Highly Similar Sequences of Mature IgA1 Proteases from Neisseria meningitidis, Neisseria gonorrhoeae and Haemophilus influenzae.
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ABSTRACT:
SUBMITTER: Karlinsky D
PROVIDER: S-EPMC9315783 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Neisseria meningitidis, Haemophilus influenzae and Streptococcus pneumoniae are major bacterial agents of meningitis. They each bind the 37/67-kDa laminin receptor (LamR) via the surface protein adhesins: meningococcal PilQ and PorA, H. influenzae OmpP2 and pneumococcal CbpA. We have previously reported that a surface-exposed loop of the R2 domain of CbpA mediates LamR-binding. Here we have identified the LamR-binding regions of PorA and OmpP2. Using truncated recombinant proteins we show that binding is dependent on amino acids 171-240 and 91-99 of PorA and OmpP2, respectively, which are predicted to localize to the fourth and second surface-exposed loops, respectively, of these proteins. Synthetic peptides corresponding to the loops bound LamR and could block LamR-binding to bacterial ligands in a dose dependant manner. Meningococci expressing PorA lacking the apex of loop 4 and H. influenzae expressing OmpP2 lacking the apex of loop 2 showed significantly reduced LamR binding. Since both loops are hyper-variable, our data may suggest a molecular basis for the range of LamR-binding capabilities previously reported among different meningococcal and H. influenzae strains.
| S-EPMC3457995 | biostudies-literature
Project description:An apparently rare Neisseria meningitidis isolate containing one copy of a Neisseria gonorrhoeae 16S rRNA gene is described herein. This isolate was identified as N. meningitidis by biochemical identification methods but generated a positive signal with Gen-Probe Aptima assays for the detection of Neisseria gonorrhoeae. Direct 16S rRNA gene sequencing of the purified isolate revealed mixed bases in signature regions that allow for discrimination between N. meningitidis and N. gonorrhoeae. The mixed bases were resolved by sequencing individually PCR-amplified single copies of the genomic 16S rRNA gene. A total of 121 discrete sequences were obtained; 92 (76%) were N. meningitidis sequences, and 29 (24%) were N. gonorrhoeae sequences. Based on the ratio of species-specific sequences, the N. meningitidis strain seems to have replaced one of its four intrinsic 16S rRNA genes with the gonococcal gene. Fluorescence in situ hybridization (FISH) probes specific for meningococcal and gonococcal rRNA were used to demonstrate the expression of the rRNA genes. Interestingly, the clinical isolate described here expresses both N. meningitidis and N. gonorrhoeae 16S rRNA genes, as shown by positive FISH signals with both probes. This explains why the probes for N. gonorrhoeae in the Gen-Probe Aptima assays cross-react with this N. meningitidis isolate. The N. meningitidis isolate described must have obtained N. gonorrhoeae-specific DNA through interspecies recombination.
| S-EPMC3811666 | biostudies-literature
Project description:Newly identified, nontypable Haemophilus influenzae (H. influenza) strains represent a serious threat to global health. Due to the increasing prevalence of antibiotic resistance, virulence factors have emerged as potential therapeutic targets that would be less likely to promote resistance. IgA1 proteases are secreted virulence factors of many Gram-negative human pathogens. These enzymes play important roles in tissue invasion as well as evasion of the immune response, yet there has been limited work on pharmacological inhibitors. Here, we report the discovery of the first small molecule, nonpeptidic inhibitors of H. influenzae IgA1 proteases. We screened over 47 000 compounds in a biochemical assay using recombinant protease and identified a hit compound with micromolar potency. Preliminary structure-activity relationships produced additional inhibitors, two of which showed improved inhibition and selectivity for IgA protease over other serine proteases. We further showed dose-dependent inhibition against four different IgA1 protease variants collected from clinical isolates. These data support further development of IgA protease inhibitors as potential therapeutics for antibiotic-resistant H. influenza strains. The newly discovered inhibitors also represent valuable probes for exploring the roles of these proteases in bacterial colonization, invasion, and infection of mucosal tissues.
| S-EPMC6625846 | biostudies-literature
Project description:Neisseria gonorrhoeae acetylates its cell wall peptidoglycan (PG) at the C-6 position on N-acetylmuramic acid. To understand the effects of PG acetylation on PG metabolism and release of PG fragments, we have made mutations in the genes responsible for PG acetylation. An insertion mutation in a putative PG acetylase gene (designated pacA) resulted in loss of PG acetylation as detected by a high-performance liquid chromatography-based assay. Sequence analysis of a naturally occurring non-acetylating strain revealed the presence of a 26-bp deletion in pacA. Introduction of the deletion mutation into wild-type gonococci resulted in lack of acetylation, and the phenotype was complemented by the addition of a wild-type copy of pacA at a distant location on the chromosome. Mutations were also introduced into three genes downstream of pacA. The gene directly downstream of pacA was required for acetylation and was designated pacB, whereas the next two genes were not required. Sequences highly similar to pacA and pacB were also found in N. meningitidis and N. lactamica strains, and an insertion in the meningococcal pacA eliminated PG acetylation. Phenotypic analyses of an N. gonorrhoeae pacA mutant did not show any decrease in lysozyme resistance or serum resistance, and the release of PG fragments during growth was unchanged. However, purified PG from the wild-type strain was significantly more resistant to the action of human lysozyme than was PG purified from the pacA mutant. Interestingly, the pacA mutant was more sensitive to EDTA, a compound known to trigger autolysis.
| S-EPMC1231103 | biostudies-literature
Project description:We evaluated two formulations of a new combined Haemophilus influenzae type b (Hib)-meningococcal serogroup C (MenC)-tetanus toxoid (TT) conjugated vaccine and two formulations of a new MenC-TT vaccine (trials 711202/001 and 711202/008; clinical trial register numbers NCT00135486 and NCT00135564 [www.ClinicalTrials.gov]). A total of 520 healthy infants were randomized to receive primary vaccination (at 2, 3, and 4 months) with either MenC-TT plus diphtheria-tetanus-acellular pertussis (DTPa)-hepatitis B virus (HBV)-inactivated poliovirus (IPV)/Hib, Hib-MenC-TT plus DTPa-HBV-IPV, or MenC-CRM(197) plus DTPa-HBV-IPV/Hib (control). At 12 to 15 months, subjects received a polysaccharide challenge with meningococcal polysaccharide C plus a DTPa-HBV-IPV/Hib booster. Immune responses were assessed 1 month after dose 2, 1 month after dose 3, and prior to and 1 month after the booster. After primary vaccination, there was no difference between groups in seroprotection rates as measured by titers of serum bactericidal antibody (SBA) to MenC (> or = 1:8) or concentrations of anti-polyribosyl ribitol phosphate (PRP) antibody (> or = 0.15 microg/ml). Prior to the booster, there was no difference between groups in SBA seroprotection rates, whereas anti-PRP seroprotection rates were significantly higher after priming with Hib-MenC-TT. Booster doses induced large increases in SBA and anti-PRP antibodies in primed groups, indicating successful priming with induction of immune memory. Reactogenicity and safety were similar in all groups during the primary and booster phases. A novel combined Hib-MenC-TT conjugate vaccine induced MenC and Hib responses comparable to those induced by licensed monovalent vaccines. A Hib-MenC-TT conjugate vaccine provides vaccination against two major pathogens in a single injection and is a suitable candidate for use in primary or booster vaccination schedules.
| S-EPMC1865605 | biostudies-literature
Project description:The safety profile of HibMenCY was compared with licensed Hib conjugate vaccines in a pooled analysis that included more than 8,500 subjects who were administered a four-dose series of HibMenCY or commercially available Hib vaccines at 2, 4, 6 and 12-15 mo of age in two primary vaccination and two fourth dose phase 3 studies. In all studies, HibMenCY or Hib vaccine was co-administered with age-appropriate, routinely recommended vaccines. In one primary and one fourth dose study (n = 4180), local and general symptoms were solicited using diary cards for 4 d after each dose. Serious adverse events (SAEs) and the occurrence of adverse events (AEs) indicating new onset of chronic disease (NOCD), rash, and conditions prompting Emergency Room (ER) visits were reported from dose 1 until 6 mo after dose 4. The incidences of solicited local and general symptoms were similar following HibMenCY and commercially available Hib vaccines. For some solicited symptoms (pain at the injection site and irritability), rates were lower in the HibMenCY group compared with the Hib control group (p value < 0.05). There were no statistically significant differences between groups in the incidences of SAEs, NOCDs, rash, or AEs leading to ER visits, with the exceptions of anemia and viral gastroenteritis, which occurred significantly less frequently in those receiving HibMenCY than those receiving commercially available Hib vaccines. In this pooled safety analysis, the safety profile of HibMenCY was similar to the safety profile of licensed monovalent Hib vaccines, despite the addition of meningococcal antigens. These studies are registered at www.clinicaltrials.gov NCT00345579 (primary vaccination study), NCT00345683 (fourth dose vaccination study) and NCT00289783 (primary and fourth dose vaccination studies).
| S-EPMC3426078 | biostudies-literature
Project description:The ppk gene, which codes for the enzyme polyphosphate kinase in Neisseria meningitidis strain BNCV, is preceded by an open reading frame coding for a protein with a predicted size of 19.2 kDa with a typical lipoprotein signal sequence of 21 amino acids. The protein has significant homology to the N-terminal portion of an outer membrane protein from Haemophilus somnus (J. Won and R. W. Griffith, Infect. Immun. 61:2813-2821, 1993). Sequencing of the same open reading frame from meningococcus strain M1080 predicted an almost identical protein. Antisera were raised against the lipoprotein, expressed in Escherichia coli as a fusion protein with glutathione S-transferase. The antisera reacted with meningococcal membrane fractions on a Western blot (immunoblot) but did not elicit complement-dependent bactericidal activity. Restriction enzyme digestion demonstrated conservation of this portion of the meningococcal and gonococcal chromosomes. However, antisera raised to the recombinant protein showed that the protein was absent from all strains of gonococcus tested. The sequences of the gene from several strains of Neisseria gonorrhoeae and N. meningitidis were compared and found to be almost identical, except that the coding sequences from all of the gonococcal strains were terminated prematurely as a result of a frameshift mutation. The significance of the remarkable conservation of these gonococcal genes is discussed.
| S-EPMC173202 | biostudies-other
Project description:BackgroundSeveral in-house PCR-based assays have been described for the detection of bacterial meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae, and Haemophilus influenzae from clinical samples. PCR-based methods targeting different bacterial genes are frequently used by different laboratories worldwide, but no standard method has ever been established. The aim of our study was to compare different in-house and a commercial PCR-based tests for the detection of bacterial pathogens causing meningitis and invasive disease in humans.MethodsA total of 110 isolates and 134 clinical samples (99 cerebrospinal fluid and 35 blood samples) collected from suspected cases of invasive disease were analyzed. Specific sets of primers frequently used for PCR-diagnosis of the three pathogens were used and compared with the results achieved using the multiplex approach described here. Several different gene targets were used for each microorganism, namely ctrA, crgA and nspA for N. meningitidis, ply for S. pneumoniae, P6 and bexA for H. influenzae.ResultsAll used methods were fast, specific and sensitive, while some of the targets used for the in-house PCR assay detected lower concentrations of genomic DNA than the commercial method. An additional PCR reaction is described for the differentiation of capsulated and non-capsulated H. influenzae strains, the while commercial method only detects capsulated strains.ConclusionsThe in-house PCR methods here compared showed to be rapid, sensitive, highly specific, and cheaper than commercial methods. The in-house PCR methods could be easily adopted by public laboratories of developing countries for diagnostic purposes. The best results were achieved using primers targeting the genes nspA, ply, and P6 which were able to detect the lowest DNA concentrations for each specific target.
| S-EPMC9427638 | biostudies-literature
Project description:BackgroundSince the appearance of COVID-19 in January 2020, invasive bacterial infections have decreased significantly worldwide. However, alterations in age and sex distributions, clinical forms, phenotypes, and genotypes of isolates have not been analyzed. Our goal is to present and discuss these data considering the current COVID-19 pandemic situation. Methods: The data of the national reference center for meningococci and Haemophilus influenzae in France were mined to examine the above aspects of invasive bacterial infection before (2018-2019) and after (2020-2021) the COVID-19 pandemic. Detailed epidemiological, clinical, and microbiological data were collected, and whole genome sequencing was carried out on meningococcal isolates (n = 1466). Results: In addition to the overall decline in the number of cases, various changes in age, sex, and phenotypes of isolates were also noted. As for N. meningitidis, more cases were observed in adults, as well as more invasive pneumopathies. Furthermore, fewer hyperinvasive meningococcal genotypes have circulated since COVID-19 emerged. The situation has been different for H. influenzae, as the number of invasive cases among adults decreased due to a reduction in non-typeable isolates. In contrast, cases due to serotypeable isolates, particularly serotypes a and b, increased in children <5 years-old. Conclusions: It is possible that measures implemented to stop COVID-19 may have reduced the circulation of N. meningitidis and H. influenzae isolates, but to a variable extent. This may be due to differences in circulation between these two species according to age groups. Vaccination schedules against these two species may have also influenced the evolution of these invasive bacterial infections since the emergence of the COVID-19 pandemic.
| S-EPMC9147110 | biostudies-literature
Project description:A single-tube 5' nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (ctrA), capsulation (bexA), and pneumolysin (ply) gene targets specific for N. meningitidis, H. influenzae, and S. pneumoniae, respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for N. meningitidis, H. influenzae, and S. pneumoniae were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The ctrA primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the ply primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the bexA primers amplified H. influenzae types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (n = 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal, H. influenzae, and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The ctrA primer set used in the multiplex PCR was found to be more sensitive (P < 0.0001) than the ctrA primers that had been used for meningococcal PCR testing at that time.
| S-EPMC87969 | biostudies-literature