Genomic Expression Analysis Reveals Strategies of Burkholderia cenocepacia to Adapt to Cystic Fibrosis Patients' Airways and Antimicrobial Therapy

Pulmonary colonization of cystic fibrosis (CF) patients with Burkholderia cenocepacia or other bacteria of the Burkholderia cepacia complex (Bcc) is associated with worse prognosis and increased risk of death. During colonization, the bacteria may evolve under the stressing selection pressures exerted in the CF lung, in particular, those resulting from challenges of the host immune defenses, antimicrobial therapy, nutrient availability and oxygen limitation. Understanding the adaptive mechanisms that promote successful colonization and long-term survival of B. cenocepacia in the CF lung is essential for an improved therapeutic outcome of chronic infections. To get mechanistic insights into these adaptive strategies a transcriptomic analysis, based on DNA microarrays, was explored in this study. The genomic expression levels in two clonal variants isolated during long-term colonization of a CF patient who died from the cepacia syndrome were compared. One of the isolates examined, IST439, is the first B. cenocepacia isolate retrieved from the patient and the other isolate, IST4113, was obtained three years later and is more resistant to different classes of antimicrobials. Approximately 1000 genes were found to be differently expressed in the two clonal variants reflecting a marked reprogramming of genomic expression. The up-regulated genes in IST4113 include those involved in translation, iron uptake (in particular, in ornibactin biosynthesis), efflux of drugs and in adhesion to epithelial lung tissue and to mucin. Alterations related with adaptation to the nutritional environment of the CF lung and to an oxygen-limited environment are also suggested to be a key feature of transcriptional reprogramming occurring during long-term colonization, antibiotic therapy and the progression of the disease

Industrial scale high-throughput screening delivers multiple fast acting macrofilaricides.

Nematodes causing lymphatic filariasis and onchocerciasis rely on their bacterial endosymbiont, Wolbachia, for survival and fecundity, making Wolbachia a promising therapeutic target. Here we perform a high-throughput screen of AstraZeneca's 1.3 million in-house compound library and identify 5 novel chemotypes with faster in vitro kill rates (<2 days) than existing anti-Wolbachia drugs that cure onchocerciasis and lymphatic filariasis. This industrial scale anthelmintic neglected tropical disease (NTD) screening campaign is the result of a partnership between the Anti-Wolbachia consortium (A∙WOL) and AstraZeneca. The campaign was informed throughout by rational prioritisation and triage of compounds using cheminformatics to balance chemical diversity and drug like properties reducing the chance of attrition from the outset. Ongoing development of these multiple chemotypes, all with superior time-kill kinetics than registered antibiotics with anti-Wolbachia activity, has the potential to improve upon the current therapeutic options and deliver improved, safer and more selective macrofilaricidal drugs

<i>Parvalbumin</i> (<i>PV</i>) specialization in dorsal arcopallial (dA) and nidopallial (dAN) nuclei in female downy woodpeckers, as well as 2 other woodpecker species.

(A, B) Female downy woodpecker. (C, D) Male red-bellied woodpecker. (E, F) Male hairy woodpecker. Shown are representative in situ hybridization microscope images (inverted black and white colormetric) of PV expression (white), with fast red as a counterstain (grey). Nido, nidopallium; Meso, mesopallium; Arco, arcopallium; Ot, optic tectum. Scale bars = 500 μm. White dashed lines in images indicate boundaries for different telencephalic regions. White dashed lines in high-magnification images indicate boundaries for different telencephalic regions (e.g., boundary between nidopallium and mesopallium), whereas blue dashed lines indicate specialized PV regions identified in woodpeckers. Photo credits: Female downy woodpecker from Ken Thomas (Public Domain via WikiMedia); male red-bellied woodpecker from Neal Lewis (Public Domain Mark 1.0 via Flickr); and male hairy woodpecker from David Whelan (Public Domain via WikiMedia).</p

Identification of forebrain structures with specialized <i>parvalbumin</i> (<i>PV</i>) mRNA expression.

(A-F) Representative radioactive in situ hybridization microscope images of PV mRNA in species representing 8 different avian orders (see S1 Fig for penguin and emu). PV-rich forebrain nuclei were present in only (E) vocal learning hummingbirds (positive control) and (F) downy woodpeckers. (Ei-iii) High magnification of 3 telencephalic “song control” nuclei in hummingbirds. (Fi) High magnification of the woodpecker drumming nucleus of the anterior nidopallium (dAN); and (Fii) drumming nucleus of arcopallium (dA). Each scale bar is equal to 2 mm. Neuroanatomical markers shown in “A” are as follows: Hyper, hyperpallium; Meso, mesopallium; Nido, nidopallium; GP, globus pallidus; T, Thalamus; Ot, optic tectum; St, striatum; Arco, arcopallium; Ento, entopallium. White dashed lines in high-magnification images (Ei-iii and Fi-ii) indicate boundaries for different telencephalic regions (e.g., boundary between nidopallium and mesopallium), whereas blue dashed lines indicate specialized PV regions identified in Anna’s hummingbirds and downy woodpeckers. Image credits: flamingo from Wilfredo Rodríguez; turaco from Edelmauswaldgeist; duck from Orso della campagna e Papera dello stagno; hawk from Cheva; hummingbird from Stickpen, and downy woodpecker from Greg Schechter. All image licenses: CC Public Domain via WikiMedia.</p

<i>Parvalbumin</i> (<i>PV</i>) mRNA staining with radioactive in situ hybridization across species (Anna’s hummingbird, <i>Calypte anna</i>; Downy woodpecker, <i>Dryobates pubescens</i>; Harris hawk, <i>Parabuteo unicinctus</i>; American flamingo, <i>Phoenicopterus ruber</i>; Red-rested turaco, <i>Tauraco erythrolophus</i>; Domestic duck, <i>Anas platyrhynchos domesticus</i>; Emu, <i>Dromaius novaehollandiae</i>; Humbolt penguin, <i>Spheniscus humboldti</i>).

PV expression present in a brain area is denoted by a plus (+) sign; PV absent in a brain area is denoted by a minus (–) sign. (DOCX)</p

Movement and hearing-induced IEG expression in telencephalic regions of woodpeckers.

(A-B) Schematic diagrams of sagittal brain sections for (A) lateral and (B) medial brain nuclei. (C) Violin plots (horizontal line denotes group median) illustrating significant differences in Arc mRNA expression in 3 movement related brain regions (AI: F3,14 = 3.99, p = 0.03, AN: F3,14 = 3.83, p = 0.03, AM: F3,14 = 10.50, p Arc expression in the (D) AI, (F) AN, or (H) AM (all p-values > 0.05). No correlation was detected between the total number of measured behaviors (drums, whinny calls, and flights) produced during the STI and Arc expression in the (E) AI (p > 0.05), but significant positive correlations were detected in the (G) AN and (I) AM (both p-values p Arc mRNA in putative drumming nuclei (e.g., dA (t13 = 3.10, p 13 = 3.73, p Arc expression (white) with fast red as a counterstain (grey) in a downy woodpecker that was (L) passively caught or (m) a bird that drummed while listening to a drumming playback during the STI. (N) Violin plots (horizontal line denotes group median) illustrating significant differences in Arc mRNA expression in 2 auditory forebrain nuclei (CMM: F3,13 = 4.91, p = 0.02; NCM: F3,13 = 4.05, p = 0.03) of downy woodpeckers caught after hearing playbacks of drumming and either being silent in response or producing different behaviors. Data for C-K and N can be found in S2 Data.</p

Parvalbumin (PV) specialization in the male and female downy woodpecker arcopallium and nidopallium.

(A-D) Representative PV mRNA expression (green) from fluorescent in situ hybridization experiments at low-magnification (tile scan) and (Ai and Ci) high-magnification illustrations of neuroanatomical regions with PV up-regulation in the male downy woodpecker brain. (E and F) Representative PV mRNA staining in the DLN, dAN, and dA of a female downy woodpecker. Blue signal is a DAPI nuclear stain. All scale bars are 1 mm. Asterisks (*) indicate folds on tissue. Photo credits: male downy woodpecker from Greg Schechter, and female downy woodpecker from Ken Thomas (CC Public Domain via WikiMedia). (TIF)</p

Assessing striatal specializations and markers for the woodpecker arcopallium.

Two markers, (A) ETV1 and (B) Lhx9 (radioactive in situ hybridization), were used to delineate the boundary of the arcopallium and nidopallium. (C-F) Representative in situ hybridization images (inverted black and white colormetric) of (C and D) FoxP1 and (E and F) RGS12 in zebra finch and downy woodpecker. Both genes are significantly enriched in the zebra finch Area X (striatal nucleus); however, neither demarcates a specialized region within the woodpecker striatum. FoxP1 allows for the clear delineation of all nidopallial-striatal boundaries. Both reveal similar patterns to zebra finches (see [23]). Data from Lhx9 was collected through radioactive in situ hybridization (see Materials and methods for details). Hyp: hyperpallium; arco: arcopallium; nido: nidopallium; Meso: mesopallium. Scale bar, 500 μm. (TIF)</p

Parvalbumin cell count data.

(CSV)</p

Coronal plane of the forebrain structures in male budgerigars (parrot) and downy woodpecker illustrating specialized <i>parvalbumin</i> (<i>PV</i>) mRNA expression, with such expression patterns absent in a Harris hawk.

(A) Representative radioactive in situ hybridization of microscope images of PV mRNA, in budgerigar (parrot) pallial song nuclei (NAO core, AAC core and shell, and NLC core and shell). Sections modified from Chakraborty and colleagues (2015) with permission from Dr. Jarvis, who is also an author on the current paper. (B) Coronal sections of the woodpecker brain showing the analogous locations for dAN in the anterior nidopallium and dNA in the arcopallium. (C) Comparable coronal sections in a Harris hawk that show PV expression in many positive control areas (see S1 Table). However, unlike the parrot and woodpecker, there was no specialized expression in the arco- or nidopallium. The in situs have cresyl violet as a counter stain. (TIFF)</p