Inhibition of bacterial swimming by heparin binding of flagellin FliC from Escherichia coli strain Nissle 1917.

Escherichia coli Nissle1917 (EcN) is a non-pathogenic probiotic strain widely used to maintain gut health, treat gastrointestinal disorders, and modulate the gut microbiome due to its anti-inflammatory and competitive exclusion effects against pathogenic bacteria. Heparin, abundant on intestinal mucosal surfaces, is a highly sulfated glycosaminoglycan primarily produced by mast cells. Currently, the interaction between EcN surface protein and heparin has remained elusive. In this study, the flagellin FliC responsible for EcN's movement was separated and characterized as a heparin binding protein by mass spectrometry (MS) analysis. The recombinant FliC protein, expressed by plasmid pET28a( +)-fliC, was further prepared to confirm the interaction between FliC and heparin. The results showed that heparin-Sepharose's ability to bind FliC was 48-fold higher than its ability to bind the negative control, bovine serum albumin (BSA). Neither the knockout of gene fliC nor the addition of heparin affects the growth of EcN, but both significantly inhibit the swimming of EcN. Adding 10 mg/ml heparin reduced the swimming diameter of the wild type and the complemented strain to 29-41% of the original, but that did not affect the swimming ability of the knockout strains. These results demonstrate that heparin interacts with EcN flagellin FliC and inhibits bacteria swimming. Exploring this interaction could improve our understanding of the relationship between hosts and microorganisms and provide a potential basis for disease treatment.

Chromosome evolution of Escherichia coli Nissle 1917 for high-level production of heparosan.

Heparosan is a crucial-polysaccharide precursor for the chemoenzymatic synthesis of heparin, a widely used anticoagulant drug. Presently, heparosan is mainly extracted with the potential risk of contamination from Escherichia coli strain K5, a pathogenic bacterium causing urinary tract infection. Here, a nonpathogenic probiotic, E. coli strain Nissle 1917 (EcN), was metabolically engineered to carry multiple copies of the 19-kb kps locus and produce heparosan to 9.1 g/L in fed-batch fermentation. Chromosome evolution driven by antibiotics was employed to amplify the kps locus, which governed the synthesis and export of heparosan from EcN at 21 mg L-1 OD-1 . The average copy number of kps locus increased from 1 to 24 copies per cell, which produced up to 104 mg L-1 OD-1 of heparosan in the shaking flask cultures of engineered strains. The following in-frame deletion of recA stabilized the recombinant duplicates of chromosomal kps locus and the productivity of heparosan in continuous culture for at least 56 generations. Fed-batch fermentation of the engineered strain EcN8 was carried out to bring the yield of heparosan up to 9.1 g/L. Heparosan from the fermentation culture was further purified at a 75% overall recovery. The structure of purified heparosan was characterized and further modified by N-sulfotransferase with 3'-phosphoadenosine-5'-phosphosulfate as the sulfo-donor. The analysis of element composition showed that heparosan was N-sulfated by over 80%. These results indicated that duplicating large DNA cassettes up to 19-kb, followed by high-cell-density fermentation, was promising in the large-scale preparation of chemicals and could be adapted to engineer other industrial-interest bacteria metabolically.

The construction of a dual-functional strain that produces both polysaccharides and sulfotransferases.

OBJECTIVES: Heparosan is used as the starting polysaccharide sulfated using sulfotransferase to generate fully elaborate heparin, a widely used clinical drug. However, the preparation of heparosan and enzymes was considered tedious since such material must be prepared in separate fermentation batches. In this study, a commonly admitted probiotic, Escherichia coli strain Nissle 1917 (EcN), was engineered to intracellularly express sulfotransferases and, simultaneously, secreting heparosan into the culture medium. RESULTS: The engineered strain EcN::T7M, carrying the λDE3 region of BL21(DE3) encoding T7 RNA polymerase, expressed the sulfotransferase domain (NST) of human N-deacetylase/N-sulfotransferase-1 (NDST-1) and the catalytic domain of mouse 3-O-sulfotransferase-1 (3-OST-1) in a flask. The fed-batch fermentation of EcN::T7M carrying the plasmid expressing NST was carried out, which brought the yield of NST to 0.21 g/L and the yield of heparosan to 0.85 g/L, respectively. Furthermore, the heparosan was purified, characterized by 1H nuclear magnetic resonance (NMR), and sulfated by NST using 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the sulfo donor. The analysis of element composition showed that over 80% of disaccharide repeats of heparosan were N-sulfated. CONCLUSIONS: These results indicate that EcN::T7M is capable of preparing sulfotransferase and heparosan at the same time. The EcN::T7M strain is also a suitable host for expressing exogenous proteins driven by tac promoter and T7 promoter.

Protein A of Staphylococcus aureus strain NCTC8325 interacted with heparin.

Heparin, known for its anticoagulant activity, is commonly used as the coatings of medical devices. The attaching of Staphylococcus aureus, a prominent human and animal pathogen, to the heparin coatings usually leads to catheter-related bloodstream infections. Hence, the study of the interaction between heparin and S. aureus surface proteins is desired. Here, we found that protein A (SpA) of S. aureus was a heparin-binding protein, contributing to the interaction between S. aureus and heparin. The cell-wall-anchored SpA was one of the most critical S. aureus virulence factors with a lysin-like motif (LysM). When SpA was mutated to remove the LysM motif, the heparin-binding capability of SpA dropped 50%. The in-frame deletion of spa also reduced the heparin-binding capability of S. aureus. There was 1.3-fold more of heparin bound to wild type S. aureus than the Δspa::Em strain. These results would help understand the host-microbe interaction and the infection by S. aureus.

Heparin stimulates biofilm formation of Escherichia coli strain Nissle 1917.

OBJECTIVES: Escherichia coli strain Nissle 1917 (EcN), a gut probiotic competing with pathogenic bacteria, has been used to attenuate various intestinal dysfunctions. Heparin is a sulfated glycosaminoglycan enriched in the human and animal intestinal mucosa, which has a close connection with bacterial biofilm formation. However, the characteristics of heparin affecting bacterial biofilm formation remain obscure. In this study, we investigated the influence of heparin and its derivatives on EcN biofilm formation. RESULTS: Here, we found that heparin stimulated EcN biofilm formation in a dose-dependent manner. With the addition of native heparin, the EcN biofilm formation increased 6.9- to 10.8-fold than that without heparin, and was 1.4-, 3.1-, 3.0-, and 3.8-fold higher than that of N-desulfated heparin (N-DS), 2-O-desulfated heparin (2-O-DS), 6-O-desulfated heparin (6-O-DS), and N-/2-O-/6-O-desulfated heparin (N-/2-O-/6-O-DS), respectively. Depolymerization of heparin produced chain-shortened heparin fragments with decreased molecular weight. The depolymerized heparins did not stimulate EcN biofilm formation. The OD570 value of EcN biofilm with the addition of chain-shortened heparin fragments was 8.7-fold lower than that of the native heparin. Furthermore, the biofilm formation of Salmonella enterica serovar Typhimurium was also investigated with the addition of heparin derivatives, and the results were consistent with that of EcN biofilm formation. CONCLUSIONS: We conclude that heparin stimulated EcN biofilm formation. Both the sulfation and chain-length of heparin contributed to the enhancement of EcN biofilm formation. This study increases the understanding of how heparin affects biofilm formation, indicating the potential role of heparin in promoting intestinal colonization of probiotics that antagonize pathogen infections.

Chemoenzymatic quantification for monitoring unpurified polysaccharide in rich medium.

The heparosan polysaccharide serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The previous quantification methods for heparosan rely on time-consuming purification or expensive instruments not readily available for many labs. Here, a chemoenzymatic approach is developed to monitor the production of heparosan in rich medium without purification. After removing the interfering small molecules by ultrafiltration, heparosan was decomposed into oligosaccharides using heparin lyase III. The oligosaccharides were separated from large molecules by ultrafiltration and quantitatively determined by the anthrone-sulfuric acid assay using a spectrophotometer. Based on the different substrate specificity of heparin lyases, the study showed that the concentration of heparosan and heparin in a mixture was discriminatively determined by the two-step chemoenzymatic assay. Furthermore, the anthrone-sulfuric acid assay was observed to be more reliable than the phenol-sulfuric acid assay under these conditions. Besides heparosan and heparin, the chemoenzymatic assay may be adapted to quantify other types of polysaccharides if the specific lyases were available.

Pb disrupts autophagic flux through inhibiting the formation and activity of lysosomes in neural cells.

Lead (Pb) has long been known as a metallic toxin to exert detrimental effects on human health, particularly on the central nervous system (CNS). Misregulated autophagy was regularly associated with multiple cellular dysfunctions and human diseases. However, the role of autophagy underlying Pb-induced neurotoxicity remains to be elucidated. In this study, we demonstrated that Pb promoted the accumulation of autophagosomes in PC12 cells, and subsequent findings revealed that this autophagosome accumulation was primarily caused by the inhibition of autophagic flux. Moreover, the results showed that Pb affected autophagy course through increasing Beclin 1 and ATG5 expression levels. Specifically, by double labeling with LC3-II (a marker of autophagosome) and LAMP-1 (a marker of lysosome), Pb impaired fusion between autophagosomes and lysosomes. Additionally, Pb exposure significantly reduced the number or size of lysosomes via decreasing the level of LAMP1, which is confirmed by the LysoTracker Red staining. Furthermore, the impairment of lysosomal activity was also signaled by the altered pH value of this acidic organelle. Overall, Pb exposure led to injuries of autophagy of neural cells through inhibiting the genesis and activity of lysosomes. The data provides insight with the neurotoxicity of Pb in a novel perspective, autophagy.

Chemoenzymatic synthesis of 3'-phosphoadenosine-5'-phosphosulfate coupling with an ATP regeneration system.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a commonly used anticoagulant drug. Previously, using ATP as the substrate, we had developed a one-pot synthesis to prepare PAPS with 47% ATP conversion efficiency. During the reaction, 47% of ATP was converted into the by-product, ADP. Here, to increase the conversion ratio of ATP to PAPS, an ATP regeneration system was developed to couple with PAPS synthesis. In the ATP regeneration system, the chemical compound, monopotassium phosphoenolpyruvate (PEP-K+), was synthesized and used as the phospho-donor. By using 3-bromopyruvic acid as the starting material, the total yield of PEP-K+ synthesis was over 50% at low cost. Then, the enzyme PykA from Escherichia coli was overexpressed, purified, and used to convert the by-product ADP into ATP. When coupled the ATP regeneration system with PAPS synthesis, the higher ratio of PEP-K+ to ADP was associated with higher ATP conversion efficiency. By using the ATP regeneration system, the conversion ratio of ATP to PAPS was increased to 98% as determined by PAMN-HPLC analysis, and 5 g of PAPS was produced in 1 L of the reaction mixture. Furthermore, the chemoenzymatic synthesized PAPS was purified and freeze-dried without observed decomposition. However, the powdery PAPS was more unstable than the PAPS sodium salt in aqueous solution at ambient temperature. This developed chemoenzymatic approach of PAPS production will contribute to the synthesis of heparin, in which PAPS is necessary as the individual sulfo-donor.

Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917.

Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription. (1)H nuclear magnetic resonance (NMR) analysis was further performed to determine the Escherichia coli strain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains.

Hydrolysis of by-product adenosine diphosphate from 3'-phosphoadenosine-5'-phosphosulfate preparation using Nudix hydrolase NudJ.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) is the obligate cosubstrate and source of the sulfonate group in the chemoenzymatic synthesis of heparin, a clinically used anticoagulant drug. Previously, we have developed a method to synthesize PAPS with Escherichia coli crude extracts, which include three overexpressed enzymes and a fourth unidentified protein. The unknown protein degrades adenosine diphosphate (ADP), the by-product of PAPS synthesis reaction. To further understand and control the process of in vitro enzymatic PAPS synthesis, we decide to identify the fourth protein and develop a defined method to synthesize PAPS using purified enzymes. Here, we show that the purified Nudix hydrolase NudJ degrades ADP at high efficiency and serves as the fourth enzyme in PAPS synthesis. Under the defined condition of PAPS synthesis, all of the 10-mM ADP is hydrolyzed to form adenosine monophosphate (AMP) in a 15-min reaction. ADP is a better substrate for NudJ than adenosine triphosphate (ATP). Most importantly, the purified NudJ does not cleave the product PAPS. The removal of ADP makes the PAPS peak more separable from other components in the chromatographic purification process. This developed enzymatic approach of PAPS production will contribute to the chemoenzymatic synthesis of heparin.

Rapeseed polysaccharides as prebiotics on growth and acidifying activity of probiotics in vitro.

In vitro digestibility, prebiotic activity and chemical composition of polysaccharides from rapeseed were deliberately studied in this paper. After preliminary treatments, two fractions of polysaccharides (RP1 and RP2) were obtained after purification by DEAE-cellulose and Sephadex G-100. Their primary structural feature and molecule weights were characterized. Furthermore, their digestibility was also evaluated by artificial gastric juice and α-amylase. Finally, their proliferative effect on bifidobacteria and lactobacilli and acid production of the resulting probiotics in vitro were investigated. The results showed that RP1 and RP2 were homogeneously protein-bound polysaccharides with molecular weights of 28.51 and 6.55 kDa, respectively. They were resistant to hydrolysis by artificial gastric juice and α-amylase. Moreover, they could also significantly stimulate the tested probiotics to proliferate and produce organic acids. These findings clearly suggest the polysaccharides from rapeseed are potential to be exploited as novel prebiotics.

Oxygen promotes biofilm formation of Shewanella putrefaciens CN32 through a diguanylate cyclase and an adhesin.

Although oxygen has been reported to regulate biofilm formation by several Shewanella species, the exact regulatory mechanism mostly remains unclear. Here, we identify a direct oxygen-sensing diguanylate cyclase (DosD) and reveal its regulatory role in biofilm formation by Shewanella putrefaciens CN32 under aerobic conditions. In vitro and in vivo analyses revealed that the activity of DosD culminates to synthesis of cyclic diguanylate (c-di-GMP) in the presence of oxygen. DosD regulates the transcription of bpfA operon which encodes seven proteins including a large repetitive adhesin BpfA and its cognate type I secretion system (TISS). Regulation of DosD in aerobic biofilms is heavily dependent on an adhesin BpfA and the TISS. This study offers an insight into the molecular mechanism of oxygen-stimulated biofilm formation by S. putrefaciens CN32.

Neutralizing the anticoagulant activity of ultra-low-molecular-weight heparins using N-acetylglucosamine 6-sulfatase.

Heparin has been the most commonly used anticoagulant drug for nearly a century. The drug heparin is generally categorized into three forms according to its molecular weight: unfractionated (UF, average molecular weight 13 000), low molecular weight (average molecular weight 5000) and ultra-low-molecular-weight heparin (ULMWH, average molecular weight 2000). An overdose of heparin may lead to very dangerous bleeding in patients. Protamine sulfate may be administered as an antidote to reverse heparin's anticoagulant effect. However, there is no effective antidote for ULMWH. In the current study, we examine the use of human N-acetylglucosamine 6-sulfatase (NG6S), expressed in Chinese hamster ovary cells, as a reversal agent for ULMWH. NG6S removes a single 6-O-sulfo group at the non-reducing end of the ULMWH Arixtra(®) (fondaparinux), effectively removing its ability to bind to antithrombin and preventing its inhibition of coagulation factor Xa. These results pave the way to developing human NG6S as an antidote for neutralizing the anticoagulant activity of ULMWHs.

Expression of heparan sulfate sulfotransferases in Kluyveromyces lactis and preparation of 3'-phosphoadenosine-5'-phosphosulfate.

Heparan sulfate (HS) belongs to a major class of glycans that perform central physiological functions. Heparin is a specialized form of HS and is a clinically used anticoagulant drug. Heparin is a natural product isolated from pig intestine. There is a strong demand to replace natural heparin with a synthetic counterpart. Although a chemoenzymatic approach has been employed to prepare synthetic heparin, the scale of the synthesis is limited by the availability of sulfotransferases and the cofactor, 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Here, we present a novel method to produce secreted forms of sulfotransferases in the yeast cells, Kluyveromyces lactis. Five sulfotransferases including N-sulfotransferase, 2-O-sulfotransferase, 3-O-sulfotransferase 1 and 6-O-sulfotransferases 1 and 3 were expressed using this method. Unlike bacterial-expressed sulfotransferases, the yeast proteins can be directly used to modify polysaccharides without laborious purification. The yeast-expressed sulfotransferases also tend to have higher specific activity and thermostability. Furthermore, we demonstrated the possibility for the gram-scale synthesis of PAPS from adenosine 5'-triphosphate at only 1/5000th of the price purchased from a commercial source. Our results pave the way to conduct the enzymatic synthesis of heparin in large quantities.

Chemoenzymatic design of heparan sulfate oligosaccharides.

Heparan sulfate is a sulfated glycan that exhibits essential physiological functions. Interrogation of the specificity of heparan sulfate-mediated activities demands a library of structurally defined oligosaccharides. Chemical synthesis of large heparan sulfate oligosaccharides remains challenging. We report the synthesis of oligosaccharides with different sulfation patterns and sizes from a disaccharide building block using glycosyltransferases, heparan sulfate C(5)-epimerase, and sulfotransferases. This method offers a generic approach to prepare heparan sulfate oligosaccharides possessing predictable structures.

LsrR-binding site recognition and regulatory characteristics in Escherichia coli AI-2 quorum sensing.

In quorum sensing (QS) process, bacteria regulate gene expression by utilizing small signaling molecules called autoinducers in response to a variety of environmental cues. Autoinducer 2 (AI-2), a QS signaling molecule proposed to be involved in interspecies communication, is produced by many species of gram-negative and gram-positive bacteria. In Escherichia coli and Salmonella typhimurium, the extracellular AI-2 is imported into the cell by a transporter encoded by the lsr operon. Upstream of the lsr operon, there is a divergently transcribed gene encoding LsrR, which was reported previously to repress the transcription of the lsr operon and itself. Here, we have demonstrated for the first time that LsrR represses the transcription of the lsr operon and itself by directly binding to their promoters using gel shift and DNase I footprinting assays. The beta-galactosidase reporter assays further suggest that two motifs in both the lsrR and lsrA promoter regions are crucial for the LsrR binding. Furthermore, in agreement with the conclusion that phosphorylated AI-2 can relieve the repression of LsrR in previous studies, our data show that phospho-AI-2 renders LsrR unable to bind to its own promoter in vitro.

An EAL domain protein and cyclic AMP contribute to the interaction between the two quorum sensing systems in Escherichia coli.

Quorum sensing (QS) is a bacterial cell-cell communication process by which bacteria communicate using extracellular signals called autoinducers. Two QS systems have been identified in Escherichia coli K-12, including an intact QS system 2 that is stimulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex and a partial QS system 1 that consists of SdiA (suppressor of cell division inhibitor) responding to signals generated by other microbial species. The relationship between QS system 1 and system 2 in E. coli, however, remains obscure. Here, we show that an EAL domain protein, encoded by ydiV, and cAMP are involved in the interaction between the two QS systems in E. coli. Expression of sdiA and ydiV is inhibited by glucose. SdiA binds to the ydiV promoter region in a dose-dependent, but nonspecific, manner; extracellular autoinducer 1 from other species stimulates ydiV expression in an sdiA-dependent manner. Furthermore, we discovered that the double sdiA-ydiV mutation, but not the single mutation, causes a 2-fold decrease in intracellular cAMP concentration that leads to the inhibition of QS system 2. These results indicate that signaling pathways that respond to important environmental cues, such as autoinducers and glucose, are linked together for their control in E. coli.