No abstract

SUMMARY Babesiosis is an emerging, tick-transmitted, zoonotic disease caused by hematotropic parasites of the genus Babesia. Babesial parasites (and those of the closely related genus Theileria) are some of the most ubiquitous and widespread blood parasites in the world, second only to the trypanosomes, and consequently have considerable worldwide economic, medical, and veterinary impact. The parasites are intraerythrocytic and are commonly called piroplasms due to the pear-shaped forms found within infected red blood cells. The piroplasms are transmitted by ixodid ticks and are capable of infecting a wide variety of vertebrate hosts which are competent in maintaining the transmission cycle. Studies involving animal hosts other than humans have contributed significantly to our understanding of the disease process, including possible pathogenic mechanisms of the parasite and immunological responses of the host. To date, there are several species of Babesia that can infect humans, Babesia microti being the most prevalent. Infections with Babesia species generally follow regional distributions; cases in the United States are caused primarily by B. microti, whereas cases in Europe are usually caused by Babesia divergens. The spectrum of disease manifestation is broad, ranging from a silent infection to a fulminant, malaria-like disease, resulting in severe hemolysis and occasionally in death. Recent advances have resulted in the development of several diagnostic tests which have increased the level of sensitivity in detection, thereby facilitating diagnosis, expediting appropriate patient management, and resulting in a more accurate epidemiological description.

Dinitrogenase, the enzyme capable of catalyzing the reduction of N2, is a heterotetramer (␣2␤2) and contains the iron-molybdenum cofactor (FeMo-co) at the active site of the enzyme. Mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase, which is enzymatically inactive but can be activated in vitro by the addition of purified FeMo-co. Apodinitrogenase from certain mutant strains of Azotobacter vinelandii has a subunit composition of ␣ 2 ␤ 2 ␥ 2 . The ␥ subunit has been implicated as necessary for the efficient activation of apodinitrogenase in vitro.Characterization of ␥ protein in crude extracts and partially pure fractions has suggested that it is a chaperone-insertase required by apodinitrogenase for the insertion of FeMo-co. There are three major forms of ␥ protein detectable by Western analysis of native gels. An apodinitrogenase-associated form is found in extracts of nifB or nifNE strains and dissociates from the apocomplex upon addition of purified FeMo-co. A second form of ␥ protein is unassociated with other proteins and exists as a homodimer. Both of these forms of ␥ protein can be converted to a third form by the addition of purified FeMo-co. This conversion requires the addition of active FeMo-co and correlates with the incorporation of iron into ␥ protein. Crude extracts that contain this form of ␥ protein are capable of donating FeMo-co to apodinitrogenase, thereby activating the apodinitrogenase. These data support a model in which ␥ protein is able to interact with both FeMo-co and apodinitrogenase, facilitate FeMo-co insertion into apodinitrogenase, and then dissociate from the activated dinitrogenase complex.Nitrogenase is comprised of two components: dinitrogenase (also known as component I or the MoFe protein) and dinitrogenase reductase (also known as component II, NifH, or the Fe protein). Nitrogenase catalyzes the ATP-and reductant-dependent reduction of N 2 and other substrates. Dinitrogenase is a 240-kDa ␣ 2 ␤ 2 tetramer encoded by nifKD (1). Dinitrogenase contains two types of metal centers: the P-cluster (8Fe-8S) (2, 3), which bridges the ␣ and ␤ subunits, and a unique ironmolybdenum cofactor (FeMo-co) 1 (4), which is buried within the ␣ subunit (3, 5) and is the site of substrate reduction (6). The dinitrogenase complex has two of each of these metal centers.Dinitrogenase reductase is a 60-kDa dimer encoded by nifH. It specifically reduces dinitrogenase, apparently transferring electrons to the P-cluster, which then channels them to FeMoco. In addition to this catalytic role, dinitrogenase reductase is also involved in the biosynthesis of FeMo-co and the maturation of dinitrogenase (reviewed in Ref.

Dinitrogenase is a heterotetrameric (␣ 2 ␤ 2 ) enzyme that catalyzes the reduction of dinitrogen to ammonium and contains the iron-molybdenum cofactor (FeMo-co) at its active site. Certain Azotobacter vinelandii mutant strains unable to synthesize FeMo-co accumulate an apo form of dinitrogenase (lacking FeMo-co), with a subunit composition ␣ 2 ␤ 2 ␥ 2 , which can be activated in vitro by the addition of FeMo-co. The ␥ protein is able to bind FeMo-co or apodinitrogenase independently, leading to the suggestion that it facilitates FeMo-co insertion into the apoenzyme. In this work, the non-nif gene encoding the ␥ subunit (nafY) has been cloned, sequenced, and found to encode a NifY-like protein. This finding, together with a wealth of knowledge on the biochemistry of proteins involved in FeMo-co and FeV-co biosyntheses, allows us to define a new family of iron and molybdenum (or vanadium) cluster-binding proteins that includes NifY, NifX, VnfX, and now ␥. In vitro FeMo-co insertion experiments presented in this work demonstrate that ␥ stabilizes apodinitrogenase in the conformation required to be fully activable by the cofactor. Supporting this conclusion, we show that strains containing mutations in both nafY and nifX are severely affected in diazotrophic growth and extractable dinitrogenase activity when cultured under conditions that are likely to occur in natural environments. This finding reveals the physiological importance of the apodinitrogenase-stabilizing role of which both proteins are capable. The relationship between the metal cluster binding capabilities of this new family of proteins and the ability of some of them to stabilize an apoenzyme is still an open matter.Nitrogenase catalyzes the reduction of nitrogen gas to ammonium, in an ATP-and reductant-dependent reaction. It is one of the best characterized metalloenzymes and is an excellent model for elucidating metalloprotein assembly. Nitrogenase is composed of two oxygen-labile metalloproteins: dinitrogenase and dinitrogenase reductase (1, 2). Dinitrogenase (also termed component I or molybdenum-iron protein) is a 240-kDa ␣ 2 ␤ 2 tetramer of the nifD and nifK gene products (3). Each ␣␤ nitrogenase dimer contains an iron-molybdenum cofactor (FeMo-co) 1 and a P cluster (3, 4). Dinitrogenase reductase (also termed component II or iron protein) is a 60-kDa ␣ 2 dimer of the nifH gene product which contains a single 4Fe-4S center coordinated between the two subunits (5). NifH has at least three roles in the nitrogenase enzyme system (6): first, it serves as electron donor to nitrogenase; second, it participates in the biosynthesis of FeMo-co; and third, it is required for maturation of apodinitrogenase to a FeMo-co-activable form.

Most reported U.S. zoonotic cases of babesiosis have occurred in the Northeast and been caused by Babesia microti. In Washington State, three cases of babesiosis have been reported previously, which were caused by WA1 (for “Washington 1”)-type parasites. We investigated a case of babesiosis in Washington in an 82–year-old man whose spleen had been removed and whose parasitemia level was 41.4%. The complete 18S ribosomal RNA gene of the parasite was amplified from specimens of his whole blood by polymerase chain reaction. Phylogenetic analysis showed the parasite is most closely related, but not identical, to B. divergens (similarity score, 99.5%), a bovine parasite in Europe. By indirect fluorescent-antibody testing, his serum reacted to B. divergens but not to B. microti or WA1 antigens. This case demonstrates that babesiosis can be caused by novel parasites detectable by manual examination of blood smears but not by serologic or molecular testing for B. microti or WA1-type parasites.

Apodinitrogenase, which lacks the iron-molybdenum cofactor at its active site, is an oligomer that contains an additional protein not found in the active dinitrogenase tetramer. This associated protein in Kiebsiella pneumoniae is shown to be the product of the nifY gene. When apodinitrogenase is activated by the addition of the iron-molybdenum cofactor, NifY dissociates from the apodinitrogenase complex. The conditions for this dissociation are described. Finally, there are aspects of the dissociation and insertion process in K. pneumoniae that are different from that in Azotobacter vinelandii. (18).The active site of component I, the iron-molybdenum cofactor (FeMo-co), is synthesized by the nifgene products, including those of nifQ, -B, -V, -N, -E, and -H (18). Many mutations in nifB and nifNE result in strains that are unable to fix N2 (13) and accumulate a form of component I without the active site. Addition of purified FeMo-co to this apocomponent I (Apo I) in vitro yields an enzyme that is catalytically active (15).When Apo I was purified from a nifB Azotobacter vinelandii mutant, another protein of approximately 20 kDa copurified with it (11). A variety of efforts to dissociate this protein without destroying Apo I proved unsuccessful, demonstrating that the complex was very tight (11).Lacking either a good genetic or biochemical perspective on this associated protein from A. vinelandii, we examined the Apo I from Kiebsiella pneumoniae, reasoning that if it was important to the biochemistry of nitrogenase, a similar factor might be detected in that organism also. In fact, a previous publication describing the purification of Apo I from K pneumoniae had already reported the presence of a 20-kDa contaminant (5). Very recently, NifY has been detected in partially purified samples of Apo I from K pneumoniae, but the nature and role of this association were unclear (19). In this report, we show that the protein associated with pure Apo I from K pneumoniae is the product of nifY. We further characterize the nature and role of the proteins associated with Apo I from both K pneumoniae and A. vinelandii.The initial indication that the associated factor in K pneumoniae is NifY came from sequence analysis of the purified protein.To isolate the protein, we purified Apo I from K pneumoniae UN1217 (nifN4536) through the hy-* Corresponding author.droxylapatite column step as outlined previously (11), except that K pneumoniae Apo I was eluted from the DEAEcellulose column at 0.25 M NaCl. The reactivatible fractions were then analyzed on an alkyl superose fast protein liquid chromatography column and eluted at 0.63 M (NH4)2SO4 in 0.025 M MOPS (morpholinepropanesulfonic acid) (pH 7.4) containing 1.7 mM Na2S204. At this point, the Apo I protein is homogeneously pure. To isolate the associated protein from Apo I, the resulting protein preparation was separated on a sodium dodecyl sulfate-13% polyacrylamide gel electrophoresis (SDS-PAGE) gel prerun with 0.25% (wt/vol) thioglycolate in the buffer and transferred to an Immobil...

Blood donors seropositive for B. microti and A. phagocytophila are present in Connecticut and Wisconsin. Donors readily recall previous tick bites, but self-reported bites are not reliable indicators of serologic status. The exposure of blood donors to tick-borne pathogens does suggest a need to better understand the transfusion transmission potential of these agents.

Infection of severe combined immunodeficient mice with Babesia sp. strain WA1 was studied to assess the contributions of innate and adaptive immunity in resistance to acute babesiosis. The scid mutation showed little effect in genetically susceptible C3H mice and did not decrease the inherent resistance of C57BL/6 mice to the infection, suggesting that innate immunity plays a central role in determining the course of Babesia infection in these strains. In contrast, the scid mutation dramatically impaired resistance in moderately susceptible BALB/c mice, suggesting that acquired immunity may play an important secondary role. In comparison to their female counterparts, male mice of different genetic backgrounds showed increased resistance to the infection, indicating that the gender of the host may influence protection against babesiosis.