Peptidases compartmentalized to the Ascaris suum intestinal lumen and apical intestinal membrane

The nematode intestine is a tissue of interest for developing new methods of therapy and control of parasitic nematodes. However, biological details of intestinal cell functions remain obscure, as do the proteins and molecular functions located on the apical intestinal membrane (AIM), and within the intestinal lumen (IL) of nematodes. Accordingly, methods were developed to gain a comprehensive identification of peptidases that function in the intestinal tract of adult female Ascaris suum. Peptidase activity was detected in multiple fractions of the A. suum intestine under pH conditions ranging from 5.0 to 8.0. Peptidase class inhibitors were used to characterize these activities. The fractions included whole lysates, membrane enriched fractions, and physiological- and 4 molar urea-perfusates of the intestinal lumen. Concanavalin A (ConA) was confirmed to bind to the AIM, and intestinal proteins affinity isolated on ConA-beads were compared to proteins from membrane and perfusate fractions by mass spectrometry. Twenty-nine predicted peptidases were identified including aspartic, cysteine, and serine peptidases, and an unexpectedly high number (16) of metallopeptidases. Many of these proteins co-localized to multiple fractions, providing independent support for localization to specific intestinal compartments, including the IL and AIM. This unique perfusion model produced the most comprehensive view of likely digestive peptidases that function in these intestinal compartments of A. suum, or any nematode. This model offers a means to directly determine functions of these proteins in the A. suum intestine and, more generally, deduce the wide array functions that exist in these cellular compartments of the nematode intestine

Communication in bacteria: an ecological and evolutionary perspective

Individual bacteria can alter their behaviour through chemical interactions between organisms in microbial communities - this is generally referred to as quorum sensing. Frequently, these interactions are interpreted in terms of communication to mediate coordinated, multicellular behaviour. We show that the nature of interactions through quorum-sensing chemicals does not simply involve cooperative signals, but entails other interactions such as cues and chemical manipulations. These signals might have a role in conflicts within and between species. The nature of the chemical interaction is important to take into account when studying why and how bacteria react to the chemical substances that are produced by other bacteria

A Potential New Pathway for Staphylococcus aureus Dissemination: The Silent Survival of S. aureus Phagocytosed by Human Monocyte-Derived Macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3–4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-γ at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in α-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular α-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection

Cathepsin B-like cysteine proteases confer intestinal cysteine protease activity in Haemonchus contortus

Cathepsin B-like cysteine protease genes (cbls) constitute large multigene families in parasitic and nonparasitic nematodes. Although expressed in the intestine of some nematodes, the biological and biochemical functions of the CBL proteins remain unresolved. Di- and tetra-oligopeptides were used as fluorogenic substrates and irreversible/competitive inhibitors to establish CBL functions in the intestine of the parasitic nematode Haemonchus contortus. Cysteine protease activity was detected against diverse substrates including the cathepsin B/L substrate FR, the caspase 1 substrate YVAD, the cathepsin B substrate RR, but not the CED-3 (caspase 3) substrate DEVD. The pH at which maximum activity was detected varied according to substrate and ranged from pH 5.0 to 7.0. Individual CBLs were affinity isolated using FA and YVAD substrates. pH influenced CBL affinity isolation in a substrate-specific manner that paralleled pH effects on individual substrates. N-terminal sequencing identified two isolated CBLs as H. contortus GCP-7 (33 kDa) and AC-4 (37 kDa). N termini of each began at a position consistent with proregion cleavage and protease activation. Isolation of the GCP-7 band by each peptide was preferentially inhibited when competed with a diazomethane-conjugated inhibitor, Z-FA-CHN(2), demonstrating one functional difference among CBLs and among inhibitors. Substrate-based histological analysis placed CBLs on the intestinal microvilli. Data indicate that CBLs are responsible for cysteine protease activity described from H. contortus intestine. Results also support a role of CBLs in nutrient digestion

Preliminary observations on the development of a latex, slide agglutination test and its use for serodiagnosis of heartwater (Cowdria ruminantium infection) in small ruminants

The possibility of detecting Cowdria ruminantium antibodies and antigen in sera and plasma of experimentally infected goats, using latex beads coated with crude Cowdria antigen (latex slide agglutination antibody test) and latex beads coated with rabbit anti-Major antigenic protein-2 (aMAP2) serum (latex slide agglutination antigen test) was investigated. In the Latex slide agglutination antibody test, Cowdria antibodies were detected in sera of goats which suffered a non fatal disease, but not in sera of goats which suffered an acute and fatal disease. Out of 81 field goat sera tested, 19(23.4 percent) were positive, 11(13.6 percent) weak positive (border line) and 51 (63 percent) negative. The Latex slide agglutination antigen test was unable to detect Cowdria antigen in blood of the infected goats. However, when solubilized elementary bodies (EBs) and intact EBs were tested using the Latex slide agglutination antigen test, weak agglutination was observed with the solubilized EBs and there was no agglutination observed with the intact EBs. The present study has demonstrated that the Latex slide agglutination test, if improved, has the potential of being a useful tool for routine diagnosis of heartwater in the field

Non-immunologic methods of diagnosis of babesiosis

The diagnosis of tick-borne diseases such as babesiosis still depends on observing the parasite in the infected erythrocyte. Microscopic observation is tedious and often problematic in both early and carrier infections. Better diagnostic methods are needed to prevent clinical disease, especially when susceptible cattle are being moved into disease enzootic areas. This study evaluates two techniques for early diagnosis of Babesia bovis infections in cattle, DNA probes specific for the organism and fluorescent probes specific nucleic acid. The radioisotopically labeled DNA probes are used in slot blot hybridizations whith lysed blood samples, not purified DNA. Thusfar, the probe is specific for B. bovis and can detect as few as 1000 B. bovis parasites in 10µl of blood. The specificity of the fluorescent probe depends on the characteristic morphology of the babesia in whole blood samples, as determined microscopically. The fluorescent probe detects as afew as 10,000 B. bovis parasites in 10*l as blood. The application of each method for alboratory and field use is discussed