Frederick K. Chu

Antibody-mediated elimination of the obligate intracellular bacterial pathogen Ehrlichia chaffeensis during active infection

ABSTRACT It is generally accepted that cellular, but not humoral immunity, plays an important role in host defense against intracellular bacteria. However, studies of some of these pathogens have provided evidence that antibodies can provide immunity if present during the initiation of infection. Here, we examined immunity against infection byEhrlichia chaffeensis, an obligate intracellular bacterium that causes human monocytic ehrlichiosis. Studies with mice have demonstrated that immunocompetent strains are resistant to persistent infection but that SCID mice become persistently and fatally infected. Transfer of immune serum or antibodies obtained from immunocompetent C57BL/6 mice to C57BL/6 scid mice provided significant although transient protection from infection. Bacterial clearance was observed when administration occurred at the time of inoculation or well after infection was established. The effect was dose dependent, occurred within 2 days, and persisted for as long as 2 weeks. Weekly serum administration prolonged the survival of susceptible mice. Although cellular immunity is required for complete bacterial clearance, the data show that antibodies can play a significant role in the elimination of this obligate intracellular bacterium during active infection and thus challenge the paradigm that humoral responses are unimportant for immunity to such organisms.

Outer Membrane Protein-Specific Monoclonal Antibodies Protect SCID Mice from Fatal Infection by the Obligate Intracellular Bacterial Pathogen Ehrlichia chaffeensis

Previous studies of Ehrlichia chaffeensis infection in the mouse have demonstrated that passive transfer of polyclonal Abs from resistant immunocompetent mice to susceptible SCID mice ameliorated infection and disease, even when Abs were administered during established infection. To identify particular Abs that could mediate bacterial clearance in vivo, E. chaffeensis-specific mAbs were generated and administered to infected SCID mice. Bacterial infection in the livers was significantly lowered after administration of either of two Abs of different isotypes (IgG2a and IgG3). Moreover, repeated administration of one Ab (Ec56.5; IgG2a) rescued mice from an otherwise lethal infection for at least 5 wk. Both protective Abs recognized the E. chaffeensis major outer membrane protein (OMP)-1g. Further studies revealed that both Abs recognized closely related epitopes within the amino terminus of the first hypervariable region of OMP-1g. Analyses of human sera showed that E. chaffeensis-infected patients also generated serological responses to OMP-1g hypervariable region 1, indicating that humans and mice recognize identical or closely related epitopes. These studies demonstrate that OMP-specific mAbs can mediate bacterial elimination in SCID mice, and indicate that Abs, in the absence of cell-mediated immunity, can play a significant role in host defense during infection by this obligate intracellular bacterium.

Antibodies Highly Effective in SCID Mice During Infection by the Intracellular Bacterium Ehrlichia chaffeensis Are of Picomolar Affinity and Exhibit Preferential Epitope and Isotype Utilization

Although often considered to be ineffective against intracellular bacteria, Abs, in the absence of lymphocytes, have been shown previously to protect SCID mice from lethal infection by the obligate intracellular bacterium Ehrlichia chaffeensis, even when administered well after infection has been established. To identify characteristics of Abs that are critical for host defense during this intracellular infection, a panel of Ehrlichia-specific mAbs was generated and analyzed. Among 100 Abs recovered, 39 recognized an amino-terminal hypervariable region of an outer membrane protein (OMP), demonstrating that the OMPs are both antigenically variable and immunodominant. A subset of 16 representative OMP-specific Abs was further examined to identify characteristics that were essential for in vivo efficacy. The highly effective Abs recognized a linear epitope within the first hypervariable region of OMP-1g. Only IgG were found to be effective, and among the effective IgG, the following hierarchy was observed: IgG2a > IgG3 = IgG2b. The most striking characteristics of the highly effective Abs were their picomolar binding affinities and long binding t1/2. Thus, although epitope recognition and isotype use may contribute to efficacy, high affinity may be a critical characteristic of Abs that can act effectively during this intracellular bacterial infection.

Processing of the intron-containing thymidylate synthase (td) gene of phage T4 is at the RNA level.

The interrupted T4 phage td gene, which encodes thymidylate synthase, is the first known example of an intron-containing prokaryotic structural gene. Analysis of td-encoded transcripts provides evidence in favor of maturation at the RNA level. Northern blotting with T4 RNA and with region-specific probes revealed three classes of RNA: diffuse premessage (ca. 2.5 kb), a low-abundance mature mRNA (ca. 1.3 kb), and an abundant free intron RNA (ca. 1.0 kb). The existence of covalently joined mature mRNA was suggested by hybridization and S1 protection experiments and was confirmed by primer extension analysis of the splice junction. In analogy to expression of interrupted eukaryotic genes, these results are consistent with an RNA processing model that would account for the direct gene transcript serving as precursor for both free intron RNA and a spliced mRNA that is colinear with the thymidylate synthase product.

Factors affecting the oligomeric structure of yeast external invertase

It has been assumed that yeast external invertase is a dimer, with each subunit composed of a 60-kDa polypeptide chain. We now present evidence that at its optimal pH of 5.0, the predominant form of external invertase is an octamer with an average size of 8 X 10(5) Da. During ultracentrifugation the octamer dissociated to lower molecular weight forms, including a hexamer, tetramer, and dimer. All forms of the enzyme were shown to possess identical specific activities and to contain a similar carbohydrate to protein ratio. Although the monomer subunits (1 X 10(5) Da) were heterogenous in carbohydrate content, each subunit possessed nine oligosaccharide chains. When stained for protein and enzyme activity following sodium dodecyl sulfate-polyacrylamide gel electrophoresis, only the oligomeric form of the enzyme appeared to be active. Thus, on partially inactivating invertase with 4 M guanidine hydrochloride both octamer and monomer were evident on the gels but only the former was active. Similarly, incubating at pH 2.5 in the presence of sodium dodecyl sulfate yielded only inactive monomer. The monomer, unlike the active oligomeric aggregate, was unable to hydrolyze sucrose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Consistent with the in vitro studies, freshly prepared yeast lysate was shown to contain the octameric species of external invertase as the major active form of this enzyme. From these studies and others which employed deglycosylated invertase, it is concluded that the carbohydrate component of external invertase contributes not only to stabilizing enzyme activity, but also to maintaining its oligomeric structure.

EXPERIMENTAL INFECTION OF THE HUMAN GRANULOCYTIC EHRLICHIOSIS AGENT IN HORSES

Human blood collected from two patients from Westchester County, New York with human granulocytic ehrlichia (HGE) infection was inoculated into two ponies. Inoculated ponies developed clinical signs similar to a previous report (Madigan et al., 1995). Histopathological changes involved follicular hyperplasia of lymphoid tissues. HGE DNA was detected by PCR in muscle, fascia, peritoneum, and adrenal gland after the ponies produced a high level of antibodies to HGE. We suggest that HGE may reside in poorly vascularized connective tissues, where the antibodies may have some difficulties to penetrate, resulting in persistent infection. Since HGE and E. equi cause very similar diseases in both humans and horses, they may be the same organism with minor genetic differences.

The use of iodinated lectins for determining the degree of deglycosylation of high-mannose glycoproteins by endo-β-N-acetylglucosaminidase H

Abstract A method which demonstrates that the removal of polymannosyl chains from glycoproteins by endo-β- N -acetylglucosaminidase H can be monitored reliably using only submicrogram quantities of glycoprotein is described. Glycoproteins and their endoglycosidase-treated forms are subjected to electrophoresis on SDS-polyacrylamide gels, which are then overlaid with [ 125 I]concanavalin A or [ 125 I]wheat germ agglutinin. The degree to which these lectins bind is measured by autoradiography. The complete loss of [ 125 I]concanavalin A binding by glycoproteins such as deoxyribonuclease I, ovalbumin, carboxypeptidase Y, and invertase is associated with the removal of their oligosaccharide chains. Invertase, unlike the above mannose-containing glycoproteins, acquires the capacity to bind [ 125 I]wheat germ agglutinin only upon partial or complete deglycosylation, a finding substantiated by wheat germ agglutinin-Sepharose column chromatography. In addition to providing a procedure for monitoring the enzymatic deglycosylation of mannose-containing glycoproteins, the lectin-gel binding technique is shown to provide an estimate of the mannose content of neutral glycoproteins at levels which cannot be detected by conventional methods. In some cases, this method may be useful in distinguishing between N - and O -glycosidic linkages where the oligosaccharide is predominantly mannosyl.

Stabilization of the structure and activity of yeast carboxypeptidase Y by its high-mannose oligosaccharide chains

Abstract Sodium dodecyl sulfate was shown to promote both the inactivation and proteolytic degradation of the yeast glycoprotein, carboxypeptidase Y, with the former effect occurring six times faster than the latter. Although the proteolysis, as judged by polyacrylamide gel electrophoresis, was inhibited by pepstatin, which implicates the presence of proteinase A, the possibility of autodigestion could not be ruled out. A contributing role of the enzymes carbohydrate moiety to these two processes was revealed by treating carboxypeptidase Y with endo-β- N -acetylglucosaminidase H. This treatment removes all four of the enzymes Oligosaccharide chains in sodium dodecyl sulfate and as a consequence increases the rate of inactivation of the resulting carboxypeptidase Y by twofold and its proteolytic degradation by threefold relative to that of untreated enzyme. It thus appears that carboxypeptidase Y is a glycoprotein whose structural integrity and functional activity are influenced by its associated carbohydrate component.

Human Granulocytic Ehrlichiosis Presenting as Facial Diplegia in a 42-Year-Old Woman

Neurologic manifestations of human ehrlichiosis are unusual and have been described almost exclusively in human monocytic ehrlichiosis associated with Ehrlichia chaffeensis. We report here a case of a previously healthy 42-year-old woman who developed bilateral facial nerve palsies in association with infection by the agent of human granulocytic ehrlichiosis (aoHGE). The diagnosis was made by specific polymerase chain reaction amplification of aoHGE sequences from samples of the patients blood and cerebrospinal fluid (CSF), as well as propagation of aoHGE in culture of HL60 cells inoculated with the patients CSF. To our knowledge, this is the first report directly demonstrating the presence of aoHGE in CSF, and it underscores the importance of considering HGE in patients presenting with a nonspecific febrile illness and unexplained neurologic manifestations. HGE should also be considered in the differential diagnosis of bilateral facial palsy-a rare occurrence.

Prevalence of Borrelia burgdorferi in Small Mammals in New York State

Abstract Intensive small mammal trapping was conducted in 12 counties in New York state during 1998–2000 to investigate the prevalence and site specificity of the Lyme disease spirochete, Borrelia burgdorferi in, and presence of the blacklegged tick, Ixodes scapularis Say on, the wild mice Peromyscus leucopus Rafinesque and Peromyscus maniculatus Wagner and other small mammal species. Previously captured mice (1992–1997) from throughout New York state also were recruited into the study, providing a total of 3,664 Peromyscus from 107 sites in 31 counties. Infection with B. burgdorferi was determined by polymerase chain reaction testing of ear tissue, and rates were determined by species, counties, and regions of the state. B. burgdorferi was detected in 10 small mammal species captured during 1998–2000. Peromyscus captured from Dutchess County in the lower Hudson Valley had the highest infection rate of 21%. The next highest infection rates were in counties within the Capital District: Albany (18%), Rensselaer (17%), and Columbia (13%). From 4,792 small animals examined, we recovered 2,073 ticks representing six species from 414 individuals of 12 mammal species, including 1,839 I. scapularis collected from 315 Peromyscus trapped in five counties. I. scapularis were most often collected from animals trapped in Albany, Rensselear, and Dutchess counties. We used protein electrophoresis of salivary amylase to distinguish between P. leucopus and P. maniculatus species. I. scapularis burdens were 5.7 ticks per P. leucopus and 14.3 ticks per P. maniculatus.