Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

Light intensity and social communication between hens.

1. The effect of light intensity on social communication in laying hens was investigated experimentally by comparing the transmission and detection of social signals between familiar and unfamiliar hens of similar or unequal rank in a competition for food. 2. The experimental method consisted of mutual inspection by a pair of hens at short range (approximately 24 cm), followed by a competition at a feed trough from which only one hen could eat a favoured food. The relative rank of a hen was inferred from success in this competition. 3. The relative rank of individual hens within 5 groups, each of 6 adult laying hens, was determined to identify those of high and low rank within their home group. 4 Social communication between pairs of either unfamiliar or familiar hens of either similar or unequal rank (highest and lowest ranking within their home groups) was then assessed under light intensities of 1, 5, 20 and 100 lux with all other cues present. Only the dimmest light of 1 lux perturbed some aspects of the competition for food. 5. The findings provide scientific justification, in part, for the current legal requirement in England for 'all hens to see other hens' by specifying a minimum light intensity of at least 5 lux for hens kept in close proximity to each other.

In vivo pharmacodynamic characterization of a novel plectasin antibiotic, NZ2114, in a murine infection model.

NZ2114 is a novel plectasin derivative with potent activity against gram-positive bacteria, including multiply drug-resistant strains. We used the neutropenic murine thigh infection model to characterize the time course of antimicrobial activity of NZ2114 and determine which pharmacokinetic/pharmacodynamic (PK/PD) index and magnitude best correlated with efficacy. Serum drug levels following administration of three fourfold-escalating single-dose levels of NZ2114 were measured by microbiologic assay. Single-dose time-kill studies following doses of 10, 40, and 160 mg/kg of body weight demonstrated concentration-dependent killing over the dose range (0.5 to 3.7 log(10) CFU/thigh) and prolonged postantibiotic effects (3 to 15 h) against both Staphylococcus aureus and Streptococcus pneumoniae. Mice had 10(6.3) to 10(6.8) CFU/thigh of strains of S. pneumoniae or S. aureus at the start of therapy when treated for 24 h with 0.625 to 160 mg/kg/day of NZ2114 fractionated for 4-, 6-, 12-, and 24-h dosing regimens. Nonlinear regression analysis was used to determine which PK/PD index best correlated with microbiologic efficacy. Efficacies of NZ2114 were similar among the dosing intervals (P = 0.99 to 1.0), and regression with the 24-h area under the concentration-time curve (AUC)/MIC index was strong (R(2), 0.90) for both S. aureus and S. pneumoniae. The maximum concentration of drug in serum/MIC index regression was also strong for S. pneumoniae (R(2), 0.96). Studies to identify the PD target for NZ2114 utilized eight S. pneumoniae and six S. aureus isolates and an every-6-h regimen of drug (0.156 to 160 mg/kg/day). Treatment against S. pneumoniae required approximately twofold-less drug for efficacy in relationship to the MIC than did treatment against S. aureus. The free drug 24-h AUCs/MICs necessary to produce a stasis effect were 12.3 +/- 6.7 and 28.5 +/- 11.1 for S. pneumoniae and S. aureus, respectively. The 24-h AUC/MIC associated with a 1-log killing endpoint was only 1.6-fold greater than that needed for stasis. Resistance to other antimicrobial classes did not impact the magnitude of the PD target required for efficacy. The PD target in this model should be considered in the design of clinical trials with this novel antibiotic.

Antimicrobial activity of the recombinant designer host defence peptide P-novispirin G10 in infected full-thickness wounds of porcine skin.

OBJECTIVES: The growing number of patients with impaired wound healing and the development of multidrug-resistant bacteria demand the investigation of alternatives in wound care. The antimicrobial activity of naturally occurring host defence peptides and their derivatives could be one alternative to the existing therapy options for topical treatment of wound infection. Therefore, the aim of this study was to investigate the antimicrobial activity of proline-novispirin G10 (P-novispirin G10) in vitro and in the infected porcine titanium wound chamber model. METHODS: The new derived designer host defence peptide P-novispirin G10 was tested in vitro against Gram-positive and Gram-negative bacterial strains. Additionally, cytotoxicity and haemolytic activities of P-novispirin G10 and protegrin-1 were measured. For in vivo studies, six wound chambers were implanted on each flank of Göttinger minipigs (n = 2, female, 6 months old, 15-20 kg). Eleven wound chambers were inoculated 8 days post-operatively with 5 x 10(8) of Staphylococcus aureus; one wound chamber remained uninfected as a system control. After wound infection had been established (4 days after inoculation), each wound chamber was topically treated with P-novispirin G10, protegrin-1 or carrier control. Wound fluid was harvested every hour for a total follow up of 3 h. RESULTS: P-novispirin G10 demonstrated broad-spectrum antimicrobial activity with moderate haemolytic and cytotoxic activities compared with protegrin-1. In the infected wound chamber model P-novispirin G10 demonstrated a 4 log(10) reduction in bacterial counts. CONCLUSIONS: This implicates the potential of P-novispirin G10 as an alternative in future antimicrobial wound care. However, more studies are necessary to further define clinical applications and potential side effects in greater detail.

Leg health and performance of broiler chickens reared in different light environments.

1. The effects of light source and intensity on leg health and performance of female ROSS 308 broiler chickens were investigated in a 2 x 2 experimental design (8 groups of 275 chicks) of two light sources (Osram biolux and Osram warm-white) and two light intensities (5 and 100 clux, adjusted to fowl-perceived illuminance). 2. At 41 d of age, body weight, gait-score, footpad dermatitis and hock-burn were measured on 50 birds from each light environment. In addition, weekly feed intake and body weight were determined on a group basis and mortality was recorded continuously. 3. The light environment did not affect the severity of the gait-score or hock-burns. The risk of moderate to severe lameness and hock-burns increased with body weight. Birds weighing more than 2400 g had an increased probability of moderate footpad lesions in biolux light. 4. Weight and gait-score, as well as gait-score and hock-burn were positively correlated. Podo-dermatitis was weakly correlated with hock-burn, which contradicts earlier findings. The light environment did not affect feed intake, body weight or mortality. 5. The light sources and intensities employed in this study did not adversely affect production or leg health of broiler chickens reared semi-commercially.

Improving on nature's defenses: optimization & high throughput screening of antimicrobial peptides.

Antimicrobial peptides (AMPs) are ubiquitous in nature where they play important roles in host defense and microbial control. Despite their natural origin, antimicrobial spectrum and potency, the lead peptide candidates that so far have entered pharmaceutical development have all been further optimized by rational or semi-rational approaches. In recent years, several high throughput screening (HTS) systems have been developed to specifically address optimization of AMPs. These include a range of computational in silico systems and cell-based in vivo systems. The in silico-based screening systems comprise several computational methods such as Quantitative Structure/Activity Relationships (QSAR) as well as simulation methods mimicking peptide/membrane interactions. The in vivo-based systems can be divided in cis-acting and trans-acting screening systems. The cis-acting pre-screens, where the AMP exerts its antimicrobial effect on the producing cell, allow screening of millions or even billions of lead candidates for their basic antimicrobial or membrane-perturbating activity. The trans-acting screens, where the AMP is secreted or actively liberated from the producing cell and interacts with cells different from the producing cell, allow for screening under more complex and application-relevant conditions. This review describes the application of HTS systems employed for AMPs and lists advantages as well as limitations of these systems.

The use of olfactory and other cues for social recognition by juvenile pigs.

Social recognition is essential for the maintenance of a stable group structure. Failure to recognise familiar conspecifics in social groups of juvenile pigs may initiate agonistic encounters that can compromise welfare and productivity. Current housing systems may allow build up of atmospheric ammonia that might, in turn, interfere with the olfactory system and compromise olfactory perception. In the present study, 16 juvenile pigs were housed in fresh air while another 16 pigs were kept in an ammoniated atmosphere (approximately 36ppm) for 1 week prior to test and another week during testing. We then assessed the role of olfaction in social recognition and determined whether chronic exposure to ammonia compromised discrimination based on olfactory perception by comparing the pigs' responses to selected cues from a familiar and an unfamiliar pig presented simultaneously in separate chambers of a modified Y-maze in each of two test situations (near, remote). Visual, auditory, olfactory, and tactile cues were all provided in the "near" test situation; here, the stimulus pigs were presented in two separate chambers behind clear perspex walls containing an aperture that allowed nose-to-nose contact between the test and stimulus pigs. On the other hand, the "remote" test provided only olfactory cues via air passed from the chambers containing the stimulus pigs into the test chamber. Each test lasted 5min and the pigs' behaviour was recorded via overhead video cameras; we then measured the accumulated times spent near and the numbers of visits made to the familiar and the unfamiliar stimulus pigs as well as the transitions between them. Overall, pigs made more visits to and spent significantly longer near both the stimulus pigs in the near test than in the remote one (ANOVA, P<0.001). They also made more transitions between the stimuli in the former test than the latter (P<0.001). Pigs from both the ammonia and the fresh-air treatment groups showed social discrimination. However, pigs that had received chronic exposure to ammonia visited the familiar pig more often and spent longer near it than the unfamiliar one regardless of the test situation (P<0.05) whereas those reared in fresh air spent longer near the unfamiliar animal (P<0.05). The present results suggest that pigs from both treatment groups employed olfactory cues in social recognition, but that chronic exposure to ammonia did not interfere with this ability. However, ammonia treatment seemingly affected social preferences, thus indicating an unknown and more fundamental effect of living in ammoniated atmospheres.

Exploring drug-induced alterations in gene expression in Mycobacterium tuberculosis by microarray hybridization.

Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium, Mycobacterium tuberculosis. Although currently available drugs kill most isolates of M. tuberculosis, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the M. tuberculosis genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in M. tuberculosis gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug's mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and fbpC, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes, efpA, fadE23, fadE24, and ahpC, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.

Design of CytR regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA polymerase-promoter interactions modulate the efficiency of CytR repression.

In CytR regulated promoters in Escherichia coli, the cAMP-CRP complex acts as a transcriptional activator as well as a co-repressor for the CytR protein. Repression by CytR depends on the formation of nucleoprotein complexes in which CytR binds cooperatively to the DNA with one or two cAMP-CRP complexes. Here, we demonstrate that in order to establish CytR regulation in a cAMP-CRP dependent class II promoter with a single CRP site (CRP site centred around position -40.5) in which the CytR operator is located upstream of the CRP site, high affinity binding sites for both regulators are required. The efficiency of CytR regulation was observed to be modulated by RNA polymerase (RNAP)-promoter interactions. Specifically, in class II promoters with a single CRP site, the efficiency of CytR regulation was found to correlate inversely with cAMP-CRP independent promoter activity. These observations can be reconciled in a competition model for CytR regulation in which CytR and RNAP compete for cooperative binding with cAMP-CRP to the promoters in vivo. In this model, two mutually exclusive ternary complexes can be formed: a CytR/cAMP-CRP/promoter repression complex and an RNAP/cAMP-CRP/promoter activation complex. Thus, CytR regulation critically depends on formation of a repression complex that binds the promoter with sufficiently high affinity to exclude formation of the competing activation complex. We suggest that the transition from repression to activation involves a switch in the protein-protein interactions made by cAMP-CRP from CytR to RNAP. On the basis of the regulatory features of the promoters analysed here, we speculate about the advantages offered by the structural complexity of natural CytR/cAMP-CRP regulated promoters.

CytR/cAMP-CRP nucleoprotein formation in E. coli: the CytR repressor binds its operator as a stable dimer in a ternary complex with cAMP-CRP.

The CytR repressor protein relies on protein-protein interactions to the cAMP-CRP complex to bind its operators with sufficiently high affinity to repress transcription. Here, the quaternary structure of CytR and the mechanism underlying the cooperative binding of CytR and cAMP-CRP have been analyzed. Using a modified Ferguson analysis in which protein-DNA complexes are separated in a non-denaturing gel system, we show that CytR binds its operators as a dimer alone as well as in a ternary complex with cAMP-CRP. Analyses of DNA binding of CytR at low protein concentrations indicate that CytR is a dimer in solution at physiological concentrations. Moreover, the CytR inducer cytidine was found not to have any effect on the oligomerization of free CytR or DNA bound CytR. Thus, these data rule out the possibility that the cooperative DNA binding of CytR and cAMP-CRP involves induced dimerization of CytR, and they suggest that cytidine interrupts the cooperative binding of CytR and cAMP-CRP solely by perturbing the protein-protein interactions between the two proteins.