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Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

The objective of this study is to determine whether DNA signature recovery of Bacillus anthracis strains from different environmental substrates correlates with pathogen cell surface hydrophobicity and induction of host cell death. We compared recovery of DNA signatures from a panel of B. anthracis strains collected from two environmental substrates, non-porous surfaces and soil, using real-time qPCR. We further assessed both cell surface hydrophobicity of the B. anthracis strains by contact angle measurements and host cell viability in response to B. anthracis infection in a mouse macrophage cell model system. Our studies demonstrated correlation between reduced B. anthracis sample recovery from environmental substrates and increased cell surface hydrophobicity. Surprisingly, the most hydrophilic strain, K4596, which exhibited the highest level of recovery from the environmental surfaces, induced the highest level of host cell cytotoxicity compared to more hydrophobic B. anthracis strains in the panel. Our results suggest that cell surface hydrophobicity may play a leading role in mediating pathogen adherence to environmental surfaces. These findings can contribute to the optimization of pathogen detection efforts by understanding how bacterial parameters such as hydrophobicity and induction of host cell death affect bacterial adherence to environmental surfaces.

Numerous bird species are highly susceptible to North American strains of West Nile virus (WNV), and although domestic chickens are relatively resistant to WNV-associated disease, this species currently represents the most practical avian model for immune responses to WNV infection. Knowledge of the immunomodulation of susceptibility to WNV in birds is important for understanding taxonomic differences in infection outcomes. While focusing on immunophenotyping of CD3+, CD4+, CD8+, and CD45+ lymphocyte subpopulations, we compared lymphocyte subpopulations, blood chemistries, cloacal temperatures, IgM and IgG antibody titers, and differential whole-blood cell counts of WNV-infected and uninfected hens. Total blood calcium and lymphocyte numbers were lower in WNV-infected chickens compared with uninfected chickens. The heterophil-to-lymphocyte ratio increased over time from 2 to 22 d postinoculation (DPI) in uninfected chickens and from 2 to 8 DPI in WNV-infected chickens, although levels declined from 8 to 22 DPI in the latter group. No significant differences were found in the remaining immunological and hematological variables of the WNV-infected and uninfected groups. Our results reaffirm that chickens are resistant to WNV infection, and demonstrated that the heterophil-to-lymphocyte ratio differed between groups, allowing for sorting of infection status. Similar patterns in immune responses over time in both infected and uninfected hens may be related to age (i.e., 10 wk) and associated immune development.

A pulsed-field gel electrophoresis (PFGE) method was developed for discriminating Bacillus anthracis from B. cereus and B. thuringiensis . A worldwide collection of 25 B. anthracis isolates showed high-profile homology, and these isolates were unambiguously distinguished from B. cereus and B. thuringiensis isolates by cluster analysis of the whole-genome macrorestriction enzyme digestion patterns generated by NotI.

The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% ± 2%) and FCM (4% ± 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD PFGE ] = 3% ± 2% and RSD FCM = 1.2% ± 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional band-based methods.

Background Bacterial small RNAs (sRNAs) regulate gene expression by base-pairing with downstream target mRNAs to attenuate translation of mRNA into protein at the post-transcriptional level. In response to specific environmental changes, sRNAs can modulate the expression levels of target genes, thus enabling adaptation of cellular physiology. Results We profiled sRNA expression in the Gram-negative bacteria Burkholderia thailandensis cultured under 54 distinct growth conditions using a Burkholderia-specific microarray that contains probe sets to all intergenic regions greater than 90 bases. We identified 38 novel sRNAs and performed experimental validation on five sRNAs that play a role in adaptation of Burkholderia to cell stressors. In particular, the trans-encoded BTH_s1 and s39 exhibited differential expression profiles dependent on growth phase and cell stimuli, such as antibiotics and serum. Furthermore, knockdown of the highly-expressed BTH_s39 by antisense transcripts reduced B. thailandensis cell growth and attenuated host immune response upon infection, indicating that BTH_s39 functions in bacterial metabolism and adaptation to the host. In addition, expression of cis-encoded BTH_s13 and s19 found in the 5′ untranslated regions of their cognate genes correlated with tight regulation of gene transcript levels. This sRNA-mediated downregulation of gene expression may be a conserved mechanism of post-transcriptional gene dosage control. Conclusions These studies provide a broad analysis of differential Burkholderia sRNA expression profiles and illustrate the complexity of bacterial gene regulation in response to different environmental stress conditions. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-385) contains supplementary material, which is available to authorized users.

Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.

The endogenous metabolite, 2-methoxyestradiol (2ME), is an inhibitor of tubulin polymerization and is therefore toxic to dividing fast-growing tumor cells. Transformed cells are not equally susceptible to the effects of 2ME. In this study the effects of 1-2 microM doses of 2ME on cell cycle progression, apoptosis induction and on p53 levels were evaluated using flow cytometry in cells with different p53 status. No effect of 2ME was seen in normal human skin fibroblast strain HSF43 with wild-type (wt) p53. However, in SV40 T antigen transformed HSF43 cells (line E8T4), 2ME caused a prominent G2/M arrest, with subsequent micronuclei formation followed by apoptosis. Increased p53 levels were present in the G2/M cells. Our results suggest that 2ME, being a microtubule poison, may release the bound p53 from T antigen, and that this p53 may enhance the apoptotic effects. Two lymphoblast cell lines derived from the same donor, TK6, expressing low levels of wt p53, and WTK1, expressing high levels of mutant p53, showed similar moderate responses to 2ME at 37 degrees C. The effects included enhanced apoptosis and a modest G2/M block. No increase in p53 levels was seen. However, at the permissive temperature of 30 degrees C marked increases in apoptosis and a prominent G2/M-phase block, similar to that seen in the E8T4 cells, were present in the WTK1 cells, indicating that the high levels of mutant p53 have now become functional, enhancing the apoptotic effects initiated by 2ME.

The objective of this study is to determine whether DNA signature recovery of Bacillus anthracis strains from different environmental substrates correlates with pathogen cell surface hydrophobicity and induction of host cell death. We compared recovery of DNA signatures from a panel of B. anthracis strains collected from two environmental substrates, non-porous surfaces and soil, using real-time qPCR. We further assessed both cell surface hydrophobicity of the B. anthracis strains by contact angle measurements and host cell viability in response to B. anthracis infection in a mouse macrophage cell model system. Our studies demonstrated correlation between reduced B. anthracis sample recovery from environmental substrates and increased cell surface hydrophobicity. Surprisingly, the most hydrophilic strain, K4596, which exhibited the highest level of recovery from the environmental surfaces, induced the highest level of host cell cytotoxicity compared to more hydrophobic B. anthracis strains in the panel. Our results suggest that cell surface hydrophobicity may play a leading role in mediating pathogen adherence to environmental surfaces. These findings can contribute to the optimization of pathogen detection efforts by understanding how bacterial parameters such as hydrophobicity and induction of host cell death affect bacterial adherence to environmental surfaces.

There was an error in the by-line in the online version of the article. The sixth author, Peter Pavlik, was mistakenly excluded.

A total of 41 Clostridium botulinum serotype E strains from different geographic regions, including Canada, Denmark, Finland, France, Greenland, Japan, and the United States, were compared by multilocus sequence typing (MLST), amplified fragment length polymorphism (AFLP) analysis, variable-number tandem-repeat (VNTR) analysis, and botulinum neurotoxin ( bont ) E gene sequencing. The strains, representing environmental, food-borne, and infant botulism samples collected from 1932 to 2007, were analyzed to compare serotype E strains from different geographic regions and types of botulism and to determine whether each of the strains contained the transposon-associated recombinase rarA , involved with bont / E insertion. MLST examination using 15 genes clustered the strains into several clades, with most members within a cluster sharing the same BoNT/E subtype (BoNT/E1, E2, E3, or E6). Sequencing of the bont / E gene identified two new variants (E7, E8) that showed regions of recombination with other E subtypes. The AFLP dendrogram clustered the 41 strains similarly to the MLST dendrogram. Strains that could not be differentiated by AFLP, MLST, or bont gene sequencing were further examined using three VNTR regions. Both intact and split rarA genes were amplified by PCR in each of the strains, and their identities were confirmed in 11 strains by amplicon sequencing. The findings suggest that (i) the C. botulinum serotype E strains result from the targeted insertion of the bont / E gene into genetically conserved bacteria and (ii) recombination events (not random mutations) within bont / E result in toxin variants or subtypes within strains.

Aim: We will validate sample collection methods for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. Methods and Results: We evaluated the sample recovery efficiencies of two collection methods – swabs and wipes – for both nonvirulent and virulent strains of Bacillus anthracis and Yersinia pestis from four types of nonporous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than nonvirulent strains. For the two nonvirulent strains, collection efficiency was similar between all four surfaces, albeit B. anthracis Sterne exhibited higher levels of recovery compared to Y. pestis A1122. In contrast, recovery of B. anthracis Ames spores and Y. pestis CO92 from the hydrophilic glass or stainless steel surfaces was generally more efficient compared to collection from the hydrophobic vinyl and plastic surfaces. Conclusions: Our results suggest that surface hydrophobicity may play a role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. Significance and Impact of the Study: These findings contribute to the validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

Petrobactin is the primary siderophore synthesized by Bacillus anthracis str Sterne and is required for virulence of this organism in a mouse model. The siderophore’s biosynthetic machinery was recently defined and gene homologues of this operon exist in several other Bacillus strains known to be mammalian pathogens, but are absent in several known to be harmless such as B. subtilis and B. lichenformis. Thus, a common hypothesis regarding siderophore production in Bacillus species is that petrobactin production is exclusive to pathogenic isolates. In order to test this hypothesis, siderophores produced by 106 strains of an in-house library of the Bacillus cereus sensu lato group were isolated and identified using a MALDI-TOF-MS assay. Strains were selected from a previously defined phylogenetic tree of this group in order to include both known pathogens and innocuous strains. Petrobactin is produced by pathogenic strains and innocuous isolates alike, and thus is not itself indicative of virulence.

T-cell lymphocyte populations can be delineated into subsets based on expression of cell surface proteins that can be measured in peripheral blood by monoclonal antibodies and flow cytometry percentages of the lymphocyte subpopulations. In order to accurately assess immunocompetence in birds, natural variability in both avian immune function and the methodology must be understood. Our objectives were to (1) further develop flow cytometry for estimating subpopulations of lymphocytes in peripheral blood from poultry, (2) estimate repeatability and variability in the methodology with respect to poultry in a free-range and environmentally diverse situation, and (3) estimate the best antibody and cell marker combination for estimating lymphocyte subpopulations. This work demonstrated the repeatability of using flow cytometry for measurements of peripheral blood in chickens using anti-chicken antibodies for lymphocyte subpopulations. Immunofluorescence staining of cells isolated from peripheral blood revealed that the CD3(+) antibodies reacted with an average of approximately 12-24% of the lymphoid cells in the blood, depending on the fluorescence type. The CD4(+) and CD8(+) molecules were expressed in a range of 4-31% and 1-10% of the lymphoid cells in the blood, respectively. Both fluorescence label and antibody company contribute to the variability of results and should be considered in future flow cytometry studies in poultry.

Chronic beryllium disease (CBD) appears to arise from a combination of both exposure and genetic risk factors. A distinguishing feature of CBD is beryllium hypersensitivity, which can be measured in vitro by a lymphocyte proliferation test. The objective of this study was to determine whether certain allelic variations of the HLA-DPB1 gene, which had been observed previously in CBD, could be found in a group of individuals having beryllium hypersensitivity, but no symptoms of CBD. A flow cytometry-based Lymphocyte Proliferation Test combined with immunophenotyping (Immuno-LPT) was used to detect CD4+ and CD8+ T cell proliferation in response to in vitro stimulation with beryllium. The HLA-DPB1 haplotypes of the same individuals were determined by automated DNA sequencing. Twenty-two out of 25 beryllium-sensitive, non-CBD individuals were found to be carriers of the HLA-DPB1 gene having a substitution of a glutamic acid at position 69 in Exon 2 (Glu69), and a significantly high percentage (24%) were Glu69 homozygotes. Most of the CD4+ responders on the Immuno-LPT (10/14) carried rare, non-*0201 Glu69 DPB1 alleles; while most of the non-CD4+ responders (9/11) were common Glu69 carriers (*0201 or *0202) or non-Glu69 individuals (non-Glu69/non-Glu69). This is the first direct evidence that HLA-DP genotype is linked to a phenotypic response that occurs in beryllium sensitization in the absence of clinical CBD.

Measurement of lymphocyte proliferation to detect hypersensitivity to beryllium (Be-LPT) in vitro is done presently using a method based on tritiated thymidine incorporation. Although this method is sensitive it gives no information on cell viability or responding lymphocyte subsets. We have developed reliable and simple flow cytometric assays for lymphocyte proliferation testing (Immuno-Be-LPT) by combining immunophenotyping with bromodeoxyuridine (BrdU) incorporation or DNA content using propidium iodide (PI) or 4'6'-diimidazolin-2-phenylindole (DAPI). Evaluation of beryllium-induced lymphocyte proliferation in blood cells from seven patients with chronic beryllium disease (CBD) and 120 beryllium workers by both the Bc-LPT and the Immuno-Be-LPT showed agreement between the tests. The Immuno-Bc-LPT provided additional information about the specific type of lymphocytes responding. CD4+ lymphocytes proliferated in response to beryllium in blood samples from all seven CBD individuals and CD8+ lymphocytes proliferated in six of the seven. Four beryllium workers without CBD had positive responses to beryllium primarily in the CD8+ cells. The use of the individual's own plasma supported a greater beryllium or tetanus-induced proliferation of CD4+ lymphocytes when compared to commercial human serum. The response of CD4+ lymphocytes measured in the Immuno-Be-LPT may provide a new marker for the diagnosis of CBD.

The effects of arachidonic acid (AA) and gamma-linolenic acid (GLA) on cell cycle progression and apoptosis induction, using flow cytometry, were compared on normal human skin fibroblasts, strain HSF43 with wild type (wt) p53, large T antigen transformed HSF43 cells (line E8T4) with non functional p53, and on two lymphoblast cell lines, TK6 with wt p53 and WTK1 with mutant p53. AA and GLA caused similar dose (50, 75 and 100 μg/ml AA and GLA) and time dependent (24 and 48 h) induction of apoptosis in each cell line. The degrees of the response of the four cell lines were, however, different. The normal HSF43 cells were most resistant against apoptosis induction and the WTK1 cells most susceptible. The apoptosis induction appeared to be independent of functional p53. Cell cycle progression was also similarly affected by AA and GLA in the two cell types. In the fibroblast type cells (HSF43 and E8T4) S- and G 2 /M-phase arrests were evident after 48 h exposure to AA and GLA, and in the lymphoblast cell lines (TK6 and WTK1) the cells were arrested in the G 1 -phase.