Structure-function relationships of periplasmic membrane-derived oligosaccharides in salmonella growth and virulence

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Membrane-derived oligosaccharides (MDO) consist of branched substituted β-linked sugar chains that are present in the periplasmic space of Escherichia coli and other gram-negative bacteria. Their common features are the presence of glucose as a major constituent sugar and their increased levels in low-osmolarity media. In several phytopathogenic bacteria, mutants defective in MDO synthesis failed to incite disease on the host plant. Very little is known about the role of MDO from Salmonella in virulence and osmotolerance. I have studied the structure-function relationship of MDO to understand if they play a role in Salmonella growth and virulence.

MDO defective mutants of Salmonella Typhimurium were generated using a gene specific mutagenesis protocol and MDO were isolated and their glycosyl composition analyzed. The fractions containing the major peak from Sephadex G-10 gel filtration chromatography were pooled and subjected to DEAE-cellulose anion exchange chromatography to separate charged and neutral MDO. Compositional analysis revealed that MDO of wild-type consist of 94% glucosyl residues (hereafter referred to as glucose) in Salmonella Typhimurium FIRN while MDO of the delta mdoG mutant was comprised of only 24% glucose. Rhamnose, mannose, and galactose accounted for the rest. We also found that MDO composition varies in different chromosomal backgrounds. For example, glucose accounted for 41% of sugar residues in MDO of Salmonella Typhimurium SL1344. This proportion was further reduced to 24% in the delta mdoG mutant.

Salmonella Typhimurium delta mdoG mutants (in FIRN as well as SL1344 chromosomal backgrounds) displayed reduced virulence in mice. Salmonella Typhimurium SL1344 delta mdoG mutant strain was recovered from the intestinal tissues and systemic tissues at a lower frequency than its parental wild-type strain and displayed a reduced ability of intracellular replication in macrophages. This defect in the delta mdoG mutant could be associated with the altered MDO composition. The delta mdoG mutant also invaded macrophages at a reduced efficiency and showed lower respiration rate under conditions mimicking acidic environments, such as stomach and phagosomes (pH 5.0). Correspondingly ATP level in the delta mdoG mutant was significantly reduced compared to the wild-type. These results support an important role for MDO in the virulence of S. Typhimurium.

In competition assays using a mouse host, the delta mdoG mutant had a reduced capacity to colonize the mouse tissues. On the contrary, competitive assays on laboratory media showed that the mdoG mutation enhanced the growth of bacteria. A mixed pictured emerged when competition assays were performed by artificially inoculating fresh-cut produce. On tomato and cucumber, the wild-type cells emerged as dominant population after 3 days of growth, while no one strain dominated during the growth on honeydews, cantaloupes, watermelons, as well as acidic fruits such as apples. Together, these data demonstrate that specific wild-type MDO are required for efficient colonization and optimal virulence in mice. For environmental survival under different niches, no evidence was found for a specific need for MDO with particular sugar composition. Enteric pathogens with altered MDO (and reduced virulence) may serve as better "live vaccines".