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Molecular heterogeneity of Ehrlichia chaffeensis isolates determined by sequence analysis of the 28-kilodalton outer membrane protein genes and other regions of the genome.

ABSTRACT: Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, is responsible for human monocytic ehrlichiosis (HME). In this study, we genetically mapped 10 isolates obtained from HME patients. Sequence analysis of the 28-kDa outer membrane protein (OMP) multigene locus spanning 6 of the 22 tandemly arranged genes identified three distinct genetic groups with shared homology among isolates within each group. Isolates in Groups I and III contained six genes each, while Group II isolates had a gene deletion. There were two regions on the locus where novel gene deletion or insertion mutations occurred, resulting in the net loss of one gene in Group II isolates. Numerous nucleotide differences among genes in isolates of each group also were detected. The shared homology among isolates in each group for the 28-kDa OMP locus suggests the derivation of clonal lineages. Transcription and translation analysis of the locus revealed differences in the expressed genes of different group isolates. Analysis of the 120-kDa OMP gene and variable-length PCR target gene showed size variations resulting from loss or gain of long, direct repeats within the protein coding sequences. To our knowledge this is the first study that looked at several regions of the genome simultaneously, and we provide the first evidence of heterogeneity resulting from gene deletion and insertion mutations in the E. chaffeensis genome. Diversity in different genomic regions could be the result of a selection process or of independently evolved genes.

SUBMITTER: Cheng C

PROVIDER: S-EPMC143425 | biostudies-literature | 2003 Jan

REPOSITORIES: biostudies-literature

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Molecular heterogeneity of Ehrlichia chaffeensis isolates determined by sequence analysis of the 28-kilodalton outer membrane protein genes and other regions of the genome.

Cheng Chuanmin C Paddock Christopher D CD Reddy Ganta Roman R

Infection and immunity 20030101 1

Ehrlichia chaffeensis, a tick-transmitted rickettsial agent, is responsible for human monocytic ehrlichiosis (HME). In this study, we genetically mapped 10 isolates obtained from HME patients. Sequence analysis of the 28-kDa outer membrane protein (OMP) multigene locus spanning 6 of the 22 tandemly arranged genes identified three distinct genetic groups with shared homology among isolates within each group. Isolates in Groups I and III contained six genes each, while Group II isolates had a gene ...[more]

PMID: 12496165

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Project description:The Ehrlichia chaffeensis 28-kDa outer membrane protein (p28) gene was sequenced completely by genomic walking with adapter PCR. The DNA sequence of the p28 gene was nearly identical to the previously reported sequence (N. Ohashi, N. Zhi, Y. Zhang, and Y. Rikihisa, Infect. Immun. 66:132-139, 1998), but analysis of a further 75 bp on the 5' end of the gene revealed DNA that encoded a 25-amino-acid signal sequence. The leader sequence was removed from the N terminus of a 30-kDa precursor to generate the mature p28 protein. A monoclonal antibody (MAb), 1A9, recognizing four outer membrane proteins of E. chaffeensis (Arkansas strain) including the 25-, 26-, 27-, and 29-kDa proteins (X.-J. Yu, P. Brouqui, J. S. Dumler, and D. Raoult, J. Clin. Microbiol. 31:3284-3288, 1993) reacted with the recombinant p28 protein. This result indicated that the four proteins recognized by MAb 1A9 were encoded by the multiple genes of the 28-kDa protein family. DNA sequence alignment analysis revealed divergence of p28 among all five human isolates of E. chaffeensis. The E. chaffeensis strains could be divided into three genetic groups on the basis of the p28 gene. The first group consisted of the Sapulpa and St. Vincent strains. They had predicted amino acid sequences identical to each other. The second group contained strain 91HE17 and strain Jax, which only showed 0.4% divergence from each other. The third group contained the Arkansas strain only. The amino acid sequences of p28 differed by 11% between the first two groups, by 13.3% between the first and third groups, and by 13.1% between the second and third groups. The presence of antigenic variants of p28 among the strains of E. chaffeensis and the presence of multiple copies of heterogeneous genes suggest a possible mechanism by which E. chaffeensis might evade the host immune defenses. Whether or not immunization with the p28 of one strain of E. chaffeensis would confer cross-protection against other strains needs to be investigated.

Project description:Ehrlichia chaffeensis, a cholesterol-rich and cholesterol-dependent obligate intracellular bacterium, partially lacks genes for glycerophospholipid biosynthesis. We found here that E. chaffeensis is dependent on host glycerolipid biosynthesis, as an inhibitor of host long-chain acyl CoA synthetases, key enzymes for glycerolipid biosynthesis, significantly reduced bacterial proliferation. E. chaffeensis cannot synthesize phosphatidylcholine or cholesterol but encodes enzymes for phosphatidylethanolamine (PE) biosynthesis; however, exogenous NBD-phosphatidylcholine, Bodipy-PE, and TopFluor-cholesterol were rapidly trafficked to ehrlichiae in infected cells. DiI (3,3'-dioctadecylindocarbocyanine)-prelabeled host-cell membranes were unidirectionally trafficked to Ehrlichia inclusion and bacterial membranes, but DiI-prelabeled Ehrlichia membranes were not trafficked to host-cell membranes. The trafficking of host-cell membranes to Ehrlichia inclusions was dependent on both host endocytic and autophagic pathways, and bacterial protein synthesis, as the respective inhibitors blocked both infection and trafficking of DiI-labeled host membranes to Ehrlichia In addition, DiI-labeled host-cell membranes were trafficked to autophagosomes induced by the E. chaffeensis type IV secretion system effector Etf-1, which traffic to and fuse with Ehrlichia inclusions. Cryosections of infected cells revealed numerous membranous vesicles inside inclusions, as well as multivesicular bodies docked on the inclusion surface, both of which were immunogold-labeled by a GFP-tagged 2×FYVE protein that binds to phosphatidylinositol 3-phosphate. Focused ion-beam scanning electron microscopy of infected cells validated numerous membranous structures inside bacteria-containing inclusions. Our results support the notion that Ehrlichia inclusions are amphisomes formed through fusion of early endosomes, multivesicular bodies, and early autophagosomes induced by Etf-1, and they provide host-cell glycerophospholipids and cholesterol that are necessary for bacterial proliferation.

Project description:Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fc?-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30?min) nuclear accumulation of NF-?B and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.

Dataset Information

Molecular heterogeneity of Ehrlichia chaffeensis isolates determined by sequence analysis of the 28-kilodalton outer membrane protein genes and other regions of the genome.

Publications

Molecular heterogeneity of Ehrlichia chaffeensis isolates determined by sequence analysis of the 28-kilodalton outer membrane protein genes and other regions of the genome.

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