Contribution of SARS-CoV-2 Accessory Proteins to Viral Pathogenicity in K18 Human ACE2 Transgenic Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University's COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, -6, -7a, -7b, and -8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.

Host- and Age-Dependent Transcriptional Changes in Mycobacterium tuberculosis Cell Envelope Biosynthesis Genes after Exposure to Human Alveolar Lining Fluid.

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 into 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 shows 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 a 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.

Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis.

The current emergence of multi-, extensively-, extremely-, and total-drug resistant strains of Mycobacterium tuberculosis poses a major health, social, and economic threat, and stresses the need to develop new therapeutic strategies. The notion of phage therapy against bacteria has been around for more than a century and, although its implementation was abandoned after the introduction of drugs, it is now making a comeback and gaining renewed interest in Western medicine as an alternative to treat drug-resistant pathogens. Mycobacteriophages are genetically diverse viruses that specifically infect mycobacterial hosts, including members of the M. tuberculosis complex. This review describes general features of mycobacteriophages and their mechanisms of killing M. tuberculosis, as well as their advantages and limitations as therapeutic and prophylactic agents against drug-resistant M. tuberculosis strains. This review also discusses the role of human lung micro-environments in shaping the availability of mycobacteriophage receptors on the M. tuberculosis cell envelope surface, the risk of potential development of bacterial resistance to mycobacteriophages, and the interactions with the mammalian host immune system. Finally, it summarizes the knowledge gaps and defines key questions to be addressed regarding the clinical application of phage therapy for the treatment of drug-resistant tuberculosis.

Cell membrane integrity and distinguishing between metabolically active and inactive cells as a means of improving viability PCR.

Viability PCR uses cell membrane integrity to differentiate live cells from dead. Our new approach improves viability PCR by enabling it to also discriminate between cells with an intact cell membrane and the ability to actively maintain bacterial homeostasis and cells that have an intact membrane but are metabolically inactive.

Evolution of a self-inducible cytolethal distending toxin type V-encoding bacteriophage from Escherichia coli O157:H7 to Shigella sonnei.

Some cdt genes are located within the genome of inducible or cryptic bacteriophages, but there is little information about the mechanisms of cdt transfer because of the reduced number of inducible Cdt phages described. In this study, a new self-inducible Myoviridae Cdt phage (ΦAA91) was isolated from a nonclinical O157:H7 Shiga toxin-producing Escherichia coli strain and was used to lysogenize a cdt-negative strain of Shigella sonnei. We found that the phage induced from S. sonnei (ΦAA91-ss) was not identical to the original phage. ΦAA91-ss was used to infect a collection of 57 bacterial strains, was infectious in 59.6% of the strains, and was able to lysogenize 22.8% of them. The complete sequence of ΦAA91-ss showed a 33,628-bp genome with characteristics of a P2-like phage with the cdt operon located near the cosR site. We found an IS21 element composed of two open reading frames inserted within the cox gene of the phage, causing gene truncation. Truncation of cox does not affect lytic induction but could contribute to phage recombination and generation of lysogens. The IS21 element was not present in the ΦAA91 phage from E. coli, but it was incorporated into the phage genome after its transduction in Shigella. This study shows empirically the evolution of temperate bacteriophages carrying virulence genes after infecting a new host and the generation of a phage population with better lysogenic abilities that would ultimately lead to the emergence of new pathogenic strains.

Persistence of infectious Shiga toxin-encoding bacteriophages after disinfection treatments.

In Shiga toxin-producing Escherichia coli (STEC), induction of Shiga toxin-encoding bacteriophages (Stx phages) causes the release of free phages that can later be found in the environment. The ability of Stx phages to survive different inactivation conditions determines their prevalence in the environment, the risk of stx transduction, and the generation of new STEC strains. We evaluated the infectivity and genomes of two Stx phages (Φ534 and Φ557) under different conditions. Infectious Stx phages were stable at 4, 22, and 37°C and at pH 7 and 9 after 1 month of storage but were completely inactivated at pH 3. Infective Stx phages decreased moderately when treated with UV (2.2-log10 reduction for an estimated UV dose of 178.2 mJ/cm(2)) or after treatment at 60 and 68°C for 60 min (2.2- and 2.5-log10 reductions, respectively) and were highly inactivated (3 log10) by 10 ppm of chlorine in 1 min. Assays in a mesocosm showed lower inactivation of all microorganisms in winter than in summer. The number of Stx phage genomes did not decrease significantly in most cases, and STEC inactivation was higher than phage inactivation under all conditions. Moreover, Stx phages retained the ability to lysogenize E. coli after some of the treatments.

Tuberculosis and COVID-19 in the elderly: factors driving a higher burden of disease.

Mycobacterium tuberculosis (M.tb) and SARS-CoV-2 are both infections that can lead to severe disease in the lower lung. However, these two infections are caused by very different pathogens (Mycobacterium vs. virus), they have different mechanisms of pathogenesis and immune response, and differ in how long the infection lasts. Despite the differences, SARS-CoV-2 and M.tb share a common feature, which is also frequently observed in other respiratory infections: the burden of disease in the elderly is greater. Here, we discuss possible reasons for the higher burden in older adults, including the effect of co-morbidities, deterioration of the lung environment, auto-immunity, and a reduced antibody response. While the answer is likely to be multifactorial, understanding the main drivers across different infections may allow us to design broader interventions that increase the health-span of older people.

Acute inflammation alters lung lymphocytes and potentiates innate-like behavior in young mouse lung CD8 T cells, resembling lung CD8 T cells from old mice.

Inflammation plays a significant role in lung infection including that caused by Mycobacterium tuberculosis, in which both adaptive and innate lymphocytes can affect infection control. How inflammation affects infection is understood in a broad sense, including inflammaging (chronic inflammation) seen in the elderly, but the explicit role that inflammation can play in regulation of lymphocyte function is not known. To fill this knowledge gap, we used an acute lipopolysaccharide (LPS) treatment in young mice and studied lymphocyte responses, focusing on CD8 T cell subsets. LPS treatment decreased the total numbers of T cells in the lungs of LPS mice while also increasing the number of activated T cells. We demonstrate that lung CD8 T cells from LPS mice became capable of an antigen independent innate-like IFN-γ secretion, dependent on IL-12p70 stimulation, paralleling innate-like IFN-γ secretion of lung CD8 T cells from old mice. Overall, this study provides information on how acute inflammation can affect lymphocytes, particularly CD8 T cells, which could potentially affect immune control of various disease states.

Immunization with Recombinant Accessory Protein-Deficient SARS-CoV-2 Protects against Lethal Challenge and Viral Transmission.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a worldwide coronavirus disease 2019 (COVID-19) pandemic. Despite the high efficacy of the authorized vaccines, there may be uncertain and unknown side effects or disadvantages associated with current vaccination approaches. Live-attenuated vaccines (LAVs) have been shown to elicit robust and long-term protection by the induction of host innate and adaptive immune responses. In this study, we sought to verify an attenuation strategy by generating 3 double open reading frame (ORF)-deficient recombinant SARS-CoV-2s (rSARS-CoV-2s) simultaneously lacking two accessory ORF proteins (ORF3a/ORF6, ORF3a/ORF7a, and ORF3a/ORF7b). We report that these double ORF-deficient rSARS-CoV-2s have slower replication kinetics and reduced fitness in cultured cells compared with their parental wild-type (WT) counterpart. Importantly, these double ORF-deficient rSARS-CoV-2s showed attenuation in both K18 hACE2 transgenic mice and golden Syrian hamsters. A single intranasal dose vaccination induced high levels of neutralizing antibodies against SARS-CoV-2 and some variants of concern and activated viral component-specific T cell responses. Notably, double ORF-deficient rSARS-CoV-2s were able to protect, as determined by the inhibition of viral replication, shedding, and transmission, against challenge with SARS-CoV-2 in both K18 hACE2 mice and golden Syrian hamsters. Collectively, our results demonstrate the feasibility of implementing the double ORF-deficient strategy to develop safe, immunogenic, and protective LAVs to prevent SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Live-attenuated vaccines (LAVs) are able to induce robust immune responses, including both humoral and cellular immunity, representing a very promising option to provide broad and long-term immunity. To develop LAVs for SARS-CoV-2, we engineered attenuated recombinant SARS-CoV-2 (rSARS-CoV-2) that simultaneously lacks the viral open reading frame 3a (ORF3a) in combination with either ORF6, ORF7a, or ORF7b (Δ3a/Δ6, Δ3a/Δ7a, and Δ3a/Δ7b, respectively) proteins. Among them, the rSARS-CoV-2 Δ3a/Δ7b was completely attenuated and able to provide 100% protection against an otherwise lethal challenge in K18 hACE2 transgenic mice. Moreover, the rSARS-CoV-2 Δ3a/Δ7b conferred protection against viral transmission between golden Syrian hamsters.

Exposure of Mycobacterium tuberculosis to human alveolar lining fluid shows temporal and strain-specific adaptation to the lung environment.

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.

Stability and infectivity of cytolethal distending toxin type V gene-carrying bacteriophages in a water mesocosm and under different inactivation conditions.

Two cytolethal distending toxin (Cdt) type V-encoding bacteriophages (Φ62 and Φ125) were induced spontaneously from their wild-type Escherichia coli strains and from the lysogens generated in Shigella sonnei. The stability of Cdt phages was determined at various temperatures and pH values after 1 month of storage by means of infectivity tests using a plaque blot assay and analysis of phage genomes using real-time quantitative PCR (qPCR): both were highly stable. We assessed the inactivation of Cdt phages by thermal treatment, chlorination, UV radiation, and in a mesocosm in both summer and winter. The results for the two Cdt phages showed similar trends and were also similar to the phage SOM23 used for reference, but they showed a much higher persistence than Cdt-producing E. coli. Cdt phages showed maximal inactivation after 1 h at 70°C, 30 min of UV radiation, and 30 min of contact with a 10-ppm chlorine treatment. Inactivation in a mesocosm was higher in summer than in winter, probably because of solar radiation. The treatments reduced the number of infectious phages but did not have a significant effect on the Cdt phage particles detected by qPCR. Cdt phages were quantified by qPCR in 73% of river samples, and these results suggest that Cdt phages are a genetic vehicle and the natural reservoir for cdt in the environment.

Genome Sequence Analysis and Characterization of Shiga Toxin 2 Production by Escherichia coli O157:H7 Strains Associated With a Laboratory Infection.

A laboratory-acquired E. coli O157:H7 infection with associated severe sequelae including hemolytic uremic syndrome occurred in an individual working in the laboratory with a mixture of nalidixic acid-resistant (NalR) O157:H7 mutant strains in a soil-biochar blend. The patient was hospitalized and treated with an intravenous combination of metronidazole and levofloxacin. The present study investigated the source of this severe laboratory acquired infection and further examined the influence of the antibiotics used during treatment on the expression and production of Shiga toxin. Genomes of two Stx2a-and eae-positive O157:H7 strains isolated from the patient's stool were sequenced along with two pairs of the wt strains and their derived NalR mutants used in the laboratory experiments. High-resolution SNP typing determined the strains' individual genetic relatedness and unambiguously identified the two laboratory-derived NalR mutant strains as the source of the researcher's life-threatening disease, rather than a conceivable ingestion of unrelated O157:H7 isolates circulating at the same time. It was further confirmed that in sublethal doses, the antibiotics increased toxin expression and production. Our results support a simultaneous co-infection with clinical strains in the laboratory, which were the causative agents of previous O157:H7 outbreaks, and further that the administration of antibiotics may have impacted the outcome of the infection.

Pathogenomes and variations in Shiga toxin production among geographically distinct clones of Escherichia coli O113:H21.

Infections with globally disseminated Shiga toxin-producing Escherichia coli (STEC) of the O113:H21 serotype can progress to severe clinical complications, such as hemolytic uremic syndrome (HUS). Two phylogeographically distinct clonal complexes have been established by multi locus sequence typing (MLST). Infections with ST-820 isolates circulating exclusively in Australia have caused severe human disease, such as HUS. Conversely, ST-223 isolates prevalent in the US and outside Australia seem to rarely cause severe human disease but are frequent contaminants. Following a genomic epidemiology approach, we wanted to gain insights into the underlying cause for this disparity. We examined the plasticity in the genome make-up and Shiga toxin production in a collection of 20 ST-820 and ST-223 strains isolated from produce, the bovine reservoir, and clinical cases. STEC are notorious for assembly into fragmented draft sequences when using short-read sequencing technologies due to the extensive and partly homologous phage complement. The application of long-read technology (LRT) sequencing yielded closed reference chromosomes and plasmids for two representative ST-820 and ST-223 strains. The established high-resolution framework, based on whole genome alignments, single nucleotide polymorphism (SNP)-typing and MLST, includes the chromosomes and plasmids of other publicly available O113:H21 sequences and allowed us to refine the phylogeographical boundaries of ST-820 and ST-223 complex isolates and to further identify a historic non-shigatoxigenic strain from Mexico as a quasi-intermediate. Plasmid comparison revealed strong correlations between the strains' featured pO113 plasmid genotypes and chromosomally inferred ST, which suggests coevolution of the chromosome and virulence plasmids. Our pathogenicity assessment revealed statistically significant differences in the Stx2a-production capabilities of ST-820 as compared to ST-223 strains under RecA-induced Stx phage mobilization, a condition that mimics Stx-phage induction. These observations suggest that ST-820 strains may confer an increased pathogenic potential in line with the strain-associated epidemiological metadata. Still, some of the tested ST-223 cultures sourced from contaminated produce or the bovine reservoir also produced Stx at levels comparable to those of ST-820 isolates, which calls for awareness and for continued surveillance of this lineage.

Pathogenome comparison and global phylogeny of Escherichia coli ST1485 strains.

Escherichia coli ST1485 strains belong to the clinically important phylogroup F and have disseminated worldwide in humans, animals, and the environment. Here, we elucidated the pathogenome of a global collection of E. coli ST1485 isolates from diverse sources retrieved from public databases and a high-quality sequenced complete genome of colistin-resistant E. coli strain CFSAN061771 isolated from raw milk cheese which designated as a reference strain. CFSAN061771 belongs to O83:H42-ST1485 pathotype and carries a conjugative ColV plasmid, pCFSAN061771_01, combining extraintestinal virulence genes (ompt, sitA, iroN, etsC, traT, cvaC, hylF, iss, tsh, mchf, iucC, iutA) with a multidrug resistance island (blaTEM-1, aph(6)-Id, aph(3″)-Ib, sul2, dfrA14). Comparative genomic analysis revealed a high frequency of pCFSAN061771_01-like plasmids in E. coli ST1485. A notable evolutionary genetic event in E. coli ST1485 strains is the acquisition of a pCFSAN061771_02-like plasmid, which confers resistance to several antimicrobials, tellurium, and quaternary ammonium compounds. The identical virulence and antibiotic resistance profiles identified in some human and animal strains are worrisome. This is the first study to emphasize the significance of E. coli ST1485 as a global high-risk virulent and multidrug-resistant clone with zoonotic potential.

Immunization with recombinant accessory protein-deficient SARS-CoV-2 protects against lethal challenge and viral transmission.

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.

The Aging Human Lung Mucosa: A Proteomics Study.

The older adult population, estimated to double by 2050, is at increased risk of respiratory infections and other pulmonary diseases. Biochemical changes in the lung alveolar lining fluid (ALF) and in alveolar compartment cells can alter local immune responses as we age, generating opportunities for invading pathogens to establish successful infections. Indeed, the lung alveolar space of older adults is a pro-inflammatory, pro-oxidative, dysregulated environment that remains understudied. We performed an exploratory, quantitative proteomic profiling of the soluble proteins present in ALF, developing insight into molecular fingerprints, pathways, and regulatory networks that characterize the alveolar space in old age, comparing it to that of younger individuals. We identified 457 proteins that were significantly differentially expressed in older adult ALF, including increased production of matrix metalloproteinases, markers of cellular senescence, antimicrobials, and proteins of neutrophilic granule origin, among others, suggesting that neutrophils in the lungs of older adults could be potential contributors to the dysregulated alveolar environment with increasing age. Finally, we describe a hypothetical regulatory network mediated by the serum response factor that could explain the neutrophilic profile observed in the older adult population.

Modeling SARS-CoV-2 and influenza infections and antiviral treatments in human lung epithelial tissue equivalents.

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 viruses and develop new antiviral treatments. Here we describe the cellular complexity of human alveolar and tracheobronchial air liquid interface (ALI) tissue models during SARS-CoV-2 and influenza A virus (IAV) infections. Our results showed that both SARS-CoV-2 and IAV effectively infect these ALI tissues, with SARS-CoV-2 exhibiting a slower replication peaking at later time-points compared to IAV. We detected tissue-specific chemokine and cytokine storms in response to viral infection, including well-defined biomarkers in severe SARS-CoV-2 and IAV infections such as CXCL10, IL-6, and IL-10. Our single-cell RNA sequencing analysis showed similar findings to that found in vivo for SARS-CoV-2 infection, including dampened IFN response, increased chemokine induction, and inhibition of MHC Class I presentation not observed for IAV infected tissues. Finally, we demonstrate the pharmacological validity of these ALI tissue models as antiviral drug screening assay platforms, with the potential to be easily adapted to include other cell types and increase the throughput to test relevant pathogens.

Bacteriophage-encoding cytolethal distending toxin type V gene induced from nonclinical Escherichia coli isolates.

Cytolethal distending toxin (Cdt) is produced by a variety of pathogenic bacteria, including pathogenic serotypes of Shiga toxin-producing Escherichia coli (STEC). The Cdt family comprises five variants (Cdt-I to Cdt-V) encoded by three genes located within the chromosome or plasmids or, in the case of Cdt-I, within bacteriophages. In this study, we evaluated the occurrence of the cdt gene in a collection of 140 environmental STEC isolates. cdt was detected in 12.1% of strains, of which five strains carried inducible bacteriophages containing the Cdt-V variant. Two Cdt-V phages of the Siphoviridae morphology lysogenized Shigella sonnei, generating two lysogens: a single Cdt phage lysogen and a double lysogen, containing a Cdt phage and an Stx phage, both from the wild-type strain. The rates of induction of Cdt phages were evaluated by quantitative PCR, and spontaneous induction of Cdt-V phage was observed, whereas induction of Stx phage in the double lysogen was mitomycin C dependent. The Cdt distending effect was observed in HeLa cells inoculated with the supernatant of the Cdt-V phage lysogen. A ClaI fragment containing the cdt-V gene of one phage was cloned, and sequencing confirmed the presence of Cdt-V, as well as a fragment downstream from the cdt homolog to gpA, encoding a replication protein of bacteriophage P2. Evaluation of Cdt-V phages in nonclinical water samples showed densities of 10(2) to 10(9) gene copies in 100 ml, suggesting the high prevalence of Cdt phages in nonclinical environments.

Evolution of Drug-Resistant Mycobacterium tuberculosis Strains and Their Adaptation to the Human Lung Environment.

In the last two decades, multi (MDR), extensively (XDR), extremely (XXDR) and total (TDR) drug-resistant Mycobacterium tuberculosis (M.tb) strains have emerged as a threat to public health worldwide, stressing the need to develop new tuberculosis (TB) prevention and treatment strategies. It is estimated that in the next 35 years, drug-resistant TB will kill around 75 million people and cost the global economy $16.7 trillion. Indeed, the COVID-19 pandemic alone may contribute with the development of 6.3 million new TB cases due to lack of resources and enforced confinement in TB endemic areas. Evolution of drug-resistant M.tb depends on numerous factors, such as bacterial fitness, strain's genetic background and its capacity to adapt to the surrounding environment, as well as host-specific and environmental factors. Whole-genome transcriptomics and genome-wide association studies in recent years have shed some insights into the complexity of M.tb drug resistance and have provided a better understanding of its underlying molecular mechanisms. In this review, we will discuss M.tb phenotypic and genotypic changes driving resistance, including changes in cell envelope components, as well as recently described intrinsic and extrinsic factors promoting resistance emergence and transmission. We will further explore how drug-resistant M.tb adapts differently than drug-susceptible strains to the lung environment at the cellular level, modulating M.tb-host interactions and disease outcome, and novel next generation sequencing (NGS) strategies to study drug-resistant TB.

New Developments and Insights in the Improvement of Mycobacterium tuberculosis Vaccines and Diagnostics Within the End TB Strategy.

PURPOSE OF REVIEW: The alignment of sustainable development goals (SDGs) with the End Tuberculosis (TB) strategy provides an integrated roadmap to implement key approaches towards TB elimination. This review summarizes current social challenges for TB control, and yet, recent developments in TB diagnosis and vaccines in the context of the End TB strategy and SDGs to transform global health. RECENT FINDINGS: Advances in non-sputum based TB biomarkers and whole genome sequencing technologies could revolutionize TB diagnostics. Moreover, synergistic novel technologies such as mRNA vaccination, nanovaccines and promising TB vaccine models are key promising developments for TB prevention and control. SUMMARY: The End TB strategy depends on novel developments in point-of-care TB diagnostics and effective vaccines. However, despite outstanding technological developments in these fields, TB elimination will be unlikely achieved if TB social determinants are not fully addressed. Indeed, the End TB strategy and SDGs emphasize the importance of implementing sustainable universal health coverage and social protection.