Pore characteristics of nontypeable Haemophilus influenzae outer membrane protein P5 in planar lipid bilayers

The structure of outer membrane protein P5 of NTHi, a homolog of Escherichia coli OmpA, was investigated by observing its pore characteristics in planar lipid bilayers. Recombinant NTHi P5 was overexpressed in E. coli and purified using ionic detergent, LDS-P5, or nonionic detergent, OG-P5. LDS-P5 and OG-P5 could not be distinguished by their migration on SDS-PAGE gels; however, when incorporated into planar bilayers of DPhPC between symmetric aqueous solutions of 1 M KCl at 22 degrees C, LDS-P5 formed narrow pores (58 +/- 6 pS) with low open probability, whereas OG-P5 formed large pores (1.1 +/- 0.1 nS) with high open probability (0.99). LDS-P5 narrow pores were gradually and irreversibly transformed into large pores, indistinguishable from those formed by OG-P5, at temperatures >or=40 degrees C; the process took 4-6 h at 40 degrees C or 35-45 min at 42 degrees C. Large pores were stable to changes in temperatures; however, large pores were rapidly converted to narrow pores when exposed to LDS at room temperatures, indicating acute sensitivity of this conformer to ionic detergent. These studies suggest that narrow pores are partially denatured forms and support the premise that the native conformation of NTHi P5 is that of a large monomeric pore.     

A high-conductance mode of a poly-3-hydroxybutyrate/calcium/polyphosphate channel isolated from competent Escherichia coli cells

Reconstitution into planar lipid bilayers of a poly-3-hydroxybutyrate/calcium/polyphosphate (PHB/Ca(2+)/polyP) complex from Escherichia coli membranes yields cationic-selective, 100 pS channels (Das, S., Lengweiler, U.D., Seebach, D. and Reusch, R.N. (1997) Proof for a non-proteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate. Proc. Natl. Acad. Sci. USA 94, 9075-9079). Here, we report that this complex can also form larger, weakly selective pores, with a maximal conductance ranging from 250pS to 1nS in different experiments (symmetric 150mM KCl). Single channels were inhibited by lanthanum (IC(50)=42+/-4microM, means+/-S.E.M.) with an unusually high Hill coefficient (8.4+/-1.2). Transition to low-conductance states (<250pS) was favored by increased membrane polarization (/V/ >or=50mV). High conductance states (>250pS) may reflect conformations important for genetic transformability, or "competence", of the bacterial cells, which requires the presence of the PHB/Ca(2+)/polyP complex in the membrane.     

lgtC expression modulates resistance to C4b deposition on an invasive nontypeable Haemophilus influenzae

We have previously shown that C3 binding to serum-resistant nontypeable Haemophilus influenzae (NTHi) strain R2866 is slower than C3 binding to a serum-sensitive strain. Ab-dependent classical pathway activation is required for complement-dependent killing of NTHi. To further characterize the mechanism(s) of serum resistance of R2866, we compared binding of complement component C4b to R2866 with a serum-sensitive variant, R3392. We show that C4b binding to R2866 relative to R3392 was delayed, suggesting regulation of the classical pathway of complement. Increased C4b deposition on R3392 was independent of the amount and subclass of Ab binding, suggesting that an impediment to C4b binding existed on R2866. Immunoblotting and mass spectrometry indicated that lipooligosaccharide and outer membrane proteins P2 and P5 were targets for C4b. P2 and P5 sequences and expression levels were similar in both strains. Insertional inactivation of the phase-variable lipooligosaccharide biosynthesis gene lgtC in R2866 augmented C4b deposition to levels seen with R3392 and rendered the bacteria sensitive to serum and whole blood. These results suggest a direct role of lgtC expression in the inhibition of C4b deposition and consequent serum resistance of R2866. Alteration of surface glycans of NTHi may be a critical event in determining the ability of a strain to evade host defenses and cause disseminated infection.     

Streptomyces lividans potassium channel contains poly-(R)-3-hydroxybutyrate and inorganic polyphosphate

The Streptomyces lividans KcsA potassium channel, a homotetramer of 17.6 kDa subunits, was found to contain two nonproteinaceous polymers, namely, poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP). PHB and polyP are ubiquitous cellular constituents with a demonstrated capacity for cation selection and transport. PHB was detected in both tetramer and monomer species of KcsA by reaction to anti-PHB IgG on Western blots, and estimated as 28 monomer units of PHB per KcsA tetramer by a chemical assay in which PHB is converted to its unique degradation product, crotonic acid. PolyP was detected in KcsA tetramers, but not in monomers, by metachromatic reaction to o-toluidine blue stain on SDS-PAGE gels. A band of free polyP was also visible, suggesting that polyP is released when tetramers dissociate. The exopolyphosphatase of Saccharomyces cerevisiae degraded the free polyP, but tetramer-associated polyP was not affected, indicating it was inaccessible to the enzyme. PolyP in KcsA was estimated as 15 monomer units per tetramer by an enzymatic assay in which polyphosphate kinase is used to transfer phosphates from polyP to [(14)C]ADP, yielding [(14)C]ATP. The experimentally determined isoelectric point of KcsA tetramer was 6.5-7.5, substantially more acidic than the theoretical pI of 10.3, and consistent with the inclusion of a polyanion. The results suggest that PHB is covalently bound to KcsA subunits while polyP is held within tetramers by ionic forces. It is posited that KcsA protein creates an environment in which PHB/polyP is selective for K(+). The basic amino acids attenuate the negative charge density of polyP, thereby transforming the cation binding preference from multivalent to monovalent, and discrimination between K(+) and Na(+) is accomplished by adjusting the ligand geometry in cation binding cavities formed by PHB and polyP.     

Intranasal Immunization with Nontypeable Haemophilus influenzae Outer Membrane Vesicles Induces Cross-Protective Immunity in Mice

Haemophilus influenzae is a Gram-negative human-restricted bacterium that can act as a commensal and a pathogen of the respiratory tract. Especially nontypeable H. influenzae (NTHi) is a major threat to public health and is responsible for several infectious diseases in humans, such as pneumonia, sinusitis, and otitis media. Additionally, NTHi strains are highly associated with exacerbations in patients suffering from chronic obstructive pulmonary disease. Currently, there is no licensed vaccine against NTHi commercially available. Thus, this study investigated the utilization of outer membrane vesicles (OMVs) as a potential vaccine candidate against NTHi infections. We analyzed the immunogenic and protective properties of OMVs derived from various NTHi strains by means of nasopharyngeal immunization and colonization studies with BALB/c mice. The results presented herein demonstrate that an intranasal immunization with NTHi OMVs results in a robust and complex humoral and mucosal immune response. Immunoprecipitation revealed the most important immunogenic proteins, such as the heme utilization protein, protective surface antigen D15, heme binding protein A, and the outer membrane proteins P1, P2, P5 and P6. The induced immune response conferred not only protection against colonization with a homologous NTHi strain, which served as an OMV donor for the immunization mixtures, but also against a heterologous NTHi strain, whose OMVs were not part of the immunization mixtures. These findings indicate that OMVs derived from NTHi strains have a high potential to act as a vaccine against NTHi infections.     

Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At < or = 25 degrees C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg(2+) cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 degrees C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.     

A method for the purification and refolding of a recombinant form of the nontypeable Haemophilus influenzae P5 outer membrane protein fused to polyhistidine

Nontypeable Haemophilus influenzae (NTHi) is an opportunistic pathogen, commonly associated with otitis media and exacerbations of chronic bronchitis. Studies concerning the pathogenesis of NTHi have proposed an important function for P5, an outer membrane protein believed to play a role in the initiation of infection by mediating adherence to respiratory mucin. P5 has also generated interest as a potential vaccine candidate. In a previous study, an NTHi library screen with antibodies raised against P5 purified from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the purified protein was contaminated with closely migrating proteins. Consequently, the aim of this study was to express P5 in a heterologous system to overcome potential contamination with NTHi proteins that may complicate analytical or vaccine studies. Recombinant P5, with an N terminal extension of 10 residues that included six histidines, was cloned and expressed in Escherichia coli. The rP5 was purified with the Talon metal affinity resin in a denatured form and then refolded by incorporation into mixed-detergent micelles of octylglucoside and SDS. Circular dichroism of the refolded rP5 demonstrated 55% beta-strand content, which is consistent with the beta-strand content of native P5 and the homologous E. coli protein OmpA.     

Identification of a novel Haemophilus influenzae protein important for adhesion to epithelial cells

Non-typable Haemophilus influenzae (NTHi) is an important human-specific respiratory pathogen colonizing the mucosa of the upper respiratory tract. The bacterium is a common cause of acute otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease (COPD). An immunoglobulin (Ig) D-lambda myeloma protein was found to detect a 16 kDa surface protein that we designated protein E (PE). The pe gene was cloned using an NTHi genomic DNA library, and a truncated PE-derived protein lacking the endogenous signal peptide (PE22-160) was synthesized and produced in large amounts in Escherichia coli. Interestingly, PE was expressed at the bacterial surface of NTHi as revealed by flow cytometry using the IgD-lambda myeloma protein or PE-specific polyclonal antibodies. A PE-deficient NTHi mutant was produced and lost 50% of its adhesive capacity as compared to the wild-type counterpart when analysed for adhesion to type II lung alveolar epithelial cells. In parallel, E. coli expressing full-length PE1-160 adhered significantly more efficiently to epithelial cells as compared to wild-type E. coli. Recombinant IgD that recognized the chemical dansyl-chloride did not interact with PE indicating that the IgD-lambda myeloma protein most likely was an antibody directed against the H. influenzae surface epitope. In conclusion, we have discovered a novel NTHi outer membrane protein with adhesive properties using an IgD-myeloma protein.     

Identification of a bifunctional lipopolysaccharide sialyltransferase in Haemophilus influenzae: incorporation of disialic acid

The lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) can be substituted at various positions by N-acetylneuraminic acid (Neu5Ac). LPS sialylation plays an important role in pathogenesis. The only LPS sialyltransferase characterized biochemically to date in H. influenzae is Lic3A, an alpha-2,3-sialyltransferase responsible for the addition of Neu5Ac to a lactose acceptor (Hood, D. W., Cox, A. D., Gilbert, M., Makepeace, K., Walsh, S., Deadman, M. E., Cody, A., Martin, A., M?nsson, M., Schweda, E. K., Brisson, J. R., Richards, J. C., Moxon, E. R., and Wakarchuk, W. W. (2001) Mol. Microbiol. 39, 341-350). Here we describe a second sialyltransferase, Lic3B, that is a close homologue of Lic3A and present in 60% of NTHi isolates tested. A recombinant form of Lic3B was expressed in Escherichia coli and purified by affinity chromatography. We used synthetic fluorescent acceptors with a terminal lactose or sialyllactose to show that Lic3B has both alpha-2,3- and alpha-2,8-sialyltransferase activities. Structural analysis of LPS from lic3B mutant strains of NTHi confirmed that only monosialylated species were detectable, whereas disialylated species were detected upon inactivation of lic3A. Furthermore, introduction of lic3B into a lic3B-deficient strain background resulted in a significant increase in sialylation in the recipient strain. Mass spectrometric analysis of LPS indicated that glycoforms containing two Neu5Ac residues were evident that were not present in the LPS of the parent strain. These findings characterize the activity of a second sialyltransferase in H. influenzae, responsible for the addition of di-sialic acid to the LPS. Modification of the LPS by di-sialylation conferred increased resistance of the organism to the killing effects of normal human serum, as compared with mono-sialylated or non-sialylated species, indicating that this modification has biological significance.     

pH regulates cation selectivity of poly-(R)-3-hydroxybutyrate/polyphosphate channels from E. coli in planar lipid bilayers

Poly-(R)-3-hydroxybutyrate/polyphosphate (PHB/polyP) complexes, whether isolated from the plasma membranes of bacteria or prepared from the synthetic polymers, form ion channels in planar lipid bilayers that are highly selective for Ca(2+) over Na(+) at physiological pH. This preference for divalent over monovalent cations is attributed to a high density of negative charge along the polyP backbone and the higher binding energies of divalent cations. Here we modify the charge density of polyP by varying the pH, and observe the effect on cation selectivity. PHB/polyP complexes, isolated from E. coli, were incorporated into planar lipid bilayers, and unitary current-voltage relations were determined as a function of pH. When Ca(2+) was the sole permeant cation, conductance diminished steadily from 97 +/- 6 pS at pH 7.4 to 47 +/- 3 pS at pH 5.5. However, in asymmetric solutions of Ca(2+) and Na(+), there was a moderate increase in conductance from 98 +/- 4 at pH 7.4 to 129 +/- 4 pS at pH 6.5, and a substantially larger increase to 178 +/- 6 pS at pH 5.6, signifying an increase in Na(+) permeability or disorganization of channel structure. Reversal potentials point to a sharp decrease in preference for Ca(2+) over Na(+) over a relatively small decrease in pH. Ca(2+) was strongly favored over Na(+) at physiological pH, but the channels became nonselective near the pK(2) of phosphate (approximately 6.8), and displayed weak selectivity for Na(+) over Ca(2+) at acidic pH. Evidently, PHB/polyP complexes are versatile ion carriers whose selectivity may be modulated by small adjustments of the local pH. The results may be relevant to the physiological function of PHB/polyP channels in bacteria and the role of PHB and polyP in the Streptomyces lividans potassium channel.     

The structural heterogeneity of the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance. NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen. Recently it was found that a detoxified lipooligo-saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect. Immun.,64, 4047-4053; X. -X.Gu et al., 1997., Infect. Immun.,65, 4488-4493). In this paper we report the chemical character-ization of the LOS from NTHi 9274 LOS. NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro-metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro-metry (MALDITOF-MS), and tandem MS/MS. At least 17 different OS molecules were observed. These contained variable glycosyl residues, phosphate (P), and phospho-ethanolamine (PEA) substituents. These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues. The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep. Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex(1)-(7)Hep(4)Kdo(1). Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kdo(1)were the major constituents. Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures where Hex is either a Glc or Gal residue.     

Structural profiling of lipopolysaccharide glycoforms expressed by non-typeable Haemophilus influenzae: phenotypic similarities between NTHi strain 162 and the genome strain Rd

Non-typeable Haemophilus influenzae (NTHi) is a significant cause of otitis media in children. We have employed single and multiple step electrospray ionization mass spectrometry (ESIMS) and NMR spectroscopy to profile and elucidate lipopolysaccharide (LPS) structural types expressed by NTHi strain 162, a strain obtained from an epidemiological study in Finland. ESIMS on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples of LPS provided information on the composition and relative abundance of glycoforms differing in the number of hexoses linked to the conserved inner-core element, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A of H. influenzae LPS. The strain examined was found to elaborate Hex2 to Hex5 LPS glycoform populations having structures identical to those observed for H. influenzae strain Rd [Risberg, A.; Masoud, H.; Martin, A.; Richards, J.C.; Moxon, E.R.; Schweda, E.K.H. Eur. J. Biochem. 1999, 261, 171-180], the strain for which the complete genome has been sequenced. In addition, sialyllactose-containing glycoforms previously identified in strain Rd as well as several NTHi strains, were identified as minor components. Multiple step tandem ESIMS (MS(n)) on dephosphorylated and permethylated OS provided information on the arrangement of glycoses within the major population of glycoforms and on the existence of additional isomeric glycoforms. Minor Hex1 and Hex6 glycoforms were detected and characterized where the Hex6 glycoform was comprised of a dihexosamine-containing pentasaccharide chain attached at the proximal heptose residue of the inner-core unit. LPS structural motifs present in the NTHi strain 162 are expressed by a genetically diverse set of disease causing isolates, providing the basis for a vaccine strategy against NTHi otitis media.     

Inorganic Polyphosphate Modulates TRPM8 Channels

Polyphosphate (polyP) is an inorganic polymer built of tens to hundreds of phosphates, linked by high-energy phosphoanhydride bonds. PolyP forms complexes and modulates activities of many proteins including ion channels. Here we investigated the role of polyP in the function of the transient receptor potential melastatin 8 (TRPM8) channel. Using whole-cell patch-clamp and fluorescent calcium measurements we demonstrate that enzymatic breakdown of polyP by exopolyphosphatase (scPPX1) inhibits channel activity in human embryonic kidney and F-11 neuronal cells expressing TRPM8. We demonstrate that the TRPM8 channel protein is associated with polyP. Furthermore, addition of scPPX1 altered the voltage-dependence and blocked the activity of the purified TRPM8 channels reconstituted into planar lipid bilayers, where the activity of the channel was initiated by cold and menthol in the presence of phosphatidylinositol 4,5-biphosphate (PtdIns(4,5)P₂). The biochemical analysis of the TRPM8 protein also uncovered the presence of poly-(R)-3-hydroxybutyrate (PHB), which is frequently associated with polyP. We conclude that the TRPM8 protein forms a stable complex with polyP and its presence is essential for normal channel activity.     

Differential effects of temperature on E. coli and synthetic polyhydroxybutyrate/polyphosphate channels

Complexes of poly-(R)-3-hydroxybutyrate and inorganic polyphosphate (PHB/polyP), isolated from the plasma membranes of Escherichia coli or prepared synthetically (HB(128)/polyP(65)), form Ca(2+)-selective ion channels in planar lipid bilayers that exhibit indistinguishable gating and conductance characteristics at 22 degrees C. Here we examine the gating and conductance of E. coli and synthetic PHB/polyP complexes in planar lipid bilayers as a function of temperature from 15 to 45 degrees C. E. coli PHB/polyP channels remained effectively open throughout this range, with brief closures that became more rare at higher temperatures. Conversely, as temperatures were gradually increased, the open probability of HB(128)/polyP(65) channels progressively decreased. The effect was fully reversible. Channel conductance exhibited three distinct phases. Below 25 degrees C, as PHB approached its glass temperature (ca. 10 degrees C), the conductance of both E. coli and synthetic channels remained at about the same level (95-105 pS). Between 25 degrees C and ca. 40 degrees C, the conductance of E. coli and synthetic channels increased gradually with temperature coefficients (Q(10)) of 1.45 and 1.42, respectively. Above 40 degrees C, E. coli channel conductance increased sharply, whereas the conductance of HB(128)/polyP(65) channels leveled off. The discontinuities in the temperature curves for E. coli channels coincide with discontinuities in thermotropic fluorescence spectra and specific growth rates of E. coli cells. It is postulated that E. coli PHB/polyP complexes are associated with membrane components that inhibit their closure at elevated temperatures.     

Bactericidal antibody response against P6, protein D, and OMP26 of nontypeable Haemophilus influenzae after acute otitis media in otitis-prone children

The bactericidal antibody response to three nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6, and OMP26) was studied in 24 otitis-prone children (aged 7-28?months) after an acute otitis media (AOM) caused by NTHi. The study was carried out to understand the contribution of antigen-specific bactericidal antibody responses in the class of children who are most vulnerable to recurrent otitis media infections. Levels of protein D (P?=?0.005) and P6 (P?=?0.026) but not OMP26 antibodies were higher in bactericidal sera compared with nonbactericidal sera. For five (24%) and 16 (76%) of 21 bactericidal sera tested, removal of anti-protein D and P6 antibody, respectively, resulted in a two- to fourfold drop in bactericidal antibody. Antibodies to OMP26 did not make any contribution to the overall bactericidal activity in any serum samples. Eleven of 21 sera (52%) had bactericidal activity against a heterologous NTHi (86-028 NP) strain but the titers were significantly lower (P?相似文献   

A new structural type for Haemophilus influenzae lipopolysaccharide. Structural analysis of the lipopolysaccharide from nontypeable Haemophilus influenzae strain 486.

Structural elucidation of the sialylated lipopolysaccharide (LPS) of non-typeable Haemophilus influenzae (NTHi) strain 486 has been achieved by the application of high-field NMR techniques and ESI-MS along with composition and linkage analyses on O-deacylated LPS and oligosaccharide samples. It was found that the LPS contains the common element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A, but instead of glycosyl substitution of the terminal heptose residue (HepIII) at the O2 position observed in other H. influenzae strains, HepIII is chain elongated at the O3 position by either lactose or sialyllactose (i.e. alpha-Neu5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp). The LPS is substituted by an O-acetyl group linked to the O2 position of HepIII and phosphocholine (PCho) which was located at the O6 position of a terminal alpha-D-Glcp residue attached to the central heptose, a molecular environment different from what has been reported earlier for PCho. In addition, minor substitution by O-linked glycine to the LPS was observed. By investigation of LPS from a lpsA mutant of NTHi strain 486, it was demonstrated that the lpsA gene product also is responsible for chain extension from HepIII in this strain. The involvement of lic1 in expression of PCho was established by investigation of a lic1 mutant of NTHi strain 486.     

Persister-promoting bacterial toxin TisB produces anion-selective pores in planar lipid bilayers

We studied membrane activity of the bacterial peptide TisB involved in persister cell formation. TisB and its analogs form multi-state ion-conductive pores in planar lipid bilayers with all states displaying similar anionic selectivity. TisB analogs differing by ±1 elementary charges show corresponding changes in selectivity. Probing TisB pores with poly-(ethylene glycol)s reveals only restricted partitioning even for the smallest polymers, suggesting that the pores are characterized by a relatively small diameter. These findings allow us to suggest that TisB forms clusters of narrow pores that are essential for its mechanism of action.