Jian Zhi Zhang

Gene Sequence-Based Criteria for Identification of New Rickettsia Isolates and Description of Rickettsia heilongjiangensis sp. nov.

ABSTRACT We propose genetic guidelines for the classification of rickettsial isolates at the genus, group, and species levels by using sequences of the 16S rRNA (rrs) gene and four protein-coding genes, the gltA, ompA, and ompB genes and gene D. To be classified as a member of the genus Rickettsia, an isolate should exhibit degrees of rrs and gltA homology with any of the 20 Rickettsia species studied of ≥98.1 and ≥86.5%, respectively. A member of the typhus group should fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of either Rickettsia typhi or Rickettsia prowazekii of ≥99.4, ≥96.6, ≥92.4, and ≥91.6%, respectively. A member of the spotted fever group should either possess the ompA gene or fulfill at least two of the following four criteria: pairwise nucleotide sequence homologies with rrs, gltA, ompB, and gene D of any member of this group of ≥98.8, ≥92.7, ≥85.8, and ≥82.2%, respectively. The existence of a distinct “ancestral” group should be questioned. To be classified as a new Rickettsia species, an isolate should not exhibit more than one of the following degrees of nucleotide similarity with the most homologous validated species: ≥99.8 and ≥ 99.9% for the rrs and gltA genes, respectively, and, when amplifiable, ≥98.8, ≥99.2, and ≥99.3% for the ompA and ompB genes and gene D, respectively. By use of our classification scheme, “Rickettsia heilongjiangii” belongs to a new species for which we officially propose the name Rickettsia heilongjiangensis sp. nov.

The developmental cycle of Ehrlichia chaffeensis in vertebrate cells

Ehrlichia chaffeensis, an obligatory intracellular bacterium, has two forms in mammalian cells: small dense‐cored cells (DC) with dense nucleoid and larger reticulate cells (RC) with uniformly dispersed nucleoid. We have determined by electron microscopy that DC but not RC attaches to and enters into the host cells and RC but not DC multiples inside the host cells. Analysis of outer membrane protein expression by confocal microscopy showed that RC expressed the 28 kDa outer membrane protein (p28), the intermediate form, which were transforming from RC to DC, expressed both gp120 and p28, and the mature DC expressed gp120 only. The TCID50 of DC is 6 log10 higher than RC. We conclude that E. chaffeensis has a developmental cycle, in which the DC attaches to and enters into the host cells, and transforms into RC and the RC multiplies by binary fission for 48 h and then matures into DC at 72 h.

Evaluation of Transovarial Transmission and Transmissibility of Ehrlichia chaffeensis (Rickettsiales: Anaplasmataceae) in Amblyomma americanum (Acari: Ixodidae)

Abstract It has long been assumed that Ehrlichia chaffeensis (Anderson, Dawson & Wilson), is not transmitted transovarially in the lone star tick vector Amblyomma americanum (L.). To test this hypothesis, three beagle dogs, Canis familiaris (L.) (Carnivora: Canidae), were subcutaneously infected with E. chaffeensis (Arkansas strain). Uninfected nymphal lone star ticks were placed on the infected dogs and allowed to feed to repletion. These nymphal ticks were allowed to molt, and five of five adult female ticks sampled were confirmed to be infected by polymerase chain reaction (PCR). Forty infected adult ticks, the majority of which were female with several males included to stimulate feeding, were then placed on two uninfected dogs. Fourteen females were removed early and the rest were allowed to feed to repletion. After feeding to repletion, the six remaining females detached and two of these females were tested preoviposition, whereas the remaining four were allowed to lay eggs. All six of these fully engorged females tested negative by PCR. The egg clutches laid by four engorged adult females, and the larvae that hatched from these eggs along with larvae from a previously untested egg clutch, were shown to be uninfected by PCR. This seems to support the long-held assumption of lack of transovarial transmission. Also of interest, we found that these PCR-confirmed infected adult ticks reared in the laboratory did not transmit E. chaffeensis to uninfected dogs.

Expression of Members of the 28-Kilodalton Major Outer Membrane Protein Family of Ehrlichia chaffeensis during Persistent Infection

ABSTRACT The 28-kDa immunodominant outer membrane proteins (P28 OMPs) of Ehrlichia chaffeensis are encoded by a multigene family. As an indirect measure of the in vivo expression of the members of the p28 multigene family of E. chaffeensis, sera from two beagle dogs experimentally infected with E. chaffeensis were evaluated for the presence of specific antibodies to P28 OMPs by enzyme-linked immunosorbent assay. Antigenic peptides unique to each of the P28s were identified within the first hypervariable region of each P28 OMP. Serological responses to peptides derived from all P28 OMPs were detected from day 30 postinoculation to day 468 and from day 46 until day 159 in the two beagles. Although antibody titers to the peptides fluctuated, the peak response to all of the peptides appeared simultaneously in each dog. The antibody responses to another outer membrane protein of E. chaffeensis (GP120) showed similar temporal and quantitative changes. These data suggest that the P28 OMPs are expressed concurrently during persistent Ehrlichia infection.

Genetic diversity of chlamydia trachomatis and the prevalence of trachoma

Background. Considerable variation in the outer membrane protein (ompA) of Chlamydia trachomatis has been uncovered by immunotyping and, more recently, by genotyping. This diversity may assist Chlamydia in evading the human immune system; organisms may have a competitive advantage if they infect a host who has previously been infected only by other strains. If so, a diverse set of strains may attain a higher prevalence in a community than a single strain. We determined the predominant strains of ocular C. trachomatis in trachoma-endemic villages of Nepal and tested the hypothesis that strain diversity is associated with the prevalence of infection. Methods. Major outer membrane protein gene sequences of chlamydial isolates were determined from ligase chain reaction-positive eye swab samples collected from 10 villages. The diversity of genovars was determined for each village, using Simpson’s index. Results. Two genovar families (Ba and C) and nine genovars were detected, with a single genovar (C1) comprising more than one-half of the samples. The prevalence of clinically active trachoma was significantly associated with the genetic diversity in a village, controlling for village size and number of samples taken in a village. Conclusion. Genetic diversity of C. trachomatis is associated with the prevalence of infection in a community, consistent with the hypothesis that diversity may be necessary to attain a high prevalence in a community.

L-selectin and E-selectin expressed on monocytes mediating Ehrlichia chaffeensis attachment onto host cells

Ehrlichia chaffeensis, the agent of human monocytic ehrlichiosis, is an obligatory intracellular bacterium that exhibits monocytic host cell tropism. Ehrlichiae must enter the host cell, and then establish infection. The tropism of E. chaffeensis for monocytes suggests that the cell contains some specific surface components that mediate E. chaffeensis attachment and entry into host cells. In this study, host cell surface components that play a role in ehrlichial attachment were identified using a human monocyte/macrophage cell line, THP-1. E. chaffeensis attachment to THP-1 cells was partially blocked in the presence of antibodies to E-selectin and L-selectin, but not by antibodies to P-selectin, integrin alpham, integrin alphax, or normal mouse IgG as determined by real time polymerase chain reaction. Conversely, in HeLa cells that do not exhibit surface expression of E-selectin and L-selectin, antibodies to these cell surface proteins did not inhibit E. chaffeensis attachment. These findings indicate that E-selectin and L-selectin are cell surface proteins that might act as co-receptors and contribute to E. chaffeensis attachment and entry into THP-1.

Obligate intracellular bacterium Ehrlichia inhibiting mitochondrial activity

Ehrlichia are obligately intracellular bacteria that reside in a vacuole in the cytoplasm of phagocytes. We determined by confocal microscopy the interaction between Ehrlichia and mitochondria in DH82 cells to investigate the mechanism of Ehrlichia survival inside the phagocyte. The most remarkable finding of our study was that Ehrlichia morulae interacted with mitochondria and inhibited mitochondrial metabolism. We showed that in Ehrlichia chaffeensis-infected DH82 cells, mitochondria did not incorporate BrdU and transcriptional level of the mitochondrial gene NADPH2 was significantly reduced, indicating the inhibition of mitochondrial metabolism. This study demonstrates that Ehrlichia are able to inhibit mitochondrial activities, and it opens up a new avenue for the study of Ehrlichia pathogenesis.

Proteomic Analysis of the Ehrlichia chaffeensis Phagosome in Cultured DH82 Cells

Ehrlichia chaffeensis is an obligately intracellular bacterium that resides and multiplies within cytoplasmic vacuoles of phagocytes. The Ehrlichia-containing vacuole (ECV) does not fuse with lysosomes, an essential condition for Ehrlichia to survive inside phagocytes, but the mechanism of inhibiting the fusion of the phagosome with lysosomes is not clear. Understanding the ECV molecular composition may decipher the mechanism by which Ehrlichia inhibits phagosome-lysosome fusion. In this study, we obtained highly purified ECVs from E. chaffeensis-infected DH82 cells by sucrose density gradient centrifugation and analyzed their composition by mass spectrometry-based proteomics. The ECV composition was compared with that of phagolysosomes containing latex beads. Lysosomal proteins such as cathepsin D, cathepsin S, and lysosomal acid phosphatase were not detected in E. chaffeensis phagosome preparations. Some small GTPases, involved in membrane dynamics and phagocytic trafficking, were detected in ECVs. A notable finding was that Rab7, a late endosomal marker, was consistently detected in E. chaffeensis phagosomes by mass spectrometry. Confocal microscopy confirmed that E. chaffeensis phagosomes contained Rab7 and were acidified at approximately pH 5.2, suggesting that the E. chaffeensis vacuole was an acidified late endosomal compartment. Our results also demonstrated by mass spectrometry and immunofluorescence analysis that Ehrlichia morulae were not associated with the autophagic pathway. Ehrlichia chaffeensis did not inhibit phagosomes containing latex beads from fusing with lysosomes in infected cells. We concluded that the E. chaffeensis vacuole was a late endosome and E. chaffeensis might inhibit phagosome-lysosome fusion by modifying its vacuolar membrane composition, rather than by regulating the expression of host genes involved in trafficking.