The Potential of TaqMan Array Cards for Detection of Multiple Biological Agents by Real-Time PCR

The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis) were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result) were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels)

Börjeson–Forssman–Lehmann syndrome: Delineating the clinical and allelic spectrum in 14 new families

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability syndrome caused by variants in the PHF6 gene. We ascertained 19 individuals from 15 families with likely pathogenic or pathogenic PHF6 variants (11 males and 8 females). One family had previously been reported. Six variants were novel. We analysed the clinical and genetic findings in our series and compared them with reported BFLS patients. Affected males had classic features of BFLS including intellectual disability, distinctive facies, large ears, gynaecomastia, hypogonadism and truncal obesity. Carrier female relatives of affected males were unaffected or had only mild symptoms. The phenotype of affected females with de novo variants overlapped with the males but included linear skin hyperpigmentation and a higher frequency of dental, retinal and cortical brain anomalies. Complications observed in our series included keloid scarring, digital fibromas, absent vaginal orifice, neuropathy, umbilical hernias, and talipes. Our analysis highlighted sex-specific differences in PHF6 variant types and locations. Affected males often have missense variants or small in-frame deletions while affected females tend to have truncating variants or large deletions/duplications. Missense variants were found in a minority of affected females and clustered in the highly constrained PHD2 domain of PHF6. We propose recommendations for the evaluation and management of BFLS patients. These results further delineate and extend the genetic and phenotypic spectrum of BFLS

Whole genome transcriptomics reveals global effects including up-regulation of Francisella pathogenicity island gene expression during active stringent response in the highly virulent Francisella tularensis subsp. tularensis SCHU S4

During conditions of nutrient limitation bacteria undergo a series of global gene expression changes to survive conditions of amino acid and fatty acid starvation. Rapid reallocation of cellular resources is brought about by gene expression changes coordinated by the signalling nucleotides’ guanosine tetraphosphate or pentaphosphate, collectively termed (p)ppGpp and is known as the stringent response. The stringent response has been implicated in bacterial virulence, with elevated (p)ppGpp levels being associated with increased virulence gene expression. This has been observed in the highly pathogenic Francisella tularensis sub spp. tularensis SCHU S4, the causative agent of tularaemia. Here, we aimed to artificially induce the stringent response by culturing F. tularensis in the presence of the amino acid analogue L-serine hydroxamate. Serinehydroxamate competitively inhibits tRNAser aminoacylation, causing an accumulation of uncharged tRNA. The uncharged tRNA enters the A site on the translating bacterial ribosome and causes ribosome stalling, in turn stimulating the production of (p)ppGpp and activation of the stringent response. Using the essential virulence gene iglC, which is encoded on the Francisella pathogenicity island (FPI) as a marker of active stringent response, we optimized the culture conditions required for the investigation of virulence gene expression under conditions of nutrient limitation. We subsequently used whole genome RNA-seq to show how F. tularensis alters gene expression on a global scale during active stringent response. Key findings included up-regulation of genes involved in virulence, stress responses and metabolism, and down-regulation of genes involved in metabolite transport and cell division. F. tularensis is a highly virulent intracellular pathogen capable of causing debilitating or fatal disease at extremely low infectious doses. However, virulence mechanisms are still poorlyunderstood. The stringent response is widely recognized as a diverse and complex bacterial stress response implicated in virulence. This work describes the global gene expression profile of F. tularensis SCHU S4 under active stringent response for the first time. Herein we provide evidence for an association of active stringent response with FPI virulence gene expression. Our results further the understanding of the molecular basis of virulence and regulation thereof in F. tularensis. These results also support research into genes involved in (p)ppGpp production and polyphosphate biosynthesis and their applicability as targets for novel antimicrobials

Biochemical and structural characterization of polyphosphate kinase 2 from the intracellular pathogen Francisella tularensis

The metabolism of polyphosphate is important for the virulence of a wide range of pathogenic bacteria and the enzymes of polyphosphate metabolism have been proposed as an antibacterial target. In the intracellular pathogen Francisella tularensis , the product of the gene FTT1564 has been identified as a polyphosphate kinase from the PPK2 family. The isogenic deletion mutant was defective for intracellular growth in macrophages and was attenuated in mice, indicating an important role for polyphosphate in the virulence of Francisella . Herein we report the biochemical and structural characterization of F. tularensis polyphosphate kinase ( Ft PPK2) with a view to characterizing the enzyme as a novel target for inhibitors. Using an HPLC based activity assay the substrate specificity of Ft PPK2 was found to include purine but not pyrimidine nucleotides. The activity was also measured using 31P NMR. Ft PPK2 has been crystallized and the structure determined to 2.23 Å resolution. The structure consists of a 6- stranded parallel ? sheet surrounded by 12 ? helices, with a high degree of similarity to other members of the PPK2 family and the thymidylate kinase superfamily. Residues proposed to be important for substrate binding and catalysis have been identified in the structure, including a lid-loop and the conserved Walker A and B motifs. The ?FTT1564 strain showed significantly increased sensitivity to a range of antibiotics in a manner independent of the mode of action of the antibiotic. This combination of biochemical, structural and microbiological data provide a sound foundation for future studies targeting the development of PPK2 small molecule inhibitors

Schematic representation of detection of 100 fg of <i>Y. pestis</i> DNA in seven Array Card channels across one Array Card.

a<p>Numbered PCR represents assays evaluated in an alternate study. Named PCRs this study.</p>b<p>C_T value: PCR cycle number at which fluorescence first detected. # = negative result.</p

Summary of PCR results from Array Card and Singleplex PCR experiments.

a<p><i>B. mallei</i> genome calculation performed using ATCC 23344 genome size. <i>B. pseudomallei</i> calucalation using 1106B genome size. Other genome calculations performed using genome sizes of stated strains.</p>b<p>No. of PCR positives from <i>n</i> replicates. nt - Not tested.</p>c<p>C_T value: PCR cycle number at which fluorescence first detected. Mean of positives only. Variance of mean in paranthesis.</p>d<p>Detection rate defined as at least one agent PCR positive replicate in the Array Card channel.</p

Quantification of B. anthracis (Ames) DNA by pXO2 MGB PCR replicates incorporated within the TaqMan Array Cards.

<p>Trendline and R2 value generated from mean CT values by Microsoft Excel.</p