Mark Tarshis

Destruction of Bacteria in the Digestive Tract of the Maggot of Lucilia sericata (Diptera: Calliphoridae)

Abstract Green fluorescent protein-producing Escherichia coli were used to investigate the fate of bacteria in the alimentary tract of sterile grown maggots, Lucilia sericata (Meigen), using a laser scanning confocal microscope. A computer program was used to analyze the intensity of the fluorescence and to quantify the number of bacteria. The crop and the anterior midgut were the most heavily infected areas of the intestine. A significant decrease in the amount of bacteria was observed in the posterior midgut. The number of bacteria decreased even more significantly in the anterior hindgut and practically no bacteria were seen in the posterior end, near the anus. The viability of bacteria in the different gut sections was examined. It was shown that 66.7% of the crops, 52.8% of the midguts, 55.6% of the anterior hindguts, and 17.8% of posterior hindguts harbored living bacteria. In conclusion, during their passage through the digestive tract the majority of E. coli was destroyed in the midgut. Most of the remaining bacteria were killed in the hindgut, indicating that the feces were either sterile or contained only small numbers of bacteria.

Mycoplasma fermentans Binds to and Invades HeLa Cells: Involvement of Plasminogen and Urokinase

ABSTRACT Adherence of Mycoplasma fermentans to HeLa cells followed saturation kinetics, required a divalent cation, and was enhanced by preincubation of the organism at 37°C for 1 h in a low-osmolarity solution. Proteolytic digestion, choline phosphate, or anti-choline phosphate antibodies partially inhibited the adherence, supporting the notion that M. fermentans utilizes at least two surface components for adhesion, a protease-sensitive surface protein and a phosphocholine-containing glycolipid. Plasminogen binding to M. fermentans greatly increased the maximal adherence of the organism to HeLa cells. Anti-plasminogen antibodies and free plasminogen inhibited this increase. These observations suggest that in the presence of plasminogen the organism adheres to novel sites on the HeLa cell surface, which are apparently plasminogen receptors. Plasminogen-bound M. fermentans was detected exclusively on the cell surface of the infected HeLa cells. Nevertheless, plasminogen binding in the presence of the urokinase-type plasminogen activator (uPA) promoted the invasion of HeLa cells by M. fermentans. The latter finding indicates that the invasiveness of M. fermentans does not result from binding plasminogen but from activation of the bound plasminogen to plasmin. Cholesterol depletion and sequestration with β-cyclodextrin and filipin, respectively, did not affect the capacity of M. fermentans to adhere, but invasion of HeLa cells by uPA-activated plasminogen-bound M. fermentans was impaired, suggesting that lipid rafts are implicated in M. fermentans entry.

Cholesterol is required for the fusion of single unilamellar vesicles with Mycoplasma capricolum

Small unilamellar vesicles (SUV) were prepared from the total lipid extract of Mycoplasma capricolum. The SUV were labeled with the fluorescent probe octadecylrhodamine B chloride (R18) to a level at which the R18 fluorescence was self-quenched. At pH 7.4 and 37 degrees C, and in the presence of 5% polyethylene glycol, an increase in the R18 fluorescence with time was observed when the R18-labeled SUV were introduced to a native M. capricolum cell suspension. The fluorescence dequenching resulting from dilution of the R18 into the unlabeled membranes of M. capricolum, was interpreted as a result of lipid mixing during fusion between the SUV and the mycoplasma cells. The presence of cholesterol in the SUV was found to be obligatory to allow SUV-mycoplasma fusion to occur. Adaptation of M. capricolum cells to grow in a medium containing low cholesterol concentration provided cells in which the unesterified cholesterol content was as low as 17 micrograms/mg cell protein. The fusion activity of the adapted cells was very low or nonexistent. Nonetheless, when an early exponential phase culture of the adapted cells was transferred to a cholesterol-rich medium, the cells accumulated cholesterol and regained their fusogenic activity. The cholesterol requirement for fusion in the target mycoplasma membrane was met by a variety of planar sterols having a free beta-hydroxyl group, but differing in the aliphatic side chain, e.g., beta-sitosterol or ergosterol, even though these sterols, having a bulky side chain, are preferentially localized in the outer leaflet of the lipid bilayer. It is suggested that the role of cholesterol in mycoplasma-SUV fusion is not at the level of bulk bilayer viscosity but rather, affecting local lipid-lipid or lipid-protein interactions that are relevant to the fusion event.

Invasion of Melanoma Cells by Mycoplasma hyorhinis: Enhancement by Protease Treatment

ABSTRACT Mycoplasma hyorhinis (strain MCLD) was recently isolated from a melanoma cell culture. Growth of MCLD was considerably improved by 24 serial passages in a modified Hayflicks mycoplasma medium. Transmission electron microscopy showed that MCLD exhibits a polymorphic appearance, with ovoid or elongated cells frequently harboring an electron-dense core at one of the poles. Adherence of M. hyorhinis to melanoma cells followed saturation kinetics. Furthermore, although M. hyorhinis has been considered to remain attached to the surface of the host cells, we show for the first time, qualitatively by confocal laser scanning microscopy and quantitatively by a gentamicin resistance assay, that MCLD is able to invade melanoma cells. The ingested mycoplasmas were randomly distributed in the cytoplasm, tending to concentrate near the plasma membrane. Both adherence to and invasion of melanoma cells by M. hyorhinis strain MCLD were dramatically enhanced by mild proteolytic digestion with proteinase K (2.5 μg/mg cell protein for 2.5 min at 37°C) that affected the surface-exposed proteins of this organism, mainly the major 47-kDa lipoprotein. We suggest that the intracellular location of M. hyorhinis strain MCLD is a privileged niche, which may explain the survival of M. hyorhinis in tissue cultures. The enhanced binding to and invasion of melanoma cells by protease treatment may be due to either the activation or the enhanced exposure of an adhesin(s) on the mycoplasmal cell surface.

Polyhydroxylated fatty alcohols derived from avocado suppress inflammatory response and provide non-sunscreen protection against UV-induced damage in skin cells

Exposing skin to ultraviolet (UV) radiation contributes to photoaging and to the development of skin cancer by DNA lesions and triggering inflammatory and other harmful cellular cascades. The present study tested the ability of unique lipid molecules, polyhydroxylated fatty alcohols (PFA), extracted from avocado, to reduce UVB-induced damage and inflammation in skin. Introducing PFA to keratinocytes prior to their exposure to UVB exerted a protective effect, increasing cell viability, decreasing the secretion of IL-6 and PGE2, and enhancing DNA repair. In human skin explants, treating with PFA reduced significantly UV-induced cellular damage. These results support the idea that PFA can play an important role as a photo-protective agent in UV-induced skin damage.

Mycoplasma fermentans (incognitus strain) induces TNFα and IL-1 production by human monocytes and murine macrophages

We have demonstrated that Mycoplasma fermentans (incognitus strain), as well as M. fermentans KL4, PG 18 and IM 1 strains have the ability to activate human peripheral blood monocytes and murine macrophages of two inbred strains to secrete a high level of tumor necrosis factor alpha (TNF alpha) in a dose-dependent manner. Secretion of interleukin-1 (IL-1) was also stimulated following the incubation of human monocytes with the organism. We suggest that cytokine secretion following infection with M. fermentans (incognitus strain) that was detected in AIDS patients may contribute to the pathological manifestations, including cachexia, in this disease.

Mycoplasma fermentans glycolipid triggers inflammatory response in rat astrocytes

Mycoplasma fermentans glycolipid (MfGL-II) is a major lipid in the membranes of this AIDS-associated mycoplasma and constituting up to 20% of the total phospholipids of this organism. It was recently shown that MfGL-II, mainly through its phosphocholine moiety, is responsible for the attachment of M. fermentans to host cells. We now show that MfGL-II is also associated with the secretion of inflammatory mediators by cells of the central nervous system. Stimulation of primary rat astrocytes by MfGL-II caused activation of protein kinase C, secretion of nitric oxide (NO) and prostaglandin E2, and augmented glucose utilization and lactate formation in a dose-dependent manner. In an attempt to define the minimal structural requirements for MfGL-II activity, the two O-acylated fatty acids in the molecule were removed. Deacylation pronouncedly reduced the stimulatory activity of the glycolipid, suggesting that the fatty acyl residues are essential. Incubation of MfGL-II with polyclonal anti-MfGL-II antiserum or with monoclonal anti-phosphocholine antibody diminished NO release, whereas incubation of MfGL-II with normal rabbit serum had no effect. It is, therefore, likely that the terminal phosphocholine moiety plays an important role in MfGL-IIs stimulation of glial cells.

Adhesion and biofilm formation of Mycoplasma pneumoniae on an abiotic surface

We demonstrated that when M. pneumoniae was grown on an abiotic surface of either glass or polystyrene with a serum-containing medium, the bacteria adhered to the surface and formed highly differentiated volcano-like biofilm structures. As adherence to the surface and/or biofilm formation was totally inhibited by anti-P1 polyclonal monospecific antibodies, we suggest that the adherence of M. pneumoniae to the abiotic surface and/or biofilm formation is associated with P1, the major tip organelle protein of this organism. Furthermore, adherence and/or biofilm formation was markedly inhibited by treating the serum component of the growth medium with neuraminidase or by growing the bacteria in the presence of sialyllactose, suggesting that the initial step in the adherence to and/or biofilm formation by M. pneumoniae on an abiotic surface is the interaction of the bacterium through its tip organelle with sialic acid residues of serum glycoproteins.

Body Louse Remains Found in Textiles Excavated at Masada, Israel

Abstract A leg of the body louse, Pediculus humanus humanus L. (Phthiraptera: Pediculidae), was found in a storeroom at Masada build during the reign of King Herod the Great. The unearthed culture material clearly indicates that the room was occupied by the rebels during the first Jewish revolt against the Romans (AD 66–73/4). The context of the textiles associated with the louse and their nature suggest a rebel origin. An exuvium of a leg of the third nymphal stage of body louse was found. The first three parts of the leg (tarsus, including the tarsal claw, tibia and femur) were preserved entirely, while the fourth segment of the leg, the trochanter, was only partially conserved. The comparison of the tarsus of this specimen with the legs of present day third instar nymphs of body and head lice revealed that the leg found within the archaeological debris belongs to a body louse.

Intracellular Location and Survival of Mycoplasma penetrans Within HeLa Cells

Mycoplasma penetrans invades HeLa cells and survives within them for prolonged periods of time. The intracellular distribution of M. penetrans within HeLa cells was studied utilizing the acidotropic dye LysoTracker (green), which permeates cell membranes and upon protonation remains trapped in acidic compartments. The excitation and emission spectra of the green LysoTracker are suitable for colocalization studies with rabbit anti-M. penetrans antibodies and red Cy5 goat anti-rabbit IgG. The images collected by confocal laser scanning microscopy revealed that in the infected HeLa cells almost all Cy5 fluorescent foci (red) were located within the LysoTrack-labelled intracellular compartments, apparently endosomes. Viable mycoplasmas were detected within endosomes for prolonged periods of time, apparently due to a potent antioxidant activity detected in M. penetrans.