Pilus Phase Variation Research Articles

The Pilin N-terminal Domain Maintains Neisseria gonorrhoeae Transformation Competence during Pilus Phase Variation.

The obligate human pathogen Neisseria gonorrhoeae is the sole aetiologic agent of the sexually transmitted infection, gonorrhea. Required for gonococcal infection, Type IV pili (Tfp) mediate many functions including adherence, twitching motility, defense against neutrophil killing, and natural transformation. Critical for immune escape, the gonococcal Tfp undergoes antigenic variation, a recombination event at the pilE locus that varies the surface exposed residues of the major pilus subunit PilE (pilin) in the pilus fiber. This programmed recombination system has the potential to produce thousands of pilin variants and can produce strains with unproductive pilin molecules that are completely unable to form Tfp. Saturating mutagenesis of the 3’ third of the pilE gene identified 68 unique single nucleotide mutations that each resulted in an underpiliated colony morphology. Notably, all isolates, including those with undetectable levels of pilin protein and no observable surface-exposed pili, retained an intermediate level of transformation competence not exhibited in ΔpilE strains. Site-directed, nonsense mutations revealed that only the first 38 amino acids of the mature pilin N-terminus (the N-terminal domain or Ntd) are required for transformation competence, and microscopy, ELISAs and pilus purification demonstrate that extended Tfp are not required for competence. Transformation in strains producing only the pilin Ntd has the same genetic determinants as wild-type transformation. The Ntd corresponds to the alternative product of S-pilin cleavage, a specific proteolysis unique to pathogenic Neisseria. Mutation of the S-pilin cleavage site demonstrated that S-pilin cleavage mediated release of the Ntd is required for competence when a strain produces unproductive pilin molecules that cannot assemble into a Tfp through mutation or antigenic variation. We conclude that S-pilin cleavage evolved as a mechanism to maintain competence in nonpiliated antigenic variants and suggest there are alternate forms of the Tfp assembly apparatus that mediate various functions including transformation.

The Long and Winding Road (Apologies to the Beatles).

The Long and Winding Road (Apologies to the Beatles).

Neisseria gonorrhoeae MutS affects pilin antigenic variation through mismatch correction and not by pilE guanine quartet binding.

Many pathogens use homologous recombination to vary surface antigens to avoid immune surveillance. Neisseria gonorrhoeae achieves this in part by changing the properties of its surface pili in a process called pilin antigenic variation (AV). Pilin AV occurs by high-frequency gene conversion reactions that transfer silent pilS sequences into the expressed pilE locus and requires the formation of an upstream guanine quartet (G4) DNA structure to initiate this process. The MutS and MutL proteins of the mismatch correction (MMC) system act to correct mismatches after replication and prevent homeologous (i.e., partially homologous) recombination, but MutS orthologs can also bind to G4 structures. A previous study showed that mutation of MutS resulted in a 3-fold increase in pilin AV, which could be due to the loss of MutS antirecombination properties or loss of G4 binding. We tested two site-directed separation-of-function MutS mutants that are both predicted to bind to G4s but are not able to perform MMC. Pilus phase variation assays and DNA sequence analysis of pilE variants produced in these mutants showed that all three mutS mutants and a mutL mutant had similar increased frequencies of pilin AV. Moreover, the mutS mutants all showed similar increased levels of pilin AV-dependent synthetic lethality. These results show that antirecombination by MMC is the reason for the effect that MutS has on pilin AV and is not due to pilE G4 binding by MutS. Neisseria gonorrhoeae continually changes its outer surface proteins to avoid recognition by the immune system. N. gonorrhoeae alters the antigenicity of the pilus by directed recombination between partially homologous pilin copies in a process that requires a guanine quartet (G4) structure. The MutS protein of the mismatch correction (MMC) system prevents recombination between partially homologous sequences and can also bind to G4s. We confirmed that loss of MMC increases the frequency of pilin antigenic variation and that two MutS mutants that are predicted to separate the two different functions of MutS inhibit pilin variation similarly to a complete-loss-of-function mutant, suggesting that interaction of MutS with the G4 structure is not a major factor in this process.

Single Cell Stochastic Regulation of Pilus Phase Variation by an Attenuation-like Mechanism

The molecular triggers leading to virulence of a number of human-adapted commensal bacteria such as Streptococcus gallolyticus are largely unknown. This opportunistic pathogen is responsible for endocarditis in the elderly and associated with colorectal cancer. Colonization of damaged host tissues with exposed collagen, such as cardiac valves and pre-cancerous polyps, is mediated by appendages referred to as Pil1 pili. Populations of S. gallolyticus are heterogeneous with the majority of cells weakly piliated while a smaller fraction is hyper piliated. We provide genetic evidences that heterogeneous pil1 expression depends on a phase variation mechanism involving addition/deletion of GCAGA repeats that modifies the length of an upstream leader peptide. Synthesis of longer leader peptides potentiates the transcription of the pil1 genes through ribosome-induced destabilization of a premature stem-loop transcription terminator. This study describes, at the molecular level, a new regulatory mechanism combining phase variation in a leader peptide-encoding gene and transcription attenuation. This simple and robust mechanism controls a stochastic heterogeneous pilus expression, which is important for evading the host immune system while ensuring optimal tissue colonization.

Pilus Phase Variation Switches Gonococcal Adherence to Invasion by Caveolin-1-Dependent Host Cell Signaling

Many pathogenic bacteria cause local infections but occasionally invade into the blood stream, often with fatal outcome. Very little is known about the mechanism underlying the switch from local to invasive infection. In the case of Neisseria gonorrhoeae, phase variable type 4 pili (T4P) stabilize local infection by mediating microcolony formation and inducing anti-invasive signals. Outer membrane porin PorBIA, in contrast, is associated with disseminated infection and facilitates the efficient invasion of gonococci into host cells. Here we demonstrate that loss of pili by natural pilus phase variation is a prerequisite for the transition from local to invasive infection. Unexpectedly, both T4P-mediated inhibition of invasion and PorBIA-triggered invasion utilize membrane rafts and signaling pathways that depend on caveolin-1-Y14 phosphorylation (Cav1-pY14). We identified p85 regulatory subunit of PI3 kinase (PI3K) and phospholipase Cγ1 as new, exclusive and essential interaction partners for Cav1-pY14 in the course of PorBIA-induced invasion. Active PI3K induces the uptake of gonococci via a new invasion pathway involving protein kinase D1. Our data describe a novel route of bacterial entry into epithelial cells and offer the first mechanistic insight into the switch from local to invasive gonococcal infection.

Genetic Characterization of the Nucleotide Excision Repair System of Neisseria gonorrhoeae

Nucleotide excision repair (NER) is universally used to recognize and remove many types of DNA damage. In eubacteria, the NER system typically consists of UvrA, UvrB, UvrC, the UvrD helicase, DNA polymerase I, and ligase. In addition, when DNA damage blocks transcription, transcription-repair coupling factor (TRCF), the product of the mfd gene, recruits the Uvr complex to repair the damage. Previous work using selected mutants and assays have indicated that pathogenic Neisseria spp. carry a functional NER system. In order to comprehensively examine the role of NER in Neisseria gonorrhoeae DNA recombination and repair processes, the predicted NER genes (uvrA, uvrB, uvrC, uvrD, and mfd) were each disrupted by a transposon insertion, and the uvrB and uvrD mutants were complemented with a copy of each gene in an ectopic locus. Each uvr mutant strain was highly sensitive to UV irradiation and also showed sensitivity to hydrogen peroxide killing, confirming that all of the NER genes in N. gonorrhoeae are functional. The effect of RecA expression on UV survival was minor in uvr mutants but much larger in the mfd mutant. All of the NER mutants demonstrated wild-type levels of pilin antigenic variation and DNA transformation. However, the uvrD mutant exhibited higher frequencies of PilC-mediated pilus phase variation and spontaneous mutation, a finding consistent with a role for UvrD in mismatch repair. We conclude that NER functions are conserved in N. gonorrhoeae and are important for the DNA repair capabilities of this strict human pathogen.

Mismatch Correction Modulates Mutation Frequency and Pilus Phase and Antigenic Variation in Neisseria gonorrhoeae

The mismatch correction (MMC) system repairs DNA mismatches and single nucleotide insertions or deletions postreplication. To test the functions of MMC in the obligate human pathogen Neisseria gonorrhoeae, homologues of the core MMC genes mutS and mutL were inactivated in strain FA1090. No mutH homologue was found in the FA1090 genome, suggesting that gonococcal MMC is not methyl directed. MMC mutants were compared to a mutant in uvrD, the helicase that functions with MMC in Escherichia coli. Inactivation of MMC or uvrD increased spontaneous resistance to rifampin and nalidixic acid, and MMC/uvrD double mutants exhibited higher mutation frequencies than any single mutant. Loss of MMC marginally enhanced the transformation efficiency of DNA carrying a single nucleotide mismatch but not that of DNA with a 1-kb insertion. Unlike the exquisite UV sensitivity of the uvrD mutant, inactivating MMC did not affect survival after UV irradiation. MMC and uvrD mutants exhibited increased PilC-dependent pilus phase variation. mutS-deficient gonococci underwent an increased frequency of pilin antigenic variation, whereas uvrD had no effect. Recombination tracts in the mutS pilin variants were longer than in parental gonococci but utilized the same donor pilS loci. These results show that gonococcal MMC repairs mismatches and small insertion/deletions in DNA and also affects the recombination events underlying pilin antigenic variation. The differential effects of MMC and uvrD in gonococci unexpectedly reveal that MMC can function independently of uvrD in this human-specific pathogen.

LeuX tRNA‐dependent and ‐independent mechanisms of Escherichia coli pathogenesis in acute cystitis

Uropathogenic Escherichia coli (UPEC) contain multiple horizontally acquired pathogenicity-associated islands (PAI) implicated in the pathogenesis of urinary tract infection. In a murine model of cystitis, type 1 pili-mediated bladder epithelial invasion and intracellular proliferation are key events associated with UPEC virulence. In this study, we examined the mechanisms by which a conserved PAI contributes to UPEC pathogenesis in acute cystitis. In the human UPEC strain UTI89, spontaneous excision of PAI II(UTI89) disrupts the adjacent leuX tRNA locus. Loss of wild-type leuX-encoded tRNA(5)(Leu) significantly delayed, but did not eliminate, FimB recombinase-mediated phase variation of type 1 pili. FimX, an additional FimB-like, leuX-independent recombinase, was also found to mediate type 1 pili phase variation. However, whereas FimX activity is relatively slow in vitro, it is rapid in vivo as a non-piliated strain lacking the other fim recombinases rapidly expressed type 1 pili upon experimental infection. Finally, we found that disruption of leuX, but not loss of PAI II(UTI89) genes, reduced bladder epithelial invasion and intracellular proliferation, independent of type 1 piliation. These findings indicate that the predominant mechanism for preservation of PAI II(UTI89) during the establishment of acute cystitis is maintenance of wild-type leuX, and not PAI II(UTI89) gene content.

Solution Structure of Escherichia coli PapI, a Key Regulator of the Pap Pili Phase Variation

Pyelonephritis-associated pili (pap) allow uropathogenic Escherichia coli to bind to epithelial cells and play an important role in urinary tract infection. Expression of pap is controlled by a phase-variation mechanism, based on the two distinct heritable states that are the result of adenine N6-methylation in either of the two GATC sequences in its regulatory region. The methylation status of these two sequences is sensed by the action of two proteins, Lrp and PapI, and they play a central role in determining pap gene expression in both phase-ON and phase-OFF cells. We used modern NMR techniques to determine the solution structure and backbone dynamics of PapI. We found its overall fold resembles closely that of the winged helix-turn-helix family of DNA-binding proteins. We determined that PapI possesses its own DNA-binding activity, albeit non-sequence-specific, independent of Lrp. PapI appears to bind to DNA with a K d in the 10 μM range. Possible mechanisms by which PapI might participate in the regulation of the pap operon are discussed in light of these new findings.

Stochastic gene expression in fluctuating environments.

Stochastic mechanisms can cause a group of isogenic bacteria, each subject to identical environmental conditions, to nevertheless exhibit diverse patterns of gene expression. The resulting phenotypic subpopulations will typically have distinct growth rates. This behavior has been observed in several contexts, including sugar metabolism and pili phase variation. Under fixed environmental conditions, the net growth rate of the population is maximized when all cells are of the fastest growing phenotype, so it is unclear what fitness advantage is conferred by population heterogeneity. However, unlike ideal laboratory conditions, natural environments tend to fluctuate, either periodically or randomly. Here we use a stochastic population model to show that, during growth in such fluctuating environments, a dynamically heterogenous bacterial population can sometimes achieve a higher net growth rate than a homogenous one. By using stochastic mechanisms to sample several distinct phenotypes, the bacteria are able to anticipate and take advantage of sudden changes in their environment. However, this heterogeneity is beneficial only if the bacterial response rate is sufficiently low. Our results could be useful in the design of artificial evolution experiments and in the optimization of fermentation processes.

The recX gene potentiates homologous recombination in Neisseria gonorrhoeae.

In the pathogen Neisseria gonorrhoeae (Gc), the RecA protein is necessary for DNA repair, DNA transformation and pilus antigenic variation. Many bacteria contain a gene, recX, which has been suggested to downregulate recA through an unknown mechanism. To investigate the possible role of recX in Gc, we cloned and insertionally inactivated the recX gene. The recX loss-of-function mutant showed decreases in pilus phase variation, DNA transformation and DNA repair ability compared with wild type. We were able to complement all these deficiencies by supplying a functional copy of recX elsewhere in the chromosome. The recX mutant still showed increases in pilus phase variation under conditions of iron starvation, and the recX mutant showed levels of RecA protein equivalent to wild type. Although the precise role of recX in recombination remains unclear, RecX aids all RecA-related processes in Gc, and this is the first demonstration of a role for recX in homologous recombination in any organism.

Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli

We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E. coli. Two Dam methyltransferase sites, GATC1028 and GATC1130, were shown previously to be differentially methylated in phase ON versus phase OFF cells. In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. Our results show that methylation of GATC1028 blocks formation of the ON state by inhibiting the binding of Lrp and PapI regulatory proteins to this site. Conversely, methylation of GATC1130 is required for the ON state. Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter. A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

Analysis of the fimB promoter region involved in type 1 pilus phase variation in Escherichia coli.

Many strains of Escherichia coli express type 1 pili which undergo phase variation during growth in broth, but become uniformly nonpiliated when passaged on agar media. In a previous analysis of these agar phase locked strains, we demonstrated that fimB and fimE, which mediate the site-specific DNA rearrangement involved in phase variation, are differentially transcribed under the two growth conditions. In this study the fimB promoter region was sequenced and characterized in agar phase locked strain J96. Primer extension analysis of the fimB gene identified three putative transcription initiation sites. Transcription starting from two of the sites (P1 and P2) would produce an mRNA that approximates the transcript size of fimB previously detected by Northern blot hybridizations. Gel mobility shift studies revealed the presence of promoter binding activity in broth-grown cell extracts, but not in extracts from agar-grown cells, that reacted specifically with DNA fragments upstream of the P1 and P2 promoter regions. This putative binding protein may be involved in the regulation of fimB, perhaps by acting as a transcriptional activator.

Methylation patterns in pap regulatory DNA control pyelonephritis-associated pili phase variation in E. coli

We have examined the roles of pap DNA methylation patterns in the regulation of the switch between phase ON and OFF pyelonephritis-associated pili (Pap) expression states in E. coli. Two Dam methyltransferase sites, GATC 1028 and GATC 1130, were shown previously to be differentially methylated in phase ON versus phase OFF cells. In work presented here, these sites were mutated so that they could not be methylated, and the effects of these mutations on Pap phase variation were examined. Our results show that methylation of GATC 1028 blocks formation of the ON state by inhibiting the binding of Lrp and Papl regulatory proteins to this site. Conversely, methylation of GATC 1130 is required for the ON state. Evidence indicates that this occurs by the inhibition of binding of Lrp to sites overlapping the pilin promoter. A model describing how the transition between the phase ON and OFF methylation states might occur is presented.

Potential role of phase variation of type 1 pili in urinary tract infection and bacterial prostatitis

Adherence of Escherichia coli to uroepithelial cells is an important step in the pathogenesis of urinary tract infection. The process is frequently mediated by pili which undergo phase variation in response to changes in environmental growth conditions in vitro. It is possible that phase variation of pili and other bacterial virulence factors may play a role in the pathogenesis of bacterial prostatitis. The obvious differences in the environmental conditions of urethra, bladder, and prostate could favor strains that have the ability to adapt. For example, adhesion to the urethral and bladder mucosa could be facilitated by pili and their expression could be advantageous in the early phase of prostatitis. However, after the prostate ducts are colonized, pili may be detrimental since they can render E. coli more susceptible to phagocytosis. Further studies on phase variation of virulence factors of bacteria that cause prostatitis appear warranted.

Phase Variation of Pili of Corynebacterium pilosum

Whether or not (?) phase variation occurs in the pili of Corynebacterium pilosum was biologically examined using the colony enzyme-linked immunosorbent assay (ELISA) blot test with anti-pili immune serum. From the densely piliated clone (35P+) of C. pilosum 35, non-piliated variants were isolated at a frequency of 2.45 x 10(-3). From one of the non-piliated variants (designated as P(-11)/35P+), a piliated variant was isolated at a frequency of 4.68 x 10(-4), one log less frequently than the non-piliated variant. From this piliated variant as designated (P+4/P(-11)/35P+), a non-piliated variant was isolated at a frequency of 3.86 x 10(-3). C. pilosum was thus alternated between piliated and non-piliated at a fairly high frequency, suggesting that the pili may undergo phase variation. This is the first finding of phase variation of the pili of Gram-positive bacteria.

Gene transfer in Neisseria gonorrhoeae

The ability of bacteria to exchange deoxyribonucleic acid (DNA) endows these organisms with greater genetic variability and increased capability to adapt to changing environments. Many bacteria have evolved transformation and conjugation systems to effect this exchange, whereas others achieve it through the action of bacteriophages. In Neisseria gonorrhoeae no transducing bacteriophages have been identified, but conjugation and transformation both occur (10). Gonococci are extremely autolytic and therefore release DNA to neighboring cells (19). Transformation with chromosomal markers has been demonstrated between strains in laboratory-grown mixed cultures (32); similar transformation probably occurs in nature. Quite recently, antigenic and phase variation of gonococcal pili has been shown to be due in large part to release of DNA from autolyzing cells, with subsequent transformation of other competent cells in the population by variant pil sequences (32a; C. Haas and T. Meyer, personal communication). Gonococcal transformation has been studied extensively and has proven useful in the construction of isogenic strains for mapping antibiotic resistance genes and biosynthetic auxotrophs, and in studies of molecules implicated in the virulence of this organism (10, 34). Conjugation in N. gonorrhoeae is important because it results in mobilization of antibiotic resistance plasmids, but chromosomal genes cannot be transferred by conjugation (3, 10, 27, 31, 37, 44J. Thus, in the laboratory and in nature, transformation is the primary means of transfer of chromosomal genes. In this brief review, mechanisms of gene exchange will be emphasized, and some speculative comments on possible future developments are included.

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.

Gene conversion involving the pilin structural gene correlates with pilus + ⇄ pilus − changes in Neisseria gonorrhoeae

Gonococci (Gc) exhibit pilus + → pilus − “phase transitoins” 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 + ⇄ 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.

Intragenic recombination leads to pilus antigenic variation in Neisseria gonorrhoeae.

The pilus of the bacterium Neisseria gonorrhoeae is a fimbriate surface structure which promotes attachment of the bacterium to host epithelial cells. Gonococcal pilus phase variation is characterized by a rapid on/off switch in which piliated (P+) cells throw off non-piliated (P-) variants and vice versa. Two regions of the gonococcal chromosome (pilE1 and pilE2) act as pilin expression loci, reminiscent of the MAT locus in the yeast Saccharomyces cerevisiae, while several other chromosomal regions contain silent (non-expressing) pilin sequences. Biochemical and antigenic diversity is seen in pili from a wide variety of clinical isolates. Pilins (pilus subunits) are composed of conserved N-terminal and variable C-terminal regions; the conserved region of gonococcal pilin is also found in pilins produced by widely disparate bacteria. We show here that the gonococcal pilin undergoes antigenic variation in vitro and in vivo. The protein consists of constant, semi-variable and hypervariable regions. This antigenic variation probably involves gene conversion of mini-cassettes of pilin information.