Acid phosphatases (Acp) of intracellular pathogens have recently been implicated as virulence factors that enhance intracellular survival through suppression of the respiratory burst. We describe here the identification, purification, characterization, and sequencing of a novel burst-inhibiting acid phosphatase from the facultative intracellular bacterium, Francisella tularensis. Similar to other the burst-inhibiting Acps, F. tularensis Acp (AcpA) is tartrate-resistant and has broad substrate specificity. The AcpA enzyme is unique, however, in that it is easily released from the bacterial cell in soluble form, is a basic enzyme, suppresses the respiratory burst of not only fMet-Leu-Phe but also phorbol 12-myristate 13-acetate-stimulated neutrophils and does not fit into any of the three currently recognized classes of acid phosphatase. We also report the complete nucleotide sequence of the gene acpA, encoding AcpA, and the deduced primary structure of its encoded polypeptide. Comparative sequence analyses of AcpA is discussed. To our knowledge, this is the first report describing the cloning and sequencing of a burst-inhibiting acid phosphatase.

Human milk (HM) is rich in oligosaccharides (HMO) that exert prebiotic and anti-infective activities. HM feeding reduces the incidence of rotavirus (RV) infection in infants. Herein, the anti-RV activity of oligosaccharides was tested in an establishedin vitrosystem for assessing cellular binding and viral infectivity/replication, and also tested in a newly developed, acute RV infection,in situpiglet model. For thein vitrowork, crude HMO isolated from pooled HM, neutral HMO (lacto-N-neotetraose, LNnT; 2′-fucosyllactose) and acidic HMO (aHMO, 3′-sialyllactose, 3′-SL; 6′-sialyllactose, 6′-SL) were tested against the porcine OSU strain and human RV Wa strain. The RV Wa strain was not inhibited by any oligosaccharides. However, the RV OSU strain infectivity was dose-dependently inhibited by sialic acid (SA)-containing HMO. 3′-SL and 6′-SL concordantly inhibited¹²⁵I-radiolabelled RV cellular binding and infectivity/replication. For thein situstudy, a midline laparotomy was performed on 21-d-old formula-fed piglets and six 10 cm loops of ileum were isolatedin situ.Briefly, 2 mg/ml of LNnT, aHMO mixture (40 % 6′-SL/10 % 3′-SL/50 % SA) or media with or without the RV OSU strain (1 × 10⁷focus-forming units) were injected into the loops and maintained for 6 h. The loops treated with HMO treatments+RV had lower RV replication, as assessed by non-structural protein-4 (NSP4) mRNA expression, than RV-treated loops alone. In conclusion, SA-containing HMO inhibited RV infectivityin vitro; however, both neutral HMO and SA with aHMO decreasedNSP4replication during acute RV infectionin situ.

We examined gliding motility and cell invasion by an early-branching apicomplexan,Cryptosporidium parvum, which causes diarrheal disease in humans and animals. Real-time video microscopy demonstrated thatC. parvumsporozoites undergo circular and helical gliding, two of the three stereotypical movements exhibited byToxoplasma gondiitachyzoites.C. parvumsporozoites moved more rapidly thanT. gondiisporozoites, which showed the same rates of motility as tachyzoites. Motility byC. parvumsporozoites was prevented by latrunculin B and cytochalasin D, drugs that depolymerize the parasite actin cytoskeleton, and by the myosin inhibitor 2,3-butanedione monoxime. Imaging of the initial events in cell entry byCryptosporidiumrevealed that invasion occurs rapidly; however, the parasite does not enter deep into the cytosol but rather remains at the cell surface in a membrane-bound compartment. Invasion did not stimulate rearrangement of the host cell cytoskeleton and was inhibited by cytochalasin D, even in host cells that were resistant to the drug. Our studies demonstrate thatC. parvumrelies on a conserved actin-myosin motor for motility and active penetration of its host cell, thus establishing that this is a widely conserved feature of the Apicomplexa.