We demonstrate that the twin arginine translocation (Tat) system contributes to bacterial resistance to cationic antimicrobial peptides (CAMPs). Our results show that a deletion at the tatC gene, which encodes a subunit of the Tat complex, caused Salmonella and Escherichia coli to become susceptible to protamine. We screened chromosomal loci that encode known and predicted Tat-dependent proteins and found that two N-acetylmuramoyl-l-alanine amidases, encoded by amiA and amiC, elevated bacterial resistance to protamine and α-helical peptides magainin 2 and melittin but not to β-sheet defensin HNP-1 and lipopeptide polymyxin B. Genetic analysis suggests that transcription of both amiA and amiC loci in Salmonella is up-regulated by the CpxR/CpxA two-component system when nlpE is overexpressed. A footprinting analysis reveals that CpxR protein can interact with amiA and amiC promoters at the CpxR box, which is localized between the predicted -10 and -35 regions but present on different strands in these two genes. In addition, our results show that activation of the CpxR/CpxA system can facilitate protamine resistance because nlpE overexpression elevates this resistance in the wild-type strain but not the cpxR deletion mutant. Thus, we uncover a new transcriptional regulation pathway in which the Cpx envelope stress response system modulates the integrity of the cell envelope in part by controlling peptidoglycan amidase activity, which confers bacterial resistance to protamine and α-helical CAMPs. Our studies have important implications for understanding transcriptional regulation of peptidoglycan metabolism and also provide new insights into the role of the bacterial envelope in CAMP resistance.
ABSTRACT We have developed a regulated delayed antigen synthesis (RDAS) system for use in recombinant attenuated Salmonella vaccine (RASV) strains to enhance immune responses by reducing the adverse effects of high-level antigen synthesis. This system includes a chromosomal repressor gene, lacI , expressed from the arabinose-regulated araC P BAD promoter. LacI serves to regulate expression from a plasmid promoter, P trc , that directs antigen synthesis. In the presence of arabinose LacI is produced, which binds to P trc , blocking antigen synthesis. In vivo , an arabinose-poor environment, the concentration of LacI decreases with each cell division, allowing increased antigen synthesis. To optimize the system and for comparison, we altered the lacI ribosome-binding site, start codon, and/or codon content to construct RDAS strains χ9095, χ9959, and χ9241, synthesizing from low to high levels of LacI, respectively, and non-RDAS strain χ9555 as a control. We evaluated this system with two test antigens, the green fluorescent protein for initial in vitro assessment and the Streptococcus pneumoniae PspA protein for validation of our system in mice. All RASV strains expressing PspA generated high antilipopolysaccharide antibody titers, indicating that expression of lacI did not interfere with the capacity to induce an immune response. Strain χ9241 induced significantly higher anti-PspA IgG and IgA antibody titers than strain χ9555, which expressed PspA constitutively. Anti-PspA antibody titers were inversely correlated to the level of LacI synthesis. Strain χ9241 also induced significantly greater protective efficacy against challenge with virulent S. pneumoniae . These results suggest that regulated delayed antigen synthesis is useful for improving immunogenicity of RASV strains.
Keywords:: antigen synthesisbiological containmentlysisrecombinant attenuated Salmonella vaccinesregulated delayed attenuationType II secretion systemType III secretion system
Ningbo municipal government existed only for short period of three years from its founding in July,1927 to January 1931 when it was abolished and integrated into Yin County.It was due to the fact that the local people abolished the municipal government,to a large extent,it was related with the bad government policy.This paper attempts to analyze the weakness of the government management in terms of Butcher Strike in 1930.
Recombinant attenuated Salmonella vaccines (RASVs) have been constructed to deliver antigens from other pathogens to induce immunity to those pathogens in vaccinated hosts. The attenuation means should ensure that the vaccine survives following vaccination to colonize lymphoid tissues without causing disease symptoms. This necessitates that attenuation and synthesis of recombinant gene encoded protective antigens do not diminish the ability of orally administered vaccines to survive stresses encountered in the gastrointestinal tract. We have eliminated these problems by using RASVs with regulated delayed expression of attenuation and regulated delayed synthesis of recombinant antigens. These changes result in RASVs that colonize effector lymphoid tissues efficiently to serve as "factories" to synthesize protective antigens that induce higher protective immune responses than achieved when using previously constructed RASVs. We have devised a biological containment system with regulated delayed lysis to preclude RASV persistence in vivo and survival if excreted. Attributes were added to reduce the mild diarrhea sometimes experienced with oral live RASVs and to ensure complete safety in newborns. These collective technologies have been used to develop a novel, low-cost, RASV-synthesizing, multiple-protective Streptococcus pneumoniae antigens that will be safe for newborns/infants and will induce protective immunity to diverse S. pneumoniae serotypes after oral immunization.
To investigate the prevalence of the HPV16 subtypes in Tongliao area in Inner Mongolia and Changchun city in Jilin province in China,the sequences of HPV16 E6 gene were amplified and sequenced from 30 HPV 16 positive cervical cancer samples isolated from Tongliao area in Inner Mongolia and Changchun city.A total of 14 variants were detected from 11 nucleotide sites in HPV16 E6 gene in 30 cervical cancer samples.The nucleotide variant T459G has not been reported previously.Phylogenetic analysis showed that all variants detected in this study belong to HPV16 E-Class.Together with the data from previous HPV16 subtype studies in China,the results indicate that HPV16 E-Clas is the most predominant in China,European subtype and East-Asian subtype distributed unregularly in different areas in China.
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