J Med Microbiol 56 (2007), 165-171; DOI: 10.1099/jmm.0.46823-0
© 2007 Society for General Microbiology
ISSN 1473-5644
Acinetobacter baumannii lipopolysaccharides are potent stimulators of human monocyte activation via Toll-like receptor 4 signalling
Clett Erridge1,
,
Olga L. Moncayo-Nieto2,,
Robert Morgan2,
Michelle Young2 and
Ian R. Poxton2
1 Department of Bioscience, 204 George Street, University of Strathclyde, Glasgow G1 1XW, UK
2 Medical Microbiology, Centre for Infectious Diseases, University of Edinburgh College of Medicine and Veterinary Medicine, Chancellor's Building, 49 Little France Crescent, Edinburgh EH16 4SB, UK
Correspondence
Ian R. Poxton
i.r.poxton{at}ed.ac.uk
Received 7 July 2006
Accepted 15 October 2006
Acinetobacter baumannii is a major nosocomial pathogen and frequent cause of hospital-acquired pneumonia, surgical wound infections and sepsis. As very little is known of the endotoxic potential of A. baumannii lipopolysaccharide (LPS) with respect to human cells or of its ability to stimulate inflammatory signalling via human Toll-like receptors (TLRs), the biological activity of these endotoxins was investigated in human monocytic THP-1 cells and in TLR-deficient HEK-293 cells transfected with human TLR2 and TLR4 constructs. Endotoxins derived from five clinical isolates of A. baumannii and one of Acinetobacter ‘genomospecies 9’ showed high potency, which was comparable to that of Escherichia coli strain R1 NCTC 13114 LPS, in the induction of the Limulus amoebocyte reaction and interleukin 8 and tumour necrosis factor alpha release from THP-1 cells. Whole UV-killed cells of A. baumannii and Acinetobacter ‘genomospecies 9’ stimulated both TLR2- and TLR4-dependent signalling, whereas pure endotoxins of all investigated strains induced signalling via TLR4, but not TLR2.
Abbreviations: ARDS, acute respiratory distress syndrome; DIC, disseminated intravascular coagulation; FCS, fetal calf serum; IL, interleukin; ITS, inter-spacer; LAL, Limulus amoebocyte lysate; LPS, lipopolysaccharide; SIRS, systemic inflammatory response syndrome; TLR, Toll-like receptor; TNF, tumour necrosis factor.
These authors contributed equally to this work. 
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INTRODUCTION
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Acinetobacter baumannii is an opportunistic bacterial pathogen that has been increasingly identified as a cause of hospital-acquired pneumonia, wound infections and sepsis (Forster & Daschner, 1998; Maragakis et al., 2004; Koprnova et al., 2001). A. baumannii is notable for the frequent development of antimicrobial resistance (Jain & Danziger, 2004; Hanlon, 2005) and is an occasional cause of a fulminant form of community-acquired pneumonia associated with a particularly high mortality rate (Leung et al., 2006; Chen et al., 2001). In some individuals, A. baumannii infection can induce systemic inflammatory response syndrome (SIRS) together with septic shock, disseminated intravascular coagulation (DIC) and acute respiratory distress syndrome (ARDS) (Leung et al., 2006; Chen et al., 2001). The mechanisms responsible for the unusually severe systemic inflammatory reaction in response to A. baumannii infection in some individuals remain poorly understood. However, as this opportunistic organism does not produce any known cytotoxins and is thought to possess only a limited number of virulence factors (Andrews, 1986), it has been suggested that the fulminant course of disease might be related to an exaggerated host response to endotoxin [lipopolysaccharide (LPS)] released from the outer membrane (Leung et al., 2006).
The ability of endotoxins from other bacteria, notably those of Escherichia coli, Neisseria meningitidis and Salmonella enterica serovar Minnesota, to induce systemic inflammation in response to low-dose challenge has been well established (Pier et al., 1981; Brandtzaeg et al., 2001; Taveira da Silva et al., 1993; Rietschel et al., 1994). When present in the blood, endotoxins from these organisms stimulate circulating white blood cells to release pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-
) and interleukin 1ß (IL1ß), thereby contributing to a wide range of deleterious effects, including DIC, ARDS, shock and multiple organ failure (Opal et al., 1999; Wakefield et al., 1998). Detection of these endotoxins occurs via the pattern-recognition receptors Toll-like receptor (TLR)-4 and MD-2 (Hirschfeld et al., 2000; Poltorak et al., 1998), though the endotoxins of certain strains of several non-enterobacterial species, such as Bacteroides fragilis, Pseudomonas aeruginosa, Porphyromonas gingivalis and Rhodobacter sphaeroides, interact only very weakly or not at all with human TLR4/MD2 and have low biological activity (Lindberg et al., 1990; Goldberg & Pier, 1996; Coats et al., 2003; Kirkland et al., 1991; Erridge et al., 2004).
As very little is known of the endotoxic potential of A. baumannii LPS with respect to human cells or of its ability to stimulate signalling via human TLRs, the biological activity of Acinetobacter endotoxins was investigated in human cells and their interactions with human TLR2 and TLR4 were studied using endotoxins extracted from six well-characterized clinical isolates of A. baumannii.
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METHODS
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Identification of strains.
A total of six strains from an established library of clinically derived Acinetobacter cultures maintained at the Royal Infirmary of Edinburgh Clinical Microbiology Laboratory (Edinburgh, UK) was investigated. Three strains were obtained originally from blood cultures, two were from sputum and one was from broncho-alveolar lavage (Table 1
). Strains were identified provisionally by API 20NE and Vitek (bioMérieux) prior to molecular characterization.
16S ribosomal gene and ITS region sequencing.
PCR was performed on boiled lysates of each A. baumannii culture. To amplify the 16S ribosomal region, primer sequences provided by E. Kuijper (Leiden University Medical Centre, Leiden, The Netherlands) were used: P0 243BAC (5'-GGCTCAGATTGAACGCTGGC-3') and P4 249BAC (5'-TCGTTGCGGGACTTAACCCAAC-3'). Conditions were: 94 °C for 3 min, followed by 35 cycles of 94 °C for 20 s, 60 °C for 23 s, 72 °C for 2 min, and a final extension at 72 °C for 10 min. Products were sequenced using primers P1 244BAC (5'-TAACACATGCAAGTCGAACG-3'), P2 245BAC (5'-CCCATTGTGCAATATTCCCC-3') and P3 248BAC (5'-GGATTAGATACCCTGGTAGTCC-3') and P4 249BAC (5'-TCGTTGCGGGACTTAACCCAAC-3'). NCBI nucleotide BLAST analysis was performed and sequence alignments made with BLOEDIT software. PCR was performed as described by Chang et al. (2005) to amplify the inter-spacer (ITS) region, which includes small fragments of the 16S rRNA and the 23S rRNA genes, using universal primers 1512F (5'-GTCGTAACAAGGTAGCCGTA-3') and 6R (5'-GGGTTYCCCCRTTCRGAAAT-3') (where Y is C or T and R is A or G). Conditions were: 94 °C for 2 min, followed by 35 cycles of 94 °C for 1 min, 62 °C for 1 min, 72 °C for 1 min, and a final extension step at 72 °C for 7 min. PCR products were sequenced using primers for the ITS (5''-ACGAAAGATT-3' and 3'-GGGGTTGTAT-5') and analysis performed as above.
UV irradiation of bacteria.
Bacteria were grown in 25 ml nutrient broth for 24 h, centrifuged at 5 000 g for 5 min, then washed twice in 0.9 % saline. Thin films of bacterial suspensions were exposed to bactericidal UV radiation for 40 min in glass Petri dishes. Viability was assessed by culturing UV-exposed cells on blood agar for 48 h.
LPS extraction.
Endotoxins of each strain were extracted by the aqueous phenol extraction method originally developed by Westphal & Luderitz (1954) with modifications as described by Hancock & Poxton (1988). Samples were run on 10 % SDS-PAGE gel and silver stained to show chemotype as described by Hancock & Poxton (1988). For the removal of protein and lipoprotein contaminants from crude primary endotoxin extracts, samples were repurified according to the method of Hirschfeld et al. (2000). Briefly, concentrations of triethylamine and sodium deoxycholate in the LPS samples were adjusted to 0.2 and 0.5 %, respectively, and samples were subjected to two rounds of aqueous phenol re-extraction. Aqueous phases were then pooled and ethanol and sodium acetate concentrations were adjusted to 75 % and 30 mM, respectively, to allow precipitation at –20 °C for 1 h. The LPS was harvested by centrifugation (10 min at 10 000 g), washed in 1 ml cold 100 % ethanol, air-dried and resuspended in the original volume of 0.2 % triethylamine. Recovery of repurified LPS was assumed to be 100 %. Following SDS-PAGE and electro-transfer to nitrocellulose membranes, colloidal gold staining of repurified endotoxin confirmed that lipoprotein contaminants could not be detected following re-extraction (data not shown)
Cells and transfection assays.
THP-1 and HEK-293 cell lines were maintained in RPMI 1640 supplemented with 10 % fetal calf serum (FCS) or Dulbecco's modified Eagle's medium/10 % FCS (Sigma), respectively. For transfection assays, HEK-293 cells were plated in 96-well plates at 8x103 cells per well and transfected after 24 h using Genejuice (Novagen) according to the manufacturer's instructions. Amounts of construct per well were 30 ng plasmids expressing human TLR2 or 30 ng human TLR4 co-expressing MD-2 (Invivogen), 30 ng pCD14 (a kind gift from Professor C. Gregory, University of Edinburgh, Edinburgh, UK) and 10 ng luciferase reporter construct driven by the NF-
B-dependent E-selectin promoter cloned into pGL3 (Promega) as described by Schindler & Baichwal (1994), with the balance made up with empty pCDNA3. Cells were grown for 2–3 days post-transfection prior to 18 h challenge. Reporter levels were normalized to co-transfected renilla expression (Promega Dual-Glo). Promoter expression is represented as the mean (±SD) of triplicate determinations of the degree of induction of the normalized signal relative to cells cultured in medium alone. Endogenous expression of TLRs in HEK-293 was ruled out by RT-PCR (data not shown). Repurified LPS of E. coli core-type R1 NCTC 13114 served as positive control for TLR4 signalling and repurified LPS of Porphyromonas gingivalis MPRL-1675 or the synthetic bacterial lipopeptide mimetic Pam3CSK4 (Invivogen) was used as positive control for TLR2 signalling.
Challenge of THP-1 cells and determination of IL8 and TNF- expression.
THP-1 cells were plated at 5x104 cells per well in 96-well plates and differentiated with 0.1 µM dihydroxy-vitamin D3 for 72 h before challenge with 0.1–100 ng ml–1 of each endotoxin preparation in triplicate in RPMI supplemented with 1 % FCS. As pilot experiments showed that maximal cytokine release was achieved in response to 10 ng E. coli LPS ml–1, this was the maximum concentration examined for this endotoxin (data not shown). Supernatant TNF- and IL8 concentrations were measured at 4 and 18 h, respectively, by ELISA (R&D). All results are presented as means of triplicate wells and are representative of at least three independent experiments.
Limulus amoebocyte lysate (LAL) assay.
Limulus amoebocyte lysate (Chromo-LAL; Quadratech) assay was performed in triplicate on samples of A. baumannii strain 4801 and E. coli R1 LPS serially diluted 3.2-fold in pyrogen-free water. Chromogen development was halted after 20 min with 1 % H2SO4.
Statistical analysis.
All results were compared by ANOVA (Tukey's test). Differences were considered significant at P<0.05.
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RESULTS AND DISCUSSION
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Identification of bacterial strains
Accurate identification of A. baumannii is unreliable by conventional phenotypic methods (Gerner-Smidt et al., 1991). Therefore, strains that were provisionally identified by API 20NE and Vitek were confirmed by 16S and 16S–23S ITS region RNA gene sequencing. API 20NE suggested that five of the six library strains were A. baumannii, but that strain 4800 was Acinetobacter lwoffii (Table 1
). 16S rRNA gene sequencing confirmed that all strains were Acinetobacter species, though as sequencing of this region cannot discriminate between Acinetobacter species, the 16S–23S ITS was investigated as this highly conserved region provides further discrimination between Acinetobacter species (Chang et al., 2005). Five of the six strains were identified as Acinetobacter genomic species 13TU, which is part of the A. baumannii complex (Chang et al., 2005), whereas strain 4800 was reclassified as Acinetobacter ‘genomospecies 9’.
LPS chemotype
The six LPS preparations on silver-stained 10 % SDS-PAGE gels were identical in appearance and gave patterns typical of R-form LPS. Bands were only found at the front of the gel, in contrast to the E. coli LPS control, which gave a typical smooth ladder pattern (results not shown). In the non-purified samples, faint bands were apparent that were probably proteins or lipoproteins. These disappeared after repurification and were absent in blots stained with colloidal gold. LPS of many A. baumannii strains are smooth in character when stained with silver (Pantophlet et al., 1999) and the ladder pattern can also be revealed by Western blotting with specific antibodies to the O-antigen. Without such antibodies, we cannot be certain that the LPSs were not smooth as the sugars of the O-antigen may be resistant to periodate oxidation, the first step of silver staining.
A. baumannii LPS is a potent stimulator of the LAL reaction
As the biological activity of A. baumannii LPS remains relatively poorly investigated, endotoxin of strain 4801 was examined using the LAL assay. This assay has been widely used to examine the potency of other endotoxins (Rietschel et al., 1994), thereby allowing comparison with endotoxins of other bacteria. Unlike many other non-enterobacterial endotoxins, which demonstrate very low activity in this assay compared to E. coli LPS (Rietschel et al., 1994; Lindberg et al., 1990; Kirkland et al., 1991), the activity of the endotoxin of A. baumannii strain 4801 was roughly comparable in this assay to that of E. coli (Fig. 1), which is the endotoxin most widely used to standardize the assay.
A. baumannii LPS is a potent inducer of pro-inflammatory cytokine expression in THP-1 cells
A. baumannii LPS has been shown to stimulate inflammatory signalling in murine cells (Garcia et al., 1999; Knapp et al., 2006). However, the potency of certain types of LPS in murine systems occasionally differs dramatically from the potency of the same endotoxins when applied to human cells (Hajjar et al., 2002; Akashi et al., 2001). For this reason, and because monocytes are thought to play a key role in the recognition of endotoxin and pathology of sepsis (Wakefield et al., 1998), the biological activity of A. baumannii LPS was examined in the human monocytic cell-line THP-1. The minimum concentration required by E. coli and each Acinetobacter endotoxin to stimulate significant (P<0.05) IL8 release from these cells was 0.1 ng ml–1, except for LPS of strains 4800 and 4808, which stimulated significant IL8 release at 1 ng ml–1 (Fig. 2a). Similarly, the minimum concentration required by each of the Acinetobacter endotoxins to stimulate significant TNF- release was the same as that of E. coli LPS, 1 ng ml–1 (Fig. 2b).
As several previous studies of the relative potency of endotoxins have used the minimum concentration required to stimulate cells as a marker of bioactivity (Lindberg et al., 1990; Goldberg & Pier, 1996; Erridge et al., 2004), the relative potency of Acinetobacter endotoxin determined in these assays can be compared with those of other non-enterobacterial endotoxins. For example, in a previous study using similar experimental systems and the same control E. coli LPS, it was shown that certain non-enterobacterial endotoxins, such as those of B. fragilis and Pseudomonas aeruginosa, stimulate human monocyte activation only at concentrations equal to or greater than 10–100 ng ml–1 (Erridge et al., 2004). Since concentrations of A. baumannii LPS as low as 0.1 ng ml–1 were sufficient to induce some degree of cellular activation, it is concluded that the potency of A. baumannii LPS is closer to that of E. coli LPS than it is to less active endotoxins such as those of B. fragilis. Together with the results from the LAL assay and the findings of earlier studies, these data suggest that LPS of both A. baumannii and Acinetobacter ‘genomospecies 9’ fall into the group of the most highly active endotoxins, which includes those of E. coli, S. enterica Minnesota and N. meningitidis (Rietschel et al., 1994; Brandtzaeg et al., 2001; Taveira da Silva et al., 1993). This observation therefore supports the hypothesis that A. baumannii endotoxin may contribute to the particularly strong inflammatory responses observed during certain forms of A. baumannii infection (Leung et al., 2006; Chen et al., 2001). Indeed, it may be of relevance that comparatively low concentrations of both A. baumannii and ‘genomospecies 9’ endotoxin induced release of the inflammatory cytokine TNF-, production of which is thought to be a major contributor to the pathology of SIRS, sepsis and DIC (Wakefield et al., 1998) (Fig. 2b).
Role of human TLR2 and TLR4 in the detection of UV-killed A. baumannii
Detection of bacterial infection is achieved by the use of pattern recognition receptors. A major family of these, the TLRs, play a critical role in the host defence against bacteria (Underhill & Ozinsky, 2002). In particular, TLR2 and TLR4 are the most significant determinants of host detection of and defence against bacterial infection (Takeuchi et al., 1999). Therefore, the further potential pathways for the production of pro-inflammatory cytokines by A. baumannii and ‘genomospecies 9’ and the potential of each to stimulate cellular signalling via human TLR2 and TLR4 were investigated.
Although a recent study has identified that TLR4 is the major receptor required for clearance of A. baumannii infection in the mouse (Knapp et al., 2006), it should be noted that the specificities of human and murine TLR4/MD2 are occasionally seen to be quite different. For example, the partial lipid-A structure lipid-IVa is a potent activator of murine TLR4, yet is an inhibitor of human TLR4 signalling (Akashi et al., 2001) and whereas penta-acyl Pseudomonas aeruginosa LPS is an agonist of murine TLR4/MD2, it is an antagonist of human TLR4/MD2 (Hajjar et al., 2002). Therefore, to avoid such species-specific disparities, A. baumannii-induced signalling was investigated by transfection of the TLR-deficient human cell-line HEK-293 with human TLR2 and TLR4 constructs.
Using this assay, it was found that whole UV-killed A. baumannii and ‘genomospecies 9’ stimulated both TLR2- and TLR4-dependent signalling, although they did not stimulate NF-B signalling in the absence of TLRs (Fig. 3). There were no significant differences between the strains in terms of capacity to induce TLR2 stimulation, although the TLR4 signal induced by strain 4801 was significantly lower than that induced by the other strains. Interestingly, a recent study has shown that TLR4 is critical to bacterial clearance in a murine model of A. baumannii infection (Knapp et al., 2006). Moreover, the surprising finding was also made that functional TLR2 appeared to reduce the magnitude of host inflammatory responses to bacterial challenge (Knapp et al., 2006). Our findings suggest that, in human cells at least, whole A. baumannii stimulates pro-inflammatory signalling, at least as measured by NF-B signalling, via both TLR2- and TLR4-dependent recognition of these bacteria. The proposal that TLR2-dependent anti-inflammatory pathways may exist that modify responses to these bacteria (Knapp et al., 2006) will require further investigation before this issue can be clarified.
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Fig. 3. Determination of TLR-signalling pathways initiated by whole UV-killed Acinetobacter. TLR-deficient HEK-293 cells were transfected with CD14 alone (white bars) or with CD14 plus TLR2 (grey bars) or TLR4/MD2 (black bars). Cells were then challenged with 105 UV-killed bacteria ml–1 for 18 h. The degree of induction of a co-transfected NF-B-sensitive reporter construct (pELAM) is shown, relative to cells cultured in medium alone (ctrl). Results are presented as means of triplicate wells and are representative of at least three independent experiments. Ec, Repurified E. coli LPS; Pg, repurified Porphyromonas gingivalis LPS.
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A. baumannii and ‘genomospecies 9’ endotoxins stimulate human TLR4-dependent signalling
As Acinetobacter endotoxin has been proposed to be a possible virulence factor for this organism (Leung et al., 2006), the role of TLR signalling in response to endotoxins of the chosen strains was investigated. A recent study has shown that A. baumannii LPS stimulates signalling in murine cells via TLR4 (Knapp et al., 2006) though, as discussed earlier, it should be noted that endotoxins that are agonists of the murine TLR4/MD2 complex are occasionally antagonists of human TLR4/MD2 (Hajjar et al., 2002; Akashi et al., 2001). Moreover, there is evidence that TLR2 may play a more prominent role in the detection of certain non-classical endotoxins in human cells, in a manner that is independent of lipoprotein contamination (Darveau et al., 2004; Werts et al., 2001; Erridge et al., 2004).
For these reasons, the potential of A. baumannii and ‘genomospecies 9’ LPS to stimulate human TLR2 and TLR4 signalling was investigated. All of the Acinetobacter endotoxins stimulated TLR4-dependent signalling, though it was found that crude endotoxin extracts from four of the six strains also stimulated significant TLR2-dependent signalling (Fig. 4a). Early studies of enterobacterial LPS revealed that the TLR2-dependent signal in crude endotoxin preparations can be due to lipoprotein contamination and that repurification of these crude extracts by phenol-water re-extraction removed TLR2-stimulating contaminants completely (Hirschfeld et al., 2000). It is likely that the crude endotoxin extracts of strains 4800–4803 contained more TLR2-stimulating contaminants than extracts of strains 4808 and 4809, as no statistically significant TLR2 signal remained following repurification (Fig. 4b). The differences in TLR2 signalling between the crude preparations are more likely to reflect variation in purification efficiency than levels of TLR2 stimulants present in the parent bacteria as TLR2 responses to each strain of whole bacteria were equivalent (Fig. 3). Likewise, statistical analysis of the TLR4 dependent signalling of the repurified endotoxins showed that strains 4800–4803 stimulated significantly less TLR4 signalling than E. coli LPS, whereas that of strain 4809 stimulated significantly more TLR4 signalling than E. coli LPS (Fig. 4b). However, given that there was no significant difference between TLR4 signalling of the crude extracts and that of E. coli LPS (Fig. 4a), it is likely that the differences between the repurified endotoxins represent variation in efficiency of LPS recovery during the repurification process rather than genuine differences in potency of the native endotoxins.
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Fig. 4. Determination of TLR-signalling pathways initiated by A. baumannii endotoxin. TLR-deficient HEK-293 cells were transfected with CD14 alone (white bars) or with CD14 plus TLR2 (grey bars) or TLR4/MD2 (black bars). Cells were then challenged with 10 ng ml–1 of crude extracts of Acinetobacter endotoxin (a) or with 10 ng ml–1 of the same endotoxins after phenol-water re-extraction (b) for 18 h. The degree of induction of a co-transfected NF-B-sensitive reporter construct (pELAM) is shown, relative to cells cultured in medium alone (ctrl). Results are presented as means of triplicate wells and are representative of at least three independent experiments. Ec, Repurified E. coli LPS; Pg, repurified Porphyromonas gingivalis LPS. *TLR2 signal significantly higher than cells cultured in medium alone (P<0.05), #TLR4 signal significantly different from TLR4 response to 10 ng E. coli LPS ml–1 (P<0.05).
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Nevertheless, these results place the endotoxins of the examined Acinetobacter strains in the group of endotoxins that interact strongly with human TLR4/MD2, which previous studies have indicated include those of E. coli, S. enterica and N. meningitidis (Trent et al., 2006), but not those of B. fragilis, Porphyromonas gingivalis or Pseudomonas aeruginosa (Coats et al., 2003; Erridge et al., 2004). It is possible that the strong interaction of the Acinetobacter endotoxins with TLR4/MD2 contributes to the potent toxicity of these endotoxins in human cells (Coats et al., 2005; Rietschel et al., 1994).
To our knowledge, this study is the first to investigate the biological activity and receptor utilization of Acinetobacter endotoxins in human cells. Evidence is presented to demonstrate that the endotoxins of both A. baumannii and Acinetobacter ‘genomospecies 9’ are potent stimulators of inflammatory signalling in human monocytic cells and that responses to these bacteria are dependent on both TLR2 and TLR4. If, as expected, an exaggerated innate immune response to the endotoxins of these organisms contributes to the pathology of Acinetobacter infection, further investigation of the interaction of these endotoxins with the TLR4/MD2 complex may be warranted as a potential drug target for the amelioration of inflammatory signalling.
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ACKNOWLEDGEMENTS
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We are grateful to Professor Sebastian Amyes for assistance in sourcing strains, Professor Christopher Gregory for CD14 construct, Dr Kate Templeton for her assistance with PCR and sequencing protocols, and Stella Yan and Rebecca Lamb for assistance in the preparation of preliminary results.
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REFERENCES
|
|---|
Akashi, S., Nagai, Y., Ogata, H., Oikawa, M., Fukase, K., Kusumoto, S., Kawasaki, K., Nishijima, M., Hayashi, S. & other authors (2001). Human MD-2 confers on mouse Toll-like receptor 4 species-specific lipopolysaccharide recognition. Int Immunol 13, 1595–1599.[Abstract/Free Full Text]
Andrews, H. J. (1986). Acinetobacter bacteriocin typing. J Hosp Infect 7, 169–175.[CrossRef][Medline]
Brandtzaeg, P., Bjerre, A., Ovstebo, R., Brusletto, B., Joo, G. B. & Kierulf, P. (2001). Neisseria meningitidis lipopolysaccharides in human pathology. J Endotoxin Res 7, 401–420.[CrossRef]
Chang, H., Wei, Y., Dijkshoorn, L., Vaneechoutte, M. T., Tang, C. T. & Chang, T. C. (2005). Species-level identification of isolates of the Acinetobacter calcoaceticus-Acinetobacter baumannii complex by sequence analysis of the 16S-23S rRNA gene spacer region. J Clin Microbiol 43, 1632–1639.[Abstract/Free Full Text]
Chen, M. Z., Hsueh, P. R., Lee, L. N., Yu, C. J., Yang, P. C. & Luh, K. T. (2001). Severe community-acquired pneumonia due to Acinetobacter baumannii. Chest 120, 1072–1077.
Coats, S. R., Reife, R. A., Bainbridge, B. W., Pham, T. T. & Darveau, R. P. (2003). Porphyromonas gingivalis lipopolysaccharide antagonizes Escherichia coli lipopolysaccharide at Toll-like receptor 4 in human endothelial cells. Infect Immun 71, 6799–6807.[Abstract/Free Full Text]
Coats, S. R., Pham, T. T., Bainbridge, B. W., Reife, R. A. & Darveau, R. P. (2005). MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex. J Immunol 175, 4490–4498.[Abstract/Free Full Text]
Darveau, R. P., Pham, T. T., Lemley, K., Reife, R. A., Bainbridge, B. W., Coats, S. R., Howald, W. N., Way, S. S. & Hajjar, A. M. (2004). Porphyromonas gingivalis lipopolysaccharide contains multiple lipid A species that functionally interact with both toll-like receptors 2 and 4. Infect Immun 72, 5041–5051.[Abstract/Free Full Text]
Erridge, C., Pridmore, A., Eley, A., Stewart, J. & Poxton, I. R. (2004). Lipopolysaccharides of Bacteroides fragilis, Chlamydia trachomatis and Pseudomonas aeruginosa signal via toll-like receptor 2. J Med Microbiol 53, 735–740.[Abstract/Free Full Text]
Forster, D. H. & Daschner, F. D. (1998). Acinetobacter species as nosocomial pathogens. Eur J Clin Microbiol Infect Dis 17, 73–77.[Medline]
Garcia, A., Salgado, F., Solar, H., Gonzalez, C. L., Zemelman, R. & Onate, A. (1999). Some immunological properties of lipopolysaccharide from Acinetobacter baumannii. J Med Microbiol 48, 479–483.[Abstract/Free Full Text]
Gerner-Smidt, P., Tjernberg, I. & Ursing, J. (1991). Reliability of phenotypic tests for identification of Acinetobacter species. J Clin Microbiol 29, 277–282.[Abstract/Free Full Text]
Goldberg, J. B. & Pier, G. B. (1996). Pseudomonas aeruginosa lipopolysaccharides and pathogenesis. Trends Microbiol 4, 490–494.[CrossRef][Medline]
Hajjar, A. M., Ernst, R. K., Tsai, J. H., Wilson, C. B. & Miller, S. I. (2002). Human Toll-like receptor 4 recognises host-specific LPS modifications. Nat Immunol 3, 354–359.[CrossRef][Medline]
Hancock, I. C. & Poxton, I. R. (1988). Bacterial Cell Surface Techniques. Chichester: Wiley.
Hanlon, G. W. (2005). The emergence of multidrug resistant Acinetobacter species: a major concern in the hospital setting. Lett Appl Microbiol 41, 375–378.[CrossRef][Medline]
Hirschfeld, M., Ma, Y., Weis, J. H., Vogel, S. N. & Weis, J. J. (2000). Cutting edge: repurification of lipopolysaccharide eliminates signaling through both human and murine toll-like receptor 2. J Immunol 165, 618–622.[Abstract/Free Full Text]
Jain, R. & Danziger, L. H. (2004). Multidrug-resistant Acinetobacter infections: an emerging challenge to clinicians. Ann Pharmacother 38, 1449–1459.[Abstract/Free Full Text]
Kirkland, T. N., Qureshi, N. & Takayama, K. (1991). Diphosphoryl lipid A derived from lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides inhibits activation of 70Z/3 cells by LPS. Infect Immun 59, 131–136.[Abstract/Free Full Text]
Knapp, S., Wieland, C. W., Florquin, S., Pantophlet, R., Dijkshoorn, L., Tshimbalanga, N., Akira, S. & van der Poll, T. (2006). Differential roles of CD14 and Toll-like receptors 4 and 2 in murine Acinetobacter pneumonia. Am J Respir Crit Care Med 173, 122–129.[Abstract/Free Full Text]
Koprnova, J., Svetlansky, I., Babel'a, R., Bilikova, E., Hanzen, J., Zuscakova, I. J., Milovsky, V., Masar, O., Kovacicova, G. & other authors (2001). Prospective study of antibacterial susceptibility, risk factors and outcome of 157 episodes of Acinetobacter baumannii bacteremia in 1999 in Slovakia. Scand J Infect Dis 33, 891–895.[CrossRef][Medline]
Leung, W. S., Chu, C. M., Tsang, K. Y., Lo, F. H., Lo, K. F. & Ho, P. L. (2006). Fulminant community-acquired Acinetobacter baumannii pneumonia as a distinct clinical syndrome. Chest 129, 102–109.
Lindberg, A. A., Weintraub, A., Zahringer, U. & Rietschel, E. T. (1990). Structure-activity relationships in lipopolysaccharides of Bacteroides fragilis. Rev Infect Dis 12, S133–S141.
Maragakis, L. L., Cosgrove, S. E., Song, X., Kim, D., Rosenbaum, P., Ciesla, N., Srinivasan, A., Ross, T., Carroll, K. & Perl, T. M. (2004). An outbreak of multidrug-resistant Acinetobacter baumannii associated with pulsatile lavage wound treatment. JAMA 292, 3006–3011.[Abstract/Free Full Text]
Opal, S. M., Scannon, P. J., Vincent, J. L., White, M., Carroll, S. F., Palardy, J. E., Parejo, N. A., Pribble, J. P. & Lemke, J. H. (1999). Relationship between plasma levels of lipopolysaccharide (LPS) and LPS-binding protein in patients with severe sepsis and septic shock. J Infect Dis 180, 1584–1589.[CrossRef][Medline]
Pantophlet, R., Brade, L. & Brade, H. (1999). Identification of Acinetobacter baumannii strains with monoclonal antibodies against the O antigens of their lipopolysaccharides. Clin Diagn Lab Immunol 6, 323–329.
Pier, G. B., Markham, R. B. & Eardley, D. (1981). Correlation of the biologic responses of C3H/HEJ mice to endotoxin with the chemical and structural properties of the lipopolysaccharides from Pseudomonas aeruginosa and Escherichia coli. J Immunol 127, 184–191.[Abstract]
Poltorak, A., He, X., Smirnova, I., Liu, M. Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E., Silva, M. & other authors (1998). Defective LPS signaling in C3H/HEJ and C57Bl/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088.[Abstract/Free Full Text]
Rietschel, E. T., Kirikae, T., Schade, F. U., Mamat, U., Scmidt, G., Loppnow, H., Ulmer, A. J., Zahringer, U., Seydel, U. & other authors (1994). Bacterial endotoxin: molecular relationships of structure to activity and function. FASEB J 8, 217–225.[Abstract]
Schindler, U. & Baichwal, V. R. (1994). Three NF-B binding sites in the human E-selectin gene required for maximal tumor necrosis factor alpha-induced expression. Mol Cell Biol 14, 5820–5831.[Abstract/Free Full Text]
Takeuchi, O., Hoshino, K., Kawei, T., Sanjo, H., Takada, H., Ogawa, T., Takeda, K. & Akira, S. (1999). Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity 11, 443–451.[CrossRef][Medline]
Taveira da Silva, A. M., Kaulbach, H. C., Chuidian, F. S., Lambert, D. R., Suffredini, A. F. & Danner, R. L. (1993). Brief report: shock and multiple-organ dysfunction after self-administration of Salmonella endotoxin. N Engl J Med 328, 1457–1460.[Free Full Text]
Trent, M. S., Stead, C. M., Tran, A. X. & Hankins, J. V. (2006). Diversity of endotoxin and its impact on pathogenesis. J Endotoxin Res 12, 205–223.[CrossRef]
Underhill, D. M. & Ozinsky, A. (2002). Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol 14, 103–110.[CrossRef][Medline]
Wakefield, C. H., Barclay, G. R., Fearon, K. C. H., Goldie, A. S., Ross, J. A., Grant, I. S., Ramsay, G. & Howie, J. C. (1998). Proinflammatory mediator activity, endogenous antagonists and the systemic inflammatory response in intra-abdominal sepsis. Br J Surg 85, 818–825.[CrossRef][Medline]
Werts, C., Tapping, R. I., Mathison, J. C., Chuang, T. H., Kravchenko, V., Saint Girons, I., Haake, D. A., Godowski, P. J., Hayashi, F. & other authors (2001). Leptospiral lipopolysaccharide activates cells through a TLR2-dependent mechanism. Nat Immunol 2, 346–352.[CrossRef][Medline]
Westphal, O. & Luderitz, O. (1954). Chemische erforschung von lipopolysaccharides gramnegativer bakterium. Agnew Chem 66, 407–417.[CrossRef]
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TOP
INTRODUCTION
METHODS
, E. coli LPS; 
, A. baumannii LPS.
, 4801; 
, 4802; 
, 4803; 
, 4808; X, 4809.