No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The Centers for Disease Control (CDC) and World Health Organizations (WHO) have highlighted six species of highly drug-resistant bacteria, commonly termed the ESKAPE pathogens, that new antibacterials are urgently needed to treat). The ESKAPE pathogens account for over two-million infections and have healthcare costs upwards of $20 billion dollars annually. Over the past several decades, pharmaceutical companies have drastically reduced their research programs for developing new antibacterial agents. As well, bacteria are predisposed to rapidly generate resistance against these “me too” drugs, making this strategy a temporary stop-gap in our ability to fight these pathogens. This has left the burden to identify new antibiotics that function through fundamentally unique mechanisms of action to academia. Towards this goal, we are developing a unique antibacterial strategy that functions through targeting the bacterial GroEL chaperonin systems. GroEL is a molecular chaperone that helps fold proteins into their functional states. Being an essential protein, inhibiting GroEL activity leads to global aggregation and bacterial cell death. We previously reported a high-throughput screening effort that identified 235 GroEL inhibitors. A subsequent study with a subset of these inhibitors identified several that kill bacteria. To follow-up, we have synthesized 43 analogs of a hit-to-lead molecule, compound 1, containing systematic deletions of substituents and substructures to determine the essential parts of the scaffold for inhibiting GroEL and killing bacteria. Along with inhibiting GroEL, several compound 1 analogs exhibit >50-fold therapeutic windows between antibacterial efficacy and cytotoxicity to human liver and kidney cells in cell culture. Evaluation of two lead candidates (1 and 11) in a gain-of-resistance assay indicated that MRSA bacteria were not able to easily generate resistance to this compound class. Compound 1 also exhibited the ability to permeate through already established S. aureus biofilms and maintain its bactericidal effects, whereas vancomycin could not. Having established initial structure-activity relationships for the compound 1 substituents and substructures in this study, future efforts will focus on optimizing the antibacterial effects of lead candidates and reducing their off-target toxicity to human cells.

The 105aa lambda S protein is the prototype holin, S accumulates in the cytoplasmic membrane during late gene expression until, at a time programmed into its primary structure, it disrupts the membrane and allows the lambda lysozyme, R, to attack the cell wall. In this study, a zwitterionic detergent Empigen BB, was used to extract and purify the lambda holin S. In Empigen BB, CD analysis on S gave 54% alpha helical content, consistent with 3 TM domains, which has been reported by other in vivo studies. Empigen BB-purified S can be exchanged into a chaotropic solution by dialysis and reconstituted into preformed lipid vesicles for activity assays. When diluted to fluorescein-loaded suspensions of liposomes, different chaotrope-solubilized S alleles caused dye release reflective of their in vivo phenotypes. The problem was the low efficiency of delivery of S to the liposomes. Unfortunately, dye loaded liposomes are highly sensitive to any detergent, making it necessary to find other ways to solubilize S. GroEL, a chaperonin from E. coli, is responsible for folding and refolding globular proteins in vitro. It has also been reported that GroEL improves the ability of a membrane protein synthesized in vitro to insert post-translationally into liposomes. This work will investigate the behavior of GroEL towards membrane proteins. The first of two membrane proteins studied in this respect is Bacteriorhodopsin (BR), a membrane proton pump, from H. halibium. The second is the105aa S protein, a prototype holin from bacteriophage lambda. Holin and BR subjected to detergent removal in the presence of GroEL remained in solution, while in the control sample (without GroEL) S and BR precipitated. "GroELsolubilized" holin still retained its lesion forming activity and solubilized BR maintained its proton pumping ability, detected by using a liposome dye activity assay unique to each protein. This approach may be applicable to other systems requiring detergent- or chaotrope-free preparation of membrane proteins. Finally, these results suggest that GroEL may be involved in the insertion of integral membrane proteins into the lipid bilayer, a role heretofore unsuspected.

The intracellular pathogen Legionella pneumophila replicates in a membrane-bound compartment known as the Legionella-containing vacuole (LCV) where it abundantly releases its chaperonin HtpB, suggesting that HtpB may have virulence-related functions. To assess these functions, I attempted to construct an L. pneumophila ?htpBmutant but was unable to do so, likely because htpB is essential. In the absence of genetic deletion, functional tests were used to study the released HtpB. A small portion of the HtpB in L. pneumophila-infected cells was found in the cytoplasm of the infected cells, as judged by the CyaA reporter assay. To identify potential functions of the HtpB present in the eukaryotic cytoplasm, htpB was ectopically expressed in Saccharomyces cerevisiae. HtpB induced pseudohyphal growth (PHG) in yeast, suggesting it interacts with eukaryotic targets. A yeast two-hybrid screen showed that HtpB interacted with SAMDC, an essential yeast enzyme encoded by SPE2 that is required for polyamine biosynthesis. Overexpression of SPE2 induced PHG in S. cerevisiae, suggesting that HtpB induces PHG by activating polyamine synthesis, and that L. pneumophila may require exogenous polyamines for growth. A pharmacological inhibitor of SAMDC reduced L. pneumophila replication in host cells, whereas exogenous polyamines enhanced intracellular growth. Bioinformatics revealed that most known enzymes required for polyamine biosynthesis in bacteria are absent in L. pneumophila, suggesting that L. pneumophila depends on exogenous polyamines transported from host cells. L. pneumophila possesses only one putative operon,potABCD, which encodes a polyamine transporter. Using GFP as a reporter of potABCD promoter (PpotA), we found that PpotA activity was turned on during exponential phase of growth in vitro. To test the potential function of this transporter in pathogenesis, potD was deleted.Although deletion of potD did not affect L. pneumophila growth in vitro, it reduced L. pneumophila attachment to phagocytic cells, intracellular growth, and the ability of the LCV to recruit vesicles. Collectively, these findings have contributed to a better understanding of the biology of L. pneumophila by suggesting that HtpB and PotD might collaborate to ensure a supply of polyamines required for the optimal intracellular growth of L. pneumophila.

Understanding how antimicrobial growth promoters (AGPs) affect small intestinal microbiota may help to discover effective alternatives. The impact of dietary supplementation with tylosin phosphate on small intestinal microbial profiles was investigated in growing pigs, and compared with the microbial profile of pigs fed flaxseed or its fractions. Eighteen ileal-cannulated barrows (33.1 +/- 2.4 kg) received either the control diet (C, wheat, peas and soybean meal), or C plus 22 mg/kg tylosin phosphate (T), 20% whole flaxseed (WF), 18% hot-water extracted flaxseed (HWE), 4% flaxseed hulls (H), or 8% flaxseed oil (O) during three 21-d periods in a change over design. Ileal digesta (100 mL) was collected on d 16 and 17 of each period. Two chaperonin-60 universal target (cpn60 UT) libraries were constructed from pooled ileal digesta DNA extracted from the C and T diets. A total of 1634 nucleotide sequences were determined, and 117 different cpn60 UT sequences identified. Microbial diversity was greatest in the C library compared to T. Taxonomic composition between libraries differed, and included Lactobacillales (94% of C and 86% of T sequences), Enterobacteriaceae (3% of C and 13% of T), Clostridiales, Bacillales and Bifidobacterium taxa. T had a reduced ratio of Lactobacillales: Enterobacteriaceae sequences (6:1) compared to C (35:1). Lactobacilli: enterobacteria plate count ratios were highest in WF compared to C or T diets. Lactobacillus johnsonii genomes detected by qPCR were increased by 17.2 and 12%, in T and WF diets, respectively, compared to C. Numbers of L. amylovorus genomes were 25% lower in the H diet compared to C. Numbers of Escherichia coli and Streptococcus alactolyticus genomes were unaffected by dietary treatment, despite differences in library clone frequency for these species. Increased L. johnsonii colonization with tylosin suggests possible probiotic properties of this bacterium. Only inclusion of whole flaxseed resulted in a similar increase in L. johnsonii. Overall, ileal microbial profiles of growing pigs were similar and remained mostly unaffected by dietary tylosin or flaxseed inclusion.

Understanding how antimicrobial growth promoters (AGPs) affect small intestinal microbiota may help to discover effective alternatives. The impact of dietary supplementation with tylosin phosphate on small intestinal microbial profiles was investigated in growing pigs, and compared with the microbial profile of pigs fed flaxseed or its fractions. Eighteen ileal-cannulated barrows (33.1 +/- 2.4 kg) received either the control diet (C, wheat, peas and soybean meal), or C plus 22 mg/kg tylosin phosphate (T), 20% whole flaxseed (WF), 18% hot-water extracted flaxseed (HWE), 4% flaxseed hulls (H), or 8% flaxseed oil (O) during three 21-d periods in a change over design. Ileal digesta (100 mL) was collected on d 16 and 17 of each period. Two chaperonin-60 universal target (cpn60 UT) libraries were constructed from pooled ileal digesta DNA extracted from the C and T diets. A total of 1634 nucleotide sequences were determined, and 117 different cpn60 UT sequences identified. Microbial diversity was greatest in the C library compared to T. Taxonomic composition between libraries differed, and included Lactobacillales (94% of C and 86% of T sequences), Enterobacteriaceae (3% of C and 13% of T), Clostridiales, Bacillales and Bifidobacterium taxa. T had a reduced ratio of Lactobacillales: Enterobacteriaceae sequences (6:1) compared to C (35:1). Lactobacilli: enterobacteria plate count ratios were highest in WF compared to C or T diets. Lactobacillus johnsonii genomes detected by qPCR were increased by 17.2 and 12%, in T and WF diets, respectively, compared to C. Numbers of L. amylovorus genomes were 25% lower in the H diet compared to C. Numbers of Escherichia coli and Streptococcus alactolyticus genomes were unaffected by dietary treatment, despite differences in library clone frequency for these species. Increased L. johnsonii colonization with tylosin suggests possible probiotic properties of this bacterium. Only inclusion of whole flaxseed resulted in a similar increase in L. johnsonii. Overall, ileal microbial profiles of growing pigs were similar and remained mostly unaffected by dietary tylosin or flaxseed inclusion.

TRiC (TCP-1 ring complex), a type II chaperonin, facilitates protein folding, and we previously showed that TRiC crosslinks to ribosome-bound actin and luciferase nascent chains. Here, it was found that actin and luciferase nascent chains were adjacent to more than one TRiC subunit at different stages of translation. Six and seven out of the eight TRiC subunits were photocrosslinked to the luciferase and actin nascent chains, respectively. Actin nascent chains with widely-spaced, site-specific probe locations were adjacent to the same three TRiC subunits (a, b and e) at different stages of translation. The exposure of other TRiC subunits to nascent chains varied with the length and identity of the nascent chain. In addition, the presence or absence of ATP influences the photocrosslinking yields. This suggests that ATP alters the conformation of the subunits and/or their affinity for the nascent chain. Photocrosslinking also revealed that TRiC is in close proximity to the exit site of the ribosomal tunnel, presumably to create a protected folding environment for the nascent chain. Immunoprecipitations under native conditions revealed that prefoldin photocrosslinks to the actin nascent chain and that these prefoldin-containing photoadducts are coimmunoprecipitated with antibodies specific for the TRiC a subunit. This result suggests that prefoldin and TRiC bind simultaneously to the same actin nascent chain. Photocrosslinking studies with probes at position 68 in the actin nascent chain revealed that prefoldin binds to the nascent chain subsequently to TRiC binding. An unknown protein with an apparent molecular mass of 105 kDa was shown to photocrosslink to the luciferase nascent chain in a length-dependent manner at specific probe locations close to the N-terminus of the nascent chain. Thus, the nascent chain sees a variety of proteins in its immediate environment as it emerges from the ribosomal tunnel and undergoes its chaperonin-assisted folding.

No description available.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2010. / Härtill 3 uppsatser.

The complete cDNA sequence of a mitochondrial protein from Chinese hamster ovary cells has been determined. This protein, designated P1, was originally identified in cells resistant to the microtubule inhibitor podophyllotoxin (Gupta, 1981). The mutant cell line contained an alteration of the P1 protein that gave rise to a new, more acidic protein, designated M1 (Gupta et al., 1982). The P1 protein was determined to be microtubule-related based on the cross-resistance pattern of the mutants to other microtubule inhibitors, and corelease with tubulin under conditions which cause microtubule depolymerization (Gupta et al., 1982). Subcellular fractionation studies localized this protein to the matrix of the mitochondria (Gupta and Austin, 1987). Antibodies raised against P1 were used to isolate a cDNA clone from human cells (Jindal et al.,1989). The human cDNA clone was used as a probe to screen for clones of the P1 protein in bacteriophage (lambda)gt10/(lambda)gt11 cDNA libraries prepared from CHO cells. The P1 cDNA encodes a protein of 573 amino acids with a relative molecular mass of 60,983 daltons. The first 26 amino acids meet the requirements of a mitochondrial matrix targeting sequence. The mature protein is 547 residues in length with a relative molecular mass of 57,949 daltons. The deduced amino acid sequence shows 97% identity to the the human P1 protein. More interestingly, the amino acid sequence shows extensive homology (42 to 55% identical residues and an additional 15 to 20% conservative replacements) to the chaperonin class of molecular chaperones. This class of proteins includes the hsp60 protein of yeast, the groEL protein of Escherichia coli, the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit binding protein of plant chloroplasts, and the 62-65-kDa major antigenic protein of mycobacteria and Coxiella burnetii. The homology of P1 with the above proteins begins after the putative mitochondrial presequence and extends to the c-terminal end. Several regions throughout the protein sequence are highly conserved and are proposed to be functional domains of the protein. Also highly conserved is a Gly-Gly-Met repeating motif at the carboxy-terminus. The function of this sequence is undetermined, as yet. A dendrogram was constructed from the sequence homology data. It suggested that mitochondrial P1 evolved from purple bacteria which is the endosymbiont which gave rise to mitochondria. The chaperonin class of proteins have been shown to assist in the assembly of oligomeric protein structures. It is suggested that the P1 protein may play a similar role in mammalian cells. The high degree of homology between P1 and the 65-kDa mycobacterial antigen also suggests that P1 may be involved in certain autoimmune diseases. / Thesis / Master of Science (MS)