DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.     

Roles of the volatile terpene, 1,8-cineole,in plant–herbivore interactions: a foraging odor cue as well as a toxin?

Olfaction is an important sense for many animals, yet its role in foraging by herbivores is poorly known. Many plants contain volatile compounds, such as terpenes, that are not only volatile but can be toxic if ingested. Volatile terpenes can be used by herbivores to assess leaf quality, but there is little evidence for whether they are also used as a searching cue. We applied the giving-up density (GUD) framework to examine fine-scale foraging by two free-ranging mammalian herbivores, the brush-tail possum (Trichosurus vulpecula) and the swamp wallaby (Wallabia bicolor), using patches with food and an inedible matrix that varied in content of a volatile terpene, 1,8-cineole. We tested the effect of (1) increasing dietary cineole concentration, and (2) masking the food odor by adding cineole to the inedible matrix, thus overriding the smell released by the diet. In both species GUD was affected by dietary cineole; a high cineole concentration raised GUD, consistent with its role as a toxin. There was a significant effect of masking on GUD for wallabies but not for possums, suggesting that odor was an important foraging cue at the feeding patch only for the former. Differences in ecological niche and diet may explain this pattern. We suggest that herbivores, such as the swamp wallaby, opportunistically eavesdrop on plant volatiles, i.e., take advantage of the signal proffered for a different function. The cost of this eavesdropping for plants, however, is presumably counteracted by other ecological benefits of these volatiles, including a reduction in leaf consumption as a function of toxicity.     

The α-test: rapid cell-free CD4 enumeration using whole saliva

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides. Thus, identification of cell-free correlates that directly regulate the number of CD4(+) T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates. The number of stem cells that enter blood and are destined to become circulating CD4(+) T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLE(CS)) and the HLE(CS)-reactive active α(1)proteinase inhibitor (α(1)PI, α(1)antitrypsin, SerpinA1). In HIV-1 disease, α(1)PI is inactivated due to disease processes. In the early asymptomatic categories of HIV-1 disease, active α(1)PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 μM, and to achieve normal levels during the symptomatic categories. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with >220 CD4 cells/μl, CD4 counts were correlated with serum levels of active α(1)PI (r(2)=0.93, p<0.0001, n=26) and inactive α(1)PI (r(2)=0.91, p<0.0001, n=26). Administration of α(1)PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting α(1)PI participates in regulating the number of CD4(+) T cells in blood. With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active α(1)PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum α(1)PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to α(1)PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA(3)NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active α(1)PI in saliva. The resulting inhibition of PPE by active α(1)PI can be measured by adding the PPE substrate SA(3)NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/μl), the concentration of α(1)PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable. Thus, active α(1)PI in saliva is calculated as a ratio to saliva protein content and is termed the α(1)PI Index. Results presented herein demonstrate that the α(1)PI Index provides an accurate and precise physiologic method for calculating CD4 counts.     

Heterogeneity of multifunctional IL-17A producing S. Typhi-specific CD8+ T cells in volunteers following Ty21a typhoid immunization

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, continues to cause significant morbidity and mortality world-wide. CD8+ T cells are an important component of the cell mediated immune (CMI) response against S. Typhi. Recently, interleukin (IL)-17A has been shown to contribute to mucosal immunity and protection against intracellular pathogens. To investigate multifunctional IL-17A responses against S. Typhi antigens in T memory subsets, we developed multiparametric flow cytometry methods to detect up to 6 cytokines/chemokines (IL-10, IL-17A, IL-2, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1β (MIP-1β)) simultaneously. Five volunteers were immunized with a 4 dose regimen of live-attenuated S. Typhi vaccine (Ty21a), peripheral blood mononuclear cells (PBMC) were isolated before and at 11 time points after immunization, and CMI responses were evaluated. Of the 5 immunized volunteers studied, 3 produced detectable CD8+ T cell responses following stimulation with S. Typhi-infected autologous B lymphoblastoid cell lines (B-LCL). Additionally, 2 volunteers had detectable levels of intracellular cytokines in response to stimulation with S. Typhi-infected HLA-E restricted cells. Although the kinetics of the responses differed among volunteers, all of the responses were bi- or tri-phasic and included multifunctional CD8+ T cells. Virtually all of the IL-17A detected was derived from multifunctional CD8+ T cells. The presence of these multifunctional IL-17A+ CD8+ T cells was confirmed using an unsupervised analysis program, flow cytometry clustering without K (FLOCK). This is the first report of IL-17A production in response to S. Typhi in humans, indicating the presence of a Tc17 response which may be important in protection. The presence of IL-17A in multifunctional cells co-producing Tc1 cytokines (IL-2, IFN-γ and TNF-α) may also indicate that the distinction between Tc17 and Tc1 responses in humans is not as clearly delineated as suggested by in vitro experiments and animal models.     

Spatial Analysis of Antibiotic Resistance Along Metal Contaminated Streams

The spatial pattern of antibiotic resistance in culturable sediment bacteria from four freshwater streams was examined. Previous research suggests that the prevalence of antibiotic resistance may increase in populations via indirect or coselection from heavy metal contamination. Sample bacteria from each stream were grown in media containing one of four antibiotics—tetracycline, chloramphenicol, kanamycin, and streptomycin—at concentrations greater than the minimum inhibitory concentration, plus a control. Bacteria showed high susceptibilities to the former two antibiotics. We summarized the latter two more prevalent (aminoglycoside) resistance responses and ten metals concentrations per sediment sample, by Principal Components Analysis. Respectively, 63 and 58% of the variability was explained in the first principal component of each variable set. We used these multivariate summary metrics [i.e., first principal component (PC) scores] as input measures for exploring the spatial correlation between antibiotic resistance and metal concentration for each stream sampled. Results show a significant and negative correlation between metals PC scores versus aminoglycoside resistance scores and suggest that selection for metal tolerance among sediment bacteria may influence selection for antibiotic resistance differently in sediments than in the water column. Our most important finding comes from geostatistical cross-variogram analysis, which shows that increasing metal concentration scores are spatially associated with decreasing aminoglycoside resistance scores—a negative correlation, but holds for contaminated streams only. We suspect our field results are influenced by metal bioavailability in the sediments and by a contaminant promoted interaction or “cocktail effect” from complex combinations of pollution mediated selection agents.     

Engineering of complex polyketide biosynthesis — insights from sequencing of the monensin biosynthetic gene cluster

The biosynthesis of complex reduced polyketides is catalysed in actinomycetes by large multifunctional enzymes, the modular Type I polyketide synthases (PKSs). Most of our current knowledge of such systems stems from the study of a restricted number of macrolide-synthesising enzymes. The sequencing of the genes for the biosynthesis of monensin A, a typical polyether ionophore polyketide, provided the first genetic evidence for the mechanism of oxidative cyclisation through which polyethers such as monensin are formed from the uncyclised products of the PKS. Two intriguing genes associated with the monensin PKS cluster code for proteins, which show strong homology with enzymes that trigger double bond migrations in steroid biosynthesis by generation of an extended enolate of an unsaturated ketone residue. A similar mechanism operating at the stage of an enoyl ester intermediate during chain extension on a PKS could allow isomerisation of an E double bond to the Z isomer. This process, together with epoxidations and cyclisations, form the basis of a revised proposal for monensin formation. The monensin PKS has also provided fresh insight into general features of catalysis by modular PKSs, in particular into the mechanism of chain initiation. Journal of Industrial Microbiology & Biotechnology (2001) 27, 360–367. Received 18 March 2001/ Accepted in revised form 09 July 2001     

Air movement and heat loss from sheep. I. Boundary layer insulation of a model sheep, with and without fleece

A model sheep, made from metal cylinders and hemispheres, was heated electrically. Heat loss by forced convection in a wind tunnel was analysed in terms of the dependence of the Nusselt number (Nu) on Reynolds number (Re). For a bare trunk Nu = 0.095 Re0.684, but with fleece covering the trunk to a depth of 3.5 cm, Nu = 0.0112 Re0.875 when the mean radiative temperature of the the coat was taken as the surface temperature. Heat transfer by convection from the whole body, including legs, was described by Nu = 0.029 Re0.80. However, a bulk Nesselt number should not be used to estimate heat loss from a live sheep in a hot environment if the windspeed is below about 4 m s-1 because the relation between mean surface temperature, Nusselt number and convective heat flux is not unique.     

Influence of Vesicular-Arbuscular Mycorrhizal Fungi on the Response of Potato to Phosphorus Deficiency

Morphological and biochemical interactions between a vesicular-arbuscular mycorrhizal (VAM) fungus (Glomus fasciculatum [Thaxt. sensu Gerdemann] Gerdemann and Trappe) and potato (Solanum tuberosum L.) plants during the development of P deficiency were characterized. Nonmycorrhizal (NM) plants grown for 63 d with low abiotic P supply (0.5 mM) produced 34, 52, and 73% less root, shoot, and tuber dry matter, respectively, than plants grown with high P (2.5 mM). The total leaf area and the leaf area:plant dry weight ratio of low-P plants were substantially lower than those of high-P plants. Moreover, a lower shoot:root dry weight ratio and tuber:plant dry weight ratio in low-P plants than in high-P plants characterized a major effect of P deficiency stress on dry matter partitioning. In addition to a slower rate of growth, low-P plants accumulated nonreducing sugars and nitrate. Furthermore, root respiration and leaf nitrate reductase activity were lower in low-P plants than in high-P plants. Low abiotic P supply also induced physiological changes that contributed to the greater efficiency of P acquisition by low-P plants than by high-P plants. For example, allocation of dry matter and P to root growth was less restricted by P deficiency stress than to shoot and tuber growth. Also, the specific activities of root acid phosphatases and vanadate-sensitive microsomal ATPases were enhanced in P-deficient plants. The establishment of a VAM symbiosis by low-P plants was essential for efficient P acquisition, and a greater root infection level for P-stressed plants indicated increased compatibility to the VAM fungus. By 63 d after planting, low-P VAM plants had recovered 42% more of the available soil P than low-P NM plants. However, the VAM fungus only partially alleviated P deficiency stress and did not completely compensate for inadequate abiotic P supply. Although the specific activities of acid phosphatases and microsomal ATPases were only marginally influenced by VAM infection, VAM roots characteristically had a higher protein concentration and, consequently, enhanced microsomal ATPase and acid phosphatase activities on a fresh weight basis compared with NM roots. Morphological and ultrastructural details of VAM plants are discussed in relation to the influence of the VAM symbiosis on P nutrition of potato.     

Spatial and temporal variability of antibiotic resistance in freshwater bacterial assemblages

Abstract Spatial and temporal variability in antibiotic resistance was examined in bacterial assemblages from streams and ponds on the US Department of Energy's Savannah River Site (SRS) in South Carolina. Sites sampled have been impacted to varying degrees by contamination with organic compounds, heavy metals, and radioactive materials because of production of nuclear materials on the site. Antibiotic resistance in the culturable portion of the bacterial assemblage was determined from coloby formation on media containing antibiotics. Eight antibiotics, chloramphenicol, cycloserine, kanamycin, neomycin, novobiocin, rifampicin, streptomycin, and tetracycline, were used at concentrations of 50 and 200 μg ml⁻¹. Statistically significant differences in frequency of antibiotic resistance were observed among sites and among dates at a single site. Bacterial densities (total and culturable), dissolved organic carbon (DOC) concentration, and human impact also varied among sites but bore no overall relationship to resistance frequency. SRS operations did not have a detectable impact on antibiotic resistance.