[Show abstract][Hide abstract] ABSTRACT: Ehrlichia chaffeensis is an obligately intracellular gram-negative bacterium and is the etiologic agent of human monocytic ehrlichiosis (HME). Although E. chaffeensis induces the generation of several cytokines and chemokines by leukocytes, E. chaffeensis lacks lipopolysaccharide and peptidoglycan. Bioinfomatic analysis of the E. chaffeensis genome, however, predicted genes encoding 15 lipoproteins and 3 posttranslational lipoprotein-processing enzymes. The present study showed that by use of multidimensional liquid chromatography followed by tandem mass spectrometry, all predicted lipoproteins as well as lipoprotein-processing enzymes were expressed by E. chaffeensis cultured in the human promyelocytic leukemia cell line HL-60. Consistent with this observation, a signal peptidase II inhibitor, globomycin, was found to inhibit E. chaffeensis infection and lipoprotein processing in HL-60 cell culture. To study in vivo E. chaffeensis lipoprotein expression and host immune responses to E. chaffeensis lipoproteins, 13 E. chaffeensis lipoprotein genes were cloned into a mammalian expression vector. When the DNA constructs were inoculated into naïve dogs, or when dogs were infected with E. chaffeensis, the animals developed delayed-type hypersensitivity reactions at cutaneous sites of the DNA construct deposition and serum antibodies to these lipoproteins. This is the first demonstration of lipoprotein expression and elicitation of immune responses by a member of the order Rickettsiales. Multiple lipoproteins expressed by E. chaffeensis in vitro and in vivo may play key roles in pathogenesis and immune responses in HME.
Infection and immunity 09/2008; 76(8):3405-14. DOI:10.1128/IAI.00056-08 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ehrlichia chaffeensis, an obligatory intracellular gram-negative bacterium, must take up various nutrients and metabolic compounds because it lacks many genes involved in metabolism. Nutrient uptake by a gram-negative bacterium occurs primarily through pores or channels in the bacterial outer membrane. Here we demonstrate that isolated E. chaffeensis outer membranes have porin activities, as determined by a proteoliposome swelling assay. The activity was partially blocked by an antibody that recognizes the two most abundant outer membrane proteins, P28/OMP-19 and OMP-1F/OMP-18. Both proteins were predicted to have structural features characteristic of porins, including 12 transmembrane segments comprised of amphipathic and antiparallel beta-strands. The sodium dodecyl sulfate stability of the two proteins was consistent with a beta-barrel structure. Isolated native P28 and OMP-1F exhibited porin activities, with pore sizes similar to and larger than, respectively, that of OprF, which is the porin with the largest pore size known to date. E. chaffeensis experiences temperature changes during transmission by ticks. During the intracellular development of E. chaffeensis, both P28 and OMP-1F were expressed mostly in the mid-exponential growth phase at 37 degrees C and the late-exponential growth phase at 28 degrees C. The porin activity of proteoliposomes reconstituted with proteins from the outer membrane fractions derived from bacteria in the mid- and late-exponential growth phases at 28 degrees C and 37 degrees C correlated with the expression levels of P28 and OMP-1F. These results imply that P28 and OMP-1F function as porins with large pore sizes, suggesting that the differential expression of these two proteins might regulate nutrient uptake during intracellular E. chaffeensis development at both temperatures.
Journal of bacteriology 06/2008; 190(10):3597-605. DOI:10.1128/JB.02017-07 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus, A. phagocytophilum needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of A. phagocytophilum has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of L-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of A. phagocytophilum. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel beta-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100 p44 paralogs and go through extensive antigenic variation. The 16-transmembrane-domain beta-strands consist of conserved P44 N- and C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of A. phagocytophilum is incomplete and requires the exogenous acquisition of L-glutamine or L-glutamate for function. Efficient diffusion of L-glutamine across the outer membrane suggests that the porin feeds the Anaplasma TCA cycle and that the relatively large pore size provides Anaplasma with the necessary metabolic intermediates from the host cytoplasm.
Journal of Bacteriology 04/2007; 189(5):1998-2006. DOI:10.1128/JB.01548-06 · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Ehrlichia canis (E. canis) is a lipopolysaccharide (LPS)-deficient obligatory intracellular bacterium that causes canine monocytic ehrlichiosis, a chronic febrile disease accompanied with hematological abnormality. This study analyzed temporal expression levels of IL-1beta, IL-2, IL-6, IL-8, IFN-gamma, and TNF-alpha mRNA by peripheral blood leukocytes from dogs experimentally infected with a new virulent strain of E. canis by using real-time RT-PCR. Relative levels of IL-1beta and IL-8 transcripts normalized by the beta-actin transcript levels, were significantly upregulated, whereas those of TNF-alpha and IFN-gamma transcripts were only weakly upregulated in all three infected dogs, starting from 2 days up to 52 days post inoculation. The expressions of IL-2 and IL-6 genes were extremely low compared with the positive control (ConA-stimulated canine peripheral blood leukocytes). This study showed that E. canis can induce chronic expression of a subset of proinflammatory cytokine genes: balance, timing, and duration of these cytokine generations may contribute to the progression of canine ehrlichiosis.
Annals of the New York Academy of Sciences 11/2006; 1078(1):482-6. DOI:10.1196/annals.1374.090 · 4.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Blood specimens from clinically normal military dogs and their trainers, in Lara, Venezuela were screened for Anaplasma platys, A. phagocytophilum, or Ehrlichia ewingii using 16S rRNA PCR tests. Sixteen percent (7/43) of dog specimens were positive by A. platys PCR test followed by sequencing of the PCR products, and all human blood specimens  were negative. All specimens from these dogs and humans were PCR negative for E. ewingii or A. phagocytophilum. Twelve Rhipicephalus sanguineus ticks removed from these dogs were negative for A. platys by reverse transcription PCR test. Almost the entire 16S rRNA gene (1,364 bp) and groESL operon (1,646 bp) sequences of A. platys isolated from a dog were determined, revealing that both sequences were closely related to the sequences of an A. platys strain detected in R. sanguineus ticks from the Democratic Republic of Congo.
[Show abstract][Hide abstract] ABSTRACT: Ehrlichia canis virB9 was cloned and expressed. The sequences of virB9 from six geographic locations were identical. virB9 was transcribed by E. canis in dogs, ticks, and cell culture. Infected dogs had antibodies to recombinant VirB9, indicating that VirB9 was produced by E. canis in dogs and was antigenic.
Infection and Immunity 11/2003; 71(10):6063-7. DOI:10.1128/IAI.71.10.6063-6067.2003 · 4.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We previously culture isolated a strain of Ehrlichia canis, the causative agent of canine ehrlichiosis, from a human in Venezuela. In the present study, we examined whether dogs and ticks are infected with E. canis in Venezuela and, if so, whether this is the same strain as the human isolate. PCR analysis using E. canis-specific primers revealed that 17 of the 55 dog blood samples (31%) and all three pools of four Rhipicephalus sanguineus ticks each were positive. An ehrlichial agent (Venezuelan dog Ehrlichia [VDE]) was isolated and propagated in cell culture from one dog sample and was further analyzed to determine its molecular and antigenic characteristics. The 16S rRNA 1,408-bp sequence of the new VDE isolate was identical to that of the previously reported Venezuelan human Ehrlichia isolate (VHE) and was closely related (99.9%) to that of E. canis Oklahoma. The 5' (333-bp) and 3' (653-bp) sequences of the variable regions of the 16S rRNA genes from six additional E. canis-positive dog blood specimens and from three pooled-tick specimens were also identical to those of VHE. Western blot analysis of serum samples from three dogs infected with VDE by using several ehrlichial antigens revealed that the antigenic profile of the VDE was similar to the profiles of VHE and E. canis Oklahoma. Identical 16S rRNA gene sequences among ehrlichial organisms from dogs, ticks, and a human in the same geographic region in Venezuela and similar antigenic profiles between the dog and human isolates suggest that dogs serve as a reservoir of human E. canis infection and that R. sanguineus, which occasionally bites humans residing or traveling in this region, serves as a vector. This is the first report of culture isolation and antigenic characterization of an ehrlichial agent from a dog in South America, as well as the first molecular characterization of E. canis directly from naturally infected ticks.