Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine.     

Identification of Pseudomonas aeruginosa flagellin as an adhesin for Muc1 mucin

We reported previously that Muc1 mucin on the epithelial cell surface is an adhesion site for Pseudomonas aeruginosa (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181-L187, 2001). The present study was designed to identify the adhesin(s) responsible for bacterial binding to Muc1 mucin using genetic and biochemical approaches. Chinese hamster ovary (CHO) cells stably transfected with a Muc1 cDNA (CHO-Muc1) or empty plasmid (CHO-X) were compared for adhesion of P. aeruginosa strain PAK. Our results showed that 1) wild-type PAK and isogenic mutant strains lacking pili (PAK/NP) or flagella cap protein (PAK/fliD) demonstrated significantly increased binding to CHO-Muc1 cells, whereas flagellin-deficient (PAK/fliC) bacteria were no more adherent to CHO-Muc1 than CHO-X cells, and 2) P. aeruginosa adhesion was blocked by pretreatment of bacteria with antibody to flagellin or pretreatment of CHO-Muc1 cells with purified flagellin. We conclude that flagellin is an adhesin of P. aeruginosa responsible for its binding to Muc1 mucin on the epithelial cell surface.     

Cutting edge: myeloid differentiation factor 88 is essential for pulmonary host defense against Pseudomonas aeruginosa but not Staphylococcus aureus

Myeloid differentiation factor 88 (MyD88) is an adapter molecule required for signal transduction via Toll-like receptors (TLRs) and receptors of the IL-1 family. Consequently, MyD88-deficient mice are highly susceptible to bacterial infections, including systemic infection with Staphylococcus aureus. To determine the role of MyD88 in innate immunity to bacterial pneumonia, we exposed MyD88-deficient and wild-type mice to aerosolized Pseudomonas aeruginosa or S. aureus. As predicted, MyD88-deficient mice failed to mount an early cytokine or inflammatory response or to control bacterial replication after infection with P. aeruginosa, which resulted in necrotizing pneumonia and death. By contrast, MyD88-deficient mice controlled S. aureus infection despite blunted local cytokine and inflammatory responses. Thus, whereas MyD88-dependent signaling is integral to the initiation of cytokine and inflammatory responses to both pathogens following infection of the lower respiratory tract, MyD88 is essential for innate immunity to P. aeruginosa but not S. aureus.     

Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa

Recently, we cloned and characterized a full-length cDNA of the hamster Muc1 gene, the expression of which appears to be associated with secretory cell differentiation (Park HR, Hyun SW, and Kim KC. Am J Respir Cell Mol Biol 15: 237-244, 1996). The role of Muc1 mucins in the airway, however, is unknown. In this study, we investigated whether cell surface mucins are adhesion sites for Pseudomonas aeruginosa. Chinese hamster ovary (CHO) cells not normally expressing Muc1 mucin were stably transfected with the hamster Muc1 cDNA, and binding to P. aeruginosa was examined. Our results showed that 1) stably transfected CHO cells expressed both Muc1 mRNA and Muc1 mucins based on Northern and Western blot analyses, 2) Muc1 mucins present on the cell surface were degraded by neutrophil elastase, and 3) expression of Muc1 mucins on the cell surface resulted in a significant increase in adhesion of P. aeruginosa that was completely abolished by either proteolytic cleavage with neutrophil elastase or deletion of the extracellular domain by mutation. We conclude that Muc1 mucins expressed on the surface of CHO cells serve as adhesion sites for P. aeruginosa, suggesting a possible role for these glycoproteins in the early stage of airway infection and providing a model system for studying epithelial cell responses to bacterial adhesion that leads to airway inflammation in general and cystic fibrosis in particular.     

CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung

CD1d-restricted T cells are implicated as key players in host defense against various microbial infections. However, the mechanisms involved and the role they play, if any, at the mucosal surfaces where pathogenic infections are initiated is unknown. In a murine pneumonia model established by intranasal application of Pseudomonas aeruginosa, CD1d(-/-) mice showed markedly reduced pulmonary eradication of P. aeruginosa compared with wild-type mice; this was associated with significantly lower amounts of macrophage inflammatory protein-2 and reduced numbers of neutrophils within the bronchoalveolar lavage fluid. Corollarily, treatment of mice with alpha-galactosylceramide--a lipid that activates CD1d-restricted T cells--increased the amount of interferon-gamma; this was associated with rapid pulmonary clearance through enhanced phagocytosis of P. aeruginosa by alveolar macrophages. These results reveal a crucial role played by CD1d-restricted T cells in regulating the antimicrobial immune functions of macrophages at the lung mucosal surface.     

Cystic fibrosis lung disease following infection with Pseudomonas aeruginosa in Cftr knockout mice using novel non-invasive direct pulmonary infection technique

To better understand the mechanism of lung infection with Pseudomonas aeruginosa (P. aeruginosa), many techniques have been developed in order to establish lung infection in rodents. A model of chronic lung infection, using tracheotomy to inoculate the bacteria, has been extensively used in the cystic fibrosis (CF) mouse model of lung infection. The cystic fibrosis transmembrane channel (Cftr) knockout (KO) mice are smaller than normal mice and are more sensitive to housing and nutritional conditions, leading to small amounts of animals being available for experiments. Because of these characteristics, and because of the invasiveness of the infection procedure which we, and others, have been using to mimic the lung infection, we sought to find an alternative way to study the inflammatory response during lung P. aeruginosa infection. The technique we describe here consists of the injection of bacterial beads directly into the lungs through the mouth without the need of any tracheal incisions. This technique of direct pulmonary delivery enables much faster infection of the animals compared with the intratracheal technique previously used. The use of this less invasive technique allows the exclusion of the surgery-related inflammation. Our results show that, using the direct pulmonary delivery technique, the KO mice were more susceptible to P. aeruginosa lung infection compared with their wild-type (WT) controls, as shown by their increased weight loss, higher bacterial burden and more elevated polymorphonuclear (PMN) alveolar cell recruitment into the lungs. These differences are consistent with the pathological profiles observed in CF patients infected with P. aeruginosa. Overall, this method simplifies the infection procedure in terms of its duration and invasiveness, and improves the survival rate of the KO mice when compared with the previously used intratracheal procedure.     

Pseudomonas aeruginosa stimulates phosphorylation of the airway epithelial membrane glycoprotein Muc1 and activates MAP kinase

We reported previously that Muc1 on the surface of epithelial cells was a receptor for Pseudomonas aeruginosa (Lillehoj EP, Kim BT, and Kim KC. Am J Physiol Lung Cell Mol Physiol 282: L751-L756, 2002). Other studies showed that the Muc1 cytoplasmic tail (CT) contains multiple phosphorylation sites, some of which are phosphorylated constitutively and associated with signaling proteins. However, the relationship between extracellular P. aeruginosa binding and intracellular signaling is unknown. To investigate the signaling mechanism of Muc1, this study examined phosphorylation of its CT and activation of the extracellular signal-regulated kinase (ERK) in response to stimulation by P. aeruginosa or purified flagellin. Our results showed 1) the Muc1 CT was phosphorylated constitutively on serine and tyrosine, 2) serine phosphorylation was stimulated by bacterial cells or flagellin, and 3) binding of P. aeruginosa or flagellin to Muc1 induced phosphorylation of ERK. These results are the first to demonstrate Muc1 CT phosphorylation and ERK activation in response to a clinically important airway pathogen.     

Alternative mechanisms of respiratory syncytial virus clearance in perforin knockout mice lead to enhanced disease

Virus-specific cytotoxic T lymphocytes are key effectors for the clearance of virus-infected cells and are required for the normal clearance of respiratory syncytial virus (RSV) in mice. Although perforin/granzyme-mediated lysis of infected cells is thought to be the major molecular mechanism used by CD8(+) cytotoxic T lymphocytes for elimination of virus, its role in RSV has not been reported. Here, we show that viral clearance in perforin knockout (PKO) mice is slightly delayed but that both PKO and wild-type mice clear virus by day 10, suggesting an alternative mechanism of RSV clearance. Effector T cells from the lungs of both groups of mice were shown to lyse Fas (CD95)-overexpressing target cells in greater numbers than target cells expressing low levels of Fas, suggesting that Fas ligand (CD95L)-mediated target cell lysis was occurring in vivo. This cell lysis was associated with a delay in RSV-induced disease in PKO mice compared to the time of disease onset for wild-type controls, which correlated with increased and prolonged production of gamma interferon and tumor necrosis factor alpha levels in PKO mice. We conclude that while perforin is not necessary for the clearance of primary RSV infection, the use of alternative CTL target cell killing mechanisms is less efficient and can lead to enhanced disease.     

Muc2和DCN基因敲除小鼠食子现象的初步研究

目的探讨Muc2和DCN基因敲除小鼠繁殖能力和食子现象的异同。方法分别将Muc2和DCN基因敲除纯合子雌雄小鼠按1∶1或1∶2的合笼,观察1～3胎产仔量、胎次间隔时间、出生存活率和食子现象。结果Muc2基因敲除小鼠平均产子量5.80±0.95只,平均胎次间隔时间（42.29±2.28）d;DCN基因敲除小鼠平均产子量3.85±0.76只,平均胎次间隔时间（24.86±10.42）d。Muc2和DCN基因敲除小鼠在产仔量、胎次间隔时间、出生存活率和食子率差异均存在显著性。结论两组基因敲除小鼠繁殖性能有差异,揭示可能与Muc2和DCN基因有关。     

Role of cystic fibrosis transmembrane conductance regulator in pulmonary clearance of Pseudomonas aeruginosa in vivo

Cystic fibrosis (CF)2 is a fatal genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) that is commonly associated with chronic pulmonary infections with mucoid Pseudomonas aeruginosa (PA). To test the hypothesis that CFTR plays a direct role in PA adhesion and clearance, we have used mouse lines expressing varying levels of human (h) or mouse (m) CFTR. A subacute intratracheal dose of 3 x 10(6) bacteria was cleared with similar kinetics in control wild-type (WT) and transgenic mice overexpressing hCFTR in the lung from the surfactant protein C (SP-C) promoter (SP-C-hCFTR+/-). In a second series of experiments, the clearance of an acute intratracheal dose of 1.5 x 10(7) PA bacteria was also similar in WT, hemizygous SP-C-hCFTR+/-, and bitransgenic gut-corrected FABP-hCFTR+/+-mCFTR-/-, the latter lacking expression of mCFTR in the lung. However, a small but significant decrease in bacterial killing was observed in lungs of homozygote SP-C-hCFTR+/+ mice. Lung pathology in both WT and SP-C-hCFTR+/+ mice was marked by neutrophilic inflammation and bacterial invasion of perivascular and subepithelial compartments. Bacteria were associated primarily with leukocytes and were not associated with alveolar type II or bronchiolar epithelial cells, the cellular sites of SP-C-hCFTR+/+ transgene expression. The results indicate that there is no direct correlation between levels of CFTR expression and bacterial clearance or association of bacteria with epithelial cells in vivo.     

Cutting edge: transplantation tolerance through enhanced CTLA-4 expression

Knockout and blocking studies have shown a critical role for CTLA-4 in peripheral tolerance, however, it is unknown whether augmenting CTLA-4 expression actually promotes tolerance. Here we demonstrate a specific and requisite role for CTLA-4 and its up-regulation in tolerance through anti-CD45RB. First, long-term murine islet allograft survival induced by anti-CD45RB is prevented by CTLA4-Ig, which interferes with B7:CTLA-4 interactions. Second, anti-CD45RB is ineffective in recipients lacking CTLA-4, B7-1, and B7-2. In contrast, CTLA4-Ig, which targets B7 on allogeneic cells, promotes long-term engraftment in these mice. Moreover, anti-CD45RB was effective in B7-deficient controls expressing CTLA-4. Finally, in wild-type mice, CTLA-4 expression returned to baseline 17 days after receiving anti-CD45RB, and was refractory to further increase. Transplantation and anti-CD45RB therapy at this time could neither augment CTLA-4 nor prolong engraftment. These data demonstrate a specific role for CTLA-4 in anti-CD45RB-mediated tolerance and indicate that CTLA-4 up-regulation can directly promote allograft survival.     

Transgenic cystic fibrosis mice exhibit reduced early clearance of Pseudomonas aeruginosa from the respiratory tract

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) has been proposed to be an epithelial cell receptor for Pseudomonas aeruginosa involved in bacterial internalization and clearance from the lung. We evaluated the role of CFTR in clearing P. aeruginosa from the respiratory tract using transgenic CF mice that carried either the DeltaF508 Cftr allele or an allele with a Cftr stop codon (S489X). Intranasal application achieved P. aeruginosa lung infection in inbred C57BL/6 DeltaF508 Cftr mice, whereas DeltaF508 Cftr and S489X Cftr outbred mice required tracheal application of the inoculum to establish lung infection. CF mice showed significantly less ingestion of LPS-smooth P. aeruginosa by lung cells and significantly greater bacterial lung burdens 4.5 h postinfection than C57BL/6 wild-type mice. Microscopy of infected mouse and rhesus monkey tracheas clearly demonstrated ingestion of P. aeruginosa by epithelial cells in wild-type animals, mostly around injured areas of the epithelium. Desquamating cells loaded with P. aeruginosa could also be seen in these tissues. No difference was found between CF and wild-type mice challenged with an LPS-rough mucoid isolate of P. aeruginosa lacking the CFTR ligand. Thus, transgenic CF mice exhibit decreased clearance of P. aeruginosa and increased bacterial burdens in the lung, substantiating a key role for CFTR-mediated bacterial ingestion in lung clearance of P. aeruginosa.     

Syndecan-1 shedding is enhanced by LasA, a secreted virulence factor of Pseudomonas aeruginosa

Microbial pathogens frequently take advantage of host systems for their pathogenesis. Shedding of cell surface molecules as soluble extracellular domains (ectodomains) is one of the host responses activated during tissue injury. In this study, we examined whether pathogenic bacteria can modulate shedding of syndecan-1, the predominant syndecan of host epithelia. Our studies found that overnight culture supernatants of Pseudomonas aeruginosa and Staphylococcus aureus enhanced the shedding of syndecan-1 ectodomains, whereas culture supernatants of several other Gram-negative and Gram-positive bacteria had only low levels of activity. Because supernatants from all tested strains of P. aeruginosa (n = 9) enhanced syndecan-1 shedding by more than 4-fold above control levels, we focused our attention on this Gram-negative bacterium. Culture supernatants of P. aeruginosa increased shedding of syndecan-1 in both a concentration- and time-dependent manner, and augmented shedding by various host cells. A 20-kDa shedding enhancer was partially purified from the supernatant through ammonium sulfate precipitation and gel chromatography, and identified by N-terminal sequencing as LasA, a known P. aeruginosa virulence factor. LasA was subsequently determined to be a syndecan-1 shedding enhancer from the findings that (i) immunodepletion of LasA from the partially purified sample resulted in abrogation of its activity to enhance shedding and (ii) purified LasA increased shedding in a concentration-dependent manner. Our results also indicated that LasA enhances syndecan-1 shedding by activation of the host cell's shedding mechanism and not by direct interaction with syndecan-1 ectodomains. Enhanced syndecan-1 shedding may be a means by which pathogenic bacteria take advantage of a host mechanism to promote their pathogenesis.     

Oxidation of 1-Tetradecene by Pseudomonas aeruginosa

Pseudomonas aeruginosa strain Sol 20 was grown on 1-tetradecene as sole carbon source, and a vinyl-unsaturated 14-carbon monocarboxylic acid, 13-tetradecenoic acid, was identified from culture fluid. This acid was not produced when n-tetradecane served as substrate for growth. Oxidation of the methyl group represents one method of attack on the 1-alkene by this organism. Tentative identification of 2-tetradecanol indicates that an attack on the double bond is also occurring. α, ω-Dienes would not support growth.     

Cutting edge: Attenuated experimental autoimmune encephalomyelitis in eta-1/osteopontin-deficient mice

Recent studies indicate that early T lymphocyte activation 1 (Eta-1), also known as osteopontin, is a cytokine contributing to the development of Th1 immunity. In the present report, the role of Eta-1 in experimental autoimmune encephalomyelitis (EAE), a disease associated with Th1 immunity, was examined by analysis of disease progression in Eta-1-deficient (Eta-1-/-) mice. Although incidence and onset of peptide-induced EAE were found to be similar in Eta-1-/- and Eta-1+/+ mice, Eta-1-/- mice displayed significantly lower mean maximal clinical score and faster recovery without spontaneous relapses. Accordingly, decreased inflammatory infiltration and demyelination were observed in the spinal cords of Eta-1-/- mice. Furthermore, in comparison to Eta-1+/+, Eta-1-/- CD4+ T cells had reduced expression of IFN-gamma and TNF-alpha upon ex vivo restimulation. Taken together, these results suggest that Eta-1 may sustain autoimmune responses by assisting in maintenance of Th1 immunity during EAE.     

Abnormal spine morphology and enhanced LTP in LIMK-1 knockout mice

In vitro studies indicate a role for the LIM kinase family in the regulation of cofilin phosphorylation and actin dynamics. In addition, abnormal expression of LIMK-1 is associated with Williams syndrome, a mental disorder with profound deficits in visuospatial cognition. However, the in vivo function of this family of kinases remains elusive. Using LIMK-1 knockout mice, we demonstrate a significant role for LIMK-1 in vivo in regulating cofilin and the actin cytoskeleton. Furthermore, we show that the knockout mice exhibited significant abnormalities in spine morphology and in synaptic function, including enhanced hippocampal long-term potentiation. The knockout mice also showed altered fear responses and spatial learning. These results indicate that LIMK-1 plays a critical role in dendritic spine morphogenesis and brain function.     

Kinase suppressor of Ras-1 protects against pulmonary Pseudomonas aeruginosa infections

Pseudomonas aeruginosa is a Gram-negative pathogen that causes severe infections in immunocompromised individuals and individuals with cystic fibrosis or chronic obstructive pulmonary disease (COPD). Here we show that kinase suppressor of Ras-1 (Ksr1)-deficient mice are highly susceptible to pulmonary P. aeruginosa infection accompanied by uncontrolled pulmonary cytokine release, sepsis and death, whereas wild-type mice clear the infection. Ksr1 recruits and assembles inducible nitric oxide (NO) synthase (iNOS) and heat shock protein-90 (Hsp90) to enhance iNOS activity and to release NO upon infection. Ksr1 deficiency prevents lung alveolar macrophages and neutrophils from activating iNOS, producing NO and killing bacteria. Restoring NO production restores the bactericidal capability of Ksr1-deficient lung alveolar macrophages and neutrophils and rescues Ksr1-deficient mice from P. aeruginosa infection. Our findings suggest that Ksr1 functions as a previously unknown scaffold that enhances iNOS activity and is therefore crucial for the pulmonary response to P. aeruginosa infections.     

Stimulus-permeability coupling in rat pulmonary macrophages challenged by Pseudomonas aeruginosa

Summary Electron probe X-ray microanalysis (XRMA) of freeze-dried ultrathin sections provides the capability of measuring intracellular elemental content. This methodology was used to investigate the stimulus-permeability coupling responses associated with phagocytosis of Pseudomonas aeruginosa by cultured pulmonary alveolar macrophages (PAMs) of rats. PAMs were challenged with P. aeruginosa suspended in Gey's buffer at a bacteria to PAM ratio of 501 for 1 h at 37° C. A 1-mm³ pellet of the unchallenged control PAMs, challenged PAMs and P. aeruginosa alone was quench-frozen in nitrogen-cooled, liquid propane, and 0.1-m cryosections were cut at -100° C. X-ray spectra were collected for nucleus and cytoplasm of 39 control PAMs, 36 challenged PAMs and 40 P. aeruginosa. Concentrations (mmole/kg dry weight) were obtained for Na, Cl, K, Ca, Mg, P, S for each cell. In the control PAMs, the content was similar to other mammalian cells. Moreover, there were no differences in elemental content between nucleus and cytoplasm. In the challenged PAMs, Na concentration was 4 times that of control PAMs (p<0.001) whereas Cl was double (p<0.001), K was 29% lower (p<0.001), and Ca was 4 times higher (p<0.05). The elemental concentration profile in the P. aeruginosa was distinctly different from that of the PAMs: higher Na, Ca, Mg, but lower Cl and K values. These results demonstrate elemental content changes in cultured PAMs challenged with P. aeruginosa that indicate a stimulus-permeability response by membranes associated with the phagocytic process.