Plasmodium species mixed infections in two areas of Manhiça district, Mozambique

We compared the distribution patterns of individual Plasmodium species and mixed-species infections in two geographically close endemic areas, but showing environmental differences. Comparisons concerned circulating Plasmodium infections in both human and mosquito vector populations in the dry and wet seasons, at a micro-epidemiological level (households). Both areas revealed a very high overall prevalence of infection, all year-round and in all age groups. Plasmodium falciparum was the predominant species, being found in the vast majority of infected individuals regardless of the presence of other species. Plasmodium malariae and Plasmodium ovale occurred almost exclusively in mixed infections. Seasonal variation in P. malariae prevalence was observed in one area but not in the other. A decrease in P. malariae prevalence concurred with a marked increase of P. falciparum prevalence. However this was strongly dependent on age and when analysing infections at the individual level, a different pattern between co-infecting species was unveiled. Regarding transmission patterns, in both areas, P. falciparum gametocytes predominated in single infections regardless of age and P. malariae gametocyte carriage increased when its overall prevalence decreased.     

Biochemical and molecular characterization of Saccharomyces cerevisiae strains obtained from sugar-cane juice fermentations and their impact in cachaça production

Saccharomyces cerevisiae strains from different regions of Minas Gerais, Brazil, were isolated and characterized aiming at the selection of starter yeasts to be used in the production of cachaça, the Brazilian sugar cane spirit. The methodology established took into account the screening for biochemical traits desirable in a yeast cachaça producer, such as no H₂S production, high tolerance to ethanol and high temperatures, high fermentative capacity, and the abilities to flocculate and to produce mycocins. Furthermore, the yeasts were exposed to drugs such as 5,5′,5"-trifluor-d,l-leucine and cerulenin to isolate those that potentially overproduce higher alcohols and esters. The utilization of a random amplified polymorphic DNA-PCR method with primers based on intron splicing sites flanking regions of the COX1 gene, as well as microsatellite analysis, was not sufficient to achieve good differentiation among selected strains. In contrast, karyotype analysis allowed a clear distinction among all strains. Two selected strains were experimentally evaluated as cachaça producers. The results suggest that the selection of strains as fermentation starters requires the combined use of biochemical and molecular criteria to ensure the isolation and identification of strains with potential characteristics to produce cachaça with a higher quality standard.Cachaça (pronounced “kha-sha-ssa”), the sugar cane spirit, is the most popular distilled beverage produced in Brazil. The annual production reaches 1.3 billion liters, with 15% being produced in more than 8,500 distilleries in the state of Minas Gerais.Traditional cachaça production relies on a spontaneous fermentative process that is mediated by the microbiota present in the cane juice wort and on the surface of equipments used in the productive process. It has been already demonstrated that in such systems there occurs a succession of yeasts, with Saccharomyces cerevisiae being the predominant species. Cachaça quality depends on the ecology of the microbial populations during an initial spontaneous fermentation (18, 29, 31, 32, 39). The fermentative process occurs through a continuously open fermentative process which is completed within 24 h and generally takes place from May to November, corresponding to the sugar cane harvesting period.Considering the conditions of production usually found in the cachaça distilleries, fermenting yeast populations have to face different types of stress (osmotic, high temperature, and high ethanol concentration). Besides, they might also present some characteristics such as a good fermentative power, no H₂S production, killer activity, flocculation ability, and production of flavoring compounds. Taking all of these factors into account, we have developed a strategy to select yeast strains with appropriated characteristics to produce cachaça with potentially higher-quality standards (52).Parallel to the selection and development of S. cerevisiae strains toward ethanolic fermentations, molecular methods were developed and validated to study the evolution of yeast flora in spontaneous but also in inoculated fermentations. Chromosomal karyotyping by pulsed-field gel electrophoresis is a complex and time-consuming process but is very efficient for the delimitation of S. cerevisiae strains since it makes it possible to distinguish strains at both the intra- and the interspecies levels. Numerous other methods of typing based on DNA polymorphism allow differentiating closely related yeast strains (8, 36, 43, 50). Restriction fragment length polymorphism (RFLP) analysis of mitochondrial DNA (mtDNA) is faster and easier than other methods (23, 31). Digestion of mtDNA with restriction enzymes such as HinfI or RsaI is associated with a high rate of polymorphism and has been used to study the authenticity of commercial wine yeast strains (14, 40, 41). A PCR method based on variations found on the mitochondrial COX1 (a gene coding for cytochrome oxidase) intron number and position has been validated to distinguish S. cerevisiae strains allowing researchers to monitor the evolution of wine fermentations conducted by commercial active dry yeast (27). Moreover, in many other studies, RAPD [random(ly) amplified polymorphic DNA]-PCR with different primers such as EL1 and LA1 has been successfully used to discriminate between wine yeast strains (2) and for differentiation of Saccharomyces species isolated in Brazil (18). In the last few years, fingerprinting of microsatellite or simple sequence repeat loci, short (1 to 10 nucleotides) DNA tandem repeats dispersed throughout the genome and with a high degree of variability, has proven to be useful for distinguishing S. cerevisiae strains (16, 20, 34, 40, 45). These loci exhibit a substantial level of polymorphism and have been used in humans for paternity tests and forensic medicine but also for the demonstration of population structures among indigenous S. cerevisiae strains (24, 42, 43).We describe here the isolation and characterization of S. cerevisiae strains from cachaça and the use of two of these strains in cachaça production. The strains were isolated from local producers and characterized by growth in high-alcohol, high-sugar environments and in the presence of 5,5′,5"-trifluor-d,l-leucine (TFL) and cerulenin to detect overproducers of flavor compounds. We also used molecular methods to evaluate the polymorphisms of yeast strains in the fermentative process. Our results demonstrate that some methodologies based on DNA polymorphism are insufficient to evaluate the diversity of S. cerevisiae population during the cachaça production. The utilization of specific biochemical tests is necessary in order to permit a more precise characterization of the dynamics of the selected strains during the fermentative process. Only by combining biochemical and molecular methods were we able to select strains that showed a suitable profile to be used as starters. The cachaça produced with these strains was also evaluated, and the results demonstrate that the quality of the final product is better than that obtained with a commercial strain also used to produce cachaça.     

Trends in antibiotic resistance in Prevotella species from patients of the University Hospital of Maxillofacial Surgery,Sofia, Bulgaria,in 2003–2009

Head-and-neck infections often involve anaerobes such as Prevotella species. Aim of the present study was to assess the evolution and the factors associated with resistance in Prevotella species to penicillin, clindamycin, metronidazole, tetracycline and β-lactams/β-lactamase inhibitors (BL/BLIs). In total, 192 Prevotella strains, isolated from patients with oral and head-and-neck infections, were evaluated. Common isolates were Prevotella intermedia and Prevotella melaninogenica within the pigmented species as well as Prevotella oris and Prevotella oralis group within the non-pigmented species. Overall resistance was 43.2% for penicillin, 10.9% for clindamycin, 0% for metronidazole. Nonsusceptibility to tetracycline was 29.1% without significant differences in resistance rates between pigmented and other species. Penicillin resistant strains were β-lactamase positive. From 2003–2004 to 2007–2009, penicillin resistance rates increased about four-fold (from 15.4% to 60.6%). Clindamycin resistance did not show evolution, whereas tetracycline nonsusceptibility decreased from 43.3% in 2003–2004 to 20.7% in 2007–2009. Except for one (0.5%) P. oralis strain with intermediate susceptibility to BL/BLIs, the other strains were susceptible to the agents. In conclusion, in Prevotella strains from patients with head-and-neck infections, the resistance rate to penicillin increased, that to clindamycin remained stable and the nonsusceptibility rate to tetracycline decreased during the period. Activity against >99% of Prevotella strains was observed with metronidazole and BL/BLIs. The penicillin resistance and tetracycline nonsusceptibility were associated with the year of study, national antibiotic consumption and possibly with previous treatment (for tetracycline). The evolution of penicillin resistance in Prevotella strains was highly dynamic.     

Virulence and molecular characterization of Toxoplasma gondii isolated from goats in Ceará, Brazil

One hundred and sixty-nine fragments of heart muscle collected from goats in the State of Ceará, Brazil, were analyzed by mouse bioassay with the aim of isolating Toxoplasma gondii. Two T. gondii isolates, named G1 and G2, were obtained and were characterized by PCR-RFLP. In addition, their virulence was evaluated by experimental inoculation into BALB/c mice. The G1 isolate presented high virulence leading to 100% mortality of mice after inoculations with 10¹, 10², and 10³ tachyzoites. The G2 isolate presented low virulence and none of the doses tested lead to mortality of mice. The PCR-RFLP analysis showed that the two isolates are recombinants of the types I/III. However, they differ in the haplotype combination for the genotypes analyzed.     

Quantification and characterization of β-lactam resistance genes in 15 sewage treatment plants from East Asia and North America

The emerging antibiotic resistance genes in the aquatic environment have aroused public concern. As β-lactam is the most widely used group of antibiotics, β-lactam resistance genes were selected to investigate their distribution and diversity in the activated sludge from 15 geographically different sewage treatment plants (STPs) of China, Singapore, USA, and Canada. Specific PCR and quantitative real-time PCR (q-PCR) were used to investigate the occurrence and abundance of nine β-lactam resistance genes. Five genes (OXA-1, OXA-2, OXA-10, ampC, and TEM-1) were detected in most of the sludge collected, while three genes (mecA, CTX-M-1, and SME) were not found in any sludge sample. The total abundances of the six detected β-lactam resistance genes in the 15 STPs varied from 5.34?×?10(1) copies/ng DNA (ampC) to 5.49?×?10(4) copies/ng DNA (OXA-1). Overall, OXA-1 had the highest total concentration, followed by IMP and OXA-10. Noticeably, the abundances of TEM-1 in Chinese STPs were generally higher than those in the STPs of other countries, while the abundances of OXA-2 and IMP in the STPs of North America were much greater than those of East Asia. A total of 78 clones carrying β-lactam resistance genes were randomly selected from six clone libraries for phylogenetic diversity analysis; the similarity of these cloned genes to known β-lactam resistance genes with sequence identities ranged from 96% to 100%. Furthermore, OXA-1, ampC, and IMP were found to be more diverse than the other β-lactam resistance genes.     

Correlation between molecular and conventional genealogies in Aicuña: a rural population from Northwestern Argentina

Aicu?a is a village in the northwest of Argentina, located about 300 km south of La Rioja city, in the province of La Rioja. The population of Aicu?a derives from a founder couple established in the uninhabited Aicu?a valley in the early years of the 17th century. Due to land ownership litigation, the descendants maintained a well-documented genealogy that extends for 12 generations, comprising more than 8,000 individuals. From the historical pedigree of Aicu?a, we selected 14 males with direct patrilineal descent from the 2 most ancient male founders, and 23 donors (9 females and 14 males) with direct matrilineal descent from the most ancient female founder. All 3 founders lived in the 17th century. We collected DNA from buccal swabs and characterized the mitochondrial DNA (mtDNA) and Y haplotypes using 14 Y-specific markers, 11 mtDNA polymorphic markers and sequencing of the mt hypervariable regions 1 and 2. We found four different Y haplotypes: Y1 and Y2 haplotypes of European origin corresponding to the founder ancestors Francisco Páez de Espinoza and Apolinario Orme?o, which were shared by 6 and 3 donors, respectively. Three males selected as Orme?o patrilineal descendants showed a different Y haplotype (Y3), probably originated by erroneous paternity registration due to illegitimacy. The remaining case (haplotype Y4), also assumed to belong to the Orme?o lineage, was probably also due to an erroneously registered paternity. Twenty-two donors showed an association of mtDNA markers corresponding to the Amerindian haplotype A2. The founder of this matrilineage could be traced back for more than 14 generations. The haplotype B of one remaining female did not correspond with the historical pedigree and could be due to an error in the genealogy registration. Our results showed an 85% agreement between conventional and molecular genealogies, with mtDNA markers being Amerindian, and Y markers being European. The methodology used in this report is a tool which could potentially be employed as a precedent for land ownership by Aicu?a villagers and Amerindian populations.     

Cloning, characterization and molecular docking of a highly thermostable β-1,4-glucosidase from Thermotoga petrophila

A genomic DNA fragment, encoding a thermotolerant β-glucosidase, of the obligate anaerobe Thermotoga petrophila RKU-1 was cloned after PCR amplification into Escherichia coli strain BL21 CodonPlus. The purified cloned enzyme was a monomeric, 51.5?kDa protein (by SDS-PAGE) encoded by 1.341?kb gene. The estimated K (m) and V (max) values against p-nitrophenyl-β-D-glucopyranoside were 2.8?mM and 42.7?mmol?min(-1)?mg(-1), respectively. The enzyme was also active against other p-nitrophenyl substrates. Possible catalytic sites involved in hydrolyzing different p-nitrophenyl substrates are proposed based on docking studies of enzyme with its substrates. Because of its unique characters, this enzyme is a potential candidate for industrial applications.     

Physiological and molecular characterisation of Saccharomyces cerevisiae cachaça strains isolated from different geographic regions in Brazil

In this work, 74 Saccharomyces cerevisiae strains isolated from cachaça fermentation of six different geographic regions in Brazil were characterized by mitochondrial DNA restriction fragment length polymorphism (mtDNA-RFLP) and by their ability to grow on stress conditions occurring during the cachaça fermentation process. Cachaça S. cerevisiae strains showed high mtDNA-RFLP polymorphism with the occurrence of 32 different molecular patterns. The S. cerevisiae strains presenting prevalent mtDNA were able to grow better in the stress conditions than strains represented by infrequent patterns. The principal coordinate analysis on 17 stress conditions revealed that the major source of growth variation were high ethanol concentrations and low temperatures. These results indicate that the stress conditions occurring in the fermentation process influence the prevalence of the most adapted S. cerevisiae strains in each distillery. The physiological tests used in our study can be used as a criterion for rapidly selecting autochthonous yeast strains for further purposes such as the selection of fermentative starters of S. cerevisiae strains.     

Epidemiologic and molecular characterization of β-lactamase-producing multidrug-resistant uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients

Uropathogenic Escherichia coli (UPECs) are the predominant cause of asymptomatic bacteriuria (ABU) and symptomatic UTI. In this study, multidrug-resistant (MDR) ABU-UPECs from hospitalized patients of Kolkata, India, were characterized with respect to their ESBL phenotype, acquisition of β-lactamase genes, mobile genetic elements (MGEs), phylotype property, ERIC-PCR profile, sequence types (STs), clonal complexes (CCs) and evolutionary and quantitative relationships and compared to the symptomatic ones to understand their epidemiology and evolutionary origin. Statistically significant incidence of ESBL producers, β-lactamase genes, MGEs and novel phylotype property (NPP) among ABU-UPECs similar to the symptomatic ones indicated the probable incidence of chromosomal plasticity on resistance gene acquisition through MGEs due to indiscriminate drug usage. ERIC-PCR typing and MLST analysis showed clonal heterogeneity and predominance of ST940 (CC448) among asymptomatic isolates akin to symptomatic ones along with the evidence of zoonotic transmissions. Minimum spanning tree analysis showed a close association between ABU-UPEC with known and unidentified STs having NPPs with isolates that belonged to phylogroups clade I, D, and B2. This is the first study that reported the occurrence of MGEs and NPPs among ABU-UPECs with the predominance of ESBL production which displayed the deleterious effect of MDR among this pathogen demanding alternative therapeutic interventions. Moreover, this study for the first time attempted to introduce a new approach to ascertain the phylotype property of unassigned UPECs. Withal, increased recognition, proper understanding and characterization of ABU-UPECs with the implementation of appropriate therapeutic measures against them when necessary are the need of the era which otherwise might lead to serious complications in the vulnerable population.

     

Saccharomyces cerevisiae strains from traditional fermentations of Brazilian cachaça: trehalose metabolism, heat and ethanol resistance

Nine indigenous cachaça Saccharomyces cerevisiae strains and one wine strain were compared for their trehalose metabolism characteristics under non-lethal (40°C) and lethal (52°C) heat shock, ethanol shock and combined heat and ethanol stresses. The yeast protection mechanism was studied through trehalose concentration, neutral trehalase activity and expression of heat shock proteins Hsp70 and Hsp104. All isolates were able to accumulate trehalose and activate neutral trehalase under stress conditions. No correlation was found between trehalose levels and neutral trehalase activity under heat or ethanol shock. However, when these stresses were combined, a positive relationship was found. After pre-treatment at 40°C for 60 min, and heat shock at 52°C for 8 min, eight strains maintained their trehalose levels and nine strains improved their resistance against lethal heat shock. Among the investigated stresses, heat treatment induced the highest level of trehalose and combined heat and ethanol stresses activated the neutral trehalase most effectively. Hsp70 and Hsp104 were expressed by all strains at 40°C and all of them survived this temperature although a decrease in cell viability was observed at 52°C. The stress imposed by more than 5% ethanol (v/v) represented the best condition to differentiate strains based on trehalose levels and neutral trehalase activity. The investigated S. cerevisiae strains exhibited different characteristics of trehalose metabolism, which could be an important tool to select strains for the cachaça fermentation process.     

Identification and characterization of the carboxy-terminal region of Sip-1, a novel autoantigen in Behçet's disease

Given the lack of a serological test specific for Behçet's disease, its diagnosis rests upon clinical criteria. The clinical diagnosis is nevertheless difficult because the disease manifestations vary widely, especially at the onset of disease. The aim of this study was to identify molecules specifically recognized by serum autoantibodies in patients with Behçet's disease and to evaluate their diagnostic value. We screened a cDNA library from human microvascular endothelial cells with serum IgG from two patients with Behçet's disease and isolated a reactive clone specific to the carboxy-terminal subunit of Sip1 (Sip1 C-ter). Using ELISA, we measured IgG, IgM and IgA specific to Sip1 C-ter in patients with various autoimmune diseases characterized by the presence of serum anti-endothelial cell antibodies, such as Behçet's disease, systemic lupus erythematosus, systemic sclerosis and various forms of primary vasculitis, as well as in patients with diseases that share clinical features with Behçet's disease, such as inflammatory bowel disease and uveitis. IgM immunoreactivity to Sip1 C-ter was significantly higher in patients with Behçet's disease and in patients with primary vasculitis than in the other groups of patients and healthy subjects tested (P < 10^-4 by Mann-Whitney test). ELISA detected IgG specific to Sip1 C-ter in sera from 11/56 (20%) patients with Behçet's disease, IgM in 23/56 (41%) and IgA in 9/54 (17%). No sera from patients with systemic lupus erythematosus, systemic sclerosis, inflammatory bowel disease, uveitis or healthy subjects but 45% of sera from patients with primary vasculitis contained IgM specific to Sip1 C-ter. Serum levels of soluble E-selectin, a marker of endothelial activation and inflammation, correlated with levels of serum IgM anti Sip-1 C-ter in patients with Behçet's disease (r = 0.36, P = 0.023). In conclusion, Sip1 C-ter is a novel autoantigen in Behçet's disease. IgM specific to Sip1 C-ter might be useful in clinical practice as an immunological marker of endothelial dysfunction in vasculitis.     

1H, 13C and 15N resonance assignment of the oxidized form (Cys67–Cys70) of the N-terminal domain of PilB from Neisseria meningitidis

We report the nearly complete ¹H, ¹³C and ¹⁵N resonance assignments of the oxidized form (Cys₆₇–Cys₇₀) of the N-terminal domain of PilB from Neisseria meningitidis. Secondary structure determination using CSI method and TALOS leads mainly to the prediction of 7 α-helical and 5 β-sheet parts.     

Molecular characterization of nitrite reductase gene (aniA) and gene product in Neisseria meningitidis isolates: is aniA essential for meningococcal survival?

The present study evaluates sequence conservation in the gene coding for nitrite reductase (aniA) and AniA expression from a panel of Neisseria meningitidis isolates. Sequence analysis of the coding region in 19 disease-associated and 4 carrier strains notwithstanding a high degree of sequence similarity showed a number of nucleotide changes, some of which possibly resulted in premature translation termination or function loss. In particular, in one disease-associated strain a 9-residues insertion was found to be located close to the type I Cu-site and a catalytic histidine at position 280 was mutated into a leucine. In two strains from carriers, a sequence corresponding to a portion of a transposase gene within the aniA was also found. The AniA protein was always expressed, except for these two carriers strains and for other two strains in which the presence of the premature stop codons was recognized. The biochemical properties of the cloned soluble domain of the enzyme (sAniA) from N. meningitidis reference MC58 strain and from a clinical invasive isolate were studied. In particular, biochemical analysis of sAniA from MC58 demonstrated a clear dependence of its catalytic activity upon acidification, while the clinical isolate-derived sAniA was not functional. Thus, the results obtained suggest that the presence of a conserved and functional aniA gene is not essential for meningococcal survival.     

Identification and molecular mapping of PdR1, a primary resistance gene to Pierce’s disease in Vitis

A major quantitative trait locus (QTL) controlling resistance to Pierce’s disease (PD) of grape, caused by the bacterium Xylella fastidiosa (Xf), was identified on a Vitis linkage map and denoted as ‘Pierce’s disease resistance 1’ (PdR1). Placement of the locus was accomplished by evaluating a family of full-sib progeny from a cross of two PD-resistant interspecific hybrids with resistance inherited from Vitis arizonica. Resistance was measured under greenhouse conditions by direct quantification of Xf numbers in stem tissues as well as by evaluation of disease symptoms based on leaf scorch and a cane maturation index (CMI). A large QTL (LOD 17.2) accounting for 72% of the phenotypic variance in bacterial numbers was localized to linkage group 14 of the male parent F8909-17. The approximate 95% confidence interval around the QTL peak extended 5.7 cM when using composite interval mapping. The other disease evaluation methods (leaf scorch and CMI, respectively) placed the resistance QTL to the same region on linkage group 14, although at wider 95% confidence intervals (6.0 and 7.5 cM), lower peak LOD scores (11.9 and 7.7) and accounting for less phenotypic variance (59 and 42%). This is the first report of an Xf resistance QTL mapped in any crop species. The relevance of the markers located in the region spanning the QTL will be discussed, addressing their usefulness for the development of PD-resistant grape cultivars.     

Molecular characterization of a 21.4 kilobase antibiotic resistance plasmid from an α-hemolytic Escherichia coli O108:H- human clinical isolate

This study characterizes the 21.4 kilobase plasmid pECTm80 isolated from Escherichia coli strain 80, an α hemolytic human clinical diarrhoeal isolate (serotype O108:H-). DNA sequence analysis of pECTm80 revealed it belonged to incompatibility group X1, and contained plasmid partition and toxin-antitoxin systems, an R6K-like triple origin (ori) replication system, genes required for replication regulation, insertion sequences IS1R, ISEc37 and a truncated transposase gene (Tn3-like ΔtnpA) of the Tn3 family, and carried a class 2 integron. The class 2 integron of pECTm80 contains an intact cassette array dfrA1-sat2, encoding resistance to trimethoprim and streptothricin, and an aadA1 gene cassette truncated by the insertion of IS1R. The complex plasmid replication system includes α, β and γ origins of replication. Pairwise BLASTn comparison of pECTm80 with plasmid pE001 reveals a conserved plasmid backbone suggestive of a common ancestral lineage. Plasmid pECTm80 is of potential clinical importance, as it carries multiple genes to ensure its stable maintenance through successive bacterial cell divisions and multiple antibiotic resistance genes.     

Epidemiology of Chagas disease in Jaguaruana, Ceará, Brazil. I. Presence of triatomines and index of Trypanosoma cruzi infection in four localities of a rural area

In order to assay the triatomine infestation and domiciliation in the rural area of Jaguaruana district, state of Ceará, Brazil, we studied, from November 2000 to April 2002, 4 localities comprising 158 domiciles as a whole, with an average of 4 inhabitants/house, who are dwelling in there for more than 7 years. Most houses have tile-covered roofs and the walls built with plaster-covered bricks (57%), followed by bricks without plaster (33%), and mud walls (7.5%). A total of 3082 triatomines were captured from different locations, according to the following capture plan: (a). intradomiciles: 238 Triatoma brasiliensis, 6 T. pseudomaculata, 9 Rhodnius nasutus, and 2 Panstrongylus lutzi; (b). peridomiciles (annexes): 2069 T. brasiliensis, 223 T. pseudomaculata, 121 R. nasutus, and 1 P. lutzi; (c). wild, in carnauba palms (Copernicia prunifera): 413 R. nasutus. From the captured triatomines, 1773 (57.5%) were examined. The natural index of Trypanosoma cruzi infection ranged from 10.8% to 30.2% (average of 17%), depending on the species and the location from where the triatomines were captured.     

Natural Phosphoryl and Acyl Variants of Lipid A from Neisseria meningitidis Strain 89I Differentially Induce Tumor Necrosis Factor-�� in Human Monocytes

The native lipooligosaccharide (LOS) from Neisseria meningitidis strain 89I was analyzed by matrix-assisted laser desorption ionization (MALDI) time-of-flight (TOF) mass spectrometry and the spectrum compared with that of the LOS after O-deacylation and hydrogen fluoride treatment. The data are consistent with the presence of natural variations in the LOS, which include a triphosphorylated lipid A (LA) with and without a phosphoethanolamine group, and both hexa- and pentaacylated LA molecules. Thin-layer chromatography was performed on 89I LA produced by hydrolysis of the LOS, and the purified LA molecules were analyzed by MALDI-TOF and tested for their relative ability to induce the secretion of tumor necrosis factor-α by human monocytic THP-1 cells and primary human monocytes. The potency of tumor necrosis factor-α induction varied by ∼2–10-fold, depending on the state of acylation and phosphorylation. The results highlight the significance of phosphorylation along with acylation of the LA component of LOS in stimulation of inflammatory signaling, and suggest that natural strain variation in these moieties may be a feature of meningococcal bacteria, which is of critical importance to the progression of the infection.The lipid A (LA)² portion of the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) of Gram-negative bacteria is an inflammatory, pathogenic component of the bacterial outer membrane (1–3). Our interests lie in the Neisserial LA as it is implicated as a significant contributor to the pathogenesis of infections due to Neisseria meningitidis and Neisseria gonorrhoeae, which are of major public health concern around the world. In particular, N. meningitidis is the leading cause of epidemic meningitis and fatal sepsis in otherwise healthy individuals (4). On average more than 500,000 cases of meningococcal infection occur annually leading to ∼50,000 deaths, and large epidemic outbreaks can cause periodic spikes in occurrence. N. gonorrhoeae is a major cause of sexually transmitted infections, which can lead to pelvic inflammatory disease in 10–20% of infected women who can suffer from chronic pain, infertility, and ectopic pregnancy as a result (5). In addition, a growing number of studies have shown that gonococcal infection can facilitate the transmission of HIV (6).Numerous studies of LA signaling through the toll-like receptor 4 (TLR4) have increased our knowledge of the relationship between the LA structure and its inflammatory and immunogenic activity. The affinity of LA for monomeric binding to MD-2, which is a critical determinant in the agonist activity of LA for TLR4, has been found to be most potent in the hexaacylated compared with penta- or tetraacylated forms (7, 8). More recently, we and others have shown that triggering receptor expressed on myeloid cells-2 binds LOS and LPS in both myeloid and non-myeloid cells and initiates an inflammatory cytokine response (9, 10).Neisserial LOS lacks the repeating O-antigens of the LPS of the Gram-negative enteric bacteria, and differs also in the position, number, and chain length of the acyl groups on the LA. There can be differences in the acyl groups on the LA moieties within individual strains as well as between strains and species of Gram-negative bacteria (11, 12). The tetraacylated lipid IVa, which is a precursor of Escherichia coli LA has been found to be a TLR4 antagonist in human cells (13).In both LPS and LOS the number of phosphate (P) and phosphoethanolamine (PEA) groups on LA can vary (14), which impacts on the bioactivity of the molecule for innate immune responses. Recent work has shown that whereas hexaacyl monophosphoryl LA was restricted to the myeloid differentiation factor 88 (MyD88)-independent pathway, which resulted in T cell activation, the diphosphoryl LA also engaged the MyD88-dependent pathway, which activated NF-κB resulting in the production of TNF-α and other inflammatory cytokines (15). Because of its immunogenic properties, monophosphoryl LA has been approved for use as an adjuvant in a hepatitis B vaccine in Europe (16).We have observed major differences in the induction of proinflammatory cytokines through TLR4-mediated signaling elicited by various LOS purified from meningococcal and gonococcal strains (17). Little is known about the extent and biological significance of the natural structural variation in the LA of LOS occurring within a Neisserial strain. Recently, we reported that structural analyses of native LOS from different Neisserial strains indicated that differences in both acylation and phosphorylation of LA correlated significantly with the potency of LA to induce inflammatory cytokines (18). In this study, we used thin-layer chromatography (TLC) and matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF) mass spectrometry (MS) to investigate the heterogeneity and inflammatory activity of the structural variants of the LA from N. meningitidis strain 89I, the LOS of which we found previously to be the most potent inducer of TNF-α among a group of seven Neisserial strains studied (17).