Association of adenovirus with the microtubule organizing center

Adenoviruses (Ad) must deliver their genomes to the nucleus of the target cell to initiate an infection. Following entry into the cell and escape from the endosome, Ad traffics along the microtubule cytoskeleton toward the nucleus. In the final step in Ad trafficking, Ad must leave the microtubule and establish an association with the nuclear envelope. We hypothesized that in cells lacking a nucleus, the capsid moves to and associates with the microtubule organizing center (MTOC). To test this hypothesis, we established an experimental system to examine Ad trafficking in enucleated cells compared to Ad trafficking in intact, mock-enucleated cells. Enucleation of a monolayer of A549 human lung epithelial cells was accomplished by depolymerization of the actin cytoskeleton followed by centrifugation. Upon infection of enucleated cells with Cy3-labeled Ad, the majority of Ad capsid trafficked to a discrete, centrally located site which colocalized with pericentrin, a component of the MTOC. MTOC-associated Ad had escaped from endosomes and thus had direct access to MTOC components. Ad localization at this site was sensitive to the microtubule-depolymerizing agent nocodazole, but not to the microfilament-depolymerizing agent cytochalasin B, indicating that intact microtubules were required to maintain the localization with the MTOC. Ad localization to the MTOC in the enucleated cells was stable, as demonstrated by continuing Ad localization with pericentrin for more than 5 h after infection, a strong preference for Ad arrival at rather than Ad departure from the MTOC, and minimal redistribution of Ad between MTOCs within a single cell. In summary, the data demonstrate that the Ad capsid establishes a stable interaction with the MTOC when a nucleus is not present, suggesting that dissociation of Ad from microtubules likely requires nuclear factors.     

Glutathione s-transferase omega 1-1 is a target of cytokine release inhibitory drugs and may be responsible for their effect on interleukin-1beta posttranslational processing

Stimulus-induced posttranslational processing of human monocyte interleukin-1beta (IL-1beta) is accompanied by major changes to the intracellular ionic environment, activation of caspase-1, and cell death. Certain diarylsulfonylureas inhibit this response, and are designated cytokine release inhibitory drugs (CRIDs). CRIDs arrest activated monocytes so that caspase-1 remains inactive and plasma membrane latency is preserved. Affinity labeling with [(14)C]CRIDs and affinity chromatography on immobilized CRID were used in seeking potential protein targets of their action. Following treatment of intact human monocytes with an epoxide-bearing [(14)C]CRID, glutathione S-transferase (GST) Omega 1-1 was identified as a preferred target. Moreover, labeling of this polypeptide correlated with irreversible inhibition of ATP-induced IL-1beta posttranslational processing. When extracts of human monocytic cells were chromatographed on a CRID affinity column, GST Omega 1-1 bound selectively to the affinity matrix and was eluted by soluble CRID. Recombinant GST Omega 1-1 readily incorporated [(14)C]CRID epoxides, but labeling was negated by co-incubation with S-substituted glutathiones or by mutagenesis of the catalytic center Cys(32) to alanine. Peptide mapping by high performance liquid chromatography-mass spectrometry also demonstrated that Cys(32) was the site of modification. Although S-alkylglutathiones did not arrest ATP-induced IL-1beta posttranslational processing or inhibit [(14)C]CRID incorporation into cell-associated GST Omega 1-1, a glutathione-CRID adduct effectively demonstrated these attributes. Therefore, the ability of CRIDs to arrest stimulus-induced IL-1beta posttranslational processing may be attributable to their interaction with GST Omega 1-1.     

Leishmania EF-1alpha activates the Src homology 2 domain containing tyrosine phosphatase SHP-1 leading to macrophage deactivation

The human leishmaniasis are persistent infections of macrophages caused by protozoa of the genus Leishmania. The chronic nature of these infections is in part related to induction of macrophage deactivation, linked to activation of the Src homology 2 domain containing tyrosine phosphatase-1 (SHP-1) in infected cells. To investigate the mechanism of SHP-1 activation, lysates of Leishmania donovani promastigotes were subjected to SHP-1 affinity chromatography and proteins bound to the matrix were sequenced by mass spectrometry. This resulted in the identification of Leishmania elongation factor-1alpha (EF-1alpha) as a SHP-1-binding protein. Purified Leishmania EF-1alpha, but not host cell EF-1alpha, bound directly to SHP-1 in vitro leading to its activation. Three independent lines of evidence indicated that Leishmania EF-1alpha may be exported from the phagosome thereby enabling targeting of host SHP-1. First, cytosolic fractions prepared from macrophages infected with [(35)S]methionine-labeled organisms contained Leishmania EF-1alpha. Second, confocal, fluorescence microscopy using Leishmania-specific antisera detected Leishmania EF-1alpha in the cytosol of infected cells. Third, co-immunoprecipitation showed that Leishmania EF-1alpha was associated with SHP-1 in vivo in infected cells. Finally, introduction of purified Leishmania EF-1alpha, but not the corresponding host protein into macrophages activated SHP-1 and blocked the induction of inducible nitric-oxide synthase expression in response to interferon-gamma. Thus, Leishmania EF-1alpha is identified as a novel SHP-1-binding and activating protein that recapitulates the deactivated phenotype of infected macrophages.     

Activation of NOTCH1 or NOTCH3 Signaling Skews Human Airway Basal Cell Differentiation toward a Secretory Pathway

Airway basal cells (BC) function as stem/progenitor cells capable of differentiating into the luminal ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. The objective of this study was to define the role of Notch signaling in regulating human airway BC differentiation into a pseudostratified mucociliated epithelium. Notch inhibition with γ-secretase inhibitors demonstrated Notch activation is essential for BC differentiation into secretory and ciliated cells, but more so for the secretory lineage. Sustained cell autonomous ligand independent Notch activation via lentivirus expression of the intracellular domain of each Notch receptor (NICD1-4) demonstrated that the NOTCH2 and 4 pathways have little effect on BC differentiation into secretory and ciliated cells, while activation of the NOTCH1 or 3 pathways has a major influence, with persistent expression of NICD1 or 3 resulting in a skewing toward secretory cell differentiation with a parallel decrease in ciliated cell differentiation. These observations provide insights into the control of the balance of BC differentiation into the secretory vs ciliated cell lineage, a balance that is critical for maintaining the normal function of the airway epithelium in barrier defense against the inhaled environment.     

The Efficacy of the BCG Vaccine against Newly Emerging Clinical Strains of Mycobacterium tuberculosis

To date, most new vaccines against Mycobacterium tuberculosis, including new recombinant versions of the current BCG vaccine, have usually been screened against the laboratory strains H37Rv or Erdman. In this study we took advantage of our recent work in characterizing an increasingly large panel of newly emerging clinical isolates [from the United States or from the Western Cape region of South Africa], to determine to what extent vaccines would protect against these [mostly high virulence] strains. We show here that both BCG Pasteur and recombinant BCG Aeras-422 [used here as a good example of the new generation BCG vaccines] protected well in both mouse and guinea pig low dose aerosol infection models against the majority of clinical isolates tested. However, Aeras-422 was not effective in a long term survival assay compared to BCG Pasteur. Protection was very strongly expressed against all of the Western Cape strains tested, reinforcing our viewpoint that any attempt at boosting BCG would be very difficult to achieve statistically. This observation is discussed in the context of the growing argument made by others that the failure of a recent vaccine trial disqualifies the further use of animal models to predict vaccine efficacy. This viewpoint is in our opinion completely erroneous, and that it is the fitness of prevalent strains in the trial site area that is the centrally important factor, an issue that is not being addressed by the field.     

Serum Metabolite Biomarkers Discriminate Healthy Smokers from COPD Smokers

COPD (chronic obstructive pulmonary disease) is defined by a fixed expiratory airflow obstruction associated with disordered airways and alveolar destruction. COPD is caused by cigarette smoking and is the third greatest cause of mortality in the US. Forced expiratory volume in 1 second (FEV1) is the only validated clinical marker of COPD, but it correlates poorly with clinical features and is not sensitive enough to predict the early onset of disease. Using LC/MS global untargeted metabolite profiling of serum samples from a well-defined cohort of healthy smokers (n = 37), COPD smokers (n = 41) and non-smokers (n = 37), we sought to discover serum metabolic markers with known and/or unknown molecular identities that are associated with early-onset COPD. A total of 1,181 distinct molecular ions were detected in 95% of sera from all study subjects and 23 were found to be differentially-expressed in COPD-smokers vs. healthy-smokers. These 23 putative biomarkers were differentially-correlated with lung function parameters and used to generate a COPD prediction model possessing 87.8% sensitivity and 86.5% specificity. In an independent validation set, this model correctly predicted COPD in 8/10 individuals. These serum biomarkers included myoinositol, glycerophopshoinositol, fumarate, cysteinesulfonic acid, a modified version of fibrinogen peptide B (mFBP), and three doubly-charged peptides with undefined sequence that significantly and positively correlate with mFBP levels. Together, elevated levels of serum mFBP and additional disease-associated biomarkers point to a role for chronic inflammation, thrombosis, and oxidative stress in remodeling of the COPD airways. Serum metabolite biomarkers offer a promising and accessible window for recognition of early-stage COPD.     

Defective interactions of protein partner with ion channels and transporters as alternative mechanisms of membrane channelopathies

The past twenty years have revealed the existence of numerous ion channel mutations resulting in human pathology. Ion channels provide the basis of diverse cellular functions, ranging from hormone secretion, excitation–contraction coupling, cell signaling, immune response, and trans-epithelial transport. Therefore, the regulation of biophysical properties of channels is vital in human physiology. Only within the last decade has the role of non-ion channel components come to light in regard to ion channel spatial, temporal, and biophysical regulation in physiology. A growing number of auxiliary components have been determined to play elemental roles in excitable cell physiology, with dysfunction resulting in disorders and related manifestations. This review focuses on the broad implications of such dysfunction, focusing on disease-causing mutations that alter interactions between ion channels and auxiliary ion channel components in a diverse set of human excitable cell disease. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé     

Restoration of Vagal Tone: A Possible Mechanism for Functional Abdominal Pain

Functional abdominal pain (FAP) causes disruption of daily activities/missed school days, over utilization of healthcare, unnecessary surgeries, and anxiety in 10–15% of children. Its etiology is not clearly understood, however the success of several clinical protocols suggests that autonomic dysregulation is a factor. In this study autonomic activity, including heart rate variability (HRV), was compared between children with FAP and a comparison group. Twenty children with FAP and 10 children without FAP between the ages of 5 and 17 years old were compared on autonomic regulation using an ambulatory system at baseline and 8 weeks later. Children with FAP participated in 6 sessions of HRV biofeedback aimed at normalizing autonomic balance. At baseline, children with FAP appear to have more autonomic dysregulation than children without FAP. After completing HRV biofeedback, the FAP group was able to significantly reduce their symptoms in relation to significantly increasing their autonomic balance. In a sample of children with FAP, it appears that HRV biofeedback treatment improved their symptoms and that a change in vagal tone was a potential mediator for this improvement. The present study appears to point to excessive vagal withdrawal as an underlying mechanism of FAP.     

Experimental Adaptation of Burkholderia cenocepacia to Onion Medium Reduces Host Range

It is unclear whether adaptation to a new host typically broadens or compromises host range, yet the answer bears on the fate of emergent pathogens and symbionts. We investigated this dynamic using a soil isolate of Burkholderia cenocepacia, a species that normally inhabits the rhizosphere, is related to the onion pathogen B. cepacia, and can infect the lungs of cystic fibrosis patients. We hypothesized that adaptation of B. cenocepacia to a novel host would compromise fitness and virulence in alternative hosts. We modeled adaptation to a specific host by experimentally evolving 12 populations of B. cenocepacia in liquid medium composed of macerated onion tissue for 1,000 generations. The mean fitness of all populations increased by 78% relative to the ancestor, but significant variation among lines was observed. Populations also varied in several phenotypes related to host association, including motility, biofilm formation, and quorum-sensing function. Together, these results suggest that each population adapted by fixing different sets of adaptive mutations. However, this adaptation was consistently accompanied by a loss of pathogenicity to the nematode Caenorhabditis elegans; by 500 generations most populations became unable to kill nematodes. In conclusion, we observed a narrowing of host range as a consequence of prolonged adaptation to an environment simulating a specific host, and we suggest that emergent pathogens may face similar consequences if they become host-restricted.Some emergent pathogens, such as Pseudomonas and Burkholderia species, persist in a wide range of plant and animal hosts, suggesting that the virulence factors needed to infect plants and animals are similar (5, 40). Yet whether adaptation to a new niche tends to compromise niche breadth or, in this case, host range is an open question. Adaptation to a novel host may restrict host range to various degrees, whether by diminishing host-specific virulence traits without affecting host colonization or by reducing the ability to initiate infection in alternative hosts. However, if factors needed to colonize plant and animal hosts are similar, then why are some bacterial populations restricted to a narrow host range while others are not? One explanation for a limited host range may be the result of genetic trade-offs associated with adaptation to a specific host (7, 18). Another explanation may be that prolonged adaptation to a specific host casts a “selective shadow” over unused functions that are relevant to colonizing other hosts but decay by genetic drift (7, 18). To address these possibilities, we quantified the direct and correlated effects of specific host adaptation by the opportunistic pathogen Burkholderia cenocepacia.Members of the Burkholderia cepacia complex (Bcc), which are ubiquitous in the environment, were once used as biocontrol and bioremedial agents but now are banned from these applications because of the potential of some members to cause plant and human disease (39). The type species B. cepacia is well known as a pathogen of the common yellow onion, Allium cepa, in which it causes a characteristic yellow or brown rot. Another species, B. cenocepacia, can also infect onions as well as a range of plants and animals, including humans (2, 6, 26, 36). Bcc bacteria can cause serious infection in the lungs of cystic fibrosis (CF) patients (6, 26). These infections, called “cepacia syndrome,” are highly contagious among CF patients, and infections produce many negative effects on an already poor quality of life, including longer hospital stays, removal from lung transplant lists, blood poisoning, and eventual death (24). B. cenocepacia, one of the two Bcc species most commonly isolated from lung infections, is especially threatening and is associated with more severe cepacia syndrome (35). However, the mechanisms allowing B. cenocepacia to adapt to colonize both human and plant hosts are unclear. Several putative virulence mechanisms have been identified by random mutagenic screens or by knockouts of candidate genes (2, 12, 20, 25, 29, 35, 43, 46), but these mechanisms generally have not been shown to function in host adaptation. One way to directly study adaptation of bacterial populations to susceptible hosts is by experimental evolution, in which bacterial populations evolve in a controlled laboratory setting that enables study of the adaptive process over time (7).We experimentally evolved populations of B. cenocepacia HI2424 to study the extent to which adaptation to the common yellow onion A. cepa affects host range. B. cenocepacia HI2424 is a soil isolate and is classified as part of the PHDC strain lineage, the strain first characterized as responsible for an outbreak of Bcc infections in large treatment centers located in the mid-Atlantic region of the United States (33). We found that adaptation of B. cenocepacia to the onion model was associated with reduced virulence but did not compromise the capacity to colonize (or be consumed by) the nematode Caenorhabditis elegans, and the coincidence of these events suggests that a genetic trade-off (antagonistic pleiotropy) between fitness in onion medium and nematode virulence exists. We also characterized several phenotypes potentially associated with adaptation to the onion or nematode virulence. Most phenotypes varied significantly among replicate populations, suggesting that adaptation to the onion model may follow several different pathways.