Cloning and sequencing of a gene involved in the synthesis of the capsular polysaccharide of Streptococcus pneumoniae type 3

A 4.5 kb ScaI chromosomal DNA fragment of a clinical isolate of Streptococcus pneumoniae serotype 3 was cloned in Escherichia coli. Combined genetic and molecular analyses have allowed the localization, in a 781 by EcoRV subfragment, of a gene (cap3-1) directly responsible for the transformation of an unencapsulated, serotype 3 mutant to the capsulated phenotype. Comparison of the deduced amino acid sequence of CAP3-1 with the protein sequences compiled in the data banks revealed that the CAP3-1 polypeptide was highly similar to the amino-terminus of the GDP-mannose dehydrogenase of Pseudomonas aeruginosa, an enzyme that participates in the synthesis of the mucoid polysaccharide of this species. In addition, the 32 N-terminal amino acids of CAP3-1 perfectly matched structures common to NAD⁺-binding domains of many dehydrogenases. Our results indicate that the 4.5 kb ScaI fragment might also contain genes common to 13 different pneumococcal serogroups or scrotypes tested. To the best of our knowledge, this is the first time that a gene of the capsular complex of S. pneumoniae has been cloned and sequenced. The findings reported here provide new insights for the study of the molecular biology of the main virulence factor responsible for the pathogenesis of pneumococcal infections and might represent a basic step in the identification of cross-reactive antigens that should allow the preparation of new and improved vaccines.     

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.     

The lytA Gene and the DNA Region Located Downstream of This Gene Are Not Involved in the Formation of the Type 3 Capsular Polysaccharide of Streptococcus pneumoniae

Streptococcus pneumoniae strain M31, an unencapsulated, serotype 2 (S2⁻) mutant having a deletion of at least 5.5 kb containing the gene lytA that encodes the main pneumococcal autolysin, was transformed to the encapsulated serotype 3 (S3⁺) with DNA from the clinical pneumococcal strain 406. Hybridization analysis revealed that the S3⁺ transformants also have the deletion demonstrating that lytA and the DNA region located downstream of this gene are not involved in the encapsulation of S. pneumoniae. Received: 29 February 1996 / Accepted: 1 March 1996     

Genome Analysis of a Highly Virulent Serotype 1 Strain of Streptococcus pneumoniae from West Africa

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and bacteremia, estimated to cause 2 million deaths annually. The majority of pneumococcal mortality occurs in developing countries, with serotype 1 a leading cause in these areas. To begin to better understand the larger impact that serotype 1 strains have in developing countries, we characterized virulence and genetic content of PNI0373, a serotype 1 strain from a diseased patient in The Gambia. PNI0373 and another African serotype 1 strain showed high virulence in a mouse intraperitoneal challenge model, with 20% survival at a dose of 1 cfu. The PNI0373 genome sequence was similar in structure to other pneumococci, with the exception of a 100 kb inversion. PNI0373 showed only15 lineage specific CDS when compared to the pan-genome of pneumococcus. However analysis of non-core orthologs of pneumococcal genomes, showed serotype 1 strains to be closely related. Three regions were found to be serotype 1 associated and likely products of horizontal gene transfer. A detailed inventory of known virulence factors showed that some functions associated with colonization were absent, consistent with the observation that carriage of this highly virulent serotype is unusual. The African serotype 1 strains thus appear to be closely related to each other and different from other pneumococci despite similar genetic content.     

Prevalence and Clonal Distribution of pcpA, psrP and Pilus-1 among Pediatric Isolates of Streptococcus pneumoniae

Streptococcus pneumoniae is the leading cause of vaccine-preventable deaths globally. The objective of this study was to determine the distribution and clonal type variability of three potential vaccine antigens: Pneumococcal serine-rich repeat protein (PsrP), Pilus-1, and Pneumococcal choline binding protein A (PcpA) among pneumococcal isolates from children with invasive pneumococcal disease and healthy nasopharyngeal carriers. We studied by Real-Time PCR a total of 458 invasive pneumococcal isolates and 89 nasopharyngeal pneumococcal isolates among children (total = 547 strains) collected in Barcelona, Spain, from January 2004 to July 2010. pcpA, psrP and pilus-1 were detected in 92.8%, 51.7% and 14.4% of invasive isolates and in 92.1%, 48.3% and 18% of carrier isolates, respectively. Within individual serotypes the prevalence of psrP and pilus-1 was highly dependent on the clonal type. pcpA was highly prevalent in all strains with the exception of those belonging to serotype 3 (33.3% in serotype 3 isolates vs. 95.1% in other serotypes; P<.001). psrP was significantly more frequent in those serotypes that are less apt to be detected in carriage than in disease; 58.7% vs. 39.1% P<.001. Antibiotic resistance was associated with the presence of pilus-1 and showed a negative correlation with psrP. These results indicate that PcpA, and subsequently Psrp and Pilus-1 together might be good candidates to be used in a next-generation of multivalent pneumococcal protein vaccine.     

The Relevance of a Novel Quantitative Assay to Detect up to 40 Major Streptococcus pneumoniae Serotypes Directly in Clinical Nasopharyngeal and Blood Specimens

For epidemiological and surveillance purposes, it is relevant to monitor the distribution and dynamics of Streptococcus pneumoniae serotypes. Conventional serotyping methods do not provide rapid or quantitative information on serotype loads. Quantitative serotyping may enable prediction of the invasiveness of a specific serotype compared to other serotypes carried. Here, we describe a novel, rapid multiplex real-time PCR assay for identification and quantification of the 40 most prevalent pneumococcal serotypes and the assay impacts in pneumonia specimens from emerging and developing countries. Eleven multiplex PCR to detect 40 serotypes or serogroups were optimized. Quantification was enabled by reference to standard dilutions of known bacterial load. Performance of the assay was evaluated to specifically type and quantify S. pneumoniae in nasopharyngeal and blood samples from adult and pediatric patients hospitalized with pneumonia (n = 664) from five different countries. Serogroup 6 was widely represented in nasopharyngeal specimens from all five cohorts. The most frequent serotypes in the French, South African, and Brazilian cohorts were 1 and 7A/F, 3 and 19F, and 14, respectively. When both samples were available, the serotype in blood was always present as carriage with other serotypes in the nasopharynx. Moreover, the ability of a serotype to invade the bloodstream may be linked to its nasopharyngeal load. The mean nasopharyngeal concentration of the serotypes that moved to the blood was 3 log-fold higher than the ones only found in the nasopharynx. This novel, rapid, quantitative assay may potentially predict some of the S. pneumoniae serotypes invasiveness and assessment of pneumococcal serotype distribution.     

A new variant of the capsule 3 cluster occurs in Streptococcus pneumoniae from deceased wild chimpanzees

The presence of new Streptococcus pneumoniae clones in dead wild chimpanzees from the Taï National Park, Côte d''Ivoire, with previous respiratory problems has been demonstrated recently by DNA sequence analysis from samples obtained from the deceased apes. In order to broadenour understanding on the relatedness of these pneumococcal clones to those from humans, the gene locus responsible for biosynthesis of the capsule polysaccharide (CPS) has now been characterized. DNA sequence analysis of PCR fragments identified a cluster named cps3_Taï containing the four genes typical for serotype 3 CPS, but lacking a 5′-region of ≥2 kb which is degenerated in other cps3 loci and not required for type 3 biosynthesis. CPS3 is composed of a simple disaccharide repeat unit comprising glucose and glucuronic acid (GlcUA). The two genes ugd responsible for GlcUA synthesis and wchE encoding the type 3 synthase are essential for CPS3 biosynthesis, whereas both, galU and the 3′-truncated gene pgm are not required due to the presence of homologues elsewhere in the genome. The DNA sequence of cps3_Taï diverged considerably from those of other cps3 loci. Also, the gene pgm _Taï represents a full length version with a nonsense mutation at codon 179. The two genes ugd _Taï and wchE _Taï including the promoter region were transformed into a nonencapsulated laboratory strain S. pneumoniae R6. Transformants which expressed type 3 capsule polysaccharide were readily obtained, documenting that the gene products are functional. In summary, the data indicate that cps3_Taï evolved independent from other cps3 loci, suggesting the presence of specialized serotype 3 S. pneumoniae clones endemic to the Taï National Park area.     

Role of Nucleotide-Binding Oligomerization Domain-Containing (NOD) 2 in Host Defense during Pneumococcal Pneumonia

Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Nucleotide-binding oligomerization domain-containing (NOD) 2 is a pattern recognition receptor located in the cytosol of myeloid cells that is able to detect peptidoglycan fragments of S. pneumoniae. We here aimed to investigate the role of NOD2 in the host response during pneumococcal pneumonia. Phagocytosis of S. pneumoniae was studied in NOD2 deficient (Nod2 ^-/-) and wild-type (Wt) alveolar macrophages and neutrophils in vitro. In subsequent in vivo experiments Nod2 ^-/- and Wt mice were inoculated with serotype 2 S. pneumoniae (D39), an isogenic capsule locus deletion mutant (D39Δcps) or serotype 3 S. pneumoniae (6303) via the airways, and bacterial growth and dissemination and the lung inflammatory response were evaluated. Nod2 ^-/- alveolar macrophages and blood neutrophils displayed a reduced capacity to internalize pneumococci in vitro. During pneumonia caused by S. pneumoniae D39 Nod2 ^-/- mice were indistinguishable from Wt mice with regard to bacterial loads in lungs and distant organs, lung pathology and neutrophil recruitment. While Nod2 ^-/- and Wt mice also had similar bacterial loads after infection with the more virulent S. pneumoniae 6303 strain, Nod2 ^-/- mice displayed a reduced bacterial clearance of the normally avirulent unencapsulated D39Δcps strain. These results suggest that NOD2 does not contribute to host defense during pneumococcal pneumonia and that the pneumococcal capsule impairs recognition of S. pneumoniae by NOD2.     

Streptococcus pneumoniae serotype 3 genotypes in invasive isolates from Colombia

Introduction: Streptococcus pneumoniae serotype 3 is an important cause of pneumonia, bacteremia, and meningitis.Objective: To establish the circulating genotypes of S. pneumoniae serotype 3 isolates recovered from the invasive disease between 1994 to 2015 in Colombia.Materials and methods: Of the 365 S. pneumoniae serotype 3 isolates recovered through the laboratory national surveillance program, 117 isolates were analyzed. Pulsed-field gel electrophoresis was used for genotyping, and multilocus sequence typing was determined in representative isolates.Results: The frequency of this serotype increased from 2.7% between 1994 and 1998 to 9.1% between 2011 and 2015 (p=0.000); 91.7% of the isolates showed a genetic similarity greater than 77% and were related to the Netherlands³-31(PMEN31) clone CC180. Several subtypes were identified, two of which showed antimicrobial resistance.Conclusion: In Colombia, the pneumococcal population of the capsular type 3 shows a continuous and homogeneous circulation relating to the clonal group ST-180.     

Identification of glycosylated regions in pneumococcal PspA conjugated to serotype 6B capsular polysaccharide

Conjugate vaccines are being widely used since their introduction. Nowadays the interest in these vaccines is still growing and new antigens and conjugate chemistry are being studied and developed. Pneumococcal surface protein A (PspA) is one of the most studied pneumococcal antigens and is an important vaccine candidate. One approach to broaden the conjugate vaccine coverage could be the conjugation of the polysaccharide to a pneumococcal protein such as PspA. Previous results have shown that conjugated recombinant fragment of PspA (rPspA) not only maintained but also in some conjugates improved the induction of protective antibodies raised against the protein carrier. We describe here a characterization study to identify the domains of Streptococcus pneumoniae recombinant PspA (rPspA), from family 1 clade 1 and family 2 clade 3, involved in the conjugation with serotype 6B capsular polysaccharide.     

Molecular cloning, expression, and characterization of dnaK in Streptococcus pneumoniae

DnaK is known to be highly conserved in all species and is a major immunogen in Streptococcus pneumoniae. To elucidate the role of dnaK in S. pneumoniae, dnaK was cloned in Escherichia coli using a homologous dnaK probe generated by PCR. The His-tagged DnaK was overexpressed in soluble form and purified from E. coli. Alignment of the deduced DnaK amino acid sequence from nucleotide sequences of the cloned dnaK revealed high homology with DnaK analogs in E. coli (53%) and Staphylococcus aureus (73%). However, anti-pneumococcal DnaK antiserum did not crossreact with DnaK analogs in E. coli, S. aureus and human cells suggesting that pneumococcal DnaK might be a good candidate as a vaccine.     

Streptococcus pneumoniae in Saliva of Dutch Primary School Children

While nasopharyngeal sampling is the gold standard for the detection of Streptococcus pneumoniae carriage, historically seen, saliva sampling also seems highly sensitive for pneumococcal detection. We investigated S. pneumoniae carriage in saliva from fifty schoolchildren by conventional and molecular methods. Saliva was first culture-enriched for pneumococci, after which, DNA was extracted from all bacterial growth and tested by quantitative-PCR (qPCR) for pneumococcus-specific genes lytA and piaA. Next, serotype composition of the samples was determined by serotype-specific qPCRs, conventional-PCRs (cPCR) and sequencing of cPCR amplicons. Although only 2 (4%) of 50 samples were positive by conventional diagnostic culture, 44 (88%) were positive for pneumococci by qPCR. In total, we detected the presence of at least 81 pneumococcal strains representing 20 serotypes in samples from 44 carriers with 23 carriers (52%) positive for multiple (up to 6) serotypes. The number of serotypes detected per sample correlated with pneumococcal abundance. This study shows that saliva could be used as a tool for future pneumococcal surveillance studies. Furthermore, high rates of pneumococcal carriage and co-carriage of multiple pneumococcal strains together with a large number of serotypes in circulation suggests a ubiquitous presence of S. pneumoniae in saliva of school-aged children. Our results also suggest that factors promoting pneumococcal carriage within individual hosts may weaken competitive interactions between S. pneumoniae strains.     

Estimating the Burden of Pneumococcal Pneumonia among Adults: A Systematic Review and Meta-Analysis of Diagnostic Techniques

Background

Pneumococcal pneumonia causes significant morbidity and mortality among adults. Given limitations of diagnostic tests for non-bacteremic pneumococcal pneumonia, most studies report the incidence of bacteremic or invasive pneumococcal disease (IPD), and thus, grossly underestimate the pneumococcal pneumonia burden. We aimed to develop a conceptual and quantitative strategy to estimate the non-bacteremic disease burden among adults with community-acquired pneumonia (CAP) using systematic study methods and the availability of a urine antigen assay.

Methods and Findings

We performed a systematic literature review of studies providing information on the relative yield of various diagnostic assays (BinaxNOW® S. pneumoniae urine antigen test (UAT) with blood and/or sputum culture) in diagnosing pneumococcal pneumonia. We estimated the proportion of pneumococcal pneumonia that is bacteremic, the proportion of CAP attributable to pneumococcus, and the additional contribution of the Binax UAT beyond conventional diagnostic techniques, using random effects meta-analytic methods and bootstrapping. We included 35 studies in the analysis, predominantly from developed countries. The estimated proportion of pneumococcal pneumonia that is bacteremic was 24.8% (95% CI: 21.3%, 28.9%). The estimated proportion of CAP attributable to pneumococcus was 27.3% (95% CI: 23.9%, 31.1%). The Binax UAT diagnosed an additional 11.4% (95% CI: 9.6, 13.6%) of CAP beyond conventional techniques. We were limited by the fact that not all patients underwent all diagnostic tests and by the sensitivity and specificity of the diagnostic tests themselves. We address these resulting biases and provide a range of plausible values in order to estimate the burden of pneumococcal pneumonia among adults.

Conclusions

Estimating the adult burden of pneumococcal disease from bacteremic pneumococcal pneumonia data alone significantly underestimates the true burden of disease in adults. For every case of bacteremic pneumococcal pneumonia, we estimate that there are at least 3 additional cases of non-bacteremic pneumococcal pneumonia.     

Functional Analysis of the Two-Gene Lysis System of the Pneumococcal Phage Cp-1 in Homologous and Heterologous Host Cells

The two lysis genes cph1 and cpl1 of the Streptococcus pneumoniae bacteriophage Cp-1 coding for holin and lysozyme, respectively, have been cloned and expressed in Escherichia coli. Synthesis of the Cph1 holin resulted in bacterial cell death but not lysis. The cph1 gene was able to complement a lambda Sam mutation in the nonsuppressing E. coli HB101 strain to produce phage progeny, suggesting that the holins encoded by both phage genes have analogous functions and that the pneumococcal holin induces a nonspecific lesion in the cytoplasmic membrane. Concomitant expression of both holin and lysin of Cp-1 in E. coli resulted in cell lysis, apparently due to the ability of the Cpl1 lysozyme to hydrolyze the peptidoglycan layer of this bacterium. The functional analysis of the cph1 and cpl1 genes cloned in a pneumococcal mutant with a complete deletion of the lytA gene, which codes for the S. pneumoniae main autolysin, provided the first direct evidence that, in this gram-positive-bacterium system, the Cpl1 endolysin is released to its murein substrate through the activity of the Cph1 holin. Demonstration of holin function was achieved by proving the release of pneumolysin to the periplasmic fraction, which strongly suggested that the holin produces a lesion in the pneumococcal membrane.     

Mycophenolate mofetil inhibits the development of Coxsackie B3-virus-induced myocarditis in mice

Background

Streptococcus pneumoniae possesses large zinc metalloproteinases on its surface. To analyse the importance in virulence of three of these metalloproteinases, intranasal challenge of MF1 outbred mice was carried out using a range of infecting doses of wild type and knock-out pneumococcal mutant strains, in order to compare mice survival.

Results

Observation of survival percentages over time and detection of LD₅₀s of knock out mutants in the proteinase genes in comparison to the type 4 TIGR4 wild type strain revealed two major aspects: i) Iga and ZmpB, present in all strains of S. pneumoniae, strongly contribute to virulence in mice; (ii) ZmpC, only present in about 25% of pneumococcal strains, has a lower influence on virulence in mice.

Conclusions

These data suggest Iga, ZmpB and ZmpC as candidate surface proteins responsible for pneumococcal infection and potentially involved in distinct stages of pneumococcal disease.     

Cloning of a new FLO gene from the flocculating Saccharomyces cerevisiae IM1-8b strain

A flocculation conferring gene was cloned from a genomic library of the flocculating strain Saccharomyces cerevisiae IM1-8b as a 5 kb DNA fragment. The shortest DNA fragment (XbaI-XbaI) able to confer the flocculating phenotype was 3.1 kb. Southern analysis revealed that this gene was not homologous to the already reported FLO1 gene since strong hybridization signals were obtained when chromosomes IV and XII were probed with a digoxygenin-labelled fragment and no signal at all was detected for chromosome I. Partial sequencing data unequivocally ascribed the cloned fragment to chromosome XII. The gene was detected in a variety of S. cerevisiae strains regardless of their being phenotypically flocculating. This gene which, we propose as FLO2, is able to complement the flo1 mutation and is suppressed by suppressors (fsu3) that do not affect other FLO genes.     

A novel gene—samfR involved in early stage ofStreptomyces ansochromogenes differentiation

A 4.6 kb DNA fragment was cloned from the DNA library ofStreptomyces ansochromogenes using a partial DNA fragment located in the downstream of promoter-P_TH4 as probe. The experiments revealed that this DNA fragment consists ofsaw D gene and a 1.4 kbPvu II fragment which can accelerate mycelium formation ofS. ansochromogenes. The nucleotide sequence of 1.4 kb DNA fragment was determined and analysed; the result indicated that the fragment contains one complete open reading frame (ORF) which encodes a protein with 213 amino acids, and this gene was designated assamfR. The deduced protein has 36% amino acid identities and 52% amino acid similarities in comparison with that encoded byhppR gene, which is involved in the regulation of catabolism for 3-(3-hydroxyphenyl) propionate (3HPP) inRhodococcus globerulus. The function ofsamfR gene was studied using strategy of gene disruption, and the resultingsamfR mutant failed to form aerial hyphae and spores, its development and differentiation stopped at the stage of substrate mycelium in contrast with wild type strain. The results showed that thesamfR gene is closely related toS. ansochromogenes differentiation.     

Serotype Distribution and Antibiotic Susceptibility of Streptococcus pneumoniae Strains Carried by Children Infected with Human Immunodeficiency Virus

Background

We studied the serotype distribution and antibiotic susceptibility of Streptococcus pneumoniae isolates carried by children infected with HIV in Jakarta, Indonesia.

Methods

Nasopharyngeal swabs were collected from 90 HIV infected children aged 4 to 144 months. S. pneumoniae was identified by conventional and molecular methods. Serotyping was performed with sequential multiplex PCR and antibiotic susceptibility with the disk diffusion method.

Results

We identified S. pneumoniae carriage in 41 children (46%). Serotype 19F was most common among 42 cultured strains (19%) followed by 19A and 6A/B (10% each), and 23F (7%). Most isolates were susceptible to chloramphenicol (86%), followed by clindamycin (79%), erythromycin (76%), tetracycline (43%), and sulphamethoxazole/trimethoprim (41%). Resistance to penicillin was most common with only 33% of strains being susceptible. Strains of serotypes targeted by the 13-valent pneumococcal conjugate polysaccharide vaccine (PCV13) were more likely to be multidrug resistant (13 of 25 or 52%) compared to non-PCV13 serotype isolates (3 of 17 or 18%; Fisher exact test p = 0.05).

Conclusion

Our study provides insight into the epidemiology of pneumococcal carriage in young HIV patients in Indonesia. These findings may facilitate potential preventive strategies that target invasive pneumococcal disease in Indonesia.