In this study, we investigated the transcriptomic response of Streptococcus pneumoniae D39 to sialic acid (N-acetylneuraminic acid [Neu5Ac]). Transcriptome comparison of wild-type D39 grown in M17 medium with and without sialic acid revealed the elevated expression of various genes and operons, including the nan gene cluster (nan operon I and nanA gene). Our microarray analysis and promoter-lacZ fusion studies showed that the transcriptional regulator NanR acts as a transcriptional activator of nan operon I and the nanA gene in the presence of sialic acid. The putative regulatory site of NanR in the promoter region of nan operon I is predicted and confirmed by promoter truncation experiments. Furthermore, the role of CcpA in the regulation of the nan gene cluster is demonstrated through microarray analysis and promoter-lacZ fusion studies, suggesting that in the presence of sialic acid and glucose, CcpA represses the expression of nan operon I while the expression of the nanA gene is CcpA independent.T he low-GC Gram-positive bacterium Streptococcus pneumoniae is a major human pathogen and the causal agent of many diseases, including pneumonia, sepsis, meningitis, otitis media, and conjunctivitis, which result in over a million deaths each year worldwide (1, 2). S. pneumoniae colonizes the human nasopharynx during the first few months of life (3). For survival in the host, bacteria rely not only on the different virulence factors they possess but also on the appropriate use of nutrients available in their habitat (4, 5). The ability of S. pneumoniae to utilize a variety of carbohydrate sources is one of the crucial factors in successful colonization and in causing pneumococcal infections (6). Metabolic selection enables a bacterium to choose a preferred source of carbon over a nonpreferred one through a mechanism called carbon catabolite repression (CCR) (5). CcpA (carbon catabolite protein A) is a transcription factor that mediates CCR in the presence of a preferred sugar source, e.g., glucose, by binding to catabolite repression elements (cre boxes) found in the promoter regions of CcpA-targeted genes (5, 7-9). Despite the importance of carbohydrates in the pathogenesis of S. pneumoniae, research concerning metabolic pathways of S. pneumoniae still demands more attention.Sialic acid is one of the most important carbohydrates for S. pneumoniae, since it plays a vital role as a carbon/energy source, a receptor for adhesion and invasion, and a molecular signal for the promotion of biofilm formation, nasopharyngeal carriage, and invasion of the lungs (10). It has been shown that S. pneumoniae can utilize sialic acid as a carbon source, which results in improved pneumococcal biofilm formation in vitro, at concentrations comparable to those of free sialic acid in human saliva (11,12). Of the 43 known naturally occurring derivatives of the nine-carbon sugar neuraminic acid, N-acetylneuraminic acid (Neu5Ac) is the only one found in humans (13). Another form of sialic acid is N-glycolylneuraminic acid (Neu5Gc), which i...
Here, we analyze the transcriptomic response of Streptococcus pneumoniae D39 to N-acetylgalactosamine (NAGa). Transcriptome comparison of S. pneumoniae D39 grown in NAGaM17 (0.5% NAGa + M17) to that grown in GM17 (0.5% Glucose + M17) revealed the elevated expression of various carbon metabolic genes/operons, including a PTS operon (denoted here as the aga operon), which is putatively involved in NAGa transport and utilization, in the presence of NAGa. We further studied the role of a GntR-family transcriptional regulator (denoted here as AgaR) in the regulation of aga operon. Our transcriptome and RT-PCR data suggest the role of AgaR as a transcriptional repressor of the aga operon. We predicted a 20-bp operator site of AagR (5′-ATAATTAATATAACAACAAA-3′) in the promoter region of the aga operon (PbgaC), which was further verified by mutating the AgaR operator site in the respective promoter. The role of CcpA in the additional regulation of the aga operon was elucidated by further transcriptome analyses and confirmed by quantitative RT-PCR.
Background Bacillus subtilis is widely used as a cell factory for numerous heterologous proteins of commercial value and medical interest. To explore the possibility of further enhancing the secretion potential of this model bacterium, a library of engineered strains with modified cell surface components was constructed, and the corresponding influences on protein secretion were investigated by analyzing the secretion of α-amylase variants with either low-, neutral- or high- isoelectric points (pI).ResultsRelative to the wild-type strain, the presence of overall anionic membrane phospholipids (phosphatidylglycerol and cardiolipin) increased dramatically in the PssA-, ClsA- and double KO mutants, which resulted in an up to 47% higher secretion of α-amylase. Additionally, we demonstrated that the appropriate net charge of secreted targets (AmyTS-23, AmyBs and AmyBm) was beneficial for secretion efficiency as well.ConclusionsIn B. subtilis, the characteristics of cell membrane phospholipid bilayer and the pIs of heterologous α-amylases appear to be important for their secretion efficiency. These two factors can be engineered to reduce the electrostatic interaction between each other during the secretion process, which finally leads to a better secretion yield of α-amylases.
Background/Introduction: Plasma protein binding (PPB) continues to be a key aspect of antibiotic development and clinical use. PPB is essential to understand several properties of drug candidates, including antimicrobial activity, drug-drug interaction, drug clearance, volume of distribution, and therapeutic index. Focus areas of the review: In this review, we discuss the basics of PPB, including the main drug binding proteins i.e., Albumin and α-1-acid glycoprotein (AAG). Furthermore, we present the effects of PPB on the antimicrobial activity of antibiotics and the current role of PPB in in vitro pharmacodynamic (PD) models of antibiotics. Moreover, the effect of PPB on the PK/PD of antibiotics has been discussed in this review. A key aspect of this paper is a concise evaluation of PPB between animal species (dog, rat, mouse, rabbit and monkey) and humans. Our statistical analysis of the data available in the literature suggests a significant difference between antibiotic binding in humans and that of dogs or mice, with the majority of measurements from the pre-clinical species falling within five-fold of the human plasma value. Conversely, no significant difference in binding was found between humans and rats, rabbits, or monkeys. This information may be helpful for drug researchers to select the most relevant animal species in which the metabolism of a compound can be studied for extrapolating the results to humans. Furthermore, state-of-the-art methods for determining PPB such as equilibrium dialysis, ultracentrifugation, microdialysis, gel filtration, chromatographic methods and fluorescence spectroscopy are highlighted with their advantages and disadvantages.
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