Sarah Glenn

Characterization of relA and codY mutants of Listeria monocytogenes: Identification of the CodY regulon and its role in virulence

Listeria monocytogenes is a Gram‐positive intracellular parasite and the causative organism of human listeriosis. In this article we demonstrate that L. monocytogenes encodes a functional member of the CodY family of global regulatory proteins that is responsive to both GTP and branched chain amino acids. By transcript analyses we identified the CodY regulon in L. monocytogenes and demonstrated that it comprises genes involved in amino acid metabolism, nitrogen assimilation as well as genes involved in sugar uptake and incorporation, indicating a role for CodY in L. monocytogenes in both carbon and nitrogen assimilation. A ΔrelA mutation reduced expression of the CodY regulon in early stationary phase and introduction of a ΔcodY mutation into a ΔrelA strain restored virulence. These data indicate that the avirulence of the ΔrelA mutant can in part be explained by the continued repression of the CodY regulon. The phenotypes of ΔrelA and ΔcodY mutants were studied in J774.A1 and Caco‐2 cells and the ΔrelA mutation shown to effect intracellular growth. These results provide the first direct evidence that the activity of a CodY‐type protein influences pathogenesis and provides new information on the physiological adaptation of L. monocytogenes to post‐exponential phase growth and virulence.

The combined actions of the copper-responsive repressor CsoR and copper-metallochaperone CopZ modulate CopA-mediated copper efflux in the intracellular pathogen Listeria monocytogenes

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.

Two Zinc Uptake Systems Contribute to the Full Virulence of Listeria monocytogenes during Growth In Vitro and In Vivo

ABSTRACT 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 Biofilm-Associated Protein (BapL) May Contribute to Surface Attachment of L. monocytogenes but Is Absent from Many Field Isolates

ABSTRACT 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.

Respiratory Syncytial Virus Increases the Virulence of Streptococcus pneumoniae by Binding to Penicillin Binding Protein 1a. A New Paradigm in Respiratory Infection

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.

The combined actions of the copper-responsive repressor CsoR and copper-metallochaperone CopZ modulate CopA-mediated copper efflux in the intracellular pathogen listeria monocytogenes

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.

The In Vivo Environment Accelerates Generation of Resuscitation-Promoting Factor–Dependent Mycobacteria

To the Editor: It is believed that one-third of the entire world population is latently infected with Mycobacterium tuberculosis, which has previously been attributed to the ability of the tubercle bacillus to survive for years without replication (1). The documented examples of tuberculosis reactivation after years of absence of disease illustrate the capacity of M. tuberculosis to reversibly switch from a nonreplicating state to active growth (2). The presence of M. tuberculosis in lungs of latently infected patients has been confirmed by molecular and immunological techniques (3), yet growth in culture of the bacteria associated with latent infection has not been demonstrated. Therefore, it has been hypothesized that during latent infection M. tuberculosis produces nonreplicating forms, which require resuscitation under specialized cultivation conditions to produce growth (4). However, nongrowing persister-like M. tuberculosis can also be detected in sputum collected from patients with active tuberculosis (5–7). The majority of these bacilli required specific growth conditions and could be cultivated only in the presence of recombinant resuscitation-promoting factor (Rpf) or culture supernatant (6). Moreover, these Rpf-dependent mycobacteria were more tolerant to rifampicin and accumulated during chemotherapy while other organisms were eliminated (6), confirming the production of physiologically distinct M. tuberculosis forms during tuberculosis infection or during transition to sputum. Because numbers of Rpf-dependent mycobacteria varied between patients (6), it is plausible to suggest the importance of specific host factors for the development of Rpf dependency. The molecular mechanisms underlying the formation of Rpf-dependent bacteria recovered from sputum remain unknown. Rpf-dependent cells could be generated in loci of infection (e.g., lungs) in high numbers and subsequently gradually released into sputum; alternatively, mycobacteria may rapidly develop Rpf dependency during transition from lung to sputum under the influence of certain, but yet unknown, stimuli. Our previous identification of Rpf-dependent bacteria in patients with active tuberculosis points to the presence of a heterogeneity in growth states within the bacterial populations residing in sputum. However, how and when these adaptions arise remains unknown and in this regard we propose two possibilities: (1) Considering that tubercle bacilli traffic out of necrotic cavities and into the airways, the accompanying changes in the extracellular environment and short exposure to sputum may induce an adaptive response that results in Rpf dependency; (2) Rpf dependency may arise in response to stresses imposed by the immune system and longer stationary exposure to the tissue or cavity environment. We previously found that direct exposure of M. tuberculosis to sputum did not result in Rpf dependency (6), which suggested that the extracellular environment in sputum cannot be the sole inducer of this adaptive response in M. tuberculosis. Consequently, in this study we investigated whether Rpf-dependent bacilli are produced in pulmonary infection in an animal model. Nine-week-old BALB/c mice were infected intranasally with Mycobacterium bovis BCG Glaxo at a dose of 2 × 105 bacteria per mouse, and numbers of mycobacteria in lungs were monitored for 6 weeks. For this, we employed growth assays previously developed for investigation of mycobacterial populations in sputum (6). We quantified numbers of mycobacteria that were able to grow either on 7H10 agar (colony-forming unit [CFU] counts) or in liquid 7H9 medium (using the most probable number [MPN] assay). The numbers of Rpf-dependent mycobacteria (RDM) were assessed by MPN assay in liquid 7H9 medium, containing culture supernatant from growing bacteria. At 24 hours postinfection, CFU, MPN, and RDM counts of mycobacteria recovered from lungs of infected animals were not significantly different (P > 0.05, one-way analysis of variance), suggesting that the preparation of bacteria for infection and initial adaptation in vivo did not induce Rpf dependency. However, during the course of infection there was a dramatic 2.5 log10 reduction in CFU and MPN counts of mycobacteria in the lungs of infected animals (Figure 1A). These results are in good accordance with previously reported survival patterns of M. bovis BCG in BALB/c mice (8, 9). In contrast, the number of mycobacteria grown with culture supernatant changed only at the beginning of infection (a 0.5 log10 reduction 1 wk postinfection) and at later stages it remained constant, suggesting that more than 98% of mycobacteria recovered from lungs at 6 weeks postinfection required special conditions for cultivation (Figure 1A). To confirm that bacteria recovered in the presence of culture supernatant were indeed Rpf dependent, further experiments were performed. In these experiments numbers of mycobacteria grown in culture supernatant treated with specific inhibitors of Rpf (10), or in culture supernatant prepared from a quintuple M. tuberculosis mutant missing all five Rpfs (11), were assessed. As shown in Figure 1B, both Rpf inhibitors completely eliminated the resuscitation activity of culture supernatant, and Rpf-negative supernatant also failed to resuscitate nonculturable bacteria. Both of these control experiments confirm that the nonculturable mycobacteria recovered were indeed Rpf dependent. Figure 1. Generation of resuscitation-promoting factor (Rpf)-dependent Mycobacterium bovis (BCG) in murine lungs. (A and B) Nine-week-old BALB/c mice were infected intranasally with 2 × 105 bacterial colony-forming units (CFUs) per animal. ... Incubation of mycobacteria in lung homogenates did not result in the development of Rpf dependency (data not shown). We therefore investigated whether exposure of mycobacteria to murine serum would stimulate production of Rpf-dependent forms. We incubated growing M. bovis BCG bacteria in phosphate-buffered saline (PBS) containing 25% (vol/vol), 50% (vol/vol), or undiluted murine serum, obtained from mice infected with M. bovis BCG for 24 hours, at 37°C without shaking. CFU and MPN counts were taken after 1 and 3 days of incubation. However, incubation of mycobacteria in PBS containing serum did not result in any statistically significant loss of culturability or generation of Rpf-dependent forms (Figure 1C). Sera from uninfected mice showed similar effects. This could be because cell-mediated immunity is essential for the generation of Rpf-dependent bacteria. This study demonstrates that the in vivo environment changes mycobacterial physiological characteristics and accelerates the generation of Rpf-dependent mycobacteria. Our results suggest that Rpf-dependent mycobacteria are generated in murine lungs soon after infection and represent significant proportions of the bacteria present. Therefore it is plausible to suggest that Rpf-dependent mycobacteria recovered from the sputum of infected patients are generated in the lungs during infection rather than during the transition into sputum. The precise factors responsible for the formation of Rpf-dependent bacteria remain unknown. Previously, it was demonstrated that M. tuberculosis bacilli developed Rpf dependency during prolonged incubation in the stationary phase (11) or on gradual acidification of medium (12). Nonculturable and Rpf-dependent cells of M. tuberculosis have been recovered from peritoneal macrophages of infected mice after several days of infection (13). However, exposure of mycobacteria to murine serum did not stimulate Rpf dependency, suggesting that a yet unknown combination of environmental conditions not identified (or present) in in vitro experiments or certain immune factors may trigger transition to the Rpf-dependent state. Our findings have important implications for the diagnosis of tuberculosis (14, 15) and development of models for testing the bactericidal activity of novel drugs. The presence of a high number of Rpf-dependent bacteria in vivo must be taken into account in the design of drugs for the treatment of tuberculosis, and compounds should also be tested for activity against Rpf-dependent forms. Our data suggest that significant proportions of mycobacteria may remain undetected in animal infection experiments and during clinical trials of new treatments.

Antimicrobial Treatment Improves Mycobacterial Survival in Nonpermissive Growth Conditions

ABSTRACT Antimicrobials targeting cell wall biosynthesis are generally considered inactive against nonreplicating bacteria. Paradoxically, we found that under nonpermissive growth conditions, exposure of Mycobacterium bovis BCG bacilli to such antimicrobials enhanced their survival. We identified a transcriptional regulator, RaaS (for regulator of antimicrobial-assisted survival), encoded by bcg1279 (rv1219c) as being responsible for the observed phenomenon. Induction of this transcriptional regulator resulted in reduced expression of specific ATP-dependent efflux pumps and promoted long-term survival of mycobacteria, while its deletion accelerated bacterial death under nonpermissive growth conditions in vitro and during macrophage or mouse infection. These findings have implications for the design of antimicrobial drug combination therapies for persistent infectious diseases, such as tuberculosis.

Recombinant plants provide a new approach to the production of bacterial polysaccharide for vaccines.

Bacterial polysaccharides have numerous clinical or industrial uses. Recombinant plants could offer the possibility of producing bacterial polysaccharides on a large scale and free of contaminating bacterial toxins and antigens. We investigated the feasibility of this proposal by cloning and expressing the gene for the type 3 synthase (cps3S) of Streptococcus pneumoniae in Nicotinia tabacum, using the pCambia2301 vector and Agrobacterium tumefaciens-mediated gene transfer. In planta the recombinant synthase polymerised plant-derived UDP-glucose and UDP-glucuronic acid to form type 3 polysaccharide. Expression of the cps3S gene was detected by RT-PCR and production of the pneumococcal polysaccharide was detected in tobacco leaf extracts by double immunodiffusion, Western blotting and high-voltage paper electrophoresis. Because it is used a component of anti-pneumococcal vaccines, the immunogenicity of the plant-derived type 3 polysaccharide was tested. Mice immunised with extracts from recombinant plants were protected from challenge with a lethal dose of pneumococci in a model of pneumonia and the immunised mice had significantly elevated levels of serum anti-pneumococcal polysaccharide antibodies. This study provides the proof of the principle that bacterial polysaccharide can be successfully synthesised in plants and that these recombinant polysaccharides could be used as vaccines to protect against life-threatening infections.

Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin

Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation.