In Silico Exploration of Mycobacterium tuberculosis Metabolic Networks Shows Host-Associated Convergent Fluxomic Phenotypes

Mycobacterium tuberculosis, the causative agent of tuberculosis, is composed of several lineages characterized by a genome identity higher than 99%. Although the majority of the lineages are associated with humans, at least four lineages are adapted to other mammals, including different M. tuberculosis ecotypes. Host specificity is associated with higher virulence in its preferred host in ecotypes such as M. bovis. Deciphering what determines the preference of the host can reveal host-specific virulence patterns. However, it is not clear which genomic determinants might be influencing host specificity. In this study, we apply a combination of unsupervised and supervised classification methods on genomic data of ~27,000 M. tuberculosis clinical isolates to decipher host-specific genomic determinants. Host-specific genomic signatures are scarce beyond known lineage-specific mutations. Therefore, we integrated lineage-specific mutations into the iEK1011 2.0 genome-scale metabolic model to obtain lineage-specific versions of it. Flux distributions sampled from the solution spaces of these models can be accurately separated according to host association. This separation correlated with differences in cell wall processes, lipid, amino acid and carbon metabolic subsystems. These differences were observable when more than 95% of the samples had a specific growth rate significantly lower than the maximum achievable by the models. This suggests that these differences might manifest at low growth rate settings, such as the restrictive conditions M. tuberculosis suffers during macrophage infection

Legionellosis Outbreak Associated with Asphalt Paving Machine, Spain, 2009

TOC Summary: The source was untreated spring water in the machine’s water tank

Molecular epidemiology and whole genome sequencing analysis of clinical Mycobacterium bovis from Ghana.

BACKGROUND: Bovine tuberculosis (bTB) caused by Mycobacterium bovis is a re-emerging problem in both livestock and humans. The association of some M. bovis strains with hyper-virulence, MDR-TB and disseminated disease makes it imperative to understand the biology of the pathogen. METHODS: Mycobacterium bovis (15) among 1755 M. tuberculosis complex (MTBC) isolated between 2012 and 2014 were characterized and analyzed for associated patient demography and other risk factors. Five of the M. bovis isolates were whole-genome sequenced and comparatively analyzed against a global collection of published M. bovis genomes. RESULTS: Mycobacterium bovis was isolated from 3/560(0.5%) females and 12/1195(1.0%) males with pulmonary TB. The average age of M. bovis infected cases was 46.8 years (7-72years). TB patients from the Northern region of Ghana (1.9%;4/212) had a higher rate of infection with M. bovis (OR = 2.7,p = 0.0968) compared to those from the Greater Accra region (0.7%;11/1543). Among TB patients with available HIV status, the odds of isolating M. bovis from HIV patients (2/119) was 3.3 higher relative to non-HIV patients (4/774). Direct contact with livestock or their unpasteurized products was significantly associated with bTB (p<0.0001, OR = 124.4,95% CI = 30.1-508.3). Two (13.3%) of the M. bovis isolates were INH resistant due to the S315T mutation in katG whereas one (6.7%) was RIF resistant with Q432P and I1491S mutations in rpoB. M. bovis from Ghana resolved as mono-phyletic branch among mostly M. bovis from Africa irrespective of the host and were closest to the root of the global M. bovis phylogeny. M. bovis-specific amino acid mutations were detected among MTBC core genes such as mce1A, mmpL1, pks6, phoT, pstB, glgP and Rv2955c. Additional mutations P6T in chaA, G187E in mgtC, T35A in Rv1979c, S387A in narK1, L400F in fas and A563T in eccA1 were restricted to the 5 clinical M. bovis from Ghana. CONCLUSION: Our data indicate potential zoonotic transmission of bTB in Ghana and hence calls for intensified public education on bTB, especially among risk groups

Adaptive advantage of deletion repair in the N-terminal domain of the SARS-CoV-2 spike protein in variants of concern

Mutations within the N-terminal domain (NTD) of the spike (S) protein play a pivotal role in the emergence of successful SARS-CoV-2 viral lineages. This study investigates the influence on viral success of novel combinations of NTD lineage-defining mutations found in the Alpha, Delta, and Omicron variants. We performed comparative genomics of more than 10 million public SARS-CoV-2 samples to decipher the transmission success of different combinations of NTD markers. Additionally, we characterized the viral phenotype of such markers in a surrogate in vitro system. Alpha viruses bearing repaired deletions S:ΔH69/V70 and S:ΔY144 in Alpha background were associated with increased transmission relative to other combinations of NTD markers. After the emergence of the Omicron BA.1 lineage, Alpha viruses harbouring both repaired deletions still showed increased transmission compared to their BA.1 analogues. Moreover, repaired deletions were more frequently observed among older individuals infected with Alpha, but not with BA.1. In vitro biological characterization of Omicron BA.1 spike deletion repair patterns also revealed substantial differences with Alpha. In BA.1, S:ΔV143/Y145 repair enhanced fusogenicity and susceptibility to neutralization by vaccinated individuals’ sera. In contrast, the S:ΔH69/V70 repair did not significantly alter these traits but reduced viral infectivity. Simultaneous repair of both deletions led to lower fusogenicity. These findings highlight the intricate genotype-phenotype landscape of the spike NTD in SARS-CoV-2, which impacts viral biology, transmission efficiency, and susceptibility to neutralization. Overall, this study advances our understanding of SARS-CoV-2 evolution, carrying implications for public health and future research.This research work was funded by the European Commission – NextGenerationEU (Regulation EU 2020/2094), through CSIC's Global Health Platform (PTI+ Salud Global) to MC, RG, IC and FGC. MAH is supported by the Generalitat Valenciana and the European Social Fund “ESF Investing in your future” through grant CIACIF/2022/333. This work was also a part of projects CNS2022-135116 (MC) and CNS2022-135100 (RG) funded by MCIN/AEI/10.13039/501100011033 and the European Union NextGenerationEU/PRTRN

Combinatorial analysis of deletion repair in SARS-CoV-2 variants of concern

Resumen del póster presentado a las III Jornadas Científicas PTI+ Salud Global, celebradas en el Centro de Ciencias Humanas y Sociales (CCHS), CSIC (Madrid) del 20 al 22 de noviembre de 2023.[Background] The spike protein of SARS-CoV-2 is a key determinant of viral fitness and immune evasion, and its N-terminal domain (NTD) is prone to mutations that may confer fitness advantages to the virus. Most variants of concern (VOCs), including Alpha, Delta, and Omicron, have harbored distinct NTD lineage-defining mutations. However, the relationship between genotype and the impact on viral transmission and viral phenotype is not yet fully understood.[Methods] We analyzed over 10 million SARS-CoV-2 genomes from GISAID to investigate the prevalence and estimate the transmission of different combinations of NTD mutations across the Alpha and the Omicron variants. Additionally, we characterized the viral phenotype of deletion repair events in a surrogate in vitro system, assessing their infectivity, fusogenicity, thermal stability, protein surface expression, and neutralization sensitivity.[Results] Some NTD mutations, such the repair of deleted amino acids at sites S:69/70 and S:144 in Alpha viruses, were associated with an increased transmission rate and higher frequency among older age groups. These deletion repairs were also detected in Omicron, but with different patterns and effects. For instance, the repair of deletion at site S:143/145 in Omicron enhanced viral fusogenicity and neutralization by sera from vaccinated individuals. However, the repair of the deletion at site S:69/70 reduced viral infectivity and did not affect these traits. The co-occurrence of both repairs resulted in reduced fusogenicity.[Conclusions] Our study reveals the complex interplay between NTD mutations, including those that lead to deletion repair, and viral success in SARS-CoV-2. This may have implications for viral transmission, immunity, and vaccine efficacy. Our findings improve our understanding of SARS- CoV-2 evolution, and provide insights for future research and public health interventions.Peer reviewe

Genomic determinants associated with SARS-CoV-2 virulence

Trabajo presentado al 31st European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), celebrado online del 9 al 12 de julio de 2021.This work was funded by the Instituto de Salud Carlos III project COV2o/oo140 and COV2o/ o0437,Spanish National Research Council project CSIC­ COV19·0l1 and CSIC-COVID19-082,and the Generalitat Valenciana (SEJI/2019/011 and Covid_19-SCI).Action co-financed by the European Union through the Operatianal Program of the European Regional Development Fund (ERDF) of the Valencian Community 2014-2020.MC is supported by Ramón y Cajal program from Ministerio de Ciencia,grant RTI2018-094399-A-I00.Peer reviewe

Bioinformatics analysis of mutations in SARSCoV- 2 and clinical phenotypes

1 p.-1 fig.-8 tab.Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initially reported in Wuhan (China) hasspread worldwide. Like other viruses, SARS-CoV-2 accumulates mutations with each cycle of replication by continuously evolving a viral strain with one or more single nucleotide variants (SNVs). However, SNVs that cause severe COVID-19 or lead to immune escape or vaccine failure are not well understood. We aim to identify SNVs associated with severe clinical phenotypes.Methods: In this study, 27429 whole-genome aligned consensus sequences of SARS-CoV-2 were collected from genomic epidemiology of SARS-CoV-2 project in Spain (SeqCOVID) [1]. These samples were obtained from patients who required hospitalization and/or intensive care unit admission (ICU), excluding those registered in the first pandemic wave.Besides, 248 SARS-CoV-2 genomes were isolated from COVID-19 hospitalized patients from Gregorio Marañon General University Hospital (GMH) of which 142 were fully vaccinated. Bioinformatics tools using R and Python programming languages were developed and implemented comparing those to SARS-CoV-2 Wuhan-Hu-1 (reference genome).Results: Using a selection threshold mutational frequency 10%, 27 SNVs were expected to have association with hospitalization and ICU risk. The reference haplotype differing at the SNV coding for lysine at the residue 203 (N:R203K) was found to have negative association with COVID-19 hospitalization risk (p = 5.37 x 10-04). Similarly, a negative association was observed when the residue at 501 is replaced by tyrosine (S:N501Y) (p = 1.33 x 10-02). The application of a Chi-square test suggested that SNV-haplotypes coding for mutants residues such as (S:A222V, N:A220V, ORF10:V30L) and (ORF1a:T1001I, ORF1a:I2230T, S:N501Y, S:T716S, S:S982A, ORF8:Q27*, N:R203K, N:S235F) have negative associations with COVID-19 hospitalization risk (p = 6.58 x 10-07 and p = 2.27 x 10-16, respectively) and COVID-19 ICU risk (p = 1.15 x 10-02 and p = 2.51 x 10-02, respectively). Focusing on the SNV-haplotype coding the mutations (S:A222V, N:A220V, N:D377Y, ORF10:V30L) were observed to increase the risk of COVID-19 hospitalization (p = 2.71 x 10-04). Results from SARS-CoV-2 genomes analysis from GMH showed 63 coding SNVs which met the established threshold value. Applying a Chi-square test, the SNV-haplotype carrying coding variants for mutant residues in 5 ORF proteins and surface and membrane glycoprotein and nucleocapsid phosphoprotein was significantly associated with vaccine failure in hospitalized COVID-19 patients (p = 7.91 x 10-04).Conclusions: SNV-haplotypes carrying variants lead to non-synonymous mutations located along SARS-CoV-2 wholeproteome may influence COVID-19 severity and vaccine failure suggesting a functional role in the clinical outcome for COVID-19 patients.This research work was funded by the European Commission-NextGenerationEU (Regulation EU 2020/2094), through CSIC’s Global Health Platform (PTI Salud Global)Peer reviewe

Phylogenomics of Mycobacterium africanum reveals a new lineage and a complex evolutionary history

Coscolla et al.Human tuberculosis (TB) is caused by members of the Mycobacterium tuberculosis complex (MTBC). The MTBC comprises several human-adapted lineages known as M. tuberculosis sensu stricto, as well as two lineages (L5 and L6) traditionally referred to as Mycobacterium africanum . Strains of L5 and L6 are largely limited to West Africa for reasons unknown, and little is known of their genomic diversity, phylogeography and evolution. Here, we analysed the genomes of 350 L5 and 320 L6 strains, isolated from patients from 21 African countries, plus 5 related genomes that had not been classified into any of the known MTBC lineages. Our population genomic and phylogeographical analyses showed that the unclassified genomes belonged to a new group that we propose to name MTBC lineage 9 (L9). While the most likely ancestral distribution of L9 was predicted to be East Africa, the most likely ancestral distribution for both L5 and L6 was the Eastern part of West Africa. Moreover, we found important differences between L5 and L6 strains with respect to their phylogeographical substructure and genetic diversity. Finally, we could not confirm the previous association of drug-resistance markers with lineage and sublineages. Instead, our results indicate that the association of drug resistance with lineage is most likely driven by sample bias or geography. In conclusion, our study sheds new light onto the genomic diversity and evolutionary history of M. africanum , and highlights the need to consider the particularities of each MTBC lineage for understanding the ecology and epidemiology of TB in Africa and globally.M.C. is supported by the Ramón y Cajal programme from the Ministerio de Ciencia, Innovación y Universidades. This work was supported by the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) (research award to M.C.), Ministerio de Ciencia, Innovación y Universidades (grant number RTI2018-094399-A-I00 to M.C.) and Consellería de Educació de la Generalitat Valenciana (grant number SEJI/2019/011 to M.C.), the Swiss National Science Foundation (grants 310030_188888, IZRJZ3_164171, IZLSZ3_170834 and CRSII5_177163 to S. G.), the European Research Council (883582-ECOEVODRTB to S. G.) and Wellcome (grant number 097134/Z/11/Z to D. Y.-M).Peer reviewe

Comparative genomics of Mycobacterium africanum Lineage 5 and Lineage 6 from Ghana suggests distinct ecological niches.

Mycobacterium africanum (Maf) causes a substantial proportion of human tuberculosis in some countries of West Africa, but little is known on this pathogen. We compared the genomes of 253 Maf clinical isolates from Ghana, including N = 175 Lineage 5 (L5) and N = 78 Lineage 6 (L6). We found that the genomic diversity of L6 was higher than in L5 despite the smaller sample size. Regulatory proteins appeared to evolve neutrally in L5 but under purifying selection in L6. Even though over 90% of the human T cell epitopes were conserved in both lineages, L6 showed a higher ratio of non-synonymous to synonymous single nucleotide variation in these epitopes overall compared to L5. Of the 10% human T cell epitopes that were variable, most carried mutations that were lineage-specific. Our findings indicate that Maf L5 and L6 differ in some of their population genomic characteristics, possibly reflecting different selection pressures linked to distinct ecological niches