Monitoring of Bordetella isolates circulating in Saint Petersburg, Russia between 2001 and 2009

Bordetella isolates in the Saint Petersburg region have been monitored since 1998. Over the past ten years, concomitant with the increase in pertussis whole-cell vaccine coverage, the incidence of whooping cough has decreased. However, this decrease exists only for Bordetella pertussis infections, as the incidence of Bordetella parapertussis confirmed cases has remained stable, suggesting that pertussis-vaccine-induced immunity is not protective against parapertussis, as expected. B. pertussis and B. parapertussis clinical isolates were analyzed using serotyping, immunoblotting, pulsed-field gel electrophoresis of chromosomal DNA (after digestion with XbaI) and sequencing of virulence genes. The bacterial population is now similar to that observed in other European countries.     

Characterization of recombinant Bordetella pertussis adenylate cyclase toxins carrying passenger proteins

Bordetella pertussis secretes a calmodulin-activated adenylate cyclase toxin, CyaA, that is able to deliver its N-terminal catalytic domain (400 amino acid residues) into the cytosol of eukaryotic target cells, directly through the cytoplasmic membrane. We have previously shown that CyaA can be used as a vehicle to deliver CD8⁺ T-cell epitopes, inserted within the catalytic domain of the toxin, into antigen-presenting cells and can trigger specific class I-restricted cytotoxic T-cell (CTL) responses in vivo. To explore the tolerance of CyaA to insertion of polypeptides of larger size, we constructed and characterized different recombinant CyaA toxins with protein inserts of 87 to 206 amino acids in length. Several of these recombinant CyaA toxins were found to be invasive. Furthermore, we showed that the unfolding of the passenger protein is a prerequisite for the translocation of the recombinant toxins into eukaryotic cells. Our results highlight the remarkable tolerance of the CyaA toxin and suggest that CyaA might be used to deliver proteins into eukaryotic cells.     

Genomic analysis of the adenylate cyclase-hemolysin C-terminal region of Bordetella pertussis, Bordetella parapertussisand Bordetella bronchiseptica

Adenylate cyclase-hemolysin plays an important role in the virulence of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica species. Its C-terminal region carries protective epitopes and receptor binding site for human cells. Genomic analyses of this region indicate no polymorphism in B. pertussis and B. parapertussis regions, but substantial variability in B. bronchiseptica that might be linked to the various niches of this species.     

Protective activity of Bordetella adenylate cyclase-hemolysin against bacterial colonization.

Bordetella pertussis synthesizes several factors. It has been suggested that one of these factors, the adenylate cyclase-hemolysin (AC-Hly), directly penetrates target cells and impairs their normal functions by elevating intracellular cAMP. In the present study, we show that active immunization with purified B. pertussis AC-Hly or AC (a fragment of the AC-Hly molecule carrying only the adenylate cyclase activity but no toxin activity in vitro) protects mice against B. pertussis intranasal infection. Immunization with AC-Hly or AC significantly shortens the period of bacterial colonization of the mouse respiratory tract. Furthermore, B. parapertussis AC-Hly or AC are also protective antigens against B. parapertussis colonization; their protective activities are equivalent to that of the whole-cell vaccine. These results suggest that AC-Hly may play an important role in Bordetella pathogenesis, in a murine model. If this factor plays a similar role in the human disease, its use as a protective antigen could reduce not only the incidence of the disease, but also the asymptomatic human reservoir by limiting bacterial carriage.     

Characterization of fimbrial subunits from Bordetella species

Using antisera raised against serotype 2 and 3 fimbrial subunits from Bordetella pertussis, serologically related polypeptides were detected in Bordetella bronchiseptica, Bordetella parapertussis and Bordetella avium strains. The two B. pertussis fimbrial subunits, and three of the serologically related B. bronchiseptica polypeptides, were shown to be very similar in amino acid composition and N-terminal amino acid sequence. Homology was observed between the N-termini of these polypeptides, and fimbrial subunits from Escherichia coli, Haemophilus influenzae and Proteus mirabilis. A synthetic oligonucleotide probe, derived from the N-terminal sequence of the B. pertussis serotype 2 fimbrial subunit, was used to identify fimbrial genes in genomic Southern blots. The results suggested the presence of multiple fimbrial subunit genes in B. pertussis, B. bronchiseptica and B. parapertussis. The DNA probe was used to clone one of the three tentative fimbrial subunit genes detected in B. pertussis.     

Antifungal activity of Pterocaulon species (Asteraceae) against Sporothrix schenckii

Plants of the genus Pterocaulon (Asteraceae) are popularly used in the treatment of skin diseases caused by fungi and bacteria. The aim of this work was to investigate the in vitro activity of the crude methanolic extracts obtained from the aerial parts of Pterocaulon polystachyum, P. balansae, P. lorentzii, P. lanatum, and P. cordobense against 24 Sporothrix schenckii clinical isolates and determine the minimum inhibitory concentration (MIC) and the minimum fungicidal concentration (MFC). MIC were performed by the broth microdilution method according guidelines recommended by Clinical and Laboratory Standards Institute for filamentous fungi and MFC were determined for transference of aliquots of the well that showed 100% of growth inhibition into tubes with culture medium. The extract from P. polystachyum was the most active sample, presenting MIC range of 156 and 312 μg/mL. The popular use of these plants corroborates the importance of ethnopharmacological surveys and opens the possibility for finding new clinically effective antifungal agents.     

Rapid molecular genetic assay for direct identification of Bordetella from patients specimens

Biological diagnosis of whooping cough is increasingly necessary to confirm respiratory tract infection. Indeed, clinical symptoms are variable especially in adolescents and adults who contaminate newborns too young to be vaccinated. The PCR assay was proven highly sensitive for the diagnosis of pertussis. In this study, we reported the use of a new test (GenoQuick^® Bordetella [GQB], Hain Life Science, Germany) which permits the fast molecular genetic identification of Bordetella pertussis and parapertussis directly from patients specimens, i.e. swabs from nose or throat. The test was performed over a three months period on 40 specimens from patients (1 month to 65 years old), most of them were young children admitted in paediatric emergency with paroxysmal cough or prolonged cough.     

The Bpel locus encodes type III secretion machinery in Bordetella pertussis

Type III secretory genes(Bscl, J, K, L, N and O) have recently been identified in Bordetella bronchiseptica and shown to be under the control of the BvgAS locus. We examined a 35 616 byte DNA sequence amplified from Bordetella pertussis Tohama I for homology with known type III secretory genes in Yersinia spp. and Pseudomonas sppand a total of 20 homologous open reading frames were detected. Putative type III secretion proteins in B. pertussis were designated according to their homology with type III secretion proteins in B. bronchiseptica, Yersinia and Pseudomonas. These ORFs were arranged in two putative operons, which together we have designated as the BpeI locus. The first spans nucleotides 23385–7888 and encodes the putative proteins LcrH1, BopD, BopB, LcfH2, BscI, BscJ, BscK, BscL, BscN, BscO, BscQ, BscR, BscS, BscT, BscU, and BscC, in this order. The second spans nucleotides 23580–29863 and encodes the putative proteins LcrE, LcrD, BscD and BscF, in this order. The homology of these proteins to type III secretory proteins was B. bronchiseptica (73–99%),Yersinia spp. (17–65%), Pseudomonas spp. (18–64%). The B. pertussis proteins were similar to their homologues in B. bronchiseptica, Yersinia and Pseudomonas in terms of length, molecular weight and isoelectric point. Coiled-coil domains were detected in putative translocation proteins, BopB and BopD. BopB and BopD were similar to each other, to the RTX toxin family and to cyaA, cyaB, cyaD and cyaE. The percentage G+C content of the sequence analysed was 66.16%, which is similar to the published percentage G+C (67–70%) for the B. pertussis chromosome.     

Effect of asphyxia on adenylate cyclase activity in cat brain cortex

Effect of 1–5-min asphyxia on adenylate cyclase activity in cat brain cortex is studied. Adenylate cyclase activity is measured in cortex specimens obtainedex vivo after 1, 2.5, and 5 min of asphyxia, and 30 and 60 min of reoxygenation by radioassay. Stimulating effects of norepinephrine and NaF on adenylate cyclase activity are assessed. Five-min asphyxia induces phasic changes in adenylate cyclase activity: on the 1st min basal activity of the enzyme increases by 97%, after 2.5 min it returns to the initial level, and increases again by 55% on the 5th min of asphyxia. On the 30th and 60th min of reoxygenation after 2.5- and 5-min asphyxia, basal adenylate cyclase activity does not differ from the initial activity. The stimulating effect of norepinephrine and NaF on enzyme activity is weakened after 5 min of asphyxia and 30 min of reoxygenation after 2.5- and 5-min asphyxia. Even short-term asphyxia affects adenylate cyclase activity and modifies the mechanisms of adrenergic signal transduction in the brain cortex in response to oxygen deficiency and probably to hypercapnia as well as during the early reoxygenation period. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 124, No. 8, pp. 131–134, August, 1997     

Cholesterol-rich domains are involved in Bordetella pertussis phagocytosis and intracellular survival in neutrophils

Bordetella pertussis-specific antibodies protect against whooping cough by facilitating host defense mechanisms such as phagocytosis. However, the mechanism involved in the phagocytosis of the bacteria under non-opsonic conditions is still poorly characterized. We report here that B. pertussis binding and internalization is cholesterol dependent. Furthermore, we found cholesterol to be implicated in B. pertussis survival upon interaction with human neutrophils. Pre-treatment of PMN with cholesterol sequestering drugs like nystatin or methyl-β-cyclodextrin (MβCD) resulted in a drastic decrease of uptake of non-opsonized B. pertussis. Conversely, phagocytosis of opsonized bacteria was not affected by these drugs, showing that cholesterol depletion affects neither the viability of PMN nor the route of entry of opsonized B. pertussis. Additionally, intracellular survival rate of non-opsonized bacteria was significantly decreased in cholesterol-depleted PMN. Accordingly, confocal laser microscopy studies showed that non-opsonized B. pertussis co-localized with lysosomal markers only in cholesterol-depleted PMN but not in normal PMN. Our results indicate that B. pertussis docks to molecules that eventually prevent cellular bactericidal activity.     

RNAi knock-down of the Litopenaeus vannamei Toll gene (LvToll) significantly increases mortality and reduces bacterial clearance after challenge with Vibrio harveyi

In this study, we used real-time PCR to simultaneously monitor the responses of 12 key genes of the shrimp innate immune system in Litopenaeus vannamei after challenge with Vibrio harveyi. In the proPO activating system, we found that proPO was up-regulated (3.3× control at 36 hpi). The hemolymph clotting genes transglutaminase (TGase) and clotting protein were also up-regulated, as were 5 genes in the antimicrobial peptide system (ALF, Crustin, Lyz, PEN2 and PEN4), with only PEN3 showing no significant changes. In the antioxidant defense system, SOD was slightly elevated while GPx was substantially down-regulated. In the pattern recognition receptor system, at 24 hpi, the Toll gene (LvToll) showed the highest relative increase in expression level of all the investigated genes (15× greater than the sterile seawater control). In the second part of this study, when LvToll was knocked down by RNAi silencing, there was no effect on either survival rates or bacterial number in unchallenged shrimp. There was also no difference in mortality rates between control shrimp and LvToll-silenced shrimp when these two groups were challenged with a viral pathogen (white spot syndrome virus; WSSV). However, when LvToll-silenced shrimp were challenged by V. harveyi, there was a significant increase in mortality and bacterial CFU counts. We note that the increase in bacterial CFU count occurred even though treatment with EGFP dsRNA had the opposite effect of reducing the CFU counts. We conclude that LvToll is an important factor in the shrimp innate immune response to acute V. harveyi infection, but not to WSSV.     

The role of the O-antigen lipopolysaccharide on the colonization in vivo of the germfree chicken gut by Klebsiella pneumoniae

We isolated lipopolysaccharide and capsular polysaccharide (K antigen)-defective mutants from two Klebsiella pneumoniae parental strains, and compared their ability to colonize in vivo the germfree chicken gut. The high-molecular weight lipopolysaccharide (LPS) (O antigen) was found necessary for the colonization while the capsular polysaccharide (K2 or K29) was not of importance.     

Allosteric modification of adenylate deaminase activity initiation of adenosine deaminase activity by potassium ions

Activation of purified adenylate deaminase from the duck myocardium by K⁺ ions is accompanied by a change in the substrate specificity of the enzyme and appearance of ability to deaminate adenosine and adenine. Adenosine deaminase activity appears with K⁺ in a concentration exhibiting maximal stimulating effect (0.15 M) and it increases with an increase in the K⁺ concentration, parallel with a decrease in Hill's coefficient. It can be concluded from the pH dependence, the character of inhibition by phosphate, and the effect of cations of the alkali metals that deamination of adenosine takes place at natural combining sites of adenylate deaminase, the conformation of which is modified by the activator.Laboratory of Biochemistry of Amines and Other Nitrogenous Bases, Institute of Biological and Medical Chemistry, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. N. Orekhovich.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 11, pp. 535–537, November, 1978.     

Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis

A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration>1 microg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations<0.5 microg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 microg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 microg/ml, whereas a approximately 500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high beta-sheet content or a beta-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations.     

Expression of P.69/ppertactin from Bordetella pertussis in a baculovirus/insect cell expression system: protective properties of the recombinant protein

The surface antigen P.69/pertactin of Bordetella pertussis has been expressed using the polyhedron promoter of baculovirus in cultured insect cells. Either full-length or truncated prn DNA was used to express P.69 pertactin. The full-length gene gave rise to low levels of P.93 precursor protein, some of which was processed to P.69. The shortened prn expressed P.69 pertactin directly at levels up to 3.5 mg per litre. P.69 vaccinated animals were protected against aerosol challenge with virulent B. pertussis bacteria.     

Effects of Trifolium pratense and Cimicifuga racemosa on the endometrium of wistar rats

Objective

The aim of this study was to evaluate the effects of Trifolium pratense and Cimicifuga racemosa upon the endometrium of castrated female Wistar rats, comparing these results with a placebo and estradiol valerate.

Methods

Thirty-two adult castrated female Wistar rats were divided into four groups (eight rats per group) that receiving either tap water, estradiol valerate, isoflavones from T. pratense or deoxyactein from C. racemosa daily. The doses used were equivalent to normal doses used in humans. The results were analyzed by endometrial histology and the expression of α-estrogen receptor and protein Ki67. Both α-receptor and Ki67 expressions were determined by immunohistochemical and morphometric analysis.

Results

Endometrium histology stayed atrophic with both herbal extracts, but T. pratense supplementation increased the expression of α-estrogen receptors when compared to the placebo group, without protein Ki67 expression enhancement. Both herbal extracts presented a lower Ki67 expression when compared to the placebo group.

Conclusion

T. pratense presented α-estrogen receptor stimulation in the endometrium without increasing cell proliferation. Both herbal extracts reduced endometrial proliferation in comparison to the placebo group.     

Apolipophorin-III in Galleria mellonella potentiates hemolymph lytic activity

Heat-inactivated serum of non-immune Galleria mellonella larvae enhanced the lytic activity of larval cell-free hemolymph against Micrococcus lysodeikticus. The increase in bacterial lysis was due to a 17.2 kDa protein known previously to bind to bacterial lipopolysaccharides. The protein enhanced the lytic activity of insect cell-free hemolymph and hen lysozyme in vitro and insect hemolymph in vivo. The hydrophobic protein, which adhered to M. lysodeikticus, was identified by its amino acid sequence homology as apolipophorin-III. The titer of apolipophorin-III in 200–250 mg last instar larvae was 8.7 mg/ml of hemolymph. Apolipophorin-III did not bind to lysozyme. A possible mode of action of apolipophorin-III with lysozyme in the insect is proposed.     

Serum dependent expression of Enterococcus faecalis adhesins involved in the colonization of heart cells

Our previous studies have shown that the adhesive ability of Enterococcus faecalis is dependent on the strain and is further modified by growth in serum. The data reported here demonstrate that E. faecalis adherence is mediated by carbohydrate residues present on the bacterial cell surface. Some of these ( -mannose and -glucose) are expressed by strains isolated from both urinary tract infections (UTI) and endocarditis (EN) when the cells are grown in brain-heart infusion broth (BHIB), and mediate adherence to either urinary tract epithelial cells or the Girardi Heart (GH) cell line. Other residues are present only on EN strains ( -galactose and -fucose) and mainly mediate adherence to GH cells. These ligands can also be expressed by UTI isolates after growth in serum. -galactose-bearing adhesins also seem to be involved in internalization of serum grown UTI strains and BHIB or serum grown EN isolates into GH cells.