Host Genetics and Chlamydia Disease: Prediction and Validation of Disease Severity Mechanisms

Genetic mapping studies may provide association between sequence variants and disease susceptibility that can, with further experimental and computational analysis, lead to discovery of causal mechanisms and effective intervention. We have previously demonstrated that polymorphisms in immunity-related GTPases (IRG) confer a significant difference in susceptibility to Chlamydia psittaci infection in BXD recombinant mice. Here we combine genetic mapping and network modeling to identify causal pathways underlying this association. We infected a large panel of BXD strains with C. psittaci and assessed host genotype, IRG protein polymorphisms, pathogen load, expression of 32 cytokines, inflammatory cell populations, and weight change. Proinflammatory cytokines correlated with each other and were controlled by a novel genetic locus on chromosome 1, but did not affect disease status, as quantified by weight change 6 days after infection In contrast, weight change correlated strongly with levels of inflammatory cell populations and pathogen load that were controlled by an IRG encoding genetic locus (Ctrq3) on chromosome 11. These data provided content to generate a predictive model of infection using a Bayesian framework incorporating genotypes, immune system parameters, and weight change as a measure of disease severity. Two predictions derived from the model were tested and confirmed in a second round of experiments. First, strains with the susceptible IRG haplotype lost weight as a function of pathogen load whereas strains with the resistant haplotype were almost completely unaffected over a very wide range of pathogen load. Second, we predicted that macrophage activation by Ctrq3 would be central in conferring pathogen tolerance. We demonstrated that macrophage depletion in strains with the resistant haplotype led to neutrophil influx and greater weight loss despite a lower pathogen burden. Our results show that genetic mapping and network modeling can be combined to identify causal pathways underlying chlamydial disease susceptibility

Inferring Regulatory Networks From Mixed Observational Data Using Directed Acyclic Graphs

Construction of regulatory networks using cross-sectional expression profiling of genes is desired, but challenging. The Directed Acyclic Graph (DAG) provides a general framework to infer causal effects from observational data. However, most existing DAG methods assume that all nodes follow the same type of distribution, which prohibit a joint modeling of continuous gene expression and categorical variables. We present a new mixed DAG (mDAG) algorithm to infer the regulatory pathway from mixed observational data containing both continuous variables (e.g. expression of genes) and categorical variables (e.g. categorical phenotypes or single nucleotide polymorphisms). Our method can identify upstream causal factors and downstream effectors closely linked to a variable and generate hypotheses for causal direction of regulatory pathways. We propose a new permutation method to test the conditional independence of variables of mixed types, which is the key for mDAG. We also utilize an L1 regularization in mDAG to ensure it can recover a large sparse DAG with limited sample size. We demonstrate through extensive simulations that mDAG outperforms two well-known methods in recovering the true underlying DAG. We apply mDAG to a cross-sectional immunological study of Chlamydia trachomatis infection and successfully infer the regularity network of cytokines. We also apply mDAG to a large cohort study, generating sensible mechanistic hypotheses underlying plasma adiponectin level. The R package mDAG is publicly available from CRAN at https://CRAN.R-project.org/package=mDAG

Recombinant Inbred Mice as Models for Experimental Precision Medicine and Biology

Recombinant inbred rodents form immortal genome-types that can be resampled deeply at many stages, in both sexes, and under multiple experimental conditions to model genome-environment interactions and to test genome-phenome predictions. This allows for experimental precision medicine, for which sophisticated causal models of complex interactions among DNA variants, phenotype variants at many levels, and innumerable environmental factors are required. Large families and populations of isogenic lines of mice and rats are now available and have been used across fields of biology. We will use the BXD recombinant inbred family and their derived diallel cross population as an example for predictive, experimental precision medicine and biology

STRUCTURAL AND FUNCTIONAL STUDIES OF THREE PROTEINS OF UNKNOWN FUNCTION ENCODED BY CHLAMYDIA TRACHOMATIS

Infections by chlamydial species are of significant impact to global public health, causing sexually transmitted infections, blinding trachoma and pneumonia. Despite its importance, there are many aspects of chlamydial biology that are not completely understood, including the mechanisms by which it infects, persists and replicates in its host cells. The reason for this ignorance of basic chlamydial biological processes is because there is an abundance of Open Reading Frames (ORFs) of unknown function present in chlamydial genomes, almost 30% of the entire genome in many species. This is likely due to the relatively large phylogenetic distance between Chlamydiae and better-understood bacteria such as E. coli and B. subtilis. Current strategies of genome annotation rely on the presence of homology to genes of known function and these approaches have not been effective in annotating chlamydial genomes. In an effort gain insight into the function of these chlamydial ORFs of unknown function, I utilized structural information (both computational and experimentally derived) about three proteins of interest. Based on these structural studies, hypotheses concerning the functions of these proteins were formed and then tested. Together, my findings not only provide valuable information about these proteins of unknown function, but they also serve to demonstrate both the strengths and shortcomings of the overall approach of utilizing structural information for functional prediction. One example of this approach is my work on the chlamydial ORF CT296. Although this protein was annotated as having an unknown function (due to insignificant homology to proteins of known function), it had been experimentally characterized as an iron-dependent transcription factor. Having an experimentally characterized function allowed me to test my approach of utilizing structural information to predict function on a protein with a relatively well-understood function. Surprisingly, structural information of this protein suggested that it functions as a Fe(II) 2-oxoglutarate-dependent enzyme and not as a transcription factor. Subsequent functional analyses of the protein were unable to reproduce previous reports of its DNA binding. Together, my findings suggest that this protein may not function as a transcription factor. A second example of my structure-function approach was applied to the chlamydial protein CT584. This protein was first experimentally described as interacting with the chlamydial Type Three Secretion System (T3SS) needle protein in an interactome study. This observation, combined with a subsequent biophysical characterization of the protein lead to an initial hypothesis that the protein may be a chlamydial T3SS needle tip protein. However, results of structural studies on the protein reveal a structure that is not similar to any of the known T3SS needle tip proteins. Additionally, functional studies on the protein focusing on identifying its localization on chlamydial organisms revealed localization patterns not consistent with its proposed role as a T3SS needle protein. Together, my studies suggest that this protein may not function as a needle tip protein. A final example of the utility of structural information for informing function concerns chlamydial ORF CT009. This protein was annotated in many chlamydial species as a protein of unknown function; however, bioinformatics analyses had identified it as a helix-turn-helix containing transcription factor. Results of computational and experimental structures of this protein show structural similarity to the protein RodZ, a key component of the bacterial morphogenic complex. Subsequent functional analyses of CT009 demonstrate that this protein has the characteristics of a chlamydial homolog to RodZ

Interacting networks of resistance, virulence and core machinery genes identified by genome-wide epistasis analysis.

Recent advances in the scale and diversity of population genomic datasets for bacteria now provide the potential for genome-wide patterns of co-evolution to be studied at the resolution of individual bases. Here we describe a new statistical method, genomeDCA, which uses recent advances in computational structural biology to identify the polymorphic loci under the strongest co-evolutionary pressures. We apply genomeDCA to two large population data sets representing the major human pathogens Streptococcus pneumoniae (pneumococcus) and Streptococcus pyogenes (group A Streptococcus). For pneumococcus we identified 5,199 putative epistatic interactions between 1,936 sites. Over three-quarters of the links were between sites within the pbp2x, pbp1a and pbp2b genes, the sequences of which are critical in determining non-susceptibility to beta-lactam antibiotics. A network-based analysis found these genes were also coupled to that encoding dihydrofolate reductase, changes to which underlie trimethoprim resistance. Distinct from these antibiotic resistance genes, a large network component of 384 protein coding sequences encompassed many genes critical in basic cellular functions, while another distinct component included genes associated with virulence. The group A Streptococcus (GAS) data set population represents a clonal population with relatively little genetic variation and a high level of linkage disequilibrium across the genome. Despite this, we were able to pinpoint two RNA pseudouridine synthases, which were each strongly linked to a separate set of loci across the chromosome, representing biologically plausible targets of co-selection. The population genomic analysis method applied here identifies statistically significantly co-evolving locus pairs, potentially arising from fitness selection interdependence reflecting underlying protein-protein interactions, or genes whose product activities contribute to the same phenotype. This discovery approach greatly enhances the future potential of epistasis analysis for systems biology, and can complement genome-wide association studies as a means of formulating hypotheses for targeted experimental work

Major histocompatibility complex and coronary artery disease : Special emphasis on Chlamydia pneumoniae, and periodontitis

Most of the genes in the MHC region are involveed in adaptive and innate immunity, with essential function in inflammatory reactions and in protection against infections. These genes might serve as a candidate region for infection and inflammation associated diseases. CAD is an inflammatory disease. The present set of studies was performed to assess whether the MHC region harbors genetic markers for CAD, and whether these genetic markers explain the CAD risk factors: e.g., C. pneumoniae, periodontitis, and periodontal pathogens. Study I was performed using two separate patient materials and age- and sex-matched healthy controls, categorizing them into two independent studies: the HTx and ACS studies. Both studies consistently showed the HLA-A3– B35– DR1 (35 ancestral haplotype) haplotype as a susceptible MHC genetic marker for CAD. HLA-DR1 alone was associated not only with CAD, but also with CAD risk factor diseases, e.g., diabetes mellitus, and hyperlipidemia. The ACS study further showed the HLA-B*07 and -DRB1*15 -related haplotype as a protective MHC haplotype for CAD. Study II showed that patients with CAD showed signs of chronic C. pneumoniae infection when compared to age- and sex-matched healthy controls. HLA-B*35 or -related haplotypes associated with the C. pneumoniae infection markers. Among these haplotype carriers, males and smokers associated with elevated C. pneumoniae infection markers. Study III showed that CAD patients with periodontitis had elevated serum markers of P. gingivalis and occurrence of the pathogen in saliva. LTA+496C strongly associated with periodontitis, while HLA-DRB1*01 with periodontitis and with the elevated serum antibodies of P. gingivalis. Study IV showed that the increased level of C3/C4 ratio was a new risk factor and was associated with recurrent cardiovascular end-points. The increased C3 and decreased C4 concentrations in serum explained the increased level of the C3/C4 ratio. Both the higher than cut-off value (4.53) and the highest quartile of the C3/C4 ratio were also associated with worst survival, increased end-points, and C4 null alleles. The presence of C4 null alleles associated with decreased serum C4 concentration, and increased C3/C4 ratio. In conclusion, the present studies show that the CAD susceptibility haplotype (HLA-A3− B35− DR1 -related haplotypes, Study I) partially explains the development of CAD in patients possessing several recognized and novel risk factors: diabetes mellitus, increased LDL, smoking, C4B*Q0, C. pneumnoiae, periodontitis, P. gingivalis, and complement C3/C4 ratio (Study II, III, and IV).Human leukocyte antigen (HLA)-geenit sijaitsevat kromosomin 6 Major Histocompatibility Complex (MHC)-alueella. Monet näistä geeneistä säätelevät hankittua ja luonnollista immuunivastetta sekä tulehdusreaktioita. HLA-geenit sopivatkin ehdokasgeeneiksi monille tulehdus- ja infektiosairauksille, myös sepelvaltimotaudille. Tämän väitöskirjan tavoitteena oli selvittää altistavatko MHC-alueen geenit tai geenipoikkevuudet sepelvaltimotaudille. Lisäksi haluttiin tutkia onko sepelvaltimotautiin liittyvillä tulehduksilla (Chlamydia pneumoniae -bakteeri tai hampaan juurikalvon tulehduksella) ja MHC-alueen geeneillä yhdessä yhteyttä sepelvaltimotautiin. Kirjan ensimmäisessä osatyössä käytettiin kahta potilasmateriaalia, jossa ensimmäisessä tutkittiin eri indikaatioilla hoidettuja sydänsiirtopotilaita ja toisessa vertailtiin akuutti koronaarisyndroomapotilaita ikä- ja sukupuolivakioituihin kontrolleihin. Molemmat tutkimukset osoittivat johdonmukaisesti, että haplotyyppi HLA-A3–B35–DR1 altistaa sepelvaltimotaudille. Tämän lisäksi osoitettiin, että HLA-DR1 liittyi sepelvaltimotaudin lisäksi myös sen riskitekijöihin, kuten diabetekseen, veren korkeaan kolesterolipitoisuuteen ja tupakointiin sekä C4B-puutokseen. Akuutti koronaarisyndrooma -työssä todettiin myös, että sepelvaltimotaudilta suojaavissa haplotyypeissä olivat HLA-B*07 ja -DRB1*15 alleelit. Toisessa osajulkaisussa tutkittiin Chlamydia pneumoniae-infektion yhteyttä HLA-alueeseen ja sepelvaltimotautiin. Chlamydia pneumoniae vasta-ainemääriä vertailtiin akuutti koronaarisyndrooma-potilaiden ja ikä- ja sukupuolivakioitujen verrokkien välillä. Tutkimus osoitti, että HLA-B*35 liittyi sepelvaltimotautipotilailla Chlamydia pneumoniae-infektioon. Lisäksi todettiin, että miessukupuoli ja tupakointi voimistivat tätä yhteyttä. HLA-B*35 saattaa olla yhteinen tekijä sepelvaltimotaudin ja Chlamydia.pneumoniae-infektion välillä. Kolmas osatyö keskittyi sepelvaltimotautipotilaisiin, joilla oli hampaan juurikalvon tulehdus eli parodontiitti. Aineistona oli 106 akuutti koronaarisyndroomapotilasta, joiden hampaat oli tutkittu sairaalaan tulovaiheessa panoramisella tomografialla ja potilaista oli otettu sylki- ja seeruminäytteet. Syljestä viljeltiin bakteerit, seerumista määritettiin muun muassa Porphyromonas gingivalis vasta-aineet ja parodontiiti määritettiin tomografialla. Löydöksiä verrattiin MHC-alueen geeneihin. Tärkeimpänä löydöksenä oli se, että LTA+496C tekijä assosioitui parodontiittiin. HLA-DRB1*01 esiintyi puolestaan potilailla, joilla oli sekä paradontiittia että Porphyromonas gingivalis -vasta-ainetta seerumissa. Neljännessä osatyössä selvitettiin akuutti koronaarisyndroomapotilailla seerumin komplementtiproteiinien C3 ja C4-suhteen (C3/C4-suhde) merkitystä sepelvaltimotautikohtausten uusiutumiseen. Tutkimuksessa pystyttiin osoittamaan, että kohonnut C3/C4-suhde on uusi riskitekijä uusivalle akuutille koronaarisyndroomalle. Sekä kohonnut C3- että alentunut C4 -pitoisuuden vaikuttivat kohonneeseen C3/C4-suhteeseen. Tämän lisäksi C4-puutokset alentuneen C4 -pitoisuus kanssa nostivat C3/C4-suhdetta. Väitöskirjan työt osoittivat, että haplotyyppi HLA-A3– B35– DR1 altistaa sepelvaltimotaudille ja liittyy niin perinteisiin kuin uusiin sepelvaltimotaudin riskitekijöihin, joita ovat mm. diabetes, veren suuri kolesterolipitoisuus, tupakointi, C4B*Q0-alleeli, Chlamydia pneumoniae, paradontiitti, Porphyromonas gingivalis, sekä komplementin C3/C4 -suhde

The characterization of novel transgenic murine models of Neisseria gonorrhoeae infection and development of a natural outer membrane vesicle anti-gonococcal vaccine candidate

Untreatable gonorrhea, caused by fully antimicrobial resistant Neisseria gonorrhoeae (GC), is a major global health threat. While a vaccine would greatly help address this crisis, development of a GC vaccine is complicated by the lack of lab models of symptomatic gonorrhea. We hypothesized that overt disease in animal models of gonorrhea is limited by the human-restriction of gonococcal virulence factors, and the impact of the reproductive hormone cycle (estrus and diestrus phases). We tested these hypotheses by examining the host response to infection in transgenic mice expressing targets of bacterial adhesion, human carcinoembryonic antigen-related cell adhesion molecules (hCEACAMs), in uterine versus vaginal infections, and in different phases of the reproductive cycle (estrus and diestrus phases). hCEACAM expression most impacted estrus phase infections, prolonging colonization in vaginal infection and inducing greater inflammation in uterine. Reproductive phase greatly influenced host response to uterine infection as diestrus infection was more inflammatory than estrus. Phase differences in uterine infection were driven by greater activation of a chemokine-centric common anti gonococcal response and unique induction of type 1 interferons in diestrus. These findings suggest that symptomatic uterine and vaginal GC infection can be modeled by transcervically infected wild-type diestrus mice and transgenic, vaginally-infected estrus mice, respectively. A novel approach to GC vaccine development is also needed. Mono-antigenic vaccines have failed to produce immunity suggesting a poly-antigenic antigen, like natural outer membrane vesicles (nOMVs) may be necessary. It has been shown that any GC vaccine must lack the bacterioprotective antigen, reduction modifiable protein (RMP), and no such nOMV has been previously described. Here we report successful isolation of RMP-deficient nOMVs through sequential size and weight restrictive filtration. Vesicle morphology, proteomics, and bioactivity was characterized via various methods. nOMVs were found to be consistent in size, shape and antigenic load. As antigens, nOMVs induced high serum titers and measurable vaginal levels of antigen and GC specific IgG that recognized several nOMV immunogens supporting the vaccine potential of GC nOMVs. These findings lay the groundwork for protective studies of nOMV vaccines in novel models of active gonorrhea moving the field closer to discovering the mechanism of protective anti-gonococcal immunity

Public health surveillance of multidrug-resistant clones of Neisseria gonorrhoeae in Europe: a genomic survey.

BACKGROUND: Traditional methods for molecular epidemiology of Neisseria gonorrhoeae are suboptimal. Whole-genome sequencing (WGS) offers ideal resolution to describe population dynamics and to predict and infer transmission of antimicrobial resistance, and can enhance infection control through linkage with epidemiological data. We used WGS, in conjunction with linked epidemiological and phenotypic data, to describe the gonococcal population in 20 European countries. We aimed to detail changes in phenotypic antimicrobial resistance levels (and the reasons for these changes) and strain distribution (with a focus on antimicrobial resistance strains in risk groups), and to predict antimicrobial resistance from WGS data. METHODS: We carried out an observational study, in which we sequenced isolates taken from patients with gonorrhoea from the European Gonococcal Antimicrobial Surveillance Programme in 20 countries from September to November, 2013. We also developed a web platform that we used for automated antimicrobial resistance prediction, molecular typing (N gonorrhoeae multi-antigen sequence typing [NG-MAST] and multilocus sequence typing), and phylogenetic clustering in conjunction with epidemiological and phenotypic data. FINDINGS: The multidrug-resistant NG-MAST genogroup G1407 was predominant and accounted for the most cephalosporin resistance, but the prevalence of this genogroup decreased from 248 (23%) of 1066 isolates in a previous study from 2009-10 to 174 (17%) of 1054 isolates in this survey in 2013. This genogroup previously showed an association with men who have sex with men, but changed to an association with heterosexual people (odds ratio=4·29). WGS provided substantially improved resolution and accuracy over NG-MAST and multilocus sequence typing, predicted antimicrobial resistance relatively well, and identified discrepant isolates, mixed infections or contaminants, and multidrug-resistant clades linked to risk groups. INTERPRETATION: To our knowledge, we provide the first use of joint analysis of WGS and epidemiological data in an international programme for regional surveillance of sexually transmitted infections. WGS provided enhanced understanding of the distribution of antimicrobial resistance clones, including replacement with clones that were more susceptible to antimicrobials, in several risk groups nationally and regionally. We provide a framework for genomic surveillance of gonococci through standardised sampling, use of WGS, and a shared information architecture for interpretation and dissemination by use of open access software. FUNDING: The European Centre for Disease Prevention and Control, The Centre for Genomic Pathogen Surveillance, Örebro University Hospital, and Wellcome

A study of the molecular and spatial determinants of ocular Chlamydia trachomatis infection on the trachoma-hyperendemic Bijagós Archipelago of Guinea Bissau, West Africa

Chlamydia trachomatis is the leading infectious cause of preventable blindness and the most common sexually transmitted bacterium worldwide. Trachoma presents an environment in which to investigate chlamydial pathogenicity, the conjunctivae serving as an accessible model with an objectively observable phenotype. The Bijagós Archipelago is a unique setting where trachoma is hyperendemic. The primary aims of this study were to use novel molecular, bioinformatic and geostatistical approaches in conjunction with population-based clinical and epidemiological metadata to investigate the micro-epidemiology of ocular C. trachomatis and active trachoma in this population. The prevalence of trachoma and ocular C. trachomatis infection have been documented, and socio-environmental risk factors have been identified that may be important in the implementation of trachoma elimination activities in these communities. A strong association was found between C. trachomatis ocular load (estimated using droplet digital PCR) and the level of conjunctival inflammation. Geostatistical analyses suggest that ocular C. trachomatis load may be important in transmission, as spatial clusters of high load infections were identified, whilst spatial clusters of low load infections were absent. This study includes the first population-based pathogen genome-wide association scan (GWAS) for C. trachomatis, using high quality next generation whole genome sequence data obtained directly from clinical samples. The genomewide associations with conjunctival inflammation (incE) and C. trachomatis load (mutY and CTA_0271) present genes involved in specific biological characteristics of C. trachomatis, the functions of which suggest that early interactions with host cells are important in C. trachomatis pathogenesis. Pathogen GWAS, applied in this context, is a powerful approach in the identification of multiple targets for further study in pathogenesis and directed study of potential vaccine candidates, allowing a greater understanding of association and interaction of genes on a genome-wide scale. Following a single round of mass drug treatment with oral azithromycin (MDA) in these communities the prevalence of active trachoma and ocular C. trachomatis were significantly reduced. Individual and median loads of C. trachomatis were reduced and the highest burden of disease and infection were concentrated in young children. Spatial clustering of infection identified using geostatistical tools was intensified following MDA, but the number of clusters of high load infections was reduced. The severity of conjunctival inflammation was reduced following MDA. This study suggests that chlamydial load is important in disease pathogenesis and may be important in transmission of infection. Geospatial tools may be useful in the context of MDA to identify clusters of infection and thresholds of C. trachomatis bacterial load that may be important foci of transmission. The association between conjunctival inflammation and C. trachomatis load may reflect pathogen virulence. This is supported by the presence of genome-wide associations with C. trachomatis load and conjunctival inflammation identified by pathogen GWAS. Further epidemiological, in vitro and in silico studies are required to provide a more complete picture of the relationship between disease severity, bacterial load and chlamydial diversity in the context of transmission and elimination dynamics

In vitro expanded human CD4+CD25+ regulatory T cells suppress effector T cell proliferation.

Regulatory T cells (Tregs) have been shown to be critical in the balance between autoimmunity and tolerance and have been implicated in several human autoimmune diseases. However, the small number of Tregs in peripheral blood limits their therapeutic potential. Therefore, we developed a protocol that would allow for the expansion of Tregs while retaining their suppressive activity. We isolated CD4+CD25 hi cells from human peripheral blood and expanded them in vitro in the presence of anti-CD3 and anti-CD28 magnetic Xcyte Dynabeads and high concentrations of exogenous Interleukin (IL)-2. Tregs were effectively expanded up to 200-fold while maintaining surface expression of CD25 and other markers of Tregs: CD62L, HLA-DR, CCR6, and FOXP3. The expanded Tregs suppressed proliferation and cytokine secretion of responder PBMCs in co-cultures stimulated with anti-CD3 or alloantigen. Treg expansion is a critical first step before consideration of Tregs as a therapeutic intervention in patients with autoimmune or graft-versus-host disease