In Solution Identification of the Lysine-Cysteine Redox Switch with a NOS Bridge in Transaldolase by Sulfur K-Edge X-ray Absorption Spectroscopy.

A novel covalent post-translational modification (lysine-NOS-cysteine) was discovered in proteins, initially in the enzyme transaldolase of Neisseria gonorrhoeae (NgTAL) [Nature 2021, 593, 460-464], acting as a redox switch. The identification of this novel linkage in solution was unprecedented until now. We present detection of the NOS redox switch in solution using sulfur K-edge X-ray absorption spectroscopy (XAS). The oxidized NgTAL spectrum shows a distinct shoulder on the low-energy side of the rising edge, corresponding to a dipole-allowed transition from the sulfur 1s core to the unoccupied σ* orbital of the S-O group in the NOS bridge. This feature is absent in the XAS spectrum of reduced NgTAL, where Lys-NOS-Cys is absent. Our experimental and calculated XAS data support the presence of a NOS bridge in solution, thus potentially facilitating future studies on enzyme activity regulation mediated by the NOS redox switches, drug discovery, biocatalytic applications, and protein design.

Synthesis and Biological Evaluation of 1,3-Dideazapurine-Like 7-Amino-5-Hydroxymethyl-Benzimidazole Ribonucleoside Analogues as Aminoacyl-tRNA Synthetase Inhibitors.

Aminoacyl-tRNA synthetases (aaRSs) have become viable targets for the development of antimicrobial agents due to their crucial role in protein translation. A series of six amino acids were coupled to the purine-like 7-amino-5-hydroxymethylbenzimidazole nucleoside analogue following an optimized synthetic pathway. These compounds were designed as aaRS inhibitors and can be considered as 1,3-dideazaadenine analogues carrying a 2-hydroxymethyl substituent. Despite our intentions to obtain N1-glycosylated 4-aminobenzimidazole congeners, resembling the natural purine nucleosides glycosylated at the N9-position, we obtained the N3-glycosylated benzimidazole derivatives as the major products, resembling the respective purine N7-glycosylated nucleosides. A series of X-ray crystal structures of class I and II aaRSs in complex with newly synthesized compounds revealed interesting interactions of these "base-flipped" analogues with their targets. While the exocyclic amine of the flipped base mimics the reciprocal interaction of the N3-purine atom of aminoacyl-sulfamoyl adenosine (aaSA) congeners, the hydroxymethyl substituent of the flipped base apparently loses part of the standard interactions of the adenine N1 and the N6-amine as seen with aaSA analogues. Upon the evaluation of the inhibitory potency of the newly obtained analogues, nanomolar inhibitory activities were noted for the leucine and isoleucine analogues targeting class I aaRS enzymes, while rather weak inhibitory activity against the corresponding class II aaRSs was observed. This class bias could be further explained by detailed structural analysis.

Effect of Lipidation on the Localization and Activity of a Lysozyme Inhibitor in Neisseria gonorrhoeae.

The Gram-negative pathogen Neisseria gonorrhoeae (gonococcus [Gc]) colonizes lysozyme-rich mucosal surfaces. Lysozyme hydrolyzes peptidoglycan, leading to bacterial lysis. Gc expresses two proteins, SliC and NgACP, that bind and inhibit the enzymatic activity of lysozyme. SliC is a surface-exposed lipoprotein, while NgACP is found in the periplasm and also released extracellularly. Purified SliC and NgACP similarly inhibit lysozyme. However, whereas mutation of ngACP increases Gc susceptibility to lysozyme, the sliC mutant is only susceptible to lysozyme when ngACP is inactivated. In this work, we examined how lipidation contributes to SliC expression, cellular localization, and resistance of Gc to killing by lysozyme. To do so, we mutated the conserved cysteine residue (C18) in the N-terminal lipobox motif of SliC, the site for lipid anchor attachment, to alanine. SliC(C18A) localized to soluble rather than membrane fractions in Gc and was not displayed on the bacterial surface. Less SliC(C18A) was detected in Gc lysates compared to the wild-type protein. This was due in part to some release of the C18A mutant, but not wild-type, protein into the extracellular space. Surprisingly, Gc expressing SliC(C18A) survived better than SliC (wild type)-expressing Gc after exposure to lysozyme. We conclude that lipidation is not required for the ability of SliC to inhibit lysozyme, even though the lipidated cysteine is 100% conserved in Gc SliC alleles. These findings shed light on how members of the growing family of lysozyme inhibitors with distinct subcellular localizations contribute to bacterial defense against lysozyme.IMPORTANCENeisseria gonorrhoeae is one of many bacterial species that express multiple lysozyme inhibitors. It is unclear how inhibitors that differ in their subcellular localization contribute to defense from lysozyme. We investigated how lipidation of SliC, an MliC (membrane-bound lysozyme inhibitor of c-type lysozyme)-type inhibitor, contributes to its localization and lysozyme inhibitory activity. We found that lipidation was required for surface exposure of SliC and yet was dispensable for protecting the gonococcus from killing by lysozyme. To our knowledge, this is the first time the role of lipid anchoring of a lysozyme inhibitor has been investigated. These results help us understand how different lysozyme inhibitors are localized in bacteria and how this impacts resistance to lysozyme.

Disulfide Cross-linking of a Multidrug and Toxic Compound Extrusion Transporter Impacts Multidrug Efflux.

Multidrug and toxic compound extrusion (MATE) transporters contribute to multidrug resistance by extruding different drugs across cell membranes. The MATE transporters alternate between their extracellular and intracellular facing conformations to propel drug export, but how these structural changes occur is unclear. Here we combine site-specific cross-linking and functional studies to probe the movement of transmembrane helices in NorM from Neiserria gonorrheae (NorM-NG), a MATE transporter with known extracellular facing structure. We generated an active, cysteine-less NorM-NG and conducted pairwise cysteine mutagenesis on this variant. We found that copper phenanthroline catalyzed disulfide bond formation within five cysteine pairs and increased the electrophoretic mobility of the corresponding mutants. Furthermore, copper phenanthroline abolished the activity of the five paired cysteine mutants, suggesting that these substituted amino acids come in spatial proximity during transport, and the proximity changes are functionally indispensable. Our data also implied that the substrate-binding transmembrane helices move up to 10 Å in NorM-NG during transport and afforded distance restraints for modeling the intracellular facing transporter, thereby casting new light on the underlying mechanism.

Requirements for the import of neisserial Omp85 into the outer membrane of human mitochondria.

ß-Barrel proteins are present only in the outer membranes of Gram-negative bacteria, chloroplasts and mitochondria. Fungal mitochondria were shown to readily import and assemble bacterial ß-barrel proteins, but human mitochondria exhibit certain selectivity. Whereas enterobacterial ß-barrel proteins are not imported, neisserial ones are. Of those, solely neisserial Omp85 is integrated into the outer membrane of mitochondria. In this study, we wanted to identify the signal that targets neisserial ß-barrel proteins to mitochondria. We exchanged parts of neisserial Omp85 and PorB with their Escherichia coli homologues BamA and OmpC. For PorB, we could show that its C-terminal quarter can direct OmpC to mitochondria. In the case of Omp85, we could identify several amino acids of the C-terminal ß-sorting signal as crucial for mitochondrial targeting. Additionally, we found that at least two POTRA (polypeptide-transport associated) domains and not only the ß-sorting signal of Omp85 are needed for its membrane integration and function in human mitochondria. We conclude that the signal that directs neisserial ß-barrel proteins to mitochondria is not conserved between these proteins. Furthermore, a linear mitochondrial targeting signal probably does not exist. It is possible that the secondary structure of ß-barrel proteins plays a role in directing these proteins to mitochondria.

Crystal structures of two solute receptors for L-cystine and L-cysteine, respectively, of the human pathogen Neisseria gonorrhoeae.

ATP-binding cassette (ABC) transporters are integral membrane proteins that carry a variety of substrates across biological membranes at the expense of ATP. The here considered prokaryotic canonical importers consist of three entities: an extracellular solute receptor, two membrane-intrinsic proteins forming a translocation pathway, and two cytoplasmic ATP-binding subunits. The ngo0372-74 and ngo2011-14 gene clusters from the human pathogen Neisseria gonorrhoeae were predicted by sequence homology as ABC transporters for the uptake of cystine and cysteine, respectively, and chosen for structural characterization. The structure of the receptor component Ngo0372 was obtained in a ligand-free "open" conformation and in a "closed" conformation when co-crystallized with L-cystine. Our data provide the first structural information of an L-cystine ABC transporter. Dissociation constants of 21 and 33 nM for L-cystine and L-selenocystine, respectively, were determined by isothermal titration calorimetry. In contrast, L-cystathionine and L-djenkolic acid are weak binders, while no binding was detectable for S-methyl-L-cysteine. Mutational analysis of two residues from the binding pocket, Trp97 and Tyr59, revealed that the latter is crucial for L-cystine binding. The structure of the Ngo2014 receptor was obtained in closed conformation in complex with co-purified L-cysteine. The protein binds L-cysteine with a K(d) of 26 nM. Comparison of the structures of both receptors and analysis of the ligand binding sites shed light on the mode of ligand recognition and provides insight into the tight binding of both substrates. Moreover, since L-cystine limitation leads to reduction in virulence of N. gonorrhoeae, Ngo0372 might be suited as target for an antimicrobial vaccine.

Dynamics of gonococcal type IV pili during infection.

Type IV pili are important bacterial virulence factors that mediate attachment to mammalian host cells and elicit downstream signals. When adhered to abiotic surfaces, the human pathogen Neisseria gonorrhoeae generates force by retracting these polymeric cell appendages. We recently found that single pili generate stalling forces that exceed 100 pN, but it is unclear whether bacteria generate force once they adhere to their human host cells. Here, we report that pili retract very actively during infection of human epithelial cells. The retraction velocity is bimodal and the high velocity mode persisted at higher forces in contrast to an abiotic environment. Bacteria generate considerable force during infection, but the maximum force is reduced from 120+/-40 pN on abiotic surfaces to 70+/-20 pN on epithelial cells, most likely due to elastic effects. Velocity and maximum force of pilus retraction are largely independent of the infection period within 1 h and 24 h post-infection. Thus, the force generated by type IV pili during infection is high enough to induce cytoskeletal rearrangements in the host cell.

Hijacking transferrin bound iron: protein-receptor interactions involved in iron transport in N. gonorrhoeae.

Neisseria gonorrhoeae has the capacity to acquire iron from its human host by removing this essential nutrient from serum transferrin. The transferrin binding proteins, TbpA and TbpB constitute the outer membrane receptor complex responsible for binding transferrin, extracting the tightly bound iron from the host-derived molecule, and transporting iron into the periplasmic space of this Gram-negative bacterium. Once iron is transported across the outer membrane, ferric binding protein A (FbpA) moves the iron across the periplasmic space and initiates the process of transport into the bacterial cytosol. The results of the studies reported here define the multiple steps in the iron transport process in which TbpA and TbpB participate. Using the SUPREX technique for assessing the thermodynamic stability of protein-ligand complexes, we report herein the first direct measurement of periplasmic FbpA binding to the outer membrane protein TbpA. We also show that TbpA discriminates between apo- and holo-FbpA; i.e. the TbpA interaction with apo-FbpA is higher affinity than the TbpA interaction with holo-FbpA. Further, we demonstrate that both TbpA and TbpB individually can deferrate transferrin and ferrate FbpA without energy supplied from TonB resulting in sequestration by apo-FbpA.

Sulfate as a synergistic anion facilitating iron binding by the bacterial transferrin FbpA: the origins and effects of anion promiscuity.

The ferric binding protein, FbpA, has been demonstrated to facilitate the transport of naked Fe3+ across the periplasmic space of several Gram-negative bacteria. The sequestration of iron by FbpA is facilitated by the presence of a synergistic anion, such as phosphate or sulfate. Here we report the sequestration of Fe3+ by FbpA in the presence of sulfate, at an assumed periplasmic pH of 6.5 to form FeFbpA-SO4 with K'(eff) = 1.7 x 10(16) M(-1) (at 20 degrees C, 50 mM MES, 200 mM KCl). The iron affinity of the FeFbpA-SO4 protein assembly is 2 orders of magnitude lower than when bound with phosphate and is the lowest of any of the FeFbpA-X assemblies yet reported. Iron reduction at the cytosolic membrane receptor may be an essential aspect of the periplasmic iron-transport process, and with an E(1/2) of -158 mV (NHE), FeFbpA-SO4 is the most easily reduced of all FeFbpA-X assemblies yet studied. The variation of FeFbpA-X assembly stability (K'(eff)) and ease of reduction (E(1/2)) with differing synergistic anions X(n-) are correlated over a range of 14 kJ, suggesting that the variations in redox potentials are due to stabilization of Fe3+ in FeFbpA-X by X(n-). Anion promiscuity of FbpA in the diverse composition of the periplasmic space is illustrated by the ex vivo exchange kinetics of FeFbpA-SO4 with phosphate and arsenate, where first-order kinetics with respect to FeFbpA-SO4 (k = 30 s(-1)) are observed at pH 6.5, independent of entering anion concentration and identity. Anion lability and influence on the iron affinity and reduction potential for FeFbpA-X support the hypothesis that synergistic anion exchange may be an important regulator in iron delivery to the cytosol. This structural and thermodynamic analysis of anion binding in FeFbpA-X provides additional insight into anion promiscuity and importance.

Identification of a new OmpA-like protein in Neisseria gonorrhoeae involved in the binding to human epithelial cells and in vivo colonization.

Outer membrane protein As (OmpAs) are highly conserved proteins within the Enterobacteriaceae family. OmpA contributes to the maintenance of structural membrane integrity and invasion into mammalian cells. In Escherichia coli K1 OmpA also contributes to serum resistance and is involved in the virulence of the bacterium. Here we describe the identification of an OmpA-like protein in Neisseria gonorrhoeae (Ng-OmpA). We show that the gonococcal OmpA-like protein, similarly to E. coli OmpA, plays a significant role in the adhesion and invasion into human cervical carcinoma and endometrial cells and is required for entry into macrophages and intracellular survival. Furthermore, the isogenic knockout ompA mutant demonstrates reduced recovery in a mouse model of infection when compared with the wild-type strain, suggesting that Ng-OmpA plays an important role in the in vivo colonization. All together, these data suggest that the newly identified surface exposed protein Ng-OmpA represents a novel virulence factor of gonococcus.

Effect of gonococcal lipooligosaccharide variation on human monocytic cytokine profile.

BACKGROUND: Neisseria gonorrhoeae is an obligate human pathogen that causes significant worldwide morbidity. N. gonorrhoeae expresses lipooligosaccharide (LOS), a phase variable molecule that plays an important role during pathogenesis of the organism. Alteration in the structure of gonococcal LOS correlates with altered disease presentation. In addition, LOS sialylation occurs readily in vivo, though the role of this sialylation during disease is unknown. RESULTS: Challenge of human monocytes with purified LOS preparations isolated from strains expressing distinct structurally defined LOSs resulted in identical production of the proinflammatory cytokines tumor necrosis factor alpha (TNFalpha) and interleukin-12 (IL-12). Similar results were seen when monocytes were challenged with either live or gentamicin-killed whole cell gonococcal variants expressing these LOS structures, although greater cytokine production was observed in comparison with challenge by purified LOS. Challenge of a human primary monocyte model with distinct LOS variants resulted in similar production of TNFalpha, IL-12, interleukin-10 (IL-10), and interleukin-8 (IL-8). A cytokine array was employed to allow measurement of a broad range of cytokines in samples challenge with gonococcal LOS variants as well as variants expressing sialylated LOS. Challenge of primary monocytes with sialylated gonococci was shown to elicit the production of more MCP-2 (monocyte chemoattractant protein-2) in comparison with challenge by unsialylated gonococci. CONCLUSION: We demonstrated that while alterations in the carbohydrate moiety of LOS do not impact the production of most cytokines by human monocytes, whole-cell bacterial challenge is more stimulatory than challenge with purified LOS, implying that other gonococcal cell surface antigens are important for the elicitation of cytokines. Challenge with gonococci expressing sialylated LOS resulted in elicitation of more of the chemokine MCP-2 from challenged cells in comparison with gonococci expressing unsialylated LOS. As MCP-2 is an important chemoattractant, this indicates that in vivo sialylation may play an important role during the pathogenesis of N. gonorrhoeae.

The thioredoxin domain of Neisseria gonorrhoeae PilB can use electrons from DsbD to reduce downstream methionine sulfoxide reductases.

The PilB protein from Neisseria gonorrhoeae is located in the periplasm and made up of three domains. The N-terminal, thioredoxin-like domain (NT domain) is fused to tandem methionine sulfoxide reductase A and B domains (MsrA/B). We show that the alpha domain of Escherichia coli DsbD is able to reduce the oxidized NT domain, which suggests that DsbD in Neisseria can transfer electrons from the cytoplasmic thioredoxin to the periplasm for the reduction of the MsrA/B domains. An analysis of the available complete genomes provides further evidence for this proposition in other bacteria where DsbD/CcdA, Trx, MsrA, and MsrB gene homologs are all located in a gene cluster with a common transcriptional direction. An examination of wild-type PilB and a panel of Cys to Ser mutants of the full-length protein and the individually expressed domains have also shown that the NT domain more efficiently reduces the MsrA/B domains when in the polyprotein context. Within this frame-work there does not appear to be a preference for the NT domain to reduce the proximal MsrA domain over MsrB domain. Finally, we report the 1.6A crystal structure of the NT domain. This structure confirms the presence of a surface loop that makes it different from other membrane-tethered, Trx-like molecules, including TlpA, CcmG, and ResA. Subtle differences are observed in this loop when compared with the Neisseria meningitidis NT domain structure. The data taken together supports the formation of specific NT domain interactions with the MsrA/B domains and its in vivo recycling partner, DsbD.

Conformation of the cell division regulator MinE: evidence for interactions between the topological specificity and anti-MinCD domains.

Symmetric division of Gram-negative bacteria depends on the combined action of three proteins that ensure correct positioning of the cell division septum, namely, MinC, MinD, and MinE. To achieve this function, MinC and MinD form a membrane-bound complex that blocks cell division at all potential sites. Opposing this inhibition is MinE, which interacts with MinD via its N-terminal anti-MinCD domain to site-specifically counter the action of the MinCD complex. The anti-MinCD domain has been proposed to bind MinD in a helical conformation; however, little is actually known about the structure of this functionally critical region. To understand how MinE can perform its anti-MinCD function, we have therefore investigated the conformation of the full-length MinE from Neisseria gonorrhoeae by solution NMR. Although solubility considerations required the use of sample conditions that limit the observation of amide resonances to regions that are protected from solvent exchange, backbone chemical shifts from both N- and C-terminal domains could be assigned. In contrast to previous models, secondary chemical shift analysis of these solvent-protected regions shows that parts of the N-terminal anti-MinCD domain are stably folded with many functionally important residues localizing to a beta-structure. In addition, this N-terminal domain may be interacting with the C-terminal topological specificity domain, since mutations made in one domain led to NMR spectral changes in both domains. The nonfunctional MinE mutant L22D showed even larger evidence of structural perturbations in both domains, with significant destabilization of the entire MinE structure. Overall, these results suggest that there is an intimate structural association between the anti-MinCD and topological specificity domains, allowing the functional properties of the two domains to be modulated through this interaction.

Specificity of the synergistic anion for iron binding by ferric binding protein from Neisseria gonorrhoeae.

Ferric binding protein in Neisseria gonorrhoeae (nFbpA) transports iron from outer membrane receptors for host proteins across the periplasm to a permease in an alternative pathway to the use of siderophores in some pathogenic bacteria. Phosphate and nitrilotriacetate, both at pH 8, and vanadate at pH 9 are shown to be synergistic in promoting ferric binding to nFbpA, in contrast to carbonate and sulfate. Interestingly, only phosphate produces the fully closed conformation of nFbpA as defined by native electrophoresis. The role of phosphate was probed by constructing three mutants: Q58E, Q58R, and G140H. The anion and iron binding properties of the Q58E mutant are similar to the wild-type protein, implying that one phosphate oxygen is a hydrogen bond donor and may in part define the specificity of nFbpA for phosphate over sulfate. Phosphate is a weakly synergistic anion in the Q58R and G140H mutants, and these mutants do not form completely closed structures. Ferric binding was investigated by both isothermal titration and differential scanning calorimetry. The apparent affinity of nFbpA for iron in a solution of 30 mM citrate is 1 order of magnitude larger in the presence (K(app)= 1.7 x 10(5) M(-1)) of phosphate than in its absence (K(app) = 1.6 x 10(4) M(-1)) at pH 7. Similar results were obtained at pH 8. This increase in affinity with phosphate as well as the formation of closed structure allows nFbpA to compete for free ferric ions in solution and suggests that ferric binding to nFbpA is regulated by the synergistic phosphate anion at sites of iron uptake.

The pilus-retraction protein PilT: ultrastructure of the biological assembly.

PilT is a biological motor required for the retraction of bacterial type IV pili. Nesseria gonorrhoeae PilT has been purified and its ultrastructure has been examined by freeze-etch electron microscopy, revealing a 115 A outer diameter, 15-35 A inner diameter ring. Aquifex aeolicus PilT crystals were obtained in a primitive hexagonal space group (unit-cell parameters a = b = 107.3, c = 68.5 A) and diffract to a minimum Bragg spacing of 2.8 A when PilT is co-crystallized with adenine nucleotides. Initial phases to 3.5 A resolution have been determined by multiwavelength anomalous dispersion and density modification. Resulting electron-density maps show a hexameric A. aeolicus PilT ring 105 A wide by 55 A high, with an inner cavity that varies in shape and width from 20 to 40 A over the height of the complex. Both PilT ultrastructures are very similar to type II and type IV secretion ATPases in overall shape, size and assembly.

A mutant form of the Neisseria gonorrhoeae pilus secretin protein PilQ allows increased entry of heme and antimicrobial compounds.

A spontaneous point mutation in pilQ (pilQ1) resulted in phenotypic suppression of a hemoglobin (Hb) receptor mutant (hpuAB mutant), allowing gonococci to grow on Hb as the sole source of iron. PilQ, formerly designated OMP-MC, is a member of the secretin family of proteins located in the outer membrane and is required for pilus biogenesis. The pilQ1 mutant also showed decreased piliation and transformation efficiency. Insertional inactivation of pilQ1 resulted in the loss of the Hb utilization phenotype and decreased entry of free heme. Despite the ability of the pilQ1 mutant to use Hb for iron acquisition and porphyrin, there was no demonstrable binding of Hb to the cell surface. The pilQ1 mutant was more sensitive to the toxic effect of free heme in growth medium and hypersensitive to the detergent Triton X-100 and multiple antibiotics. Double mutation in pilQ1 and tonB had no effect on these phenotypes, but a double pilQ1 pilT mutant showed a reduction in Hb-dependent growth and decreased sensitivity to heme and various antimicrobial agents. Insertional inactivation of wild-type pilQ also resulted in reduced entry of heme, Triton X-100, and some antibiotics. These results show that PilQ forms a channel that allows entry of heme and certain antimicrobial compounds and that a gain-of function point mutation in pilQ results in TonB-independent, PilT-dependent increase of entry.

Transcription activation at Escherichia coli FNR-dependent promoters by the gonococcal FNR protein: effects of a novel S18F substitution and comparisons with the corresponding substitution in E. coli FNR.

The Neisseria gonorrhoeae genome encodes a homologue of the Escherichia coli FNR protein (the fumarate and nitrate reductase regulator). Despite its similarity to E. coli FNR, the gonococcal FNR only partially complemented an E. coli fnr mutation. After error-prone PCR mutagenesis of the gonococcal fnr gene, we identified four mutant fnr derivatives carrying the same S18F substitution, and we showed that the mutant FNR could activate transcription from a range of class I and class II FNR-dependent promoters in E. coli. Prompted by the similarities between gonococcal and E. coli FNR, we made changes in gonococcal fnr that created substitutions that are equivalent to previously characterized substitutions in E. coli FNR. First, our experiments showed that cysteine, C116, in the gonococcal FNR, equivalent to C122 in E. coli FNR, is essential, presumably because, as in E. coli FNR, it binds to an iron-sulfur center. Second, the L22H and D148A substitutions in gonococcal FNR were made. These changes are equivalent to the L28H and D154A changes in E. coli FNR, which had been shown to increase FNR activity in the presence of oxygen. We show that the effects of these substitutions in gonococcal FNR are distinct from those of the S18F substitution. Similarly, substitutions in the putative activating regions of gonococcal FNR were made. We show that the activity of gonococcal FNR in E. coli can be increased by transplanting certain activating regions from E. coli FNR. The effects of these substitutions are additive to those due to S18F. From these data, we conclude that the effects of the S18F substitution in gonococcal FNR are distinct from the effects of the other substitutions. S18 is immediately adjacent to one of three N-terminal cysteine residues that coordinate the iron-sulfur center, and thus the S18F substitution is most likely to stabilize this center. Support for this came from complementary experiments in which we created the S24F substitution in E. coli FNR, which is equivalent to the S18F substitution in gonococcal FNR. Our results show that the S24F substitution changes the activity of E. coli FNR and that the changes are distinct from those due to previously characterized substitutions.

Receptor-mediated endocytosis of Neisseria gonorrhoeae into primary human urethral epithelial cells: the role of the asialoglycoprotein receptor.

Urethral epithelial cells are invaded by Neisseria gonorrhoeae during gonococcal infection in men. To understand further the mechanisms of gonococcal entry into host cells, we used the primary human urethral epithelial cells (PHUECs) tissue culture system recently developed by our laboratory. These studies showed that human asialoglycoprotein receptor (ASGP-R) and the terminal lactosamine of lacto-N-neotetraose-expressing gonococcal lipooligosaccharide (LOS) play an important role in invasion of PHUECs. Microscopy studies showed that ASGP-R traffics to the cell surface after gonococcal challenge. Co-localization of ASGP-R with gonococci was observed. As ASGP-R-mediated endocytosis is clathrin dependent, clathrin localization in PHUECs was examined after infection. Infected PHUECs showed increased clathrin recruitment and co-localization of clathrin and gonococci. Preincubating PHUECs in 0.3 M sucrose or monodansylcadaverine (MDC), which both inhibit clathrin-coated pit formation, resulted in decreased invasion. N. gonorrhoeae strain 1291 produces a single LOS glycoform that terminates with Gal(beta1-4)GlcNac(beta1-3)Gal(beta1-4)Glc (lacto-N-neotetraose). Invasion assays showed that strain 1291 invades significantly more than four isogenic mutants expressing truncated LOS. Sialylation of strain 1291 LOS inhibited invasion significantly. Preincubation of PHUECs in asialofetuin (ASF), an ASGP-R ligand, significantly reduced invasion. A dose-response reduction in invasion was observed in PHUECs preincubated with increasing concentrations of NaOH-deacylated 1291 LOS. These studies indicated that an interaction between lacto-N-neotetraose-terminal LOS and ASGP-R allows gonococcal entry into PHUECs.

Proteome and transcriptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein.

Bacillus subtilis and related Bacillus species are frequently used as hosts for the industrial production of recombinant proteins. In this study the cellular response of B. subtilis to the overproduction of an insoluble heterologous protein was investigated. For this purpose PorA, an outer membrane protein from Neisseria meningitidis, which accumulates after overexpression in the cytoplasm of B. subtilis mainly in the form of inclusion bodies, was used. The molecular response to overexpression of porA has been analysed at the transcriptional level using the DNA macro array technique and at the translational level by two-dimensional polyacrylamide gel electrophoresis. It was found that the expression of the heat shock genes of class I (dnaK, groEL and grpE) and class III (clpP and clpC) are increased under overproducing conditions. Furthermore, the protein levels of the two ribosomal proteins RpsB and RplJ are increased in the PorA overproducing cells. The transcriptome analysis indicated that mRNA levels of genes encoding pyrimidine and purine synthesis enzymes but also from ribosomal protein genes have elevated levels under overproducing conditions. Finally, the association of the protease ClpP and its ATPase subunits ClpC and ClpX with the PorA inclusion bodies was demonstrated by means of the immunogold labelling technique.

Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules.

The human pathogens Neisseria meningitidis and Neisseria gonorrhoeae express a family of variable outer membrane opacity-associated (Opa) proteins that recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N-terminal domain of the CD66 family of adhesion molecules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidis strain C751 have the dual capacity to interact with both receptors. In addition, all three Opa proteins of C751 bind equally well to HeLa cells transfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgroup of the CD66 family, but show distinct tropism for CGM1- (CD66d) and NCA (CD66c)-expressing cells. Because the C751 Opa proteins make up distinct structures via the surface-exposed hypervariable domains (HV-1 and HV-2), these combinations appear to be involved in tropism for the distinct CD66 subgroups. To define the determinants of receptor recognition, we used mutant proteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at several predicted exposed sites in the N-domain and compared their interactions with several Opa proteins of both N. meningitidis and N. gonorrhoeae. The observations applied to the molecular model of the BGP N-domain that we constructed show that the binding of all Opa proteins tested occurs at the non-glycosylated (CFG) face of the molecule and, in general, appears to require Tyr-34 and Ile-91. Further, efficient interaction of distinct Opa proteins depends on different non-adjacent amino acids. In the three-dimensional model, these residues lie in close proximity to Tyr-34 and Ile-91 at the CFG face, making continuous binding domains (adhesiotopes). The epitope of the monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identified within the Opa adhesiotopes on the N-domain. These studies define the molecular basis that directs the Opa specificity for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups.