Invasive potential of nonencapsulated disease isolates of Neisseria meningitidis

The capsule of Neisseria meningitidis is the major virulence factor that enables this bacterium to overcome host immunity elicited by complement and phagocytes, rendering it capable of surviving in blood. As such, nonencapsulated N. meningitidis isolates are generally considered nonpathogenic. Here, we consider the inherent virulence of two nonencapsulated N. meningitidis isolates obtained from our national surveillance of infected blood cultures in Canada. Capsule deficiency of both strains was confirmed by serology and PCR for the ctrA to ctrD genes and siaA to siaC genes, as well as siaD genes specific to serogroups B, C, Y, and W135. In both strains, the capsule synthesis genes were replaced by the capsule null locus, cnl-2. In accordance with a lack of capsule, both strains were fully susceptible to killing by both human and baby rabbit complement. However, in the presence of cytidine-5' monophospho-N-acetylneuraminic acid (CMP-NANA), allowing for lipooligosaccharide (LOS) sialylation, a significant increase of resistance to complement killing was observed. Mass spectrometry of purified LOS did not reveal any uncommon modifications that would explain their invasive phenotype. Finally, in a mouse intraperitoneal challenge model, these nonencapsulated isolates displayed enhanced virulence relative to an isogenic mutant of serogroup B strain MC58 lacking capsule (MC58ΔsiaD). Virulence of all nonencapsulated isolates tested was below that of encapsulated serogroup B strains MC58 and B16B6. However, whereas no mortality was observed with MC58ΔsiaD, 5/10 mice succumbed to infection with strain 2275 and 2/11 mice succumbed to strain 2274. Our results suggest the acquisition of a new virulence phenotype by these nonencapsulated strains.     

Genetic mechanisms for loss of encapsulation in polysialyltransferase-gene-positive meningococci isolated from healthy carriers

Encapsulated Neisseria meningitidis expressing serogroups A, B, C, W-135, or Y remain a major cause of morbidity and mortality globally. This bacterium is, however, a common commensal inhabitant of the human nasopharynx that causes disease infrequently. Isolates obtained from healthy carriers are frequently unencapsulated and therefore essentially avirulent. The lack of capsule can be due to inactivation of capsule synthesis genes by a variety of genetic mechanisms, or the absence of capsule synthesis genes. Analysis of inactivation mechanisms was undertaken in a diverse but representative set of 166 acapsulate meningococci isolated from carriage that possessed capsule synthesis genes. Slipped strand mispairing in the siaA and siaD genes of the capsule synthesis locus was observed in 39 isolates. Insertion sequence (IS) elements (IS1016-like, IS1106 and IS1301) were responsible for the loss of encapsulation in 46 isolates. Irreversible gene silencing events (insertions, deletions, base exchanges) were found in 47 isolates. Two non-synonymous mutations were identified in close vicinity of the putative active site of the UDP-N-acetylglucosamine 2-epimerase encoded by the siaA gene. The mechanisms for loss of encapsulation were not associated with particular meningococcal genotypes. There was no evidence for successive gene silencing events in the capsule genes, suggesting that the irreversible inactivation events observed were the result of short-term, within-host evolution. These observations are consistent with the postulate that particular meningococcal clonal complexes are associated with possession of a capsule and that this association is important for transmission success.     

Capsule acetylation does not impair recognition of serogroup C, W-135 and Y meningococci by human dendritic cells

The capsule polysaccharide of several Neisseria meningitidis serogroups can be modified by O-acetylation in vivo. As capsule expression is a major determinant of the interaction between N. meningitidis and human dendritic cells (DCs), the influence of the capsule polysaccharide acetylation status on activation of DCs was investigated. For serogroup C, W-135 and Y, mutations resulting in a lack of capsule acetylation did not interfere with recognition and phagocytosis of N. meningitidis, induction of DC maturation or triggering of cytokine release. Therefore, acetylation of the meningococcal capsule does not modify the activation of dendritic cells by this pathogen.     

Organization of heat shock dnaK and groE operons of the nosocomial pathogen Enterococcus faecium

Enterococcus faecium is a frequently antibiotic-resistant opportunistic pathogen that is commonly recovered from hospitalized patients. The genetic organization of the dnaK operon was analyzed and was shown to consist of at least four heat shock genes, hrcA-grpE-dnaK-dnaJ. The dnaK/J intergenic region was 140 bp shorter than in E. faecalis. The dnaK operon was expressed from a putative sigma(A)-type promoter (PhrcA) upstream of the hrcA start codon and was preceded by two conserved CIRCE sequences. Northern hybridization revealed the presence of multiple mRNAs in the dnaK operon. Conversely, the groE operon was transcribed as a single mRNA. Induction of dnaK and groEL genes occurred in response to either heat shock or exposure to other stress agents.     

Role of lipopolysaccharide in wheat germ agglutinin-mediated agglutination of Neisseria meningitidis and Neisseria gonorrhoeae.

Wheat germ agglutinin, having specificity for N-acetyl glucosamine, agglutinated known nonencapsulated Neisseria meningtidis strains, but failed to agglutinate encapsulated strains of all serogroups tested Presence of a capsule, therefore, blocked wheat germ agglutinin agglutination of N. meningitidis strains. In contrast, Neisseria gonorrhoeae strains were strongly agglutinated, providing additional evidence for nonencapsulation of N. meningitidis strains. In contrast, Neisseria gonorrhoeae strains were strongly agglutinated, providing additional evidence for nonencapsulation of N. gonorrhoeae. Purified lipopolysaccharide from a single N. meningitidis and N. gonorrhoeae strains tested. Thus, in absence of capsular polysaccharide wheat germ agglutinin agglutinates Niesseria strains through interaction with lipoplysaccharide in the outer membrane. Of 34 nongroupable throat N. meningitidis isolates, 10 failed to agglutinate in wheat germ agglutinin, suggesing that at least some nongroupable. N. meningitidis strains may possess capsule-like materials.     

Development of a multiplex PCR assay for detection and genogrouping of Neisseria meningitidis

Neisseria meningitidis is a leading pathogen of epidemic bacterial meningitis and fulminant sepsis worldwide. Twelve different N. meningitidis serogroups have been identified to date based on antigenic differences in the capsular polysaccharide. However, more than 90% of human cases of N. meningitidis meningitis are the result of infection with just five serogroups, A, B, C, W135, and Y. Efficient methods of detection and genogrouping of N. meningitidis isolates are needed, therefore, in order to monitor prevalent serogroups as a means of disease control and prevention. The capsular gene complex regions have been sequenced from only seven out of the 12 serogroups. In this study, the capsular gene complexes of the remaining five serogroups were sequenced and analyzed. Primers were designed that were specific for N. meningitidis species and for the 12 individual serogroups, and a multiplex PCR assay using these specific primers was developed for N. meningitidis detection and genogrouping. The assay was tested using 15 reference strains covering all 12 serogroups, 143 clinical isolates, and 21 strains from closely related species or from species that cause meningitis. The assay could detect N. meningitidis serogroups and was shown to be specific, with a detection sensitivity of 1 ng of genomic DNA (equivalent to ～4 × 10(5) genomes) or 3 × 10(5) CFU/ml in noncultured mock cerebrospinal fluid (CSF) specimens. This study, therefore, describes for the first time the development of a molecular protocol for the detection of all N. meningitidis serogroups. This multiplex PCR-based assay may have use for the clinical diagnosis and epidemiological surveillance of N. meningitidis.     

Molecular analysis of Mycobacterium tuberculosis phosphate specific transport system in Mycobacterium smegmatis. Characterization of recombinant 38 kDa (PstS-1)

The functionality of the putative Mycobacterium tuberculosis phosphate transport operon was studied by operon- lacZ promoterless fusions in Mycobacterium smegmatis. The expression of the operon genes was evaluated in transformed M. smegmatis growing in medium with low and high phosphate concentration. Although the gene fusions expressed beta-galactosidase in medium with phosphate, a higher activity was detected in bacteria growing in medium with low phosphate. In contrast, alkaline phosphatase activity from M. smegmatis was detected only in bacteria growing in medium with low phosphate. The expression of the operon genes was driven by a promoter located 5' upstream from the start codon of the pstB gene. A second putative internal promoter 5' upstream of the pstS-1 gene was also detected. Furthermore, comparative analysis between the native and recombinant PstS-1 proteins showed that they were very similar. Like the native protein, the recombinant protein was also secreted to the culture medium as a glycosylated band. The results show that M. smegmatis recognized phosphate regulatory signals of the M. tuberculosis phosphate transport operon genes, and open the possibility to study gene phosphate regulation in mycobacteria.     

The phenotypic relationship of Neisseria polysaccharea to commensal and pathogenic Neisseria spp

Eight of 22 non-capsulate strains of Neisseria meningitidis previously isolated from primary school children were re-identified as N. polysaccharea by aminopeptidase reactions and polysaccharide production. N. polysaccharea was not identified amongst 91 non-capsulate strains of N. meningitidis isolated from adults attending the Genito-urinary Medicine clinic, Westminster Hospital, London. The biochemical reactions of N. polysaccharea strains were similar to those of N. lactamica and N. gonorrhoeae, but N. polysaccharea could be distinguished from these organisms by examination of beta-galactosidase activity, carbohydrate reactions and polysaccharide production. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed closer similarity of N. polysaccharea to N. lactamica than to the pathogenic Neisseria spp. An additional finding was variation in the position of one of the major proteins of N. lactamica in the 34-39-Kda region.     

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.     

The prevalence of Staphylococcus aureus with mucoid phenotype in the airways of patients with cystic fibrosis—A prospective study

Background: Staphylococcus aureus is one of the most frequently isolated pathogens in the respiratory tract of CF patients. Recently, we characterized peculiar mucoid S. aureus isolates, which are excessive biofilm formers and which carried a 5bp-deletion within the intergenic region of the ica operon. In this prospective study, we determined the prevalence of mucoid S. aureus-isolates in the airways of CF-patients during a 3-months period.Methods: We analyzed specimens (sputa, throat swabs) from 81 CF patients who attended two CF centers in Münster, Germany. Ten S. aureus isolates were randomly picked from every S. aureus-positive airway specimen and evaluated for mucoidy using Congo Red agar and phenotypic tests. Mucoid isolates were characterized by spa sequence typing, biofilm production and sequencing of the intergenic region of the ica operon to screen for the 5bp-deletion.Results: In 7 of 81 examined patients (8.6%), we detected mucoid S. aureus phenotypes (37 out of 1050 isolates; 3.5%). Twenty-five mucoid isolates carried the 5bp-deletion. Mucoid isolates produced excessive biofilm and were significantly more resistant to certain antibiotics.Conclusions: In our prospective study, mucoid S. aureus was present in 8.6% of S. aureus–positive CF-patients. In 6 of 7 patients, mucoid isolates carried the 5bp-deletion, indicating that also other so far not identified mechanisms cause excessive biofilm formation. Further studies are necessary to ascertain the clinical impact of mucoid S. aureus phenotypes on the severity of the CF disease.     

Translocation and surface expression of lipidated serogroup B capsular Polysaccharide in Neisseria meningitidis

The capsule of N. meningitidis serogroup B, (alpha2-->8)-linked polysialic acid and the capsules of other meningococcal serogroups and of other gram-negative bacterial pathogens are anchored in the outer membrane through a 1,2-diacylglycerol moiety. Previous work on the meningococcal cps complex in Escherichia coli K-12 indicated that deletion of genes designated lipA and lipB caused intracellular accumulation of hyperelongated capsule polymers lacking the phospholipid substitution. To better understand the role of lip and lipB in capsule expression in a meningococcal background, the location, sequence, and relationship to related bacterial capsule genes were defined and specific mutations in lipA and lipB were generated in the serogroup B meningococcal strain NMB. The lipA and lipB genes are located on the 3' end of the ctr operon and are most likely transcribed independently. Inactivation of lipA, lipB, and both resulted in the same total levels of capsular polymer production as in the parental controls; however, these mutants were as sensitive as an unencapsulated mutant to killing by normal human serum. Immunogold electron microscopy and flow cytometric analyses revealed intracellular inclusions of capsular polymers in lipA, lipB, and lipA lipB mutants. Capsular polymers purified from lipA, lipB, and lipA lipB mutants were lipidated. The phospholipid anchor was shown by gas chromatography-mass spectroscopy analysis to be a phosphodiester-linked 1,2-dipalmitoyl (C16:0) glycerol moiety and was identical in structure to that found on the wild-type meningococcal capsule polymers. Thus, lipA and lipB do not encode proteins responsible for diacylglycerophosphatidic acid substitution of the meningococcal capsule polymer; rather, they are required for proper translocation and surface expression of the lipidated polymer.