Flagellar antibody stimulated opsonophagocytosis of Pseudomonas aeruginosa associated with response to either a- or b-type flagellar antigen

Pseudomonas aeruginosa exhibit one of two flagella types: a homogeneous b type, with molecular weight of 53,000, or a heterogeneous a type (subtypes a0, a1, a2, a3, and a4), with molecular weights ranging from 45,000 to 52,000. Pseudomonas aeruginosa flagellar antiserum was shown to promote uptake of radiolabeled bacteria by mouse polymorphonuclear leukocytes. Bacteria were detected directly associated with washed leukocytes and visualized, by electron microscopy, internalized in polymorphonuclear leukocytes. Phagocytosis was specific for the flagella type (a or b) in that homologous flagella serum enhanced uptake three to four times greater than heterologous serum or normal rabbit serum. An a-type antiserum was shown to enhance phagocytosis of four different a-type strains with varying subantigen types, indicating the presence of a common cross-reactive a0 antigen in this flagella type. Phagocytic killing of internalized bacteria was not seen with the addition of only flagellar antiserum.     

Monoclonal IgM antibody protection in mice against infection with an encapsulated strain of Staphylococcus epidermidis.

Passive protective activities of three different classes of monoclonal antibodies in mice against challenge with strain ATCC 31432 (capsular type I) of Staphylococcus epidermidis were examined. Monoclonal IgM antibody passively protected mice against challenge with the homologous strain, whereas monoclonal IgG1 and IgG2b antibodies did not. The protective activity of IgM was absorbed by the cell surface antigen extracted from the homologous strain but not by the antigen from heterologous strains. Rapid reduction of viable cells took place in the peritoneal cavity of mice immunized with monoclonal IgM as early as 6 h after the challenge with the homologous strain. An enzyme-linked immunosorbent inhibition assay showed there was remarkable inhibition with the homologous cell surface antigen but not with heterologous preparations from other strains. Results suggest that in the mouse the major passive protection against the S. epidermidis strain is provided by the IgM antibody to the cell surface antigen.     

Analysis of common antigen of flagella in Bacillus cereus and Bacillus thuringiensis

Abstract Flagellar antigen of Bacillus cereus H.1 was purified and tested for serodiagnostic antigen by ELISA. The antibody against the flagellar antigen of B. cereus H.1 reacted not only with the homologous specific antigen but also reacted with the flagellar antigens of 23 strains of B. cereus . This common flagellar antigen of B. cereus was found to be due to 61-kDa protein by SDS-PAGE and immunoblot assay. Monoclonal antibody H15A5 against common antigenic epitope of B. cereus also reacted with flagellar antigens of 21 strains of Bacillus thuringiensis by ELISA. This monoclonal antibody reacted with the 61-kDa protein of the flagella of B. cereus H.1 and H.2 and B. thuringiensis Kurstaki HD1, Alesti and Aizawai juroi by immunoblot analysis. These results indicated that the common antigenic epitope of the 61-kDa protein existed in the flagella both of B. cereus and B. thuringiensis .     

Tipping and mating-structure activation induced in Chlamydomonas gametes by flagellar membrane antisera

Antisera raised against vegetative and gametic flagella of Chlamydomonas reinhardi have been used to probe dynamic properties of the flagellar membranes. The antisera, which agglutinate cells via their flagella, associate with antigens that are present on both vegetative and gametic membranes and on membranes of both mating types (mt+ and mt-). Gametic cells respond to antibody presentation very differently from vegetative cells, mobilizing even high concentrations of antibody towards the flagellar tips; the possibility is discussed that such "tipping" ability reflects a differentiated gametic property relevant to sexual agglutinability. Gametic cells also respond to antibody agglutination by activating their mating structures, the mt+ reaction involving a rapid polymerization of microfilaments. Several impotent mt+ mutant strains that fail to agglutinate sexually are also activated by the antisera and procede to form zygotes with normal mt- gametes. Fusion does not occur between activated cells of like mating type. Monovalent (Fab) preparations of the antibody fail to activate mt+ gametes, suggesting that the cross-linking properties of the antisera are essential for their ability to mimic, or bypass, sexual agglutination.     

Serological Study of Bacterial Flagellar Hooks

Bacterial hooks were partially purified from flagella isolated from Salmonella SJ25, by treatment with heat to depolymerize flagellar filaments and with n-butanol and calcium chloride to remove membranes. Antihook serum was obtained from a rabbit inoculated with a preparation of hooks. The serum contained antibodies directed against the flagellar filament and cell membrane. These antibodies could be removed from the serum by absorption with purified flagellar filaments and cells of a nonflagellated mutant strain. It was shown by electron microscopy that anti-SJ25-hook antibody reacts with hooks from a number of strains of Salmonella which differed from SJ25 in H and O antigens, flagellar shape, and motility. Hooks possessed by various strains of Salmonella have a common antigenicity. In addition, anti-SJ25-hook cross-reacted with hooks from Escherichia coli W3110 but did not react at all which those from strains of Serratia, Proteus, Aerobacter, and Klebsiella. It is well known that bacteria stop moving upon addition of antiflagella serum to the medium. However, the addition of purified antihook was found to have little effect on motility. At physiological ionic strength and pH, flagellin (Salmonella) can polymerize into flagellar filaments only in the presence of seeds. It was shown that a crude preparation of hooks was able to initiate in vitro polymerization of flagellin.     

Step-wise loss of bacterial flagellar torsion confers progressive phagocytic evasion

Phagocytosis of bacteria by innate immune cells is a primary method of bacterial clearance during infection. However, the mechanisms by which the host cell recognizes bacteria and consequentially initiates phagocytosis are largely unclear. Previous studies of the bacterium Pseudomonas aeruginosa have indicated that bacterial flagella and flagellar motility play an important role in colonization of the host and, importantly, that loss of flagellar motility enables phagocytic evasion. Here we use molecular, cellular, and genetic methods to provide the first formal evidence that phagocytic cells recognize bacterial motility rather than flagella and initiate phagocytosis in response to this motility. We demonstrate that deletion of genes coding for the flagellar stator complex, which results in non-swimming bacteria that retain an initial flagellar structure, confers resistance to phagocytic binding and ingestion in several species of the gamma proteobacterial group of Gram-negative bacteria, indicative of a shared strategy for phagocytic evasion. Furthermore, we show for the first time that susceptibility to phagocytosis in swimming bacteria is proportional to mot gene function and, consequently, flagellar rotation since complementary genetically- and biochemically-modulated incremental decreases in flagellar motility result in corresponding and proportional phagocytic evasion. These findings identify that phagocytic cells respond to flagellar movement, which represents a novel mechanism for non-opsonized phagocytic recognition of pathogenic bacteria.     

Influence on motility of Escherichia coli and Salmonella typhimurium by a naturally occurring conjugative plasmid.

In a collection of 45 R-plasmids, one was found to be associated with loss of motility of its Escherichia coli K-12 and Salmonella typhimurium host bacteria when tested in conventional motility agar. Genetic experiments, as well as analyses of deoxyribonucleic acid, showed that inhibition of motility was caused by a conjugative plasmid that was separate from the R-plasmid. This second plasmid, named pUM5, was fi- and mediated the same type of sex pilus (F-like) as the accompanying R-plasmid but lacked resistance determinants. Preliminary studies indicated that bacterial cells carrying the motility inhibition plasmid pUM5 were still equipped with flagella. The mechanism by which flagellar action is disturbed by the plasmid is presently not known.     

Demonstration of protective antigen carried by flagella of Clostridium chauvoei

The protective antigen present on the flagella of Clostridium chauvoei was studied by the mouse protection test. A partially purified flagella preparation (PPF) showed protective antigenicity after two intraperitoneal injections of 2 micrograms as protein, while the protective antigenicity of nonflagellated mutants (NFM) was 100-fold less than that of the flagellated parent strain. Although the protective effect of antisera against the whole cells and PPF, in terms of ED50 values, was mostly lost after absorption with the parent strain, that of antisera after absorption with NFMs showed no appreciable loss. These results suggest that the flagella of Cl. chauvoei play some role in inducing protective immunity in mice.     

Characterization of Protective Immune Responses Induced by Pneumococcal Surface Protein A in Fusion with Pneumolysin Derivatives

Pneumococcal surface protein A (PspA) and Pneumolysin derivatives (Pds) are important vaccine candidates, which can confer protection in different models of pneumococcal infection. Furthermore, the combination of these two proteins was able to increase protection against pneumococcal sepsis in mice. The present study investigated the potential of hybrid proteins generated by genetic fusion of PspA fragments to Pds to increase cross-protection against fatal pneumococcal infection. Pneumolisoids were fused to the N-terminus of clade 1 or clade 2 pspA gene fragments. Mouse immunization with the fusion proteins induced high levels of antibodies against PspA and Pds, able to bind to intact pneumococci expressing a homologous PspA with the same intensity as antibodies to rPspA alone or the co-administered proteins. However, when antibody binding to pneumococci with heterologous PspAs was examined, antisera to the PspA-Pds fusion molecules showed stronger antibody binding and C3 deposition than antisera to co-administered proteins. In agreement with these results, antisera against the hybrid proteins were more effective in promoting the phagocytosis of bacteria bearing heterologous PspAs in vitro, leading to a significant reduction in the number of bacteria when compared to co-administered proteins. The respective antisera were also capable of neutralizing the lytic activity of Pneumolysin on sheep red blood cells. Finally, mice immunized with fusion proteins were protected against fatal challenge with pneumococcal strains expressing heterologous PspAs. Taken together, the results suggest that PspA-Pd fusion proteins comprise a promising vaccine strategy, able to increase the immune response mediated by cross-reactive antibodies and complement deposition to heterologous strains, and to confer protection against fatal challenge.     

Partial purification of presynaptic plasma membrane by immunoadsorption

Chlamydomonas flagella exhibit force transduction in association with their surface. This flagellar surface motility is probably used both for whole cell gliding movements (flagella-substrate interaction) and for reorientation of flagella during mating (flagella-flagella interaction). The present study seeks to identify flagellar proteins that may function as exposed adhesive sites coupled to a motor responsible for their translocation in the plane of the plasma membrane. The principal components of the flagellar membrane are a pair of glycoproteins (approximately 350,000 mol wt), with similar mobility on SDS polyacrylamide gels. A rabbit IgG preparation has been obtained which is specific for these two glycoproteins; this antibody preparation binds to and agglutinates cells by their flagellar surfaces only. Treatment of cells with 0.1 mg/ml pronase results in a loss of motility-coupled flagellar membrane adhesiveness. This effect is totally reversible, but only in the presence of new protein synthesis. The major flagellar protein modified by this pronase treatment is the faster migrating of the two high molecular weight glycoproteins; the other glycoprotein does not appear to be accessible to external proteolytic digestion. Loss and recovery of flagella surface binding sites for the specific antibody parallels the loss and recovery of the motility-coupled flagellar surface adhesiveness, as measured by the binding and translocation of polystyrene microspheres. These observations suggest, but do not prove, that the faster migrating of the major high molecular weight flagellar membrane glycoproteins may be the component which provides sites for substrate interaction and couples these sites to the cytoskeletal components responsible for force transduction.     

Flagellated ectosymbiotic bacteria propel a eucaryotic cell

A devescovinid flagellate from termites exhibits rapid gliding movements only when in close contact with other cells or with a substrate. Locomotion is powered not by the cell's own flagella nor by its remarkable rotary axostyle, but by the flagella of thousands of rod bacteria which live on its surface. That the ectosymbiotic bacteria actually propel the protozoan was shown by the following: (a) the bacteria, which lie in specialized pockets of the host membrane, bear typical procaryotic flagella on their exposed surface; (b) gliding continues when the devescovinid's own flagella and rotary axostyle are inactivated; (c) agents which inhibit bacterial flagellar motility, but not the protozoan's motile systems, stop gliding movements; (d) isolated vesicles derived from the surface of the devescovinid rotate at speeds dependent on the number of rod bacteria still attached; (e) individual rod bacteria can move independently over the surface of compressed cells; and (f) wave propagation by the flagellar bundles of the ectosymbiotic bacteria is visualized directly by video-enhanced polarization microscopy. Proximity to solid boundaries may be required to align the flagellar bundles of adjacent bacteria in the same direction, and/or to increase their propulsive efficiency (wall effect). This motility-linked symbiosis resembles the association of locomotory spirochetes with the Australian termite flagellate Mixotricha (Cleveland, L. R., and A. V. Grimstone, 1964, Proc. R. Soc. Lond. B Biol. Sci., 159:668-686), except that in our case propulsion is provided by bacterial flagella themselves. Since bacterial flagella rotate, an additional novelty of this system is that the surface bearing the procaryotic rotary motors is turned by the eucaryotic rotary motor within.     

The flagellar cytoskeleton of the spirochetes

The recent discoveries of prokaryotic homologs of all three major eukaryotic cytoskeletal proteins (actin, tubulin, intermediate filaments) have spurred a resurgence of activity in the field of bacterial morphology. In spirochetes, however, it has long been known that the flagellar filaments act as a cytoskeletal protein structure, contributing to their shape and conferring motility on this unique phylum of bacteria. Therefore, revisiting the spirochete cytoskeleton may lead to new paradigms for exploring general features of prokaryotic morphology. This review discusses the role that the periplasmic flagella in spirochetes play in maintaining shape and producing motility. We focus on four species of spirochetes: Borrelia burgdorferi, Treponema denticola, Treponema phagedenis and Leptonema (formerly Leptospira) illini. In spirochetes, the flagella reside in the periplasmic space. Rotation of the flagella in the above species by a flagellar motor induces changes in the cell morphology that drives motility. Mutants that do not produce flagella have a markedly different shape than wild-type cells.     

Induction of Resistance in Mice by the Capsular Polysaccharide Antigens of Staphylococcus aureus

Mice actively immunized with capsular polysaccharides extracted from capsular type strains A, B, C, and D, determined by the serum-soft agar technique, were protected against lethal infection by homologous strains, but no animals survived infection by heterologous substance immunization even with at high doses. Passive protective antibody in rabbit antisera prepared using these strains was absorbed out only by homologous capsular polysaccharide in mice. These results indicated that resistance was specific for capsular polysaccharide. The substance contained mainly neutral sugar, small amounts of hexosamine, methyl-pentose, and phosphate although these amounts varied depending on the capsular types strains.     

The cell biology of peritrichous flagella in Bacillus subtilis

Bacterial flagella are highly conserved molecular machines that have been extensively studied for assembly, function and gene regulation. Less studied is how and why bacteria differ based on the number and arrangement of the flagella they synthesize. Here we explore the cell biology of peritrichous flagella in the model bacterium Bacillus subtilis by fluorescently labelling flagellar basal bodies, hooks and filaments. We find that the average B. subtilis cell assembles approximately 26 flagellar basal bodies and we show that basal body number is controlled by SwrA. Basal bodies are assembled rapidly (< 5 min) but the assembly of flagella capable of supporting motility is rate limited by filament polymerization (> 40 min). We find that basal bodies are not positioned randomly on the cell surface. Rather, basal bodies occupy a grid‐like pattern organized symmetrically around the midcell and that flagella are discouraged at the poles. Basal body position is genetically determined by FlhF and FlhG homologues to control spatial patterning differently from what is seen in bacteria with polar flagella. Finally, spatial control of flagella in B. subtilis seems more relevant to the inheritance of flagella and motility of individual cells than the motile behaviour of populations.     

Characterizing the flagellar filament and the role of motility in bacterial prey-penetration by Bdellovibrio bacteriovorus

The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments.     

Experimental dissection of flagellar surface motility in chlamydomonas

Experiments have explored the possible relationships between the flagellar surface motility of chlamydomonas, visualized as translocation of polystyrene beads by paralyzed (pf) mutants (Bloodgood, 1977, J. Cell Biol. 15:983-989), and the capacity of gametic flagella to participate in the mating reaction. While vegetative and gametic flagella bind beads with equal efficiencies and are capable of transporting them along entire flagellar lengths, beads on vegetative flagella are primarily associated with the proximal half of the flagella whereas those of gametic flagella exhibit no such preference. This difference may relate to the "tipping" response of gametes during sexual flagellar agglutination (Goodenough and Jurivich, 1978, J. Cell Biol. 79:680-693). Colchicine, vinblastine, chymotrypsin, cytochalasins B and D, and anti-β-tubulin antiserum are all able to inhibit the binding of beads to the flagellar suface. Trysin digestion and an antiserum directed against whole chlamydomonas flagella have no effect on the ability of flagella to bind beads, but the beads remain immobile. These results suggest that at least two flagellar activities participate in surface motility: (a) bead binding, which may involve a tubulin-like component at the flagellar surface; and (b) bead translocation, which may depend on a second component (e.g. an ATPase) of the flagellar surface. Surface motility is shown to be distinct from gametic adhesiveness per se, but it may participate in concentrating dispersed agglutinins, in driving them toward the flagellar tips, and/or in generating a signal-to-fuse from the flagellar tips to the cell body. Directly supporting these concepts is the observation that bound beads remain immobilized at the flagellar tips during the "tip-locking" stage of pf x pf matings, and the observation that bound ligands such as antibody fail to be tipped by trypsinized flagella.     

Correlation between bacteriophage chi adsorption and mode of flagellar rotation of Escherichia coli chemotaxis mutants

We studied the adsorption of phage chi to various behavioral mutants (che mutants) of Escherichia coli having different swimming modes. Bacteriophage chi infects only bacteria with active flagella, and it was therefore of interest to examine whether the mode of swimming has an effect on the susceptibility of the bacteria to the phage. Neither the mode of swimming (smooth swimming or tumbling) nor the direction of flagellar rotation affected the degree of chi adsorption to the bacterial cells. Furthermore, the tumbling frequency, the rotation speed (tethered cells of all of the strains examined had the same average speed of rotation), the time proportion of rotation, and the reversal frequency were not important in determining susceptibility to chi. The only variable that influenced chi adsorption was the fraction of the population whose flagella rotated incessantly. A direct, linear correlation was found between chi adsorption and the fraction of unceasing rotation in each population. It seems, therefore, that an individual bacterium whose flagella pause periodically and briefly during rotation is not susceptible to irreversible adsorption of the phage. Pausing of rotation thus seems to be a new feature of motility that is prevalent especially in che mutants. It is concluded that irreversible chi adsorption can serve as a quantitative assay only for incessant flagellar rotation of E. coli.     

Spirochete periplasmic flagella and motility

Spirochetes have a unique structure, and as a result their motility is different from that of other bacteria. They also have a special attribute: spirochetes can swim in a highly viscous, gel-like medium, such as that found in connective tissue, that inhibits the motility of most other bacteria. In spirochetes, the organelles for motility, the periplasmic flagella, reside inside the cell within the periplasmic space. A given periplasmic flagellum is attached only at one end of the cell, and depending on the species, may or may not overlap in the center of the cell with those attached at the other end. The number of periplasmic flagella varies from species to species. These structures have been shown to be directly involved in spirochete motility, and they function by rotating within the periplasmic space. The mechanics of motility also vary among the spirochetes. In Leptospira, a motility model developed several years ago has been extensively tested, and the evidence supporting this model is convincing. Borrelia burgdorferi swims differently, and a model of its motility has been recently put forward. This model is based on analyzing the motion of swimming cells, high voltage electron microscopy of fixed cells, and mutant analysis. To better understand spirochete motility on a more molecular level, the proteins and genes involved in motility are being analyzed. Spirochete periplasmic flagellar filaments are among the most complex of bacterial flagella. They are composed of the FlaA sheath proteins, and in many species, multiple FlaB core proteins. Allelic exchange mutagenesis of the genes which encode these proteins is beginning to yield important information with respect to periplasmic flagellar structure and function. Although we are at an early stage with respect to analyzing the function, organization, and regulation of many of the genes involved in spirochete motility, unique aspects have already become evident. Future studies on spirochete motility should be exciting, as only recently have complete genome sequences and tools for allelic exchange mutagenesis become available.     

Giardia flagellar motility is not directly required to maintain attachment to surfaces

Giardia trophozoites attach to the intestinal microvilli (or inert surfaces) using an undefined "suction-based" mechanism, and remain attached during cell division to avoid peristalsis. Flagellar motility is a key factor in Giardia's pathogenesis and colonization of the host small intestine. Specifically, the beating of the ventral flagella, one of four pairs of motile flagella, has been proposed to generate a hydrodynamic force that results in suction-based attachment via the adjacent ventral disc. We aimed to test this prevailing "hydrodynamic model" of attachment mediated by flagellar motility. We defined four distinct stages of attachment by assessing surface contacts of the trophozoite with the substrate during attachment using TIRF microscopy (TIRFM). The lateral crest of the ventral disc forms a continuous perimeter seal with the substrate, a cytological indication that trophozoites are fully attached. Using trophozoites with two types of molecularly engineered defects in flagellar beating, we determined that neither ventral flagellar beating, nor any flagellar beating, is necessary for the maintenance of attachment. Following a morpholino-based knockdown of PF16, a central pair protein, both the beating and morphology of flagella were defective, but trophozoites could still initiate proper surface contacts as seen using TIRFM and could maintain attachment in several biophysical assays. Trophozoites with impaired motility were able to attach as well as motile cells. We also generated a strain with defects in the ventral flagellar waveform by overexpressing a dominant negative form of alpha2-annexin::GFP (D122A, D275A). This dominant negative alpha2-annexin strain could initiate attachment and had only a slight decrease in the ability to withstand normal and shear forces. The time needed for attachment did increase in trophozoites with overall defective flagellar beating, however. Thus while not directly required for attachment, flagellar motility is important for positioning and orienting trophozoites prior to attachment. Drugs affecting flagellar motility may result in lower levels of attachment by indirectly limiting the number of parasites that can position the ventral disc properly against a surface and against peristaltic flow.