Contribution of genomics and transcriptomics to the understanding of the biological role of DNases in Streptococcus agalactiae

Streptococcus agalactiae is the leading cause of neonatal pneumonia, sepsis, and meningitis. Strains III/ST17 emerged as a hypervirulent clone mostly associated with meningitis during late-onset disease. Production of extracellular DNases and biofilm formation have been proposed to explain the leading role of this clone in neonatal meningitis. The aim of this thesis was to shed some light on the genetic background of DNase production in S. agalactiae strains isolated in humans presenting diverse cell tropism (invasive vs carriage). Genomic approaches were used to decipher the molecular basis of the differential production of DNases, through the analysis of the “core-genome” and the “pan-genome” of ST17 and ST19 strains. All ST17 strains, except one, displayed DNase activity which was observed in only one ST19 strain. ST17 DNase(-) revealed an exclusive amino acid change in NucA, a TnGBS2.3 homolog and an intact phage without homology in S. agalactiae. ST19 DNase(+) revealed an exclusive amino acid change alteration in nuclease GBS0609. A transcriptomic approach allowed the analysis of the level of expression, by functional category, of genes of S. agalactiae reference strain NEM316 at the exponential growth phase; exposure to human DNA did not affect the transcriptome. The optimal conditions for S. agalactiae biofilm assembly were determined for ST17 and ST19 strains, contributing to the standardization of experimental procedures, thus allowing the comparison of results between different laboratories. In addition, the enzymatic digestion of mature biofilms for the three strongest biofilm producers, evidenced that proteins were the predominant component of the extracellular polymeric matrix. Overall, the findings presented in this Ph.D. thesis may contribute for the knowledge on the production of extracellular DNases, and provide new insights into biofilm formation, genomics and transcriptomics for ST17 and ST19 S. agalactiae lineages.Streptococcus agalactiae é a principal causa de pneumonia neonatal, sépsis e meningite. O clone hipervirulento III/ST17, tem sido associado à meningite neonatal de início tardio, tendo a atividade de DNases extracelulares e a formação de biofilmes sido apontados como fatores de virulência nessas estirpes. O objetivo da presente dissertação de doutoramento foi contribuir para a descodificação da base genómica de produção de DNases em estirpes de S. agalactiae, isoladas em humanos, com diferentes tropismos celulares (invasivo vs colonização). A produção diferencial de DNases de um conjunto de estirpes pertencentes às linhagens ST17 e ST19 foi investigada através da análise do “core-genome” e do “pan-genome”. As estirpes ST17, exceto uma, apresentaram atividade nucleásica contrariamente às estirpes ST19, em que apenas uma revelou um fenótipo DNase(+). A estirpe ST17 DNase(-) exibiu uma alteração aminoacídica exclusiva na proteína NucA e a presença de um homólogo do tranposão TnGBS2.3, para além de um fago intacto sem homologia em S. agalactiae. A estirpe ST19 DNase(+) revelou um aminoácido alterado na nuclease GBS0609. A análise transcriptómica permitiu avaliar os níveis de expressão génica, por categoria funcional, da estirpe de referência S. agalactiae NEM316, durante a fase exponencial de crescimento; a exposição ao DNA humano não induziu alterações no transcriptoma. Foram determinadas as condições ótimas para a formação de biofilmes em estirpes de S. agalactiae de ST17 e ST19, contribuindo para a padronização dos procedimentos experimentais do estudo de produção de biofilmes nesta espécie bacteriana. A digestão enzimática de biofilmes maduros, para as três estirpes mais produtoras de biofilmes, evidenciou que o componente predominante da matriz extracelular são as proteínas. Globalmente, a presente dissertação de doutoramento contribui para o conhecimento sobre a produção das DNases extracelulares e a capacidade de formação de biofilmes das linhagens ST17 e ST19 de S. agalactiae, providenciando novos dados genómicos e transcriptómicos

Genetic Control of Survival and Weight Loss during Pneumonic Burkholderia pseudomallei (Bp) Infection

Burkholderia pseudomallei (Bp) is a saprophytic, gram-negative aerobe and the causative agent of the disease melioidosis. Melioidosis is an infectious disease that occurs in humans and animals and is prevalent in Southeast Asia, northern Australia and other tropical areas. Transmission occurs through direct contact with the organism via ingestion, inhalation, or through open wounds and skin abrasions. Clinical presentation is extremely variable and can range from acute septicemia with bacterial dissemination to distant sites, to an isolated pulmonary infection. Treatment of melioidosis can be problematic because it is often difficult to diagnose and Bp is resistant to a diverse group of antibiotics. Even with treatment, the mortality rate of melioidosis is 20 to 50%. While this pathogen is not widespread in the United States, it is a significant U.S. health issue because of its high potential for use as a biological weapon. The US Centers for Disease Control and Prevention (CDC) and The Department of Health and Human Services (DHHS) has recognized Bp as a Tier 1 Select Agent, meaning there is no effective vaccine against this high morbidity/mortality disease. Furthermore, according the CDC, other factors including aerosol infectivity, the severity of infection, and the global distribution of this pathogen makes it a potential bioterrorism agent that poses a threat to national security, if intentionally released into populated areas. There has been little investigation into the role of host genetic background as it relates to susceptibility and/or resistance to Bp infection. It has been known for some time that inbred and recombinant inbred mice exhibit differences in susceptibility to many intracellular pathogens and are thought to provide excellent models for acute and chronic human melioidosis. Therefore, this project set out to identify host genetic elements that contribute to differential immune responsiveness and/or susceptibility to a Bp infection using a murine pneumonic challenge model. There is a high potential for discovery of host genes/genetic networks that confer resistance or sensitivity to this dangerous and emerging pathogen using inbred and BXD mouse strains as an in vivo modeling system of melioidosis. We have utilized recombinant inbred BXD mice and a powerful array of complementary computer-based modeling algorithms and databases collectively known as GeneNetwork. We infected parental mice and 32 BXD strains with 50-100 CFU of Bp (strain 1026b) and monitored survival and weight retention each day over an eleven-day time course. Initial studies revealed that Bp infection elicits phenotypically distinct innate immune responses in terms of survival and weight loss following pneumonic infection in parental and BXD mice. Using the computational tools in GeneNetwork, we performed genome-wide linkage analysis of our survival and weight loss phenotypic data to identify that survival is a complex trait involving loci on chromosomes 5 and 7 and weight retention involves loci on chromosome 12. We then analyzed and ranked several potential candidate genes within the significant and/or suggestive interval on these chromosomes that appear to correlate with differential susceptibility to Bp infection. Several genes have intriguing connections with innate immunity, regulation of Nf-kβ, apoptosis, cell cycle regulation, nervous system development, calcium homeostasis, lipid transport, host cell growth and development, and autophagy. To date, there have been few published studies that have identified specific host genetic elements that correlate with resistance and/or susceptibility to the acute form of melioidosis. Therefore, the identification of key host genetic factors that control resistance or susceptibility to Bp is of much importance. Results generated from this work will increase our understanding of the interactions between Bp and its genetically diverse hosts, which will enhance the understanding of Bp pathogenesis and increase the biological knowledge to help guide the progression and development of preventative and therapeutic measures to a Bp infection as well as other high morbidity/mortality respiratory pathogens

Streptococcal collagen-like protein 1, Scl1, modulates group a Streptococcus adhesion, biofilm formation and virulence

Background: The collagens comprise a large family of versatile proteins found in all three domains of life. The streptococcal collagen-like protein 1, scl1, of group A Streptococcus (GAS) binds extracellular matrix components (ECM), cellular fibronectin and laminin, via the surface-exposed globular domain. GAS strains express scl1 and form biofilm in vitro, except for M3-type strains that are particularly invasive to humans. Hypothesis: Lack of scl1 adhesin in M3 GAS results in decreased adherence and biofilm formation, and increased virulence. Results and Discussion : First crystal structure of the globular domain revealed a unique six-helical bundle fold, consisting of three pairs of alpha helices connected by variable loops. ECM binding by Scl1 promotes the formation of stable tissue microcolonies, which was demonstrated in vitro during infection of wounded human skin equivalents. A conserved nonsense mutation was identified in the scl1 allele of the M3-type strains (scl1.3) that truncates the coding sequence, presumably resulting in a secreted Scl1 variant. Absence of Scl1 on the surface of M3-type GAS was demonstrated experimentally, as well as diminished expression of the scl1 transcript in M3 strains relative to other M-types. Therefore, M3-type strains have reduced biofilm capacity on ECM coatings relative to other M-types. Constructed full-length recombinant Scl1.3 protein displayed binding capacity to cellular fibronectin and laminin, and M3 strains complemented with functional Scl1.3 adhesin displayed increased biofilm formation. The isoallelic M3 strain, carrying a rare carrier allele encoding cell-associated Scl1.3 variant, showed decreased pathology in mice, compared to the invasive M3 strain. Similarly, scl1 inactivation in biofilm-capable M28- and M41-type GAS led to increased lesion size during subcutaneous infection. Conclusions: The studies presented here demonstrate the importance of surface Scl1 in modulating biofilm formation and virulence of GAS, and provide insight into the structure and function of Scl proteins

Clinical, genetic and molecular aspects of membranous nephropathy

Membranous Nephropathy (MN) is one of the leading causes of end-stage renal disease (ESRD). MN is an autoimmune disease in which autoantibodies target antigens at the level of the glomerular basement membrane. The nature of these antibodies and the reason why they develop are not fully understood. One of the strategies towards a better understanding of the disorder is genetic analysis, of which two approaches have been attempted: linkage mapping, based on a family suggestive for X-linked transmission of the MN trait; and whole genome association mapping, based on three case-control cohorts. The first cohort (335 cases and ethnically matched controls from the UK) was genotyped using SNP markers and analysed in an exploratory study which led to the identification of two highly significant loci of association. Two cohorts (146 biopsy proven MN cases and ethnically matched controls from the Dutch research group in Nijmegen and 75 biopsy proven cases and ethnically matched controls from the French research group in Paris) were used to successfully replicate the results. The two loci which we identified and independently confirmed are located on chromosome 2 and on chromosome 6. The chromosome 2 locus includes the PLA2R gene, confirming the hypothesis of Beck et al. which identified PLA2R as a key antigen in idiopathic MN by using an immunological approach [1]. The chromosome 6 locus lies within the extended Human Leukocyte Antigene (HLA) system locus, with the highest significance for association reached by alleles of HLA-DQA1. Our results suggest that the susceptibility to membranous nephropathy is associated to genetic variants at the level of both PLA2R1 and HLA loci. The causative variants could be some of the polymorphisms captured by the genotyping array which was analysed or, more likely variants (single nucleotide or copy number variant type) situated nearby (and therefore in linkage disequilibrium)

Host and pathogen genetics associated with pneumococcal meningitis

Meningitis is an infection of the meninges, a layer of tissue surrounding the brain. In cases of pneumococcal meningitis (where the bacterium Streptococcus pneumoniae is the causat- ive agent) this causes severe inflammation, requiring intensive care and rapid antibiotic treatment. The contribution of variation in host and pathogen genetics to pneumococcal meningitis is unknown. In this thesis I develop and apply statistical genetics techniques to identify genomic variation associated with the various stages of pneumococcal meningitis, including colonisation, invasion and severity. I start by describing the development of a method to perform genome-wide association studies (GWAS) in bacteria, which can find variation in bacterial genomes associated with bacterial traits such as antibiotic resistance and virulence. I then applied this method to longitudinal samples from asymptomatic carriage, and found lineages and specific variants associated with altered duration of carriage. To assess meningitis versus carriage samples I applied similar analysis techniques, and found that the bacterial genome is crucial in determining invasive potential. As well as bacterial serotype, which I found to be the main effect, I discovered many independent sequence variants associated with disease. Separately, I analysed within host-diversity during the invasive phase of disease and found it to be of less relevance to disease progression. Finally, I analysed host genotype data from four independent studies using GWAS and heritability estimates to determine the contribution of human sequence variation to pneumococcal meningitis. Host sequence accounted for some variation in susceptibility to and severity of meningitis. The work concludes with a combined analysis of pairs of bacterial and human sequences from meningitis cases, and finds variation correlated between the two.This work was funded by Wellcome and Medical Research Council studentship grant (1365620

Grand Celebration: 10th Anniversary of the Human Genome Project

In 1990, scientists began working together on one of the largest biological research projects ever proposed. The project proposed to sequence the three billion nucleotides in the human genome. The Human Genome Project took 13 years and was completed in April 2003, at a cost of approximately three billion dollars. It was a major scientific achievement that forever changed the understanding of our own nature. The sequencing of the human genome was in many ways a triumph for technology as much as it was for science. From the Human Genome Project, powerful technologies have been developed (e.g., microarrays and next generation sequencing) and new branches of science have emerged (e.g., functional genomics and pharmacogenomics), paving new ways for advancing genomic research and medical applications of genomics in the 21st century. The investigations have provided new tests and drug targets, as well as insights into the basis of human development and diagnosis/treatment of cancer and several mysterious humans diseases. This genomic revolution is prompting a new era in medicine, which brings both challenges and opportunities. Parallel to the promising advances over the last decade, the study of the human genome has also revealed how complicated human biology is, and how much remains to be understood. The legacy of the understanding of our genome has just begun. To celebrate the 10th anniversary of the essential completion of the Human Genome Project, in April 2013 Genes launched this Special Issue, which highlights the recent scientific breakthroughs in human genomics, with a collection of papers written by authors who are leading experts in the field

Detailed genomic and antimicrobial resistance comparison of UK Streptococcus agalactiae isolates from adults to those of diverse global origins

Invasive group B Streptococcus (GBS), a leading cause of illness and death among infants in the first week of life is also an important infectious agent able to cause invasive infections in adults. Serious life-threatening invasive GBS infections are increasingly recognized in the elderly and individuals compromised by underlying diseases such as diabetes, cirrhosis, and cancer. The significance of GBS as a cause of severe infections among adults is not widely appreciated. In adults, the modes of transmission and acquisition are less identified. Penicillin is the antibiotic of choice for treatment of GBS infection however, resistance to multiple antibiotics is increasing in Europe and worldwide in these organisms, making them increasingly difficult to treat, but also making them a potential danger as a silent resource for donating resistance genes to more aggressive pathogens. To date, there are no guidelines for the prevention of adult GBS disease; vaccines in development may hold promise. GBS emerged rapidly all over the world during the 1970s to become the leading cause of neonatal sepsis. However, several reports in recent years suggest that the incidence of GBS disease is also increasing among adults. The driving force behind this change has not been fully explained, and recent trends in disease incidence in adults have not been characterized in any systematic reviews due to inadequate data available on adult GBS disease.The aim of the work presented in this thesis was to characterize the population structure of human source isolated invasive and non-invasive GBS from adults in United Kingdom, and to evaluate the genetic diversity of isolates recovered from invasive disease in adult patients of Brazil and from vaginal and rectal colonization in Australian pregnant women with ≤22 weeks gestations to contribute to the global epidemiology of GBS and our understanding of GBS population biology. For this a set of conventional techniques were employed including antimicrobial susceptibility testing using disc diffusion test, serotyping using software Geneious (Biomatters ltd., New Zealand) and published primers for ten different GBS capsular types, multilocus sequence typing (MLST) and surface protein gene profiling using SRST2 v2.2. In combination, these methods allowed the identification of the main genetic lineages circulating in UK, Brazil, and Australia, providing the means for an appropriate comparison of Brazilian and Australian GBS to UK GBS population. In addition, analysis of WGS to determine GBS potential to switch capsule, antimicrobial resistance (AMR) genes associated with mobile genetic elements (MGEs), examining relatedness of the GBS strains using whole genome phylogeny and correlate serotyping, patient age group to AMR genes and pan genome wide association study (pan-GWAS) on UK, Brazil, and publicly available genomes of GBS isolates from Canada and the United States was performed. These analyses revealed a reasonable number of isolates with potential capsular switch including some cases switched from current vaccine (under trail) covered capsular type to non-vaccine covered serotypes. Further a diverse group of MGEs were identified with a capacity to disseminate the resistance phenotype, the association between strains clustered in a group based on patients age group they isolated from and the AMR genes they carry were studied. An intense pan-GWAS was performed to discover Clonal Complex (CC) specific genes that may play role in increased colonization, invasiveness, pathogenicity, and better survival of GBS in the host cell. The thesis work started with investigating 193 clinical GBS strains isolated from adults submitted to the UK national reference laboratory (179 invasive; 13 non-invasive; 1 with no information provided) for capsule type, MLST, presence of virulence factors, antimicrobial resistance genes, phylogeny, and genetic recombination. The genetic lineages defined by MLST identified very diverse populations but consistent in terms of serotypes prevalence and clonal structure identified previously in GBS invasive disease in United Kingdom. The prevalence of serotype III in this population, regardless of age, highlighted the importance of this serotype in GBS pathogenesis as a leading cause of invasive infections in adults. Macrolide resistance is disseminated in UK by both a multiclonal mechanism resulting from the spread of resistance genes throughout most serotypes and genetic backgrounds, as well as by clonal expansion of specific lineages, such as the serotype V ST1/alp3. Attachment and invasion of host cells are key steps in GBS pathogenesis, strong associations were identified between serotypes and virulence genes, such as serotype V/alp3, serotype II and III/bca+cba, serotype Ia/bibA predominantly clustered in CC1, CC8/CC10 and CC23, respectively, whereas serotype III/rib clustered in CC17 and CC19 demonstrating GBS strains belonging to a particular CC differ in their abilities to attach and invade to host cell types and express key virulence genes that are relevant to the disease process. A major finding includes a high number of capsular serotype-CC mismatches (14/179, 7.8%) iGBS identified with a concerning recombination of hypervirulent hvgA core genome expressing a non-vaccine covered serotype IV capsule. The mechanism for these genetic transfer events involved the replacement of the whole capsular locus instead of the previously proposed genetic transfer of only the serotype specific genes. The consequent analysis of MGEs carrying multidrug resistance genes in 41/193 GBS isolates revealed a diverse group of MGES used three different insertion sites (rumA, rplL and rpsI) to disseminate phenotypic resistance in GBS isolated from adult patients of United Kingdom. Out of 41 isolates, only one isolate carried the macrolide resistance (ermT) gene was on a plasmid, while for 4 isolates fluoroquinolone resistance was mediated by double point somatic mutation in parC and gyrA; for all other isolates ARGs were acquired by MGEs including five novel MGEs identified in this study, including ICESag84 and ICESag100414 carrying ermA alone, ICESag662 containing ermB, tetS, ant(6-Ia) and aph(3'-III), ICESag71 carrying ermB and tetO, and ICESag139 containing ermA and the high gentamicin level resistance gene aac(6')-aph(2"). The Tn916 and Tn5801 belonging to Tn916/Tn1545 family harboured majority of tetM genes (88%, 154/175) found in UK tetracycline resistant GBS isolates and were significantly associated to CC1, CC8/10, CC19 and CC17 and CC23 isolates, respectively suggesting these ICEs are clonally related, acquired through limited and rare insertion events and led to expansion of these lineages, also supporting the earlier interpretations [1]. In addition, the Tn916/Tn1545 family ICE identified in this study were mostly integrated at two insertion sites, adjacent to two target genes (TGs) – rumA for Tn916 and guaA for Tn5801 demonstrating their integration preferences at hot spot regions. The high prevalence of MGEs carrying ARGs in UK adults GBS isolates implied that in GBS, these MGEs probably act like a reservoir of ARGs, and play a central role in the dissemination of resistance genes via horizontal gene transfer. Antibiotic susceptibility testing (AST) of GBS isolates recovered from UK (n=193) and Brazil (n=26) adults, and from vaginal and rectal sites of Australian pregnant women (n=171) revealed that all tested GBS populations were sensitive to ampicillin, vancomycin, and gentamicin, except a single UK strain that conferred high level gentamicin resistance through aminoglycoside modifying enzyme encoding gene aac(6')-aph(2"). Further all UK and Brazil GBS isolates carried five penicillin binding protein (PBPs) types with amino acid substitutions that did not appear to be associated with decreased β-lactam susceptibility suggesting penicillin as the first choice and vancomycin as the second choice of drug for GBS disease treatment as currently recommended by Centers for Disease Control and Prevention (CDC) and Royal College of Obstetricians and Gynaecologists (RCOG) guidelines [2, 3]. Accelerating resistance rates to erythromycin and clindamycin were observed in UK, which in comparison found less in Australian pregnant women colonized GBS and Brazilian GBS populations suggesting routine susceptibility testing of erythromycin and clindamycin for penicillin allergic patients to ensure effective treatment. A very few UK and Australian GBS strains were found resistant to chloramphenicol and/or levofloxacin while all Brazilian GBS were susceptible to these two antibiotics. High resistance rate to tetracycline was detected in UK, Brazilian and Australian GBS isolates and the tetM gene was found widespread and carried predominantly by Tn916/Tn1545 family elements. Three distinctive variants of Tn916 were observed in UK GBS strains, respectively including two Tn916 variants carrying tetM as a single resistance gene with additional 9 orfs in conjugation module, while the third Tn916 variant carried tetracycline efflux MFS transporter (tetL) gene near the tetM gene. The pangenome wide association study (pan-GWAS) was conducted on 447 GBS genomes, including this study sequenced UK (n=193) and Brazil (n=26) GBS strains, in addition to deliberately selected serotype V and III publicly available genomes from Canada (n=134) and the United States (n=94) to study genes specific to CC and their role in pathogenicity and invasiveness, since these two serotypes are universally found highly associated with adults and neonatal diseases, respectively. Each CC was characterized by specific genes that provides selective advantage to GBS for improved colonization, invasion, virulence, and survival within host. This analysis identified 97 CC-specific genes associated (excluding hypothetical proteins) with virulence, metabolism, and regulation of cellular mechanisms that may explain the differential virulence potential of the CCs. Among CC17 and CC23 GBS isolates, micronutrient uptake proteins (iron and manganese), two component systems, accessory secondary proteins, pilus and quorum-sensing genes were identified which were absent in less invasive lineages (CC1, CC8 and CC19). Metal resistance genes (arsenic, cadmium, and copper) and CRISPR associated genes (cas1/cas2) were confined to CC8 whereas the type IV secretory protein (VirD4) was significantly associated to CC19. Collectively this analysis underlines the lineage-specific basis of GBS niche adaptation and virulence. In summary, in this thesis GBS shown to have an evident stable clonal structure both temporally and geographically. Interestingly, capsular switching occurred across multiple serotypes and among strains with dissimilar genomic backgrounds in high numbers demonstrated ongoing GBS diversification due to recombination and highlights the importance of ongoing surveillance of GBS and may have implications for vaccine development strategies. Regardless of increasing information on invasive disease and maternal colonization, a thorough understanding of colonization in adults and natural reservoirs of GBS is required for the appropriate management of the GBS infections

Preclinical Animal Modeling in Medicine

The results of preclinical animal research have been successfully implemented in various medical and biological practices. The use of animals in medicine is based on significant anatomical, physiological, and molecular similarities between humans and animals. Particularly, mammals that have vast biological commonalities with humans represent not only a valuable model to explore the mechanisms of varied human diseases, but also to define new diagnostic and treatment strategies. This book covers broad but important aspects of animal modeling for scientific medicine as well as for translational systems and biological sciences. Alternative methods such as cell culture and in vitro experiments that do not require the sacrifice of an animal are encouraged for scientific and medical studies