Detection of tuberculosis in cynomolgus macaques (Macaca fascicularis) using a supplementary Monkey Interferon Gamma Releasing Assay (mIGRA).

Cynomolgus monkeys (Macaca fascicularis; MF) are commonly used as nonhuman primate models for pharmaceutical product testing. In their habitat range, monkeys have close contact with humans, allowing the possibility of bidirectional transmission of tuberculosis (TB) between the two species. Although the intradermal tuberculin skin test (TST) is used for TB detection in MF, it has limitations. Herein, we established the mIGRA, combining human QuantiFERON-TB Gold-Plus and monkey IFN-γ ELISApro systems, and used it to investigate 39 captive MF who were cage-mates or lived in cages located near a monkey who died from the naturally TB infection. During a 12-month period of study, 14 (36%), 10 (26%), and 8 (21%) monkeys showed TB-positive results using the mIGRA, the TST, and TB culture, respectively. Among the 14 mIGRA-positive monkeys, 8 (57.1%) were TST-positive and 7 (50%) were culture-positive, indicating early TB detection in the latent and active TB stages with the mIGRA. Interestingly, 3 (37.5%) of the TST-negative monkeys were culture-positive. Our study showed that the mIGRA offers many advantages, including high sensitivity and high throughput, and it requires only one on-site visit to the animals. The assay may be used as a supplementary tool for TB screening in MF.

Footprinting with an automated capillary DNA sequencer.

Footprinting is a valuable tool for studying DNA-protein contacts. However, it usually involves expensive, tedious and hazardous steps such as radioactive labeling and analyses on polyacrylamide sequencing gels. We have developed an easy four-step footprinting method involving (i) the generation and purification of a PCR fragment that is fluorescently labeled at one end with 6-carboxyfluorescein; (ii) brief exposure of the fragment to a DNA-binding protein and then DNase I; (iii) spin-column purification; and (iv) analysis of partial digestion products on the ABI Prism 310 capillary DNA sequencer/genetic analyzer. Very detailed and sensitive footprints of large (> 400 bp) DNA fragments can be easily obtained, as illustrated by our use of this method to characterize binding of PhcA, a LysR-type activator, to two sites greater than 100 bp apart in the 5' untranslated region of xpsR, one of its regulated target genes. The advantages of this new method are that it (i) uses long-lived, safe and easy-to-make fluorescently labeled target fragments; (ii) uses sensitive, robust and highly reproducible fragment analysis using an automated DNA sequencer, instead of gel electrophoresis and autoradiography; and (iii) is cost effective.

Multicomponent transcriptional regulation at the complex promoter of the exopolysaccharide I biosynthetic operon of Ralstonia solanacearum.

High-level transcription of eps, an operon encoding biosynthesis of an exopolysaccharide virulence factor of the phytopathogen Ralstonia (Pseudomonas) solanacearum, requires the products of at least seven regulatory genes (phcA, phcB, xpsR, vsrA-vsrD, and vsrB-vsrC), which are organized in three converging signal transduction cascades. Because xpsR and the vsrB-vsrC two-component system are the most downstream cascade components required for activation of eps, we explored how these components control transcription from the eps promoter (P(eps)). Deletion and PCR mutagenesis identified an upstream region of P(eps) (nucleotides -82 to -62) that is critical for transcription activation by VsrB-VsrC and XpsR and also is required for negative control of P(eps) by the putative eps regulator EpsR. Using PCR mutagenesis we generated the vsrC1 allele that encodes a response regulator that constitutively activates P(eps) in the absence of its cognate sensor, VsrB. However, activation of P(eps) by vsrC1 still required xpsR. Unexpectedly, the amino acid substitution conferring the constitutive phenotype on VsrC1 is 12 residues from its C terminus, outside the known functional domains of response regulators. Finally, a modified DNase I footprinting method was used to demonstrate specific binding of both VsrC1 and VsrC to the -72 to -62 upstream region of P(eps).

Evidence that Ralstonia eutropha (Alcaligenes eutrophus) contains a functional homologue of the Ralstonia solanacearum Phc cell density sensing system.

In the phytopathogen Ralstonia (Pseudomonas) solanacearum, control of many virulence genes is partly mediated by the Phc cell density sensing system. Phc uses a novel self-produced signal molecule [3-hydroxypalmitic acid methyl ester (3-OH PAME)], an atypical two-component system (PhcS/PhcR), and a LysR-type activator (PhcA) to regulate a reversible switching between two different physiological states. While Phc is present in most R. solanacearum strains, it is apparently absent from other pseudomonad plant pathogens and prokaryotic genomes that have been sequenced. Here, we report discovery of a phcA orthologue in the non-pathogenic, facultative chemolithoautotroph Ralstonia eutropha (Alcaligenes eutrophus) that fully complements R. solanacearum phcA mutants. We also demonstrate that some R. eutropha produce an extracellular factor that complements R. solanacearum mutants deficient in production of the 3-OH PAME signal molecule that controls phcA. Additionally, Southern blot hybridization analysis suggested that R. eutropha harbours other Phc components, such as PhcB (a biosynthetic enzyme for 3-OH PAME) and PhcS (a 3-OH PAME-responsive sensor kinase). Analysis of a phcA-null mutant of R. eutropha showed that phcA (and probably Phc) positively activates motility, in contrast to R. solanacearum where it represses motility. Similarly, the R. eutropha phcA mutant was unaffected in siderophore production, whereas inactivation of phcA in R. solanacearum increases siderophore production. Although our data strongly suggest that R. eutropha has a functional Phc-like system and support the phylogeny of Ralstonia, it implies that Phc may have a different physiological and ecological function in R. eutropha.

Joint transcriptional control of xpsR, the unusual signal integrator of the Ralstonia solanacearum virulence gene regulatory network, by a response regulator and a LysR-type transcriptional activator.

Ralstonia (Pseudomonas) solanacearum is a soil-borne phytopathogen that causes a wilting disease of many important crops. It makes large amounts of the exopolysaccharide EPS I, which it requires for efficient colonization, wilting, and killing of plants. Transcription of the eps operon, encoding biosynthetic enzymes for EPS I, is controlled by a unique and complex sensory network that responds to multiple environmental signals. This network is comprised of the novel transcriptional activator XpsR, three distinct two-component regulatory systems (VsrAD, VsrBC, and PhcSR), and the LysR-type regulator PhcA, which is under the control of PhcSR. Here we show that the xpsR promoter (PxpsR) is simultaneously controlled by PhcA and VsrD, permitting XpsR to act like a signal integrator, simultaneously coordinating signal input into the eps promoter from both VsrAD and PhcSR. Additionally, we used in vivo expression analysis and in vitro DNA binding assays with substitution and deletion mutants of PxpsR to show the following. (i) PhcA primarily interacts with a typical 14-bp LysR-type consensus sequence around position -77, causing a sixfold activation of PxpsR; a weaker, less-defined binding site between -183 and -239 likely enhances PhcA binding and activation via the -77 site another twofold. (ii) Full 70-fold activation of PxpsR requires the additional interaction of the VsrD response regulator (or its surrogate) with a 14-bp dyadic sequence centered around -315 where it enhances activation (and possibly binding) by PhcA; however, VsrD alone cannot activate PxpsR. (iii) Increasing the distance between the putative VsrD binding site from that of PhcA by up to 232 bp did not dramatically affect PxpsR activation or regulation.