Characterization of L-Serine Deaminase and its Potential Role in the Pathogenicity of Pseudomonas aeruginosa

All pathogens require high energetic influxes to counterattack the host immune system and without this energy bacterial infections are easily cleared. This study is an investigation into one highly bioenergetic pathway in Pseudomonas aeruginosa involving the amino acid L-serine and the enzyme L-serine deaminase (L-SD). P. aeruginosa is an opportunistic pathogen causing infections in patients with compromised immune systems as well as patients with cystic fibrosis. Recent evidence has linked L-SD directly to the pathogenicity of several organisms including but not limited to Campylobacter jejuni, Mycobacterium bovis, Streptococcus pyogenes, and Yersinia pestis. We hypothesized that P. aeruginosa L-SD is likely to be critical for its virulence. Genome sequence analysis revealed the presence of two L-SD homo logs encoded by sdaA and sdaB. We analyzed the ability of P. aeruginosa to utilize serine and the role of SdaA and SdaB in serine deamination by comparing mutant strains of sdaA (PAOsdaA) and sdaB (PAOsdaB) with their isogenic parent P. aeruginosa P AO 1. We demonstrated that P. aeruginosa is unable to use serine as a sole carbon source. However, serine utilization is enhanced in the presence of glycine and this glycine-dependent induction of L-SD activity requires the inducer serine. The amino acid leucine was shown to inhibit L-SD activity from both SdaA and SdaB and the net contribution to L-serine deamination by SdaA and SdaB was ascertained at 34% and 66 %, respectively. These results suggest that P. aeruginosa LSD is quite different from the characterized E. coli L-SD that is glycine-independent but leucine-dependent for activation. Growth mutants able to use serine as a sole carbon source were also isolated and in addition, suicide vectors were constructed which allow for selective mutation of the sdaA and sdaB genes on any P. aeruginosa strain of interest. Future studies with a double mutant will reveal the importance of these genes for pathogenicity.

Pseudomonas aeruginosa AMPR transcriptional regulatory network

In Enterobacteriaceae, the transcriptional regulator AmpR, a member of the LysR family, regulates the expression of a chromosomal β-lactamase AmpC. The regulatory repertoire of AmpR is broader in Pseudomonas aeruginosa, an opportunistic pathogen responsible for numerous acute and chronic infections including cystic fibrosis. Previous studies showed that in addition to regulating ampC, P. aeruginosa AmpR regulates the sigma factor AlgT/U and production of some quorum sensing (QS)-regulated virulence factors. In order to better understand the ampR regulon, the transcriptional profiles generated using DNA microarrays and RNA-Seq of the prototypic P. aeruginosa PAO1 strain with its isogenic ampR deletion mutant, PAOΔampR were analyzed. Transcriptome analysis demonstrates that the AmpR regulon is much more extensive than previously thought influencing the differential expression of over 500 genes. In addition to regulating resistance to β-lactam antibiotics via AmpC, AmpR also regulates non-β-lactam antibiotic resistance by modulating the MexEF-OprN efflux pump. Virulence mechanisms including biofilm formation, QS-regulated acute virulence, and diverse physiological processes such as oxidative stress response, heat-shock response and iron uptake are AmpR-regulated. Real-time PCR and phenotypic assays confirmed the transcriptome data. Further, Caenorhabditis elegans model demonstrates that a functional AmpR is required for full pathogenicity of P. aeruginosa. AmpR, a member of the core genome, also regulates genes in the regions of genome plasticity that are acquired by horizontal gene transfer. The extensive AmpR regulon included other transcriptional regulators and sigma factors, accounting for the extensive AmpR regulon. Gene expression studies demonstrate AmpR-dependent expression of the QS master regulator LasR that controls expression of many virulence factors. Using a chromosomally tagged AmpR, ChIP-Seq studies show direct AmpR binding to the lasR promoter. The data demonstrates that AmpR functions as a global regulator in P. aeruginosa and is a positive regulator of acute virulence while negatively regulating chronic infection phenotypes. In summary, my dissertation sheds light on the complex regulatory circuit in P. aeruginosa to provide a better understanding of the bacterial response to antibiotics and how the organism coordinately regulates a myriad of virulence factors.

Characterization of the poxAB Operon Encoding a Class D Carbapenemase in Pseudomonas aeruginosa,

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.

Análise da imunoexpressão de OCT4 e CD44 em neoplasias de glândulas salivares menores e maiores

Salivary gland neoplasms exhibit a wide variety of biological behavior and a high morphological diversity raises the interest in researching these lesions. The stem cells are the main source for the generation and maintenance of cell diversity, disorders in the regulation of these cells can lead to the production of altered stem cells, termed cancer stem cells capable of generate the tumor. Researches on cancer stem cells and associated proteins have been developed in some oral cancers; however, their role in salivary gland neoplasms is not well established. Thus, the aim of this study was to identify the tumor parenchyma cells exhibiting stem cell characteristics, by evaluating the immunoreactivity of OCT4 and CD44, in a number of cases of salivary gland neoplasms. The sample consisted of 20 pleomorphic adenomas, 20 mucoepidermoid carcinomas and 20 adenoid cystic carcinoma located in minor and major salivary glands. The expression of OCT4 and CD44 was evaluated by the percentage of positive cells (PP) and the intensity of expression (IE), it is realized the sum of the scores, resulting in the total score immunostaining (PIT) ranging 0-7. All studied cases showed positive expression of OCT4 and CD44 and higher values than the control groups. It was observed that for OCT4 luminal cells and non-luminal were immunostained in the case of pleomorphic adenomas and adenoid cystic carcinoma. Already the immunoreactivity of CD44 was particularly evident in the non-luminal cells of these lesions. In mucoepidermoid carcinomas for both markers, there was immunoreactivity in squamous and intermediate cells and absence of staining mucous cells. For both markers, a statistically significant higher immunostaining was verified in neoplasms located in the major salivary glands compared with lesions in the minor salivary (p<0.001). At the total sample and in the group of minor salivary glands, malignant neoplasms exhibited higher immunoreactivity for OCT4 than pleomorphic adenoma. However, there was no statistically significant difference between the lesions and between their classifications histomorphologic. Analyzing the correlation between OCT4 and CD44 immunoexpressions, a statistically significant moderate positive correlation (r = 0.444) was observed. The high expression of OCT4 and CD44 may indicate that these proteins play an important role in identifying cancer stem cells, allowing a prediction of biological behavior of salivary gland neoplasms.

An investigation of the molecular interactions between statins and bacterial pathogens, and their combined impact on the human immune system

Statins are a class of drug that inhibits cholesterol biosynthesis, and are used to treat patients with high serum cholesterol levels. They exert this function by competitively binding to the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA reductase (HMGR), which catalyses the formation of mevalonate, a rate-limiting step in cholesterol biosynthesis. In addition, statins have what are called “pleiotropic effects”, which include the reduction of inflammation, immunomodulation, and antimicrobial effects. Statins can also improve survival of patients with sepsis and pneumonia. Cystic fibrosis (CF) is the most common recessive inherited disease in the Caucasian population, which is characterised by factors including, but not limited to, excessive lung inflammation and increased susceptibility to infection. Therefore, the overall objective of this study was to examine the effects of statins on CFassociated bacterial pathogens and the host response. In this work, the prevalence of HMGR was examined in respiratory pathogens, and several CF-associated pathogens were found to possess homologues of this enzyme. HMGR homology was analysed in Staphylococcus aureus, Burkholderia cenocepacia and Streptococcus pneumoniae, and the HMGR of B. cenocepacia was found to have significant conservation to that of Pseudomonas mevalonii, which is the most widely-characterised bacterial HMGR. However, in silico analysis revealed that, unlike S. aureus and S. pneumoniae, B. cenocepacia did not possess homologues of other mevalonate pathway proteins, and that the HMGR of B. cenocepacia appeared to be involved in an alternative metabolic pathway. The effect of simvastatin was subsequently tested on the growth and virulence of S. aureus, B. cenocepacia and S. pneumoniae. Simvastatin inhibited the growth of all 3 species in a dose-dependent manner. In addition, statin treatment also attenuated biofilm formation of all 3 species, and reduced in vitro motility of S. aureus. Interestingly, simvastatin also increased the potency of the aminoglycoside antibiotic gentamicin against B. cenocepacia. The impact of statins was subsequently tested on the predominant CF-associated pathogen Pseudomonas aeruginosa, which does not possess a HMGR homologue. Mevastatin, lovastatin and simvastatin did not influence the growth of this species. However, sub-inhibitory statin concentrations reduced the swarming motility and biofilm formation of P. aeruginosa. The influence of statins was also examined on Type 3 toxin secretion, quorum sensing and chemotaxis, and no statin effect was observed on any of these phenotypes. Statins did not appear to have a characteristic effect on the P. aeruginosa transcriptome. However, a mutant library screen revealed that the effect of statins on P. aeruginosa biofilm was mediated through the PvrR regulator and the Cup fimbrial biosynthesis genes. Furthermore, proteomic analysis demonstrated that 6 proteins were reproducibly induced by simvastatin in the P. aeruginosa swarming cells. The effect of statins on the regulation of the host-P. aeruginosa immune response was also investigated. Statin treatment increased expression of the pro-inflammatory cytokines IL-8 and CCL20 in lung epithelial cells, but did not attenuate P. aeruginosa-mediated inflammatory gene induction. In fact, simvastatin and P. aeruginosa caused a synergistic effect on CCL20 expression. The expression of the transcriptional regulators KLF2 and KLF6 was also increased by statins and P. aeruginosa, with the induction of KLF6 by simvastatin proving to be a novel effect. Interestingly, both statins and P. aeruginosa were capable of inducing alternative splicing of KLF6. P. aeruginosa was found to induce KLF6 alternative splicing by way of the type 3 secreted toxin ExoS. In addition, a mechanistic role was elucidated for KLF6 in the lung, as it was determined that statin-mediated induction of this protein was responsible for the induction of the host response genes CCL20 and iNOS. Moreover, statin treatment caused a slight increase in infection-related cytotoxicity, and increased bacterial adhesion to cells. Taken together, these data demonstrate that statins can reduce the virulence of CFassociated bacterial pathogens and alter host response effectors. Furthermore, novel statin effectors were identified in both bacterial and host cells.

Genetic medicines for CF: hype versus reality

Since identification of the CFTR gene over 25 years ago, gene therapy for cystic fibrosis (CF) has been actively developed. More recently gene therapy has been joined by other forms of “genetic medicines” including mRNA delivery, as well as genome editing and mRNA repair-based strategies. Proof-of-concept that gene therapy can stabilize the progression of CF lung disease has recently been established in a Phase IIb trial. An early phase study to assess the safety and explore efficacy of CFTR mRNA repair is ongoing, while mRNA delivery and genome editing-based strategies are currently at the pre-clinical phase of development. This review has been written jointly by some of those involved in the various CF “genetic medicine” fields and will summarize the current state-of-the-art, as well as discuss future developments. Where applicable, it highlights common problems faced by each of the strategies, and also tries to highlight where a specific strategy may have an advantage on the pathway to clinical translation. We hope that this review will contribute to the ongoing discussion about the hype versus reality of genetic medicine-based treatment approaches in CF.

Eradication and phenotypic tolerance of Burkholderia cenocepacia biofilms exposed to atmospheric pressure non-thermal plasma.

Chronic lung infection with bacteria from the Burkholderia cepacia complex (BCC), and in particular B. cenocepacia, is associated with significant morbidity and mortality in patients with cystic fibrosis (CF). B. cenocepacia can spread from person to person and exhibits intrinsic broad-spectrum antibiotic resistance. Recently, atmospheric pressure non-thermal plasmas (APNTPs) have gained increasing attention as a novel approach to the prevention and treatment of a variety of hospital-acquired infections. In this study, we evaluated an in-house-designed kHz-driven plasma source for the treatment of biofilms of a number of clinical CF B. cenocepacia isolates. The results demonstrated that APNTP is an effective and efficient tool for the eradication of B. cenocepacia biofilms but that efficacy is highly variable across different isolates. Determination of phenotypic differences between isolates in an attempt to understand variability in plasma tolerance revealed that isolates which are highly tolerant to APNTP typically produce biofilms of greater biomass than their more sensitive counterparts. This indicates a potential role for biofilm matrix components in biofilm tolerance to APNTP exposure. Furthermore, significant isolate-dependent differences in catalase activity in planktonic bacteria positively correlated with phenotypic resistance to APNTP by isolates grown in biofilms.

Genome-wide association studies in oesophageal adenocarcinoma and Barrett's oesophagus: a large-scale meta-analysis

Background: Esophageal adenocarcinoma (EA) is one of the fastest rising cancers in western countries. Barrett’s Esophagus (BE) is the premalignant precursor of EA. However, only a subset of BE patients develop EA, which complicates the clinical management in the absence of valid predictors. Genetic risk factors for BE and EA are incompletely understood. This study aimed to identify novel genetic risk factors for BE and EA.Methods: Within an international consortium of groups involved in the genetics of BE/EA, we performed the first meta-analysis of all genome-wide association studies (GWAS) available, involving 6,167 BE patients, 4,112 EA patients, and 17,159 representative controls, all of European ancestry, genotyped on Illumina high-density SNP-arrays, collected from four separate studies within North America, Europe, and Australia. Meta-analysis was conducted using the fixed-effects inverse variance-weighting approach. We used the standard genome-wide significant threshold of 5×10-8 for this study. We also conducted an association analysis following reweighting of loci using an approach that investigates annotation enrichment among the genome-wide significant loci. The entire GWAS-data set was also analyzed using bioinformatics approaches including functional annotation databases as well as gene-based and pathway-based methods in order to identify pathophysiologically relevant cellular pathways.Findings: We identified eight new associated risk loci for BE and EA, within or near the CFTR (rs17451754, P=4·8×10-10), MSRA (rs17749155, P=5·2×10-10), BLK (rs10108511, P=2·1×10-9), KHDRBS2 (rs62423175, P=3·0×10-9), TPPP/CEP72 (rs9918259, P=3·2×10-9), TMOD1 (rs7852462, P=1·5×10-8), SATB2 (rs139606545, P=2·0×10-8), and HTR3C/ABCC5 genes (rs9823696, P=1·6×10-8). A further novel risk locus at LPA (rs12207195, posteriori probability=0·925) was identified after re-weighting using significantly enriched annotations. This study thereby doubled the number of known risk loci. The strongest disease pathways identified (P<10-6) belong to muscle cell differentiation and to mesenchyme development/differentiation, which fit with current pathophysiological BE/EA concepts. To our knowledge, this study identified for the first time an EA-specific association (rs9823696, P=1·6×10-8) near HTR3C/ABCC5 which is independent of BE development (P=0·45).Interpretation: The identified disease loci and pathways reveal new insights into the etiology of BE and EA. Furthermore, the EA-specific association at HTR3C/ABCC5 may constitute a novel genetic marker for the prediction of transition from BE to EA. Mutations in CFTR, one of the new risk loci identified in this study, cause cystic fibrosis (CF), the most common recessive disorder in Europeans. Gastroesophageal reflux (GER) belongs to the phenotypic CF-spectrum and represents the main risk factor for BE/EA. Thus, the CFTR locus may trigger a common GER-mediated pathophysiology.

Angiomatoid fibrous histiocytoma: comparison of fluorescence in situ hybridization and reverse transcription polymerase chain reaction as adjunct diagnostic modalities

Angiomatoid fibrous histiocytoma (AFH) is a rare soft tissue neoplasm of intermediate biologic potential and uncertain differentiation, most often arising in the extremities of children and young adults. Although it has characteristic histologic features of a lymphoid cuff surrounding nodules of ovoid cells with blood-filled cystic cavities, diagnosis is often difficult due to its morphologic heterogeneity and lack of specific immunoprofile. Angiomatoid fibrous histiocytoma is associated with recurrent chromosomal translocations, leading to characteristic EWSR1-CREB1, EWSR1-ATF1, and, rarely, FUS-ATF1 gene fusions; fluorescence in situ hybridization (FISH), detecting EWSR1 or FUS rearrangements, and reverse transcription-polymerase chain reaction (RT-PCR) for EWSR1-CREB1 and EWSR1-ATF1 fusion transcripts have become routine ancillary tools. We present a large comparative series of FISH and RT-PCR for AFH. Seventeen neoplasms (from 16 patients) histologically diagnosed as AFH were assessed for EWSR1 rearrangements or EWSR1-CREB1 and EWSR1-ATF1 fusion transcripts. All 17 were positive for either FISH or RT-PCR or both. Of 16, 14 (87.5%) had detectable EWSR1-CREB1 or EWSR1-ATF1 fusion transcripts by RT-PCR, whereas 13 (76.5%) of 17 had positive EWSR1 rearrangement with FISH. All 13 of 13 non-AFH control neoplasms failed to show EWSR1-CREB1 or EWSR1-ATF1 fusion transcripts, whereas EWSR1 rearrangement was present in 2 of these 13 cases (which were histopathologically myoepithelial neoplasms). This study shows that EWSR1-CREB1 or EWSR1-ATF1 fusions predominate in AFH (supporting previous reports that FUS rearrangement is rare in AFH) and that RT-PCR has a comparable detection rate to FISH for AFH. Importantly, cases of AFH can be missed if RT-PCR is not performed in conjunction with FISH, and RT-PCR has the added advantage of specificity, which is crucial, as EWSR1 rearrangements are present in a variety of neoplasms in the histologic differential diagnosis of AFH, that differ in behavior and treatment.

Chronic inflammation in CF airways - a persistent issue for A20

Cystic Fibrosis (CF) is characterised by prolonged and exaggerated airways inflammation. Despite recent developments to overcome the underlying functional defect in CFTR (cystic fibrosis transmembrane conductance regulator), there is still an unmet need to reduce the inflammatory response. The NF-kB regulator A20 is a key target to normalise the inflammatory response and is reduced in CF. Here, we describe the plethora of functions of A20 as they apply to innate immune function within the airways. Pharmacological compounds can enhance A20 mRNA and protein expression, but we observed a blunted effect in CF primary epithelial cells. In CF cells pre-treatment with gibberellic acid (GA3) shows anti-inflammatory effects only in some patients. We show that cells with higher basal p38 expression respond with an increase in pro-inflammatory cytokines. Furthermore, all CF PNECs show increased p38 mRNA when stimulated in the presence of GA3. Our results suggest that those patients may benefit from therapeutics targeting p38.

The temperate Burkholderia phage AP3 of the Peduovirinae shows efficient antimicrobial activity against B. cenocepacia of the IIIA lineage

Burkholderia phage AP3 (vB_BceM_AP3) is a temperate virus of the Myoviridae and the Peduovirinae subfamily (P2likevirus genus). This phage specifically infects multidrug-resistant clinical Burkholderia cenocepacia lineage IIIA strains commonly isolated from cystic fibrosis patients. AP3 exhibits high pairwise nucleotide identity (61.7%) to Burkholderia phage KS5, specific to the same B. cenocepacia host, and has 46.7% - 49.5% identity to phages infecting other species of Burkholderia. The lysis cassette of these related phages has a similar organization (putative antiholin, putative holin, endolysin and spanins) and shows 29-98% homology between specific lysis genes, in contrast to Enterobacteria phage P2, the hallmark phage of this genus. The AP3 and KS5 lysis genes have conserved locations and high amino acid sequence similarity. The AP3 bacteriophage particles remain infective up to 5 h at pH 4-10 and are stable at 60°C for 30 min, but are sensitive to chloroform, with no remaining infective particles after 24 h of treatment. AP3 lysogeny can occur by stable genomic integration and by pseudo-lysogeny. The lysogenic bacterial mutants did not exhibit any significant changes in virulence compared to wild-type host strain when tested in the Galleria mellonella moth wax model. Moreover, AP3 treatment of larvae infected with B. cenocepacia revealed a significant increase (P < 0.0001) in larvae survival in comparison to AP3-untreated infected larvae. AP3 showed robust lytic activity, as evidenced by its broad host range, the absence of increased virulence in lysogenic isolates, the lack of bacterial gene disruption conditioned by bacterial tRNA downstream integration site, and the absence of detected toxin sequences. These data suggest the AP3 phage is a promising potent agent against bacteria belonging to most common B. cenocepacia IIIA lineage strains.

L'impact de la création littéraire, en contexte d'aide, sur la symptomatologie psychophysiologique d'adolescents atteints d'une maladie chronique : étude interdisciplinaire et exploratoire.

Thèse réalisée dans le cadre d'un Ph.D.interdisciplinaire en Psychologie, en création littéraire et en orthopédagogie. L'impact de la création littéraire a été étudié chez des adolescents atteints d'une maladie chronique au CHU Sainte-Justine de Montréal. Cette recherche est exploratoire car la création littéraire n'a jamais été étudiée dans cette perspective. Elle a été réalisée sous la direction de Catherine Mavrikakis, professeure et écrivain à la Faculté des arts et sciences au Département des littératures francophones de l'Université de Montréal et de Jean-François Saucier, psychiatre et anthropologue à la Faculté de médecine au Département de psychiatrie de l'Université de Montréal et chercheur au CHU Sainte-Justine. Interdisciplinary Study.

Neoplasia papilar cística do pâncreas

Papyllary cystic tumor of the pancreas, so-called Frantz’s tumor, is rare. Clinical presentation of this disease is usually a slowly growing abdominal mass with or without abdominal pain, affecting predominantly young females. Its pathogenesis is still unknown . Surgical resection is usually curative, and prognosis is excellent. The authors report two pancreatic tumor cases(Frantz’s tumor) in women aged 26 and 31 years old. Pre operative assessment showed a solid-cystic tumor of the tail and body of the pancreas. An extended distal pancreatectomy was performed without splenic preservation