Insight of host immune evasion mediated by two variants of group a Streptococcus Mac protein

Group A Streptococcus has evolved numerous mechanisms to evade the host immune system to survive, disseminate, and cause disease. Recently a secreted protein named Mac-1 was identified and shown to enhance survival of the pathogen. A new variant of Mac-1 (designated Mac-2) also was recently described and shown to differ from Mac-1 by approximately 50% amino acid sequence divergence in the middle one-third of the molecule. To gain new information about the role of Mac-1 and Mac-2 in host-pathogen interactions, solution binding experiments were performed using surface plasmon resonance and purified Mac proteins. Mac-1 bound the same lower hinge region of human IgG as Fc receptors with 2.5 microM affinity, which lead to proteolytic cleavage of the antibody. Similar Km (6.8-18.9 microM) and kcat (0.02-0.13 s(-1)) values of the Mac-1 endopeptidase activity were obtained for IgG1, IgG2, IgG3, and IgG4. Mac-2 variant, in contrast, bound human IgG poorly (KD = 16 mM) and had weak endopeptidase activity against IgG. Instead, Mac-2 bound FcgammaRII and FcgammaRIII with 5 and 75 microM affinity, respectively. This binding competitively blocked IgG from recognition by Fc receptors. Taken together, Mac proteins block immunoglobulin recognition by Fc receptors and degrade immunoglobulins, thereby enhancing survival of the pathogen through the inhibition of phagocytosis, endocytosis of IgG-opsonized particles, and antibody-dependent cell-mediated cytotoxicity. Consequently, these proteins may be potential therapeutic targets.     

Survey of predators and sampling method comparison in sweet corn

Natural predation is an important component of integrated pest management that is often overlooked because it is difficult to quantify and perceived to be unreliable. To begin incorporating natural predation into sweet corn, Zea mays L., pest management, a predator survey was conducted and then three sampling methods were compared for their ability to accurately monitor the most abundant predators. A predator survey on sweet corn foliage in New York between 1999 and 2001 identified 13 species. Orius insidiosus (Say), Coleomegilla maculata (De Geer), and Harmonia axyridis (Pallas) were the most numerous predators in all years. To determine the best method for sampling adult and immature stages of these predators, comparisons were made among nondestructive field counts, destructive counts, and yellow sticky cards. Field counts were correlated with destructive counts for all populations, but field counts of small insects were biased. Sticky cards underrepresented immature populations. Yellow sticky cards were more attractive to C. maculata adults than H. axyridis adults, especially before pollen shed, making coccinellid population estimates based on sticky cards unreliable. Field counts were the most precise method for monitoring adult and immature stages of the three major predators. Future research on predicting predation of pests in sweet corn should be based on field counts of predators because these counts are accurate, have no associated supply costs, and can be made quickly.     

Visualizing single molecules interacting with nuclear pore complexes by narrow-field epifluorescence microscopy

The utility of single molecule fluorescence (SMF) for understanding biological reactions has been amply demonstrated by a diverse series of studies over the last decade. In large part, the molecules of interest have been limited to those within a small focal volume or near a surface to achieve the high sensitivity required for detecting the inherently weak signals arising from individual molecules. Consequently, the investigation of molecular behavior with high time and spatial resolution deep within cells using SMF has remained challenging. Recently, we demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single cargo level. We describe here the methodological approach that yields 2 ms and approximately 15 nm resolution for a stationary particle. The spatial resolution for a mobile particle is inherently worse, and depends on how fast the particle is moving. The signal-to-noise ratio is sufficiently high to directly measure the time a single cargo molecule spends interacting with the nuclear pore complex. Particle tracking analysis revealed that cargo molecules randomly diffuse within the nuclear pore complex, exiting as a result of a single rate-limiting step. We expect that narrow-field epifluorescence microscopy will be useful for elucidating other binding and trafficking events within cells.     

Integrating biological and chemical controls in decision making: European corn borer (Lepidoptera: Crambidae) control in sweet corn as an example

As growers switch to transgenic crops and selective insecticides that are less toxic to natural enemies, natural enemies can become more important in agricultural pest management. Current decision-making guides are generally based on pest abundance and do not address pest and natural enemy toxicity differences among insecticides or the impact of natural enemies on pest survival. A refined approach to making pest management decisions is to include the impact of natural enemies and insecticides, thereby better integrating biological and chemical control. The result of this integration is a dynamic threshold that varies for each product and the level of biological control expected. To demonstrate the significance of conserved biological control in commercial production, a decision-making guide was developed that evaluates control options for European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), in sweet corn, Zea mays L., where the primary natural enemies are generalist predators. Management options are lambda-cyhalothrin (broad-spectrum insecticide), spinosad (selective insecticide), Trichogramma ostriniae (Peng and Chen) (Hymenoptera: Trichogrammatidae) (parasitoid), and Bacillus thuringiensis (Bt) sweet corn (transgenic variety). The key factors influencing thresholds for all treatments are the intended market, predator populations, and the presence of alternative foods for the predators. Treatment cost is the primary factor separating the threshold for each treatment within a common scenario, with the lowest cost treatment having the lowest pest threshold. However, when the impact of a treatment on natural enemies is projected over the 3-wk control period, the impact of the treatment on predators becomes the key factor in determining the threshold, so the lowest thresholds are for broad-spectrum treatments, whereas selective products can have thresholds > 6 times higher by the third week. This decision guide can serve as a framework to help focus future integrated pest management research and to aid in the selection of pest management tools.     

Multi high-throughput approach for highly selective identification of vaccine candidates: the Group A Streptococcus case

We propose an experimental strategy for highly accurate selection of candidates for bacterial vaccines without using in vitro and/or in vivo protection assays. Starting from the observation that efficacious vaccines are constituted by conserved, surface-associated and/or secreted components, the strategy contemplates the parallel application of three high throughput technologies, i.e. mass spectrometry-based proteomics, protein array, and flow-cytometry analysis, to identify this category of proteins, and is based on the assumption that the antigens identified by all three technologies are the protective ones. When we tested this strategy for Group A Streptococcus, we selected a total of 40 proteins, of which only six identified by all three approaches. When the 40 proteins were tested in a mouse model, only six were found to be protective and five of these belonged to the group of antigens in common to the three technologies. Finally, a combination of three protective antigens conferred broad protection against a panel of four different Group A Streptococcus strains. This approach may find general application as an accelerated and highly accurate path to bacterial vaccine discovery.     

Genome sequence of a Lancefield group C Streptococcus zooepidemicus strain causing epidemic nephritis: new information about an old disease

Outbreaks of disease attributable to human error or natural causes can provide unique opportunities to gain new information about host-pathogen interactions and new leads for pathogenesis research. Poststreptococcal glomerulonephritis (PSGN), a sequela of infection with pathogenic streptococci, is a common cause of preventable kidney disease worldwide. Although PSGN usually occurs after infection with group A streptococci, organisms of Lancefield group C and G also can be responsible. Despite decades of study, the molecular pathogenesis of PSGN is poorly understood. As a first step toward gaining new information about PSGN pathogenesis, we sequenced the genome of Streptococcus equi subsp. zooepidemicus strain MGCS10565, a group C organism that caused a very large and unusually severe epidemic of nephritis in Brazil. The genome is a circular chromosome of 2,024,171 bp. The genome shares extensive gene content, including many virulence factors, with genetically related group A streptococci, but unexpectedly lacks prophages. The genome contains many apparently foreign genes interspersed around the chromosome, consistent with the presence of a full array of genes required for natural competence. An inordinately large family of genes encodes secreted extracellular collagen-like proteins with multiple integrin-binding motifs. The absence of a gene related to speB rules out the long-held belief that streptococcal pyrogenic exotoxin B or antibodies reacting with it singularly cause PSGN. Many proteins previously implicated in GAS PSGN, such as streptokinase, are either highly divergent in strain MGCS10565 or are not more closely related between these species than to orthologs present in other streptococci that do not commonly cause PSGN. Our analysis provides a comparative genomics framework for renewed appraisal of molecular events underlying APSGN pathogenesis.     

Identification of Mycobacterium tuberculosis clinical isolates with altered phagocytosis by human macrophages due to a truncated lipoarabinomannan

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.