Chemoenzymatic synthesis of sialylated lactuloses and their inhibitory effects on <i>Staphylococcus aureus</i>

<div><p>Background</p><p>Sialylated glycoconjugates play important roles in physiological and pathological processes. However, available sialylated oligosaccharides source is limited which is a barrier to study their biological roles. This work reports an efficient approach to produce sialic acid-modified lactuloses and investigates their inhibitory effects on <i>Staphylococcus aureus</i> (<i>S</i>. <i>aureus</i>).</p><p>Methods</p><p>A one-pot two-enzyme (OPTE) sialylation system was used to efficiently synthesize sialylated lactuloses. Silica gel flash chromatography column was employed to purify the sialylated products. The purity and identity of the product structures were confirmed with mass spectrometry (MS) and nuclear magnetic resonance (NMR). The inhibitory effect of sialylated lactuloses against <i>S</i>. <i>aureus</i> was evaluated by using microplate assay, fluorescence microscopy, DAPI (4',6-diamidino-2-phenylindole) fluorescence staining and protein leakage quantification.</p><p>Results</p><p>Neu5Ac-containing sialylated lactuloses with either α2,3- or α2,6-linkages were efficiently synthesized via an efficient OPTE sialylation system using α-2,3-sialyltransferase or α-2,6-sialyltransferase, respectively. Neu5Ac-α2,3-lactulose and Neu5Ac-α2,6-lactulose significantly inhibited the growth of <i>S</i>. <i>aureus</i>. Fluorescence microscopy and DAPI fluorescence staining indicated that the sialylated lactuloses might disrupt nucleic acid synthesis of <i>S</i>. <i>aureus</i>.</p><p>Conclusions</p><p>Neu5Ac-containing sialylated lactuloses had higher antibacterial activity against <i>S</i>. <i>aureus</i> than non-sialylated lactulose. The inhibitory effect of Neu5Ac-α2,3-lactulose was superior to that of Neu5Ac-α2,6-lactulose. The sialylated lactuloses might inhibit <i>S</i>. <i>aureus</i> by causing cell membrane leakage and disrupting nucleic acid synthesis.</p></div

Protein leakage in <i>S</i>. <i>aureus</i> cells exposed to α2,3-linked and α2,6-linked sialyllactuloses.

<p>Protein leakage in <i>S</i>. <i>aureus</i> cells exposed to α2,3-linked and α2,6-linked sialyllactuloses.</p

The bacterial growing curves of <i>S</i>. <i>aureus</i> in modified medium containing α2,3-linked and α2,6-linked sialyllactuloses.

<p>The control strain was not added sialyllactulose. The results are the mean values obtained from three separate experiments. Error bars represent the standard deviation.</p

Changes of fluorescence intensity of <i>S</i>. <i>aureus</i> strains.

<p>a, b, c and d were the samples with different carbohydrates. a, control; b, lactulose; c, α2,3-linked sialyllactulose; d, α2,6-linked sialyllactulose.</p

Mass spectrum of purified Neu5Ac-α2,3-lactulose and Neu5Ac-α2,6-lactulose.

<p>Mass spectrum of purified Neu5Ac-α2,3-lactulose and Neu5Ac-α2,6-lactulose.</p

Changes of DNA content of <i>S</i>. <i>aureus with</i> α2,3-linked and α2,6-linked sialyllactuloses.

<p>Changes of DNA content of <i>S</i>. <i>aureus with</i> α2,3-linked and α2,6-linked sialyllactuloses.</p