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La toxina Cdt (“Cytolethal Distending Toxin”) fue descrita por primera vez en 1987 por Johnson y Lior como una nueva toxina producida por Escherichia coli. Es una toxina de tipo AB2 que bloquea el ciclo celular eucariota en las fases G2/M de la mitosis, cosa que provoca una distensión celular de hasta 5 veces el tamaño normal, y finalmente se produce la apoptosis. En la actualidad, la Cdt ha sido descrita en un gran número de bacterias Gram negativas como Vibrio, Helicobacter, o Campylobacter, entre otros. Está codificada por tres genes adyacentes (cdtA, cdtB y cdtC) que muestran diferentes grados de similitud entre géneros, siendo CdtB la subunidad catalítica que actúa como una ADNasa de tipo I, y CdtA y CdtC las subunidades que transportan a CdtB al núcleo. En E. coli han sido descritas 5 variantes, localizadas en el cromosoma (Cdt-II), en el plásmido pVir (Cdt-III) o rodeadas de secuencias homólogas a genes de fagos de tipo lambda o P2 (Cdt-I, Cdt-IV y Cdt-V). El hecho de que cdt se encuentre en diferentes géneros bacterianos con secuencias adyacentes homólogas a genes de fagos, así como que no aparezca asociado significativamente a otros factores de virulencia, sugiere que estos genes cdt son adquiridos por transferencia horizontal a través de fagos. Estos fagos Cdt pueden ser inducidos a partir de la cepa huésped y transmitir el gen de la toxina a otras cepas, causando la aparición de nuevas cepas virulentas. Además de en ambientes clínicos, se han encontrado fagos Cdt en agua residual y, tal como veremos en este estudio, también en agua de río, hecho que refuerza su potencial papel como transmisores de la toxina en el medio ambiente. Por tanto, un aspecto importante a tener en cuenta es la persistencia y la estabilidad de estos fagos en las diferentes condiciones ambientales. Por ello, en este trabajo, se ha estudiado la estabilidad de los fagos Cdt bajo diferentes condiciones de temperatura (4ºC, 22ºC y 37ºC) y pH (3, 7 y 9), y bajo diferentes tratamientos de inactivación: calor (60ºC y 70ºC, 30 y 60 min), luz UV (1, 5, 10 y 30 min), cloración (1, 3, 5, 10, 20 y 30 min) e inactivación natural. Para ello se han aislado dos fagos Cdt-V de muestras ambientales, y se han monitorizado mediante ensayos de infectividad (ufp/ml) y mediante cuantificación de copias genómicas por qPCR en cada una de las condiciones ensayadas. Además, se han comparado los resultados obtenidos con la estabilidad de fagos Stx, también de elevada persistencia ambiental, bajo las mismas condiciones. Por su propia naturaleza, y tal y como se ha visto en este trabajo y en estudios anteriores, los fagos son mucho más resistentes a ciertos tratamientos de desinfección e inactivación natural que las bacterias huésped, reafirmando su papel como reservorios de genes de virulencia en el medio ambiente. Además, se han descrito ciertas morfologías de fagos que permitirían una mayor persistencia a dichos tratamientos. Si los fagos consiguen sobrevivir a los tratamientos comúnmente utilizados en la eliminación de las bacterias, podrían transducir el gen de la toxina a otras cepas. Por tanto, no se puede descartar su potencial en la generación de nuevas cepas patógenas emergentes. Además de la persistencia de los fagos Cdt-V, también se ha estudiado su capacidad de inducción y de generación de lisógenos, así como su morfología y su estructura genética. Gracias a ello, se ha podido determinar a qué familia pertenecen y observar su capacidad de evolución y adaptación al lisogenizar a un nuevo huésped., Cdt toxin (“Cytolethal Distending Toxin”) was first described by Johnson and Lior in 1987. It is an AB5 toxin that blocks the eukariotic cell cycle between G2 and M phases, which distends the cells, since cell division ceases but growth continues. Cdt is produced by different Gram negative pathogenic microorganisms, including Escherichia coli, Vibrio, Campylobacter and Helicobacter, among others. It consists of three subunits (CdtA, CdtB, and CdtC) that are encoded by three adjacent genes. The catalytic subunit CdtB is homologous to DNase I and it is the most conserved gene, and the other subunits act as binding proteins that deliver CdtB into target cells. Five variants of the toxin have been reported in E. coli, located in the chromosome (Cdt-II), in the conjugative plasmid pVir (Cdt-III) or flanked by lambda-like or P2 bacteriophage genes (Cdt-I, Cdt-IV and Cdt-V). The fact that Cdt is present in different microorganisms and, in some cases flanked by bacteriophage genes, and not significantly associated to other virulence factors, suggest that cdt genes are acquired by horizontal transfer by means of bacteriophages. These Cdt phages could be induced from the host and transduce the toxin to other strains, causing the emergence of new virulent strains. Cdt phages have not only been isolated from clinical samples, but also from environmental samples. For the potential role of these phages in cdt transduction, it is important to consider and study the persistence of Cdt phages in different environmental conditions. In this work, it has been studied the stability of two Cdt phages isolated from the environment under different conditions and inactivation treatments (temperature, pH, heat treatment, UV treatment, chlorination and natural inactivation). Moreover, the results were compared with Stx phages stability (that also have a significantly environmental prevalence) under the same conditions. The fact that Cdt and Stx phages and, in general, all phages, are more resistent to all the treatments and conditions than their natural hosts, reinforces their potential role as a reservoir of virulence genes in the environment. We have also characterized Cdt phages: its capacity of induction and lisogenization, morphology and genetic structure, to understand better the performance of these phages.

Upon infection, Mycobacterium tuberculosis ( M.tb ) reaches the alveolar space and comes in close contact with human alveolar lining fluid (ALF) for an uncertain period of time prior to its encounter with alveolar cells. We showed that homeostatic ALF hydrolytic enzymes modify the M.tb cell envelope, driving M.tb -host cell interactions. Still, the contribution of ALF during M.tb infection is poorly understood. Here, we exposed 4 M.tb strains with different levels of virulence, transmissibility, and drug resistance (DR) to physiological concentrations of human ALF for 15-min and 12-h, and performed RNA sequencing. Gene expression analysis showed a temporal and strain-specific adaptation to human ALF. Differential expression (DE) of ALF-exposed vs. unexposed M.tb revealed a total of 397 DE genes associated with lipid metabolism, cell envelope and processes, intermediary metabolism and respiration, and regulatory proteins, among others. Most DE genes were detected at 12-h post-ALF exposure, with DR- M.tb strain W-7642 having the highest number of DE genes. Interestingly, genes from the KstR2 regulon, which controls the degradation of cholesterol C and D rings, were significantly upregulated in all strains post-ALF exposure. These results indicate that M.tb -ALF contact drives initial metabolic and physiologic changes in M.tb , with potential implications in infection outcome., Importance: Tuberculosis, caused by airborne pathogen Mycobacterium tuberculosis ( M.tb ), is one of the leading causes of mortality worldwide. Upon infection, M.tb reaches the alveoli and gets in contact with human alveolar lining fluid (ALF), where ALF hydrolases modify the M.tb cell envelope driving subsequent M.tb -host cell interactions. Still, the contributions of ALF during infection are poorly understood. We exposed 4 M.tb strains to ALF for 15-min and 12-h and performed RNA sequencing, demonstrating a temporal and strain-specific adaptation of M.tb to ALF. Interestingly, genes associated with cholesterol degradation were highly upregulated in all strains. This study shows for the first time that ALF drives global metabolic changes in M.tb during the initial stages of the infection, with potential implications in disease outcome. Biologically relevant networks and common and strain-specific bacterial determinants derived from this study could be further investigated as potential therapeutic candidates.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has led to a worldwide Coronavirus Disease 2019 (COVID-19) pandemic. Despite high efficacy of the authorized vaccines, protection against the surging variants of concern (VoC) was less robust. Live-attenuated vaccines (LAV) have been shown to elicit robust and long-term protection by induction of host innate and adaptive immune responses. We sought to develop a COVID-19 LAV by generating 3 double open reading frame (ORF)-deficient recombinant (r)SARS-CoV-2 simultaneously lacking two accessory open reading frame (ORF) proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). Here, we report that these double ORF-deficient rSARS-CoV-2 have slower replication kinetics and reduced fitness in cultured cells as compared to their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2 showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against different SARS-CoV-2 VoC, and also activated viral component-specific T-cell responses. Notably, the double ORF-deficient rSARS-CoV-2 were able to protect, as determined by inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2. Collectively, our results demonstrate the feasibility to implement these double ORF-deficient rSARS-CoV-2 as safe, stable, immunogenic and protective LAV for the prevention of SARS-CoV-2 infection and associated COVID-19 disease.

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis ( M . tb ), resulted in almost 1.4 million deaths in 2019 and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M . tb comes in close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M . tb upon contact, defining subsequent M . tb -host cell interactions and infection outcomes in vitro and in vivo . We also demonstrated that ALF from 60+ year old elders (E-ALF) vs . healthy 18- to 45-year-old adults (A-ALF) is dysfunctional with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay demonstrates that M . tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M . tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M . tb exposure to E-ALF shows lesser transcriptional response, with most of the M . tb genes unchanged or downregulated. Overall, this study indicates that M . tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status determined by factors such as age might play an important role in determining infection outcome.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the third coronavirus in less than 20 years to spillover from an animal reservoir and cause severe disease in humans. High impact respiratory viruses such as pathogenic beta-coronaviruses and influenza viruses, as well as other emerging respiratory viruses, pose an ongoing global health threat to humans. There is a critical need for physiologically relevant, robust and ready to use, in vitro cellular assay platforms to rapidly model the infectivity of emerging respiratory viruses and discover and develop new antiviral treatments. Here, we validate in vitro human alveolar and tracheobronchial tissue equivalents and assess their usefulness as in vitro assay platforms in the context of live SARS-CoV-2 and influenza A virus infections. We establish the cellular complexity of two distinct tracheobronchial and alveolar epithelial air liquid interface (ALI) tissue models, describe SARS-CoV-2 and influenza virus infectivity rates and patterns in these ALI tissues, the viral-induced cytokine production as it relates to tissue-specific disease, and demonstrate the pharmacologically validity of these lung epithelium models as antiviral drug screening assay platforms.