Summary Listeria monocytogenes is a Gram

Rationale: Respiratory syncytial virus (RSV) and Streptococcus pneumoniae are major respiratory pathogens. Coinfection with RSV and S. pneumoniae is associated with severe and often fatal pneumonia but the molecular basis for this remains unclear.Objectives: To determine if interaction between RSV and pneumococci enhances pneumococcal virulence.Methods: We used confocal microscopy and Western blot to identify the receptors involved in direct binding of RSV and pneumococci, the effects of which were studied in both in vivo and in vitro models of infection. Human ciliated respiratory epithelial cell cultures were infected with RSV for 72 hours and then challenged with pneumococci. Pneumococci were collected after 2 hours exposure and changes in gene expression determined using quantitative real-time polymerase chain reaction.Measurements and Main Results: Following incubation with RSV or purified G protein, pneumococci demonstrated a significant increase in the inflammatory response and bacterial adherence to human ciliated epithelial cultures and markedly increased virulence in a pneumonia model in mice. This was associated with extensive changes in the pneumococcal transcriptome and significant up-regulation in the expression of key pneumococcal virulence genes, including the gene for the pneumococcal toxin, pneumolysin. We show that mechanistically this is caused by RSV G glycoprotein binding penicillin binding protein 1a.Conclusions: The direct interaction between a respiratory virus protein and the pneumococcus resulting in increased bacterial virulence and worsening disease outcome is a new paradigm in respiratory infection.Keywords: respiratory syncytial virus; pneumococcus; cilia; virulence; G protein Respiratory syncytial virus (RSV) and Streptococcus pneumoniae, also known as the pneumococcus, are major respiratory pathogens causing a huge global healthcare burden, predominantly in young children and the elderly (1). Global RSV disease burden is estimated at 64 million cases and 160,000 deaths every year (2). Pneumococcal pneumonia results in more than 1 million deaths each year worldwide with approximately half a million in children younger than 5 years (3). There is increasing evidence that RSV and the pneumococcus interact to increase the severity of respiratory disease (4-6). Seasonal increases in infections This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org This article has embedded videos, which play in place from both the online PDF or HTML versions. If you cannot view Flash videos on your device, please try playing the videos in the online PDF, or access the original uncompressed videos at http://www.atsjournals.org/doi/suppl/10.1164/rccm.201311-2110OC. To play, mouse over the image and click on the arrow that will appear in the center or on the lower left.

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.

Summary We have characterized the csoR‐copA‐copZ copper resistance operon of the important human intracellular pathogen Listeria monocytogenes. Transcription of the operon is specifically induced by copper, and mutants lacking the P1‐type ATPase CopA have reduced copper tolerance and over‐accumulate copper relative to wild type. The copper‐responsive repressor CsoR autoregulates transcription by binding to a single 32 bp site spanning the −10 and −35 elements of the promoter. Copper co‐ordination by CsoR derepresses transcription of the operon and alters CsoR:DNA complex assembly as determined by DNase I footprinting and electrophoretic mobility shift assays, with some DNA‐binding capacity being retained in the presence of 2 mole equivalents of copper. Analysis of the CsoR copper sensory site demonstrated that substitution of Cys42 with Ala generated a CsoR variant that was unresponsive to copper. Importantly, in the absence of CopZ, copper responsiveness of csoR‐copA‐copZ expression is substantially increased, implying that CopZ reduces the access of CsoR to copper. Furthermore, CopZ is shown to confer copper resistance in mutants lacking copper‐inducible csoR‐copA‐copZ expression, thus providing protection from the deleterious effects of copper within the cytoplasm.

We report here the identification and characterization of two zinc uptake systems, ZurAM and ZinABC, in the intracellular pathogen Listeria monocytogenes. Transcription of both operons was zinc responsive and regulated by the zinc-sensing repressor Zur. Deletion of either zurAM or zinA had no detectable effect on growth in defined media, but a double zurAM zinA mutant was unable to grow in the absence of zinc supplementation. Deletion of zinA had no detectable effect on intracellular growth in HeLa epithelial cells. In contrast, growth of the zurAM mutant was significantly impaired in these cells, indicating the importance of the ZurAM system during intracellular growth. Notably, the deletion of both zinA and zurAM severely attenuated intracellular growth, with the double mutant being defective in actin-based motility and unable to spread from cell to cell. Deletion of either zurAM or zinA had a significant effect on virulence in an oral mouse model, indicating that both zinc uptake systems are important in vivo and establishing the importance of zinc acquisition during infection by L. monocytogenes. The presence of two zinc uptake systems may offer a mechanism by which L. monocytogenes can respond to zinc deficiency within a variety of environments and during different stages of infection, with each system making distinct contributions under different stress conditions.