Unravelling Chlamydia trachomatis diversity in Amhara, Ethiopia: MLVA-ompA sequencing as a molecular typing tool for trachoma.

Trachoma is the leading infectious cause of blindness worldwide and is now largely confined to around 40 low- and middle-income countries. It is caused by Chlamydia trachomatis (Ct), a contagious intracellular bacterium. The World Health Organization recommends mass drug administration (MDA) with azithromycin for treatment and control of ocular Ct infections, alongside improving facial cleanliness and environmental conditions to reduce transmission. To understand the molecular epidemiology of trachoma, especially in the context of MDA and transmission dynamics, the identification of Ct genotypes could be useful. While many studies have used the Ct major outer membrane protein gene (ompA) for genotyping, it has limitations. Our study applies a typing system novel to trachoma, Multiple Loci Variable Number Tandem Repeat Analysis combined with ompA (MLVA-ompA). Ocular swabs were collected post-MDA from four trachoma-endemic zones in Ethiopia between 2011-2017. DNA from 300 children with high Ct polymerase chain reaction (PCR) loads was typed using MLVA-ompA, utilizing 3 variable number tandem repeat (VNTR) loci within the Ct genome. Results show that MLVA-ompA exhibited high discriminatory power (0.981) surpassing the recommended threshold for epidemiological studies. We identified 87 MLVA-ompA variants across 26 districts. No significant associations were found between variants and clinical signs or chlamydial load. Notably, overall Ct diversity significantly decreased after additional MDA rounds, with a higher proportion of serovar A post-MDA. Despite challenges in sequencing one VNTR locus (CT1299), MLVA-ompA demonstrated cost-effectiveness and efficiency relative to whole genome sequencing, providing valuable information for trachoma control programs on local epidemiology. The findings suggest the potential of MLVA-ompA as a reliable tool for typing ocular Ct and understanding transmission dynamics, aiding in the development of targeted interventions for trachoma control.

HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1 allele frequencies of North Tanzanian Maasai.

Locus-specific amplicon sequencing was used to HLA type 336 participants of Maasai ethnicity at the HLA-A, -B, -C, -DRB1, -DQB1 and -DPB1 loci. Participants were recruited from three study villages in North Tanzania, for the purpose of investigating risk factors for trachomatous scarring in children. Other than HLA-A, all loci significantly deviated from Hardy-Weinberg equilibrium, possibly due to high relatedness between individuals: 238 individuals shared a house with at least one another participant. The most frequent allele for each locus were A*68:02 (14.3 %), B*53:01 (8.4 %), C*06:02 (19.2 %), DRB1*13:02 (17.7 %), DQB1*02:01 (16.9 %) and DPB1*01:01 (15.7 %), while the most common inferred haplotype was A*68:02 âˆ¼ B*18:01 âˆ¼ C*07:04 âˆ¼ DRB1*08:04 âˆ¼ DQB1*04:02 âˆ¼ DPB1*04:01 (1.3 %).

The conjunctival microbiome before and after azithromycin mass drug administration for trachoma control in a cohort of Tanzanian children.

Background: Trachoma, caused by ocular infection with Chlamydia trachomatis, is a neglected tropical disease that can lead to blinding pathology. Current trachoma control programmes have successfully used mass drug administration (MDA) with azithromycin to clear C. trachomatis infection and reduce transmission, alongside promoting facial cleanliness for better personal hygiene and environmental improvement. In areas of low-trachoma endemicity, the relationship between C. trachomatis infection and trachomatous disease weakens, and non-chlamydial bacteria have been associated with disease signs. Methods: We enrolled a cohort of children aged 6-10 years from three adjacent trachoma endemic villages in Kilimanjaro and Arusha regions, Northern Tanzania. Children were divided into four clinical groups based on the presence or absence of ocular C. trachomatis infection and clinical signs of trachomatous papillary inflammation (TP). To determine the impact of treatment on the ocular microbiome in these clinical groups, we performed V4-16S rRNA sequencing of conjunctival DNA from children 3-9 months pre-MDA (n = 269) and 3 months post-MDA (n = 79). Results: Chlamydia trachomatis PCR-negative, no TP children had the highest pre-MDA ocular microbiome alpha diversity, which was reduced in C. trachomatis infected children and further decreased in those with TP. Pre-MDA, Haemophilus and Staphylococcus were associated with C. trachomatis infection with and without concurrent TP, while Helicobacter was increased in those with TP in the absence of current C. trachomatis infection. Post-MDA, none of the studied children had ocular C. trachomatis infection or TP. MDA increased ocular microbiome diversity in all clinical groups, the change was of greater magnitude in children with pre-MDA TP. MDA effectively reduced the prevalence of disease causing pathogenic non-chlamydial bacteria, and promoted restoration of a normal, healthy conjunctival microbiome. Conclusion: We identified Helicobacter as a non-chlamydial bacterium associated with the clinical signs of TP. Further investigation to determine its relevance in other low-endemicity communities is required. MDA was shown to be effective at clearing C. trachomatis infection and other non-chlamydial ocular pathogens, without any detrimental longitudinal effects on the ocular microbiome. These findings suggest that azithromycin MDA may be valuable in trachoma control even in populations where the relationship between clinical signs of trachoma and the prevalence of current ocular C. trachomatis infection has become dissociated.

Effects of ivermectin mass drug administration for malaria vector control on ectoparasites and soil-transmitted helminths: a cluster randomized trial.

OBJECTIVES: Ivermectin, used to control several neglected tropical diseases, may also reduce malaria transmission. Mass drug administration (MDA) for malaria control therefore might have off-target impacts on neglected tropical diseases. METHODS: In The Gambia, nested in a trial of ivermectin MDA, cross-sectional surveys measuring ectoparasites and soil-transmitted helminths in children aged 3 to 14 years took place in June and November 2019 and in November 2021. RESULTS: After MDA, scabies prevalence was 41.2% (237/576) in the control and 38.2% (182/476) in the intervention arm (odds ratio [OR] 0.89 (95% confidence interval [CI] 0 67-1.2), P-value = 0.471) but by 2021, had rebounded to 38.8% (180/464) in the control and 53.2% (245/458) in the intervention arm. After MDA, prevalence of Strongyloides stercoralis was 16.8% (87/518) in the control and 9.1% (40/440) in the intervention arm (OR 0.4 (95% CI 0.16-0.94), P-value = 0.039). In 2021, it was 9.2% (38/413) in the control and 11.3% (45/399) in the intervention arm (OR 1.31 (95% CI 0.74-2.28), P-value = 0.35). CONCLUSION: Scabies prevalence was similar between the two study arms. S. stercoralis prevalence was reduced. However, this effect did not last long: the prevalence 2 years after MDA was similar between study arms.

Biannual Administrations of Azithromycin and the Gastrointestinal Microbiome of Malawian Children: A Nested Cohort Study Within a Randomized Controlled Trial.

Community-level mass treatment with azithromycin has been associated with a mortality benefit in children. However, antibiotic exposures result in disruption of the gut microbiota and repeated exposures may reduce recovery of the gut flora. We conducted a nested cohort study within the framework of a randomized controlled trial to examine associations between mass drug administration (MDA) with azithromycin and the gut microbiota of rural Malawian children aged between 1 and 59 months. Fecal samples were collected from the children at baseline and 6 months after two or four biannual rounds of azithromycin treatment. DNA was extracted from fecal samples and V4-16S rRNA sequencing used to characterize the gut microbiota. Firmicutes, Bacteroidetes, Proteobacteria and Actinobacteria were the dominant phyla while Faecalibacterium and Bifidobacterium were the most prevalent genera. There were no associations between azithromycin treatment and changes in alpha diversity, however, four biannual rounds of treatment were associated with increased abundance of Prevotella. The lack of significant changes in gut microbiota after four biannual treatments supports the use of mass azithromycin treatment to reduce mortality in children living in low- and middle-income settings.

The Mycobacterium tuberculosis sRNA F6 Modifies Expression of Essential Chaperonins, GroEL2 and GroES.

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of posttranscriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA F6 and shown it to be dependent on SigF for expression and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins GroEL2 and GroES. Our results point toward a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas. IMPORTANCE Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis. Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA. Our results demonstrate that deletion of F6 leads to dysregulation of the two essential chaperonins GroEL2 and GroES and, moreover, indicate a role for F6 in the long-term survival and persistence of M. tuberculosis in the human host.

Meningococcal carriage among Hajj pilgrims, risk factors for carriage and records of vaccination: a study of pilgrims to Mecca.

OBJECTIVE: The Saudi government requires that all pilgrims receive a quadrivalent meningococcal vaccine at least 10 days before the Hajj. We conducted a study to determine the uptake of meningococcal vaccine and antibiotic use. We also investigated risk factors of meningococcal carriage and carriage of Neisseria meningitidis pathogenic serogroups A, C, W and Y. METHODS: A cross-sectional oropharyngeal carriage survey was conducted in 2973 Hajj pilgrims in September 2017. A real-time polymerase chain reaction (rt-PCR) assay was used to identify N. meningitidis from the oropharyngeal swabs. A questionnaire investigated potential risk factors for carriage of N. meningitidis. RESULTS: Two thousand two hundred forty nine oropharyngeal swabs were obtained. The overall prevalence of carriage of N. meningitidis was 4.6% (95% CI: 3.4%-6%). Carriage of pathogenic serogroups was not associated significantly with any of the meningococcal risk factors evaluated. 77% of pilgrims were vaccinated but 22.58 % said they were carrying unofficial vaccination cards. CONCLUSION: Carriage of serogroups A, C, W and Y was not significantly associated with any of the risk factors investigated. Almost a quarter of pilgrims were unlikely to have been vaccinated, highlighting a need to strengthen compliance with the current policy of vaccination to prevent meningococcal disease outbreaks during and after the Hajj.

Prevalence, risk factors and health consequences of soil-transmitted helminth infection on the Bijagos Islands, Guinea Bissau: A community-wide cross-sectional study.

Soil-transmitted helminths (STH) are endemic and widespread across Sub-Saharan Africa. A community wide soil-transmitted helminth (STH) prevalence survey was performed on the island of Bubaque in Guinea-Bissau using both Kato-katz microscopy and qPCR methodology. Predictors of infection and morbidity indicators were identified using multivariable logistic regression, and diagnostic methods were compared using k statistics. Among 396 participants, prevalence of STH by microscopy was 23.2%, hookworm was the only species identified by this method and the mean infection intensity was 312 eggs per gram. qPCR analysis revealed an overall prevalence of any STH infection of 47.3%, with the majority A. duodenale (32.3%), followed by N. americanus (15.01%) and S. stercoralis (13.2%). A. lumbricoides, and T. trichiura infections were negligible, with a prevalence of 0.25% each. Agreement between diagnostic tests was k = 0.22, interpreted as fair agreement, and infection intensity measured by both methods was only minimally correlated (Rs = -0.03). STH infection overall was more common in females and adults aged 31-40. STH infection was associated with open defaecation, low socio-economic status and further distance to a water-source. The prevalence of anaemia (defined as a binary outcome by the WHO standards for age and sex) was 69.1%, and 44.2% of children were malnourished according to WHO child growth standards. Hookworm infection intensity by faecal egg count showed no statistically significant association with age (Rs 0.06) but S. Stercoralis infection intensity by qPCR cycle threshold was higher in pre-school aged children (Rs = 0.30, p-value 0.03) There was no statistically significant association between STH infection and anaemia (OR 1.0 p = 0.8), stunting (OR 1.9, p-value 0.5) and wasting (OR 2.0, p-value 0.2) in children. This study reveals a persistent reservoir of STH infection across the community, with high rates of anaemia and malnutrition, despite high-coverage of mebendazole mass-drug administration in pre-school children. This reflects the need for a new strategy to soil-transmitted helminth control, to reduce infections and ultimately eliminate transmission.

Viability PCR shows that non-ocular surfaces could contribute to transmission of Chlamydia trachomatis infection in trachoma.

BACKGROUND: The presence of Chlamydia trachomatis (Ct) DNA at non-ocular sites suggests that these sites may represent plausible routes of Ct transmission in trachoma. However, qPCR cannot discriminate between DNA from viable and non-viable bacteria. Here we use a propodium monoazide based viability PCR to investigate how long Ct remains viable at non-ocular sites under laboratory-controlled conditions. METHODS: Cultured Ct stocks (strain A2497) were diluted to final concentrations of 1000, 100, 10 and 1 omcB copies/µL and applied to plastic, woven mat, cotton cloth and pig skin. Swabs were then systemically collected from each surface and tested for the presence Ct DNA using qPCR. If Ct DNA was recovered, Ct viability was assessed over time by spiking multiple areas of the same surface type with the same final concentrations. Swabs were collected from each surface at 0, 2, 4, 6, 8 and 24 hours after spiking. Viability PCR was used to determine Ct viability at each timepoint. RESULTS: We were able to detect Ct DNA on all surfaces except the woven mat. Total Ct DNA remained detectable and stable over 24 hours for all concentrations applied to plastic, pig skin and cotton cloth. The amount of viable Ct decreased over time. For plastic and skin surfaces, only those where concentrations of 100 or 1000 omcB copies/µL were applied still had viable loads detectable after 24 hours. Cotton cloth showed a more rapid decrease and only those where concentrations of 1000 omcB copies/µL were applied still had viable DNA detectable after 24 hours. CONCLUSION: Plastic, cotton cloth and skin may contribute to transmission of the Ct strains that cause trachoma, by acting as sites where reservoirs of bacteria are deposited and later collected and transferred mechanically into previously uninfected eyes.

Optimising the use of molecular tools for the diagnosis of yaws.

BACKGROUND: Yaws is a neglected tropical disease and results in lesions of skin, soft tissues and bones. PCR plays an important part in surveillance. METHODS: Children suspected to have yaws were enrolled. From the largest lesion, paired swabs were collected, one in transport medium and one as a dry swab. In children with multiple lesions we collected additional swabs from up to four subsequent lesions. Swabs in transport medium were maintained in a cold chain while dry swabs were stored at ambient temperature. Swabs were tested by PCR for Treponema pallidum and Haemophilus ducreyi. RESULTS: Of 55 individuals, 10 (18%) had at least one positive PCR for T. pallidum and 12 (22%) had at least one positive result for H. ducreyi. Concordance was 100% between swabs in transport medium and dry swabs. One patient had PCR-confirmed yaws on the swab of a third lesion when both the first and second lesions were PCR-negative. CONCLUSIONS: Storing swabs in transport medium and transporting in a cold chain did not improve yield, however, detection of T. pallidum is increased by swabbing additional lesions. As the target for yaws is eradication, approaches to sample collection need revisiting to ensure cases are not missed.

Conjunctival Microbiome-Host Responses Are Associated With Impaired Epithelial Cell Health in Both Early and Late Stages of Trachoma.

Background: Trachoma, 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 hypothesized risk factors for development of active trachoma and conjunctival scarring. Methods: Ocular 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. Findings: In 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. Interpretation: In the absence of current Chlamydia trachomatis infection, changes in the ocular microbiome associate with differential expression of antimicrobial and inflammatory genes 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-2 interferon responses in scarring, whilst highlighting a common suppression of re-epithelialization with altered epithelial and bacterial adhesion, likely contributing to development of scarring pathology.

Influenza Vaccination Primes Human Myeloid Cell Cytokine Secretion and NK Cell Function.

Cytokine-induced memory-like (CIML) NK cells generated in response to proinflammatory cytokines in vitro and in vivo can also be generated by vaccination, exhibiting heightened responses to cytokine stimulation months after their initial induction. Our previous study demonstrated that in vitro human NK cell responses to inactivated influenza virus were also indirectly augmented by very low doses of IL-15, which increased induction of myeloid cell-derived cytokine secretion. These findings led us to hypothesize that IL-15 stimulation could reveal a similar effect for active influenza vaccination and influence CIML NK cell effector functions. In this study, 51 healthy adults were vaccinated with seasonal influenza vaccine, and PBMC were collected before and up to 30 d after vaccination. Myeloid and lymphoid cell cytokine secretion was measured after in vitro PBMC restimulation with low-dose IL-15, alone or in combination with inactivated H3N2 virus; the associated NK cell response was assessed by flow cytometry. PBMC collected 30 d postvaccination showed heightened cytokine production in response to IL-15 compared with PBMC collected at baseline; these responses were further enhanced when IL-15 was combined with H3N2. NK cell activation in response to IL-15 alone (CD25) and H3N2 plus IL-15 (CD25 and IFN-γ) was enhanced postvaccination. We also observed proliferation of less-differentiated NK cells with downregulation of cytokine receptors as early as 3 d after vaccination, suggesting cytokine stimulation in vivo. We conclude that vaccination-induced "training" of accessory cells combines with the generation of CIML NK cells to enhance the overall NK cell response postvaccination.

Proteomic and transcriptomic experiments reveal an essential role of RNA degradosome complexes in shaping the transcriptome of Mycobacterium tuberculosis.

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.

Active Trachoma Cases in the Solomon Islands Have Varied Polymicrobial Community Structures but Do Not Associate with Individual Non-Chlamydial Pathogens of the Eye.

BACKGROUND: Several 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. Therefore, we 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. METHODS: We 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. RESULTS: Although Ct was associated with active trachoma, the number of infections was low (cases, 3.9%; controls, 0.4%). Estimated prevalence (cases and controls, respectively) of each non-chlamydial infection was as follows: Staphylococcus aureus: 1.9 and 1.9%, Adenoviridae: 1.2 and 1.2%, coagulase-negative Staphylococcus: 5.8 and 4.3%, Haemophilus influenzae: 7.4 and 11.7%, Moraxella catarrhalis: 2.3 and 4.7%, and Streptococcus pneumoniae: 7.0 and 6.2%. There was no statistically significant association between the clinical signs of trachoma and the presence or load of any of the non-Ct infections that were assayed. Interindividual variations in the conjunctival microbiome were characterized by differences in the levels of Corynebacterium, Propionibacterium, Helicobacter, and Paracoccus, but diversity and relative abundance of these specific genera did not differ significantly between cases and controls. DISCUSSION: It is unlikely that the prevalent trachoma-like follicular conjunctivitis in this region of the Solomon Islands has a dominant bacterial etiology. 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.

Differential frequency of NKG2C/KLRC2 deletion in distinct African populations and susceptibility to Trachoma: a new method for imputation of KLRC2 genotypes from SNP genotyping data.

NKG2C is an activating receptor that is preferentially expressed on natural killer (NK) cells. The gene encoding NKG2C (killer cell lectin-like receptor C2, KLRC2) is present at different copy numbers in the genomes of different individuals. Deletion at the NKG2C locus was investigated in a case-control study of 1522 individuals indigenous to East- and West-Africa and the association with the ocular Chlamydia trachomatis infection and its sequelae was explored. The frequency of homozygous KLRC2 deletion was 13.7 % in Gambians and 4.7 % in Tanzanians. A significantly higher frequency of the deletion allele was found in West-Africans from the Gambia and Guinea-Bissau (36.2 % p = 2.105 × 10(-8), 26.8 % p = 0.050; respectively) in comparison to East-African Tanzanians where the frequency of the deletion is comparable to other human populations (20.9 %). We found no evidence for an association between the numbers of KLRC2 gene copies and the clinical manifestations of trachoma (follicular trachoma or conjunctival scarring). A new method for imputation of KLRC2 genotypes from single nucleotide polymorphism (SNP) data in 2621 individuals from the Gambia further confirmed these results. Our data suggest that NKG2C does not play a major role in trachomatous disease. We found that the deletion allele is present at different frequencies in different populations but the reason behind these differences is currently not understood. The new method offers the potential to use SNP arrays from genome wide association studies to study the frequency of KLRC2 deletion in other populations and its association with other diseases.

A small RNA encoded in the Rv2660c locus of Mycobacterium tuberculosis is induced during starvation and infection.

Enhanced transcription of the Rv2660c locus in response to starvation of Mycobacterium tuberculosis H37Rv encouraged addition of the predicted Rv2660c protein to an improved vaccine formulation. Using strand-specific RNA sequencing, we show that the up-regulated transcript is in fact a small RNA encoded on the opposite strand to the annotated Rv2660c. The transcript originates within a prophage and is expressed only in strains that carry PhiRv2. The small RNA contains both host and phage sequences and provides a useful biomarker to monitor bacterial starvation during infection and/or non-replicating persistence. Using different approaches we do not find any evidence of Rv2660c at the level of mRNA or protein. Further efforts to understand the mechanism by which Rv2660c improves efficacy of the H56 vaccine are likely to provide insights into the pathology and immunology of tuberculosis.

Important role for Mycobacterium tuberculosis UvrD1 in pathogenesis and persistence apart from its function in nucleotide excision repair.

Mycobacterium tuberculosis survives and replicates in macrophages, where it is exposed to reactive oxygen and nitrogen species that damage DNA. In this study, we investigated the roles of UvrA and UvrD1, thought to be parts of the nucleotide excision repair pathway of M. tuberculosis. Strains in which uvrD1 was inactivated either alone or in conjunction with uvrA were constructed. Inactivation of uvrD1 resulted in a small colony phenotype, although growth in liquid culture was not significantly affected. The sensitivity of the mutant strains to UV irradiation and to mitomycin C highlighted the importance of the targeted genes for nucleotide excision repair. The mutant strains all exhibited heightened susceptibility to representatives of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). The uvrD1 and the uvrA uvrD1 mutants showed decreased intracellular multiplication following infection of macrophages. Most importantly, the uvrA uvrD1 mutant was markedly attenuated following infection of mice by either the aerosol or the intravenous route.

Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid.

Thymidylate synthase (TS) enzymes catalyse the biosynthesis of deoxythymidine monophosphate (dTMP or thymidylate), and so are important for DNA replication and repair. Two different types of TS proteins have been described (ThyA and ThyX), which have different enzymic mechanisms and unrelated structures. Mycobacteria are unusual as they encode both thyA and thyX, and the biological significance of this is not yet understood. Mycobacterium tuberculosis ThyX is thought to be essential and a potential drug target. We therefore analysed M. tuberculosis thyA and thyX expression levels, their essentiality and roles in pathogenesis. We show that both thyA and thyX are expressed in vitro, and that this expression significantly increased within murine macrophages. Under all conditions tested, thyA expression exceeded that of thyX. Mutational studies show that M. tuberculosis thyX is essential, confirming that the enzyme is a plausible drug target. The requirement for M. tuberculosis thyX in the presence of thyA implies that the essential function of ThyX is something other than dTM synthesis [corrected].We successfully deleted thyA from the M. tuberculosis genome, and this deletion conferred an in vitro growth defect that was not observed in vivo. Presumably ThyX performs TS activity within M. tuberculosis ΔthyA at a sufficient rate in vivo for normal growth, but the rate in vitro is less than optimal. We also demonstrate that thyA deletion confers M. tuberculosis p-aminosalicylic acid resistance, and show by complementation studies that ThyA T202A and V261G appear to be functional and non-functional, respectively.

Sequence-based analysis uncovers an abundance of non-coding RNA in the total transcriptome of Mycobacterium tuberculosis.

RNA sequencing provides a new perspective on the genome of Mycobacterium tuberculosis by revealing an extensive presence of non-coding RNA, including long 5' and 3' untranslated regions, antisense transcripts, and intergenic small RNA (sRNA) molecules. More than a quarter of all sequence reads mapping outside of ribosomal RNA genes represent non-coding RNA, and the density of reads mapping to intergenic regions was more than two-fold higher than that mapping to annotated coding sequences. Selected sRNAs were found at increased abundance in stationary phase cultures and accumulated to remarkably high levels in the lungs of chronically infected mice, indicating a potential contribution to pathogenesis. The ability of tubercle bacilli to adapt to changing environments within the host is critical to their ability to cause disease and to persist during drug treatment; it is likely that novel post-transcriptional regulatory networks will play an important role in these adaptive responses.

The biological and structural characterization of Mycobacterium tuberculosis UvrA provides novel insights into its mechanism of action.

Mycobacterium tuberculosis is an extremely well adapted intracellular human pathogen that is exposed to multiple DNA damaging chemical assaults originating from the host defence mechanisms. As a consequence, this bacterium is thought to possess highly efficient DNA repair machineries, the nucleotide excision repair (NER) system amongst these. Although NER is of central importance to DNA repair in M. tuberculosis, our understanding of the processes in this species is limited. The conserved UvrABC endonuclease represents the multi-enzymatic core in bacterial NER, where the UvrA ATPase provides the DNA lesion-sensing function. The herein reported genetic analysis demonstrates that M. tuberculosis UvrA is important for the repair of nitrosative and oxidative DNA damage. Moreover, our biochemical and structural characterization of recombinant M. tuberculosis UvrA contributes new insights into its mechanism of action. In particular, the structural investigation reveals an unprecedented conformation of the UvrB-binding domain that we propose to be of functional relevance. Taken together, our data suggest UvrA as a potential target for the development of novel anti-tubercular agents and provide a biochemical framework for the identification of small-molecule inhibitors interfering with the NER activity in M. tuberculosis.