Motility is involved in Silicibacter sp. TM1040 interaction with dinoflagellates

Silicibacter sp. TM1040, originally isolated from a culture of the dinoflagellate Pfiesteria piscicida, senses and responds to the dinoflagellate secondary metabolite dimethylsulfoniopropionate (DMSP) by flagella-mediated chemotaxis behaviour. In this report we show that swimming motility is important for initiating the interaction between the bacterium and dinoflagellate. Following transposon mutagenesis, three mutants defective in wild-type swimming motility (Mot-) were identified. The defects in motility were found to be in homologues of cckA and ctrA, encoding a two-component regulatory circuit, and in a novel gene, flaA, likely to function in flagellar export or biogenesis. Mutation of flaA or cckA results in the loss of flagella and non-motile cells (Fla-), while CtrA- cells possess flagella, but have reduced motility due to increased cell length. All three Mot- mutants were defective in attaching to the dinoflagellate, particularly to regions that colocalized with intracellular organelles. The growth rate of the dinoflagellates was reduced in the presence of the Fla- mutants compared with Fla+ cells. These results indicate that bacterial motility is important for the Silicibacter sp. TM1040-P. piscicida interaction.     

Chemotaxis is required for virulence and competitive fitness of the bacterial wilt pathogen Ralstonia solanacearum

Ralstonia solanacearum, a soilborne plant pathogen of considerable economic importance, invades host plant roots from the soil. Qualitative and quantitative chemotaxis assays revealed that this bacterium is specifically attracted to diverse amino acids and organic acids, and especially to root exudates from the host plant tomato. Exudates from rice, a nonhost plant, were less attractive. Eight different strains from this heterogeneous species complex varied significantly in their attraction to a panel of carbohydrate stimuli, raising the possibility that chemotactic responses may be differentially selected traits that confer adaptation to various hosts or ecological conditions. Previous studies found that an aflagellate mutant lacking swimming motility is significantly reduced in virulence, but the role of directed motility mediated by the chemotaxis system was not known. Two site-directed R. solanacearum mutants lacking either CheA or CheW, which are core chemotaxis signal transduction proteins, were completely nonchemotactic but retained normal swimming motility. In biologically realistic soil soak virulence assays on tomato plants, both nonchemotactic mutants had significantly reduced virulence indistinguishable from that of a nonmotile mutant, demonstrating that directed motility, not simply random motion, is required for full virulence. In contrast, nontactic strains were as virulent as the wild-type strain was when bacteria were introduced directly into the plant stem through a cut petiole, indicating that taxis makes its contribution to virulence in the early stages of host invasion and colonization. When inoculated individually by soaking the soil, both nontactic mutants reached the same population sizes as the wild type did in the stems of tomato plants just beginning to wilt. However, when tomato plants were coinoculated with a 1:1 mixture of a nontactic mutant and its wild-type parent, the wild-type strain outcompeted both nontactic mutants by 100-fold. Together, these results indicate that chemotaxis is an important trait for virulence and pathogenic fitness in this plant pathogen.     

Chemotactic control of the two flagellar systems of Vibrio parahaemolyticus.

Vibrio parahaemolyticus synthesizes two distinct flagellar organelles, the polar flagellum (Fla), which propels the bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible for movement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluated separately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla+ Laf- (swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutions to measure swarming motility, were used to quantitate chemotaxis by the Fla+ Laf- or Fla- Laf+ mutants. The behavior of the mutants was very similar with respect to the attractant compounds and the concentrations which elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems was also examined. A locus previously shown to encode functions required for chemotactic control of the polar flagellum was cloned and mutated by transposon Tn5 insertion in Escherichia coli, and the defects in this locus, che-4 and che-5, were then transferred to the Fla+ Laf- or Fla- Laf+ strains of V. parahaemolyticus. Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatly diminished movement of bacteria over the surface of agar media or through semisolid media. We conclude that the two flagellar organelles, which consist of independent motor-propeller structures, are directed by a common chemosensory control system.     

Role of pili in the pathogenesis of Pseudomonas aeruginosa burn infection

The present study using three isogenic mutants (F+P-, F-P+, F-P-) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD50) value for burned mice inoculated with non-piliated (P-) mutant was at least ten times higher than those inoculated with piliated (P+) bacteria. Meanwhile the LD50 value for burned mice inoculated with non-flagellated (F-) mutant was at least 10(5) times higher than those inoculated with flagellated (F+) bacteria. At 24 hr after inoculation, the bacterial counts in burned skin of mice inoculated with P+ bacteria were ten times higher than those inoculated with P- bacteria; and at 48 hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24 hr after inoculation, P+ bacteria were isolated from blood, liver (F+P+), lung (F+P+), and kidney, while P- bacteria were not present in these tissues. And at 48 hr after inoculation, P+ bacteria were isolated from all tissues, while P- bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili-mediated enhancement of virulence of P. aeruginosa was attributed to pili-mediated enhanced colonization of the organisms at the burned skin surfaces.     

Protection against Pseudomonas aeruginosa infection by passive transfer of anti-flagellar serum

The specificity of adsorbed flagellar antisera for H-antigen was demonstrated in vitro by cross-agglutination assays, motility inhibition, and an ELISA. The specific flagellar antibody was determined to be an IgG. Complete protection against burn wound sepsis was achieved with flagellar antisera. Cross-protection experiments revealed that protection was not only H-antigen dependent, but specific for the flagella antigen type. Antiserum raised against b-type flagella would only protect against homologous bacterial challenge and not against a-type flagellated strains. Results using a-type antisera were consistent, giving protection only against the homologous strain. In contrast, protective capacity was selectively removed from antisera by adsorbing with Fla+ cells. Bacteria colonized the burn wounds of passively protected mice to similar levels as seen in nonprotected animals, but the colonization remained localized and did not result in systemic infection, a pattern similar to infections with motility mutants observed in other studies. Animals rendered neutropenic prior to burning were not protected with flagellar antisera. These data suggested a role for phagocytic cells in protection. Immobilization by flagellar antiserum was observed both by microscopic studies and by inhibition of colony spreading. Antiflagellar antibody is hypothesized as exerting its protective capacity possibly in two ways; first by inhibiting the motility of invading bacteria by binding to the flagellum and immobilizing the bacteria, and secondly by acting as an opsonin, targeting either immobilized or mobile cells for phagocytosis.     

Absence of membrane phosphatidylcholine does not affect virulence and stress tolerance phenotypes in the opportunistic pathogen Pseudomonas aeruginosa

During growth in presence of choline, both laboratory and clinical Pseudomonas aeruginosa strains synthesize phosphatidylcholine (PC), and PC makes up ～4% of the total membrane phospholipid content. In all the strains tested, PC synthesis occurred only when choline is provided exogenously. Mutants defective in synthesis of PC were generated in the strain backgrounds PAO1 and PA14. Minimum inhibitory concentration studies testing sensitivity of PC-deficient strains towards various antibiotics and cationic antimicrobial peptides revealed no differences as compared to wild-type strains. Mutants incapable of synthesizing PC were also found to be unaffected in motility and biofilm formation on abiotic surfaces, colonization of biotic surfaces and virulence in a mouse infection model. A global phenotypic microarray was further used to identify conditions wherein membrane PC may play a role of in P. aeruginosa. No culture conditions were identified wherein wild-type and PC-deficient mutants showed phenotypic differences. Membrane PC may serve a highly specific role during P. aeruginosa interactions with its eukaryotic hosts based on all the clinical strains tested retaining the ability to synthesize it during availability of choline.     

In vivo growth of Pseudomonas aeruginosa strains PAO1 and PA14 and the hypervirulent strain LESB58 in a rat model of chronic lung infection

Pseudomonas aeruginosa chronic lung infections are the major cause of morbidity and mortality in cystic fibrosis (CF) patients. The P. aeruginosa strains PAO1 and PA14 were compared with the Liverpool epidemic strain LESB58 to assess in vivo growth, infection kinetics, and bacterial persistence and localization within tissues in a rat model of chronic lung infection. The three P. aeruginosa strains demonstrated similar growth curves in vivo but differences in tissue distribution. The LESB58 strain persisted in the bronchial lumen, while the PAO1 and PA14 strains were found localized in the alveolar regions and grew as macrocolonies after day 7 postinfection. Bacterial strains were compared for swimming and twitching motility and for the production of biofilm. The P. aeruginosa LESB58 strain produced more biofilm than PAO1 and PA14. Competitive index (CI) analysis of PAO1, PA14, and LESB58 in vivo indicated CI values of 0.002, 0.0002, and 0.14 between PAO1-PA14, PAO1-LESB58, and LESB58-PA14, respectively. CI analysis comparing the in vivo growth of the PAO1 DeltaPA5441 mutant and four PA14 surface attachment-defective (sad) mutants gave CI values 10 to 1,000 times lower in competitions with their respective wild-type strains PAO1 and PA14. P. aeruginosa strains studied in the rat model of chronic lung infection demonstrated similar in vivo growth but differences in virulence as shown with a competitive in vivo assay. These differences were further confirmed with biofilm and motility in vitro assays, where strain LESB58 produced more biofilm but had less capacity for motility than PAO1 and PA14.     

Involvement of periplasmic fibrils in motility of spirochetes.

Nonmotile (Mot-) strains of Spirochaeta aurantia and Spirochaeta halophila were isolated with a procedure involving mutagenesis of motile wild-type cells. Electron microscopy showed that a Mot- mutant strain of S. halophia possessed incomplete periplasmic fibrils, inasmuch as most or all of the filamentous portion of the periplasmic fibrils was absent. Some of the cells of this Mot-, fibril-defective mutant strain lacked the filamentous portion of the periplasmic fibrils and formed proximal hooks, whereas other cells appeared to have a very small segment of the filamentous portion of the periplasmic fibrils attached to the proximal hooks. Motile revertants were isolated repeatedly from cultures of the Mot-, fibril-defective mutant and from S. halophila Mot- mutants that completely lacked periplasmic fibrils. The motile revertants possessed periplasmic fibrils ultrastructurally indistinguishable from wild-type periplasmic fibrils. This study indicates that periplasmic fibrils play an essential role in the motility of spirochetes.     

Drosophila as a model host for Pseudomonas aeruginosa infection

Using the fruit fly Drosophila melanogaster as model host, we have identified mutants of the bacterium Pseudomonas aeruginosa with reduced virulence. Strikingly, all strains strongly impaired in fly killing also lacked twitching motility; most such strains had a mutation in pilGHIJKL chpABCDE, a gene cluster known to be required for twitching motility and potentially encoding a signal transduction system. The pil chp genes appear to control the expression of additional virulence factors, however, since the wild-type fly-killing phenotype of a subset of mutants isolated on the basis of their compact colony morphology indicated that twitching motility itself was not required for full virulence in the fly.     

Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching.

Three flagellar genes of Salmonella typhimurium (flaAII.2, flaQ, and flaN) were found to be multifunctional, each being associated with four distinct mutant phenotypes: nonflagellate (Fla-), paralyzed (Mot-), nonchemotactic (Che-) with clockwise motor bias, and nonchemotactic (Che-) with counterclockwise motor bias. The distribution of Fla, Mot, and Che mutational sites within each gene was examined. Fla sites were fairly broadly distributed, whereas Mot and Che sites were more narrowly defined. Local subregions rich in sites of one type were not generally rich in sites of another type. Among Che sites, there was little overlap between those corresponding to a clockwise bias and those corresponding to a counterclockwise bias. Our results suggest that within the corresponding gene products there are specialized subregions for flagellar structure, motor rotation, and control of the sense of rotation.     

The cucurbit pathogenic bacterium Acidovorax citrulli requires a polar flagellum for full virulence before and after host-tissue penetration

Acidovorax citrulli causes seedling blight and bacterial fruit blotch of cucurbits. Previous reports demonstrated the contribution of type IV pili (T4P) to A. citrulli virulence and to systemic infection of melon seedlings. Microfluidic flow-chamber assays demonstrated the involvement of T4P in surface adhesion and biofilm formation, whereas polar flagella did not appear to contribute to either of these features. On the other hand, a transposon mutant impaired in the biosynthesis of polar flagella was identified in screens for reduced virulence of an A. citrulli mutant library. Further characterization of polar flagellum mutants confirmed that A. citrulli requires a polar flagellum for full virulence on melon plants. Foliage and stem inoculation experiments revealed that polar flagella contribute to A. citrulli virulence and growth in planta at both pre- and post-host-tissue penetration. Interestingly, light microscope observations revealed that almost all A. citrulli wild-type cells extracted from the xylem sap of stem-inoculated melon seedlings remained motile, supporting the importance of this organelle in virulence and colonization of the host vascular system. We also report a negative effect of polar flagellum impairment on T4P-mediated twitching motility of A. citrulli and discuss a possible co-regulation of these two motility machineries in this bacterium.     

Deficiency of mannose-binding lectin greatly increases susceptibility to postburn infection with Pseudomonas aeruginosa

Burn injury disrupts the mechanical and biological barrier that the skin presents against infection by symbionts like the Pseudomonas aeruginosa, a Gram-negative bacteria. A combination of local factors, antimicrobial peptides, and resident effector cells form the initial response to mechanical injury of the skin. This activity is followed by an inflammatory response that includes influx of phagocytes and serum factors, such as complement and mannose-binding lectin (MBL), which is a broad-spectrum pattern recognition molecule that plays a key role in innate immunity. A growing consensus from studies in humans and mice suggests that lack of MBL together with other comorbid factors predisposes the host to infection. In this study we examined whether MBL deficiency increases the risk of P. aeruginosa infection in a burned host. We found that both wild-type and MBL null mice were resistant to a 5% total body surface area burn alone or s.c. infection with P. aeruginosa alone. However, when mice were burned then inoculated s.c. with P. aeruginosa at the burn site, all MBL null mice died by 42 h from septicemia, whereas only one-third of wild-type mice succumbed (p = 0.0005). This result indicates that MBL plays a key role in containing and preventing a systemic spread of P. aeruginosa infection following burn injury and suggests that MBL deficiency in humans maybe a premorbid variable in the predisposition to infection in burn victims.     

Prophylactic treatment with fms-like tyrosine kinase-3 ligand after burn injury enhances global immune responses to infection

Severely burned patients are susceptible to infections with opportunistic organisms due to altered immune responses and frequent wound contamination. Immunomodulation to enhance systemic and local responses to wound infections may be protective after burn injury. We previously demonstrated that pretreatments with fms-like tyrosine kinase-3 (Flt3) ligand (Flt3L), a dendritic cell growth factor, increase the resistance of mice to a subsequent burn injury and wound infection by a dendritic cell-dependent mechanism. This study was designed to test the hypothesis that Flt3L administration after burn injury decreases susceptibility to wound infections by enhancing global immune cell activation. Mice were treated with Flt3L after burn injury and examined for survival, wound and systemic bacterial clearance, and immune cell activation after wound inoculation with Pseudomonas aeruginosa. To gain insight into the local effects of Flt3L at the burn wound, localization of Langerhans cells was examined. Mice treated with Flt3L had significantly greater numbers of CD25-expressing T cells and CD69-expressing T and B cells, neutrophils, and macrophages after, but not before, infection. Overall leukocyte apoptosis in response to infection was decreased with Flt3L treatment. Survival and local and systemic bacterial clearance were enhanced by Flt3L. Langerhans cells appeared in the dermis of skin bordering the burn wound, and further increased in response to wound infection. Flt3L augmented the appearance of Langerhans cells in response to both injury and infection. These data suggest that dendritic cell enhancement by Flt3L treatments after burn injury protects against opportunistic infections through promotion of local and systemic immune responses to infection.     

Pseudomonas aeruginosa virulence analyzed in a Dictyostelium discoideum host system

Pseudomonas aeruginosa is an important opportunistic pathogen that produces a variety of cell-associated and secreted virulence factors. P. aeruginosa infections are difficult to treat effectively because of the rapid emergence of antibiotic-resistant strains. In this study, we analyzed whether the amoeba Dictyostelium discoideum can be used as a simple model system to analyze the virulence of P. aeruginosa strains. The virulent wild-type strain PAO1 was shown to inhibit growth of D. discoideum. Isogenic mutants deficient in the las quorum-sensing system were almost as inhibitory as the wild type, while rhl quorum-sensing mutants permitted growth of Dictyostelium cells. Therefore, in this model system, factors controlled by the rhl quorum-sensing system were found to play a central role. Among these, rhamnolipids secreted by the wild-type strain PAO1 could induce fast lysis of D. discoideum cells. By using this simple model system, we predicted that certain antibiotic-resistant mutants of P. aeruginosa should show reduced virulence. This result was confirmed in a rat model of acute pneumonia. Thus, D. discoideum could be used as a simple nonmammalian host system to assess pathogenicity of P. aeruginosa.     

The alternative sigma factor sigma B of Staphylococcus aureus modulates virulence in experimental central venous catheter-related infections

The impact of the alternative sigma factor sigma B (SigB) on pathogenesis of Staphylococcus aureus is not conclusively clarified. In this study, a central venous catheter (CVC) related model of multiorgan infection was used to investigate the role of SigB for the pathogenesis of S. aureus infections and biofilm formation in vivo. Analysis of two SigB-positive wild-type strains and their isogenic mutants revealed uniformly that the wild-type was significantly more virulent than the SigB-deficient mutant. The observed difference in virulence was apparently not linked to the capability of the strains to form biofilms in vivo since wild-type and mutant strains were able to produce biofilm layers inside of the catheter. The data strongly indicate that the alternative sigma factor SigB plays a role in CVC-associated infections caused by S. aureus.     

Genome instability of Pseudomonas aeruginosa phages of the EL species: examination of virulent mutants

The article continues a study of pseudolysogeny in Pseudominas aeruginosa infected with phiKZ-like phages of the EL species. Analysis was performed for several newly isolated virulent mutants of EL phages (EL and RU) that were virulent (capable of causing lysis of bacteria infected with the wild-type phage) and a lower extent of opalescence of negative colonies (NCs). Wile-type recombinants were detected in crosses of virulent mutants of phages EL and RU to confirm the polygenic control of virulence. Since a deletion mutation was found in one of the virulent EL mutants and high genetic instability was characteristic of another mutant, a mobile genetic element was assumed to play a role in mutagenesis. Pseudolysogeny of bacteria provides for horizontal gene transfer between different bacterial strains. Hence, sequencing of the phage genome and demonstration of the lack of toxic gene products are insufficient for the phage to be included into a therapeutic mixture. To use live phages, it is essential to study in detail the possible consequences of their interaction with host bacteria.     

Twitching motility is essential for virulence in Dichelobacter nodosus

Type IV fimbriae are essential virulence factors of Dichelobacter nodosus, the principal causative agent of ovine foot rot. The fimA fimbrial subunit gene is required for virulence, but fimA mutants exhibit several phenotypic changes and it is not certain if the effects on virulence result from the loss of type IV fimbria-mediated twitching motility, cell adherence, or reduced protease secretion. We showed that mutation of either the pilT or pilU gene eliminated the ability to carry out twitching motility. However, the pilT mutants displayed decreased adhesion to epithelial cells and reduced protease secretion, whereas the pilU mutants had wild-type levels of extracellular protease secretion and adherence. These data provided evidence that PilT is required for the type IV fimbria-dependent protease secretion pathway in D. nodosus. It was postulated that sufficient fimbrial retraction must occur in the pilU mutants to allow protease secretion, but not twitching motility, to take place. Although no cell movement was detected in a pilU mutant of D. nodosus, aberrant motion was detected in an equivalent mutant of Pseudomonas aeruginosa. These observations explain how in D. nodosus protease secretion can occur in a pilU mutant but not in a pilT mutant. In addition, virulence studies with sheep showed that both the pilT and pilU mutants were avirulent, providing evidence that mutation of the type IV fimbrial system affects virulence by eliminating twitching motility, not by altering cell adherence or protease secretion.