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.

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.Listeria monocytogenes is a food-borne pathogen that causes serious illness, including meningitis, septicemia, and stillbirth, with a mortality rate of up to 30% (37). More recently, there have been reports of listerial gastroenteritis following the consumption of several different food types (16,34). A number of studies have demonstrated that this organism is able to persist in the food-processing environment for several months and even up to 10 years (23, 29). One of the major causes for concern about L. monocytogenes in these environments is its ability to attach to many different surfaces (2). Indeed, there is recent evidence to show that listerial biofilms formed inside the lumens of stainless steel tubes are able to withstand the shears generated by high-Reynolds-number flows (31). Biofilms, including those produced by L. monocytogenes, are more resistant to detergents and disinfectants (33) and also are a potential source of contamination within food-processing plants; hence, they pose a risk to the maintenance of product safety (27). Consequently, there is considerable interest in determining the mechanisms of attachment and biofilm formation.Our in silico analysis of the genome sequence of L. monocytogenes identified an open reading frame (lmo0435) for a protein with similarity to biofilm-associated proteins (Bap) believed to be important for the binding of staphylococci to abiotic surfaces (10). This Bap protein also has been implicated in the virulence of Staphylococcus aureus (10, 11). Thus, the aim of the current study was to establish if this protein (Lmo0435 [BapL]) of L. monocytogenes influenced biofilm formation and virulence and to determine the prevalence of the lmo0435 (bapL) gene within a selection of field isolates. MATERIALS AND METHODS Bacterial strains and plasmids.A list of the L. monocytogenes isolates and plasmids used in this study is given in Table 1. The strains were cultured in tryptone soya broth (TSB; Oxoid) or brain heart infusion agar (Oxoid) with shaking at 37°C unless otherwise stated. Escherichia coli JM...

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Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis.