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  current news   Press   selected story    
     
  23 March 2012  
 

Congratulations to IMCB’s recent PhD graduates

 
 



(To view more about each student's thesis, please click on his/her name in the image above)

PhD Graduate: Jasmine LEE
Thesis Title: Non-ribosomal peptide-based quorum sensing signal in Pseudomonas aeruginosa

Abstract
The quorum sensing (QS) network in Pseudomonas aeruginosa plays a key role in the coordination of virulence factors production and contributes to its pathogenesis. Since the las system is at the top of the QS hierarchy, knocking it out would be akin to paralyzing the intercellular communication network and disarming the pathogen. However, P. aeruginosa clinical strains isolated from chronically infected cystic fibrosis patients were often found to be bearing a defective las system, yet remain profligate producers of virulence factors such as pyocyanin and elastase. Further, under phosphate limitation conditions, a las-independent activation of the rhl QS system and pyocyanin production could be observed. This lead us to question whether there exists an alternative intercellular communication circuit that functions independent of LasIR, and whose effects are only apparent under non-standard, stressful environmental conditions, the very same conditions that P. aeruginosa is subjected to during infections. Therefore by random transposon mutagenesis, we screened for mutants with alterations in production of pyocyanin under low phosphate condition, and isolated a mutant disrupted at qrpA, a putative non-ribosomal peptide synthase gene. Subsequent in-frame deletion of qrpA and in trans complementation of the latter confirmed that QrpA positively affects the production of virulence factors pyocyanin and elastase, PQS and the rhl quorum sensing systems, and contributes to the full virulence of the bacteria towards nematodes C. elegans and zebrafish D. rerio. We have purified the putative enzyme product of the QrpA NRPS cluster and named it Qrp (quinolone regulating peptide). When exogenously added, only 10 nM of Qrp is required to restore PQS production levels and its dependent phenotypes back to twice that of wild type levels. Further, we discovered that the ferric-binding siderophore pyochelin functions as a downstream Qrp-dependent signaling intermediate and forms a tripartite relay between Qrp and PQS, with a similar minimum effective concentration of 10 nM. Lastly, we also assayed the applicability of Qrp as a las-independent activator of quorum sensing and QS-dependent phenotypes in non-standard conditions. During phosphate depletion, the ΔlasIΔqrpA double mutant shows an overall downregulation in virulence genes expression and decreases in pyocyanin, elastase and PQS production compared to the ?lasI strain, signifying its positive effects on quorum sensing systems downstream of las. The dramatic upregulation of qrpA expression observed in P. aeruginosa clinical isolates further implies that QrpA/ Qrp could represent a formidable force in driving forward the expression of quorum sensing-regulated virulence factors in the context where quorum sensors become defunct.

 
 


 
 

Figure Legend: Novel las-independent quorum sensing network of P. aeruginosa involving Qrp. Abbreviations: R, putative receptor for Qrp.

For more information on Lianhui ZHANG's Lab, please click here.


 
 


PhD Graduate: William GO
Thesis Title: The zebrafish plasma membrane Ca2+ pump Atp2b1a acts in the development of mechanoreceptors and early bone calcification.

Abstract
Tol2 enhancer-trap (ET) transgenic line is widely used for studies of development and diseases. This transposable element uses conservative ("cut and paste") transposition mechanism; meaning it is excised from the donor site when the whole element moves to the new position. Enhanced green fluorescent protein (EGFP) was used as a reporter to fully utilize the nature of live zebrafish embryos 'optical clarity. When random insertion occurred within the vicinity of an enhancer present in the genome, enhancer is metaphorically "trapped" to activate reporter gene expression even at considerable distance in an orientation-independent manner and ideally, in a tissue specific manner. In this work, I have focused on one such transgenic line, due to its untapped potential in representing its trapped gene. SqET4 is the ET line, where EGFP expression takes place in mechanosensory hair cells (Parinov et al., 2004; Choo et al., 2006). The "Achilles' heel" of the SqET4 line, however, is the lack of information about the gene(s) expression pattern it mimics, and therefore the current deficit in understanding the developmental process reflected by EGFP expression in vivo. We showed that it represents Tg:atp2b1a-EGFP. This provided a possibility to study a function of Atp2b1a/PMCA1, a plasma membrane Ca2+ ATPase mainly required by cells for Ca2+ extrusion. Combining the morpholino mediated-knockdown and small molecule inhibition, I showed that deficiency of Atp2b1a results in auditory and vestibular defects due to defects of Ca2+ export by mechanoreceptors. Acting downstream of the transcriptional regulator Atoh1a, Atp2b1a plays a crucial role in division of terminally determined mechanoreceptor progenitors. Serendipitously, another domain of EGFP/atp2b1a expression was identified in the ultimobranchial bodies (UB) of SqET4. In mammals UB give rise to the parafollicular cells of the thyroid gland, which is the main source of a blood Ca2+-reducing hormone, calcitonin. Since in zebrafish UB remains a separate organ away from thyroid lobes, SqET4 has concurrently became a useful tool to study both the anatomy and function of during UB's development. In particular, a novel connection between UB and developing pharyngeal dentition and function of Atp2b1a during bones calcification was demonstrated.

 
 


 
 

Figure Legend: Atp2b1a is required for Ca2+ extrusion from mechanosensory hair cell.
Calcium Crimson AM (CC) was used as an in vivo indicator of intracellular Ca2+ level in SqET4 transgenics. Ca2+ is predominantly localized within the cupula. (Ai & Aii) Orthogonal sections of control L1 neuromast (55 hpf; hours post fertilization); (Aiii) 3D projection (42º along Y-axis) of the same neuromast depicting the spread of Ca2+ along cupula. (Bi & Bii) Orthogonal sections of atp2b1a morphant L1 neuromast (55 hpf), (Biii) 3D projection of the same neuromast. (Ci & Cii) Orthogonal sections of 5(6)-Carboxyeosin-treated (CE; Atp2b1a inhibitor of Ca2+ clearance treatment) L1 neuromast (55 hpf), (Ciii) 3D projection of the same neuromast. Green and red box indicates plane of orthogonal section; blue box indicates z-depth of the confocal stack. In controls, intracellular Ca2+ is kept low. (Di & Dii) Orthogonal sections of a neuromast after atp2b1a mRNA rescue. (Diii) 3D projection of the neuromast. (E) Intensity profiling (x-axis: measured distance (μm) versus y-axis: florescence intensity) of EGFP v. CC in 5 dpf control L1 neuromast across two planes of measurement (white dashed lines). Within hair cells CC was detected only at low level (Ei). The intensity of CC peaks (Eii) at the level of the stereocilia. (F) In morphants intracellular Ca2+ is high (3-fold difference). (Fi, Fii) Intensity profiling of EGFP v. CC in 5 dpf morphant L1 neuromast across the plane of measurement. The intensity of CC at the level of hair cells is high. (N = 10). Intensity profiling of EGFP v. CC in 5 dpf 5(6)-carboxyeosin (CE) treated embryo (Gi, Gii). The intensity of CC at the level of hair cells is high. (N = 18). (Hi, Hii) Intensity profiling of EGFP v. CC in 5 dpf atp2b1a mRNA rescued-morphant L1 neuromast across two planes of measurement. CC was low within hair cells (Hi). The intensity of CC peaks (Hii) at the level of stereocilia. (N = 10). (I-L) The integrity of stereocilia of L1 neuromast for control and morphant larvae was assessed by anti-acetylated tubulin antibody (green) which labels kinocilium, and Alexa Fluor 635 phalloidin (red) which marks actin in the stereocilia. Length of kinocilium was negatively affected by atp2b1a knockdown and CE treatment. Ii, Ji, Ki & Li shows merged signals of kinocilium and actin-rich cuticular plate. Iii, Jii, Kii & Lii are enlarged images of actin signal (white dashed box), revealing reduced number of actin-rich cuticular plates and lack of hair cell polarity as seen in control and rescued larvae (yellow dashed line). Scale bar at 5 μM.

This part of the study was published in Cell Calcium (2010) 48:302-313.

For more information on Vladimir KORZH's Lab, please click here.