A Type IV Secretion System Contributes to Intracellular Survival and Replication of Burkholderia cenocepacia

Burkholderia cenocepacia is an important respiratory pathogen in persons with cystic fibrosis (CF). Recent studies indicate that B. cenocepacia survives within macrophages and airway epithelial cells in vitro by evading endosome-lysosome fusion. We investigated the role of a plasmid-encoded type IV secretion system in the intracellular survival, replication, and processing of B. cenocepacia. Both a wild-type strain (K56-2) and its type IV secretion system mutant (designated LC101) entered and replicated in CF airway epithelial cells and monocyte-derived macrophages. However, significantly more intracellular K56-2 than LC101 bacteria were found in both cell types at 24 h postinfection. Colocalization of bacteria with markers of the classical endocytic pathway indicated that although both K56-2 and LC101 reside transiently in early endosomes, a greater proportion of the mutant bacteria are targeted to lysosomal degradation. In contrast, wild-type bacteria escape from the classical endocytic pathway and traffic to the endoplasmic reticulum, where they replicate. Our results show that the intracellular processing of B. cenocepacia is similar in both professional and nonprofessional phagocytes and that a functional plasmid-encoded type IV secretion system contributes to the survival and replication of B. cenocepacia in eukaryotic cells

H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM‐1 and TLR3 expression

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/154674/1/fsb2fj065806fje.pd

MDA5 and TLR3 Initiate Pro-Inflammatory Signaling Pathways Leading to Rhinovirus-Induced Airways Inflammation and Hyperresponsiveness

Rhinovirus (RV), a single-stranded RNA picornavirus, is the most frequent cause of asthma exacerbations. We previously demonstrated in human bronchial epithelial cells that melanoma differentiation-associated gene (MDA)-5 and the adaptor protein for Toll-like receptor (TLR)-3 are each required for maximal RV1B-induced interferon (IFN) responses. However, in vivo, the overall airway response to viral infection likely represents a coordinated response integrating both antiviral and pro-inflammatory pathways. We examined the airway responses of MDA5- and TLR3-deficient mice to infection with RV1B, a minor group virus which replicates in mouse lungs. MDA5 null mice showed a delayed type I IFN and attenuated type III IFN response to RV1B infection, leading to a transient increase in viral titer. TLR3 null mice showed normal IFN responses and unchanged viral titers. Further, RV-infected MDA5 and TLR3 null mice showed reduced lung inflammatory responses and reduced airways responsiveness. Finally, RV-infected MDA5 null mice with allergic airways disease showed lower viral titers despite deficient IFN responses, and allergic MDA5 and TLR3 null mice each showed decreased RV-induced airway inflammatory and contractile responses. These results suggest that, in the context of RV infection, binding of viral dsRNA to MDA5 and TLR3 initiates pro-inflammatory signaling pathways leading to airways inflammation and hyperresponsiveness

Host evasion by Burkholderia cenocepacia

Burkholderia cenocepacia is an opportunistic respiratory pathogen of individuals with cystic fibrosis (CF). It is one of the highly transmissible species of Burkholderia cepacia complex and very resistant to almost all the antibiotics. Approximately 1/3rd of B. cenocepacia infected CF patients go on to develop fatal ‘cepacia syndrome’. During the last two decades, substantial progress has been made with regards to evasion of host innate defense mechanisms by B. cenocepacia. Almost all strains of B. cenocepacia has capacity to survive and replicate intracellularly in both airway epithelial cells and macrophages, which are primary centennials of the lung and play a pivotal role in clearance of infecting bacteria. Some strains of B. cenocepaica, which express cable pili and the associated 22kDa adhesin are also capable of transmigrating across airway epithelium and persist in mouse models of infection. In this review, we will discuss how this type of interaction between B. cenocepacia and host may lead to persistence of bacteria and contribute to lung inflammation in CF patients

Responses of Well-Differentiated Airway Epithelial Cell Cultures from Healthy Donors and Patients with Cystic Fibrosis to Burkholderia cenocepacia Infection

Well-differentiated cultures established from airway epithelia of patients with cystic fibrosis (CF cultures) exhibited goblet cell hyperplasia, increased secretion of mucus, and higher basal levels of interleukin-8 than similarly cultured cells from healthy donors. Upon apical infection with low doses (10(4) to 10(5) CFU) of Burkholderia cenocepacia isolate BC7, the two cultures gave different responses. While normal cultures trapped the added bacteria in the mucus layer, killed and/or inhibited bacterial replication, and prevented bacterial invasion of the cells, CF cultures failed to kill and/or supported the growth of bacteria, leading to invasion of underlying epithelial cells, compromised transepithelial permeability, and cell damage. Depletion of the surface mucus layer prior to bacterial infection rendered the normal cultures susceptible to bacterial invasion, but the invading bacteria were mainly confined to vacuoles within the cells and appeared to be nonviable. In contrast, bacteria that invaded cells in CF cultures were found free in the cytoplasm surrounded by intermediate filaments and also between cells. Cultured CF airway epithelium was therefore more susceptible to infection than normal epithelium. This mimics CF tissue in vivo and illustrates differences in the way epithelia in CF patients and normal subjects handle bacterial infection. In addition, we found that the CF and normal cell cultures responded differently not only to isolate BC7 but also to isolates of other B. cepacia complex species. We therefore conclude that this cell culture model is suitable for investigation of B. cepacia complex pathogenesis in CF patients

Effect of hydrocolloids on the rheological properties of wheat starch

The rheological properties of 5% starch alone and in the presence of 0·5% guar, locust bean or xanthan gums were studied using a Rheotest 2 viscometer at different temperatures. The 5% starch alone exhibited non-Newtonian behaviour at 30°C, but upon gelatinization it exhibited non-Newtonian shear thinning behaviour. In the presence of 0·5% guar, locust bean or xanthan gum at 30°C itself, the flow behaviour index 'n' of starch dispersions decreased and ranged between 0·83 and 0·90 indicating shear thinning or pseudoplastic behaviour of the starch-gum dispersions. After gelatinization, the effect of these gums was higher than at 30°C and the flow behaviour index 'n' decreased from 0·64 to 0·34, 0·45 and 0·25 in the presence of 0·5% guar, locust bean and xanthan gum respectively. None of the starch or starch-gum pastes studied exhibited the yield value in the range of shear rates studied

Functional properties of native and carboxymethyl guar gum

Guar gum in its native form exhibits a very good thickening capacity and hence is used as a thickener or agent for 'binding' water in food, cosmetics and toiletries, in pharmaceuticals and in the mining, paper and textile industries (Glicksman 1976). This communication reports the effect of introducing an anionic carboxymethyl group upon the thickening capacity of guar gum and the related secondary functional properties of stabilising foams and suspensions. Compared with native guar gum, carboxymethyl guar gum with a degree of substitution of 0·68 exhibited a higher viscosity and an enhanced ability to stabilise foams and suspensions. Both native and carboxymethyl guar gum induced a non-Newtonian pseudoplastic behaviour to casein (100 g litre-1) suspension. At the same time only carboxymethyl guar gum containing suspensions above 1 g litre-1 exhibited measurable yield value

Increased expression of miR146a dysregulates TLR2-induced HBD2 in airway epithelial cells from patients with COPD

Background Airway epithelial cells from patients with COPD show suboptimal innate immune responses to nontypeable Haemophilus influenzae (NTHi) and Toll-like receptor (TLR)2 ligands despite expressing TLR2 similar to normal airway epithelial cells, but the underlying mechanisms are poorly understood. Methods Normal or COPD mucociliary-differentiated airway epithelial cells were treated with TLR2 agonists or infected with NTHi and expression of β-defensin (HBD)2 was examined. Interleukin-1 receptor-associated kinase (IRAK)-1 and microRNA (miR)146a were genetically inhibited in normal and COPD airway epithelial cell cultures, respectively, and HBD2 responses to TLR2 ligands were determined. IRAK-1 expression in lung sections was determined by immunofluorescence microscopy. Results Compared to normal, COPD airway epithelial cell cultures showed impaired expression of HBD2 in response to TLR2 agonists or NTHi infection. Apical secretions from TLR2 agonist-treated normal, but not COPD, airway epithelial cells efficiently killed NTHi. Knockdown of HBD2 significantly reduced NTHi killing by apical secretions of normal airway epithelial cells. Compared to normal, COPD cells showed significantly reduced expression of IRAK-1 and this was associated with increased expression of miR146a. Inhibition of miR146a increased the expression of IRAK-1, improved the expression of HBD2 in response to TLR2 agonists in COPD cells and enhanced the killing of bacteria by apical secretions obtained from TLR2 agonist-treated COPD cells. Bronchial epithelium of COPD patients showed reduced expression of IRAK-1. Conclusions These results suggest that reduced levels of IRAK-1 due to increased expression of miR146a may contribute to impaired expression of TLR2-induced HBD2 in COPD airway epithelial cells