Versatile cell surface structures of archaea.

Archaea are ubiquitously present in nature and colonize environments with broadly varying growth conditions. Several surface appendages support their colonization of new habitats. A hallmark of archaea seems to be the high abundance of type IV pili (T4P). However, some unique non T4 filaments are present in a number of archaeal species. Archaeal surface structures can mediate different processes such as cellular surface adhesion, DNA exchange, motility and biofilm formation and represent an initial attachment site for infecting viruses. In addition to the functionally characterized archaeal T4P, archaeal genomes encode a large number of T4P components that might form yet undiscovered surface structures with novel functions. In this review, we summarize recent advancement in structural and functional characterizations of known archaeal surface structures and highlight the diverse processes in which they play a role.

Natural and Artificial Strategies To Control the Conjugative Transmission of Plasmids.

Conjugative plasmids are the main carriers of transmissible antibiotic resistance (AbR) genes. For that reason, strategies to control plasmid transmission have been proposed as potential solutions to prevent AbR dissemination. Natural mechanisms that bacteria employ as defense barriers against invading genomes, such as restriction-modification or CRISPR-Cas systems, could be exploited to control conjugation. Besides, conjugative plasmids themselves display mechanisms to minimize their associated burden or to compete with related or unrelated plasmids. Thus, FinOP systems, composed of FinO repressor protein and FinP antisense RNA, aid plasmids to regulate their own transfer; exclusion systems avoid conjugative transfer of related plasmids to the same recipient bacteria; and fertility inhibition systems block transmission of unrelated plasmids from the same donor cell. Artificial strategies have also been designed to control bacterial conjugation. For instance, intrabodies against R388 relaxase expressed in recipient cells inhibit plasmid R388 conjugative transfer; pIII protein of bacteriophage M13 inhibits plasmid F transmission by obstructing conjugative pili; and unsaturated fatty acids prevent transfer of clinically relevant plasmids in different hosts, promoting plasmid extinction in bacterial populations. Overall, a number of exogenous and endogenous factors have an effect on the sophisticated process of bacterial conjugation. This review puts them together in an effort to offer a wide picture and inform research to control plasmid transmission, focusing on Gram-negative bacteria.

Bacterial motility: secretory secrets of gliding bacteria.

Many bacteria glide over surfaces without the aid of flagella. Gliding is still somewhat mysterious, but recent studies show that it involves specialized secretory systems that assemble membrane-associated filaments, and the recognition of extracellular components that trigger movement via transmembrane transducers.

Different transferability of incompatibility (Inc) P-7 plasmid pCAR1 and IncP-1 plasmid pBP136 in stirring liquid conditions.

Self-transmissible plasmids are classified into two types based on their sex pili: short and rigid pili, and long and flexible pili. The transferability of two plasmids with different types of sex pili, pBP136 and pCAR1, was compared in stirring liquid conditions with different cell density. The most probable number method to count transconjugants could detect differences in the transfer frequency with higher resolution in comparison with the conventional CFU counting method. Both plasmids showed higher transfer frequency in high stirring rates than static liquid conditions when the donor and recipient density was 106-107 CFU mL-1. The probability of donor-initiated plasmid transfer was investigated by a single-cell-level analysis using a cell sorter. The probability was >36-fold higher for pBP136 than for pCAR1; thus, the simulated transfer frequency of pBP136 was much higher than that of pCAR1 in stirring liquid conditions. Nevertheless, the transfer frequency of pCAR1 was as high as that of pBP136 when the donor and recipient cell density was 106 CFU mL-1. This fact indicates that the lower probability of the donor pCAR1 to initiate transfer could be overcome by its high tolerance to the shearing force between donor and recipient cells under higher stirring liquid conditions. Our findings can explain the different survival strategies of these two types of plasmids based on their preferences of transfer conditions.

The Agrobacterium tumefaciens T pilus composed of cyclic T pilin is highly resilient to extreme environments.

Agrobacterium tumefaciens T pili are long semi-rigid, flexuous filaments of 10 nm diameter that are primarily composed of T pilin cyclized protein subunits. The cyclic character of T pilin apparently confers a high level of structural stability on the T pilus. Purified T pili subjected to extreme environmental conditions such as acid and alkali, including glycerol remained relatively unaffected morphologically. T pili lost their semi-rigidity when subjected to high temperatures and high pH, and dissociated into donut shaped subunits when exposed to Triton X-100. Sodium dodecyl sulfate increased the uptake of uranyl acetate exposing a 2 nm wide lumen running the length of the T pilus filament.

Interactions of Porphyromonas gingivalis with host cells: implications for cardiovascular diseases.

BACKGROUND: Recent epidemiological studies have suggested a contribution of periodontitis in atherosclerotic diseases. Two mechanisms have been proposed to explain such a connection involving general inflammatory responses and/or specific effects of periodontal bacteria on host tissues. METHODS: The role of the periodontopathogen Porphyromonas gingivalis as a potential contributor to atherosclerosis has been investigated in model systems using human umbilical vein endothelial cells (HUVEC) and murine J774 macrophage cell cultures. RESULTS: P. gingivalis 381 was demonstrated to induce foam cell formation in J774 macrophage cell cultures in the presence of low-density lipoproteins. The active bacterial component involved in this process appears to be lipopolysaccharide. This effect was not limited to these organisms as several other Gram-positive and Gram-negative oral bacteria exhibited the same property. In addition, in a more specific manner, P. gingivalis induced monocyte chemoattractant protein-1 secretion in HUVEC cultures. CONCLUSIONS: The fimbriae of strain 381 are important, but are not required, for this inductive effect. Taken together, these results suggest a potential role for P. gingivalis in several steps involved in atherosclerotic lesion formation.

Characterization of biologically active cell surface components of a periodontal pathogen. The roles of major and minor fimbriae of Porphyromonas gingivalis.

BACKGROUND: We previously reported that the presence of 2 different types of fimbriae expressed on the cell surface of Porphyromonas gingivalis ATCC 33277. The initial event in most infectious diseases involves adhesion of pathogens to host tissues and subsequent invasion by the pathogens. To define the role of fimbriae in Porphyromonas gingivalis adherence to and invasion of epithelial cells, we have constructed fimbrial mutants. The involvement of P. gingivalis fimbriae in the invasion process and alveolar bone resorption in rats was examined. METHODS: Inactivated mutants of 41-K fimbrillin gene (fimA) and/or the 67-K fimbrillin gene (mfa1) were constructed by a homologous recombination technique and compared among fimA mutant (MPG1), mfa1 mutant (MPG67), and double knockout mutant (MPG4167). Adherence and invasion of P. gingivalis was assessed in human oral epithelial KB cells. We used a rat model to examine the role of each type of fimbriae in alveolar bone loss by oral infection. RESULTS: The adherence and invasion levels of the mutants were lower than the wild-type strain. The bone loss of rats infected with the MPG1 was higher than that of those infected with MPG67. Moreover, the bone loss of rats infected with the double knockout mutant was significantly decreased compared to that of rats infected with the wild-type strain. CONCLUSIONS: Data from this study suggest that not only the 41-K fimbrial protein, but also the 67-K fimbrial protein, play important roles in the pathogenesis of periodontal disease.

Molecular interaction of Porphyromonas gingivalis with host cells: implication for the microbial pathogenesis of periodontal disease.

Porphyromonas gingivalis is a predominant periodontal pathogen, which expresses a number of potential virulence factors involved in the pathogenesis of periodontitis. Among them, fimbriae are a critical factor to mediate the bacterial interaction with host tissues, which promotes the bacterial adhesion to and invasion of the targeted sites. Fimbriae are capable of binding to human salivary components, commensal bacteria, and a variety of host cells including macrophages, epithelial cells, and fibroblasts. Human extracellular matrix (ECM) proteins such as vitronectin and fibronectin play important roles in cellular signal transduction via binding to receptor integrins. Fimbriae showed significant binding affinity to ECM proteins and clearly inhibited the molecular interactions between vitronectin/fibronectin and their receptor alphavbeta3 and alpha5beta1 integrins overexpressed on Chinese hamster ovary (CHO) cell strain. P. gingivalis fimbriae are likely to interrupt the cellular signaling via ECM proteins/integrins in periodontal regions. Fimbriae are also thought to be critically important in invasive events of the organism to host cells. The fimA genes, encoding FimA (a subunit of fimbriae), of P. gingivalis strains are classified into 5 types, I to V. Recent clinical investigations demonstrated the close relationship between the organisms with type II fimA and periodontitis development. Recombinant FimA (rFimA) proteins of types I to V were generated to compare their adhesion/invasion abilities to human gingival fibroblasts (HGF) and a human epithelial cell line (HEp-2 cells), respectively. There were no significant differences in the adhesion ability of microspheres (MS) coated with these rFimAs to HGF; however, the adhesion of type II rFimA-MS to HEp-2 cells was significantly greater than that of other rFimA types. It was also observed that the type II rFimA-MS markedly invaded the epithelial cells and accumulated around the nuclei. Collectively, these findings suggest that fimbriae of P. gingivalis, especially type II, are involved in the initiation and progression of human periodontitis.

Direct visualization of horizontal gene transfer.

Conjugation allows bacteria to acquire genes for antibiotic resistance, novel virulence attributes, and alternative metabolic pathways. Using a fluorescent protein fusion, SeqA-YFP, we have visualized this process in real time and in single cells of Escherichia coli. We found that the F pilus mediates DNA transfer at considerable cell-to-cell distances. Integration of transferred DNA by recombination occurred in up to 96% of recipients; in the remaining cells, the transferred DNA was fully degraded by the RecBCD helicase/nuclease. The acquired integrated DNA was tracked through successive replication rounds and was found to occasionally split and segregate with different chromosomes, leading to the inheritance of different gene clusters within the cell lineage. The incidence of DNA splitting corresponds to about one crossover per cell generation.

Distinct function of Pseudomonas aeruginosa type IV pili disclosed in the bacterial pass-through of membrane filter with smaller pore sizes.

Membrane filter pass-through ability of Pseudomonas aeruginosa was analyzed with isogenic mutants. A flagellum-deficient fliC mutant required two-times longer time (12 hr) to pass through a 0.45-microm pore size filter. With 0.3- and 0.22-microm filters, however, the fliC mutant showed no remarkable disability. Meanwhile a pilA mutant defective in twitching motility failed to pass through the 0.22-microm filter. Complementation of the mutant with pilA gene on a plasmid restored the twitching motility and the 0.22-microm filter pass-through activity. Thus, the distinctive role of P. aeruginosa type IV pili in infiltration into finer reticulate structures was indicated.

I-domain-containing integrins serve as pilus receptors for Neisseria gonorrhoeae adherence to human epithelial cells.

Two pilus receptors are identified for the pathogenic Neisseria, CD46 and complement receptor 3. An intimate association between the asialoglycoprotein receptor and gonococcal lipooligosaccharide mediates invasion of primary, male urethral epithelial cells (UECs); however, studies to identify pilus receptors on these cells have not been performed. Based on our previous studies we reasoned that the I-domain-containing (IDC), alpha(1)- and alpha(2)-integrins might serve as pilus receptors on UECs and on urethral tissue. Confocal microscopy revealed colocalization of pilus with alpha(1) and alpha(2) integrins on UECs and tissue. We found that recombinant I-domain and antibodies directed against the alpha(1)- and alpha(2)-integrins inhibited gonococcal association with UECs and with immortal cell lines of variable origin. Gonococcus-integrin colocalization occurred at early time points post infection, but this interaction dissociated with extended infection. Similarly, Western Blot analyses revealed that gonococcal pilin coimmunoprecipitates with alpha(1)- and alpha(2)-integrins. However, studies performed in parallel and that were designed to capture CD46-pilus immune complexes indicated that a CD46-pilus interaction did not occur. Collectively, these data suggest that while CD46 might be able to bind gonococcal pilus, IDC integrins are preferentially used as the initial docking site for gonococci on UECs, on urethral tissue and on some immortal cell lines.

Interactions of pathogenic Neisseria with host cells. Is it possible to assemble the puzzle?

Neisseria meningitidis and Neisseria gonorrhoeae are human pathogens that have to interact with mucosa and/or cellular barriers for their life cycles to progress. Even though they both give rise to dramatically different diseases, the use of in vitro models has shown that most of the mechanisms mediating cellular interactions are common to N. meningitidis and N. gonorrhoeae. This suggests that bacterial cell interactions may be essential not only for pathogenesis but also for other aspects of the bacterial life cycle that are common to both N. meningitidis and N. gonorrhoeae. This manuscript will review the most recent developments concerning the mechanisms mediating cellular interaction of pathogenic Neisseria and will then try to put them into the perspective of pathogenesis and bacterial life cycle.

Atpase activity and multimer formation of Pilq protein are required for thin pilus biogenesis in plasmid R64.

Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus. The pilQ product contains NTP binding motifs and belongs to the PulE-VirB11 family of NTPases. The pilQ gene was overexpressed with an N-terminal His tag, and PilQ protein was purified. Purified His tag PilQ protein displayed ATPase activity with a V(max) of 0.71 nmol/min/mg of protein and a K(m) of 0.26 mm at pH 6.5. By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ protein forms a homooctamer. To analyze the relationship between structure and function of PilQ protein, amino acid substitutions were introduced within several conserved motifs. Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings. Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag. The purified mutant PilQ proteins contained various levels of reduced ATPase activity. Three mutant PilQ proteins formed stable multimers similar to wild-type PilQ, whereas the PilQ D263N multimer was unstable. PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not. These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis.

Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes.

The Pseudomonas aeruginosa genes pilB-D and pilQ are necessary for the assembly of type 4 fimbriae. Homologues of these genes and of the subunit (pilin) gene have been described in various different bacterial species, but not always in association with type 4 fimbrial biosynthesis and function. Pil-like proteins are also involved in protein secretion, DNA transfer by conjugation and transformation, and morphogensis of filamentous bacteriophages. It seems likely that the Pil homologues function in the processing and export of proteins resembling type 4 fimbrial subunits, and in their organization into fimbrial-like structures. These may either be true type 4 fimbriae, or components of protein complexes which act in the transport of macromolecules (DNA or protein) into or out of the cell. Some PilB-like and PilQ-like proteins are apparently also involved in the assembly of non-type 4 polymeric structures (filamentous phage virions and conjugative pili). The diverse studies summarized in this review are providing insight into an extensive infrastructural system which appears to be utilized in the formation of a variety of cell surface-associated complexes.

Effects of bfp mutations on biogenesis of functional enteropathogenic Escherichia coli type IV pili.

Enteropathogenic Escherichia coli expresses a type IV fimbria known as the bundle-forming pilus (BFP) that is required for autoaggregation and localized adherence (LA) to host cells. A cluster of 14 genes is sufficient to reconstitute BFP biogenesis in a laboratory strain of E. coli. We have undertaken a systematic mutagenesis of the individual genes to determine the effect of each mutation on BFP biogenesis and LA. Here we report the construction and analysis of nonpolar mutations in six genes of the bfp cluster, bfpG, bfpB, bfpC, bfpD, bfpP, and bfpH, as well as the further analysis of a previously described bfpA mutant strain that is unable to express bundlin, the pilin protein. We found that mutations in bfpB, which encodes an outer membrane protein; bfpD, which encodes a putative nucleotide-binding protein; and bfpG and bfpC, which do not have sequence homologues in other type IV pilus systems, do not affect prebundlin expression or processing but block both BFP biogenesis and LA. The mutation in bfpP, the prepilin peptidase gene, does not affect prebundlin expression but blocks signal sequence cleavage of prebundlin, BFP biogenesis, and LA. The mutation in bfpH, which is predicted to encode a lytic transglycosylase, has no effect on prebundlin expression, prebundlin processing, BFP biogenesis, or LA. For each mutant for which altered phenotypes were detected, complementation with a plasmid containing the corresponding wild-type allele restored the wild-type phenotypes. We also found that association of prebundlin or bundlin with sucrose density flotation gradient fractions containing both inner and outer membrane proteins does not require any accessory proteins. These studies indicate that many bfp gene products are required for biogenesis of functional type IV pili but that mutations in the individual genes do not lead to the identification of new phases of pilus assembly.

The phase-variable pilus-associated protein PilC is commonly expressed in clinical isolates of Neisseria gonorrhoeae, and shows sequence variability among strains.

PilC is a phase-variable protein associated with pilus-mediated adherence of pathogenic Neisseria to target cells. In this study, 24 strains of Neisseria gonorrhoeae with known epidemiological data were examined for expression of PilC. All strains produced PilC independently of serovar and site of isolation. To investigate whether the PilC protein is conserved or variable among gonococcal strains, the complete nucleotide sequence of pilC in four strains, isolated from either rectum, throat or blood, was determined. The deduced amino acid sequence in these strains differed from each other and from the two PilC proteins of N. gonorrhoeae MS11. These data demonstrate that PilC is commonly expressed, but the PilC sequence may vary among gonococcal strains.

Conjugative junctions in RP4-mediated mating of Escherichia coli.

The physical association of bacteria during conjugation mediated by the IncPalpha plasmid RP4 was investigated. Escherichia coli mating aggregates prepared on semisolid medium were ultrarapidly frozen using copper block freezing, followed by freeze substitution, thin sectioning, and transmission electron microscopy. In matings where the donor bacteria contained conjugative plasmids, distinctive junctions were observed between the outer membranes of the aggregates of mating cells. An electron-dense layer linked the stiffly parallel outer membranes in the junction zone, but there were no cytoplasmic bridges nor apparent breaks in the cell walls or membranes. In control experiments where the donors lacked conjugative plasmids, junctions were not observed. Previous studies have shown that plasmid RP4 carries operons for both plasmid DNA processing (Tra1) and mating pair formation (Tra2). In matings where donor strains carried Tra2 only or Tra2 plus the pilin-processing protease TraF, junctions were found but they were shorter and more interrupted than the wild type. If the donor strain had the pilin gene knocked out (trbC), junctions were still found. Thus, it appears that the electron-dense layer between the outer membranes of the conjugating cells is not composed of pilin.

Functional implications of the expression of PilC proteins in meningococci.

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil- backswitcher isolated from the hyper-adherent variant was PilC+ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.

Salmonella typhimurium fimbrial phase variation and FimA expression.

Bacteria in a nonfimbriate phase because of continuous aeration of liquid cultures produce FimA in amounts similar to those produced by fimbriate bacteria. However, relatively low FimA production was observed in nonfimbriate-phase cultures obtained by growth on solid media or by anaerobic incubation. Regardless of the fimbrial phase of Salmonella typhimurium, the fimA promoter region was always oriented in the direction that might allow fimA transcription.