysis of membrane versus whole cell protein indicated that growth of P. aeruginosa in low magnesium resulted in altered subcellular compartmentalization of large enzyme complexes such as ribosomes. This result was confirmed by 2-D PAGE analysis of P. aeruginosa outer membrane proteins. This study shows that large-scale quantitative proteomic technology can be successfully applied to the analysis of whole bacteria and to the discovery of functionally relevant biologic phenotypes.

Bacterial Outer Membrane Proteins↗

PubMed2003

Genome mosaicism is conserved but not unique in Pseudomonas aeruginosa isolates from the airways of young children with cystic fibrosis.

Pseudomonas aeruginosa strains from the chronic lung infections of cystic fibrosis (CF) patients are phenotypically and genotypically diverse. Using strain PAO1 whole genome DNA microarrays, we assessed the genomic variation in P. aeruginosa strains isolated from young children with CF (6 months to 8 years of age) as well as from the environment. Eighty-nine to 97% of the PAO1 open reading frames were detected in 20 strains by microarray analysis, while subsets of 38 gene islands were absent or divergent. No specific pattern of genome mosaicism defined strains associated with CF. Many mosaic regions were distinguished by their low G + C content; their inclusion of phage related or pyocin genes; or by their linkage to a vgr gene or a tRNA gene. Microarray and phenotypic analysis of sequential isolates from individual patients revealed two deletions of greater than 100 kbp formed during evolution in the lung. The gene loss in these sequential isolates raises the possibility that acquisition of pyomelanin production and loss of pyoverdin uptake each may be of adaptive significance. Further characterization of P. aeruginosa diversity within the airways of individual CF patients may reveal common adaptations, perhaps mediated by gene loss, that suggest new opportunities for therapy.

Adaptation, Physiological↗