Albert Haas

Insights into the Mode of Action of Chitosan as an Antibacterial Compound

ABSTRACT Chitosan is a polysaccharide biopolymer that combines a unique set of versatile physicochemical and biological characteristics which allow for a wide range of applications. Although its antimicrobial activity is well documented, its mode of action has hitherto remained only vaguely defined. In this work we investigated the antimicrobial mode of action of chitosan using a combination of approaches, including in vitro assays, killing kinetics, cellular leakage measurements, membrane potential estimations, and electron microscopy, in addition to transcriptional response analysis. Chitosan, whose antimicrobial activity was influenced by several factors, exhibited a dose-dependent growth-inhibitory effect. A simultaneous permeabilization of the cell membrane to small cellular components, coupled to a significant membrane depolarization, was detected. A concomitant interference with cell wall biosynthesis was not observed. Chitosan treatment of Staphylococcus simulans 22 cells did not give rise to cell wall lysis; the cell membrane also remained intact. Analysis of transcriptional response data revealed that chitosan treatment leads to multiple changes in the expression profiles of Staphylococcus aureus SG511 genes involved in the regulation of stress and autolysis, as well as genes associated with energy metabolism. Finally, a possible mechanism for chitosans activity is postulated. Although we contend that there might not be a single classical target that would explain chitosans antimicrobial action, we speculate that binding of chitosan to teichoic acids, coupled with a potential extraction of membrane lipids (predominantly lipoteichoic acid) results in a sequence of events, ultimately leading to bacterial death.

The phagosome: compartment with a license to kill.

Phagosomes are fascinating subcellular structures. After all, there are only a few compartments that are born before our very eyes and whose development we can follow in a light microscope until their contents disintegrate and are completely absorbed. Yet, some phagosomes are taken advantage of by pathogenic microorganisms, which change their fate. Research into phagosome biogenesis has flourished in recent years – the purpose of this review is to give a glimpse of where this research stands, with emphasis on the cell biology of macrophage phagosomes, on new model organisms for the study of phagosome biogenesis and on intracellular pathogens and their interference with normal phagosome function.

Cellular Mechanotransduction Relies on Tension-Induced and Chaperone-Assisted Autophagy

Mechanical tension is an ever-present physiological stimulus essential for the development and homeostasis of locomotory, cardiovascular, respiratory, and urogenital systems. Tension sensing contributes to stem cell differentiation, immune cell recruitment, and tumorigenesis. Yet, how mechanical signals are transduced inside cells remains poorly understood. Here, we identify chaperone-assisted selective autophagy (CASA) as a tension-induced autophagy pathway essential for mechanotransduction in muscle and immune cells. The CASA complex, comprised of the molecular chaperones Hsc70 and HspB8 and the cochaperone BAG3, senses the mechanical unfolding of the actin-crosslinking protein filamin. Together with the chaperone-associated ubiquitin ligase CHIP, the complex initiates the ubiquitin-dependent autophagic sorting of damaged filamin to lysosomes for degradation. Autophagosome formation during CASA depends on an interaction of BAG3 with synaptopodin-2 (SYNPO2). This interaction is mediated by the BAG3 WW domain and facilitates cooperation with an autophagosome membrane fusion complex. BAG3 also utilizes its WW domain to engage in YAP/TAZ signaling. Via this pathway, BAG3 stimulates filamin transcription to maintain actin anchoring and crosslinking under mechanical tension. By integrating tension sensing, autophagosome formation, and transcription regulation during mechanotransduction, the CASA machinery ensures tissue homeostasis and regulates fundamental cellular processes such as adhesion, migration, and proliferation.

Delay of phagosome maturation by a mycobacterial lipid is reversed by nitric oxide

Mycobacterium tuberculosis is a facultative intracellular pathogen that inhibits phagosome maturation in macrophages thereby securing survival and growth. Mycobacteria reside in an early endocytic compartment of near‐neutral pH where they upregulate production of complex glycolipids such as trehalose dimycolate. Here, we report that trehalose dimycolate coated onto beads increased the bead retention in early phagosomes, i.e. at a similar stage as viable mycobacteria. Thus, a single mycobacterial lipid sufficed to divert phagosome maturation and likely contributes to mycobacterial survival in macrophages. Previous studies showed that activated macrophages promote maturation of mycobacterial phagosomes and eliminate mycobacteria through bactericidal effectors including nitric oxide generated by inducible nitric‐oxide synthase. We show that deceleration of bead phagosome maturation by trehalose dimycolate was abolished in immune‐activated wild type, but not in activated nitric‐oxide synthase‐deficient macrophages, nor when hydroxyl groups of trehalose dimycolate were chemically modified by reactive nitrogen intermediates. Thus, specific host defence effectors of activated macrophages directly target a specific virulence function of mycobacteria.

Maturation of Rhodococcus equi‐Containing Vacuoles is Arrested After Completion of the Early Endosome Stage

Rhodococcus equi is a facultative intracellular bacterium that can cause bronchopneumonia in foals and AIDS patients. Here, we have analyzed R. equi‐containing vacuoles (RCVs) in murine macrophages by confocal laser scanning microscopy, by transmission electron microscopy and by immunochemistry upon purification. We show that RCVs progress normally through the early stages of phagosome maturation acquiring PI(3)P, early endosome antigen‐1, and Rab5, and loosing all or much of them within minutes. Although mature RCVs possess the normally late endocytic markers, lysosome‐associated membrane proteins, lysobisphosphatidic acid and Rab7, they lack other hallmark features of late endocytic organelles such as possession of cathepsin D, acid β‐glucuronidase, proton‐pumping ATPase and the ability to fuse with prelabeled lysosomes. Bacterial strains possessing a virulence‐associated plasmid maintain a nonacidified compartment for 48 h, whereas isogenic strains lacking such plasmids acidify progressively. In summary, RCVs represent a novel phagosome maturation stage positioned after completion of the early endosome stage and before reaching a fully mature late endosome compartment. In addition, vacuole biogenesis can be influenced by bacterial plasmids.

Secretive bacterial pathogens and the secretory pathway.

Eukaryotic cells possess two extensive endomembrane systems, each consisting of several sub‐compartments connected by vesicular trafficking. One of these systems, the endocytic pathway, serves incoming traffic, and the other system, the secretory pathway (SP), is responsible for surface‐bound traffic of intracellularly formed vesicles. Compartments derived of either system can be colonized by intracellular pathogens. In this review, we discuss the interactions between the SP and prominent intracellular bacterial pathogens of the genera Legionella, Brucella, Chlamydia and Salmonella. We emphasize secreted bacterial effector proteins, which directly manipulate host components of this pathway.

Molecular and infection biology of the horse pathogen Rhodococcus equi

The soil actinomycete Rhodococcus equi is a pulmonary pathogen of young horses and AIDS patients. As a facultative intracellular bacterium, R. equi survives and multiplies in macrophages and establishes its specific niche inside the host cell. Recent research into chromosomal virulence factors and into the role of virulence plasmids in infection and host tropism has presented novel aspects of R. equi infection biology and pathogenicity. This review will focus on new findings in R. equi biology, the trafficking of R. equi-containing vacuoles inside host cells, factors involved in virulence and host resistance and on host-pathogen interaction on organismal and cellular levels.

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

Endolysosomal organelles play a key role in trafficking, breakdown and receptor-mediated recycling of different macromolecules such as low-density lipoprotein (LDL)-cholesterol, epithelial growth factor (EGF) or transferrin. Here we examine the role of two-pore channel (TPC) 2, an endolysosomal cation channel, in these processes. Embryonic mouse fibroblasts and hepatocytes lacking TPC2 display a profound impairment of LDL-cholesterol and EGF/EGF-receptor trafficking. Mechanistically, both defects can be attributed to a dysfunction of the endolysosomal degradation pathway most likely on the level of late endosome to lysosome fusion. Importantly, endolysosomal acidification or lysosomal enzyme function are normal in TPC2-deficient cells. TPC2-deficient mice are highly susceptible to hepatic cholesterol overload and liver damage consistent with non-alcoholic fatty liver hepatitis. These findings indicate reduced metabolic reserve of hepatic cholesterol handling. Our results suggest that TPC2 plays a crucial role in trafficking in the endolysosomal degradation pathway and, thus, is potentially involved in the homoeostatic control of many macromolecules and cell metabolites.

Necrotic Death of Rhodococcus equi-Infected Macrophages Is Regulated by Virulence-Associated Plasmids

ABSTRACT Rhodococcus equi is a gram-positive intracellular pathogen that can cause severe bronchopneumonia in foals and AIDS patients. It has been reported that advanced infection of foals is characterized by tissue necrosis, coinciding with the presence of degenerate bacteria-laden macrophages. Here, we report that the possession of the VapA-expressing plasmid, which has been previously correlated with a high level of virulence for foals and mice, strongly increases cytotoxicity of R. equi for murine macrophage-like (J774E) cells. Isolates containing different, VapB-expressing plasmids are less virulent and also have a lower cytotoxic potential. Isogenic strains lacking either plasmid are avirulent and have a very low cytotoxic potential. We show, using fluorescence-activated cell sorter analysis (annexin V/7-amino-actinomycin D and sub-G1-analysis), Western blotting [poly(ADP-ribose) polymerase processing analysis], and electron microscopy (macrophage and nucleus morphologies) that the deaths of murine macrophages are the result of necrotic rather than apoptotic events. We demonstrate that the bacteria must be alive in order to act cytotoxic. Therefore, one effect of the virulence-associated plasmids during infection with R. equi is the promotion of necrotic damage to the host.

A method to purify bacteria-containing phagosomes from infected macrophages

When small particles, such as microorganisms, are taken up by macrophages, they are wrapped with a portion of the host cell plasma membrane and ingested, creating a new organelle, the phagosome. This phagosome matures stepwise as newly formed endosomes do, finally forming a phagolysosome, a process that contributes to killing of ingested microbes and to the presentation of microbial antigens on the surface of the phagocyte. Some pathogenic bacteria, however, reprogram the phagocytic cell in such a way that the phagosome will either be arrested in an early stage of maturation or will be diverted and create an unusual, novel phagosomal compartment. To study the molecular processes that underly biogenesis of bacteria-containing phagosomes, we have established a method to isolate and to biochemically analyse bacteria- containing phagosomes. This method consists of mechanical lysis of infected macrophages, production of a postnuclear supernatant followed by fractionation in a discontinuous sucrose density gradient, separation through a Ficoll cushion, and by a final concentration step. These phagosome preparations contain very little endosomal or lysosomal contamination (the organelles of most concern when studying phagosome biogenesis) and very little Golgi- and plasma membrane-derived contamination, but do contain some mitochondrial and ER contamination. This method could also be used to study bacterial factors (proteins, RNA) produced while in phagosomes.