Contribution of Individual Ebp Pilus Subunits of Enterococcus faecalis OG1RF to Pilus Biogenesis,Biofilm Formation and Urinary Tract Infection

The endocarditis and biofilm-associated pilus (Ebp) operon is a component of the core genome of Enterococcus faecalis that has been shown to be important for biofilm formation, adherence to host fibrinogen, collagen and platelets, and in experimental endocarditis and urinary tract infection models. Here, we created single and double deletion mutants of the pilus subunits and sortases; next, by combining western blotting, immunoelectron microscopy, and using ebpR in trans to increase pilus production, we identified EbpA as the tip pilin and EbpB as anchor at the pilus base, the latter attached to cell wall by the housekeeping sortase, SrtA. We also confirmed EbpC and Bps as the major pilin and pilin-specific sortase, respectively, both required for pilus polymerization. Interestingly, pilus length was increased and the number of pili decreased by deleting ebpA, while control overexpression of ebpA in trans restored wild-type levels, suggesting a dual role for EbpA in both initiation and termination of pilus polymerization. We next investigated the contribution of each pilin subunit to biofilm formation and UTI. Significant reduction in biofilm formation was observed with deletion of ebpA or ebpC (P<0.001) while ebpB was found to be dispensable; a similar result was seen in kidney CFUs in experimental UTI (ΔebpA, ΔebpC, P≤0.0093; ΔebpB, non-significant, each vs. OG1RF). Hence, our data provide important structural and functional information about these ubiquitous E. faecalis pili and, based on their demonstrated importance in biofilm and infection, suggest EbpA and EbpC as potential targets for antibody-based therapeutic approaches.     

Link between culture zeta potential homogeneity and Ebp in Enterococcus faecalis

Enterococcus faecalis, a commensal of the gastrointestinal tract and an opportunistic pathogen, has the ability to adhere to surfaces and form biofilms. It has been shown earlier that only 10 to 20% of an E. faecalis OG1RF culture expresses endocarditis- and biofilm-associated pili (Ebp), which are involved in biofilm formation. Another study revealed that E. faecalis clinical isolates, as well as OG1RF, are heterogeneous with respect to their apparent zeta potential, which was also correlated with increased ability to form biofilm. The aim of this study was to demonstrate that the heterogeneity in the presence of Ebp is correlated to that in apparent zeta potential. Heterogeneous cultures of OG1RF showed two distinct subpopulations with the most (-38 mV) and least (-26 mV) negative zeta potential. Deletion of EbpR, the activator of the ebp operon, or the structural genes ebpABC resulted in homogeneous culture with the most negative zeta potential. Conversely, overexpression of EbpR or the structural genes ebpABC resulted in homogeneous culture with the least negative zeta potential. The results show that ebp operon expression in E. faecalis, as measured by using P(ebp)-gfp promoter fusion, is the cause of heterogeneity in zeta potential and that pilus production causes the cells to behave as the least negative particle in an electric field.     

Effects of recA Mutations on Pilus Antigenic Variation and Phase Transitions in Neisseria gonorrhoeae

Intragenic recombination between the single complete pilin gene (expression locus) and multiple, distinct, partial pilin gene copies (silent, storage loci) is thought to account for the generation of pilus antigenic diversity and piliation phase (on-off) changes exhibited by Neisseria gonorrhoeae. The mechanisms operating in the genomic rearrangements associated with these forms of pilus variation were investigated through the study of isogenic strains of gonococci bearing either wild-type or altered recA alleles. Examination of the rates of pilus phase variation and the genetic basis for changes in piliation status displayed by these strains show that recA mediated homologous recombination is required for these high frequency events and confirm that the nonpiliated state results from mutations in the expressed pilin gene. In a strain that is deficient in recA mediated homologous recombination, pilus phase variation occurs at a 100-1000-fold reduced rate and results predominantly from one class of spontaneous frameshift mutations within the pilin structural gene.     

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.     

VirB6 is required for stabilization of VirB5 and VirB3 and formation of VirB7 homodimers in Agrobacterium tumefaciens

VirB6 from Agrobacterium tumefaciens is an essential component of the type IV secretion machinery for T pilus formation and genetic transformation of plants. Due to its predicted topology as a polytopic inner membrane protein, it was proposed to form the transport pore for cell-to-cell transfer of genetic material and proteinaceous virulence factors. Here, we show that the absence of VirB6 leads to reduced cellular levels of VirB5 and VirB3, which were proposed to assist T pilus formation as minor component(s) or assembly factor(s), respectively. Overexpression of virB6 in trans restored levels of cell-bound and T pilus-associated VirB5 to wild type but did not restore VirB3 levels. Thus, VirB6 has a stabilizing effect on VirB5 accumulation, thereby regulating T pilus assembly. In the absence of VirB6, cell-bound VirB7 monomers and VirB7-VirB9 heterodimers were reduced and VirB7 homodimer formation was abolished. This effect could not be restored by expression of VirB6 in trans. Expression of TraD, a component of the transfer machinery of the IncN plasmid pKM101, with significant sequence similarity to VirB6, restored neither protein levels nor bacterial virulence but partly permitted T pilus formation in a virB6 deletion strain. VirB6 may therefore regulate T pilus formation by direct interaction with VirB5, and wild-type levels of VirB3 and VirB7 homodimers are not required.     

Gene conversion involving the pilin structural gene correlates with pilus+ in equilibrium with pilus- changes in Neisseria gonorrhoeae

Gonococci (Gc) exhibit pilus+----pilus- "phase transitions" at high frequency, but only some of the pilus- Gc can revert to pilus+ phenotype. We examined reversible phase transitions between pilus+ Gc and a particular pilus- variant (P-rp+ phenotype) whose pilin mRNA carries a unique block of nucleotides encoding an "assembly missense" pilin polypeptide. The results show that Gc pilus+ in equilibrium with P-rp+ transitions can result from intragenic recombination in which there is nonreciprocal exchange of partially homologous DNA sequences from a partial pilin gene (in silent, storage form) into the expression locus' complete pilin gene. Hence Gc pilus phase variation, like pilus antigenic variation, can occur by gene conversion of the pilin structural gene expression locus.     

Regulation and function of pilus island 1 in group B streptococcus

Group B streptococcus (GBS) pili may enhance colonization and infection by mediating bacterial adhesion to host cells, invasion across endothelial and epithelial barriers, and resistance to bacterial ingestion and killing by host phagocytes. However, it remains unclear how pilus expression is regulated and how modulation of pilus production affects GBS interactions with the human host. We investigated the regulation and function of pilus island 1 (PI-1) pili in GBS strain 2603. We found that PI-1 gene expression was controlled by the CsrRS two-component system, by Ape1, an AraC-type regulator encoded by a divergently transcribed gene immediately upstream of PI-1, and by environmental pH. The response regulator CsrR repressed expression of Ape1, which is an activator of PI-1 gene expression. In addition, CsrR repressed PI-1 gene expression directly, independent of its regulation of Ape1. In vitro assays demonstrated specific binding of both CsrR and Ape1 to chromosomal DNA sequences upstream of PI-1. Pilus gene expression was activated by acidic pH, and this effect was independent of CsrRS and Ape1. Unexpectedly, characterization of PI-1 deletion mutants revealed that PI-1 pili do not mediate adhesion of strain 2603 to A549 respiratory epithelial cells, ME180 cervical cells, or VK2 vaginal cells in vitro. PI-1 pili reduced internalization and intracellular killing of GBS by human monocyte-derived macrophages, by approximately 50%, but did not influence complement-mediated opsonophagocytic killing by human neutrophils. These findings shed new light on the complex nature of pilus regulation and function in modulating GBS interactions with the human host.     

PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer

Studies of gonococcal pilus biogenesis are fundamental to understanding organelle structure/function relationships and identifying new approaches to controlling disease. This area of research is also relevant to elucidating the basic mechanisms of outer membrane translocation of macromolecules, which requires components highly related to those involved in type IV pilus expression. Previous studies have shown that products of several ancillary pil genes are required for organelle biogenesis but of these only PilQ, a member of the GspD protein family, is a component of the outer membrane. DNA sequencing of the region upstream of pilQ revealed the presence of two open reading frames (ORFs) whose deduced polypeptides shared significant identities with proteins required for pilus expression in Pseudomonas aeruginosa and Pseudomonas syringae, the genes for which are arrayed upstream of a gene encoding a PilQ homologue. Gonococcal mutants bearing transposon insertions in these ORFs were non-piliated and failed to express pilus-associated phenotypes, and the corresponding genes were designated pilO and pilP. The piliation defects in the mutants could not be ascribed to polarity on distal pilQ expression as shown by direct measurement of PilQ antigen in those backgrounds and the use of a novel technique to create tandem duplications in the gonococcus (Gc) genome. As predicted by the presence of a consensus lipoprotein signal sequence, PilP expressed in both Escherichia coli and Gc could be labelled with [³H]-palmitic acid. PilP^? as well as PilQ^? mutants shed PilC, a protein which facilitates pilus assembly and is implicated in epithelial cell adherence, in a soluble form. Combined with the finding that levels of multimerized PilQ were greatly reduced in PilP^? mutants, the results suggest that PilP is required for PilQ function and that PilQ and PilC may interact during the terminal stages of pilus biogenesis. The findings also support the hypothesis that the Gc PilQ multimer corresponds to a physiologically relevant form of the protein required for pilus biogenesis.     

A conserved set of pilin-like molecules controls type IV pilus dynamics and organelle-associated functions in Neisseria gonorrhoeae

Type IV pili (Tfp) play central roles in prokaryotic cell biology and disease pathogenesis. As dynamic filamentous polymers, they undergo rounds of extension and retraction modelled as pilin subunit polymerization and depolymerization events. Currently, the molecular mechanisms and components influencing Tfp dynamics remain poorly understood. Using Neisseria gonorrhoeae as a model system, we show that mutants lacking any one of a set of five proteins sharing structural similarity to the pilus subunit are dramatically reduced in Tfp expression and that these defects are suppressed in the absence of the PilT pilus retraction protein. Thus, these molecules are not canonical assembly factors but rather act as effectors of pilus homeostasis by promoting extension/polymerization events in the presence of PilT. Furthermore, localization studies support the conclusion that these molecules form a Tfp-associated complex and influence levels of PilC, the epithelial cell adhesin, in Tfp-enriched shear fractions. This is the first time that the step at which individual pilin-like proteins impact on Tfp expression has been defined. The findings have important implications for understanding Tfp dynamics and fundamental Tfp structure/function relationships.     

Characterization of FimC, a periplasmic assembly factor for biogenesis of type 1 pili in Escherichia coli

Assembly of type 1 pili from Escherichia coli is mediated by FimC, a periplasmic chaperone (assembly factor) consisting of two immunoglobulin-like domains. FimC is assumed to recognize the individual pilus subunits in the periplasm mainly via their conserved C-terminal segments and to deliver the subunits to an assembly platform in the outer membrane. Here we present the first biochemical characterization of a periplasmic pilus chaperone and analyze the importance of the two chaperone domains for stability and function. Comparison of the isolated C-terminal domain with wild-type FimC revealed a strongly reduced thermodynamic stability, indicating strong interdomain interactions. The affinity of FimC toward a peptide corresponding to the 11 C-terminal residues of the type 1 pilus adhesin FimH is at least 1000-fold lower compared to binding of intact FimH, confirming that bacterial pilus chaperones, unlike other chaperones, specifically interact with folded pilus subunits.     

A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro

The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin.     

Pili of oral Streptococcus sanguinis bind to fibronectin and contribute to cell adhesion

Streptococcus sanguinis is a predominant bacterium in the human oral cavity and occasionally causes infective endocarditis. We identified a unique cell surface polymeric structure named pili in this species and investigated its functions in regard to its potential virulence. Pili of S. sanguinis strain SK36 were shown to be composed of three distinctive pilus proteins (PilA, PilB, and PilC), and a pili-deficient mutant demonstrated reduced bacterial adherence to HeLa and human oral epithelial cells. PilC showed a binding ability to fibronectin, suggesting that pili are involved in colonization by this species. In addition, ATCC10556, a standard S. sanguinis strain, was unable to produce pili due to defective pilus genes, which indicates a diversity of pilus expression among various S. sanguinis strains.     

The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype‐specific targets

Bacterial pathogens commonly show intra‐species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T‐antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT‐types), and (c) the streptokinase‐encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype‐specific manner. Parental, fasX mutant and complemented derivatives of serotype M1 (ska‐2, FCT‐2), M2 (ska‐1, FCT‐6), M6 (ska‐2, FCT‐1) and M28 (ska‐1, FCT‐4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT‐region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen‐expressing mice. Our data are the first to identify and characterize serotype‐specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence.     

Pilicides regulate pili expression in E. coli without affecting the functional properties of the pilus rod

The infectious ability of uropathogenic Escherichia coli relies on adhesive fibers, termed pili or fimbriae, that are expressed on the bacterial surface. Pili are multi-protein structures that are formed via a highly preserved assembly and secretion system called the chaperone-usher pathway. We have earlier reported that small synthetic compounds, referred to as pilicides, disrupt both type 1 and P pilus biogenesis in E. coli. In this study, we show that the pilicides do not affect the structure, dynamics or function of the pilus rod. This was demonstrated by first suppressing the expression of P pili in E. coli by pilicide treatment and, next, measuring the biophysical properties of the pilus rod. The reduced abundance of pili was assessed with hemagglutination, atomic force microscopy and Western immunoblot analysis. The biodynamic properties of the pili fibers were determined by optical tweezers force measurements on individual pili and were found to be intact. The presented results establish a potential use of pilicides as chemical tools to study important biological processes e.g. adhesion, pilus biogenesis and the role of pili in infections and biofilm formation.     

Twelve pil genes are required for biogenesis of the R64 thin pilus

The IncI1 plasmid R64 produces two kinds of sex pili: a thin pilus and a thick pilus. The thin pilus, which belongs to the type IV family, is required only for liquid matings. Fourteen genes, pilI to -V, were found in the DNA region responsible for the biogenesis of the R64 thin pilus (S.-R. Kim and T. Komano, J. Bacteriol. 179:3594-3603, 1997). In this study, we introduced frameshift mutations into each of the 14 pil genes to test their requirement for R64 thin pilus biogenesis. From the analyses of extracellular secretion of thin pili and transfer frequency in liquid matings, we found that 12 genes, pilK to -V, are required for the formation of the thin pilus. Complementation experiments excluded the possible polar effects of each mutation on the expression of downstream genes. Two genes, traBC, were previously shown to be required for the expression of the pil genes. In addition, the rci gene is responsible for modulating the structure and function of the R64 thin pilus via the DNA rearrangement of the shufflon. Altogether, 15 genes, traBC, pilK through pilV, and rci, are essential for R64 thin pilus formation and function.     

Expression of multiple types of N-methyl Phe pili in Pseudomonas aeruginosa

The nature of pili synthesized by Pseudomonas aeruginosa when plasmid-borne genes of homologous pilins from Bacteroides nodosus are introduced as thermoregulated expression systems has been ascertained. Expression of B. nodosus pili inhibited the production of indigenous P. aeruginosa pili, and an organism harbouring pilin genes from two strains of B. nodosus produced two serologically distinct populations of pili on each cell. Simultaneous production of both indigenous and foreign pili was achieved by partial induction of expression. Homogeneity in pilus structure suggests either that there is an exclusive specificity of interaction between identical pilin subunits in pilus assembly, or that each pilus is produced from the translation products of a single messenger RNA molecule, with translation and pilus assembly closely coupled.     

Opacity determinants of Neisseria gonorrhoeae: gene expression and chromosomal linkage to the gonococcal pilus gene

In N. gonorrhoeae, the expression of pilus and opacity (Op) proteins can be switched on and off and a single cell apparently has a whole repertoire of genes to express many serologically distinguishable protein types. We describe the isolation of several different Op genes and of nonexpressing gene equivalents, all derived from isogenic gonococcal variants. In the E. coli host, Op proteins identical with those made in the respective N. gonorrhoeae strain are produced. The Op genes map near the pilus expression locus. Genomic blotting experiments with an Op gene probe reveal complex hybridization patterns but little heterogeneity among the genes of Op variants. It appears that colonial variation involving the Op protein of N. gonorrhoeae is based on minor sequence alterations, in contrast to the pilus variation system, in which changes in the expression can be evoked by substantial genomic rearrangements.