Deciphering tissue-induced Klebsiella pneumoniae lipid A structure.

The outcome of an infection depends on host recognition of the pathogen, hence leading to the activation of signaling pathways controlling defense responses. A long-held belief is that the modification of the lipid A moiety of the lipopolysaccharide could help Gram-negative pathogens to evade innate immunity. However, direct evidence that this happens in vivo is lacking. Here we report the lipid A expressed in the tissues of infected mice by the human pathogen Klebsiella pneumoniae. Our findings demonstrate that Klebsiella remodels its lipid A in a tissue-dependent manner. Lipid A species found in the lungs are consistent with a 2-hydroxyacyl-modified lipid A dependent on the PhoPQ-regulated oxygenase LpxO. The in vivo lipid A pattern is lost in minimally passaged bacteria isolated from the tissues. LpxO-dependent modification reduces the activation of inflammatory responses and mediates resistance to antimicrobial peptides. An lpxO mutant is attenuated in vivo thereby highlighting the importance of this lipid A modification in Klebsiella infection biology. Colistin, one of the last options to treat multidrug-resistant Klebsiella infections, triggers the in vivo lipid A pattern. Moreover, colistin-resistant isolates already express the in vivo lipid A pattern. In these isolates, LpxO-dependent lipid A modification mediates resistance to colistin. Deciphering the lipid A expressed in vivo opens the possibility of designing novel therapeutics targeting the enzymes responsible for the in vivo lipid A pattern.

Functional Genomic Screen Identifies Klebsiella pneumoniae Factors Implicated in Blocking Nuclear Factor κB (NF-κB) Signaling.

Klebsiella pneumoniae is an etiologic agent of community-acquired and nosocomial pneumonia. It has been shown that K. pneumoniae infections are characterized by reduced early inflammatory response. Recently our group has shown that K. pneumoniae dampens the activation of inflammatory responses by antagonizing the activation of the NF-κB canonical pathway. Our results revealed that K. pneumoniae capsule polysaccharide (CPS) was necessary but not sufficient to attenuate inflammation. To identify additional Klebsiella factors required to dampen inflammation, we standardized and applied a high-throughput gain-of-function screen to examine a Klebsiella transposon mutant library. We identified 114 mutants that triggered the activation of NF-κB. Two gene ontology categories accounted for half of the loci identified in the screening: metabolism and transport genes (32% of the mutants) and envelope-related genes (17%). Characterization of the mutants revealed that the lack of the enterobactin siderophore was linked to a reduced CPS expression, which in turn underlined the NF-κB activation induced by the mutant. The lipopolysaccharide (LPS) O-polysaccharide and the pullulanase (PulA) type 2 secretion system (T2SS) are required for full effectiveness of the immune evasion. Importantly, these factors do not play a redundant role. The fact that LPS O-polysaccharide and T2SS mutant-induced responses were dependent on TLR2-TLR4-MyD88 activation suggested that LPS O-polysaccharide and PulA perturbed Toll-like receptor (TLR)-dependent recognition of K. pneumoniae. Finally, we demonstrate that LPS O-polysaccharide and pulA mutants are attenuated in the pneumonia mouse model. We propose that LPS O-polysaccharide and PulA T2SS could be new targets for the design of new antimicrobials. Increasing TLR-governed defense responses might provide also selective alternatives for the management of K. pneumoniae pneumonia.

Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes.

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.

Klebsiella pneumoniae outer membrane protein A is required to prevent the activation of airway epithelial cells.

Outer membrane protein A (OmpA) is a class of proteins highly conserved among the Enterobacteriaceae family and throughout evolution. Klebsiella pneumoniae is a capsulated gram-negative pathogen. It is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by a lack of an early inflammatory response. Data from our laboratory indicate that K. pneumoniae CPS helps to suppress the host inflammatory response. However, it is unknown whether K. pneumoniae employs additional factors to modulate host inflammatory responses. Here, we report that K. pneumoniae OmpA is important for immune evasion in vitro and in vivo. Infection of A549 and normal human bronchial cells with 52OmpA2, an ompA mutant, increased the levels of IL-8. 52145-Δwca(K2)ompA, which does not express CPS and ompA, induced the highest levels of IL-8. Both mutants could be complemented. In vivo, 52OmpA2 induced higher levels of tnfα, kc, and il6 than the wild type. ompA mutants activated NF-κB, and the phosphorylation of p38, p44/42, and JNK MAPKs and IL-8 induction was via NF-κB-dependent and p38- and p44/42-dependent pathways. 52OmpA2 engaged TLR2 and -4 to activate NF-κB, whereas 52145-Δwca(K2)ompA activated not only TLR2 and TLR4 but also NOD1. Finally, we demonstrate that the ompA mutant is attenuated in the pneumonia mouse model. The results of this study indicate that K. pneumoniae OmpA contributes to attenuate airway cell responses. This may facilitate pathogen survival in the hostile environment of the lung.

Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner.

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Subversion of inflammation is essential for pathogen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro-inflammatory mediators-induced IL-8 secretion. Klebsiella antagonizes the activation of NF-κB via the deubiquitinase CYLD and blocks the phosphorylation of mitogen-activated protein kinases (MAPKs) via the MAPK phosphatase MKP-1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti-inflammatory effect, Klebsiella engages NOD1. In NOD1 knock-down cells, Klebsiella neither induces the expression of CYLD and MKP-1 nor blocks the activation of NF-κB and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1-mediated CYLD and MKP-1 expression which in turn attenuates IL-1ß-induced IL-8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL-1ß-induced IL-8 secretion nor induces the expression of CYLD and MKP-1 thereby indicating that CPS is necessary but not sufficient to attenuate inflammation.

Evidence for a non-replicative intracellular stage of nontypable Haemophilus influenzae in epithelial cells.

Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi- or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells.

Klebsiella pneumoniae capsule polysaccharide impedes the expression of beta-defensins by airway epithelial cells.

Human beta-defensins (hBDs) contribute to the protection of the respiratory tract against pathogens. It is reasonable to postulate that pathogens have developed countermeasures to resist them. Klebsiella pneumoniae capsule polysaccharide (CPS), but not the lipopolysaccharide O antigen, mediated resistance against hBD1 and hBD2. hBD3 was the most potent hBD against Klebsiella. We investigated the possibility that as a strategy for survival in the lung, K. pneumoniae may not activate the expression of hBDs. Infection of A549 and normal human bronchial cells with 52145-Deltawca(K2), a CPS mutant, increased the expression of hBD2 and hBD3. Neither the wild type nor the lipopolysaccharide O antigen mutant increased the expression of hBDs. In vivo, 52145-Deltawca(K2) induced higher levels of mBD4 and mBD14, possible mouse orthologues of hBD2 and hBD3, respectively, than the wild type. 52145-Deltawca(K2)-dependent upregulation of hBD2 occurred via NF-kappaB and mitogen-activated protein kinases (MAPKs) p44/42, Jun N-terminal protein kinase (JNK)-dependent pathways. The increase in hBD3 expression was dependent on the MAPK JNK. 52145-Deltawca(K2) engaged Toll-like receptors 2 and 4 (TLR2 and TLR4) to activate hBD2, whereas hBD3 expression was dependent on NOD1. K. pneumoniae induced the expression of CYLD and MKP-1, which act as negative regulators for 52145-Deltawca(K2)-induced expression of hBDs. Bacterial engagement of pattern recognition receptors induced CYLD and MKP-1, which may initiate the attenuation of proinflammatory pathways. The results of this study indicate that K. pneumoniae CPS not only protects the pathogen from the bactericidal action of defensins but also impedes their expression. These features of K. pneumoniae CPS may facilitate pathogen survival in the hostile environment of the lung.

Expression of toll-like receptors 2 and 4 is upregulated during hospital admission in traumatic patients: lack of correlation with blunted innate immune responses.

BACKGROUND: There are reports with conflicting results on the expression of toll-like receptors (TLRs) in trauma patients. In addition, these studies analyzed TLR expression only at patients' hospital admission but not later when complications usually arise. OBJECTIVES: To analyze the surface expression of TLR2 and TLR4 on circulating monocytes from trauma patients during the hospitalization period and to correlate this with cytokine production after stimulation with TLR2 and TLR4 agonists. The phagocytic capacity of monocytes was analyzed at the same time points of TLR expression analysis; to correlate these molecular findings with the presence or absence of infections. METHODS: Prospective and observational study from June 2005 to June 2007. In all analysis, a control group composed of healthy subjects was included. RESULTS: We studied 70 trauma patients admitted to the intensive care unit (ICU) of a tertiary hospital, and 30 healthy volunteers. Blood samples were collected at hospital admission, on day 7 and 14. Forty-four patients (63%) developed at least one episode of infection. Monocytes from trauma patients expressed higher levels of TLR2 and TLR4 than monocytes from control subjects at all time points. Expression of TLR2 and TLR4 in monocytes from those patients who developed any infection was significantly lower than in those patients without infection but still significantly higher than in control subjects. Cellular responses to TLR4 agonist were impaired. Monocytes from traumatic patients phagocytosized less efficiently than monocytes from control subjects. CONCLUSIONS: These results indicate that trauma patients present a dysregulation of the innate immune system that persists during the first 14 days after hospital admission.

Defective B cell response to TLR9 ligand (CpG-ODN), Streptococcus pneumoniae and Haemophilus influenzae extracts in common variable immunodeficiency patients.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinaemia and antibody deficiency to both T dependent and independent antigens. Patients suffer from recurrent sinopulmonary infections mostly caused by Streptococcus pneumoniae and Haemophilus influenzae, but also gastrointestinal or autoimmune symptoms. Their response to vaccination is poor or absent. In this study we investigated B cell activation induced by the TLR9 specific ligand (CpG-ODN) and bacterial extracts from S. pneumoniae and H. influenzae known to stimulate several TLR. We found that B cells from CVID patients express lower levels of CD86 after stimulation with CpG-ODN, S. pneumoniae and H. influenzae extracts in combination with anti-IgM antibody and also display a lower proliferative index when stimulated with bacterial extracts. Our results point to a broad TLR signalling defect in B lymphocytes from CVID patients that may be related to the hypogammaglobulinaemia and poor response to vaccination characteristic of these patients.

Lack of effect of glutamine administration to boost the innate immune system response in trauma patients in the intensive care unit.

INTRODUCTION: The use of glutamine as a dietary supplement is associated with a reduced risk of infection. We hypothesized that the underlying mechanism could be an increase in the expression and/or functionality of Toll-like receptors (TLR), key receptors sensing infections. The objective of this study was to evaluate whether glutamine supplementation alters the expression and functionality of TLR2 and TLR4 in circulating monocytes of trauma patients admitted to the intensive care unit (ICU). METHODS: We designed a prospective, randomized and single-blind study. Twenty-three patients received parenteral nutrition (TPN) with a daily glutamine supplement of 0.35 g/kg. The control group (20 patients) received an isocaloric-isonitrogenated TPN. Blood samples were extracted before treatment, at 6 and 14 days. Expression of TLR2 and TLR4 was determined by flow cytometry. Monocytes were stimulated with TLR specific agonists and cytokines were measured in cell culture supernatants. Phagocytic ability of monocytes was also determined. RESULTS: Basal characteristics were similar in both groups. Monocytes from patients treated with glutamine expressed the same TLR2 levels as controls before treatment (4.9 ± 3.5 rmfi vs. 4.3 ± 1.9 rmfi, respectively; P = 0.9), at Day 6 (3.8 ± 2.3 rmfi vs. 4.0 ± 1.7 rmfi, respectively; P = 0.7) and at Day 14 (4.1 ± 2.1 rfim vs. 4.6 ± 1.9 rmfi, respectively; P = 0.08). TLR4 levels were not significantly different between the groups before treatment: (1.1 ± 1 rmfi vs 0.9 ± 0.1 rmfi respectively; P = 0.9), at Day 6 (1.1 ± 1 rmfi vs. 0.7 ± 0.4 rmfi respectively; P = 0.1) and at Day 14 (1.4 ± 1.9 rmfi vs. 1.0 ± 0.6 rmfi respectively; P = 0.8). No differences in cell responses to TLR agonists were found between groups. TLR functionality studied by phagocytosis did not vary between groups. CONCLUSIONS: In trauma patients in the intensive care unit, TPN supplemented with glutamine does not improve the expression or the functionality of TLRs in peripheral blood monocytes. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01250080.

Klebsiella pneumoniae increases the levels of Toll-like receptors 2 and 4 in human airway epithelial cells.

Airway epithelial cells act as the first barrier against pathogens. These cells recognize conserved structural motifs expressed by microbial pathogens via Toll-like receptors (TLRs) expressed on the surface. In contrast to the level of expression in lymphoid cells, the level of expression of TLR2 and TLR4 in airway epithelial cells is low under physiological conditions. Here we explored whether Klebsiella pneumoniae upregulates the expression of TLRs in human airway epithelial cells. We found that the expression of TLR2 and TLR4 by A549 cells and human primary airway cells was upregulated upon infection with K. pneumoniae. The increased expression of TLRs resulted in enhancement of the cellular response upon stimulation with Pam3CSK4 and lipopolysaccharide, which are TLR2 and TLR4 agonists, respectively. Klebsiella-dependent upregulation of TLR expression occurred via a positive IkappaBalpha-dependent NF-kappaBeta pathway and via negative p38 and p44/42 mitogen-activated protein kinase-dependent pathways. We showed that Klebsiella-induced TLR2 and TLR4 upregulation was dependent on TLR activation. An isogenic capsule polysaccharide (CPS) mutant did not increase TLR2 and TLR4 expression. Purified CPS upregulated TLR2 and TLR4 expression, and polymyxin B did not abrogate CPS-induced TLR upregulation. Although no proteins were detected in the CPS preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and colloidal gold staining, we could not rule out the possibility that traces of protein in our CPS preparation could have been responsible, at least in part, for the TLR upregulation.

Nontypeable Haemophilus influenzae clearance by alveolar macrophages is impaired by exposure to cigarette smoke.

Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.

Glutamine as a modulator of the immune system of critical care patients: effect on Toll-like receptor expression. A preliminary study.

OBJECTIVE: We evaluated the expression of Toll-like receptors 2 and 4 (TLR-2 and TLR-4) in circulating monocytes from peripheral blood of critical care patients treated with and without glutamine. Because no research has been published to date on the effect of glutamine on TLR receptors in critical patients, it was determined in an initial sample of 30 patients. METHODS: This was a prospective, randomized, single-blind study with 15 patients assigned to receive parenteral nutrition with a daily glutamine supplement of 0.35 g/kg. The control group received isocaloric-isonitrogenous parenteral nutrition. Blood samples were extracted before beginning the treatment and at 5 and 14 d. Expressions of CD14, TLR-2, and TLR-4 were determined by flow cytometry. Levels of TLRs were expressed as mean fluorescence intensity (mfi). RESULTS: Basal characteristics were similar in both groups. The expressions of TLR-2 in the treatment group with glutamine were 4.67 +/- 3.82 mfi before treatment, 3.91 +/- 2.04 mfi at 5 d, and 4.28 +/- 2.47 mfi at 14 d. The expressions of TLR-2 in the control group were 5.49 +/- 3.20 mfi before treatment, 4.48 +/- 2.15 mfi at 5 d, and 4.36 +/- 2.36 mfi at 14 d. The expressions of TLR-4 in the treatment group were 1.65 +/- 1.89 mfi before treatment, 1.23 +/- 1.10 mfi at 5 d, and 1.77 +/- 1.97 at 14 d. The expressions of TLR-4 in the control group were 1.51 +/- 1.76 mfi before treatment, 1.36 +/- 0.99 mfi at 5 d, and 1.26 +/- 0.59 mfi at 14 d. Infections were detected in 11 patients who received glutamine and 13 control patients (P = 0.51). CONCLUSION: In critical care patients, parenteral nutrition supplemented with glutamine does not increase the expression of TLR-2 or TLR-4 in peripheral blood monocytes.

Apoptosis, Toll-like, RIG-I-like and NOD-like Receptors Are Pathways Jointly Induced by Diverse Respiratory Bacterial and Viral Pathogens.

Lower respiratory tract infections are among the top five leading causes of human death. Fighting these infections is therefore a world health priority. Searching for induced alterations in host gene expression shared by several relevant respiratory pathogens represents an alternative to identify new targets for wide-range host-oriented therapeutics. With this aim, alveolar macrophages were independently infected with three unrelated bacterial (Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus) and two dissimilar viral (respiratory syncytial virus and influenza A virus) respiratory pathogens, all of them highly relevant for human health. Cells were also activated with bacterial lipopolysaccharide (LPS) as a prototypical pathogen-associated molecular pattern. Patterns of differentially expressed cellular genes shared by the indicated pathogens were searched by microarray analysis. Most of the commonly up-regulated host genes were related to the innate immune response and/or apoptosis, with Toll-like, RIG-I-like and NOD-like receptors among the top 10 signaling pathways with over-expressed genes. These results identify new potential broad-spectrum targets to fight the important human infections caused by the bacteria and viruses studied here.

Expression of Toll-like receptor 2 is up-regulated in monocytes from patients with chronic obstructive pulmonary disease.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterised by pulmonary and systemic inflammation which flare-up during episodes of acute exacerbation (AECOPD). Given the role of Toll-like receptors (TLRs) in the induction of inflammatory responses we investigated the involvement of TLRs in COPD pathogenesis. METHODS: The expression of TLR-2, TLR-4 and CD14 in monocytes was analyzed by flow cytometry. To study the functional responses of these receptors, monocytes were stimulated with peptidoglycan or lipopolysaccharide and the amounts of TNFalpha and IL-6 secreted were determined by ELISA. RESULTS: We found that the expression of TLR-2 was up-regulated in peripheral blood monocytes from COPD patients, either clinically stable or during AECOPD, as compared to never smokers or smokers with normal lung function. Upon stimulation with TLR-2 ligand monocytes from COPD patients secreted increased amounts of cytokines than similarly stimulated monocytes from never smokers and smokers. In contrast, the expressions of TLR-4 and CD14 were not significantly different between groups, and the response to lipopolysaccharide (a TLR-4 ligand) stimulation was not significantly different either. At discharge from hospital TLR-2 expression was down-regulated in peripheral blood monocytes from AECOPD patients. This could be due to the treatment with systemic steroids because, in vitro, steroids down-regulated TLR-2 expression in a dose-dependent manner. Finally, we demonstrated that IL-6, whose plasma levels are elevated in patients, up-regulated in vitro TLR-2 expression in monocytes from never smokers. CONCLUSION: Our results reveal abnormalities in TLRs expression in COPD patients and highlight its potential relationship with systemic inflammation in these patients.

Dissection of host cell signal transduction during Acinetobacter baumannii-triggered inflammatory response.

Infected airway epithelial cells up-regulate the expression of chemokines, chiefly IL-8, and antimicrobial molecules including beta-defensins (BD). Acinetobacter baumannii is a cause of hospital-acquired pneumonia. We examined whether A. baumannii induced the expressions of IL-8 and BD2 by airway epithelial cells and the receptors implicated in bacterial detection. A549 and human primary airway cells released IL-8 upon infection. A. baumannii-infected cells also increased the expression of BD2 which killed A. baummannii strains. IL-8 induction was via NF-kappaB and mitogen-activated kinases p38 and p44/42-dependent pathways. A. baumannii engaged Toll-like receptor (TLR) 2 and TLR4 pathways and A549 cells could use soluble CD14 as TLRs co-receptor. A. baumannii lipopolysaccharide stimulated IL-8 release by A549 cells and sCD14 facilitated the recognition of the lipopolysaccharide. Mass spectrometry analysis revealed that A. baumannii lipid A structure matches those with endotoxic potential. These results demonstrate that airway epithelial cells produce mediators important for A. baumannii clearance.

The uptake of a Klebsiella pneumoniae capsule polysaccharide mutant triggers an inflammatory response by human airway epithelial cells.

The means by which airway epithelial cells sense a bacterial infection and which intracellular signalling pathways are activated upon infection are poorly understood. A549 cells and human primary airway cells (NHBE) were used to investigate the response to infection with Klebsiella pneumoniae. Infection of A549 and NHBE with K. pneumoniae 52K10, a capsule polysaccharide (CPS) mutant, increased the surface levels of ICAM-1 and caused the release of IL-8. By contrast, the wild-type strain did not elicit these responses. Consistent with a functional role for these responses, there was a correlation between ICAM-1 levels and the number of adherent leukocytes on the epithelial cell surface. In addition, treatment of neutrophils with IL-8 enhanced their ability to kill K. pneumoniae. Strain 52K10 was internalized by A549 cells more efficiently than the wild-type, and when infections with 52K10 were performed in the presence of cytochalasin D the inflammatory response was abrogated. These findings suggest that cellular activation is mediated by bacterial internalization and that CPS prevents the activation through the blockage of bacterial adhesion and uptake. Collectively, the results indicate that bacterial internalization by airway epithelial cells could be the triggering signal for the activation of the innate immune system of the airway. Infection of A549 cells by 52K10 was shown to trigger the nuclear translocation of NF-kappaB. Evidence is presented showing that 52K10 activated IL-8 production through Toll-like receptor (TLR) 2 and TLR4 pathways and that A549 cells could use soluble CD14 as TLR co-receptor.

Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides.

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.