Consequences of a sortase A mutation in Streptococcus gordonii.

Sortase A (SrtA) is required for cell-wall anchoring of LPXTG-containing Gram-positive surface proteins. It was hypothesized, therefore, that disruption of the srtA gene would alter surface anchoring and functions of target LPXTG motif-bearing SspA and SspB proteins of Streptococcus gordonii. Mutant strains in srtA (V288srtA(-), DL1srtA(-)) were constructed in S.

Endocarditis and biofilm-associated pili of Enterococcus faecalis.

Increasing multidrug resistance in Enterococcus faecalis, a nosocomial opportunist and common cause of bacterial endocarditis, emphasizes the need for alternative therapeutic approaches such as immunotherapy or immunoprophylaxis. In an earlier study, we demonstrated the presence of antibodies in E. faecalis endocarditis patient sera to recombinant forms of 9 E.

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on enterocyte shedding of syndecan-1 and associated decreases in internalization of Listeria monocytogenes and Staphylococcus aureus.

Syndecan-1 is a heparan sulfate proteoglycan expressed on epithelia, and its ectodomain can be shed into the extracellular milieu, affecting a variety of cellular functions. Using two bacteria known to react with heparan sulfate, Listeria monocytogenes and Staphylococcus aureus, experiments were designed to clarify the effect of syndecan-1 shedding on bacterial internalization by human HT-29 enterocytes. Mature enterocytes were incubated with tumor necrosis factor (TNF)-alpha and/or interferon (IFN)-gamma for 16h prior to addition of bacteria.

Heparan sulfate proteoglycans mediate Staphylococcus aureus interactions with intestinal epithelium.

Staphylococcus aureus can be internalized by non-professional phagocytes, and may colonize the intestine in normal and antibiotic-treated individuals. Intestinal colonization may depend on the interactions of S. aureus with the intestinal epithelium.

Ability of the heparan sulfate proteoglycan syndecan-1 to participate in bacterial translocation across the intestinal epithelial barrier.

Although hundreds of microbial species reside in the human intestinal tract, comparatively few (e.g., Escherichia coli and other enterobacteria, Enterococcus faecalis, etc.

Selective inhibition by rottlerin of macropinocytosis in monocyte-derived dendritic cells.

We present here the analysis of fluid-phase endocytosis (FPE) in human blood monocytes and monocyte-derived dendritic cells (MDDC) facilitated by our serendipitous identification of rottlerin as an efficient inhibitor of dendritic cell FPE (IC(50) of 0.4 microM). Rottlerin was found to be an excellent tool for FPE analysis: rapid-acting, irreversible and selective for FPE (as opposed to receptor-mediated endocytosis) at concentrations of 3 microM and below.

Development of species-specific rDNA probes for Giardia by multiple fluorescent in situ hybridization combined with immunocytochemical identification of cyst wall antigens.

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts.

Impact of the indigenous flora in animal models of shock and sepsis.

Septicemia is currently the 10th leading cause of death in the United States, and shock and trauma patients are the source of much of the morbidity and mortality associated with septicemia. There is substantial evidence that the composition of the indigenous flora plays an important role in modulating outcome variables in animal models of shock and sepsis. Germ-free animals that lack an indigenous flora are not as susceptible to shock as their conventionally reared counterparts.

High-resolution visualization of the microbial glycocalyx with low-voltage scanning electron microscopy: dependence on cationic dyes.

The microbial glycocalyx is composed of a variety of polyanionic exopolysaccharides and plays important roles in microbial attachment to different substrata and to other cells. Here we report the successful use of low-voltage scanning electron microscopy (LVSEM) to visualize the glycocalyx in two microbial models (Klebsiella pneumoniae and Enterococcus faecalis biofilms) at high resolution, and also the dependence on fixation containing polycationic dyes for its visualization. Fixation in a paraformaldehyde-glutaraldehyde cocktail without cationic dyes was inadequate for visualizing the glycocalyx, whereas addition of various dyes (alcian blue, safranin, and ruthenium red) to the aldehyde cocktail appeared necessary for stabilization.

Evidence for adhesive activity of the ventrolateral flange in Giardia lamblia.

Giardia lamblia is an intestinal protozoan that inhabits the intestinal tract of man and other mammals by attaching to the mucosal surface via the contractile activity of an attachment organelle called the ventral adhesive disk. We have investigated the presence of other attachment mechanisms in G. lamblia trophozoites by using microfabricated substrates that sterically interfere with formation of the hypothesized "negative pressure" under the ventral adhesive disk that would mediate attachment to a substratum.

Chemokine stimulation of human peripheral blood T lymphocytes induces rapid dephosphorylation of ERM proteins, which facilitates loss of microvilli and polarization.

Lymphocyte microvilli mediate initial rolling-adhesion along endothelium but are lost during transmigration from circulation to tissue. However, the mechanism for resorption of lymphocyte microvilli remains unexplored. We show that chemokine stimulation of human peripheral blood T (PBT) cells is sufficient to induce rapid resorption of microvilli.

Hypoxia and extraintestinal dissemination of Candida albicans yeast forms.

Candida albicans is a pleomorphic fungus with budding yeast and filamentous forms, and is a frequent cause of complicating infections in patients who are postsurgical, in shock, and have trauma. Many cases of systemic candidiasis are thought to orginate from the intestine, but it is unclear if the filament or the yeast is the more invasive form. Because C.

Inhibition of in vitro attachment of Giardia trophozoites by mucin.

An enzyme-linked immunoabsorbent assay was developed to quantify the number of Giardia sp. trophozoites attached to a substratum. Trophozoites were allowed to attach for 5 or 10 min to untreated or modified substratum, or allowed to attach in the presence of cytoskeletal inhibitors.

Presence of human Giardia in domestic, farm and wild animals, and environmental samples suggests a zoonotic potential for giardiasis.

Giardia lamblia which parasitize humans belong to either of two genotypes, A or B, based on specific signature sequences in the 5' end of the small subunit (16S) ribosomal RNA (rRNA) gene. These two genotypes also were found in cysts from fecal samples of animal origin such as dogs, cats, some farm animals and wild animals. In addition, trophozoites recovered from cysts obtained from environmental samples belonged to these two genotypes as well, suggesting that the G.

Adherence of yeast and filamentous forms of Candida albicans to cultured enterocytes.

Objective: Systemic candidiasis is a major cause of complicating infections in intensive care units. Morbidity and mortality are high, even in those who receive appropriate antifungal therapy. Because the intestinal tract is considered a major portal of entry for systemic candidiasis, experiments were designed to clarify the ability of yeast and filamentous forms, as well as the INT1 gene product, to influence adherence of Candida albicans to the intestinal epithelium.