CD47 deficiency protects mice from lipopolysaccharide-induced acute lung injury and Escherichia coli pneumonia

CD47 modulates neutrophil transmigration toward the sites of infection or injury. Mice lacking CD47 are susceptible to Escherichia coli (E. coli) peritonitis. However, less is known concerning the role of CD47 in the development of acute lung inflammation and injury. In this study, we show that mice lacking CD47 are protected from LPS-induced acute lung injury and E. coli pneumonia with a significant reduction in pulmonary edema, lung vascular permeability, and bacteremia. Reconstitution of CD47(+/-) mice with CD47(-/-) neutrophils significantly reduced lung edema and neutrophil infiltration, thus demonstrating that CD47(+) neutrophils are required for the development of lung injury from E. coli pneumonia. Importantly, CD47-deficient mice with E. coli pneumonia had an improved survival rate. Taken together, deficiency of CD47 protects mice from LPS-induced acute lung injury and E. coli pneumonia. Targeting CD47 may be a novel pathway for treatment of acute lung injury.     

E. coli virulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model

Enteric gram-negative bacilli, such as Escherichia coli are the most common cause of nosocomial pneumonia. In this study a wild-type extraintestinal pathogenic strain of E. coli (ExPEC)(CP9) and isogenic derivatives deficient in hemolysin (Hly) and cytotoxic necrotizing factor (CNF) were assessed in vitro and in a rat model of gram-negative pneumonia to test the hypothesis that these virulence factors induce neutrophil apoptosis and/or necrosis/lysis. As ascertained by in vitro caspase-3/7 and LDH activities and neutrophil morphology, Hly mediated neutrophil apoptosis at lower E. coli titers (1 x 10(5-6) cfu) and necrosis/lysis at higher titers (> or =1 x 10(7) cfu). Data suggest that CNF promotes apoptosis but not necrosis or lysis. We also demonstrate that annexin V/7-amino-actinomycin D staining was an unreliable assessment of apoptosis using live E. coli. The use of caspase-3/7 and LDH activities and neutrophil morphology supported the notion that necrosis, not apoptosis, was the primary mechanism by which neutrophils were affected in our in vivo gram-negative pneumonia model using live E. coli. In addition, in vivo studies demonstrated that Hly mediates lung injury. Neutrophil necrosis was not observed when animals were challenged with purified lipopolysaccharide, demonstrating the importance of using live bacteria. These findings establish that Hly contributes to ExPEC virulence by mediating neutrophil toxicity, with necrosis/lysis being the dominant effect of Hly on neutrophils in vivo and by lung injury. Whether Hly-mediated lung injury is due to neutrophil necrosis, a direct effect of Hly, or both is unclear.     

Surfactant dysfunction and lung injury due to the E. coli virulence factor hemolysin in a rat pneumonia model

This study tests the hypothesis that the virulence factor hemolysin (Hly) expressed by extraintestinal pathogenic Escherichia coli contributes to surfactant dysfunction and lung injury in a rat model of gram-negative pneumonia. Rats were instilled intratracheally with CP9 (wild type, Hly-positive), CP9hlyA (Hly-minus), CP9/pEK50 (supraphysiological Hly), or purified LPS. At 6 h postinfection, rats given CP9 had a decreased percentage content of large surfactant aggregates in cell-free bronchoalveolar lavage (BAL), decreased large aggregate surface activity, decreased Pa(O2)/FiO2) ratio, increased BAL albumin/protein levels, and increased histological evidence of lung injury compared with rats given CP9hlyA or LPS. In addition, rats given CP9/pEK50 or CP9 had decreased large aggregate surface activity, decreased Pa(O2)/FiO2) ratios, and increased BAL albumin/protein levels at 2 h postinfection compared with rats given CP9hlyA. The severity of permeability lung injury based on albumin/protein levels in BAL at 2 h was ordered as CP9/pEK50 > CP9 > CP9hlyA > normal saline controls. Total lung titers of bacteria were increased at 6 h in rats given CP9 vs. CP9hlyA, but bacterial titers were not significantly different at 2 h, indicating that increased surfactant dysfunction and lung injury were associated with Hly as opposed to bacterial numbers per se. Further studies in vitro showed that CP9 could directly lyse transformed pulmonary epithelial cells (H441 cells) but that indirect lysis of H441 cells secondary to Hly-induced neutrophil lysis did not occur. Together, these data demonstrate that Hly is an important direct mediator of surfactant dysfunction and lung injury in gram-negative pneumonia.     

Leukocyte antibacterial functions are not impaired by perfluorocarbon exposure in vitro

Application of liquid, aerosolized, and vaporized perfluorocarbons (PFC) in acute lung injury has shown anti-inflammatory effects. Although this may be beneficial in states of pulmonary hyperinflammation, it also could increase susceptibility to nosocomial lung infection. We hypothesized that PFC impair cellular host defense and therefore investigated in an in vitro model the influence of perfluorohexane (PFH) on crucial mechanisms of bacterial elimination in human neutrophils and monocytes. Using scanning and transmission electron microscopy, we could show membrane-bound and ingested PFH particles that morphologically did not alter adherence and phagocytosis of Escherichia coli or leukocyte viability. The amount of adherent and phagocytosed bacteria as determined by flow cytometry was not influenced in cells only pretreated with PFH for 1 and 4 h. When PFH was present during E. coli challenge, bacterial adherence was decreased in polymorphonuclear neutrophils, but respective intracellular uptake was not impaired and was even significantly promoted in monocytes. Overall, E. coli-induced respiratory burst capacity was not reduced by PFH. Our findings provide evidence that key functions of innate host defense are not compromised by PFH treatment in vitro.     

Systemic complement activation and acute lung injury

Experimental studies of rats have provided significant evidence that intravascular complement activation after i.v. injection of cobra venom factor (CVF) or thermal injury of skin can result in acute lung injury. This has been determined by morphological changes in lung and increases in lung vascular permeability. Systemic complement activation is associated with an early appearance of C5-derived chemotactic activity in the circulation coincident with the development of transient neutropenia, followed by extensive granulocytosis and sequestration of neutrophils in lung interstitial capillaries. The acute pulmonary injury depends on availability of complement and neutrophils. Depletion of either complement or blood neutrophils before CVF injection or thermal injury will prevent development of lung injury. Interventional studies with catalase, scavengers of hydroxyl radical OH., and iron chelators have revealed that the acute pulmonary injury is related to production of oxygen-derived free radicals by activated neutrophils. OH. appears to be the key mediator involved in the acute lung microvascular injury.     

Molecular analysis of Shigella boydii O1 O-antigen gene cluster and its PCR typing

Shigella is an important human pathogen and is closely related to Escherichia coli. O-antigen is the most variable part of the lipopolysaccharide on the cell surface of Gram-negative bacteria and plays an important role in pathogenicity. The O-antigen gene cluster of S. boydii O1 was sequenced. The putative genes encoding enzymes for rhamnose synthesis, transferases, O-unit flippase, and O-unit polymerase were identified on the basis of homology. The O-antigen gene clusters of S. boydii O1 and E. coli O149, which share the same O-antigen form, were found to have the same genes and organization by adjacent gene PCR assay. Two genes specific for S. boydii O1 and E. coli O149 were identified by PCR screening against E. coli- and Shigella-type strains of the 186 known O-antigen forms and 39 E. coli clinical isolates. A PCR sensitivity of 103 to 104 CFU/mL overnight culture of S. boydii O1 and E. coli O149 was obtained. S. boydii O1 and E. coli O149 were differentiated by PCR using lacZ- and cadA-based primers.     

ICAM-1 and CD11b inhibition worsen outcome in rats with E. coli pneumonia.

We investigated whether inhibiting an endothelial adhesion molecule [intracellular adhesion molecule 1 (ICAM-1)] would alter outcome and lung injury in a similar fashion to inhibition of a leukocyte adhesion molecule (integrin CD11b) in a rat model of gram-negative pneumonia. Inhibition of ICAM-1 with monoclonal antibody (MAb) 1A29 (1 mg/kg sc or 0.2 or 2 mg/kg iv, q 12 h x 3) or of CD11b with MAb 1B6 (1 mg/kg sc, q 12 h x 3) were compared against similarly administered placebo proteins in rats challenged with intrabronchial Escherichia coli. After challenge, all animals were treated with antibiotics. ICAM-1 MAb (6 mg/kg, iv, total dose) increased mortality vs. control (P = 0.03). CD11b MAb (3 mg/kg, sc, total dose) did not significantly (P = 0.16) increase mortality rates, but this was not in a range of probability to exclude a harmful effect. All other doses of MAb had no significant effect on survival rates. ICAM-1 and CD11b MAbs had significantly different effects on the time course of lung injury, circulating white cells and lymphocytes, and lung lavage white cells and neutrophils (P = 0.04-0.003). CD11b MAb decreased, whereas ICAM-1 MAb increased these measures compared with control from 6 to 12 h after E. coli. However, from 144 to 168 h after E. coli both MAbs increased these measures compared with control rats but to a greater level with CD11b MAb. Thus both ICAM-1 and CD11b appear to be necessary for survival during E. coli pneumonia. Although these adhesion molecules may participate differently in early lung injury, with CD11b increasing and ICAM-1 decreasing inflammation and injury, both are important for the resolution of later injury. During gram-negative pneumonia the protective roles of ICAM-1 and CD11b may make their therapeutic inhibition difficult.     

Protective effect of endogenous beta-adrenergic tone on lung fluid balance in acute bacterial pneumonia in mice

Some investigators have reported that endogenous beta-adrenoceptor tone can provide protection against acute lung injury. Therefore, we tested the effects of beta-adrenoceptor inhibition in mice with acute Escherichia coli pneumonia. Mice were pretreated with propranolol or saline and then intratracheally instilled with live E. coli (10(7) colony-forming units). Hemodynamics, arterial blood gases, plasma catecholamines, extravascular lung water, lung permeability to protein, bacterial counts, and alveolar fluid clearance were measured. Acute E. coli pneumonia was established after 4 h with histological evidence of acute pulmonary inflammation, arterial hypoxemia, a threefold increase in lung vascular permeability, and a 30% increase in extravascular lung water as an increase in plasma catecholamine levels. beta-Adrenoceptor inhibition resulted in a marked increase in extravascular lung water that was explained by both an increase in lung vascular permeability and a reduction in net alveolar fluid clearance. The increase in extravascular lung water with propranolol pretreatment was not explained by an increase in systemic or vascular pressures. The increase in lung vascular permeability was explained in part by anti-inflammatory effects of beta-adrenoceptor stimulation because plasma macrophage inflammatory protein-2 levels were higher in the propranolol pretreatment group compared with controls. The decrease in alveolar fluid clearance with propranolol was explained by a decrease in catecholamine-stimulated fluid clearance. Together, these results indicate that endogenous beta-adrenoceptor tone has a protective effect in limiting accumulation of extravascular lung water in acute severe E. coli pneumonia in mice by two mechanisms: 1) reducing lung vascular injury and 2) upregulating the resolution of alveolar edema.     

Febrile-range hyperthermia augments neutrophil accumulation and enhances lung injury in experimental gram-negative bacterial pneumonia

We previously demonstrated that exposure to febrile-range hyperthermia (FRH) accelerates pathogen clearance and increases survival in murine experimental Klebsiella pneumoniae peritonitis. However, FRH accelerates lethal lung injury in a mouse model of pulmonary oxygen toxicity, suggesting that the lung may be particularly susceptible to injurious effects of FRH. In the present study, we tested the hypothesis that, in contrast with the salutary effect of FRH in Gram-negative peritonitis, FRH would be detrimental in multilobar Gram-negative pneumonia. Using a conscious, temperature-clamped mouse model and intratracheal inoculation with K. pneumoniae Caroli strain, we showed that FRH tended to reduce survival despite reducing the 3 day-postinoculation pulmonary pathogen burden by 400-fold. We showed that antibiotic treatment rescued the euthermic mice, but did not reduce lethality in the FRH mice. Using an intratracheal bacterial endotoxin LPS challenge model, we found that the reduced survival in FRH-treated mice was accompanied by increased pulmonary vascular endothelial injury, enhanced pulmonary accumulation of neutrophils, increased levels of IL-1beta, MIP-2/CXCL213, GM-CSF, and KC/CXCL1 in the bronchoalveolar lavage fluid, and bronchiolar epithelial necrosis. These results suggest that FRH enhances innate host defense against infection, in part, by augmenting polymorphonuclear cell delivery to the site of infection. The ultimate effect of FRH is determined by the balance between accelerated pathogen clearance and collateral tissue injury, which is determined, in part, by the site of infection.     

Functional characterization of UDP-glucose:undecaprenyl-phosphate glucose-1-phosphate transferases of Escherichia coli and Caulobacter crescentus

Escherichia coli K-12 WcaJ and the Caulobacter crescentus HfsE, PssY, and PssZ enzymes are predicted to initiate the synthesis of colanic acid (CA) capsule and holdfast polysaccharide, respectively. These proteins belong to a prokaryotic family of membrane enzymes that catalyze the formation of a phosphoanhydride bond joining a hexose-1-phosphate with undecaprenyl phosphate (Und-P). In this study, in vivo complementation assays of an E. coli K-12 wcaJ mutant demonstrated that WcaJ and PssY can complement CA synthesis. Furthermore, WcaJ can restore holdfast production in C. crescentus. In vitro transferase assays demonstrated that both WcaJ and PssY utilize UDP-glucose but not UDP-galactose. However, in a strain of Salmonella enterica serovar Typhimurium deficient in the WbaP O antigen initiating galactosyltransferase, complementation with WcaJ or PssY resulted in O-antigen production. Gas chromatography-mass spectrometry (GC-MS) analysis of the lipopolysaccharide (LPS) revealed the attachment of both CA and O-antigen molecules to lipid A-core oligosaccharide (OS). Therefore, while UDP-glucose is the preferred substrate of WcaJ and PssY, these enzymes can also utilize UDP-galactose. This unexpected feature of WcaJ and PssY may help to map specific residues responsible for the nucleotide diphosphate specificity of these or similar enzymes. Also, the reconstitution of O-antigen synthesis in Salmonella, CA capsule synthesis in E. coli, and holdfast synthesis provide biological assays of high sensitivity to examine the sugar-1-phosphate transferase specificity of heterologous proteins.     

Evolutionary origins and sequence of the Escherichia coli O4 O-antigen gene cluster

Escherichia coli express many types of O antigen, present in the outer membrane of the Gram-negative bacterial cell wall. O-Antigen biosynthesis genes are clustered together and differences seen in O-antigen types are due to genetic variation within this gene cluster. Sequencing of the E. coli O4 O-antigen gene cluster revealed a similar gene order and high levels of similarity to that of E. coli O26; indicating a common ancestor. These lateral transfer events observed within O-antigen gene clusters may occur as part of the evolution of the pathogenic clones.     

Genetic and structural analyses of Escherichia coli O107 and O117 O-antigens

The O-antigen, consisting of many repeats of an oligosaccharide, is an essential component of the lipopolysaccharide on the surface of Gram-negative bacteria. The O-antigen is one of the most variable cell constituents, and different O-antigen forms are almost entirely due to genetic variations in O-antigen gene clusters. In this paper, we present structural and genetic evidence for a close relationship between Escherichia coli O107 and E. coli O117 O antigens. The O-antigen of E. coli O107 has a pentasaccharide repeating unit with the following structure: →4)-β- d -Gal p NAc-(1→3)-α- l -Rha p -(1→4)-α- d -Glc p NAc-(1→4)-β- d -Gal p -(1→3)-α- d -Gal p NAc-(1→, which differs from the known repeating unit of E. coli O117 only in the substitution of d -GlcNAc for d -Glc. The O-antigen gene clusters of E. coli O107 and O117 share 98.6% overall DNA identity and contain the same set of genes in the same organization. It is proposed that one cluster was evolved from another via mutations, and the substitution of a few amino acids residues in predicted glycosyltransferases resulted in the functional change of one such protein for transferring different sugars in O107 ( d -GlcNAc) and O117 ( d -Glc), leading to different O-antigen structures. This is an example of the O-antigen alteration caused by nucleotide mutations, which is less commonly reported for O-antigen variations.     

Induction of complement sensitivity in Escherichia coli by citric acid and low pH

AIMS: The lytic functions of the complement system play an important role in the control of Gram-negative infections. Complement-resistant Escherichia coli LP1395 (O18) grown under normal conditions can survive the bactericidal action of complement present in human serum. Towards elucidating the mechanisms of complement resistance, the resistance of E. coli LP1395 grown under conditions of low pH and in the presence of citric acid was tested. METHODS AND RESULTS: E. coli LP1395 becomes sensitive to complement after growth in the presence of citric acid at pH 5. Complement resistance could be restored when the cells were transferred to pH 7 media. However, this recovery was greatly impaired when the cells were transferred to pH 7 media with chloramphenicol. This implies that protein synthesis may be involved in complement resistance. The cells exposed to citric acid at pH 5 showed no indication of a generalized outer membrane (OM) permeability when compared with those grown under normal conditions in terms of sensitivity to lysozyme, uptake of lipophilic dye, or sensitivity to a number of antibiotics. CONCLUSION: Complement-resistant LP1395 may acquire a sensitivity to complement due not to a generalized disruption of the OM barrier, but possibly to the alteration of the activity of one or more normal complement resistance factors. SIGNIFICANCE AND IMPACT OF THE STUDY: The elucidation of the mechanisms of complement resistance of Gram-negative pathogens would bring important information about bacterial infections. Complement resistance factors could also be potential targets in antimicrobial therapies.     

Structural and genetic characterization of the Escherichia coli O180 O antigen and identification of a UDP-GlcNAc 6-dehydrogenase

The O antigen is an essential component of the lipopolysaccharides on the surface of Gram-negative bacteria and its variation provides a major basis for serotyping schemes. The Escherichia coli O-antigen form O180 was first designated in 2004, and O180 strains were found to contain virulence factors and cause diarrhea. Different O-antigen forms are almost entirely due to genetic variations in the O-antigen gene clusters. In this study, the chemical structure and gene cluster of E. coli O180 O antigen were investigated. A tetrasaccharide repeating unit with the following structure: →4)-β-d-ManpNAc3NAcA-(1?→?2)-α-l-Rhap(I)-(1?→?3)-β-l-Rhap(II)-(1?→?4)-α-d-GlcpNAc-(1→was identified in the E. coli O180 O antigen, including the residue d-ManpNAc3NAcA (2,3-diacetamido-2,3-dideoxy-d-mannopyranuronic acid) that had not been hitherto identified in E. coli. Genes in the O-antigen gene cluster were assigned functions based on their similarities with those from available databases, and five genes involved in the synthesis of UDP-d-ManpNAc3NAcA (the nucleotide-activated form of d-ManpNAc3NAcA) were identified. The gnaA gene, encoding the enzyme involved in the initial step of the UDP-d-ManpNAc3NAcA biosynthetic pathway, was cloned and the enzyme product was expressed, purified and assayed for its activity. GnaA was characterized using capillary electrophoresis and electrospray ionization mass spectrometry and identified as a UDP-GlcNAc 6-dehydrogenase. The kinetic and physicochemical parameters of GnaA also were determined.     

Structural and genetic relationships of two pairs of closely related O-antigens of Escherichia coli and Salmonella enterica: E. coli O11/S. enterica O16 and E. coli O21/S. enterica O38

The O-antigen is a part of the lipopolysaccharide molecule present in the outer membrane of Gram-negative bacteria, and is essential for the full function of the microorganisms. Salmonella enterica and Escherichia coli are taxonomically closely related species. In this study, the O-antigen structures of S. enterica O16 and O38 and E. coli O11 were determined. Salmonella enterica O38 and E. coli O21 were found to have identical O-antigen structures, whereas S. enterica O16 and E. coli O11 had closely related structures, differing only in the presence of a lateral glucose residue and O-acetylation of a mannose residue in the former. The O-antigen gene clusters of S. enterica O16 and O38 and E. coli O11 were sequenced and analyzed together with that of E. coli O21 retrieved from the GenBank. Each S. enterica/E. coli pair was found to contain the same set of genes organized in the same manner and to share 56-78% overall DNA identity. These data suggest that the O-antigen gene clusters of each pair studied originated from a common ancestor. Thus, it has become evident that in the past, the degree of relatedness between the O-antigens of S. enterica and E. coli was underestimated.     

大肠杆菌O23 O-抗原基因簇序列的破译及UDP-N-乙酰葡萄糖-C4异构酶的鉴定

采用鸟枪法破译大肠杆菌O23标准株的O-抗原基因簇序列,并用生物信息学的方法进行了基因注释和分析;采用基因缺失和互补的方法鉴定了O23的UDP-GlcNAc C4异构酶(Gne);用同源建模的方法构建了O23 Gne的高级结构并对其活性位点进行了分析;分析了不同血清型大肠杆菌O-抗原基因簇中gne基因的多样性;根据O23O-抗原基因簇中的特异基因筛选出了可用于大肠杆菌O23快速检测的特异DNA序列。     

Group V phospholipase A2 in bone marrow-derived myeloid cells and bronchial epithelial cells promotes bacterial clearance after Escherichia coli pneumonia

Group V-secreted phospholipase A(2) (GV sPLA(2)) hydrolyzes bacterial phospholipids and initiates eicosanoid biosynthesis. Here, we elucidate the role of GV sPLA(2) in the pathophysiology of Escherichia coli pneumonia. Inflammatory cells and bronchial epithelial cells both express GV sPLA(2) after pulmonary E. coli infection. GV(-/-) mice accumulate fewer polymorphonuclear leukocytes in alveoli, have higher levels of E. coli in bronchoalveolar lavage fluid and lung, and develop respiratory acidosis, more severe hypothermia, and higher IL-6, IL-10, and TNF-α levels than GV(+/+) mice after pulmonary E. coli infection. Eicosanoid levels in bronchoalveolar lavage are similar in GV(+/+) and GV(-/-) mice after lung E. coli infection. In contrast, GV(+/+) mice have higher levels of prostaglandin D(2) (PGD(2)), PGF(2α), and 15-keto-PGE(2) in lung and express higher levels of ICAM-1 and PECAM-1 on pulmonary endothelial cells than GV(-/-) mice after lung infection with E. coli. Selective deletion of GV sPLA(2) in non-myeloid cells impairs leukocyte accumulation after pulmonary E. coli infection, and lack of GV sPLA(2) in either bone marrow-derived myeloid cells or non-myeloid cells attenuates E. coli clearance from the alveolar space and the lung parenchyma. These observations show that GV sPLA(2) in bone marrow-derived myeloid cells as well as non-myeloid cells, which are likely bronchial epithelial cells, participate in the regulation of the innate immune response to pulmonary infection with E. coli.     

Cathelicidin-related antimicrobial peptide is required for effective lung mucosal immunity in Gram-negative bacterial pneumonia

Cathelicidins are a family of endogenous antimicrobial peptides that exert diverse immune functions, including both direct bacterial killing and immunomodulatory effects. In this study, we examined the contribution of the murine cathelicidin, cathelicidin-related antimicrobial peptide (CRAMP), to innate mucosal immunity in a mouse model of Gram-negative pneumonia. CRAMP expression is induced in the lung in response to infection with Klebsiella pneumoniae. Mice deficient in the gene encoding CRAMP (Cnlp(-/-)) demonstrate impaired lung bacterial clearance, increased bacterial dissemination, and reduced survival in response to intratracheal K. pneumoniae administration. Neutrophil influx into the alveolar space during K. pneumoniae infection was delayed early but increased by 48 h in CRAMP-deficient mice, which was associated with enhanced expression of inflammatory cytokines and increased lung injury. Bone marrow chimera experiments indicated that CRAMP derived from bone marrow cells rather than structural cells was responsible for antimicrobial effects in the lung. Additionally, CRAMP exerted bactericidal activity against K. pneumoniae in vitro. Similar defects in lung bacterial clearance and delayed early neutrophil influx were observed in CRAMP-deficient mice infected with Pseudomonas aeruginosa, although this did not result in increased bacterial dissemination, increased lung injury, or changes in lethality. Taken together, our findings demonstrate that CRAMP is an important contributor to effective host mucosal immunity in the lung in response to Gram-negative bacterial pneumonia.     

Outer membrane protein A deficient Escherichia coli activates neutrophils to produce superoxide and shows increased susceptibility to antibacterial peptides

The outer membrane protein A (OmpA) of Gram-negative bacteria has been ascribed multiple functions including maintenance of structural membrane integrity and porin activity. OmpA has also been implicated in various host defense processes in that it contributes to bacterial serum resistance and activates certain immune cells. Recently, OmpA was shown to be the molecular target for neutrophil elastase (NE), and Escherichia coli mutants lacking OmpA were resistant to the bactericidal effects of NE. In addition to NE, neutrophils use a variety of other antibacterial effector molecules such as oxygen radicals and bactericidal peptides or proteins. The aim of this study was to investigate the role of E. coli OmpA regarding susceptibility to other neutrophil-derived defense systems. We found that OmpA-deficient (OmpA(-)), but not wild-type isogenic, E. coli activated human neutrophils to produce oxygen radicals intracellularly. This activation was found to require an intact neutrophil cytoskeleton but was independent of bacterial phagocytosis. Furthermore, we found that the OmpA(-) strain was more susceptible to membrane-acting bactericidal peptides than the wild-type strain, although the susceptibility to different oxygen radicals was independent of the presence of OmpA. Taken together, these data suggest an important role for OmpA in the context of bacteria vs. host interactions.     

Characterization of an oxygen-dependent inducible promoter,the Escherichia coli nar promoter,in gram-negative host strains

The Escherichia coli nar promoter is maximally induced under anaerobic conditions in the presence of nitrate ion or under anaerobic only conditions, depending on the genotype of the E. coli nar promoter. Previously, we found that the E. coli nar promoter has some desirable characteristics as an inducible promoter in the E. coli host strains. In this study, the E. coli nar promoter with lacZ gene at the downstream was cloned onto a broad-host-range Gram-negative vector, pBBR122. It was then induced in some other Gram-negative host strains, such as Agrobacterium, Pseudomonas, and Rhizobium, to determine whether the E. coli nar promoter could be used as an inducible promoter in these strains. From shake-flask experiments it was found that the wild-type E. coli nar promoter cloned onto pBBR122, pNW61, was suppressed under aerobic conditions in an Agrobacterium host strain, was partially induced under microaerobic only conditions, and was maximally induced under microaerobic conditions in the presence of nitrate ion. Whereas the mutant-type E. coli nar promoter cloned onto pBBR122, pNW618, was suppressed under aerobic conditions and was maximally induced under microaerobic conditions, regardless of the presence of nitrate ion. This kind of induction pattern observed for the E. coli nar promoters in the Agrobacterium host strain was similar to that observed for the E. coli nar promoters in the E. coli host strain. On the other hand, it was found that both of the E. coli nar promoters, pNW61 and pNW618, in a Pseudomonas host strain were partially induced under aerobic conditions and were maximally induced under microaerobic conditions, regardless of the presence of nitrate. Finally, it was found that both of the E. coli nar promoters in a Rhizobium host strain were minimally induced, regardless of the presence of oxygen or nitrate ion. Similar induction patterns for the three strains were also observed from fermentor experiments in which the dissolved oxygen (DO) level was tightly controlled. From an evolutionary point of view, the results from the three Gram-negative host strains indicate that the E. coli nar promoter system, including the promoter and regulatory proteins, was best conserved in the Agrobacterium host strain and the least conserved in the Rhizobium host strain. From an industrial point of view, the results indicate that the E. coli nar promoter system can be used as an oxygen-dependent inducible promoter in both Agrobacterium and Pseudomonas host strains.