Theo Hoogenboezem

Anti-Staphylococcal Humoral Immune Response in Persistent Nasal Carriers and Noncarriers of Staphylococcus aureus

BACKGROUND Persistent carriers have a higher risk of Staphylococcus aureus infections than noncarriers but a lower risk of bacteremia-related death. Here, the role played by anti-staphylococcal antibodies was studied. METHODS Serum samples from 15 persistent carriers and 19 noncarriers were analyzed for immunoglobulin (Ig) G, IgA, and IgM binding to 19 S. aureus antigens, by means of Luminex technology. Nasal secretions and serum samples obtained after 6 months were also analyzed. RESULTS Median serum IgG levels were significantly higher in persistent carriers than in noncarriers for toxic shock syndrome toxin (TSST)-1 (median fluorescence intensity [MFI] value, 11,554 vs. 4291; P < .001) and staphylococcal enterotoxin (SE) A (742 vs. 218; P < .05); median IgA levels were higher for TSST-1 (P < .01), SEA, and clumping factor (Clf) A and B (P < .05). The in vitro neutralizing capacity of anti-TSST-1 antibodies was correlated with the MFI value (R(2) = 0.93) and was higher in persistent carriers (90.6% vs. 70.6%; P < .05). Antibody levels were stable over time and correlated with levels in nasal secretions (for IgG, R(2) = 0.87; for IgA, R(2) = 0.77). CONCLUSIONS Antibodies to TSST-1 have a neutralizing capacity, and median levels of antibodies to TSST-1, SEA, ClfA, and ClfB are higher in persistent carriers than in noncarriers. These antibodies might be associated with the differences in the risk and outcome of S. aureus infections between nasal carriers and noncarriers.

CodY of Streptococcus pneumoniae: Link between Nutritional Gene Regulation and Colonization

CodY is a nutritional regulator mainly involved in amino acid metabolism. It has been extensively studied in Bacillus subtilis and Lactococcus lactis. We investigated the role of CodY in gene regulation and virulence of the human pathogen Streptococcus pneumoniae. We constructed a codY mutant and examined the effect on gene and protein expression by microarray and two-dimensional differential gel electrophoresis analysis. The pneumococcal CodY regulon was found to consist predominantly of genes involved in amino acid metabolism but also several other cellular processes, such as carbon metabolism and iron uptake. By means of electrophoretic mobility shift assays and DNA footprinting, we showed that most of the targets identified are under the direct control of CodY. By mutating DNA predicted to represent the CodY box based on the L. lactis consensus, we demonstrated that this sequence is indeed required for in vitro DNA binding to target promoters. Similar to L. lactis, DNA binding of CodY was enhanced in the presence of branched-chain amino acids, but not by GTP. We observed in experimental mouse models that codY is transcribed in the murine nasopharynx and lungs and is specifically required for colonization. This finding was underscored by the diminished ability of the codY mutant to adhere to nasopharyngeal cells in vitro. Furthermore, we found that pcpA, activated by CodY, is required for adherence to nasopharyngeal cells, suggesting a direct link between nutritional regulation and adherence. In conclusion, pneumococcal CodY predominantly regulates genes involved in amino acid metabolism and contributes to the early stages of infection, i.e., colonization of the nasopharynx.

The Streptococcal Lipoprotein Rotamase A (SlrA) Is a Functional Peptidyl-prolyl Isomerase Involved in Pneumococcal Colonization

Streptococcus pneumoniae expresses two surface-exposed lipoproteins, PpmA and SlrA, which share homology with distinct families of peptidyl-prolyl isomerases (PPIases). In this study, we demonstrated for the first time that the lipoprotein cyclophilin, SlrA, can catalyze the cis-trans isomerization of proline containing tetrapeptides and that SlrA contributes to pneumococcal colonization. The substrate specificity of SlrA is typical for prokaryotic and eukaryotic cyclophilins, with Suc-Ala-Ala-Pro-Phe-p-nitroanilide (pNA) being the most rapidly catalyzed substrate. In a mouse pneumonia model the slrA knock-out D39ΔslrA did not cause significant differences in the survival times of mice compared with the isogenic wild-type strain. In contrast, a detailed analysis of bacterial outgrowth over time in the nasopharynx, airways, lungs, blood, and spleen showed a rapid elimination of slrA mutants from the upper airways but did not reveal significant differences in the lungs, blood, and spleen. These results suggested that SlrA is involved in colonization but does not contribute significantly to invasive pneumococcal disease. In cell culture infection experiments, the absence of SlrA impaired adherence to pneumococcal disease-specific epithelial and endothelial non-professional cell lines. Adherence of the slrA mutant could not be restored by exogenously added SlrA. Strikingly, deficiency in SlrA did not reduce binding activity to host target proteins, but resulted in enhanced uptake by professional phagocytes. In conclusion, SlrA is a functional, cyclophilin-type PPIase and contributes to pneumococcal virulence in the first stage of infection, namely, colonization of the upper airways, most likely by modulating the biological function of important virulence proteins.

The Role of the Ferric Uptake Regulator (Fur) in Regulation of Helicobacter pylori Iron Uptake

Background. Availability of the essential nutrient iron is thought to vary greatly in the gastric mucosa, and thus the human gastric pathogen Helicobacter pylori requires regulatory responses to these environmental changes. Bacterial iron‐responsive regulation is often mediated by Ferric Uptake Regulator (Fur) homologs, and in this study we have determined the role of H. pylori Fur in regulation of H. pylori iron uptake.

Raman spectroscopic typing reveals the presence of carotenoids in Mycoplasma pneumoniae.

Raman spectroscopy has previously been demonstrated to be a highly useful methodology for the identification and/or typing of micro-organisms. In this study, we set out to evaluate whether this technology could also be applied as a tool to discriminate between isolates of Mycoplasma pneumoniae, which is generally considered to be a genetically highly uniform species. In this evaluation, a total of 104 strains of M. pneumoniae were analysed, including two reference strains (strains M129 and FH), and 102 clinical isolates, which were isolated between 1973 and 2005 and originated from various countries. By Raman spectral analysis (Raman typing) of this strain collection, we were able to reproducibly distinguish six different clusters of strains. An unequivocal correlation between Raman typing and P1 genotyping, which is based on sequence differences in the P1 (or MPN141) gene of M. pneumoniae, was not observed. In the two major Raman clusters that we identified (clusters 3 and 6, which together harboured 81 % of the strains), the different P1 subtypes were similarly distributed, and approximately 76 % isolates were of subtype 1, approximately 20 % of subtype 2 and approximately 5 % of variant 2a. Nevertheless, a relatively high prevalence of P1 subtype 2 strains was found in clusters 2 and 5 (100 %), as well as in cluster 1 (75 %) and cluster 4 (71 %); these clusters, however, harboured a small number of strains. Only two of the strains (2 %) could not be typed correctly. Interestingly, analysis of the Raman spectra revealed the presence of carotenoids in M. pneumoniae. This finding is in line with the identification of M. pneumoniae genes that have similarity with genes involved in a biochemical pathway leading to carotenoid synthesis, i.e. the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Therefore, we hypothesize that M. pneumoniae hosts an MEP-like pathway for carotenoid synthesis. We conclude that Raman spectroscopy is a convenient tool for discriminating between M. pneumoniae strains, and that it presents a promising supplement to the current methods for typing of this bacterium.

Identification and Characterization of a Novel Outer Membrane Protein (OMP J) of Moraxella catarrhalis That Exists in Two Major Forms

Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fishers exact test; P < 0.01). Experiments using two DeltaompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic DeltaompJ2 mutant (unpaired Students t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.

The Mycoplasma pneumoniae MPN229 gene encodes a protein that selectively binds single-stranded DNA and stimulates Recombinase A-mediated DNA strand exchange

BackgroundMycoplasma pneumoniae has previously been characterized as a micro-organism that is genetically highly stable. In spite of this genetic stability, homologous DNA recombination has been hypothesized to lie at the basis of antigenic variation of the major surface protein, P1, of M. pneumoniae. In order to identify the proteins that may be involved in homologous DNA recombination in M. pneumoniae, we set out to characterize the MPN229 open reading frame (ORF), which bears sequence similarity to the gene encoding the single-stranded DNA-binding (SSB) protein of other micro-organisms.ResultsThe MPN229 ORF has the capacity to encode a 166-amino acid protein with a calculated molecular mass of 18.4 kDa. The amino acid sequence of this protein (Mpn SSB) is most closely related to that of the protein predicted to be encoded by the MG091 gene from Mycoplasma genitalium (61% identity). The MPN229 ORF was cloned, and different versions of Mpn SSB were expressed in E. coli and purified to > 95% homogeneity. The purified protein was found to exist primarily as a homo-tetramer in solution, and to strongly and selectively bind single-stranded DNA (ssDNA) in a divalent cation- and DNA substrate sequence-independent manner. Mpn SSB was found to bind with a higher affinity to ssDNA substrates larger than 20 nucleotides than to smaller substrates. In addition, the protein strongly stimulated E. coli Recombinase A (RecA)-promoted DNA strand exchange, which indicated that Mpn SSB may play an important role in DNA recombination processes in M. pneumoniae.ConclusionThe M. pneumoniae MPN229 gene encodes a protein, Mpn SSB, which selectively and efficiently binds ssDNA, and stimulates E. coli RecA-promoted homologous DNA recombination. Consequently, the Mpn SSB protein may play a crucial role in DNA recombinatorial pathways in M. pneumoniae. The results from this study will pave the way for unraveling these pathways and assess their role in antigenic variation of M. pneumoniae.

PerR controls peroxide- and iron-responsive expression of oxidative stress defense genes in Helicobacter hepaticus.

Chronic intestinal and hepatic colonization with the microaerophilic murine pathogen Helicobacter hepaticus can lead to a range of inflammatory diseases of the lower digestive tract. Colonization is associated with an active cellular immune response and production of oxygen radicals. During colonization, H. hepaticus needs to cope with and respond to oxidative stress, and here we report on the role of the H. hepaticus PerR-regulator (HH0942) in the expression of the peroxidase-encoding katA (HH0043) and ahpC (HH1564) genes. Transcription of katA and ahpC was induced by hydrogen peroxide, and by iron restriction of growth media. This iron- and hydrogen peroxide-responsive regulation of katA and ahpC was mediated at the transcriptional level, from promoters directly upstream of the genes. Inactivation of the perR gene resulted in constitutive, iron-independent high-level expression of the katA and ahpC transcripts and corresponding proteins. Finally, inactivation of the katA gene resulted in increased sensitivity of H. hepaticus to hydrogen peroxide and reduced aerotolerance. In H. hepaticus, iron metabolism and oxidative stress defense are intimately connected via the PerR regulatory protein. This regulatory pattern resembles that observed in the enteric pathogen Campylobacter jejuni, but contrasts with the pattern observed in the closely related human gastric pathogen Helicobacter pylori.

A rare TaqI polymorphism in a human complement C4 gene is caused by an additional restriction site in the first intron

We studied the configuration of the complement C4/CYP21 (steroid 21-hydroxylase) region of the human major histocompatibility complex in patients suffering from congenital adrenal hyperplasia (CAH) and in the general population in The Netherlands, using C4 and CYP21 probes and the restriction enzymes TaqI and Bg/II. We found a rare TaqI 3.9-kb restriction fragment in the mother of a CAH patient, and present evidence that this polymorphism is caused by an additional restriction site in the first intron of a complement C4 gene.

The Role of B Cells in Carriage and Clearance of Mycoplasma pneumoniae From the Respiratory Tract of Mice

Background Carriage of Mycoplasma pneumoniae (Mp) in the nasopharynx is considered a prerequisite for pulmonary infection. It is interesting to note that Mp carriage is also detected after infection. Although B cells are known to be involved in pulmonary Mp clearance, their role in Mp carriage is unknown. Methods In this study, we show in a mouse model that Mp persists in the nose after pulmonary infection, similar to humans. Results Infection of mice enhanced Mp-specific immunoglobulin (Ig) M and IgG levels in serum and bronchoalveolar lavage fluid. However, nasal washes only contained elevated Mp-specific IgA. These differences in Ig compartmentalization correlated with differences in Mp-specific B cell responses between nose- and lung-draining lymphoid tissues. Moreover, transferred Mp-specific serum Igs had no effect on nasal carriage in B cell-deficient μMT mice, whereas this enabled μMT mice to clear pulmonary Mp infection. Conclusions We report the first evidence that humoral immunity is limited in clearing Mp from the upper respiratory tract.