Browsing by Subject "Bacterial"
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Item The contribution of quorum sensing to the pathogenesis of pseudomonas aeruginosa in burn wound infections(Texas Tech University, 2001-05) Rumbaugh, Kendra P.Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes severe infections in burned patients. In this work, we examined the contribution ofthe cell-to-cell communication systems or quorum sensing (QS) systems to the pathogenesis of P aeruginosa infection of burn wounds. For these studies, we used the thermally-injured mouse model and specific mutants that carry deletions in genes encoding specific components of the P aeruginosa QS systems {lasR, rhlR, lasl and rhll). In comparison with their parent strain (PAOl), all mutants displayed decreased lethality. The mutants were defective in their ability to spread systemically throughout the body of the mice. In addition, the lasR (PAO-Rl) and the lasl/rhll (PAO-JP2) mutants were defective in their ability to spread locally within the burned skin at 8 and 16 hours post-bum/infection. The defects in the PAO-JP2 strain were complemented upon the introduction of a plasmid carrying intact lasl and rhll genes. To determine if the defect in PAO-JP2 is due to the loss of one or more of the QScontrolled virulence factors, isogenic mutants that carry deletions in lasA, lasB, lasAAasB, toxA, lasB/toxA or rpoS were examined. With the exception of the rpoS mutant, all mutants were defective in their in vivo virulence. However, none was as severely defective as PAO-JP2. Our attempt to ascertain the role of autoinducers as virulence factors was confounded by the influence of the solvent used to purify them. Host production of cytokines in response to P aeruginosa infection in bum wounds was examined using the Multi-Probe Template/RNase protection assay. The expression of several proinflammatory and hematopoietic cytokines was up-regulated in burned mice infected with PAOl at 40 hours post-bum/infection. In contrast, the expression of most of these cytokines was not enhanced in burned mice infected with PAO-JP2. These results suggest that: (1) the QS systems play an important role in the pathogenesis of P aeruginosa in bum wound infections; (2) their effects may be contributed to by autoinducers or other, yet undefined, QS-controlled factors; and (3) QS may play a role in modulating the host immune system in response to P. aeruginosa.Item The effect of ptxR on the production of exotoxin A, exoenzyme U, and the pyoverdine chromophore in the Pseudomonas aeruginosa strain PA103(Texas Tech University, 2002-05) Carty, NancyThe production of exotoxin A and the pyoverdine siderophore m Pseudomonas aeruginosa is negatively regulated by iron. We have previously described a P aeruginosa gene ptxR that positively regulates exotoxin A and pyoverdine production. Previous studies from our laboratory and others reported that the presence of a pixR plasmid in P aeruginosa increased exotoxin A production by 4 to 5 fold and deregulated the production of the pyoverdine chromophore (pvc) with respect to iron. In this study, we examined toxA regulation by ptxR in the P. aeruginosa strain PA103, which has been extensively used in exotoxin A regulatory studies. To avoid problems associated with regulatory studies involving multiple copies of genes, we constructed a PA103 strain carrying two copies of ptxR (PA103-2R) by homologous recombination. Exotoxin A production in PA103-2R was dramatically increased and deregulated with respect to iron. The expression of the pvc operon remained deregulated with respect to iron. These effects, which were not due to ptxR overexpression, occurred at the transcnptional level. SDS-PAGE and amino acid sequencing analysis demonstrated that the type III secretion effector protein, ExoU, is over produced in PA103-2R. Further analysis confirmed that e.vof/expression in PA103-2R is deregulated with respect to calcium. We detected the same phenotype when the integrated plasmid together with adjacent PA103 chromosome (pNC105) was retrieved from PA103-2R and introduced into PA103 as a stable plasmid (pKHl). Nucleotide sequence analysis revealed that pNC105 carries genes that exist in PA103 but not PAOl. In addition, pNC105 carries an intact copy of ptxR, but is lacking 322 bp of the region 3' of it. The role of the deleted region was investigated by constructing a new plasmid with a 4 bp insertion at a unique site 3' of ptxR, pNC313. The presence of pNC313 in PA103 produced the same phenotype observed in PA103-2R. We have eliminated the possibility that changes within the region 3' of pi.xR abolished a binding site for a potential regulator. Examination of differences in the predicted mRNA secondary structure for ptxR due to the 3' changes eliminated this possibility as at least two mechanisms would be required to explain the phenotype. However, an open reading frame encoding an estimated 14 kDa protein, PtxQ, was identified 3' of ptxR. Changes in PA103-2R, pKHl, and pNC313 either eliminated p/.vg or produced a frame shift mutation within it. Based on the available results, we propose that PtxQ modulates PtxR function by forming a PtxR:PtxQ heterodimer. In the absence of PtxQ, excess molecules of PtxR increase toxA, pvc, and exoU expression beyond the repressive ability of either iron or calcium.Item The regulation of Vibrio cholerae MDR efflux Pump in a TolC mutant of Escherichia coli(Texas Tech University, 2004-05) Ramasubramian, BhagavathiThe emergence of antibiotic-resistance among bacterial pathogens is becoming a serious threat to human health and welfare. One mechanism by which bacteria become resistant to antibiotics is to remove them via a multiple drug resistant (MDR) efflux pump. The first multidrug resistance pump was described in tumor cells that were resistant to chemotherapeutic agents. More recently, MDRs have been characterized in both Gram-negative and Gram-positive bacteria. Vibrio cholerae, a Gram-negative non-invasive enteric pathogen, is the causative agent of the severe diarrheal disease cholera. The first multi-drug efflux pump of V. cholerae, VceAB, was isolated and characterized by Colmer et al. In that study the authors cloned a 6.6 kb SalI-Hind III fragment of V cholerae into pBR322, pVC2 and demonstrated that this plasmid was capable of providing both V. cholerae and an Escherichia coli to/C mutant with resistance to a variety of toxic compounds and antibiotics. The authors also demonstrated that this 6.6 kb DNA fragment contained two genes, vceA and vceB, which encoded proteins whose amino acid sequences shared a high degree of similarity with the EmrA and EmrB proteins of E.coli, respectively. Since those studies were published, our laboratory has identified the outer membrane component of this efflux pump, OepX, whose gene resides in an operon with vceA and vceB and a regulatory gene vceR, whose product is a member of the TetR/AcrR transcriptional regulator family and which negatively regulates the oepX-vceA-vceB operon. During that study, the authors constructed an in-frame vceA::phoA gene fusion in pVC2 (pi328). In this construct the phoA gene is fused in-frame to the vceA gene at position 753 I have employed this reporter fusion to examine the regulation of the oepX-vceA- vceB (i.e., VceAB MDR) operon. The activity of this gene fusion, measured by alkaline phosphatase assay was found to be up regulated in the tolC mutant strain. The purpose of this study was to decipher the cause of this regulation which seems to be a tolC effect.