Localization of adhesins on the surface of a pathogenic bacterial envelope through atomic force microscopy.

Bacterial adhesion is the first and a significant step in establishing infection. This adhesion normally occurs in the presence of flow of fluids. Therefore, bacterial adhesins must be able to provide high strength interactions with their target surface in order to maintain the adhered bacteria under hydromechanical stressing conditions. In the case of B. pertussis, a Gram-negative bacterium responsible for pertussis, a highly contagious human respiratory tract infection, an important protein participating in the adhesion process is a 220 kDa adhesin named filamentous haemagglutinin (FHA), an outer membrane and also secreted protein that contains recognition domains to adhere to ciliated respiratory epithelial cells and macrophages. In this work, we obtained information on the cell-surface localization and distribution of the B. pertussis adhesin FHA using an antibody-functionalized AFM tip. Through the analysis of specific molecular recognition events we built a map of the spatial distribution of the adhesin which revealed a non-homogeneous pattern. Moreover, our experiments showed a force induced reorganization of the adhesin on the surface of the cells, which could explain a reinforced adhesive response under external forces. This single-molecule information contributes to the understanding of basic molecular mechanisms used by bacterial pathogens to cause infectious disease and to gain insights into the structural features by which adhesins can act as force sensors under mechanical shear conditions.

Adhesin contribution to nanomechanical properties of the virulent Bordetella pertussis envelope.

Adherence to a biological surface allows bacteria to colonize and persist within the host and represents an essential first step in the pathogenesis of most bacterial diseases. Consequently, the physicochemical properties of the outer membrane in bacteria play a key role for attachment to surfaces and therefore for biofilm formation. Bordetella pertussis is a Gram-negative bacterium that colonizes the respiratory tract of humans, producing whooping cough or pertussis, a highly infectious disease. B. pertussis uses various adhesins exposed on its surface to promote cell-surface and cell-cell interactions. The most dominant adhesin function is displayed by filamentous hemagglutinin (FHA). B. pertussis Tohama I wild-type (Vir+) strain and two defective mutants, an avirulent (Vir-) and a FHA-deficient (FHA-) B. pertussis strains were studied by AFM under physiological conditions to evaluate how the presence or absence of adhesins affects the mechanical properties of the B. pertussis cell surface. Quantitative information on the nanomechanical properties of the bacterial envelope was obtained by AFM force-volume analysis. These studies suggested that the presence of virulence factors is correlated with an increase in the average membrane rigidity, which is largely influenced by the presence of FHA. Moreover, for this system we built a nanoscale stiffness map that reveals an inhomogeneous spatial distribution of Young modulus as well as the presence of rigid nanodomains on the cell surface.

Analyses of lipopolysaccharides, outer membrane proteins and DNA fingerprints reveal intraspecies diversity in Moraxella bovis isolated in Argentina.

Intra-specific diversity within Moraxella bovis was investigated analysing DNA fingerprints, outer membrane proteins (OMP) and lipopolysaccharides (LPS) profiles. Three collection strains and 57 isolates of M. bovis, collected during 3 years from cattle with infectious bovine keratoconjunctivitis (IBK) symptoms, from diverse geographical locations of Argentina, were examined. The LPS and OMP profiles were studied through SDS-PAGE analysis and genotype was determined by PCR-DNA fingerprinting. Genotyping identified five DNA types while analysis of LPS and OMP profiles identified three rough LPS types and three OMP types among the 60 isolates of M. bovis including the three collection strains. None of the three methods employed to assess diversity was discriminating when used alone because the degree of heterogeneity in each group of surface structures was limited, but when data of each typing method were combined, 15 distinct subgroups were determined. This subgrouping was clearly able to differentiate isolates of the same genotype. These typing methods appear to be useful to assess different aspects of the disease such as the diversity within a population of M. bovis associated to epidemic conditions, track the causal agent in an outbreak of the disease, monitoring vaccination programs and studies on virulence.

Effect of hydromechanical stress on cellular antigens of Bordetella pertussis.

Cells of Bordetella pertussis grown in a bioreactor under stirring conditions were studied to investigate the effect of shear stress on cellular-bound filamentous haemagglutinin (FHA). FHA attached to the bacterial surface, unlike extracellular FHA, was not affected at the shear levels tested. Moreover, no other cellular immunogen involved in the whole-cell protective activity seemed to be affected by hydromechanical forces.

Effect of hydromechanical forces on the production of filamentous haemagglutinin and pertussis toxin of Bordetella pertussis.

The production of Bordetella pertussis extracytoplasmic filamentous haemagglutinin (FHA) and pertussis toxin (PT) in a bioreactor under stirring conditions was studied in order to investigate the effect of hydromechanical forces on yields of both antigens. It was shown that FHA loses its haemagglutinin activity when the power transmitted by the agitator and the aerator per unit volume increases, whereas PT production is not affected. The loss of FHA activity can be explained by the action of shear forces on the filamentous structure of this antigen.

Effect of methyl-cyclodextrin on adenylate cyclase activity of Bordetella pertussis.

The activity of Bordetella pertussis extracytoplasmic adenylate cyclase (AC) decreased during decelerating growth phase in a Stainer-Scholte medium. Neither proteolytic activity nor virulence variation (phase variation; antigenic modulation) appears to be responsible for the observed activity fall. The addition of methyl-ß-cyclo-dextrin enhances AC activity and prevents the inhibition of AC activity by fatty acids. Cyclodextrin could entrap inhibitors increasing in this way the AC activity. These results show that the inclusion of cyclodextrin in the culture medium increases the AC activity.

Organic and inorganic nitrogen source ratio effects onBacillus thuringiensis var.israelensis delta-endotoxin production.

The influence of different organic and inorganic nitrogen source combinations and C∶N ratios was studied in connection with growth and protein production ofBacillus thuringiensis var.israelensis. Protein production was assumed to be proportional to delta-endotoxin production. Delta-endotoxin concentration increased when media were supplemented with (NH4)2SO4, but the delta-endotoxin: biomass dry weight ratio was unaffected by different C∶N ratios. Organic nitrogen source, yeast extract, could be partially replaced by (NH4)2SO4 with a significant increase in delta-endotoxin production.

Effect of the media composition on the growth parameters and biological properties ofBacillus thuringiensis var.israelensis delta-endotoxin.

The effects of media composition on growth parameters, total protein production (including delta-endotoxin) and its relation to the biological properties ofBacillus thuringiensis var.israelensis was investigated. The replacement of glucose by glycerol as the carbon source yielded higher concentrations of delta-endotoxin. This increase in toxicity was associated with increased amounts of the 130 kDa polypeptide fraction. The biocide activity was not related to haemolytic activity. specific growth rate nor spore count.

[Influence of various ions in sporulation and the formation of delta-endotoxin in Bacillus thuringiensis cultures]. / Influencia de algunos iones en la esporulación y formación de delta-endotoxina en cultivos de Bacillus thuringiensis.

This paper deals with studies related to the influence of several ions on growth, spore formation and endotoxin formation by a Bacillus thuringiensis HD-1 strain commonly used for bioinsecticide production. Two basal media (4 and 5, Table 1) containing glucose, (NH4)2 SO4, phosphates and yeast extract or bacto peptone as organic nitrogen sources were supplemented with several ions in different concentrations, as shown in table 1. The experiments were conducted in 1000 ml Erlenmeyer flasks, containing 100 ml of medium, located in a rotary shaker at 30 degrees C. Several estimations were carried out, mainly biomass by optical density and colony forming units (CFU), glucose, Mg+2 and Mn+2 consumption and delta-endotoxin by a rocket immunoelectrophoretic method. The results obtained (Table 2) clearly show the importance of the addition of Ca+2, Mg+2 and Mn+2 to the basal media, because the highest values of CFU/l and delta-endotoxin (expressed as protein in g/l) were achieved in those media supplemented with the ions mentioned. It was also proved that the supplementation with Ca+2 was also essential for maintaining the thermal stability of the spores (Table 3). It can be concluded that an adequate formulation of media mainly related with the content of Mn+2, Mg+2 and Ca+2 is essential for obtaining high yields of spore crystal production of a Bacillus thuringiensis HD-1 strain.

Influencia de algunos iones en la esporulación y formación de delta-endotoxina en cultivos de Bacillus thuringiensis. / [Influence of various ions in sporulation and the formation of delta-endotoxin in Bacillus thuringiensis cultures]

This paper deals with studies related to the influence of several ions on growth, spore formation and endotoxin formation by a Bacillus thuringiensis HD-1 strain commonly used for bioinsecticide production. Two basal media (4 and 5, Table 1) containing glucose, (NH4)2 SO4, phosphates and yeast extract or bacto peptone as organic nitrogen sources were supplemented with several ions in different concentrations, as shown in table 1. The experiments were conducted in 1000 ml Erlenmeyer flasks, containing 100 ml of medium, located in a rotary shaker at 30 degrees C. Several estimations were carried out, mainly biomass by optical density and colony forming units (CFU), glucose, Mg+2 and Mn+2 consumption and delta-endotoxin by a rocket immunoelectrophoretic method. The results obtained (Table 2) clearly show the importance of the addition of Ca+2, Mg+2 and Mn+2 to the basal media, because the highest values of CFU/l and delta-endotoxin (expressed as protein in g/l) were achieved in those media supplemented with the ions mentioned. It was also proved that the supplementation with Ca+2 was also essential for maintaining the thermal stability of the spores (Table 3). It can be concluded that an adequate formulation of media mainly related with the content of Mn+2, Mg+2 and Ca+2 is essential for obtaining high yields of spore crystal production of a Bacillus thuringiensis HD-1 strain.

[Production of glucose isomerase by Streptomyces phaeochromogenes]. / Producción de glucosa-isomerasa por Streptomyces phaechromogenes.

The production of glucose isomerase was studied in the Streptomyces phaeochromogenes NRRL B-3559 strain. The influence of the medium composition and the aeration conditions was determined Selection of colonies was necessary to obtain the best results. A grey colony proved to be the most productive one. It was necessary to use the frozen inocula in order to obtain steady reproduction values. It was possible to achieve the maximum enzymatic level (1600 UE/1 min) using a CoCl2. 6H2O concentration of 0.18 g/l. The aeration studies conducted in a mechanically stirred fermentor showed results comparable to those obtained in shaker flasks, operating at 550 rpm and using an air flow of 1 1/1 min. The culture proved to properly aerated since both the cellular oxygen uptake and oxygen demand were similar (Table 1).

Producción de glucosa-isomerasa por Streptomyces phaechromogenes. / [Production of glucose isomerase by Streptomyces phaeochromogenes]

The production of glucose isomerase was studied in the Streptomyces phaeochromogenes NRRL B-3559 strain. The influence of the medium composition and the aeration conditions was determined Selection of colonies was necessary to obtain the best results. A grey colony proved to be the most productive one. It was necessary to use the frozen inocula in order to obtain steady reproduction values. It was possible to achieve the maximum enzymatic level (1600 UE/1 min) using a CoCl2. 6H2O concentration of 0.18 g/l. The aeration studies conducted in a mechanically stirred fermentor showed results comparable to those obtained in shaker flasks, operating at 550 rpm and using an air flow of 1 1/1 min. The culture proved to properly aerated since both the cellular oxygen uptake and oxygen demand were similar (Table 1).