Gene evolutionary trajectories in Mycobacterium tuberculosis reveal temporal signs of selection

Genetic differences between different Mycobacterium tuberculosis complex (MTBC) strains determine their ability to transmit within different host populations, their latency times, and their drug resistance profiles. Said differences usually emerge through de novo mutations and are maintained or discarded by the balance of evolutionary forces. Using a dataset of ∼5,000 strains representing global MTBC diversity, we determined the past and present selective forces that have shaped the current variability observed in the pathogen population. We identified regions that have evolved under changing types of selection since the time of the MTBC common ancestor. Our approach highlighted striking differences in the genome regions relevant for host–pathogen interaction and, in particular, suggested an adaptive role for the sensor protein of two-component systems. In addition, we applied our approach to successfully identify potential determinants of resistance to drugs administered as second-line tuberculosis treatments.

The Mycobacterium tuberculosis complex (MTBC) is a genetically monomorphic group of bacteria (1, 2) whose members cause tuberculosis in humans and animals. The MTBC comprises both human-associated (L1, L2, L3, L4, L5, L6, L7, L8, and L9) and animal-associated (A1, A2, A3, and A4) clades (3–7). Due to the absence of horizontal gene transfer, plasmids, and measurable recombination among strains and other species (8–10), chromosomal mutations represent the source of MTBC genetic diversity. The maximum genetic distance between any two MTBC strains is around 2,500 single-nucleotide polymorphisms (SNPs). Strikingly, studies have highlighted large phenotypic differences between strains involving traits like gene expression, drug resistance, transmissibility, and immune response, despite this limited variation. In some cases, the mutations driving phenotypic differences have been identified—for example, nonsynonymous variants in genes, such as rpoB, katG, or gyrA, cause drug-resistant phenotypes (11–13). Furthermore, single mutations in regulatory elements can induce alterations to downstream gene expression, which can foster differential virulence characteristics (14, 15). Finally, specific gene mutations may affect transmission (9), host tropism within the complex (16), and the host immune response (17). However, many of the genomic determinants of these phenotypes remain elusive, despite robust evidence that they are driven by genetic differences between strains (18, 19).Several types of evolutionary forces play crucial roles in the fixation of mutations in bacterial populations. Previous research has provided evidence for the ongoing positive selection of specific genes and regions (9, 20–23), while other studies have reported ongoing purifying selection of specific genomic regions, especially in epitopes and essential genes (24). Additionally, there exists some evidence that genetic drift may have significant functional and evolutionary consequences (25).Detecting selection in MTBC at the genome-wide level remains a challenging task due to limited genetic diversity. The significant accumulation of nonsynonymous substitutions has been previously used to characterize patterns of mutation accumulation in large categories of genes (24, 26); however, these studies employed a limited number of strains. Of note, the number of MTBC sequences has undergone a recent and rapid expansion, with studies involving hundreds to thousands of strains. The large number of available sequences has allowed, for example, the estimation of the ratio of nonsynonymous to synonymous substitutions (dN/dS) signatures in more than 10,000 strains (27), thereby allowing the identification of targets of selection with some probably related to host–pathogen interactions. Host–pathogen interaction signals are specially challenging as they are likely obscured by the force exerted by antimicrobial therapies. Weaker signals are also expected in genes related to second-line drugs related to the relative underuse of related treatments and the low abundance of associated resistant strains in genome databases (28).We reasoned that to detect signs of selection, we should focus on when and/or where they occurred in the phylogenetic tree instead of averaging signs across the phylogeny. In this study, we developed a methodology to study temporal signs of selection in MTBC genes and identified positive selection in a larger number of genes than previously described. This allowed the identification of past and currently unknown players in the MTBC evolution, particularly two-component systems (2CSs), related to host adaptation and second-line drug resistance. This methodology can be applied to other tuberculosis settings to explore signs of selection associated with changing selective pressures and could be extremely useful to unravel hidden details in the evolution of other human pathogens.     

Efficacy of a spatial repellent for control of Aedes-borne virus transmission: A cluster-randomized trial in Iquitos,Peru

Over half the world’s population is at risk for viruses transmitted by Aedes mosquitoes, such as dengue and Zika. The primary vector, Aedes aegypti, thrives in urban environments. Despite decades of effort, cases and geographic range of Aedes-borne viruses (ABVs) continue to expand. Rigorously proven vector control interventions that measure protective efficacy against ABV diseases are limited to Wolbachia in a single trial in Indonesia and do not include any chemical intervention. Spatial repellents, a new option for efficient deployment, are designed to decrease human exposure to ABVs by releasing active ingredients into the air that disrupt mosquito–human contact. A parallel, cluster-randomized controlled trial was conducted in Iquitos, Peru, to quantify the impact of a transfluthrin-based spatial repellent on human ABV infection. From 2,907 households across 26 clusters (13 per arm), 1,578 participants were assessed for seroconversion (primary endpoint) by survival analysis. Incidence of acute disease was calculated among 16,683 participants (secondary endpoint). Adult mosquito collections were conducted to compare Ae. aegypti abundance, blood-fed rate, and parity status through mixed-effect difference-in-difference analyses. The spatial repellent significantly reduced ABV infection by 34.1% (one-sided 95% CI lower limit, 6.9%; one-sided P value = 0.0236, z = 1.98). Aedes aegypti abundance and blood-fed rates were significantly reduced by 28.6 (95% CI 24.1%, ∞); z = −9.11) and 12.4% (95% CI 4.2%, ∞); z = −2.43), respectively. Our trial provides conclusive statistical evidence from an appropriately powered, preplanned cluster-randomized controlled clinical trial of the impact of a chemical intervention, in this case a spatial repellent, to reduce the risk of ABV transmission compared to a placebo.

Aedes-borne viral diseases (ABVDs) [e.g., dengue (DENV), chikungunya, Zika (ZIKV), and yellow fever] are devastating, expanding global public health threats that disproportionally affect low- and middle-income countries. DENV, one of the most rapidly increasing vector-borne infectious diseases, results in ∼400 million infections each year (1, 2), with 4 billion people at risk for infection annually (3). Currently, the primary means for ABVD prevention is controlling the primary mosquito vector, Aedes aegypti. Existing vector control interventions, however, have failed to prevent ABV transmission and epidemics (4–6).There is an urgent need to develop evidence-based guidance for the use of new and existing ABV vector control tools. The evidence base for vector control against ABVs is weak, despite considerable government investments in World Health Organization (WHO)-recommended control of larval habitats (larviciding, container removal) and ultra-low-volume insecticide spraying (4, 5, 7–9). These strategies continue to be implemented despite the lack of rigorously generated data from controlled clinical trials demonstrating they reduce ABV infection or disease (6). The only ABV intervention with a proven epidemiological impact in a cluster-randomized control trial (cRCT) assessed community mobilization to reduce mosquito larval habitats (10). A recent test-negative trial with Wolbachia-infected mosquitoes reported a significant reduction of DENV illness in Indonesia (11).Spatial repellents (SRs) are devices that contain volatile active ingredients that disperse in air. The active ingredients can repel mosquitoes from entering a treated space, inhibit attraction to human host cues, or disrupt mosquito biting and blood-feeding behavior and, thus, interfere with mosquito–human contact (12–14). Any of these outcomes reduce the probability of pathogen transmission. Pyrethroid-based SRs have shown efficacy in reducing malaria infections in China (15) and Indonesia (16). There have, however, been no clinical trials evaluating the protective efficacy (PE) of SRs against ABV infection or disease.To generate evidence for public health consideration, we conducted a double-blinded, parallel cRCT to demonstrate and quantify the PE of a transfluthrin-based SR to reduce ABV infection incidence over 2 y in a human cohort in Iquitos, Peru.     

Investigating the Human Host—ssRNA Virus Interaction Landscape Using the SMEAGOL Toolbox

Viruses have evolved numerous mechanisms to exploit the molecular machinery of their host cells, including the broad spectrum of host RNA-binding proteins (RBPs). However, the RBP interactomes of most viruses are largely unknown. To shed light on the interaction landscape of RNA viruses with human host cell RBPs, we have analysed 197 single-stranded RNA (ssRNA) viral genome sequences and found that the majority of ssRNA virus genomes are significantly enriched or depleted in motifs for specific human RBPs, suggesting selection pressure on these interactions. To facilitate tailored investigations and the analysis of genomes sequenced in future, we have released our methodology as a fast and user-friendly computational toolbox named SMEAGOL. Our resources will contribute to future studies of specific ssRNA virus—host cell interactions and support the identification of antiviral drug targets.     

Taxonomic Characterization and Secondary Metabolite Profiling of Aspergillus Section Aspergillus Contaminating Feeds and Feedstuffs

Xerophilic fungal species of the genus Aspergillus are economically highly relevant due to their ability to grow on low water activity substrates causing spoilage of stored goods and animal feeds. These fungi can synthesize a variety of secondary metabolites, many of which show animal toxicity, creating a health risk for food production animals and to humans as final consumers, respectively. Animal feeds used for rabbit, chinchilla and rainbow trout production in Argentina were analysed for the presence of xerophilic Aspergillus section Aspergillus species. High isolation frequencies (>60%) were detected in all the studied rabbit and chinchilla feeds, while the rainbow trout feeds showed lower fungal charge (25%). These section Aspergillus contaminations comprised predominantly five taxa. Twenty isolates were subjected to taxonomic characterization using both ascospore SEM micromorphology and two independent DNA loci sequencing. The secondary metabolite profiles of the isolates were determined qualitatively by HPLC-MS. All the isolates produced neoechinulin A, 17 isolates were positive for cladosporin and echinulin, and 18 were positive for neoechinulin B. Physcion and preechinulin were detected in a minor proportion of the isolates. This is the first report describing the detailed species composition and the secondary metabolite profiles of Aspergillus section Aspergillus contaminating animal feeds.     

Massive organ embolization from primary aortic thrombosis

A 49-year-old woman was hospitalized for acute left foot arterial ischemia. Arterial Doppler revealed occlusion of the dorsalis pedis and posterior tibial arteries. A computed tomography angiography performed to assess abdominal pain showed hepatic, splenic, renal and pancreatic infarctions. A splenic artery embolism and a small aortic wall thrombus at the celiac trunk were identified. No radiological signs of aortic atherosclerosis were found. No predisposing conditions for secondary aortic thrombosis or intracardiac embolic sources were detected. It was determined that primary aortic thrombosis, a rare though potentially serious condition, was to blame. Isolated aortic mural thrombosis therapy is not well established, although systemic anticoagulation, thrombolysis, thromboaspiration, endovascular stent grafting and surgical thrombectomy have been attempted with varying success. In our patient, systemic anticoagulation therapy was initiated and resulted in aortic thrombus resolution. Close clinical follow-up is crucial, as the aortic thrombus can recur despite anticoagulation and aggressive control of the atherosclerotic risk factors.     

Salt Intake and Risk of Gastric Intestinal Metaplasia: Systematic Review and Meta-Analysis

The understanding of the association between salt intake and precancerous lesions may contribute to clarify the causal relation with gastric cancer. We systematically reviewed 17 articles addressing the association between dietary salt exposure and gastric intestinal metaplasia and conducted meta-analyses for quantitative synthesis (random effects model). Salt exposure was estimated assessing salted/salty food consumption, preference for salted/salty foods, use of table salt, or sodium urinary excretion. Heterogeneity was also large regarding food items evaluated, consumption categories, and data analysis. The combined odds ratio (OR) was 1.68 (95% confidence interval (CI) = 0.98–2.90; I² = 55.4%) for the association between salted/salty meat and intestinal metaplasia (4 studies) and the OR was 1.53 (95% CI = 0.72–3.24; I² = 76.8%) for salt preference. There was a positive, nonstatistically significant association between intestinal metaplasia and urinary sodium excretion. The heterogeneity of methodological options and results preclude quantitative synthesis or its proper interpretation, even if the available evidence may suggest a positive association between salt and intestinal metaplasia.     

Milk derived colloid as a novel drug delivery carrier for breast cancer

Triple negative breast cancer has an extremely poor prognosis when chemotherapy is no longer effective. To overcome drug resistance, novel drug delivery systems based on nanoparticles have had remarkable success. We produced a novel nanoparticle component ‘MDC’ from milk-derived colloid. In order to evaluate the anti-cancer effect of MDC, we conducted in vitro and in vivo experiments on cancer cell lines and a primary tumor derived breast xenograft. Doxorubicin (Dox) conjugated to MDC (MDC-Dox) showed higher cancer cell growth inhibition than MDC alone especially in cell lines with high EGFR expression. In a mouse melanoma model, MDC-Dox significantly suppressed tumor growth when compared with free Dox. Moreover, in a primary tumor derived breast xenograft, one of the mice treated with MDC-Dox showed partial regression, while mice treated with free Dox failed to show any suppression of tumor growth. We have shown that a novel nanoparticle compound made of simple milk-derived colloid has the capability for drug conjugation, and serves as a tumor-specific carrier of anti-cancer drugs. Further research on its safety and ability to carry various anti-cancer drugs into multiple drug-resistant primary breast models is warranted.     

Factors that positively or negatively mediate the effects of age on working memory across the adult life span

Working memory abilities significantly decrease with advancing age; hence, the search for factors that may increase or mitigate this decline is critical. Several factors have been identified that influence working memory; however, their effects have been mainly assessed separately and rarely together with other factors in the same sample. We examined 120 variables to search for factors that jointly act as mediators of working memory decay across the adult life span. A sample of 1652 healthy adults was assessed in spatial and verbal working memory domains. Structural equation modeling analyses were conducted to search for potential mediators that intervened between age and working memory. Only 14 and 10 variables reliably mediated spatial and verbal working memory, respectively. Factors from several domains remained in the models, such as individual characteristics, physiological traits, consumption habits, and regular activities. These factors are sufficiently powerful to influence working memory decline when they jointly interact, as in everyday living.