Nod1 signaling overcomes resistance of S. pneumoniae to opsonophagocytic killing

Airway infection by the Gram-positive pathogen Streptococcus pneumoniae (Sp) leads to recruitment of neutrophils but limited bacterial killing by these cells. Co-colonization by Sp and a Gram-negative species, Haemophilus influenzae (Hi), provides sufficient stimulus to induce neutrophil and complement-mediated clearance of Sp from the mucosal surface in a murine model. Products from Hi, but not Sp, also promote killing of Sp by ex vivo neutrophil-enriched peritoneal exudate cells. Here we identify the stimulus from Hi as its peptidoglycan. Enhancement of opsonophagocytic killing was facilitated by signaling through nucleotide-binding oligomerization domain-1 (Nod1), which is involved in recognition of γ-D-glutamyl-meso-diaminopimelic acid (meso-DAP) contained in cell walls of Hi but not Sp. Neutrophils from mice treated with Hi or compounds containing meso-DAP, including synthetic peptidoglycan fragments, showed increased Sp killing in a Nod1-dependent manner. Moreover, Nod1-/- mice showed reduced Hi-induced clearance of Sp during co-colonization. These observations offer insight into mechanisms of microbial competition and demonstrate the importance of Nod1 in neutrophil-mediated clearance of bacteria in vivo

Conserved Mutations in the Pneumococcal Bacteriocin Transporter Gene, blpA, Result in a Complex Population Consisting of Producers and Cheaters

All fully sequenced strains of Streptococcus pneumoniae possess a version of the blp locus, which is responsible for bacteriocin production and immunity. Activation of the blp locus is stimulated by accumulation of the peptide pheromone, BlpC, following its secretion by the ABC transporter, BlpA. The blp locus is characterized by significant diversity in blpC type and in the region of the locus containing putative bacteriocin and immunity genes. In addition, the blpA gene can represent a single large open reading frame or be divided into several smaller fragments due to the presence of frameshift mutations. In this study, we use a collection of strains with blp-dependent inhibition and immunity to define the genetic changes that bring about phenotypic differences in bacteriocin production or immunity. We demonstrate that alterations in blpA, blpC, and bacteriocin/immunity content likely play an important role in competitive interactions between pneumococcal strains. Importantly, strains with a highly conserved frameshift mutation in blpA are unable to secrete bacteriocins or BlpC, but retain the ability to respond to exogenous peptide pheromone produced by cocolonizing strains, stimulating blp-mediated immunity. These “cheater” strains can only coexist with bacteriocin-producing strains that secrete their cognate BlpC and share the same immunity proteins. The variable outcome of these interactions helps to explain the heterogeneity of the blp pheromone, bacteriocin, and immunity protein content

Field Effect Transistors for Terahertz Detection: Physics and First Imaging Applications

Resonant frequencies of the two-dimensional plasma in FETs increase with the reduction of the channel dimensions and can reach the THz range for sub-micron gate lengths. Nonlinear properties of the electron plasma in the transistor channel can be used for the detection and mixing of THz frequencies. At cryogenic temperatures resonant and gate voltage tunable detection related to plasma waves resonances, is observed. At room temperature, when plasma oscillations are overdamped, the FET can operate as an efficient broadband THz detector. We present the main theoretical and experimental results on THz detection by FETs in the context of their possible application for THz imaging.Comment: 22 pages, 12 figures, review pape

Molecular Basis of Increased Serum Resistance among Pulmonary Isolates of Non-typeable Haemophilus influenzae

Non-typeable Haemophilus influenzae (NTHi), a common commensal of the human pharynx, is also an opportunistic pathogen if it becomes established in the lower respiratory tract (LRT). In comparison to colonizing isolates from the upper airway, LRT isolates, especially those associated with exacerbations of chronic obstructive pulmonary disease, have increased resistance to the complement- and antibody-dependent, bactericidal effect of serum. To define the molecular basis of this resistance, mutants constructed in a serum resistant strain using the mariner transposon were screened for loss of survival in normal human serum. The loci required for serum resistance contribute to the structure of the exposed surface of the bacterial outer membrane. These included loci involved in biosynthesis of the oligosaccharide component of lipooligosaccharide (LOS), and vacJ, which functions with an ABC transporter encoded by yrb genes in retrograde trafficking of phospholipids from the outer to inner leaflet of the cell envelope. Mutations in vacJ and yrb genes reduced the stability of the outer membrane and were associated with increased cell surface hyrophobicity and phospholipid content. Loss of serum resistance in vacJ and yrb mutants correlated with increased binding of natural immunoglobulin M in serum as well as anti-oligosaccharide mAbs. Expression of vacJ and the yrb genes was positively correlated with serum resistance among clinical isolates. Our findings suggest that NTHi adapts to inflammation encountered during infection of the LRT by modulation of its outer leaflet through increased expression of vacJ and yrb genes to minimize recognition by bactericidal anti-oligosaccharide antibodies

Transcriptional Profile of the Escherichia coli Response to the Antimicrobial Insect Peptide Cecropin A

Cationic antimicrobial peptides are believed to exert their primary activities on anionic bacterial cell membranes; however, this model does not adequately account for several important structure-activity relationships. These relationships are likely to be influenced by the bacterial response to peptide challenge. In order to characterize the genomic aspect of this response, transcription profiles were examined for Escherichia coli isolates treated with sublethal and lethal concentrations of the cationic antimicrobial peptide cecropin A. Transcript levels for 26 genes changed significantly following treatment with sublethal peptide concentrations, and half of the transcripts corresponded to protein products with unknown function. The pattern of response is distinct from that following treatment with lethal concentrations and is also distinct from the bacterial response to nutritional, thermal, osmotic, or oxidative stress. These results demonstrate that cecropin A induces a genomic response in E. coli apart from any lethal effects on the membrane and suggest that a complete understanding of its mechanism of action may require a detailed examination of this response

Short-Sequence Tandem and Nontandem DNA Repeats and Endogenous Hydrogen Peroxide Production Contribute to Genetic Instability of Streptococcus pneumoniae

Loss-of-function mutations in the following seven pneumococcal genes were detected and analyzed: pspA, spxB, xba, licD2, lytA, nanA, and atpC. Factors associated with these mutations included (i) frameshifts caused by reversible gain and loss of single bases within homopolymeric repeats as short as 6 bases, (ii) deletions caused by recombinational events between nontandem direct repeats as short as 8 bases, and (iii) substitutions of guanine residues caused at an increased frequency by the high levels of hydrogen peroxide (>2 mM) typically generated by this species under aerobic growth conditions. The latter accounted for a frequency as high as 2.8 × 10(−6) for spontaneous mutation to resistance to optochin and was 10- to 200-fold lower in the absence of detectable levels of H(2)O(2). Some of these mutations appear to have been selected for in vivo during pneumococcal infection, perhaps as a consequence of immune pressure or oxidative stress

Identifying Mutator Phenotypes among Fluoroquinolone-Resistant Strains of Streptococcus pneumoniae Using Fluctuation Analysis▿

The occurrence of mutator phenotypes among laboratory-generated and clinical levofloxacin-resistant strains of Streptococcus pneumoniae was determined using fluctuation analysis. The in vitro selection for levofloxacin-resistant mutants of strain D39, each with point mutations in both gyrA and parC or parE, was not associated with a significant change in the mutation rate. Two of eight clinical isolates resistant to levofloxacin (MIC, >8 μg/ml) had estimated mutation rates of 1.2 × 10−7 and 9.4 × 10−8 mutations per cell division, indicating potential mutator phenotypes, compared to strain D39, which had an estimated mutation rate of 1.4 × 10−8 mutations per cell division. The levofloxacin-resistant isolates with the highest mutation rates showed evidence of dysfunctional mismatch repair and contained missense mutations in mut genes at otherwise highly conserved sites. The association of hypermutability in levofloxacin-resistant S. pneumoniae clinical isolates with mutations in DNA mismatch repair genes provides further evidence that mismatch repair mutants may have a selective advantage in the setting of antibiotic pressure, facilitating the development of further antibiotic resistance

Characterization of <i>vacJ</i> and <i>yrb</i> mutants.

<p>(<b>A</b>) Effect of mutations in <i>vacJ</i> and genes of the <i>yrb</i> ABC transporter on serum resistance of strain R2866. Survival was determined over 60 min in 5% normal human serum and expressed relative to controls in which complement was inactivated. (<b>B</b>) Representative histogram comparing the binding, as measured by fluorescence intensity (x-axis), of total IgM purified from normal human serum to parent strain (WT) or <i>vacJ</i> by flow cytometry. Control performed without IgM (<b>C</b>) Percent IgM binding for each mutant was determined by calculating the percentage of 50,000 events with an increase in mean fluorescence intensity following incubation in 5% heat-inactivated normal human serum compared to no serum controls. (<b>D</b>) Survival of mutants in 10% normal human serum in the presence (black bars) or absence (white bars) of Mg-EGTA to inhibit the classical pathway of complement activation. Values represent two independent experiments in triplicate ± SD. ^*<i>P</i><0.05, ^**<i>P</i><0.01, ^***<i>P</i><0.001.</p