Repurposing Carbamazepine To Treat Gonococcal Infection in Women: Oral Delivery for Control of Epilepsy Generates Therapeutically Effective Levels in Vaginal Secretions.

Neisseria gonorrhoeae has developed resistance to all previous antibiotics used for treatment. This highlights a crucial need for novel antimicrobials to treat gonococcal infections. We previously showed that carbamazepine (Cz), one of the most commonly prescribed antiepileptic drugs, can block the interaction between gonococcal pili and the I-domain region of human complement receptor 3 (CR3)-an interaction that is vital for infection of the female cervix. We also show that Cz can completely clear an established N. gonorrhoeae infection of primary human cervical cells. In this study, we quantified Cz in serum, saliva, and vaginal fluid collected from 16 women who were, or were not, regularly taking Cz. We detected Cz in lower reproductive tract mucosal secretions in the test group (women taking Cz) at potentially therapeutic levels using a competitive ELISA. Furthermore, we found that Cz concentrations present in vaginal fluid from women taking this drug were sufficient to result in a greater than 99% reduction (within 24 h) in the number of viable gonococci recovered from ex vivo, human, primary cervical cell infections. These data provide strong support for the further development of Cz as a novel, host-targeted therapy to treat gonococcal cervicitis.

Serum Neu5Gc biomarkers are elevated in primary cutaneous melanoma.

Cutaneous melanoma is one of the most aggressive and deadly types of skin cancer and rates of disease are continuing to increase worldwide. Currently, no serum biomarkers exist for the early detection of cutaneous melanoma. Normal human cells cannot make the sialic acid sugar, Neu5Gc, yet human tumor cells express Neu5Gc and Neu5Gc-containing glycoconjugates have been proposed as tumor biomarkers. We engineered a Neu5Gc-specific lectin based on the pentameric B-subunit of the Shiga toxigenic Escherichia coli subtilase cytotoxin, termed SubB2M. We have detected elevated Neu5Gc-containing biomarkers in the sera of ovarian and breast cancer patients in a highly sensitive surface plasmon resonance (SPR)-based assay using our SubB2M lectin. Here, we used the SubB2M-SPR assay to investigate Neu5Gc-containing glycoconjugates in the serum of cutaneous melanoma patients. We found elevated total serum Neu5Gc levels in primary (n = 24) and metastatic (n = 38) patients compared to cancer-free controls (n = 34). Serum Neu5Gc levels detected with SubB2M can distinguish cutaneous melanoma patients from cancer-free controls with high sensitivity and specificity as determined by ROC curve analysis. These data indicate that serum Neu5Gc-containing glycoconjugates are a novel class of biomarkers for cutaneous melanoma, particularly for primary melanoma, and have the potential to contribute to the early diagnosis of this disease.

N-glycolylneuraminic acid serum biomarker levels are elevated in breast cancer patients at all stages of disease.

BACKGROUND: Normal human tissues do not express glycans terminating with the sialic acid N-glycolylneuraminic acid (Neu5Gc), yet Neu5Gc-containing glycans have been consistently found in human tumor tissues, cells and secretions and have been proposed as a cancer biomarker. We engineered a Neu5Gc-specific lectin called SubB2M, and previously reported elevated Neu5Gc biomarkers in serum from ovarian cancer patients using a Surface Plasmon Resonance (SPR)-based assay. Here we report an optimized SubB2M SPR-based assay and use this new assay to analyse sera from breast cancer patients for Neu5Gc levels. METHODS: To enhance specificity of our SPR-based assay, we included a non-sialic acid binding version of SubB, SubBA12, to control for any non-specific binding to SubB2M, which improved discrimination of cancer-free controls from early-stage ovarian cancer. We analysed 96 serum samples from breast cancer patients at all stages of disease compared to 22 cancer-free controls using our optimized SubB2M-A12-SPR assay. We also analysed a collection of serum samples collected at 6 monthly intervals from breast cancer patients at high risk for disease recurrence or spread. RESULTS: Analysis of sera from breast cancer cases revealed significantly elevated levels of Neu5Gc biomarkers at all stages of breast cancer. We show that Neu5Gc serum biomarker levels can discriminate breast cancer patients from cancer-free individuals with 98.96% sensitivity and 100% specificity. Analysis of serum collected prospectively, post-diagnosis, from breast cancer patients at high risk for disease recurrence showed a trend for a decrease in Neu5Gc levels immediately following treatment for those in remission. CONCLUSIONS: Neu5Gc serum biomarkers are a promising new tool for early detection and disease monitoring for breast cancer that may complement current imaging- and biopsy-based approaches.

Specificity and utility of SubB2M, a new N-glycolylneuraminic acid lectin.

The B subunit of the subtilase cytotoxin (SubB) recognises N-glycolylneuraminic acid (Neu5Gc) containing glycans, the most prominent form of aberrant glycosylation in human cancers. We have previously engineered SubB by construction of a SubBΔS106/ΔT107 mutant (SubB2M) for greater specificity and enhanced recognition of Neu5Gc containing glycans. In this study, we further explore the utility of SubB2M as a Neu5Gc lectin by showing its improved specificity and recognition for Neu5Gc containing glycans over the wild-type SubB protein and an anti-Neu5Gc IgY antibody in a N-acetylneuraminic acid (Neu5Ac)/Neu5Gc glycan array and by surface plasmon resonance. Far-western blot analysis showed that SubB2M preferentially binds to bovine serum glycoproteins over human serum glycoproteins. SubB2M was also able to detect Neu5Gc containing bovine glycoproteins spiked into normal human serum with greater sensitivity than the wild-type SubB and the anti-Neu5Gc IgY antibody. These results suggest that SubB2M will be a useful tool for the testing of serum and other bodily fluids for cancer diagnosis and prognosis.

The cholesterol-dependent cytolysins pneumolysin and streptolysin O require binding to red blood cell glycans for hemolytic activity.

The cholesterol-dependent cytolysin (CDC) pneumolysin (Ply) is a key virulence factor of Streptococcus pneumoniae. Membrane cholesterol is required for the cytolytic activity of this toxin, but it is not clear whether cholesterol is the only cellular receptor. Analysis of Ply binding to a glycan microarray revealed that Ply has lectin activity and binds glycans, including the Lewis histo-blood group antigens. Surface plasmon resonance analysis showed that Ply has the highest affinity for the sialyl LewisX (sLeX) structure, with a K(d) of 1.88 × 10(-5) M. Ply hemolytic activity against human RBCs showed dose-dependent inhibition by sLeX. Flow cytometric analysis and Western blots showed that blocking binding of Ply to the sLeX glycolipid on RBCs prevents deposition of the toxin in the membrane. The lectin domain responsible for sLeX binding is in domain 4 of Ply, which contains candidate carbohydrate-binding sites. Mutagenesis of these predicted carbohydrate-binding residues of Ply resulted in a decrease in hemolytic activity and a reduced affinity for sLeX. This study reveals that this archetypal CDC requires interaction with the sLeX glycolipid cellular receptor as an essential step before membrane insertion. A similar analysis conducted on streptolysin O from Streptococcus pyogenes revealed that this CDC also has glycan-binding properties and that hemolytic activity against RBCs can be blocked with the glycan lacto-N-neotetraose by inhibiting binding to the cell surface. Together, these data support the emerging paradigm shift that pore-forming toxins, including CDCs, have cellular receptors other than cholesterol that define target cell tropism.

Characterisation of a multi-ligand binding chemoreceptor CcmL (Tlp3) of Campylobacter jejuni.

Campylobacter jejuni is the leading cause of human gastroenteritis worldwide with over 500 million cases annually. Chemotaxis and motility have been identified as important virulence factors associated with C. jejuni colonisation. Group A transducer-like proteins (Tlps) are responsible for sensing the external environment for bacterial movement to or away from a chemical gradient or stimulus. In this study, we have demonstrated Cj1564 (Tlp3) to be a multi-ligand binding chemoreceptor and report direct evidence supporting the involvement of Cj1564 (Tlp3) in the chemotaxis signalling pathway via small molecule arrays, surface plasmon and nuclear magnetic resonance (SPR and NMR) as well as chemotaxis assays of wild type and isogenic mutant strains. A modified nutrient depleted chemotaxis assay was further used to determine positive or negative chemotaxis with specific ligands. Here we demonstrate the ability of Cj1564 to interact with the chemoattractants isoleucine, purine, malic acid and fumaric acid and chemorepellents lysine, glucosamine, succinic acid, arginine and thiamine. An isogenic mutant of cj1564 was shown to have altered phenotypic characteristics of C. jejuni, including loss of curvature in bacterial cell shape, reduced chemotactic motility and an increase in both autoagglutination and biofilm formation. We demonstrate Cj1564 to have a role in invasion as in in vitro assays the tlp3 isogenic mutant has a reduced ability to adhere and invade a cultured epithelial cell line; interestingly however, colonisation ability of avian caeca appears to be unaltered. Additionally, protein-protein interaction studies revealed signal transduction initiation through the scaffolding proteins CheV and CheW in the chemotaxis sensory pathway. This is the first report characterising Cj1564 as a multi-ligand receptor for C. jejuni, we therefore, propose to name this receptor CcmL, Campylobacter chemoreceptor for multiple ligands. In conclusion, this study identifies a novel multifunctional role for the C. jejuni CcmL chemoreceptor and illustrates its involvement in the chemotaxis pathway and subsequent survival of this organism in the host.

Protection against Shiga-Toxigenic Escherichia coli by Non-Genetically Modified Organism Receptor Mimic Bacterial Ghosts.

Shiga-toxigenic Escherichia coli (STEC) causes severe gastrointestinal infections in humans that may lead to life-threatening systemic sequelae, such as the hemolytic uremic syndrome (HUS). Rapid diagnosis of STEC infection early in the course of disease opens a window of opportunity for therapeutic intervention, for example, by administration of agents that neutralize Shiga toxin (Stx) in the gut lumen. We previously developed a recombinant bacterium that expresses a mimic of the Stx receptor globotriaosyl ceramide (Gb3) on its surface through modification of the lipopolysaccharide (A. W. Paton, R. Morona, and J. C. Paton, Nat Med 6:265-270, 2000, http://dx.doi.org/10.1038/73111). This construct was highly efficacious in vivo, protecting mice from otherwise fatal STEC disease, but the fact that it is a genetically modified organism (GMO) has been a barrier to clinical development. In the present study, we have overcome this issue by development of Gb3 receptor mimic bacterial ghosts (BGs) that are not classified as GMOs. Gb3-BGs neutralized Stx1 and Stx2 in vitro with high efficiency, whereas alternative Gb3-expressing non-GMO subbacterial particles (minicells and outer membrane blebs) were ineffective. Gb3-BGs were highly efficacious in a murine model of STEC disease. All mice (10/10) treated with Gb3-BGs survived challenge with a highly virulent O113:H21 STEC strain and showed no pathological signs of renal injury. In contrast, 6/10 mice treated with control BGs succumbed to STEC challenge, and survivors exhibited significant weight loss, neutrophilia, and histopathological evidence of renal damage. Thus, Gb3-BGs offer a non-GMO approach to treatment of STEC infection in humans, particularly in an outbreak setting.

The essential malaria protein PfCyRPA targets glycans to invade erythrocytes.

Plasmodium falciparum is a human-adapted apicomplexan parasite that causes the most dangerous form of malaria. P. falciparum cysteine-rich protective antigen (PfCyRPA) is an invasion complex protein essential for erythrocyte invasion. The precise role of PfCyRPA in this process has not been resolved. Here, we show that PfCyRPA is a lectin targeting glycans terminating with α2-6-linked N-acetylneuraminic acid (Neu5Ac). PfCyRPA has a >50-fold binding preference for human, α2-6-linked Neu5Ac over non-human, α2-6-linked N-glycolylneuraminic acid. PfCyRPA lectin sites were predicted by molecular modeling and validated by mutagenesis studies. Transgenic parasite lines expressing endogenous PfCyRPA with single amino acid exchange mutants indicated that the lectin activity of PfCyRPA has an important role in parasite invasion. Blocking PfCyRPA lectin activity with small molecules or with lectin-site-specific monoclonal antibodies can inhibit blood-stage parasite multiplication. Therefore, targeting PfCyRPA lectin activity with drugs, immunotherapy, or a vaccine-primed immune response is a promising strategy to prevent and treat malaria.

Recombinant truncated AniA of pathogenic Neisseria elicits a non-native immune response and functional blocking antibodies.

AniA of the pathogenic Neisseria is glycosylated in its C-terminal repeat region by the pilin glycosylation (pgl) pathway. AniA appears to be unique among bacterial nitrite reductases as it contains an N-terminal extension that includes a lipid modification site as well as a C-terminal extension that is glycosylated. Immunising with various glycoforms of the AniA protein demonstrated a strong humoral immune response to the basal monosaccharide. In addition, when animals were immunised with a truncated form of AniA, completely lacking the glycosylated C-terminal region, the antibody response was directed against AniA regardless of the glycosylation state of the protein. Immuno-SEM confirmed that AniA is expressed on the cell surface in Neisseria gonorrhoeae. Antisera generated against a truncated, non-glycosylated, recombinant form of the AniA protein are capable of blocking nitrite reductase function in a whole cell assay. We propose that recombinant modified AniA has potential as a vaccine antigen for N. gonorrhoeae.

N-glycolylneuraminic acid as a carbohydrate cancer biomarker.

One of the forms of aberrant glycosylation in human tumors is the expression of N-glycolylneuraminic acid (Neu5Gc). The only known enzyme to biosynthesize Neu5Gc in mammals, cytidine-5'-monophosphate-N-acetylneuraminic acid (CMAH), appears to be genetically inactivated in humans. Regardless, low levels of Neu5Gc have been detected in healthy humans. Therefore, it is proposed that the presence of Neu5Gc in humans is from dietary acquisition, such as red meat. Notably, detection of elevated Neu5Gc levels has been repeatedly found in cancer tissues, cells and serum samples, thereby Neu5Gc-containing antigens may be exploited as a class of cancer biomarkers. Here we review the findings to date on using Neu5Gc-containing tumor glycoconjugates as a class of cancer biomarkers for cancer detection, surveillance, prognosis and therapeutic targets. We review the evidence that supports an emerging hypothesis of de novo Neu5Gc biosynthesis in human cancer cells as a source of Neu5Gc in human tumors, generated under certain metabolic conditions.

Variation of chemosensory receptor content of Campylobacter jejuni strains and modulation of receptor gene expression under different in vivo and in vitro growth conditions.

BACKGROUND: Chemotaxis is crucial for the colonisation/infection of hosts with Campylobacter jejuni. Central to chemotaxis are the group A chemotaxis genes that are responsible for sensing the external environment. The distribution of group A chemoreceptor genes, as found in the C. jejuni sequenced strains, tlp1-4, 7, 10 and 11 were determined in 33 clinical human and avian isolates. RESULTS: Group A tlp gene content varied among the strains with genes encoding tlp1 (aspartate receptor, ccaA) and tlp7 present in all strains tested, where as tlp11 was present in only one of our international collection clinical isolates, C. jejuni 520, but was more prevalent (9/13) in the freshly isolated clinical stains from patients who required hospitalisation due to C. jejuni infection (GCH1-17). Relative expression levels of the group A tlp genes were also determined in C. jejuni reference strains NCTC 11168-GS, 11168-O and 81116 using cells grown in vitro at 37°C, 42°C and maintained at room temperature and with cells isolated directly from murine and avian hosts by immune magnetic separation without subsequent culture. Gene expression of tlp genes was varied based on strain, growth conditions and in vivo isolation source. Tlp1, although the most conserved, showed the lowest and most varied mRNA expression and protein production under laboratory conditions. Tlp7 was highly expressed at most conditions tested, and gene expression was not influenced by the tlp7 gene encoding a full length protein or one expressed as separate periplasmic and cytoplasmic domains. CONCLUSION: We have shown that chemosensory receptor set variation exists among C. jejuni strains, but is not dependent on the isolation source.

Identification and characterization of the aspartate chemosensory receptor of Campylobacter jejuni.

Campylobacter jejuni is a highly motile bacterium that responds via chemotaxis to environmental stimuli to migrate towards favourable conditions. Previous in silico analysis of the C. jejuni strain NCTC11168 genome sequence identified 10 open reading frames, tlp1-10, that encode putative chemosensory receptors. We describe the characterization of the role and specificity of the Tlp1 chemoreceptor (Cj1506c). In vitro and in vivo models were used to determine if Tlp1 had a role in host colonization. The tlp1(-) isogenic mutant was more adherent in cell culture, however, showed reduced colonization ability in chickens. Specific interactions between the purified sensory domain of Tlp1 and l-aspartate were identified using an amino acid array and saturation transfer difference nuclear magnetic resonance spectroscopy. Chemotaxis assays showed differences between migration of wild-type C. jejuni cells and that of a tlp1(-) isogenic mutant, specifically towards aspartate. Furthermore, using yeast two-hybrid and three-hybrid systems for analysis of protein-protein interactions, the cytoplasmic signalling domain of Tlp1 was found to preferentially interact with CheV, rather than the CheW homologue of the chemotaxis signalling pathway; this interaction was confirmed using immune precipitation assays. This is the first identification of an aspartate receptor in bacteria other than Escherichia coli and Salmonella enterica serovar Typhimurium.

The Campylobacter jejuni chemoreceptor Tlp10 has a bimodal ligand-binding domain and specificity for multiple classes of chemoeffectors.

Campylobacter jejuni is a bacterial pathogen that is a common cause of enteritis in humans. We identified a previously uncharacterized type of sensory domain in the periplasmic region of the C. jejuni chemoreceptor Tlp10, termed the DAHL domain, that is predicted to have a bimodular helical architecture. Through two independent ligand-binding sites in this domain, Tlp10 responded to molecular aspartate, isoleucine, fumarate, malate, fucose, and mannose as attractants and to arginine, galactose, and thiamine as repellents. Tlp10 also recognized glycan ligands when present as terminal and intermediate residues of complex structures, such as the fucosylated human ganglioside GM1 and Lewisa antigen. A tlp10 mutant strain lacking the ligand-binding sites was attenuated in its ability to colonize avian caeca and to adhere to cultured human intestinal cells, indicating the potential involvement of the DAHL domain in host colonization and disease. The Tlp10 intracellular signaling domain interacted with the scaffolding proteins CheV and CheW, which couple chemoreceptors to intracellular signaling machinery, and with the signaling domains of other chemoreceptors, suggesting a key role for Tlp10 in signal transduction and incorporation into sensory arrays. We identified the DAHL domain in other bacterial signal transduction proteins, including the essential virulence induction protein VirA from the plant pathogen Agrobacterium tumefaciens Together, these results suggest a potential link between Tlp10 and C. jejuni virulence.

Binding specificity of ostreolysin A6 towards Sf9 insect cell lipids.

Oyster mushrooms (Pleurotus spp.) have recently been shown to produce insecticidal bi-component protein complexes based on the aegerolysin proteins. A role for these proteins is thus indicated for defence and protection of the mushroom, and we propose their use as new environmentally friendly bioinsecticides. These aegerolysin-based protein complexes permeabilise artificial lipid vesicles through aegerolysin binding to an insect-specific sphingolipid, ceramide phosphoethanolamine (CPE), and they are cytotoxic for the Spodoptera frugiferda (Sf9) insect cell line. Tandem mass spectrometry analysis of the Sf9 lipidome uncovered lipids not previously reported in the literature, including in particular C14 sphingosine-based CPE molecular species, which comprised ~4 mol% of the whole lipidome. Further analysis of the lipid binding specificity of an aegerolysin from P. ostreatus, ostreolysin A6 (OlyA6), to lipid vesicles composed of commercial lipids, to lipid vesicles composed of the total lipid extract from Sf9 cells, and to HPLC-separated Sf9 cell lipid fractions containing ceramides, confirmed CPE as the main OlyA6 receptor, but also highlighted the importance of membrane cholesterol for formation of strong and stable interactions of OlyA6 with artificial and natural lipid membranes. Binding assays performed with glycan arrays and surface plasmon resonance, which included invertebrate-specific glycans, excluded these saccharides as potential additional OlyA6 receptors.

All major cholesterol-dependent cytolysins use glycans as cellular receptors.

Cholesterol-dependent cytolysins (CDCs) form pores in cholesterol-rich membranes, but cholesterol alone is insufficient to explain their cell and host tropism. Here, we show that all eight major CDCs have high-affinity lectin activity that identifies glycans as candidate cellular receptors. Streptolysin O, vaginolysin, and perfringolysin O bind multiple glycans, while pneumolysin, lectinolysin, and listeriolysin O recognize a single glycan class. Addition of exogenous carbohydrate receptors for each CDC inhibits toxin activity. We present a structure for suilysin domain 4 in complex with two distinct glycan receptors, P1 antigen and αGal/Galili. We report a wide range of binding affinities for cholesterol and for the cholesterol analog pregnenolone sulfate and show that CDCs bind glycans and cholesterol independently. Intermedilysin binds to the sialyl-TF O-glycan on its erythrocyte receptor, CD59. Removing sialyl-TF from CD59 reduces intermedilysin binding. Glycan-lectin interactions underpin the cellular tropism of CDCs and provide molecular targets to block their cytotoxic activity.

Refinement of immunizing antigens to produce functional blocking antibodies against the AniA nitrite reductase of Neisseria gonorrhoeae.

The emergence of multi-drug resistant Neisseria gonorrhoeae has generated an urgent need for novel therapies or a vaccine to prevent gonococcal disease. In this study we investigate the potential of targeting the surface exposed nitrite reductase, AniA, to block activity by producing functional blocking antibodies. AniA activity is essential for anaerobic growth and biofilm formation of N. gonorrhoeae and functional blocking antibodies may prevent colonisation and disease. Seven peptides covering regions adjacent to the active site were designed based on the AniA structure. Six of the seven peptide conjugates generated immune responses. Peptide 7, GALGQLKVEGAEN, was able to elicit antibodies capable of blocking AniA activity. Antiserum raised against the peptide 7 conjugate detected AniA in 20 N. gonorrhoeae clinical isolates. Recombinant AniA protein antigens were also assessed in this study and generated high-titre, functional blocking antibody responses. Peptide 7 conjugates or truncated recombinant AniA antigens have potential for inclusion in a vaccine against N. gonorrhoeae.

Structure aided design of a Neu5Gc specific lectin.

Subtilase cytotoxin (SubAB) of Escherichia coli is an AB5 class bacterial toxin. The pentameric B subunit (SubB) binds the cellular carbohydrate receptor, α2-3-linked N-glycolylneuraminic acid (Neu5Gc). Neu5Gc is not expressed on normal human cells, but is expressed by cancer cells. Elevated Neu5Gc has been observed in breast, ovarian, prostate, colon and lung cancer. The presence of Neu5Gc is prognostically important, and correlates with invasiveness, metastasis and tumour grade. Neu5Gc binding by SubB suggests that it may have utility as a diagnostic tool for the detection Neu5Gc tumor antigens. Native SubB has 20-fold less binding to N-acetlylneuraminic acid (Neu5Ac); over 30-fold less if the Neu5Gc linkage was changed from α2-3 to α2-6. Using molecular modeling approaches, site directed mutations were made to reduce the α2-3 [Formula: see text] α2-6-linkage preference, while maintaining or enhancing the selectivity of SubB for Neu5Gc over Neu5Ac. Surface plasmon resonance and glycan array analysis showed that the SubBΔS106/ΔT107 mutant displayed improved specificity towards Neu5Gc and bound to α2-6-linked Neu5Gc. SubBΔS106/ΔT107 could discriminate NeuGc- over Neu5Ac-glycoconjugates in ELISA. These data suggest that improved SubB mutants offer a new tool for the testing of biological samples, particularly serum and other fluids from individuals with cancer or suspected of having cancer.

A direct-sensing galactose chemoreceptor recently evolved in invasive strains of Campylobacter jejuni.

A rare chemotaxis receptor, Tlp11, has been previously identified in invasive strains of Campylobacter jejuni, the most prevalent cause of bacterial gastroenteritis worldwide. Here we use glycan and small-molecule arrays, as well as surface plasmon resonance, to show that Tlp11 specifically interacts with galactose. Tlp11 is required for the chemotactic response of C. jejuni to galactose, as shown using wild type, allelic inactivation and addition mutants. The inactivated mutant displays reduced virulence in vivo, in a model of chicken colonization. The Tlp11 sensory domain represents the first known sugar-binding dCache_1 domain, which is the most abundant family of extracellular sensors in bacteria. The Tlp11 signalling domain interacts with the chemotaxis scaffolding proteins CheV and CheW, and comparative genomic analysis indicates a likely recent evolutionary origin for Tlp11. We propose to rename Tlp11 as CcrG, Campylobacter ChemoReceptor for Galactose.

A random six-phase switch regulates pneumococcal virulence via global epigenetic changes.

Streptococcus pneumoniae (the pneumococcus) is the world's foremost bacterial pathogen in both morbidity and mortality. Switching between phenotypic forms (or 'phases') that favour asymptomatic carriage or invasive disease was first reported in 1933. Here, we show that the underlying mechanism for such phase variation consists of genetic rearrangements in a Type I restriction-modification system (SpnD39III). The rearrangements generate six alternative specificities with distinct methylation patterns, as defined by single-molecule, real-time (SMRT) methylomics. The SpnD39III variants have distinct gene expression profiles. We demonstrate distinct virulence in experimental infection and in vivo selection for switching between SpnD39III variants. SpnD39III is ubiquitous in pneumococci, indicating an essential role in its biology. Future studies must recognize the potential for switching between these heretofore undetectable, differentiated pneumococcal subpopulations in vitro and in vivo. Similar systems exist in other bacterial genera, indicating the potential for broad exploitation of epigenetic gene regulation.