Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores.
Streptococcus mutans, a major etiological agent of human dental caries, produces membrane vesicles (MVs) that contain protein and extracellular DNA. In this study, functional genomics, along with in vitro biofilm models, was used to identify factors that regulate MV biogenesis. Our results showed that when added to growth medium, MVs significantly enhanced biofilm formation by S. mutans, especially during growth in sucrose. This effect occurred in the presence and absence of added human saliva. Functional genomics revealed several genes, including sfp, which have a major effect on S. mutans MVs. In Bacillus sp. sfp encodes a 4′‐phosphopantetheinyl transferase that contributes to surfactin biosynthesis and impacts vesiculogenesis. In S. mutans, sfp resides within the TnSmu2 Genomic Island that supports pigment production associated with oxidative stress tolerance. Compared to the UA159 parent, the Δsfp mutant, TW406, demonstrated a 1.74‐fold (p < .05) higher MV yield as measured by BCA protein assay. This mutant also displayed increased susceptibility to low pH and oxidative stressors, as demonstrated by acid killing and hydrogen peroxide challenge assays. Deficiency of bacA, a putative surfactin synthetase homolog within TnSmu2, and especially dac and pdeA that encode a di‐adenylyl cyclase and a phosphodiesterase, respectively, also significantly increased MV yield (p < .05). However, elimination of bacA2, a bacitracin synthetase homolog, resulted in a >1.5‐fold (p < .05) reduction of MV yield. These results demonstrate that S. mutans MV properties are regulated by genes within and outside of the TnSmu2 island, and that as a major particulate component of the biofilm matrix, MVs significantly influence biofilm formation.
The accumulation of modified proteins in aging is well documented in many aging models. For example, the deamidated isoforms of triosephosphate isomerase accumulate in: (a) old erythrocytes, (b) fibroblasts from old donors, (c) fibroblasts aged in vitro, (d) premature-aging syndromes and (e) old cells in the eye lens. However, a fundamental remaining question is: ‘Do such modified proteins interfere with cellular function?’ It has been difficult to assess this question at the molecular level using whole-organism models and equally frustrating to evaluate the physiological significance of such changes using classical cellular models. Tissue equivalent systems (TES) provide an opportunity for examining the molecular basis and physiological consequences of modified proteins during aging. TES are composed of differentiating and proliferating heterogeneous cell types with symbiotic cell-cell and cell-matrix interactions. They closely resemble, both morphologically and functionally, the tissues from which they were derived. Aging studies utilizing TES can provide information on modifications of protein structures, isozyme patterns, enzymes of the cellular environmental protection system and metabolic parameters which may regulate protein synthesis and degradation.
Our previous studies showed that brpA in S. mutans, which encodes a member of the LytR-CpsA-Psr family of proteins, can be cotranscribed with brpB upstream as a bicistronic operon, while the intergenic region also has strong promoter activity. To elucidate how brpA expression is regulated, the promoter regions were analyzed using PCR-based deletions and site-directed mutagenesis and a promoterless luciferase gene as a reporter. Allelic exchange mutagenesis was also used to examine genes encoding putative trans-acting factors, and the impact of such mutations on brpA expression was analyzed by reporter assays. Multiple elements in the short brpA promoter (nucleotide −1 to −344 relative to start cordon ATG) were shown to have a major impact on brpA expression, including an FNR-box, for putative binding site of an FNR-type of transcriptional regulator. When compared to the intact brpA promoter, mutations of the highly conserved nucleotides in FNR-box from TTGATgtttAcCtt to gcacagtttAcCtt resulted in 769-fold increases of luciferase activity (P<0.001), indicative of the FNR-box mediated repression as a major mechanism in regulation of brpA expression. When luciferase reporter was fused to the upstream brpBA promoter (nt −784 to −1144), luciferase activity was decreased by 4.5-fold (P<0.001) in the brpA mutant, TW14D, and by 67.7-fold (P<0.001) in the brpB mutant, JB409, as compared to the wild-type, UA159. However, no such effects were observed when the reporter gene was fused to the short brpA promoter and its derivatives. These results also suggest that brpA expression in S. mutans is auto-regulated through the upstream brpBA promoter.
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