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Serotype A extracellular polysaccharide produced by Pasteurella multocida is composed largely of hyaluronic acid and is an important virulence factor in avian pasteurellosis. Hyaluronidase, mechanical shearing, and serial subculturing were employed to decapsulate strains exhibiting disparate degrees of capsulation, thereby allowing an investigation of the role serotype A capsulation plays in outer cell envelope physiology and virulence of avian isolates. Experimental loss of capsular material occurred concomitantly with an overall increase in cell surface hydrophobicity values, which approached those of naturally-occurring noncapsulated strains. The electronegativity of capsulated cell surfaces was found to be significantly greater than that of both noncapsulated and experimentally decapsulated cells. Naturally-occurring noncapsulated variants were readily phagocytized by mouse peritoneal macrophages, whereas capsulated strains were not. A moderately capsulated variant was phagocytized less effectively than noncapsulated strains. Experimental decapsulation of capsulated variants did not significantly hinder their ability to resist phagocytosis. Moreover, small amounts of residual capsular material appeared to remain after experimental decapsulation as detected with the aid of light and transmission electron microscopy. These data suggest that while cell surface hydrophobicity, charge, and susceptibility to phagocytosis are influenced by the degree to which cells are capsulated with serotype A extracellular polysaccharide, other factors which are unaffected by experimental decapsulation may also be involved in protection from phagocytosis in capsulated avian isolates of P . multocida.

Actinobacillus pleuropneumoniae is an important pathogen of swine. Lipopolysaccharide (LPS) has been identified as the major adhesin of A. pleuropneumoniae and it is involved in adherence to porcine respiratory tract cells. We previously generated seven rough LPS mutants of A. pleuropneumoniae serotype 1 by using a mini-Tn 10 transposon mutagenesis system [Rioux S, Galarneau C, Harel J et al. Isolation and characterization of mini-Tn 10 lipopolysaccharide mutants of Actinobacillus pleuropneumoniae serotype 1. Can J Microbiol 1999; 45: 1017—1026]. The purpose of the present study was to characterize these mutants in order to learn more about LPS O-antigen biosynthesis genes and their organization in A. pleuropneumoniae, and to determine the surface properties and virulence in pigs of these isogenic mutants. By mini-Tn 10 insertions in rough mutants, four putative genes (ORF12, ORF16, ORF17, and ORF18) involved in O-antigen biosynthesis in A. pleuropneumoniae serotype 1 were found within a region of 18 ORFs. This region is homologous to the gene cluster of serotype-specific O-polysaccharide biosynthesis from A. actinomycetemcomitans strain Y4 (serotype b). Two mutants showed homology to a protein with identity to glycosyltransferases (ORF12); two others had the mini-Tn 10 insertion localized in genes encoding for two distinct proteins with identity to rhamnosyltransferases (ORF16 and ORF17) and three showed homology to a protein which is known to initiate polysaccharide synthesis (ORF18). These four ORFs were also present in A. pleuropneumoniae serotypes 9 and 11 that express an O-antigen that serologically cross-reacts with serotype 1. Evaluation of some biological properties of rough mutants seems to indicate that the absence of O-chains does not appear to have an influence on the virulence of the bacteria in pigs and on the overall surface hydrophobicity, charge and hemoglobin-binding activity, or on LAL activation. An acapsular mutant was included in the present study in order to compare the influence of O-chains and capsule polysaccharides on different cell surface properties. Our data suggest that capsular polysaccharides and not O-chains polysaccharides have a major influence on surface properties of A. pleuropneumoniae serotype 1 and its virulence in pigs.

Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters. Streptococcus pneumoniae (pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs) which have been associated with adhesion and virulence. The purpose of this study was to determine the contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis. Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule. Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocyte-like cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and may facilitate adherence to host cells partially through nonspecific, physicochemical interactions.

Surface interfacial physiology is particularly important to unicellular organisms with regard to maintenance of optimal cell function. Bacterial cell surfaces possess net negative electrostatic charge by virtue of ionized phosphoryl and carboxylate substituents on outer cell envelope macromolecules which are exposed to the extracellular environment. The degree of peripheral electronegativity influences overall cell surface polarity and can be assessed on the basis of zeta potential which is most often determined by estimating the electrophoretic mobility of cells in an electric field. The purpose of this review is to provide bacteriologists with assistance as they seek to better understand available instrumentation and fundamental principles concerning the estimation of zeta potential as it relates to bacterial surface physiology.

This paper examines affinity chromatography (AC) as an alternative tool for the determination of protein-ligand interactions for the particular case in which the ligand is the same protein. The methodology is less labor-intensive and more sample-efficient than traditional methods used to measure the second virial coefficient (B(22)), a parameter commonly used to evaluate protein-protein interactions. The chromatographic capacity factor (k') was studied for lysozyme and equine serum albumin for a wide range of experimental solution conditions such as crystallizing agent concentration, protein concentration and pH. Parallel experiments using AC to determine k' and static light scattering (SLS) to determine B(22) showed that the two parameters were highly correlated. Two different column volumes ( approximately 1 and approximately 0.1 mL) were tested and gave essentially the same values for k', showing the feasibility of miniaturization.

The Haas – Drenth – Wilson (HDW) (Haas et al., 1999) theoretical model was used to correlate osmotic second virial coefficient (B) values with solubility (S) values for equine serum albumin (ESA) and ovalbumin for corresponding solution conditions. The best fit from the theoretical model was compared to experimental S versus B data. B values were experimentally measured using static light scattering. Solubilities of ESA were estimated using a sitting drop method. When the experimental data for S versus B were plotted, an excellent fit for ESA was obtained according to the HDW model. The results showed that the coordination number (z) in the crystal lattice was 6, and the adjustable parameter (A) was 0.072. For ovalbumin, previously reported solubility data in aqueous ammonium sulfate solutions were utilized. The solubility data for ovalbumin were correlated with the measured B values obtained in our laboratory. The resulting best fit from the HDW model showed that z = 6 and A = 0.084.