Julia Hauser

Optimized method for preparation of DNA from pathogenic and environmental mycobacteria

ABSTRACT Genomic studies on pathogenic and environmental mycobacteria are of growing interest for understanding of their evolution, distribution, adaptation, and host-pathogen interaction. Since most mycobacteria are slow growers, material from in vitro cultures is usually scarce. The robust mycobacterial cell wall hinders both experimental cell lysis and efficient DNA extraction. Here, we compare elements of several DNA preparation protocols and describe a method that is economical and practical and reliably yields large amounts—usually 10-fold increased compared to earlier protocols—of highly pure genomic DNA for sophisticated downstream applications. This method was optimized for cultures of a variety of pathogenic and environmental mycobacterial species and proven to be suitable for direct mycobacterial DNA extraction from infected insect specimens.

Emergence of a New Epidemic Neisseria meningitidis Serogroup A Clone in the African Meningitis Belt: High-Resolution Picture of Genomic Changes That Mediate Immune Evasion

ABSTRACT In the African “meningitis belt,” outbreaks of meningococcal meningitis occur in cycles, representing a model for the role of host-pathogen interactions in epidemic processes. The periodicity of the epidemics is not well understood, nor is it currently possible to predict them. In our longitudinal colonization and disease surveys, we have observed waves of clonal replacement with the same serogroup, suggesting that immunity to noncapsular antigens plays a significant role in natural herd immunity. Here, through comparative genomic analysis of 100 meningococcal isolates, we provide a high-resolution view of the evolutionary changes that occurred during clonal replacement of a hypervirulent meningococcal clone (ST-7) by a descendant clone (ST-2859). We show that the majority of genetic changes are due to homologous recombination of laterally acquired DNA, with more than 20% of these events involving acquisition of DNA from other species. Signals of adaptation to evade herd immunity were indicated by genomic hot spots of recombination. Most striking is the high frequency of changes involving the pgl locus, which determines the glycosylation patterns of major protein antigens. High-frequency changes were also observed for genes involved in the regulation of pilus expression and the synthesis of Maf3 adhesins, highlighting the importance of these surface features in host-pathogen interaction and immune evasion. IMPORTANCE While established meningococcal capsule polysaccharide vaccines are protective through the induction of anticapsular antibodies, findings of our longitudinal studies in the African meningitis belt have indicated that immunity to noncapsular antigens plays a significant role in natural herd immunity. Our results show that meningococci evade herd immunity through the rapid homologous replacement of just a few key genomic loci that affect noncapsular cell surface components. Identification of recombination hot spots thus represents an eminent approach to gain insight into targets of protective natural immune responses. Moreover, our results highlight the role of the dynamics of the protein glycosylation repertoire in immune evasion by Neisseria meningitidis. These results have major implications for the design of next-generation protein-based subunit vaccines. While established meningococcal capsule polysaccharide vaccines are protective through the induction of anticapsular antibodies, findings of our longitudinal studies in the African meningitis belt have indicated that immunity to noncapsular antigens plays a significant role in natural herd immunity. Our results show that meningococci evade herd immunity through the rapid homologous replacement of just a few key genomic loci that affect noncapsular cell surface components. Identification of recombination hot spots thus represents an eminent approach to gain insight into targets of protective natural immune responses. Moreover, our results highlight the role of the dynamics of the protein glycosylation repertoire in immune evasion by Neisseria meningitidis. These results have major implications for the design of next-generation protein-based subunit vaccines.

Large Sequence Polymorphisms Unveil the Phylogenetic Relationship of Environmental and Pathogenic Mycobacteria Related to Mycobacterium ulcerans

ABSTRACT Mycolactone is an immunosuppressive cytotoxin responsible for the clinical manifestation of Buruli ulcer in humans. It was believed to be confined to its etiologic agent, Mycobacterium ulcerans. However, the identification of other mycolactone-producing mycobacteria (MPMs) in other species, including Mycobacterium marinum, indicated a more complex taxonomic relationship. This highlighted the need for research on the biology, evolution, and distribution of such emerging and potentially infectious strains. The reliable genetic fingerprinting analyses presented here aim at both the unraveling of phylogenetic relatedness and of dispersal between environmental and pathogenic mycolactone producers and the identification of genetic prerequisites that enable lateral gene transfer of such plasmids. This will allow for the identification of environmental reservoirs of virulence plasmids that encode enzymes required for the synthesis of mycolactone. Based on dynamic chromosomal loci identified earlier in M. ulcerans, we characterized large sequence polymorphisms for the phylogenetic analysis of MPMs. Here, we identify new insertional-deletional events and single-nucleotide polymorphisms that confirm and redefine earlier strain differentiation markers. These results support other data showing that all MPMs share a common ancestry. In addition, we found unique genetic features specific for M. marinum strain M, the genome sequence strain which is used widely in research.

A broadly-protective vaccine against meningococcal disease in sub-Saharan Africa based on generalized modules for membrane antigens (GMMA).

INTRODUCTION Neisseria meningitidis causes epidemics of meningitis in sub-Saharan Africa. These have mainly been caused by capsular group A strains, but W and X strains are increasingly contributing to the burden of disease. Therefore, an affordable vaccine that provides broad protection against meningococcal disease in sub-Saharan Africa is required. METHODS We prepared Generalized Modules for Membrane Antigens (GMMA) from a recombinant serogroup W strain expressing PorA P1.5,2, which is predominant among African W isolates. The strain was engineered with deleted capsule locus genes, lpxL1 and gna33 genes and over-expressed fHbp variant 1, which is expressed by the majority of serogroup A and X isolates. RESULTS We screened nine W strains with deleted capsule locus and gna33 for high-level GMMA release. A mutant with five-fold increased GMMA release compared with the wild type was further engineered with a lpxL1 deletion and over-expression of fHbp. GMMA from the production strain had 50-fold lower ability to stimulate IL-6 release from human PBMC and caused 1000-fold lower TLR-4 activation in Human Embryonic Kidney cells than non-detoxified GMMA. In mice, the GMMA vaccine induced bactericidal antibody responses against African W strains expressing homologous PorA and fHbp v.1 or v.2 (geometric mean titres [GMT]=80,000-200,000), and invasive African A and X strains expressing a heterologous PorA and fHbp variant 1 (GMT=20-2500 and 18-5500, respectively). Sera from mice immunised with GMMA without over-expressed fHbp v.1 were unable to kill the A and X strains, indicating that bactericidal antibodies against these strains are directed against fHbp. CONCLUSION A GMMA vaccine produced from a recombinant African N. meningitidis W strain with deleted capsule locus, lpxL1, gna33 and overexpressed fHbp v.1 has potential as an affordable vaccine with broad coverage against strains from all main serogroups currently causing meningococcal meningitis in sub-Saharan Africa.

Single nucleotide polymorphisms on the road to strain differentiation in Mycobacterium ulcerans.

ABSTRACT The genomic fine-typing of strains of Mycobacterium ulcerans, the causative agent of the emerging human disease Buruli ulcer, is difficult due to the clonal population structure of geographical lineages. Although large sequence polymorphisms (LSPs) resulted in the clustering of patient isolates originating from across the globe, differentiation of strains within continents using conventional typing methods is very limited. In this study, we analyzed M. ulcerans LSP haplotype-specific insertion sequence elements among 83 M. ulcerans strains and identified single nucleotide polymorphisms (SNPs) that differentiate between regional strains. This is the first genetic discrimination based on SNPs of M. ulcerans strains from African countries where Buruli ulcer is endemic, resulting in the highest geographic resolution of genotyping so far. The findings support the concept of genome-wide SNP analyses as tools to study the epidemiology and evolution of M. ulcerans at a local level.

Lack of Insertional-Deletional Polymorphism in a Collection of Mycobacterium ulcerans Isolates from Ghanaian Buruli Ulcer Patients

ABSTRACT Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for microepidemiological studies aiming to characterize transmission pathways. In a previous comparative genomic hybridization analysis with a microarray covering part of the M. ulcerans genome, we have found extensive insertional-deletional sequence polymorphisms among M. ulcerans isolates of diverse geographic origins that allowed us to distinguish between strains coming from different continents. Since large numbers of insertion sequences are spread over the genome of African M. ulcerans strains, we reasoned that these may drive large sequence polymorphisms in otherwise clonal local mycobacterial populations. In this study, we used a printed DNA microarray covering the whole genome of the Ghanaian M. ulcerans reference strain Agy99 for comparative genomic hybridization. The assay identified multiple regions of difference when DNA of a Japanese M. ulcerans strain was analyzed. In contrast, not a single insertional-deletional genomic variation was found within a panel of disease isolates coming from an area of Ghana where Buruli ulcer is endemic. These results indicate that, despite the expectations deduced from other mycobacterial pathogens, only analyses of single nucleotide polymorphisms will have the potential to differentiate local populations of M. ulcerans.

Optimized DNA preparation from mycobacteria.

Extraction of genomic DNA from mycobacteria requires special consideration because (i) many mycobacterial species exhibit extremely slow growth, and thus produce only small amounts of starting material, and (ii) a robust and waxy cell wall renders mycobacteria difficult to lyse. Hence, mycobacterial DNA extraction often results in low DNA yields of unsuitable quality. Published protocols for mycobacterial DNA preparations and commercially available extraction kits are mainly designed for the isolation of small amounts of genomic material suitable for polymerase chain reaction (PCR)-based applications like species identification. However, such DNA quantities and qualities are usually not sufficient for contemporary genomic analyses such as whole genome sequence analysis, single nucleotide polymorphism (SNP) detection, or DNA microarrays, or for investigations of bacterial evolution, virulence, or epidemiology on a world-wide population level. Moreover, most protocols that achieve a high standard in DNA recovery typically employ large reaction volumes and thus require milliliter-scale plasticware and centrifugal equipment as well as large amounts of chemicals, all of which are costly both in purchase and disposal. The DNA extraction method described here was established to address the challenges that result from the slow growth and distinct cell wall composition of mycobacteria, and to greatly enhance both yield and purity of mycobacterial DNA preparations in a small extraction volume. Designed to be performed using 1.5-mL reaction tubes and the corresponding equipment, the method is economical and practical, and reliably yields large amounts of pure genomic DNA--increases of at least 10-fold as compared to earlier protocols.

A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A

Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement) are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs). SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20–30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA). These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae.

Emergence and genomic diversification of a virulent serogroup W:ST-2881(CC175) Neisseria meningitidis clone in the African meningitis belt

Countries of the African ‘meningitis belt’ are susceptible to meningococcal meningitis outbreaks. While in the past major epidemics have been primarily caused by serogroup A meningococci, W strains are currently responsible for most of the cases. After an epidemic in Mecca in 2000, W:ST-11 strains have caused many outbreaks worldwide. An unrelated W:ST-2881 clone was described for the first time in 2002, with the first meningitis cases caused by these bacteria reported in 2003. Here we describe results of a comparative whole-genome analysis of 74 W:ST-2881 strains isolated within the framework of two longitudinal colonization and disease studies conducted in Ghana and Burkina Faso. Genomic data indicate that the W:ST-2881 clone has emerged from Y:ST-175(CC175) bacteria by capsule switching. The circulating W:ST-2881 populations were composed of a variety of closely related but distinct genomic variants with no systematic differences between colonization and disease isolates. Two distinct and geographically clustered phylogenetic clonal variants were identified in Burkina Faso and a third in Ghana. On the basis of the presence or absence of 17 recombination fragments, the Ghanaian variant could be differentiated into five clusters. All 25 Ghanaian disease isolates clustered together with 23 out of 40 Ghanaian isolates associated with carriage within one cluster, indicating that W:ST-2881 clusters differ in virulence. More than half of the genes affected by horizontal gene transfer encoded proteins of the ‘cell envelope’ and the ‘transport/binding protein’ categories, which indicates that exchange of non-capsular antigens plays an important role in immune evasion.

PCR Typing of Mycobacterium ulcerans Lineages Based on Large Sequence Polymorphisms

Genetic fine typing of Mycobacterium ulcerans, the causative agent of the neglected tropical disease Buruli ulcer, has long been hampered by the remarkable genetic homogeneity of patient isolates. However, large sequence polymorphisms (LSPs), mainly originating from insertional-deletional genomic events, allow the differentiation between major lineages of M. ulcerans and other mycolactone producing mycobacteria (MPMs). Here, we describe an LSP-based rapid PCR typing scheme for these mycobacteria. Application of this approach to a selection of mycobacterial strains isolated from Buruli ulcer patients in Japan shows that they belong to the M. ulcerans Asian haplotype.