Search

Inflammation plays a significant role in lung infection including that caused by Mycobacterium tuberculosis (M.tb), where 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.

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.

Infections with globally disseminated Shiga toxin-producing

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.

The elderly population is at increased risk of acute and chronic respiratory infections and other pulmonary diseases, and it is estimated that this population will double in the next 30 years. Biochemical changes in the lung alveolar mucosa and lung cells alter local immune response as we age, creating opportunities for invading pathogens to establish successful infections. Indeed, the lungs of the elderly are a pro-inflammatory, pro-oxidative, dysregulated environment but this environment has remained understudied. We performed a comprehensive, quantitative proteomic profile of the lung mucosa in the elderly, developing insight into the molecular fingerprints, pathways, and regulatory networks that characterize the lung in old age. We identified neutrophils in the lungs of elderly individuals as possible contributors to dysregulated lung tissue environment. This study establishes a baseline for future investigations to develop strategies to mitigate susceptibility to respiratory infections in the elderly.

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 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.

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.

There is emerging evidence that food of animal origin may be responsible for the spread of multidrug resistant extraintestinal pathogenic Escherichia coli in the community. Here, we describe the emergence of colistin resistance gene, mcr-1, in a strain belonging to the dominant uropathogenic E. coli ST69 lineage. E. coli strain CFSAN061770 was isolated during monitoring of the popular Egyptian raw milk cheese, karish cheese, for the presence of colistin resistance. The complete genome of E. coli strain CFSAN061770 comprises a chromosome of 5,292,297 bp with a G + C content of 50.6%. Further, three plasmids named pEGY1-MCR-1, pEGY2 and pEGY3 of 228,947 bp, 103,234 bp and 87,012 bp were detected, respectively. Plasmid pEGY1-MCR-1 belongs to the IncHI2 incompatibility group and carries the colistin resistance mcr-1 gene flanked by two ISApl1 elements and forms a composite transposon. It mediates resistance to aminoglycosides (aadA1 and aadA2), phenicol (cmlA1 and floR), sulfonamides (sul3), and tetracycline (tet(A)), and these loci were found clustered in a multidrug resistant region. Plasmid pEGY3 carries a complex multiple resistance locus (CMR) (aph(3′)-Ia, strA, strB, sul2, and blaTEM-1) encoding resistance to different classes of antibiotics. Interestingly, the closest plasmids to plasmid pEGY1-MCR-1 detected from the NCBI Blast search belonged to the incompatibility group IncHI2 and were from the Kingdom of Saudi Arabia and Qatar which suggests a dissemination of pEGY1-MCR-1-like plasmids in the Middle East. Most striking, and of great public health concern is that strain CFSAN061770 carries five virulence genes (iss, fimH, iutA, kpsMIII and kpsTIII) which were identified in clinical extraintestinal pathogenic E. coli. Besides that, it carries the astA gene, which codes for the enteroaggregative E. coli heat-stable toxin 1 (EAST1).

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), has a complex cell envelope that provides a barrier of protection to various environments. Peripheral lipids on the M.tb cell envelope act as virulent factors influencing the host immune response to infection. Of these, some phosphatidyl-myo-inositol mannosides (PIMs) and their associated lipoglycans (lipomannan; mannose-capped lipoarabinomannan) influence M.tb-host interactions by increasing phagocytosis, blocking the maturation of M.tb-containing phagosomes, and increasing the anti-inflammatory response of infected macrophages. However, there is limited knowledge regarding how M.tb regulates PIMs levels, and how this regulation influences infection outcomes. In our previous studies, we hypothesized that Rv2623 and Rv1747 work together to regulate PIMs on the M.tb cell surface. Indeed, deleting the universal stress protein Rv2623 increases PIMs levels, growth, and virulence in vivo; while deleting the ABC transporter Rv1747 decreases PIMs levels. Here we investigate the mechanistic regulation of M.tb PIMs levels and its impact on growth and pathogenesis in vitro by assessing multiple M.tb strains with Rv2623 or Rv1747 mutated at different amino acids. Depending on the mutation, Rv2623 interacts with Rv1747 to modulate M.tb cell envelope PIMs levels at different degrees. Mutants with increased PIMs levels have increased growth in macrophages, and PIMs levels on the M.tb cell envelope may also correlate to resistance to certain anti-TB drugs. Overall, our data indicate that Rv2623 negatively regulates Rv1747 to modulate PIMs levels on the M.tb cell envelope, ultimately influencing growth and M.tb-host interactions in vitro and in vivo. Supported by grants from NIH/NIAID (1R01AI146340-01A1)

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.

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.

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.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease., Here, the authors characterize tissue-level SARS-CoV-2 infection and pathogenesis in transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18-hACE2) and show that infection induces lethality, making the K18-hACE2 model suitable for vaccine and therapeutic evaluation.

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.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. Here, we show that transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2-transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2-transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 4. K18 hACE2-transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

To discern if there was a particular genotype associated with clinical enteroaggregative Escherichia coli (EAEC) strains isolated from deployed military personnel (DMP) with travelers' diarrhea (TD), we characterized a collection of EAEC from DMP deployed to Afghanistan, Djibouti, Kenya, or Honduras. Although we did not identify a specific EAEC genotype associated with TD in DMP, we found that EAEC isolated at the first clinic visit were more likely to encode the dispersin gene aap than EAEC collected at follow-up visits. A majority of the EAEC isolates were typical EAEC that adhered to HEp-2 cells, formed biofilms, and harbored genes for aggregative adherence fimbriae (AAF), AggR, and serine protease autotransporters of Enterobacteriaceae (SPATEs). A separate subset of the EAEC had aggR and genes for SPATEs but encoded a gene highly homologous to that for CS22, a fimbriae more commonly found in enterotoxigenic E. coli. None of these CS22-encoding EAEC formed biofilms in vitro or adhered to HEp-2 cells. Whole genome sequence and single nucleotide polymorphism analyses demonstrated that most of the strains were genetically diverse, but that a few were closely related. Isolation of these related strains occurred within days to more than a year apart, a finding that suggests a persistent source and genomic stability. In an ampicillin-treated mouse model we found that an agg4A+ aar- isolate formed a biofilm in the intestine and caused reduced weight gain in mice, whereas a strain that did not form an in vivo biofilm caused no morbidity. Our diverse strain collection from DMP displays the heterogeneity of EAEC strains isolated from human patients, and our mouse model of infection indicated the genotype agg4A+ aar– and/or capacity to form biofilm in vivo may correlate to disease severity.