Browsing by Subject "Septicemia"

Now showing 1 - 4 of 4
  • Thumbnail Image
    Item
    Aerobic Uptake of Cholesterol by Ergosterol Auxotrophic Strains in Candida glabrata & Random and Site-Directed Mutagenesis of ERG25 in Saccharomyces cerevisiae
    (2012-09-27) Whybrew, Jennafer Marie; Bard, Martin; Lees, N. Douglas; Blacklock, Brenda
    Candida albicans and Candida glabrata are opportunistic human pathogens that are the leading cause of fungal infections, which are increasingly becoming the leading cause of sepsis in immunosuppressed individuals. C. glabrata in particular has become a significant concern due to the increase in clinical isolates that demonstrate resistance to triazole antifungal drugs, the most prevalent treatment for such infections. Triazole drugs target the ERG11 gene product and prevent C-14 demethylation of the first sterol intermediate, lanosterol, preventing the production of the pathways end product ergosterol. Ergosterol is required by yeast for cell membrane fluidity and cell signaling. Furthermore, C. glabrata, and not C. albicans, has been reported to utilize cholesterol as a supplement for growth. Although drug resistance is known to be caused by an increase in expression of drug efflux pumps, we hypothesize a second mechanism: that the overuse of triazole drugs has lead to the increase of resistance by C. glabrata through a 2-step process: 1) the accumulation of ergosterol auxotrophic mutations and 2) mutants able to take up exogenous cholesterol anaerobically in the body acquire a second mutation allowing uptake of cholesterol aerobically. Two groups of sterol auxotrophic C. glabrata clinical isolates have been reported to take up sterol aerobically but do not produce a sterol precursor. Sterol auxotrophs have been created in C. glabrata by disrupting different essential genes (ERG1, ERG7, ERG11, ERG25, and ERG27) in the ergosterol pathway to assess which ergosterol mutants will take up sterols aerobically. Random and site-directed mutagenesis was also completed in ERG25 of Saccharmoyces cerevisiae. The ERG25 gene encodes a sterol C-4 methyloxidase essential for sterol biosynthesis in plants, animals, and yeast. This gene functions in turn with ERG26, a sterol C-3 dehydrogenase, and ERG27, a sterol C-3 keto reductase, to remove two methyl groups at the C-4 position on the sterol A ring. In S. cerevisiae, ERG25 has four putative histidine clusters, which bind non-heme iron and a C-terminal KKXX motif, which is a Golgi to ER retrieval motif. We have conducted site-directed and random mutagenesis in the S. cerevisiae wild-type strain SCY876. Site-Directed mutagenesis focused on the four histidine clusters, the KKXX C-terminal motif and other conserved amino acids among various plant, animal, and fungal species. Random mutagenesis was completed with a procedure known as gap repair and was used in an effort to find novel changes in enzyme function outside of the parameters utilized for site-directed mutagenesis. The four putative histidine clusters are expected to be essential for gene function by acting as non-heme iron binding ligands bringing in the oxygen required for the oxidation-reduction in the C-4 demethylation reaction.
  • Thumbnail Image
    Item
    Functional role of the TLR4 signaling pathway in the bone marrow response to sepsis
    (2015-03-31) Zhang, Huajia; Carlesso, Nadia; Blum, Janice S.; Cardoso, Angelo A.; Herbert, Brittney-Shea; Ivan, Mircea; Liu, Yunlong
    Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. Little is known about the changes in the bone marrow (BM) and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, we have previously reported that hematopoietic stem cells (HSC) undergo a significant expansion in the BM accompanied with myeloid suppression. This bone marrow response was Toll-like Receptor 4 (TLR4)-dependent. TLR4 is activated by bacterial lipopolysaccharide (LPS) and signals through two major independent downstream molecules: TRIF and MyD88. In the present study, I found that the TLR4/TRIF and the TLR4/MyD88 pathways contribute in a distinct manner to the BM response to P. aeruginosa's LPS. TRIF plays a major role in the expansion of the HSC pool, whereas MyD88 is required for myeloid suppression. Following LPS stimulation, HSCs enter in the cell cycle, expand and exhaust when transplanted in healthy mice. Loss of TRIF rescued completely the long-term engraftment and multilineage reconstitution potential of septic HSCs, but did not affect myeloid differentiation. Conversely, MyD88 deficiency prevented completely the myeloid suppression in the myeloid progenitors, but conferred limited protective effects on the HSC function. It is of great therapeutic value to identify the downstream molecules involved in TLR4/MyD88 dependent myeloid suppression. I found miR-21, a microRNA that is involved in inflammation, was up-regulated upon LPS challenge in a MyD88-dependent manner. However, deletion of miR-21 in the BM did not rescue LPS-induced bone marrow dysfunction, demonstrating that miR-21 is not a critical regulator in these processes. Further studies are warranted to determine the precise molecular mechanisms involved in the complex pathogenesis of BM response to sepsis. Taken together, my results show for the first time that the TLR4/TRIF signaling as a key mediator of HSC damage during acute LPS exposure and that activation of the TLR4/MyD88 signaling pathway play a dominant role in myeloid suppression. These results provide novel insights into our understanding of the molecular mechanisms underlying bone marrow injury during severe sepsis and may lead to the development of new therapeutic approaches in this disease.
  • Thumbnail Image
    Item
    Gardnerella vaginalis causing pulmonary infection in a young adult: A novel case
    (Elsevier, 2019-07-25) Bittar, Julie M.; Gazzetta, Joshua; Surgery, School of Medicine
    Gardnerella vaginalis is an anaerobic, gram-variable bacterium primarily found in vaginal microflora of women. Previous reports of G. vaginalis cultured in men are few and have primarily been limited to the gastrointestinal and genitourinary tract.2-4 Few reports of G. vaginalis causing severe infections have been reported in the literature, including septicemia7 and two cases of perinephric abscess.8,9 There has been one previously reported case of G. vaginalis causing pulmonary complications that occurred in a male alcohol abuser. In our case review, we aim to demonstrate an unusual source of a pulmonary infection and highlight the importance of proper microbial isolation to guide treatment. Our patient is a young male who presented following multiple gunshot wounds including one to his head causing an intracranial hemorrhage, hydrocephalus, and a dural sinus thrombosis. His hospital course was complicated by a decline in neurological status treated with a craniotomy and external drain placement and multiple pulmonary infections. During his fever work-ups, he found to have G. vaginalis on mini-bronchoalveolar lavage and was subsequently treated with metronidazole. After treating his G. vaginalis pneumonia and other infectious sources, namely Haemaphilus influenzae and coagulase-negative staphylococcus pneumonias, his fevers and leukocytosis resolved and he was successfully discharged to a rehabilitation facility for neurologic recovery. To our knowledge, this is the second reported case of G. vaginalis isolated from a pulmonary culture and the first in a previously healthy, immunocompetent young male outside of the urinary tract.
  • Thumbnail Image
    Item
    Guns, not roses – here’s the true story of penicillin’s first patient
    (The Conversation US, Inc., 2022-03-11) Sullivan, BIll