Control of pili and sialyltransferase expression in Neisseria gonorrhoeae is mediated by the transcriptional regulator CrgA.

Contact-regulated gene A (CrgA) is a transcriptional regulator present in the pathogenic Neisseria that functions as both an activator and a repressor of transcription following contact with host cells. While its mechanism of action has been studied extensively in Neisseria meningitidis, the specific subset of genes that CrgA targets has been debated. Although the majority of these constitute virulence genes, suggesting that CrgA is important in pathogenesis, no study to date has examined the effects of CrgA in Neisseria gonorrhoeae. In this report, we generated a knockout mutant of crgA (ΔcrgA) in the serum-sensitive gonococcal strain F62. crgA deletion resulted in a reduction in the transcript and protein levels of the primary pilin component pilE via mechanisms that were both contact-dependent and -independent. In contrast, ΔcrgA overexpressed the main determinant of serum resistance in F62, lipooligosaccharide sialyltransferase (Lst). CrgA-mediated lst repression was direct as both recombinant and native CrgA bound to the lst promoter at multiple locations in EMSA and ChIP assays respectively. The increase in Lst levels associated with crgA deletion correlated with enhanced protection against killing by normal human serum. These data suggest a role for CrgA in virulence regulation during both cell adherence and planktonic growth.

NLRP3 inflammasome is a target for development of broad-spectrum anti-infective drugs.

We describe the molecular mode of action and pharmacodynamics of a new molecular entity (NME) that induces the NLRP3 inflammasome-mediated innate immune response. This innate response reduces the pathogen load in an experimentally induced methicillin-resistant Staphylococcos aureus infection, enhances survival in an experimentally induced Gram-negative bacteremia, and overrides the escape mechanism of an obligate intracellular pathogen, viz. Chlamydia pneumoniae. Furthermore, the NME is more effective than standard-of-care antibiotic therapy in a clinically established multifactorial bacterial infection. Analysis of transcriptional regulation of inflammasome signaling genes and innate/adaptive immune genes revealed consistent and significant host changes responsible for the improved outcomes in these infections. These studies pave the way for the development of first-in-class drugs that enhance inflammasome-mediated pathogen clearance and identify the NLRP3 inflammasome as a drug target to address the global problem of emerging new infectious diseases and the reemergence of old diseases in an antibiotic-resistant form.

Rapid sporulation of Bacillus anthracis in a high iron, glucose-free medium.

Spores are the infectious form of Bacillus anthracis (BA), causing cutaneous, inhalation and gastrointestinal anthrax. Because of the possible use of BA spores in a bioterrorism attack, there is considerable interest in studying spore biology. In the laboratory, however, it takes a number of days to prepare spores. Standard sporulation protocols, such as the use of 'PA broth', allow sporulation of BA to occur in 3 to 5 days. Another method employs growth of BA on plates in the dark for several days until they have efficiently sporulated. In efforts to determine the effect of iron on gene expression in BA, we grew BA Sterne strain 7702 in a minimal defined medium (CDM; Koppisch et al., 2005) with various concentrations of iron and glucose. As part of our initial observations, we monitored BA sporulation in CDM via light microscopy. In glucose-free CDM containing 1.5mM Fe(NO(3))(3) (CDM-Fe), >95% of the BA sporulated by 30 h; a far shorter time period than expected. We pursued this observation and we further characterized spores derived from PA and CDM-Fe media. Purified spores derived from PA or CDM-Fe had similar morphologies when viewed by light or electron microscopy, and were equally resistant to harsh conditions including heat (65 degrees C), ice and fresh 30% H(2)O(2). Spore viability in long term cold storage in water was similar for the two spore preparations. Extracted spore coat proteins were evaluated by SDS-PAGE and silver staining, which revealed distinct protein profiles for PA and CDM-Fe spore coat extracts. ELISA assays were done to compare the interaction of the two spore preparations with rabbit antiserum raised against UV-killed Sterne strain 7702 spores prepared in PA medium. Spores from both media reacted identically with this antiserum. Finally, the interaction and fate of spores incubated with macrophages in vitro was very similar. In summary, BA spores induced in CDM-Fe or in PA medium are similar by several criteria, but show distinct extractable coat proteins. CDM-Fe liquid medium can be used for rapid production of BA spores, and could save considerable time in spore research studies.

1-Peptidyl-2-arachidonoyl-3-stearoyl-sn-glyceride: an immunologically active lipopeptide from goat serum (Capra hircus) is an endogenous damage-associated molecular pattern.

Experiments were undertaken to isolate a component of the serum of goat (Capra hircus) that is effective at mediating an innate immune response. This report describes the isolation and structure elucidation of 1-(N-acetyl-ALYDKGYTSKEQKDCVGI)-2-arachidonoyl-3-stearoyl glyceride (1) and its immunomodulatory activity. A dose-response relationship for inflammatory cytokine and chemokine production and release from human fibroblasts incubated with nanomolar concentrations of 1 was shown. Moreover, the membrane transport role of the diacylglycerol moiety in 1 is demonstrated with nanomolar quantities of the transfected N-acetyl peptide moiety of 1 also inducing inflammatory cytokine and chemokine production and release. The apparent EC99 for 1 was 3 ng/mL (1 nM). The likely biological role for naturally occurring 1 as a damage-associated molecular pattern is postulated.

Human alpha-defensins inhibit hemolysis mediated by cholesterol-dependent cytolysins.

Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependent cytolysins. Here, we report that human alpha-defensins HNP-1 to HNP-3 acted in a concentration-dependent manner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO), listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against the other toxins. Human alpha-defensins HNP-4 and HD-6 and human beta-defensin-1, -2, and -3 lacked protective ability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog, in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced binding and protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Abu residues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmon resonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also that solution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibited hemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or to cholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B of Clostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first time these defensins have been shown to inhibit pore-forming toxins. An "ABCDE mechanism" that can account for the ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.

Cellular functions and X-ray structure of anthrolysin O, a cholesterol-dependent cytolysin secreted by Bacillus anthracis.

Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALO exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.

Array lead zirconate titanate/glass piezoelectric microcantilevers for real-time detection of Bacillus anthracis with 10 spores/ml sensitivity and 1/1000 selectivity in bacterial mixtures.

An array of three identical piezoelectric microcantilever sensors (PEMSs) consisting of a lead zirconate titanate layer bonded to a glass layer was fabricated and examined for simultaneous, in situ, real-time, all-electrical detection of Bacillus anthracis (BA) spores in an aqueous suspension using the first longitudinal extension mode of resonance. With anti-BA antibody immobilized on the sensor surfaces all three PEMS exhibited identical BA detection resonance frequency shifts at all tested concentrations, 10-10(7) spores/ml with a standard deviation of less than 10%. The detection concentration limit of 10 spores/ml was about two orders of magnitude lower than would be permitted by flexural peaks. In blinded-sample testing, the array PEMS detected BA in three samples containing BA: (1) 3.3x10(3) spores/ml, (2) a mixture of 3.3x10(3) spores/ml and 3.3x10(5) S. aureus (SA) and P. aeruginosa (PA) per ml, and (3) a mixture of 3.3x10(3) spores/ml with 3.3x10(6) SA+PA/ml. There was no response to a sample containing only 3.3x10(6) SA+PA/ml. These results illustrate the sensitivity, specificity, reusability, and reliability of array PEMS for in situ, real-time detection of BA spores.

Passive administration of monoclonal antibodies to anthrolysin O prolong survival in mice lethally infected with Bacillus anthracis.

BACKGROUND: Bacillus anthracis has two major virulence factors: a tripartite toxin that produces lethal and edema toxins and a polyglutamic acid capsule. A recent report suggested that a toxin belonging to the cholesterol dependent cytolysin (CDC) family, anthrolysin O (ALO) was a new virulence factor for B. anthracis but subsequent studies have questioned its relevance in pathogenesis. In this study, we examined the immunogenicity of recombinant anthrolysin O (rALO) in mice. RESULTS: BALB/c mice immunized with rALO and boosted after two weeks, produce sera with strong Ab responses with a predominance of IgG1 and IgG2a. Five hybridomas to rALO were recovered representing the IgM, IgG1, and IgG2b isotypes. Passive administration of 3 of the five monoclonal antibodies (mAbs) to rALO prior to infection with lethal intravenous (i.v.) B. anthracis Sterne strain infection in mice was associated with enhanced average survival and a greater likelihood of surviving infection. A combination of two mAbs to ALO was more effective than either mAb separately. One mAb (64F8) slowed the toxicity of rALO for J774.16 macrophage-like cells. CONCLUSION: Our results suggest that ALO contributes to the virulence of B. anthracis Sterne strain in this infection model and that Ab response to ALO may contribute to protection in certain circumstances.

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.

Bactericidal activity of chlorine-loaded carbide-derived carbon against Escherichia coli and Bacillus anthracis.

The authors investigated the bactericidal activity of high-chlorine-content nanoporous carbide-derived carbon (CDC) against the Gram-positive, spore-forming bacterium Bacillus anthracis and the common Gram-negative enteric bacterium Escherichia coli. Chlorine-loaded nanoporous CDC produced by thermochemical etching of metals and metalloids by chlorination of carbides can retain up to 40 wt % of chlorine. Etching temperature and the structure and composition of carbides allow tuning the porosity of CDC. The CDC chlorine content depends on the synthesis temperature, pore size, and metal carbide used during preparation. It was observed that chlorine-loaded CDC killed up to 100% of exposed E. coli and B. anthracis spores and vegetative cells in a dose and time-dependent manner. CDC containing higher concentrations of chlorine killed bacteria to a greater extent and faster than did CDC containing lesser concentrations of chlorine. The results suggest that chlorine-loaded CDC can be used in several commercial, defense, and industrial activities and processes to kill bacteria.

The Bacillus anthracis cholesterol-dependent cytolysin, Anthrolysin O, kills human neutrophils, monocytes and macrophages.

BACKGROUND: Bacillus anthracis is an animal and human pathogen whose virulence is characterized by lethal and edema toxin, as well as a poly-glutamic acid capsule. In addition to these well characterized toxins, B. anthracis secretes several proteases and phospholipases, and a newly described toxin of the cholesterol-dependent cytolysin (CDC) family, Anthrolysin O (ALO). RESULTS: In the present studies we show that recombinant ALO (rALO) or native ALO, secreted by viable B. anthracis, is lethal to human primary polymorphonuclear leukocytes (PMNs), monocytes, monocyte-derived macrophages (MDMs), lymphocytes, THP-1 monocytic human cell line and ME-180, Detroit 562, and A549 epithelial cells by trypan blue exclusion or lactate dehydrogenase (LDH) release viability assays. ALO cytotoxicity is dose and time dependent and susceptibility to ALO-mediated lysis differs between cell types. In addition, the viability of monocytes and hMDMs was assayed in the presence of vegetative Sterne strains 7702 (ALO+), UT231 (ALO-), and a complemented strain expressing ALO, UT231 (pUTE544), and was dependent upon the expression of ALO. Cytotoxicity of rALO is seen as low as 0.070 nM in the absence of serum. All direct cytotoxic activity is inhibited by the addition of cholesterol or serum concentration as low as 10%. CONCLUSION: The lethality of rALO and native ALO on human monocytes, neutrophils, macrophages and lymphocytes supports the idea that ALO may represent a previously unidentified virulence factor of B. anthracis. The study of other factors produced by B. anthracis, along with the major anthrax toxins, will lead to a better understanding of this bacterium's pathogenesis, as well as provide information for the development of antitoxin vaccines for treating and preventing anthrax.

Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis.

Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.

Differential expression and transcriptional analysis of the alpha-2,3-sialyltransferase gene in pathogenic Neisseria spp.

Alpha-2,3-sialyltransferase (Lst) is expressed on the outer membrane of Neisseria gonorrhoeae and Neisseria meningitidis and sialylates surface lipooligosaccharide (LOS), facilitating resistance to complement-mediated killing. The enzyme is constitutively expressed from a single gene (lst) and does not undergo antigenic or phase variation. We observed that Triton X-100 extracts of N. gonorrhoeae strain F62 contain about fivefold more sialyltransferase (Stase) activity than extracts of N. meningitidis strain MC58 [symbol: see text]3 a serogroup B acapsulate mutant. We confirmed and expanded upon this observation by showing that extracts of 16 random N. gonorrhoeae isolates contain various amounts of Stase activity, but, on average, 2.2-fold-more Stase activity than extracts of 16 N. meningitidis clinical isolates, representing several serogroups and nongroupable strains. Northern and real-time reverse transcription-PCR analysis of lst transcript levels in N. gonorrhoeae and N. meningitidis revealed that N. gonorrhoeae strains express more lst transcript than N. meningitidis strains. Although transcript levels correlate with average Stase activity observed in the two species, there was not a direct correlation between lst transcript levels and Stase activity among individual isolates of each species. Comparison of lst upstream (5'lst) regions of N. gonorrhoeae and N. meningitidis revealed striking sequence differences characteristic of the two pathogens. N. gonorrhoeae 5'lst regions possess 30-bp and 13-bp elements present as single elements or as tandem repeats that exist only as single elements in the 5'lst regions of N. meningitidis isolates. In addition, the 5'lst regions of N. meningitidis strains have 105-bp transposon-like Correia elements which are absent in N. gonorrhoeae. Chromosomal N. gonorrhoeae 5'lst::lacZ translational fusions expressed 4.75 +/- 0.09-fold (n = 4) higher beta-galactosidase (beta-gal) activity than N. meningitidis 5'lst::lacZ fusions in a host-independent manner, indicating differential expression is governed at least in part by sequence variations in the 5'lst regions. Reporter fusion assays and promoter-mapping analysis revealed that N. gonorrhoeae and N. meningitidis use different promoters with different strengths to transcribe lst. In N. gonorrhoeae, a strong sigma 70 promoter 80 bp upstream of the translational start site is used to transcribe lst, whereas this promoter is inactive in N. meningitidis. In N. meningitidis, a weak sigma 70 promoter at the 3' terminus of a 105-bp Correia repeat-enclosed element 99 bp upstream of the translational start site is used to transcribe lst. We conclude that differential Stase expression between N. gonorrhoeae and N. meningitidis is due at least in part to differential lst gene transcription.

Anthrolysin O and other gram-positive cytolysins are toll-like receptor 4 agonists.

Exposure of bone marrow-derived macrophages (BMDMs) to low concentrations of Bacillus anthracis lethal toxin (LT), whose catalytic subunit is lethal factor (LF), results in induction of a robust apoptotic response dependent on activation of Toll-like receptor (TLR)4. A similar TLR4-dependent apoptotic response is observed when BMDMs are infected with live B. anthracis (Sterne strain). However, TLR4 is considered to be a specific signaling receptor for lipopolysaccharide (LPS), a typical product of gram-negative bacteria, whereas B. anthracis is gram-positive. To understand how B. anthracis can activate TLR4, we analyzed its culture supernatants and found them to contain a potent TLR4-stimulating activity that can also induce apoptosis in macrophages in which the antiapoptotic p38 MAP kinase (whose activation is prevented by LF) was inhibited. Purification of this activity suggested it consists of anthrolysin O (ALO), a member of the cholesterol-dependent cytolysin (CDC) family. We show that recombinant ALO can activate TLR4 in a manner independent of LPS contamination and, together with LT, can induce macrophage apoptosis. We also provide genetic evidence that ALO is required for induction of macrophage apoptosis in response to infection with live B. anthracis and that other CDC family members share the ability to activate TLR4.

Characterization of anthrolysin O, the Bacillus anthracis cholesterol-dependent cytolysin.

We characterized the expression of a putative toxin of Bacillus anthracis, a member of the cholesterol-dependent cytolysin (CDC) family, which includes listeriolysin O, perfringolysin O, and streptolysin O. We named this cytotoxin anthrolysin O (ALO). Although B. anthracis expresses minimal hemolytic activity in clinical settings, we show that Sterne strain 7702 expresses hemolytic activity when grown in brain heart infusion broth or in other rich bacteriologic media, but it secretes barely detectable amounts of hemolysin when grown in Luria-Bertani (LB) broth. Glucose supplementation of LB broth increases the amount of secreted hemolytic activity. Expression of hemolytic activity is maximal during mid- to late-log phase and decreases in the stationary phase. These observations are supported, in part, by semiquantitative reverse transcriptase PCR of alo mRNA. Hemolytic activity in growth supernatants was increased in the presence of reducing agent and almost totally inhibited in a dose-dependent manner by cholesterol; both of these activities are characteristic of a CDC toxin. A mutant of Sterne strain 7702, strain UT231, in which the alo gene was deleted and replaced by a kanamycin cassette, secreted barely detectable hemolytic activity into the growth medium. When strain UT231 was complemented in trans with native alo on a low-copy-number plasmid [strain UT231(pUTE554)], it regained the ability to secrete hemolytic activity, indicating that ALO is the major hemolysin secreted by this strain of B. anthracis in rich media in vitro. To further support the alo gene product being a hemolysin, recombinant B. anthracis ALO (rALO) purified from Escherichia coli was extremely active against washed human erythrocytes, with complete hemolysis detected at approximately 30 molecules of rALO per erythrocyte. Considering the virulence roles of CDCs for other gram-positive bacteria, we speculate that ALO may have a role in anthrax virulence.

Genome analysis and strain comparison of correia repeats and correia repeat-enclosed elements in pathogenic Neisseria.

Whole genome sequences of Neisseria meningitidis strains Z2491 and MC58 and Neisseria gonorrhoeae FA1090 were analyzed for Correia repeats (CR) and CR-enclosed elements (CREE). A total of 533, 516, and 256 copies of CR and 270, 261, and 102 copies of CREE were found in these three genomes, respectively. The lengths of CREE range from 28 to 348 bp, and the lengths of multicopy CREE appear mainly in the ranges of 154 to 156 bp and 105 to 107 bp. The distribution of CREE lengths is similar between the two N. meningitidis genomes, with a greater number of 154- to 156-bp CREE (163 and 152 copies in N. meningitidis strain Z2491 and N. meningitidis strain MC58, respectively) than 105- to 107-bp CREE (72 and 77 copies). In the N. gonorrhoeae strain FA1090 genome there are relatively more 105- to 107-bp CREE (51 copies) than 154- to 156-bp CREE (36 copies). The genomic distribution of 107-bp CREE also shows similarity between the two N. meningitidis strains (15 copies share the same loci) and differences between N. meningitidis strains and N. gonorrhoeae FA1090 (only one copy is located in the same locus). Detailed sequence analysis showed that both the terminal inverted repeats and the core regions of CREE are composed of distinct basic sequence blocks. Direct TA dinucleotide repeats exist at the termini of all CREE. A survey of DNA sequence upstream of the sialyltransferase gene, lst, in several Neisseria isolates showed that 5 N. meningitidis strains contain a 107-bp CREE in this region but 25 N. gonorrhoeae strains show an exact absence of a 105-bp sequence block (i.e., the 107-bp CREE without a 5' TA dinucleotide) in the same region. Whole-genome sequence analysis confirmed that this 105-bp indel exists in many homologous 107-bp CREE loci. Thus, we postulate that all CREE are made of target TA with indels of various lengths. Analysis of 107-bp CREE revealed that they exist predominantly in intergenic regions and are often near virulence, metabolic, and transporter genes. The abundance of CREE in Neisseria genomes suggests that they may have played a role in genome organization, function, and evolution. Their differential distribution in different pathogenic Neisseria strains may contribute to the distinct behaviors of each Neisseria species.

The Neisseria lipooligosaccharide-specific alpha-2,3-sialyltransferase is a surface-exposed outer membrane protein.

Neisseria gonorrhoeae and Neisseria meningitidis express an approximately 43-kDa alpha-2,3-sialyltransferase (Lst) that sialylates the surface lipooligosaccharide (LOS) by using exogenous (in all N. gonorrhoeae strains and some N. meningitidis serogroups) or endogenous (in other N. meningitidis serogroups) sources of 5'-cytidinemonophospho-N-acetylneuraminic acid (CMP-NANA). Sialylation of LOS can protect N. gonorrhoeae and N. meningitidis from complement-mediated serum killing and from phagocytic killing by neutrophils. The precise subcellular location of Lst has not been determined. We confirm and extend previous studies by demonstrating that Lst is located in the outer membrane and is surface exposed in both N. gonorrhoeae and N. meningitidis. Western immunoblot analysis of subcellular fractions of N. gonorrhoeae strain F62 and N. meningitidis strain MC58 not subset 3 (an acapsulate serogroup B strain) performed with rabbit antiserum raised against recombinant Lst revealed an approximately 43-kDa protein exclusively in outer membrane preparations of both pathogens. Inner membrane, periplasmic, cytoplasmic, and culture supernatant fractions were devoid of Lst, as determined by Western blot analysis. Consistent with this finding, outer membrane fractions of N. gonorrhoeae were significantly enriched for sialyltransferase enzymatic activity. A trace of enzymatic activity was detected in inner membrane fractions, which may have represented Lst in transit to the outer membrane or may have represented inner membrane contamination of outer membrane preparations. Subcellular preparations of an isogenic lst insertion knockout mutant of N. gonorrhoeae F62 (strain ST01) expressed neither a 43-kDa immunoreactive protein nor sialyltransferase activity. Anti-Lst rabbit antiserum bound to whole cells of N. meningitidis MC58 not subset 3 and wild-type N. gonorrhoeae F62 but not to the Lst mutant ST01, indicating the surface exposure of the enzyme. Although the anti-Lst antiserum avidly bound enzymatically active, recombinant Lst, it inhibited Lst (sialyltransferase) activity by only about 50% at the highest concentration of antibody used. On the contrary, anti-Lst antiserum did not inhibit sialylation of whole N. gonorrhoeae cells in the presence of exogenous CMP-NANA, suggesting that the antibody did not bind to or could not access the enzyme active site on the surface of viable Neisseria cells. Taken together, these results indicate that Lst is an outer membrane, surface-exposed glycosyltransferase. To our knowledge, this is the first demonstration of the localization of a bacterial glycosyltransferase to the outer membrane of gram-negative bacteria.