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Protein p60 encoded by theiapgene is regarded as an essential gene product ofListeria monocytogenes. Here we report, however, the successful construction of a viableiapdeletion mutant ofL. monocytogenesEGD. The mutant, which produces no p60, shows abnormal septum formation and tends to form short filaments and hooked forms during logarithmic growth. These abnormal bacterial cells break into almost normal sized single bacteria in the late-stationary-growth phase. Theiapmutant is strongly attenuated in a mouse model after intravenous injection, demonstrating the importance of p60 during infection, and the invasiveness of the Δiapmutant for 3T6 fibroblasts and Caco-2 epithelial cells is slightly reduced. Upon uptake by epithelial cells and macrophages, theiapmutant escapes from the phagosome into the cytosol with the same efficiency as the wild-type strain, and the mutant bacteria also grow intracellularly at a rate similar to that of the wild-type strain. Intracellular movement and cell-to-cell spread are drastically reduced in various cell lines, since theiap-negative bacteria fail to induce the formation of actin tails. However, the bacteria are covered with actin filaments. Most intracellular bacteria show a nonpolar and uneven distribution of ActA around the cell, in contrast to that for the wild-type strain, where ActA is concentrated at the old pole. In aniap+revertant strain that produces wild-type levels of p60, intracellular movement, cell-to-cell spread, and polar distribution of ActA are fully restored. In vitro analysis of ActA distribution on the filaments of the Δiapstrain shows that the loss of bacterial septum formation leads to ActA accumulation at the presumed division sites. In the light of data presented here and elswhere, we propose to renameiap(invasion-associated protein)cwhA(cell wall hydrolase A).

Human-monocyte-derived dendritic cells (MoDC) are very efficient in the uptake ofListeria monocytogenes, a gram-positive bacterium which is an important pathogen in humans and animals causing systemic infections with symptoms such as septicemia and meningitis. In this work, we analyzed the influence of blood plasma on the internalization ofL. monocytogenesinto human MoDC and compared the uptake ofL. monocytogeneswith that ofSalmonella entericaserovar Typhimurium andYersinia enterocolitica. While human plasma did not significantly influence the uptake of serovar Typhimurium andY. enterocoliticaby human MoDC, the efficiency of the uptake ofL. monocytogenesby these phagocytes was strongly enhanced by human plasma. In plasma-free medium the internalization ofL. monocytogeneswas very low, whereas the addition of pooled human immunoglobulins resulted in the internalization of these bacteria to a degree comparable to the highly efficient uptake observed with human plasma. All human plasma tested contained antibodies against the 60-kDa extracellular protein ofL. monocytogenes(p60), and anti-p60 antibodies were also found in the commercially available pooled immunoglobulins. Strikingly, in contrast toL. monocytogeneswild type, aniapdeletion mutant (totally deficient in p60) showed only a minor difference in the uptake by human MoDC in the presence or the absence of human plasma. These results support the assumption that antibodies against the listerial p60 protein may play an important role in Fc-receptor-mediated uptake ofL. monocytogenesby human MoDC via opsonization of the bacteria. This process may have a major impact in preventing systemic infection inL. monocytogenesin immunocompetent humans.

In this work we analyzed the roles of meningococcal lipooligosaccharide (LOS) and capsule expression in the interaction ofNeisseria meningitidiswith human dendritic cells (DC). Infection of DC with serogroup B wild-type meningococci induced a strong burst of the proinflammatory cytokines and chemokines tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8. In contrast, a serogroup B mutant strain lacking LOS expression barely led to cytokine induction, demonstrating that meningococcal LOS is the main mediator of the proinflammatory response in human DC. Sialylation of meningococcal LOS did not influence cytokine secretion by DC. However, we found the phagocytosis ofN. meningitidisby human DC to be inhibited by LOS sialylation. In addition, the expression of the meningococcal serogroup A, B, and C capsules dramatically reduced DC adherenceof N. meningitidisand phagocytosis to some extent. Hence, LOS sialylation and capsule expression are independent mechanisms protectingN. meningitidisfrom the phagocytic activity of human DC.