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The dlt operon of Gram-positive bacteria comprises four genes (dltA, dltB, dltC and dltD) that catalyse the incorporation of D-alanine residues into the cell wall-associated lipoteichoic acids (LTAs). In this work, we characterized the dlt operon of Listeria monocytogenes and constructed a D-Ala-deficient LTA mutant by inactivating the first gene (dltA) of this operon. The DltA- mutant did not show any morphological alterations and its growth rate was similar to that of the wild-type strain. However, it exhibited an increased susceptibility to the cationic peptides colistin, nisin and polymyxin B. The virulence of the DltA- mutant was severely impaired in a mouse infection model (4 log increase in the LD50) and, in vitro, the adherence of the mutant to various cell lines (murine bone marrow-derived macrophages and hepatocytes and a human epithelial cell line) was strongly restricted, although the amounts of surface proteins implicated in virulence (ActA, InlA and InlB) remains unaffected. We suggest that the decreased adherence of the DltA- mutant to non-phagocytic and phagocytic cells might be as a result of the increased electronegativity of its charge surface and/or the presence at the bacterial surface of adhesins possessing altered binding activities. These results show that the D-alanylation of the LTAs contributes to the virulence of the intracellular pathogen L. monocytogenes.

Peripheral blood neutrophils from germ-free (GF), specific-pathogen-free (SPF) and conventional (CV) rats were compared. Besides neutropenia and impaired superoxide anion generation as previously reported, it was found that GF rats had lower phagocytic function (70%) and generated less nitric oxide than the other rats. GF and SPF rats were injected with recombinant human granulocyte-colony-stimulating factor (rhG-CSF), or were transferred to natural environment. It was found that total number, phagocytosis, intracellular killing, ratio of phagocytosis versus killing (killing efficiency) and nitric oxide production induced by recombinant rat interferon-gamma (rrIFN-gamma) were normalized upon injection of rhG-CSF. These results indicate that rhG-CSF may stimulate neutrophil production and induce the expression of neutrophil receptors for phagocytosis and nitric oxide production in GF rats. Although lower than in CV rats, the level of superoxide produced was sufficient for normal neutrophil-killing efficiency in SPF and GF rats. In SPF rats, this could be amended by exposure to natural environment. However, neither rhG-CSF injection nor transfer to natural environment could increase the generation of superoxide anion in GF rats.