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Enterococci are major contributors of hospital-acquired infections and have emerged as important reservoirs for the dissemination of antibiotic resistance traits. The ability to form biofilms on medical devices is an important aspect of pathogenesis in the hospital environment. The Enterococcus faecalis Fsr quorum system has been shown to regulate biofilm formation through the production of gelatinase, but the mechanism has been hitherto unknown. Here we show that both gelatinase (GelE) and serine protease (SprE) contribute to biofilm formation by E. faecalis and provide clues to how the activity of these proteases governs this developmental process. Confocal imaging of biofilms suggested that GelE(-) mutants were significantly reduced in biofilm biomass compared to the parental strain, whereas the absence of SprE appeared to accelerate the progression of biofilm development. The phenotype observed in a SprE(-) mutant was linked to an observed increase in autolytic rate compared to the parental strain. Culture supernatant analysis and confocal microscopy confirmed the inability of mutants deficient in GelE to release extracellular DNA (eDNA) in planktonic and biofilm cultures, whereas cells deficient in SprE produced significantly more eDNA as a component of the biofilm matrix. DNase I treatment of E. faecalis biofilms reduced the accumulation of biofilm, implying a critical role for eDNA in biofilm development. In conclusion, our data suggest that the interplay of two secreted and coregulated proteases--GelE and SprE--is responsible for regulating autolysis and the release of high-molecular-weight eDNA, a critical component for the development of E. faecalis biofilms. 相似文献
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Merve S. Zeden Igor Kviatkovski Christopher F. Schuster Vinai C. Thomas Paul D. Fey Angelika Gründling 《Molecular microbiology》2020,113(6):1085-1100
A Staphylococcus aureus strain deleted for the c-di-AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine-betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild-type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c-di-AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c-di-AMP signalling in S. aureus. 相似文献
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Molecular basis of β-thalassemia in Thailand: analysis of β-thalassemia mutations using the polymerase chain reaction 总被引:2,自引:0,他引:2
Supan Fucharoen Goonnapa Fucharoen Winionman Sriroongrueng Vichai Laosombat Arunee Jetsrisuparb Sutthipan Prasatkaew Voravarn S. Tanphaichitr Vinai Suvatte Soodsarkorn Tuchinda Yasuyuki Fukumaki 《Human genetics》1989,84(1):41-46
Summary -Thalassemia mutations in 71 chromosomes of Thai patients from the northeast, the middle and the south of the country were investigated using dot blot hybridization of PCR (polymerase chain reaction)-amplified DNA with allelespecific oligonucleotide probes. Eight different known molecular defects were detected, at different frequencies. There was an amber mutation in codon 17, a C-T transversion at position 654 of IVS-2, a frameshift mutation between codons 71 and 72, an A-G transition at nucleotide -28 within the TATA box (known as Chinese mutations), a G-T transversion at position 1 of IVS-1 (an Indian mutation), a 4bp deletion in codons 41/42 and a G-C transversion at position 5 of IVS-1 (described as both Chinese and Indian mutations) and a Thai original mutation, an ochre mutation in codon 35. Analysis of the three unknown alleles by DNA sequencing of the cloned DNA fragment amplified by PCR revealed an A-G substitution at the second position of the codon for amino acid 19 (AAC-AGC). The analytic approach used in the present study and the characteristic distribution of mutations in each region of Thailand will prove useful for setting up a prenatal diagnosis program. 相似文献
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Lance R. Thurlow Vinai Chittezham Thomas Lynn E. Hancock 《Journal of bacteriology》2009,191(20):6203-6210
Many bacterial species produce capsular polysaccharides that contribute to pathogenesis through evasion of the host innate immune system. The gram-positive pathogen Enterococcus faecalis was previously reported to produce one of four capsule serotypes (A, B, C, or D). Previous studies describing the four capsule serotypes of E. faecalis were based on immunodetection methods; however, the underlying genetics of capsule production did not fully support these findings. Previously, it was shown that capsule production for serotype C (Maekawa type 2) was dependent on the presence of nine open reading frames (cpsC to cpsK). Using a novel genetic system, we demonstrated that seven of the nine genes in the cps operon are essential for capsule production, indicating that serotypes A and B do not make a capsular polysaccharide. In support of this observation, we showed that serotype C and D capsule polysaccharides mask lipoteichoic acid from detection by agglutinating antibodies. Furthermore, we determined that the genetic basis for the difference in antigenicity between serotypes C and D is the presence of cpsF in serotype C strains. High-pH anion-exchange chromatography with pulsed amperometric detection analysis of serotype C and D capsules indicated that cpsF is responsible for glucosylation of serotype C capsular polysaccharide in E. faecalis.Enterococcus faecalis is a gram-positive bacterium commonly found as a commensal organism in the gastrointestinal tracts of most mammals. E. faecalis is one of the leading causes of hospital-acquired urinary tract infections, bacteremia, and surgical-site infections (29). The development of multiple antibiotic resistances, including resistance to vancomycin, makes treatment of enterococcal infections difficult (11). The 2004 National Nosocomial Infections Surveillance report indicated that nearly 30% of enterococci isolated from clinical settings were resistant to vancomycin, constituting a 12% rise from the previous 5 years (26). The development of alternative therapies to treat enterococcal infections has frequently been suggested due to rising percentages of antibiotic-resistant enterococcal strains (13-15, 19).Capsular polysaccharides are major contributors to the virulence of many microorganisms. The presence of capsule allows these microbes to escape detection and clearance by the host immune system (9, 27, 30, 41). There have been several publications regarding the role of cell wall polysaccharides in the pathogenesis of enterococcal infections (10, 13, 17, 37, 43). Several attempts have been made to establish a serotyping system for E. faecalis capsular polysaccharides (16, 23, 35, 36). These serotyping schemes include differences in capsular polysaccharide antigens but are also based on differences in surface antigens, including lipoteichoic acid (16, 38). To date, only one study has linked genetic evidence with capsule production (12). Two loci that have been reported to contain putative genes for capsule production are the epa and cps operons (10, 42). The polysaccharide produced by the epa locus is thought to be the cell wall rhamnopolymer (10), but it cannot be detected on the surface of the bacterium (43). Although rhamnopolymer production is reported to be abrogated by mutation (43), the full nature of rhamnopolymer production is yet to be determined for many E. faecalis strains. Probing the genomes of serotype A and B strains with a probe specific to the cps locus, including the genes cpsA and cpsB, identified a single ClaI restriction fragment for serotypes A and B (16). However, multiple ClaI restriction fragments were identified in serotypes C and D (16), suggesting that the genes responsible for capsule production in serotypes C and D were absent in serotypes A and B. Furthermore, the hybridization pattern between serotype C and D strains indicated a single restriction fragment polymorphism, but the basis on which genes were different between the two serotypes was not fully characterized (16). Studies based on the serotyping scheme proposed by Hufnagel et al. (17) have shown that serotype C and D strains are much more resistant to opsonophagoctyosis by neutrophils in the presence of normal human serum. More recently, a study by McBride et al. indicated that serotype C clinical isolates harbored a greater repertoire of antibiotic resistance cassettes and were more likely to possess multiple virulence factors than the other serotypes, suggesting that the presence of the capsule is associated with pathogenic lineages of E. faecalis (17, 24).It is essential to understand the underlying mechanisms of capsule production in E. faecalis because of ongoing efforts to develop alternative therapies targeting capsule. Here, we used a novel vector system for creating isogenic, in-frame deletion mutants to analyze the genetic basis for capsule production and serotype specificity. Our results show that only serotype C and D strains of E. faecalis produce capsular polysaccharides, based on the observation that deletions of cpsC, cpsD, cpsE cpsG, and cpsI abolish the production of capsule. In conjunction with these observations, we also demonstrated that the presence of capsule prevents detection of lipoteichoic acid on the surface of serotype C and D strains but not on unencapsulated strains. Our data also show that CpsF is responsible for the difference in serospecificity between serotype C and D strains. 相似文献
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Yanling Yan Anna P. Shapiro Steven Haller Vinai Katragadda Lijun Liu Jiang Tian Venkatesha Basrur Deepak Malhotra Zi-jian Xie Nader G. Abraham Joseph I. Shapiro Jiang Liu 《The Journal of biological chemistry》2013,288(47):34249-34258
Cardiotonic steroids (such as ouabain) signaling through Na/K-ATPase regulate sodium reabsorption in the renal proximal tubule. We report here that reactive oxygen species are required to initiate ouabain-stimulated Na/K-ATPase·c-Src signaling. Pretreatment with the antioxidant N-acetyl-l-cysteine prevented ouabain-stimulated Na/K-ATPase·c-Src signaling, protein carbonylation, redistribution of Na/K-ATPase and sodium/proton exchanger isoform 3, and inhibition of active transepithelial 22Na+ transport. Disruption of the Na/K-ATPase·c-Src signaling complex attenuated ouabain-stimulated protein carbonylation. Ouabain-stimulated protein carbonylation is reversed after removal of ouabain, and this reversibility is largely independent of de novo protein synthesis and degradation by either the lysosome or the proteasome pathways. Furthermore, ouabain stimulated direct carbonylation of two amino acid residues in the actuator domain of the Na/K-ATPase α1 subunit. Taken together, the data indicate that carbonylation modification of the Na/K-ATPase α1 subunit is involved in a feed-forward mechanism of regulation of ouabain-mediated renal proximal tubule Na/K-ATPase signal transduction and subsequent sodium transport. 相似文献
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Kalams SA Parker S Jin X Elizaga M Metch B Wang M Hural J Lubeck M Eldridge J Cardinali M Blattner WA Sobieszczyk M Suriyanon V Kalichman A Weiner DB Baden LR;NIAID HIV Vaccine Trials Network 《PloS one》2012,7(1):e29231
Background
DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques.Methodology/Principal Findings
We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37) DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug) IL-12 DNA. However, after three doses, 44.4% (4/9) of vaccinees receiving gag DNA and intermediate dose (500 ug) of IL-12 DNA demonstrated a detectable cellular immune response.Conclusions/Significance
This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity.Trial Registration
Clinicaltrials.gov NCT00115960 NCT00111605相似文献10.
Vinai Chittezham Thomas Yasuaki Hiromasa Nathan Harms Lance Thurlow John Tomich Lynn E. Hancock 《Molecular microbiology》2009,72(4):1022-1036
Extracellular DNA (eDNA), a by-product of cell lysis, was recently established as a critical structural component of the Enterococcus faecalis biofilm matrix. Here, we describe fratricide as the governing principle behind gelatinase (GelE)-mediated cell death and eDNA release. GFP reporter assays confirmed that GBAP (gelatinase biosynthesis-activating pheromone) quorum non-responders (GelE– SprE– ) were a minority subpopulation of prey cells susceptible to the targeted fratricidal action of the quorum responsive predatorial majority (GelE+ SprE+ ). The killing action is dependent on GelE, and the GelE producer population is protected from self-destruction by the co-production of SprE as an immunity protein. Targeted gene inactivation and protein interaction studies demonstrate that extracellular proteases execute their characteristic effects following downstream interactions with the primary autolysin, AtlA. Finally, we address a mechanism by which GelE and SprE may modify the cell wall affinity of proteolytically processed AtlA resulting in either a pro- or anti-lytic outcome. 相似文献