Optical microstructure fabrication using structured polarized illumination.

A versatile system for the fabrication of surface microstructures is demonstrated by combining the photomechanical response of supramolecular azopolymers with structured polarized illumination from a high resolution spatial light modulator. Surface relief structures with periods 900 nm - 16.5 µm and amplitudes up to 1.0 µm can be fabricated with a single 5 sec exposure at 488 nm. Sinusoidal, circular, and chirped surface profiles can be fabricated via direct programming of the spatial light modulator, with no optomechanical realignment required. Surface microstructures can be combined into macroscopic areas by mechanical translation followed by exposure. The surface structures grow immediately in response to illumination, can be visually observed in real time, and require no post-exposure processing.

Arcanobacterium haemolyticum Utilizes Both Phospholipase D and Arcanolysin To Mediate Its Uptake into Nonphagocytic Cells.

Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis and wound infections. A few studies have suggested that A. haemolyticum is able to induce its uptake into nonphagocytic epithelial cells, but the bacterial factors associated with host cell invasion and the host cell processes involved have yet to be studied. We investigated how two A. haemolyticum virulence factors, arcanolysin (ALN) and phospholipase D (PLD), affect the ability of the bacteria to adhere to and subsequently invade Detroit 562 pharyngeal epithelial cells. The sphingomyelinase activity of phospholipase D was necessary to increase bacterial adherence, while the absence of a functional arcanolysin had no effect on A. haemolyticum adherence but did lead to a decrease in A. haemolyticum invasion into Detroit 562 cells. Because of the known roles of cholesterol-dependent cytolysins in disrupting calcium gradients and inducing F-actin-mediated bacterial internalization, we sought to determine whether ALN and PLD played a similar role in the ability of A. haemolyticum to invade nonphagocytic cells. Elimination of extracellular calcium and inhibition of the Arp2/3 complex or F-actin polymerization also caused a decrease in the ability of A. haemolyticum to invade Detroit 562 cells. Overall, our findings suggest that A. haemolyticum utilizes phospholipase D primarily for adherence and utilizes arcanolysin primarily for invasion into Detroit 562 cells in a process dependent on extracellular calcium and F-actin polymerization. Our work marks the first insight into how the individual activities of arcanolysin and phospholipase D affect A. haemolyticum host-pathogen interactions using the biologically relevant Detroit 562 cell line.

Personalized Bioactive Nasal Supports for Postoperative Cleft Rhinoplasty.

PURPOSE: After cleft lip and palate surgical procedures, patients often need nostril supports to help the reconstructed nostrils retain their shape during healing. Many postoperative nasal stents use a one-size-fits-all approach, in which a standard rubber tube retainer is trimmed and used to support the healing nares. The purpose of this study was to examine photogrammetry and 3-dimensional (3D) printing as a fabrication tool for postoperative patient-specific nasal supports that can be loaded with bioactive agents for localized delivery. MATERIALS AND METHODS: A "normal" right nostril injection mold was prepared from a left-sided unilateral cleft defect, and the negative-space impression was modeled using a series of photographs taken at different rotation angles with a commercial mobile phone camera. These images were "stitched" together using photogrammetry software, and the computer-generated models were reflected, joined, and digitally sculpted to generate hollow bilateral supports. Three-dimensional prints were coated with polyvinylpyrrolidone-penicillin and validated for their ability to inhibit Escherichia coli using human blood agar diffusion assays. RESULTS: The results showed that our approach had a high level of contour replication and the antibiotic coating was able to inhibit bacterial growth with a mean zone of inhibition of 15.15 ± 0.99 mm (n = 9) (P < .0001) in disc diffusion assays. CONCLUSIONS: Consumer-grade 3D printing displays potential as a fabrication method for postoperative cleft bilateral nasal supports and may support the surgically reconstructed internal contours. The results of this study suggest that such types of bioactive 3D prints may have potential applications in personalized drug-delivery systems and medical devices.

Detoxification of 7-dehydrocholesterol fatal to Helicobacter pylori is a novel role of cholesterol glucosylation.

The glucosylation of free cholesterol (FC) by Helicobacter pylori cells has various biological significances for the survival of this bacterium. H. pylori cells with glucosylated FC are capable of evading host immune systems, such as phagocytosis by macrophages and activation of antigen-specific T cells, and surviving in the gastric mucosal tissues for long periods. An additional role of cholesterol glucosylation in the survival of H. pylori which is distinct from the role of escaping the host immune system, however, has yet to be identified. This study demonstrated that 7-dehydrocholesterol (7dFC), an FC precursor, is a toxic compound fatal to H. pylori cells, but the cell membrane of H. pylori is capable of absorbing this toxic sterol via glucosylation. In contrast to the case with 7dFC, no toxicity to H. pylori cells was detected from the glucosylated 7dFC. In addition, cgt gene mutant H. pylori cells that cannot glucosylate cholesterols had higher susceptibility to the toxic action of 7dFC than wild-type H. pylori cells. These results indicate that the cgt gene product of H. pylori serves to detoxify the sterol fatal to this bacterium and to permit this toxic sterol as a cell membrane lipid component. In summary, this study defined a novel role of cholesterol glucosylation in H. pylori.

Transcriptional profiling of gastric epithelial cells infected with wild type or arginase-deficient Helicobacter pylori.

BACKGROUND: Helicobacter pylori causes acute and chronic gastric inflammation induced by proinflammatory cytokines and chemokines secreted by cells of the gastric mucosa, including gastric epithelial cells. Previous studies have demonstrated that the bacterial arginase, RocF, is involved in inhibiting T cell proliferation and CD3ζ expression, suggesting that arginase could be involved in a more general dampening of the immune response, perhaps by down-regulation of certain pro-inflammatory mediators. RESULTS: Global transcriptome analysis was performed on AGS gastric epithelial cells infected for 16 hours with a wild type Helicobacter pylori strain 26695, an arginase mutant (rocF-) or a rocF+ complemented strain. H. pylori infection triggered altered host gene expression in genes involved in cell movement, death/growth/proliferation, and cellular function and maintenance. While the wild type strain stimulates host inflammatory pathways, the rocF- mutant induced significantly more expression of IL-8. The results of the microarray were verified using real-time PCR, and the differential levels of protein expression were confirmed by ELISA and Bioplex analysis. MIP-1B was also significantly secreted by AGS cells after H. pylori rocF- mutant infection, as determined by Bioplex. Even though not explored in this manuscript, the impact that the results presented here may have on the development of gastritis, warrant further research to understand the underlying mechanisms of the relationship between H. pylori RocF and IL-8 induction. CONCLUSIONS: We conclude that H. pylori arginase modulates multiple host signaling and metabolic pathways of infected gastric epithelial cells. Arginase may play a critical role in anti-inflammatory host responses that could contribute to the ability of H. pylori to establish chronic infections.

Role of the HefC efflux pump in Helicobacter pylori cholesterol-dependent resistance to ceragenins and bile salts.

The human gastric pathogen Helicobacter pylori modifies host cholesterol via glycosylation and incorporates the glycosylated cholesterol into its membrane; however, the benefits of cholesterol to H. pylori are largely unknown. We speculated that cholesterol in the H. pylori membrane might alter the susceptibility of these organisms to membrane-disrupting antibacterial compounds. To test this hypothesis, H. pylori strains were cultured in Ham's F-12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 10 bile salts and four ceragenins, which are novel bile salt derivatives that mimic membrane-disrupting activity of antimicrobial peptides. H. pylori cultured with cholesterol was substantially more resistant to seven of the bile salts and three ceragenins than H. pylori cultured without cholesterol. In most cases, these cholesterol-dependent differences ranged from 2 to 7 orders of magnitude; this magnitude depended on concentration of the agent. Cholesterol is modified by glycosylation using Cgt, a cholesteryl glycosyltransferase. Surprisingly, a cgt knockout strain still maintained cholesterol-dependent resistance to bile salts and ceragenins, indicating that cholesterol modification was not involved in resistance. We then tested whether three putative, paralogous inner membrane efflux pumps, HefC, HefF, or HefI, played a role. While HefF and HefI appeared unimportant, HefC was shown to play a critical role in the resistance to bile salts and ceragenins by multiple methods in multiple strain backgrounds. Thus, both cholesterol and the putative bile salt efflux pump HefC play important roles in H. pylori resistance to bile salts and ceragenins.

Helicobacter pylori AlpA and AlpB bind host laminin and influence gastric inflammation in gerbils.

Helicobacter pylori persistently colonizes humans, causing gastritis, ulcers, and gastric cancer. Adherence to the gastric epithelium has been shown to enhance inflammation, yet only a few H. pylori adhesins have been paired with targets in host tissue. The alpAB locus has been reported to encode adhesins involved in adherence to human gastric tissue. We report that abrogation of H. pylori AlpA and AlpB reduces binding of H. pylori to laminin while expression of plasmid-borne alpA or alpB confers laminin-binding ability to Escherichia coli. An H. pylori strain lacking only AlpB is also deficient in laminin binding. Thus, we conclude that both AlpA and AlpB contribute to H. pylori laminin binding. Contrary to expectations, the H. pylori SS1 mutant deficient in AlpA and AlpB causes more severe inflammation than the isogenic wild-type strain in gerbils. Identification of laminin as the target of AlpA and AlpB will facilitate future investigations of host-pathogen interactions occurring during H. pylori infection.

Cholesterol enhances Helicobacter pylori resistance to antibiotics and LL-37.

The human gastric pathogen Helicobacter pylori steals host cholesterol, modifies it by glycosylation, and incorporates the glycosylated cholesterol onto its surface via a cholesterol glucosyltransferase, encoded by cgt. The impact of cholesterol on H. pylori antimicrobial resistance is unknown. H. pylori strain 26695 was cultured in Ham's F12 chemically defined medium in the presence or absence of cholesterol. The two cultures were subjected to overnight incubations with serial 2-fold dilutions of 12 antibiotics, six antifungals, and seven antimicrobial peptides (including LL-37 cathelicidin and human alpha and beta defensins). Of 25 agents tested, cholesterol-grown H. pylori cells were substantially more resistant (over 100-fold) to nine agents than were H. pylori cells grown without cholesterol. These nine agents included eight antibiotics and LL-37. H. pylori was susceptible to the antifungal drug pimaricin regardless of cholesterol presence in the culture medium. A cgt mutant retained cholesterol-dependent resistance to most antimicrobials but displayed increased susceptibility to colistin, suggesting an involvement of lipid A. Mutation of lpxE, encoding lipid A1-phosphatase, led to loss of cholesterol-dependent resistance to polymyxin B and colistin but not other antimicrobials tested. The cgt mutant was severely attenuated in gerbils, indicating that glycosylation is essential in vivo. These findings suggest that cholesterol plays a vital role in virulence and contributes to the intrinsic antibiotic resistance of H. pylori.

Arcanolysin is a cholesterol-dependent cytolysin of the human pathogen Arcanobacterium haemolyticum.

BACKGROUND: Arcanobacterium haemolyticum is an emerging human pathogen that causes pharyngitis, wound infections, and a variety of occasional invasive diseases. Since its initial discovery in 1946, this Gram positive organism has been known to have hemolytic activity, yet no hemolysin has been previously reported. A. haemolyticum also displays variable hemolytic activity on laboratory blood agar that is dependent upon which species the blood is derived. RESULTS: Here we describe a cholesterol-dependent cytolysin (CDC) secreted by A. haemolyticum, designated arcanolysin (aln), which is present in all strains (n = 52) tested by DNA dot hybridization. Among the known CDCs, ALN is most closely related to pyolysin (PLO) from Trueperella (formerly Arcanobacterium) pyogenes. The aln probe, however, did not hybridize to DNA from T. pyogenes. The aln open reading frame has a lower mol %G+C (46.7%) than the rest of the A. haemolyticum genome (53.1%) and is flanked by two tRNA genes, consistent with probable acquisition by horizontal transfer. The ALN protein (~ 64 kDa) contains a predicted signal sequence, a putative PEST sequence, and a variant undecapeptide within domain 4, which is typically important for function of the toxins. The gene encoding ALN was cloned and expressed in Escherichia coli as a functional recombinant toxin. Recombinant ALN had hemolytic activity on erythrocytes and cytolytic activity on cultured cells from human, rabbit, pig and horse origins but was poorly active on ovine, bovine, murine, and canine cells. ALN was less sensitive to inhibition by free cholesterol than perfringolysin O, consistent with the presence of the variant undecapeptide. CONCLUSIONS: ALN is a newly identified CDC with hemolytic activity and unique properties in the CDC family and may be a virulence determinant for A. haemolyticum.

Multiple caspases mediate acute renal cell apoptosis induced by bacterial cell wall components.

The stimulus for caspase-mediated renal cell apoptosis in septic acute renal failure (ARF) is unclear. To demonstrate the nephrotoxic effects of bacterial cell wall components, the anti-cellular activity of bacterial muropeptides (muramyl dipeptides), peptidoglycans, and lipopolysaccharides was investigated in rabbit kidney cells. Changes in the cell membrane (APOPercentage™ dye uptake), caspase activities, and DNA degradation were quantified colorimetrically and using densitometric assays and their inhibition by caspase-specific and pan-caspase inhibitors was determined. The onset and levels of APOPercentage™ dye-positive rabbit kidney cells, caspase activities, and DNA degradation were closely associated. Specific caspase-1, -2, -3, -4, -8, -10, and -12 inhibitors reduced caspase-3 activity by ≥40%, but only caspase-3 and -8-specific inhibitors reduced apoptotic DNA levels. Pan-caspase inhibitor Q-VD-OPh was 10-fold more effective at inhibiting rabbit kidney cell death, caspase activation, and DNA degradation than caspase-family inhibitor Z-VAD-FMK. Apoptosis was inhibited effectively by both pan-caspase inhibitors when applied early during the stimulus-to-response period. Multiple initiator and effector caspases were activated suggesting extrinsic, intrinsic, and endoplasmic reticulum/stress apoptotic pathway stimulation in rabbit kidney cells treated with bacterial cell wall components. The results provide in vitro support for bacterial cell wall-induced apoptosis as a pathogenic mechanism of renal cell death in septic ARF and support the potential prophylactic use of pan-caspase inhibitors to suppress septic ARF.

Unique host iron utilization mechanisms of Helicobacter pylori revealed with iron-deficient chemically defined media.

Helicobacter pylori chronically infects the gastric mucosa, where it can be found free in mucus, attached to cells, and intracellularly. H. pylori requires iron for growth, but the sources of iron used in vivo are unclear. In previous studies, the inability to culture H. pylori without serum made it difficult to determine which host iron sources might be used by H. pylori. Using iron-deficient, chemically defined medium, we determined that H. pylori can bind and extract iron from hemoglobin, transferrin, and lactoferrin. H. pylori can use both bovine and human versions of both lactoferrin and transferrin, contrary to previous reports. Unlike other pathogens, H. pylori preferentially binds the iron-free forms of transferrin and lactoferrin, which limits its ability to extract iron from normal serum, which is not iron saturated. This novel strategy may have evolved to permit limited growth in host tissue during persistent colonization while excessive injury or iron depletion is prevented.

Phospholipase D promotes Arcanobacterium haemolyticum adhesion via lipid raft remodeling and host cell death following bacterial invasion.

BACKGROUND: Arcanobacterium haemolyticum is an emerging bacterial pathogen, causing pharyngitis and more invasive infections. This organism expresses an unusual phospholipase D (PLD), which we propose promotes bacterial pathogenesis through its action on host cell membranes. The pld gene is found on a genomic region of reduced %G + C, suggesting recent horizontal acquisition. RESULTS: Recombinant PLD rearranged HeLa cell lipid rafts in a dose-dependent manner and this was inhibited by cholesterol sequestration. PLD also promoted host cell adhesion, as a pld mutant had a 60.3% reduction in its ability to adhere to HeLa cells as compared to the wild type. Conversely, the pld mutant appeared to invade HeLa cells approximately two-fold more efficiently as the wild type. This finding was attributable to a significant loss of host cell viability following secretion of PLD from intracellular bacteria. As determined by viability assay, only 15.6% and 82.3% of HeLa cells remained viable following invasion by the wild type or pld mutant, respectively, as compared to untreated HeLa cells. Transmission electron microscopy of HeLa cells inoculated with A. haemolyticum strains revealed that the pld mutant was contained within intracellular vacuoles, as compared to the wild type, which escaped the vacuole. Wild type-infected HeLa cells also displayed the hallmarks of necrosis. Similarly inoculated HeLa cells displayed no signs of apoptosis, as measured by induction of caspase 3/7, 8 or 9 activities. CONCLUSIONS: These data indicate that PLD enhances bacterial adhesion and promotes host cell necrosis following invasion, and therefore, may be important in the disease pathogenesis of A. haemolyticum infections.

Helicobacter pylori lipopolysaccharide modification, Lewis antigen expression, and gastric colonization are cholesterol-dependent.

BACKGROUND: Helicobacter pylori specifically takes up cholesterol and incorporates it into the bacterial membrane, yet little is currently known about cholesterol's physiological roles. We compared phenotypes and in vivo colonization ability of H. pylori grown in a defined, serum-free growth medium, F12 with 1 mg/ml albumin containing 0 to 50 mug/ml cholesterol. RESULTS: While doubling times were largely unaffected by cholesterol, other overt phenotypic changes were observed. H. pylori strain SS1 grown in defined medium with cholesterol successfully colonized the stomach of gerbils, whereas SS1 grown without cholesterol failed to colonize. H. pylori lipopolysaccharide often displays Lewis X and/or Y antigens. Expression of these antigens measured by whole-cell ELISA was markedly enhanced in response to growth of strain SS1, 26695, or G27 in cholesterol. In addition, electrophoretic analysis of lipopolysaccharide in wild type G27 and in mutants lacking the O-chain revealed structural changes within the oligosaccharide core/lipid A moieties. These responses in Lewis antigen levels and in lipopolysaccharide profiles to cholesterol availability were highly specific, because no changes took place when cholesterol was substituted by beta-sitosterol or bile salts. Disruption of the genes encoding cholesterol alpha-glucosyltransferase or lipid A phosphoethanolamine transferase had no effect on Lewis expression, nor on lipopolysaccharide profiles, nor on the cholesterol responsiveness of these properties. Disruption of the lipid A 1-phosphatase gene eliminated the effect of cholesterol on lipopolysaccharide profiles but not its effect on Lewis expression. CONCLUSIONS: Together these results suggest that cholesterol depletion leads to aberrant forms of LPS that are dependent upon dephosphorylation of lipid A at the 1-position. A tentative model for the observed effects of cholesterol is discussed in which sequential steps of lipopolysaccharide biogenesis and, independently, presentation of Lewis antigen at the cell surface, depend upon membrane composition. These new findings demonstrate that cholesterol availability permits H. pylori to modify its cell envelope in ways that can impact colonization of host tissue in vivo.

Characterization of Bacillus anthracis arginase: effects of pH, temperature, and cell viability on metal preference.

BACKGROUND: Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea. While previously characterized arginases have an alkaline pH optimum and require activation with manganese, arginase from Helicobacter pylori is optimally active with cobalt at pH 6. The arginase from Bacillus anthracis is not well characterized; therefore, this arginase was investigated by a variety of strategies and the enzyme was purified. RESULTS: The rocF gene from B. anthracis was cloned and expressed in E. coli and compared with E. coli expressing H. pylori rocF. In the native organisms B. anthracis arginase was up to 1,000 times more active than H. pylori arginase and displayed remarkable activity in the absence of exogenous metals, although manganese, cobalt, and nickel all improved activity. Optimal B. anthracis arginase activity occurred with nickel at an alkaline pH. Either B. anthracis arginase expressed in E. coli or purified B. anthracis RocF showed similar findings. The B. anthracis arginase expressed in E. coli shifted its metal preference from Ni > Co > Mn when assayed at pH 6 to Ni > Mn > Co at pH 9. Using a viable cell arginase assay, B. anthracis arginase increased dramatically when the cells were grown with manganese, even at final concentrations of <1 muM, whereas B. anthracis grown with cobalt or nickel (> or =500 microM) showed no such increase, suggesting existence of a high affinity and specificity manganese transporter. CONCLUSION: Unlike other eubacterial arginases, B. anthracis arginase displays unusual metal promiscuity. The unique properties of B. anthracis arginase may allow utilization of a specific metal, depending on the in vivo niches occupied by this organism.

Arcanobacterium haemolyticum Phospholipase D Enzymatic Activity Promotes the Hemolytic Activity of the Cholesterol-Dependent Cytolysin Arcanolysin.

Arcanolysin, produced by the human pathogen Arcanobacterium haemolyticum, is a cholesterol-dependent cytolysin. To mediate the pore-formation process, arcanolysin is secreted by A. haemolyticum and then must interact with cholesterol embedded within a host membrane. However, arcanolysin must compete with membrane components, such as the phospholipid sphingomyelin, to interact with cholesterol and form pores. Cholesterol forms transient hydrogen bonds with the extracellular portion of sphingomyelin, shielding cholesterol from extracellular factors, including arcanolysin. A. haemolyticum also produces a sphingomyelin-specific phospholipase D, which removes the choline head from sphingomyelin, leaving cyclic-ceramide phosphate and eliminating the potential for cholesterol sequestration. We hypothesized that the enzymatic activity of phospholipase D decreases sphingomyelin-mediated cholesterol sequestration and increases cholesterol accessibility for arcanolysin. Using purified arcanolysin and phospholipase D, we demonstrate that the enzymatic activity of phospholipase D is necessary to promote arcanolysin-mediated hemolysis in both time- and concentration-dependent manners. Phospholipase D promotion of arcanolysin-mediated cytotoxicity was confirmed in Detroit 562 epithelial cells. Furthermore, we determined that incubating phospholipase D with erythrocytes corresponds with an increase in the amount of arcanolysin bound to host membranes. This observation suggests that phospholipase D promotes arcanolysin-mediated cytotoxicity by increasing the ability of arcanolysin to bind to a host membrane.

Stomaching the Possibility of a Pathogenic Role for Helicobacter pylori in Parkinson's Disease.

While a small subset of Parkinson's disease cases have genetic causes, most cases are sporadic and may have an environmental contributor that has largely remained enigmatic. Remarkably, gastrointestinal symptoms in PD patients serve as a prodrome for the eventual motor dysfunctions. Herein, we review studies exploring a possible link between the gastric human pathogen Helicobacter pylori and PD. We provide plausible and testable hypotheses for how this organism might contribute to PD: 1) a toxin(s) produced by the bacteria; 2) disruption of the intestinal microbiome; 3) local inflammation that crosses the gut-brain axis, leading to neuroinflammation; and 4) manipulation of the pharmacokinetics of the PD drug levodopa by H. pylori, even in those not receiving exogenous levodopa. Key findings are: 1) people with PD are 1.5-3-fold more likely to be infected with H. pylori than people without PD; 2) H. pylori-infected PD patients display worse motor functions than H. pylori-negative PD patients; 3) eradication of H. pylori improves motor function in PD patients over PD patients whose H. pylori was not eradicated; and 4) eradication of H. pylori improves levodopa absorption in PD patients compared to that of PD patients whose H. pylori was not eradicated. Evidence is accumulating that H. pylori has a link with PD, but the mechanism is unclear. Future work should explore the effects of H. pylori on development of PD in defined PD animal models, focusing on the roles of H. pylori toxins, inflammation, levodopa absorption, and microbiome dysbiosis.

Three-Dimensional Printing Antimicrobial and Radiopaque Constructs.

Three-dimensional (3D) printing holds tremendous potential as a tool for patient-specific devices. This proof-of- concept study demonstrated the feasibility, antimicrobial properties, and computed tomography(CT) imaging characteristics of iodine/polyvinyl alcohol (PVA) 3D meshes and stents. Under scanning electron microscopy, cross-linked PVA displays smoother and more compacted filament arrangements. X-ray and transaxial CT images of iodized PVA vascular stents show excellent visibility and significantly higher Hounsfield units of radiopacity than control prints. Three-dimensional PVA prints stabilized by glutaraldehyde cross-linking and loaded with iodine through sublimation significantly suppressed Escherichia coli and Staphylococcus aureus growth in human blood agar disk diffusion assays. It is suggested that PVA 3D printing with iodine represents an important new synthetic platform for generating a wide variety of antimicrobial and high-visibility devices.

Genetic microheterogeneity and phenotypic variation of Helicobacter pylori arginase in clinical isolates.

BACKGROUND: Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid. RESULTS: The degree of variation in arginase was explored at the DNA sequence, enzyme activity and protein expression levels. To this end, arginase activity was measured from 73 minimally-passaged clinical isolates and six laboratory-adapted strains of H. pylori. The rocF gene from 21 of the strains was cloned into genetically stable E. coli and the enzyme activities measured. Arginase activity was found to substantially vary (>100-fold) in both different H. pylori strains and in the E. coli model. Western blot analysis revealed a positive correlation between activity and amount of protein expressed in most H. pylori strains. Several H. pylori strains featured altered arginase activity upon in vitro passage. Pairwise alignments of the 21 rocF genes plus strain J99 revealed extensive microheterogeneity in the promoter region and 3' end of the rocF coding region. Amino acid S232, which was I232 in the arginase-negative clinical strain A2, was critical for arginase activity. CONCLUSION: These studies demonstrated that H. pylori arginase exhibits extensive genotypic and phenotypic variation which may be used to understand mechanisms of microheterogeneity in H. pylori.

Smooth and Rough Biotypes of Arcanobacterium haemolyticum Can Be Genetically Distinguished at the Arcanolysin Locus.

Arcanobacterium haemolyticum is a Gram-positive, ß-hemolytic emerging human pathogen that is classified into smooth or rough biotypes. This bacterial species is also a rare pathogen of animals. Smooth biotypes possess smooth colony edges, are moderate to strong in ß-hemolysis, and predominately cause wound infections. In contrast, rough biotypes possess rough and irregular colony edges, have weak to no ß-hemolytic activity, and predominately cause pharyngitis. Using horse erythrocytes we confirmed that smooth isolates are generally more hemolytic than rough isolates. A hemolysin from A. haemolyticum, arcanolysin (aln/ALN), was recently discovered and is a member of the cholesterol-dependent cytolysin (CDC) family. PCR amplification of aln from all 36 smooth A. haemolyticum isolates yielded the expected 2.0 kb product. While 21 rough isolates yielded the 2.0 kb product, 16 isolates had a 3.2 kb product. The extra 1.2 kb segment was 99% identical to IS911 (insertion sequence) from Corynebacterium diphtheriae. PCR amplification and sequence analysis of the upstream region of aln revealed ~40 nucleotide polymorphisms among 73 clinical isolates from Finland, Denmark, Germany and United States (Nebraska). Remarkably, multi-sequence alignments of the aln upstream region demonstrated that ~90% of the isolates phylogenetically clustered as either smooths or roughs. Differential restriction enzyme analysis of the aln upstream region also demonstrated that the aln upstream region of most (~75%) smooth isolates was cleaved with ClaI while this region in most (~86%) rough isolates was cleaved with XcmI. We conclude that the aln upstream region can be used to genetically distinguish between smooth and rough biotypes of this important emerging pathogen.

The Helicobacter pylori flbA flagellar biosynthesis and regulatory gene is required for motility and virulence and modulates urease of H. pylori and Proteus mirabilis.

Helicobacter pylori and Proteus mirabilis ureases are nickel-requiring metallo-enzymes that hydrolyse urea to NH3 and CO2. In both H. pylori and in an Escherichia coli model of H. pylori urease activity, a high affinity nickel transporter, NixA, is required for optimal urease activity, whereas the urea-dependent UreR positive transcriptional activator governs optimal urease expression in P. mirabilis. The H. pylori flbA gene is a flagellar biosynthesis and regulatory gene that modulates urease activity in the E. coli model of H. pylori urease activity. All flbA mutants of eight strains of H. pylori were non-motile and five had a strain-dependent alteration in urease activity. The flbA gene decreased urease activity 15-fold when expressed in E. coli containing the H. pylori urease locus and the nixA gene; this was reversed by disruption of flbA. The flbA gene decreased nixA transcription. flbA also decreased urease activity three-fold in E. coli containing the P. mirabilis urease locus in a urea- and UreR-dependent fashion. Here the flbA gene repressed the P. mirabilis urease promoter. Thus, FlbA decreased urease activity of both H. pylori and P. mirabilis, but through distinct mechanisms. H. pylori wild-type strain SS1 colonised gerbils at a mean of 5.4 x 10(6) cfu/g of antrum and caused chronic gastritis and lesions in the antrum. In contrast, the flbA mutant did not colonise five of six gerbils and caused no lesions, indicating that motility mediated by flbA was required for colonisation. Because FlbA regulates flagellar biosynthesis and secretion, as well as forming a structural component of the flagellar secretion apparatus, two seemingly unrelated virulence attributes, motility and urease, may be coupled in H. pylori and P. mirabilis and possibly also in other motile, ureolytic bacteria.