Biology subjects

Holland, M. J.

Publications and source records attributed to Holland, M. J..

9 recordsLinked to original sources

bioRxiv2025

Comparative transcriptomic profiling of human conjunctival epithelial cells and macrophages in response to Chlamydia trachomatis genovars A and B in early- and mid-infection cycles

Chlamydia trachomatis (Ct), an obligate intracellular bacterium, is the primary infectious cause of blindness through trachoma. Ct undergoes a unique biphasic developmental cycle between infectious elementary bodies and replicating reticulate bodies, manipulating host cells via secreted effector proteins. Whilst previous studies have characterised host-pathogen interactions including transcriptomes of urogenital Ct genovars E and L2, limited studies exist on ocular Ct genovars. This study examined transcriptomic responses of human conjunctival epithelial (HCjE) cells and PMA-differentiated THP-1 macrophages to infection with ocular Ct strains A/2497 or B/Tunis864 (live or heat-inactivated) at 4 and 24 hours post-infection (hpi). Transcriptomic profiling was performed using Lexogen QuantSeq 3 mRNA-Seq, with differential gene expression analysis conducted using DESeq2. Gene Ontology Biological Process and KEGG pathway enrichment analyses were performed using Gene Set Enrichment Analysis via clusterProfiler to identify strain-specific and cell type-specific transcriptional signatures. HCjE cells exhibited progressive transcriptional activation, with differentially expressed genes (DEGs) increasing from 4 to 24 hpi (144 to 259; P = 0.0458), whilst THP-1 macrophages showed temporal attenuation (391 to 154; P < 0.0001). B/Tunis864 consistently elicited higher responses than A/2497 in HCjE cells at both time points (4 hpi: 152 vs. 54, P = 0.0003; 24 hpi: 259 vs. 83, P < 0.0001). Conversely, THP-1 macrophages showed higher responses to A/2497 than B/Tunis864 at both time points (4 hpi: 599 vs. 376, P < 0.0001; 24 hpi: 166 vs. 114, P = 0.0221). HCjE cells demonstrated markedly higher proportions of strain-specific DEGs and pathways compared to macrophages. B/Tunis864 infection in HCjE cells induced pronounced interferon-stimulated gene signatures, particularly at 24 hpi. This study revealed contrasting temporal patterns: THP-1 macrophages showed peak-then-decline responses, whilst HCjE cells exhibited progressive activation to 24 hpi. HCjE cells demonstrated predominantly strain-specific responses, whereas macrophages deployed strain-invariant programmes. B/Tunis864s enhanced interferon-stimulated gene and inflammatory pathway activation in HCjE cells may suggest molecular hints for genovar B-associated trachoma severity.

microbiology↗

bioRxiv2023

Genomic insights into local scale evolution of ocular Chlamydia trachomatis strains within and between individuals in Gambian trachoma-endemic villages

Trachoma, a neglected tropical disease caused by Chlamydia trachomatis (Ct) serovar A-C, is the leading infectious cause of blindness worldwide. Its impact is substantial, with approximately 1.9 million individuals suffering from visual impairment. Africa bears the highest burden, accounting for over 86% of global trachoma cases. We investigated Ct serovar A (SvA) and serovar B (SvB) whole genome sequences prior to the induction of mass antibiotic drug administration in The Gambia. Here, we explore the factors contributing to Ct strain diversification and implications for Ct evolution within the context of ocular infection. A cohort study in 2002-2003 collected ocular swab samples across nine Gambian villages during a six-month follow-up study. To explore the genetic diversity of Ct within and between individuals, we conducted Whole-Genome Sequencing (WGS) on a limited number (n=43) of Ct positive samples. WGS was performed using target enrichment with SureSelect and Illumina paired-end sequencing. Out of 43 WGS samples, 41 provided sufficient quality for further analysis. ompA analysis revealed that 11 samples had a highest identity to Ct strain A/HAR13 (NC_007429) and 30 had a highest identity to Ct strain B/Jali20 (NC_012686). While SvB genome sequences formed two distinct village-driven subclades, the heterogeneity of SvA sequences led to the formation of many individual branches within the Gambian SvA subclade. Comparing the Gambian SvA and SvB sequences with their reference strains; Ct A/HAR13 and Ct B/Jali20 indicated a single nucleotide polymorphism accumulation rate of 2.4 {xi} 10^-5/site/year for the Gambian SvA and 1.3 {xi} 10^-5/site/year for SvB variants (p<0.0001). Variant calling resulted in a total of 1,371 single nucleotide variants (SNVs) with a frequency > 25% in SvA sequences, and 438 SNVs in SvB sequences, from which 740 (62.8%), and 241 (66.4%) were non-synonymous, respectively. Of note, in SvA variants, highest evolutionary pressure was recorded on genes responsible for host cell modulation and intracellular survival mechanisms, whereas in SvB variants this pressure was mainly on genes essential for DNA replication/ repair mechanisms and protein synthesis. A comparison of the sequences between observed separate infection events (4-20 weeks between infections) suggested that the majority of the variations accumulated in genes responsible for host-pathogen interaction such as CTA_0166 (phospholipase D-like protein), CTA_0498 (TarP), and CTA_0948 (deubiquitinase). This comparison of Ct SvA and SvB variants within a trachoma endemic population focused on their local evolutionary adaptation. We found a different variation accumulation pattern in the Gambian SvA chromosomal genes compared with SvB hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies. Impact StatementChlamydia trachomatis (Ct) is a globally significant pathogen. It is the leading infectious cause of blindness--a disease called trachoma. In addition, Ct causes the majority of bacterial sexually transmitted infections. Current control measures for trachoma are based on the "SAFE" strategy; Surgery for trichiasis (S), Antibiotics (A), Facial cleanliness (F) and Environmental improvement (E). Whilst this strategy has achieved remarkable success, the target date for the global elimination of blinding trachoma as a public health problem has been pushed back from 2020 to 2030. Prior studies demonstrated evidence indicating variations in infection loads and severity among different ocular Ct serovars. However, there remains a significant knowledge gap regarding the specific genes and mechanisms responsible for these variations. We generated genetic data from two main serovars of Ct that infect human eyes: Serovar A (SvA) and Serovar B (SvB) variants collected from four villages in two different administrative regions on opposing sides of the river Gambia to elucidate (i) the factors driving the diversification of ocular Ct strains; (ii) disparities in mutation frequency/accumulation profiles; (iii) selective pressures between serovar A and B; and (iv) the dynamics of mutation accumulation within the Gambian ocular Ct positive population over a short timeframe. Our findings suggest a different variation accumulation pattern in SvA chromosomal genes compared with SvB hinting at the potential of Ct serovar-specific variation in diversification and evolutionary fitness. These findings may have implications for optimizing trachoma control and prevention strategies.

genomics↗

bioRxiv2023

A Systems Serology Approach to the Investigation of Infection-Induced Antibody Responses and Protection in Trachoma

BackgroundOcular infections with Chlamydia trachomatis serovars A-C cause the neglected tropical disease trachoma. As infection does not confer complete immunity, repeated infections are common, leading to long term sequelae such as scarring and blindness. Here we apply a systems serology approach to investigate whether systemic antibody features are associated with susceptibility to infection. MethodsSera from children in five trachoma endemic villages in The Gambia were assayed for 23 antibody features: IgG responses towards two Chlamydia trachomatis antigens and three serovars (elementary bodies and major outer membrane protein MOMP, serovars A-C), IgG responses towards five MOMP peptides (serovars A-C), neutralization and antibody-dependent phagocytosis. Participants were considered resistant if they subsequently developed infection only when over 70% of other children in the same compound were infected. ResultsThe antibody features assayed were not associated with resistance to infection (false discovery rate < 0.05). Anti-MOMP SvA IgG and neutralization titer were higher in susceptible individuals (p < 0.05 before multiple testing adjustment). Classification using partial least squares performed only slightly better than chance in distinguishing between susceptible and resistant participants based on systemic antibody profile (specificity 71%, sensitivity 36%). ConclusionsSystemic infection-induced IgG and functional antibody responses do not appear to be protective against subsequent infection. This may be due to confounding factors increasing both past and future exposure to C. trachomatis, or antibody-dependent enhancement. Ocular responses, IgA, avidity or cell-mediated responses may play a greater role in protective immunity than systemic IgG.

immunology↗

bioRxiv2020

Genomics of Ocular Chlamydia trachomatis after 5 years of SAFE interventions for trachoma in Amhara, Ethiopia

BackgroundTo eliminate trachoma as a public health problem, the WHO recommends the SAFE strategy. As part of the SAFE strategy in the Amhara Region, Ethiopia, the Trachoma Control Program distributed over 124 million doses of antibiotic between 2007 and 2015. Despite these interventions, trachoma remained hyperendemic in many districts and a considerable level of Chlamydia trachomatis (Ct) infection was evident. MethodsWe utilised residual material from Abbott m2000 Ct diagnostic tests to sequence 99 ocular Ct samples from Amhara and investigated the role of Ct genomic variation in the continued transmission of Ct following 5 years of SAFE. FindingsSequences were typical of ocular Ct, at the whole-genome level and in tissue tropism-associated genes. There was no evidence of macrolide-resistance in this Ct population. Polymorphism in a region around ompA gene was associated with village-level TF prevalence. Additionally, greater ompA diversity at the district-level was associated with increased Ct infection prevalence. InterpretationWe found no evidence for Ct genomic variation contributing to continued transmission of Ct after treatment, adding to previous evidence that azithromycin does not drive acquisition of macrolide resistance alleles in Ct. Increased Ct infection in villages and in districts with more ompA variants requires longitudinal investigation to understand what impact this may have on treatment success and host immunity. FundingEuropean Commission; Neglected Tropical Disease Support Center; International Trachoma Initiative

genomics↗

bioRxiv2019

Conjunctival microbiome-host responses are associated with impaired epithelial cell health in both early and late stages of trachoma

BackgroundTrachoma, a neglected tropical disease, is the leading infectious cause of blindness and visual impairment worldwide. Host responses to ocular chlamydial infection resulting in chronic inflammation and expansion of non-chlamydial bacteria are hypothesised risk factors for development of active trachoma and conjunctival scarring\n\nMethodsOcular swabs from trachoma endemic populations in The Gambia were selected from archived samples for 16S sequencing and host conjunctival gene expression. We recruited children with active trachoma and adults with conjunctival scarring, alongside corresponding matched controls.\n\nFindingsIn children, active trachoma was not associated with significant changes in the ocular microbiome. Haemophilus enrichment was associated with antimicrobial responses but not linked to active trachoma. Adults with scarring trachoma had a reduced ocular bacterial diversity compared to controls, with increased relative abundance of Corynebacterium. Increased abundance of Corynebacterium in scarring disease was associated with innate immune responses to the microbiota, dominated by altered mucin expression and increased matrix adhesion.\n\nInterpretationIn the absence of current C. trachomatis infection, changes in the ocular microbiome associate with antimicrobial and inflammatory responses that impair epithelial cell health. In scarring trachoma, expansion of non-pathogenic bacteria such as Corynebacterium and innate responses are coincident, warranting further investigation of this relationship. Comparisons between active and scarring trachoma supported the relative absence of type-1 interferon responses in scarring, whilst highlighting a common suppression of re-epithelialisation with altered epithelial and bacterial adhesion, likely contributing to development of scarring pathology.

microbiology↗

bioRxiv2018

Evaluation of a Chlamydia trachomatis-specific, commercial, real-time PCR for use with ocular swabs

BackgroundChlamydia trachomatis (Ct) is the most common bacterial sexually transmitted infection and the causative organism of trachoma, the leading infectious cause of blindness worldwide. Trachoma is diagnosed clinically by observation of conjunctival inflammation and/or scarring, however, there is evidence that monitoring Ct infection may be required for elimination programs. There are many commercial and in-house nucleic acid amplification tests for the detection of Ct DNA, but the vast majority have not been validated for use with ocular swabs. This study evaluated a commercial assay, the Fast-Track Vaginal swab kit, using conjunctival samples from two trachoma-endemic areas. An objective, biostatistical-based method for binary classification of continuous PCR data was also developed, to limit potential user-bias in diagnostic settings.\n\nResultsThe Fast-Track Vaginal swab assay was run on 210 ocular swab samples from Guinea-Bissau and Tanzania. Fit of individual amplification curves to exponential or sigmoid models, derivative and second derivative of the curves and final fluorescence value were examined for utility in thresholding for determining positivity. The results from the Fast-Track Vaginal swab assay were evaluated against a commercial test (Amplicor CT/NG) as well as a non-commercial test (in-house ddPCR) both of whose performance has previously been evaluated.\n\nSignificant evidence of exponential amplification (R2 > 0.99) and final fluorescence > 0.15 were combined for thresholding. This objective approach identified a population of positive samples, however there were a subset of samples that amplified towards the end of the cycling protocol (at or later than 35 cycles), which were less clearly defined. The Fast-Track Vaginal swab assay showed good sensitivity against the commercial (95.71) and non-commercial (97.18) tests. Specificity was lower against both tests (90.00 and 96.55 respectively).\n\nConclusionsThis study defined a simple, automated protocol for binary classification of continuous, real time qPCR data, for use in an end-point diagnostic test. This method identified a population of positive samples, however, as with manual thresholding, a subset of samples that amplified towards the end of the thermal cycling program were less easily classified. When used with ocular swabs, the Fast-Track Vaginal swab assay had good sensitivity but for Ct detection lower specificity than the commercial and non-commercial assays it was evaluated against, possibly leading to false positives.

molecular biology↗

bioRxiv2017

Pathways-wide genetic risks in Chlamydial infections overlap between tissue tropisms: A genome-wide association scan

Chlamydia trachomatis is the most commonly diagnosed bacterial sexually transmitted infection and can lead to tubal factor infertility, a disease characterised by fibrosis of the fallopian tubes. Genetic polymorphisms in molecular pathways involving G protein coupled receptor signalling, the Akt/PI3K cascade, the mitotic cell cycle and immune response have been identified in association with the development of trachomatous scarring, an ocular form of chlamydia-related fibrotic pathology. In this case control study, we performed genome-wide association and pathways based analysis in a sample of 71 Dutch women who attended an STI clinic who were seropositive for Chlamydia trachomatis antibodies and 169 high risk Dutch women who sought similar health services but who were seronegative. We identified two regions of within-gene SNP association with Chlamydia trachomatis serological response and found that GPCR signalling and cell cycle pathways were also associated with the trait. These pathway level associations appear to be common to immunological sequelae of Chlamydial infections in both ocular and urogenital tropisms. These pathways may be central mediators of human refractoriness to chlamydial diseases.

genetics↗

bioRxiv2017

Population-based analysis of ocular Chlamydia trachomatis in trachoma-endemic West African communities identifies genomic markers of disease severity

Chlamydia trachomatis (Ct) is the most common infectious cause of blindness and bacterial sexually transmitted infection worldwide. Using Ct whole genome sequences obtained directly from conjunctival swabs, we studied Ct genomic diversity and associations between Ct genetic polymorphisms with ocular localization and disease severity in a treatment-naive trachoma-endemic population in Guinea Bissau, West Africa. All sequences fall within the T2 ocular clade phylogenetically. This is consistent with the presence of the characteristic deletion in trpA resulting in a truncated non-functional protein and the ocular tyrosine repeat regions present in tarP associated with ocular tissue localization. We have identified twenty-one Ct non-synonymous single nucleotide polymorphisms (SNPs) associated with ocular localization, including SNPs within pmpD (OR=4.07, p*=0.001) and tarP (OR=0.34, p*=0.009). Eight SNPs associated with disease severity were found in yjfH (rlmB) (OR=0.13, p*=0.037), CTA0273 (OR=0.12, p*=0.027), trmD (OR=0.12, p*=0.032), CTA0744 (OR=0.12, p*=0.041), glgA (OR=0.10, p*=0.026), alaS (OR=0.10, p*=0.032), pmpE (OR=0.08, p*=0.001) and the intergenic region CTA0744-CTA0745 (OR=0.13, p*=0.043). This study demonstrates the extent of genomic diversity within a naturally circulating population of ocular Ct, and the first to describe novel genomic associations with disease severity. These findings direct investigation of host-pathogen interactions that may be important in ocular Ct pathogenesis and disease transmission.

genomics↗

bioRxiv2017

Active Trachoma Cases In The Solomon Islands Have Varied Polymicrobial Community Structures But Do Not Associate With Individual Non-Chlamydial Pathogens Of The Eye

BackgroundSeveral non-chlamydial microbial pathogens are associated with clinical signs of active trachoma in trachoma-endemic communities with a low prevalence of ocular Chlamydia trachomatis (Ct) infection. In the Solomon Islands, the prevalence of Ct among children is low despite the prevalence of active trachoma being moderate. We therefore set out to investigate whether active trachoma was associated with a common non-chlamydial infection or with a dominant polymicrobial community dysbiosis in the Solomon Islands.\n\nMethodsWe studied DNA from conjunctival swabs collected from 257 Solomon Islanders with active trachoma and matched controls. Droplet digital PCR was used to test for pathogens suspected to be able to induce follicular conjunctivitis. Polymicrobial community diversity and composition were studied by sequencing of hypervariable regions of the 16S ribosomal ribonucleic acid gene in a subset of 54 cases and 53 controls.\n\nResultsAlthough Ct was associated with active trachoma, the number of infections was low (cases: 3.9%, controls: 0.4%). Estimated prevalence (cases, controls) of each non-chlamydial infection was as follows: S. aureus (1.9%, 1.9%), Adenoviridae (1.2%, 1.2%), coagulase-negative Staphylococcus (5.8%, 4.3%), H. influenzae (7.4%, 11.7%), M. catarrhalis (2.3%, 4.7%) and S. pneumoniae (7.0%, 6.2%). There was no statistically significant association between clinical signs of trachoma and presence or load of any of the non-Ct infections that were assayed. Inter-individual variations in the conjunctival microbiome were characterised by differences in the levels of Corynebacterium, Proprionibacterium, Helicobacter and Paracoccus, but diversity and relative abundance of these specific genera did not differ significantly between cases and controls.\n\nDiscussionIt is unlikely that the prevalent trachoma-like follicular conjunctivitis in the Solomon Islands has a dominant bacterial aetiology. Before implementing community-wide azithromycin distribution for trachoma, policy makers should consider that clinical signs of trachoma can be observed in the absence of any detectable azithromycin-susceptible organism.

microbiology↗