Risk Management in the Water Industry

Extraordinary events have always threatened us. The continuity of drinking water supplies cannot be exposed to such risks. When dimensioning water supply systems, it is necessary to consider the use of preventive measures, which include risk management. The following article is the basic scope of this issue and presents risk management procedures in the water industry

Innate Immune Recognition: Implications for the Interaction of Francisella tularensis with the Host Immune System

The intracellular bacterial pathogen Francisella tularensis causes serious infectious disease in humans and animals. Moreover, F. tularensis, a highly infectious pathogen, poses a major concern for the public as a bacterium classified under Category A of bioterrorism agents. Unfortunately, research has so far failed to develop effective vaccines, due in part to the fact that the pathogenesis of intracellular bacteria is not fully understood and in part to gaps in our understanding of innate immune recognition processes leading to the induction of adaptive immune response. Recent evidence supports the concept that immune response to external stimuli in the form of bacteria is guided by the primary interaction of the bacterium with the host cell. Based on data from different Francisella models, we present here the basic paradigms of the emerging innate immune recognition concept. According to this concept, the type of cell and its receptor(s) that initially interact with the target constitute the first signaling window; the signals produced in the course of primary interaction of the target with a reacting cell act in a paracrine manner; and the innate immune recognition process as a whole consists in a series of signaling windows modulating adaptive immune response. Finally, the host, in the strict sense, is the interacting cell

An Attenuated Strain of the Facultative Intracellular Bacterium Francisella tularensis Can Escape the Phagosome of Monocytic Cells

The facultative intracellular bacterium Francisella tularensis is a highly virulent and contagious organism, and little is known about its intracellular survival mechanisms. We studied the intracellular localization of the attenuated human vaccine strain, F. tularensis LVS, in adherent mouse peritoneal cells, in mouse macrophage-like cell line J774A.1, and in human macrophage cell line THP-1. Confocal microscopy of infected J774A.1 cells indicated that during the first hour of infection the bacteria colocalized with the late endosomal-lysosomal glycoprotein LAMP-1, but within 3 h this colocalization decreased significantly from approximately 60% to 30%. Transmission electron microscopy revealed that >90% of bacteria were not enclosed by a phagosomal membrane after 2 h of infection, and some bacteria were in vacuoles that were only partially surrounded by a limiting membrane. Similar findings were obtained with all three host cell types. Immunoelectron microscopy performed with an F. tularensis LVS-specific polyclonal rabbit antiserum showed that the antiserum stained a thick, evenly distributed capsule-like material in bacteria grown in broth. In contrast, intracellular F. tularensis LVS cells were only marginally stained with this antiserum. Instead, most of the immunoreactive material was diffusely localized in the phagosomes or was associated with the phagosomal membrane. Our findings indicate that F. tularensis LVS is able to escape from the phagosomes of macrophages via a mechanism that may involve degradation of the phagosomal membrane

Entry of <i>Francisella tularensis</i> into Murine B Cells: The Role of B Cell Receptors and Complement Receptors

<div><p><i>Francisella tularensis</i>, the etiological agent of tularemia, is an intracellular pathogen that dominantly infects and proliferates inside phagocytic cells but can be seen also in non-phagocytic cells, including B cells. Although protective immunity is known to be almost exclusively associated with the type 1 pathway of cellular immunity, a significant role of B cells in immune responses already has been demonstrated. Whether their role is associated with antibody-dependent or antibody-independent B cell functions is not yet fully understood. The character of early events during B cell–pathogen interaction may determine the type of B cell response regulating the induction of adaptive immunity. We used fluorescence microscopy and flow cytometry to identify the basic requirements for the entry of <i>F</i>. <i>tularensis</i> into B cells within <i>in vivo</i> and <i>in vitro</i> infection models. Here, we present data showing that <i>Francisella tularensis</i> subsp. <i>holarctica</i> strain LVS significantly infects individual subsets of murine peritoneal B cells early after infection. Depending on a given B cell subset, uptake of <i>Francisella</i> into B cells is mediated by B cell receptors (BCRs) with or without complement receptor CR1/2. However, <i>F</i>. <i>tularensis</i> strain FSC200 Δ<i>iglC</i> and Δ<i>ftdsbA</i> deletion mutants are defective in the ability to enter B cells. Once internalized into B cells, <i>F</i>. <i>tularensis</i> LVS intracellular trafficking occurs along the endosomal pathway, albeit without significant multiplication. The results strongly suggest that BCRs alone within the B-1a subset can ensure the internalization process while the BCRs on B-1b and B-2 cells need co-signaling from the co receptor containing CR1/2 to initiate <i>F</i>. <i>tularensis</i> engulfment. In this case, fluidity of the surface cell membrane is a prerequisite for the bacteria’s internalization. The results substantially underline the functional heterogeneity of B cell subsets in relation to <i>F</i>. <i>tularensis</i>.</p></div

Deletion mutant <i>F</i>. <i>tularensis</i> strains failed to enter the A20 cells.

<p>A20 cells were infected with wild type <i>F</i>. <i>tularensis</i> FSC200 (FSC200), with deletion mutant <i>F</i>. <i>tularensis</i> FSC200 Δ<i>ftdsbA</i> (FSC200 ΔftdsbA), and with deletion mutant <i>F</i>. <i>tularensis</i> FSC200 Δ<i>iglC</i> (FSC200 ΔiglC), respectively, at MOI 500. The infected cells were determined by florescent microscopy. The cells were stained with DAPI to visualize nuclei and with rabbit anti-<i>F</i>. <i>tularensis sera</i> and goat anti-rabbit secondary antibody conjugated with Alexa Fluor 488 to visualize <i>F</i>. <i>tularensis</i>. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed <i>t</i>-test was used to test for significant differences between FSC200 and FSC200 ΔftdsbA and FSC200 ΔiglC. (*** <i>P</i> < 0.001). Results shown from one experiment are representative of three independent experiments.</p

<i>F</i>. <i>tularensis</i> infecting subsets of B cells <i>in vitro</i>.

<p>Subsets of B cells were infected for 3 h with unopsonized <i>F</i>. <i>tularensis</i> LVS/GFP (GFP), <i>F</i>. <i>tularensis</i> LVS/GFP opsonized with fresh un-inactivated serum (GFP+C) from naïve mice, and bacteria opsonized with heat-inactivated immune sera (GFP+Ab). The proportions of infected CD19⁺ cells from all measured cells and of infected B-1a, B-1b, and B-2 cells from CD19⁺ cells were measured by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed <i>t</i>-test was used to test for significant differences between GFP and GFP+C and GFP+Ab (*** <i>P</i> < 0.001, ** <i>P</i> < 0.01, * <i>P</i> < 0.05). Results shown from one experiment are representative of three independent experiments.</p

Disturbance of lipid rafts.

<p>For disturbing lipid rafts, the cholesterol-binding agent filipin or methyl-beta cyclodextrin (Cyclodex) was used. The peritoneal B cells were pretreated with 10 μg/mL filipin or 10 mM cyclodextrin and consequently infected with <b>(A)</b><i>F</i>. <i>tularensis</i> LVS/GFP or <b>(B)</b> opsonized <i>F</i>. <i>tularensis</i> LVS/GFP with complement. Entry into all B cells (CD19⁺) and individual B cell subsets was detected by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed <i>t</i>-test was used to test for significant differences between untreated B cells and cyclodextrin- or filipin-treated cells (*** <i>P</i> < 0.001). Results shown from one experiment are representative of three independent experiments.</p

Intracellular trafficking.

<p>A20 mouse B cell line (1 x 10⁶ per well in total volume 0.5 mL) was infected with <i>F</i>. <i>tularensis</i> LVS (MOI 500). Cells were infected for 5, 15 and 30 min, as well as 1 and 2 h. To identify intracellular trafficking, endosomal/lysosomal membrane markers EEA1, LAMP-1, and Cathepsin D were used for determining colocalization of these markers with <i>F</i>. <i>tularensis</i> LVS by fluorescent microscopy. Error bars indicate SD around the means of samples obtained from three independent experiments.</p

Blocking of FcγR.

<p>Peritoneal cells were incubated with the antibody against CD16/32 (dFcRg). Thereafter, cells were infected with <i>F</i>. <i>tularensis</i> LVS/GFP (GFP), <i>F</i>. <i>tularensis</i> LVS/GFP opsonized with antibodies (GFP+Ab), and <i>F</i>. <i>tularensis</i> LVS/GFP opsonized with murine fresh serum and antibodies (dFcRg+GFP+Ab+C) at MOI 500. Entry into all B cells (CD19⁺) and individual B cell subsets was detected 3 h after infection by flow cytometry. Error bars indicate SD around the means of samples processed in triplicate. Two-tailed <i>t</i>-test was used to test for significant differences between GFP and GFP+Ab and between GFP+Ab and dFcRg+GFP+Ab+C (*** <i>P</i> < 0.001, ** <i>P</i> < 0.01). Results shown from one experiment are representative of three independent experiments</p

Fluorescent microscopy.

<p>The representative picture was chosen to show the difference in the numbers of A20 cells infected with <b>(A)</b> unopsonized <i>F</i>. <i>tularensis</i> LVS/GFP bacteria, <b>(B)</b><i>F</i>. <i>tularensis</i> LVS/GFP opsonized with murine fresh serum, and <b>(C)</b> bacteria opsonized with immune sera. A20 cells in total volume 0.5 mL (1 x 10⁶ cells per well) were infected with <i>F</i>. <i>tularensis</i> LVS/GFP at MOI 500 for 3 h. The cell nuclei were stained with DAPI. Note: The number of infected cells was counted using flow cytometry.</p