Salmonella enterica sv. typhimurium (S. enterica sv. Typhimurium) has two metal-transporting P1-type ATPases whose actions largely overlap with respect to growth in elevated copper. Mutants lacking both ATPases over-accumulate copper relative to wild-type or either single mutant. Such duplication of ATPases is unusual in bacterial copper tolerance. Both ATPases are under the control of MerR family metal-responsive transcriptional activators. Analyses of periplasmic copper complexes identified copper-CueP as one of the predominant metal pools. Expression of cueP was recently shown to be controlled by the same metal-responsive activator as one of the P1-type ATPase genes (copA), and copper-CueP is a further atypical feature of copper homeostasis in S. enterica sv. Typhimurium. Elevated copper is detected by a reporter construct driven by the promoter of copA in wild-type S. enterica sv. Typhimurium during infection of macrophages. Double mutants missing both ATPases also show reduced survival inside cultured macrophages. It is hypothesized that elevated copper within macrophages may have selected for specialized copper-resistance systems in pathogenic microorganism such as S. enterica sv. Typhimurium.
C3 glomerulopathy (C3G) is associated with dysregulation of the alternative pathway of complement activation, and treatment options for C3G remain limited. Complement factor H (FH) is a potent regulator of the alternative pathway and might offer a solution, but the mass and complexity of FH makes generation of full-length FH far from trivial. We previously generated a mini-FH construct, with FH short consensus repeats 1-5 linked to repeats 18-20 (FH), that was effective in experimental C3G. However, the serum of FH was significantly shorter than that of serum-purified FH. We introduced the oligomerization domain of human FH-related protein 1 (denoted by R1-2) at the carboxy or amino terminus of human FH to generate two homodimeric mini-FH constructs (FH and FH, respectively) in Chinese hamster ovary cells and tested these constructs using binding, fluid-phase, and erythrocyte lysis assays, followed by experiments in FH-deficient mice. FH and FH homodimerized in solution and displayed avid binding profiles on clustered C3b surfaces, particularly FH Each construct was >10-fold more effective than FH at inhibiting cell surface complement activity and restricted glomerular basement membrane C3 deposition significantly better than FH or FH FH had a C3 breakdown fragment binding profile similar to that of FH, a >5-fold increase in serum compared with that of FH and significantly better retention in the kidney than FH or FH FH may have utility as a treatment option for C3G or other complement-mediated diseases.
Treatment of Clostridium difficile is a major problem as a hospital-associated infection which can cause severe, recurrent diarrhea. The currently available antibiotics are not effective in all cases and alternative treatments are required. In the present study, an ovine antibody-based platform for passive immunotherapy of C. difficile infection is described. Antibodies with high toxin-neutralizing titers were generated against C. difficile toxins A and B and were shown to neutralize three sequence variants of these toxins (toxinotypes) which are prevalent in human C. difficile infection. Passive immunization of hamsters with a mixture of toxin A and B antibodies protected them from a challenge with C. difficile spores in a dose-dependent manner. Antibodies to both toxins A and B were required for protection. The administration of toxin A and B antibodies up to 24 h postchallenge was found to reduce significantly the onset of C. difficile infection compared to nonimmunized controls. Protection from infection was also demonstrated with key disease isolates (ribotypes 027 and 078), which are members of the hypervirulent C. difficile clade. The ribotype 027 and 078 strains also have the capacity to produce an active binary toxin and these data suggest that neutralization of this toxin is unnecessary for the management of infection induced by these strains. In summary, the data suggest that ovine toxin A and B antibodies may be effective in the treatment of C. difficile infection; their potential use for the management of severe, fulminant cases is discussed.
Atypical hemolytic uremic syndrome (aHUS) is frequently associated in humans with loss-of-function mutations in complement-regulating proteins or gain-of-function mutations in complement-activating proteins. Thus, aHUS provides an archetypal complement-mediated disease with which to model new therapeutic strategies and treatments. Herein, we show that, when transferred to mice, an aHUS-associated gain-of-function change (D1115N) to the complement-activation protein C3 results in aHUS. Homozygous C3 p.D1115N (C3KI) mice developed spontaneous chronic thrombotic microangiopathy together with hematuria, thrombocytopenia, elevated creatinine, and evidence of hemolysis. Mice with active disease had reduced plasma C3 with C3 fragment and C9 deposition within the kidney. Therapeutic blockade or genetic deletion of C5, a protein downstream of C3 in the complement cascade, protected homozygous C3KI mice from thrombotic microangiopathy and aHUS. Thus, our data provide in vivo modeling evidence that gain-of-function changes in complement C3 drive aHUS. They also show that long-term C5 deficiency is not accompanied by development of other renal complications (such as C3 glomerulopathy) despite sustained dysregulation of C3. Our results suggest that this preclinical model will allow testing of novel complement inhibitors with the aim of developing precisely targeted therapeutics that could have application in many complement-mediated diseases.
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