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Student Number : 9906314R - MSc dissertation - School of Pathology - Faculty of Science / Inappropriate use of antimicrobial agents has been shown to select for organisms with resistance mechanisms (eg. efflux pumps), which could lead to the development of antibiotic resistance. The objective of this study was to investigate a possible link between antibiotic resistant gastrointestinal pathogens and reduced susceptibility to anti microbial agents found in commonly used household disinfectants. Tetracycline and ofloxacin resistant and sensitive Salmonella and E.coli species were isolated from fresh poultry and clinical samples. Ethidium bromide accumulation assays were performed to assess the presence of active efflux pumps. Using spectrophotometric accumulation assays, the extrusion of the active components of commercial household agents by the efflux pumps was tested. To determine changes in the efficacy of these products, in-use disinfectant testing was performed. Active efflux pumps and extrusion of the active ingredients was observed in the resistant but not in the sensitive organisms. When the household products were used at the recommended concentrations, a significant reduction of both resistant and sensitive bacteria was observed after the in-use disinfectant testing procedure. However, if the household products were used at concentrations below the recommended concentration, the resistant bacteria were not eliminated as efficiently as the sensitive bacteria.

Intrinsic antimicrobial resistance of the opportunistic human pathogen Pseudomonas aeruginosa has mainly been attributed to the presence of several chromosomally-encoded multidrug efflux systems. The MexAB-OprM system exports the largest range of structurally unrelated antimicrobial agents and its expression is modulated by multiple regulatory controls. To develop a better understanding of mexAB-oprM overexpression in nalC mutants, which characteristically produce the effector protein PA3719 that binds and disrupts MexR transcriptional repression of mexAB-oprM, the PA3719-MexR interaction domains were investigated. Using a bacterial two-hybrid system, the C-terminus of PA3719 was found to be sufficient to mediate MexR-binding, and the binding region was found to be distinct from the MexR DNA-binding motif. The two-hybrid system was also used in an attempt to understand the role of PA3720, a protein of unknown function that is also overexpressed in nalC mutants. Results from this study confirm that PA3720 does not function to bind and alleviate NalC transcriptional repression of the PA3720-PA3719 operon. This study also attempted to identify the signals involved in overexpressing PA3720-PA3719, in the hopes to elucidate the natural function of MexAB-OprM. Random transposon mutagenesis using a PA3720-PA3719 promoter-lacZ fusion containing P. aeruginosa strain was conducted, but failed to clearly identify any disrupted genes associated with PA3720-PA3719 overexpression. Using the same PA3720-PA3719 promoter-lacZ fusion, expression of these genes was assessed as a function of growth in both wildtype and nalC mutant P. aeruginosa strains. Interestingly, PA3720-PA3719 expression was found to be growth-regulated, with an increased amount of expression occurring in late log/early stationary phase, even in the absence of nalC. This suggests that another regulator(s) is/are involved in modulating PA3720-PA3719 levels in late log/early stationary phase. Since PA3719 ultimately influences mexAB-oprM expression, its involvement in mediating growth-phase mexAB-oprM expression was assessed by examining mexA expression in both wildtype and PA3719 deletion P. aeruginosa strains. PA3719 was found to be involved in some, but not all, of the growth phase control of mexAB-oprM. These results suggest that mexAB-oprM growth-phase regulation is complex, as both MexR-dependent and MexR-independent regulatory pathways seem to exist. Overall, this study has produced a better understanding of mexAB-oprM regulation in nalC mutant P. aeruginosa strains. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-09-25 19:00:42.929

abstract: Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of these three proteins (and many of their homologs) are known, the exact mechanisms of interaction are still poorly understood. By mutational analysis of the TolC turn 1 residues, a drug hypersensitive mutant has been identified which is defective in functional interactions with AcrA and AcrB. Antibiotic resistant revertants carry alterations in both TolC and AcrA act by stabilizing functional complex assembly and opening of the TolC aperture, as monitored by stability of a labile TolC mutant and sensitivity to vancomycin, respectively. Alterations in the AcrB periplasmic hairpin loops lead to a similar antibiotic hypersensitivity phenotype and destabilized complex assembly. Likewise, alterations in TolC which constitutively open the aperture suppress this antibiotic sensitivity. Suppressor alterations in AcrA and AcrB partially restore antibiotic resistance by mediating stability of the complex. The AcrA suppressor alterations isolated in these studies map to the three crystallized domains and it is concluded they alter the AcrA conformation such that it is permanently fixed in an active state, which wild type only transiently goes through when activated by AcrB. Through this genetic evidence, a direct interaction between TolC and AcrB which is stabilized by AcrA has been proposed. In addition to stabilizing the interactions between TolC and AcrB, AcrA is also responsible for triggering opening of the TolC aperture by mediating energy flow from AcrB to TolC. By permanently altering the conformation of AcrA, suppressor mutants allow defective TolC or AcrB mutants to regain functional interactions lost by the initial mutations. The data provide the genetic proof for direct interaction between AcrB and that AcrA mediated opening of TolC requires AcrB as a scaffold. / Dissertation/Thesis / Ph.D. Microbiology 2012

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The aims of this study were: (1) to determine simultaneously by means of a micro-potometric method the rates of water and various isotopic ion effluxes from isolated onion roots; (2) to determine and compare the effects of metabolic inhibitors on both processes simultaneously; (3) to ascertain the similarity or dissimilarity of mechanisms involved with these two processes; (4) to ascertain the role of active metabolism in each of these processes; (5) to shed light on possible gearing mechanism(s) between metabolic and transport processes.

The MexCD-OprJ multidrug efflux pump of Pseudomonas aeruginosa confers resistance to a range of antimicrobials. Although not expressed under normal laboratory conditions, exposure to the membrane-active biocides, chlorhexidine or benzalkonium chloride, results in mexCD-oprJ expression. This suggests that membrane disruption provides the inducing signal. Consistent with this, increased mexCD-oprJ expression was demonstrated in the presence of additional membrane-damaging agents including polymyxin B, ethanol, SDS, EDTA, the organic solvents n-hexane and p-xylene, and the antimicrobial peptides melittin, V8 and V681. MexCD-OprJ expression was initially verified through increased resistance to known MexCD-OprJ antimicrobial substrates and subsequently using a mexC-lacZ transcriptional fusion and RT-PCR. Since the P. aeruginosa sigma factor AlgU is responsive to envelope stress, it was of interest to ascertain whether AlgU is capable of mediating this increased mexCD-oprJ expression. Thus, the impact of AlgU loss on mexCD-oprJ expression in response to membrane-damaging agents was assessed in a algU strain. In contrast with above, little or no mexCD-oprJ expression (assessed using resistance to MexCD-OprJ antimicrobial substrates, the mexC-lacZ transcriptional fusion and RT-PCR) occurred in response to membrane-damaging agents in the algU strain, consistent with AlgU playing a role in the envelope stress inducibility of mexCD-oprJ. Overall, envelope stress, and the ability to react to this stress through AlgU, appears to play an important role in mexCD-oprJ induction. This suggests an important role for MexCD-OprJ in alleviating envelope stress, independent of its ability to export and provide resistance to antimicrobials. A gene, PA4596, whose product shows substantial homology to the NfxB repressor of mexCD-oprJ expression, occurs downstream of mexCD-oprJ and shows AlgU-dependence and chlorhexidine inducibility, suggesting a role in the chlorhexidine-induced, AlgU-mediated expression of mexCD-oprJ. Thus, the impact of PA4596 loss on mexCD-oprJ expression was assessed. Paradoxically, the loss of PA4596 increases mexCD-oprJ expression in wild-type cells in response to chlorhexidine treatment (as assessed through RT-PCR), while its loss compromises mexCD-oprJ expression in an nfxB mutant. Nonetheless, this suggests that PA4596 is involved in the induction of mexCD-oprJ and that its ability to induce mexCD-oprJ differs depending on the state of nfxB. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-07-31 12:03:52.535

L’intestin est un acteur majeur du métabolisme du cholestérol de part son rôle dans dans l’absorption, la sécrétion des lipoprotéines et l’efflux transintestinal de cholestérol (TICE). De plus, c’est le second organe majeur, après le foie, à exprimer la Proproteine Convertase Subtilisine Kexine de type 9 (PCSK9), un inhibiteur naturel du récepteur aux LDL. Notre analyse des mécanismes moléculaires impliqués dans l’ hypocholestérolémie induite par les chirurgies bariatriques montre que la sleeve gastrectomie induit une hypocholestérolémie transitoire et modérée lié aux modifications de prise alimentaire. En revanche, le by-pass Roux en Y (RYGB) réduit fortement la cholestérolémie, stimule significativement son élimination fécale en induisant le TICE et en réduisant l’absorption intestinale de cholestérol. La seconde partie de ma thèse visait à répondre à une controverse autour de la faculté de l’intestin à sécréter PCSK9. In vivo (souris) et ex vivo (souris et homme), il ne semble pas que les cellules intestinales matures sécrètent PCSK9. En revanche, nous confirmons que la lignée humaine Caco2 est capable de sécréter PCSK9 mais que cette sécrétion est abolie lorsque les cellules deviennent matures. Les mécanismes responsables de cette perte de sécrétion restent mal définies mais sont dues au moins à deux paramètres: 1) une réduction du contenu intracellulaire induite par un catabolisme lysosomal accru; 2) une modification posttraductionelle de PCSK9 (glycosilation) altérant les voies de sécrétion post-Golgiennes. Les cellules Caco2 constituent un outil précieux pour disséquer les mécanismes et partenaires protéiques nécessaires à la sécrétion de PCSK9. Leurs identifications pourraient permettre de développer de nouveaux inhibiteurs pour réduire la sécrétion de PCSK9, réduire l’hypercholestérolémie et lutter plus efficacement contre les maladies cardiovasculaires. / The intestine is a major actor of cholesterol metabolism from its role in absorption, secretion of lipoproteins and transintestinal cholesterol efflux (TICE). In addition, it is the second major organ, after the liver, to express the Proproteine Convertase Subtilisin Kexin type 9 (PCSK9), the natural inhibitor of the LDL receptor. Our analysis of molecular mechanism involved in hypocholesterolemia induced by bariatric surgeries shows that the gastrectomy sleeve induces a transient and moderate hypocholesterolemia linked to the modification of the food intake. In contrast, the Roux-Y by-pass (RYGB) strongly reduces cholesterol, significantly stimulates its fecal elimination by inducing TICE and decreasing intestinal absorption of cholesterol. The second part of my thesis consisted to elucidate the controversy around the faculty of the intestine to secrete PCSK9. In vivo (mice) and ex vivo (mice and human), it seems that mature enterocytes can’t secrete PCSK9. On the other hand, we confirm that the Caco2, an human intestinal cell line, is capable of secreting PCSK9, but this secretion is abolished when the cells become mature. Mechanisms responsible for this loss of secretion remain poorly defined and are, at least, due to: 1) a reduction in the intracellular content induced by increased lysosomal catabolism; 2) a post-translational modification of PCSK9 (glycosilation) altering post-Golgi secretion pathways. Caco2 cells are a powerfull tool for identify the mechanisms and partners required for the secretion of PCSK9. Their identifiers allow the development of new inhibitors to reduce the secretion of PCSK9, reduce hypercholesterolemia and fight more effectively against cardiovascular diseases.

MexCD-OprJ is an RND-type multidrug efflux pump present in P. aeruginosa and is capable of exporting, and as such providing resistance to, several clinically important antimicrobials including fluoroquinolones, cephems, macrolides, and several biocides including chlorhexidine (CHX). Expression of mexCD-oprJ is negatively regulated by NfxB, a LacI-type repressor. The promoter region of mexCD-oprJ was identified and included two inverted repeat operator sites, B1 and B2, both of which are required in order for NfxB to bind, thereby repressing mexCD-oprJ. NfxB oligomerizes into a tetramer in solution and likely functions as a dimer of NfxB homodimers. In addition to being derepressed by loss of NfxB, MexCD-OprJ is inducible by a variety of non-antibiotic membrane-damaging agents (MDAs) such as CHX. A homologue of NfxB, PA4596, was found to be induced in response to CHX-promoted envelope stress in an AlgU-dependent manner and is directly repressed by NfxB. Loss of PA4596 resulted in increased resistance to the biocide CHX, shown to be a result of increased CHX-dependent expression of mexCD-oprJ. Susceptibility to CHX was restored upon expression of PA4596 from the plasmid pAK1900 as was decreased expression of mexCD-oprJ in the presence of CHX, indicating that PA4596 contributes to mexCD-oprJ repression in the presence of CHX. PA4596 was found to form oligomers in solution, likely dimers and tetramers. In the absence of NfxB, PA4596 is unable to contribute to repression of mexCD-oprJ. However, NfxB and PA4596 interact and together form a repressor capable of regulating mexCD-oprJ expression. Screening of transposon mutants for increased resistance to erythromycin potentially indicative of increased mexCD-oprJ expression lead to the identification of several novel genes including PA0479, cupA3, faoA, PA3259, mucD, and clpA whose loss generated a multidrug resistance profile consistent with increased production of MexCD-OprJ. However, further studies are required to determine how each of these genes may be affecting expression of mexCD-oprJ. / Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-06-12 12:07:28.67