The genome of the obligately intracellular bacterium Ehrlichia canis reveals themes of complex membrane structure and immune evasion strategies.

Ehrlichia canis, a small obligately intracellular, tick-transmitted, gram-negative, alpha-proteobacterium, is the primary etiologic agent of globally distributed canine monocytic ehrlichiosis. Complete genome sequencing revealed that the E. canis genome consists of a single circular chromosome of 1,315,030 bp predicted to encode 925 proteins, 40 stable RNA species, 17 putative pseudogenes, and a substantial proportion of noncoding sequence (27%). Interesting genome features include a large set of proteins with transmembrane helices and/or signal sequences and a unique serine-threonine bias associated with the potential for O glycosylation that was prominent in proteins associated with pathogen-host interactions. Furthermore, two paralogous protein families associated with immune evasion were identified, one of which contains poly(G-C) tracts, suggesting that they may play a role in phase variation and facilitation of persistent infections. Genes associated with pathogen-host interactions were identified, including a small group encoding proteins (n = 12) with tandem repeats and another group encoding proteins with eukaryote-like ankyrin domains (n = 7).

Cold adaptation of a psychrophilic chitinase: a mutagenesis study.

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.

Engineering the properties of a cold active enzyme through rational redesign of the active site.

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.

Cloning, sequences, and characterization of two chitinase genes from the Antarctic Arthrobacter sp. strain TAD20: isolation and partial characterization of the enzymes.

Arthrobacter sp. strain TAD20, a chitinolytic gram-positive organism, was isolated from the sea bottom along the Antarctic ice shell. Arthrobacter sp. strain TAD20 secretes two major chitinases, ChiA and ChiB (ArChiA and ArChiB), in response to chitin induction. A single chromosomal DNA fragment containing the genes coding for both chitinases was cloned in Escherichia coli. DNA sequencing analysis of this fragment revealed two contiguous open reading frames coding for the precursors of ArChiA (881 amino acids [aa]) and ArChiB (578 aa). ArChiA and ArChiB are modular enzymes consisting of a glycosyl-hydrolase family 18 catalytic domain as well as two and one chitin-binding domains, respectively. The catalytic domain of ArChiA exhibits 55% identity with a chitodextrinase from Vibrio furnissii. The ArChiB catalytic domain exhibits 33% identity with chitinase A of Bacillus circulans. The ArChiA chitin-binding domains are homologous to the chitin-binding domain of ArChiB. ArChiA and ArChiB were purified to homogeneity from the native Arthrobacter strain and partially characterized. Thermal unfolding of ArChiA, ArChiB, and chitinase A of Serratia marcescens was studied using differential scanning calorimetry. ArChiA and ArChiB, compared to their mesophilic counterpart, exhibited increased heat lability, similar to other cold-adapted enzymes.

Early effects of arterial hemodynamic conditions on human saphenous veins perfused ex vivo.

Exposure to the arterial hemodynamic environment is thought to be a potential trigger for the pathological remodeling of saphenous vein grafts. Using matched pairs of freshly isolated human saphenous vein, we analyzed the early effects of ex vivo hemodynamic conditions mimicking the venous (native) compared with arterial (graft) environment on the key components of vascular remodeling, ie, matrix metalloproteinase (MMP)-9 and MMP-2 and cell proliferation. Interestingly, we found that arterial conditions halved latent MMP-9 (50+/-11%, P=0.01) and MMP-2 (44+/-6%, P=0.005) levels relative to matched vein pairs maintained ex vivo under venous perfusion for up to 3 days. Immunostaining supported decreased MMP levels in the innermost area of arterially perfused veins. Either decreased synthesis or increased posttranslational processing may decrease MMP zymogen levels. Biosynthetic radiolabeling showed that arterial perfusion actually increased MMP-9 and MMP-2 production. When we then examined potential pathways for MMP zymogen processing, we found that arterial conditions did not affect the expression of MT-MMP-1, a cell-associated MMP activator, but that they significantly increased the levels of superoxide, another MMP activator, suggesting redox-dependent MMP processing. Additional experiments indicated that increased superoxide under arterial conditions was due to diminished scavenging by decreased extracellular superoxide dismutase. Arterial perfusion also stimulated cell proliferation (by 220% to 750%) in the majority of vein segments investigated. Our observations support the hypothesis that arterial hemodynamic conditions stimulate early vein graft remodeling. Furthermore, physiological arterial flow may work to prevent pathological remodeling, particularly the formation of intimal hyperplasia, through rapid inactivation of secreted MMPs and, possibly, through preferential stimulation of cell proliferation in the outer layers of the vein wall.

Vascular superoxide production and vasomotor function in hypertension induced by deoxycorticosterone acetate-salt.

BACKGROUND: Angiotensin II-induced hypertension is associated with increased vascular superoxide production, which contributes to hypertension caused by the octapeptide. In cell culture, stretch increases endothelial and vascular smooth muscle production of reactive oxygen species (ROS). In perfused isolated vessels, elevations of pressure can increase vessel angiotensin II production. The effects of low-renin hypertension on vascular ROS production remain unclear. Furthermore, the role of ROS in vascular function and hypertension in low-renin hypertension is undefined. METHODS AND RESULTS: Rats were treated with DOCA and saline drinking water for 3 weeks. Both systolic blood pressure (189+/-4 versus 126+/-2 mm Hg) and aortic superoxide production (3972+/-257 versus 852+/-287, P<0. 05) were increased compared with controls. Relaxations of vascular segments to acetylcholine (ACh, 100+/-2% versus 75+/-2%, P<0.05) and the calcium ionophore A23187 (92+/-2% versus 72+/-3%, P<0.05) were also impaired in DOCA-salt. Heparin-binding superoxide dismutase (1200 U/d IV for 3 days) had no effect on blood pressure but significantly improved relaxations to ACh and A23187. Losartan (25 mg x kg(-1) x d(-1) PO) for 7 days did not correct the hypertension or endothelium-dependent vessel relaxation in DOCA-salt rats, excluding a role of a local renin/angiotensin II system. CONCLUSIONS: These findings indicate that increased vascular superoxide production occurs not only in angiotensin II-induced hypertension but also in hypertension known to be associated with low-renin states. Increased superoxide production alters large-vessel endothelium-dependent vascular relaxation but does not modulate blood pressure in low-renin hypertension.

Alkaline phosphatase from the Antarctic strain TAB5. Properties and psychrophilic adaptations.

The gene encoding alkaline phosphatase (AP) from the psychrophilic strain TAB5 was cloned, and its nucleotide sequence was determined. A single open reading frame consisting of 1125 base pairs which encodes a polypeptide consisting of signal peptide of 22 amino acids and a mature protein of 353 amino acids was identified. The deduced protein sequence of AP exhibits a 38% identity to the AP III and AP IV sequences of Bacillus subtilis and conserves the typical sequence motifs of the core structure and active sites of APs from various sources. Based on the crystal structure of the mutated Escerichia coli AP D153H, a homology-based 3D model of the TAB5 AP was constructed on the basis of which various features of the enzyme amino-acid sequence can be interpreted in terms of potential psychrophilic adaptations. The AP gene was expressed in E. coli BL21(DE3) cells, the recombinant protein was isolated to homogeneity from the membrane fraction of the cells and its properties were examined. The purified TAB5 AP shows typical features of a cold enzyme: high catalytic activity at low temperature and a remarkable thermosensitivity. The use of this heat-labile enzyme, for dephosphorylation of nucleic acids, simplifies dephosphorylation protocols.

Mechanical stretching of human saphenous vein grafts induces expression and activation of matrix-degrading enzymes associated with vascular tissue injury and repair.

After coronary artery bypass surgery, saphenous vein graft occlusion occurs through tissue remodeling. Although a likely trigger, the role of preparative mechanical injury incurred by the graft is not yet understood. We studied the early effects of simple mechanical injury on human saphenous vein grafts by exposing them to longitudinal stretch, a deformation which potentially occurs during surgery. We then maintained ex vivo for up to 7 days matched pairs of experimentally stretched and nonstretched (control) vein segments and examined the expression and activation of matrix metalloproteinases (MMPs) and integrin alphav, molecules implicated in vascular remodeling. At peak expression on day 3, stretched vein secreted 177 +/- 16% active MMP-2 (P < 0.01), 161 +/- 36% (P < 0.05) pro-MMP-9, and contained 206 +/- 18% (P < 0.01) alphav, a receptor for active MMP-2, compared to control. In situ gelatinase activity was present in the intima and adventitia of stretched veins, but not of control, and correlated spatially with expression of alphav. Stretch also increased severalfold cell proliferation (1.27 +/- 0.4 vs. 0.23 +/- 0.05% in control, P < 0.05), as assessed by bromodeoxyuridine incorporation. Furthermore, we found that cell proliferation colocalized with gelatinase activity and alphav in the adventitia. Our results show that a single longitudinal stretch of vein grafts produces significant changes in the expression and activation of key molecules in vascular remodeling. We also found support for the notion that the adventitial layer contributes to vein graft remodeling.

Cloning and characterization of the gene encoding PspPI methyltransferase from the Antarctic psychrotroph Psychrobacter sp. strain TA137. Predicted interactions with DNA and organization of the variable region.

The gene (pspPIM) encoding the PspPI DNA methyltransferase (MTase) associated with the PspPI restriction-modification (R-M) system (5'-GGNCC-3') of Psychrobacter species TA137 has been cloned and expressed in E. coli, and its nucleotide (nt) sequence has been determined. The coding region was 1248 nt in length and capable of specifying a 46826-Da protein of 416 amino acids (aa). The predicted sequence of the MTase protein displays ten sequence motifs characteristic of all prokaryotic m5C-MTases and shows the highest similarity to other MTases that methylate the GGNCC sequence, namely M . Eco47II and M . Sau96I. All three MTases methylate the internal cytosine within their recognition sequence. Sequence similarities between M . PspPI and its isospecific M . Eco47II and M . Sau96I as well as with four other m5C-MTases that methylate the related GGWCC sequence, namely M . SinI, M . HgiCII, M . HgiBI, M . HgiEI have been also found within the variable region of these proteins. On the basis of structural information from M . HhaI and M . HaeIII, several M . PspPI residues that are expected to interact with DNA can be predicted. Furthermore, an organization of the variable region of m5C-MTases into two segments exhibiting a pattern of conserved residues and a considerable degree of structural homologies is described.

The carboxyl-terminal zinc-binding domain of the human papillomavirus E7 protein can be functionally replaced by the homologous sequences of the E6 protein.

The carboxyl-terminus is necessary for the functional and structural integrity of the human papillomavirus (HPV) E7 oncoprotein. Since many mutations in this domain of E7 result in the formation of unstable proteins, we have evaluated the importance of this region by replacing it with structurally related domains derived from HPV E6 proteins. Biological analysis of these mutant chimeric E7/E6 proteins showed that they retained E7-specific biological activities including cooperation with the ras oncogene to transform primary baby rat kidney cells and transcriptional activation of an E2F responsive reporter plasmid. One of the chimeric proteins was impaired in its ability to physically disrupt pRB/E2F complexes in vitro suggesting that there are defined molecular determinants in the carboxyl-terminus of E7 for this activity. In contrast, none of these proteins exhibited E6-like properties including binding to p53 and/or degradation of associated proteins.