Early host immune responses in a human organoid-derived gallbladder monolayer to Salmonella Typhi strains from patients with acute and chronic infections: a comparative analysis.

Salmonella enterica serovar Typhi (S. Typhi), a human-restricted pathogen, invades the host through the gut to cause typhoid fever. Recent calculations of the typhoid fever burden estimated that more than 10 million new typhoid fever cases occur in low and middle-income countries, resulting in 65,400-187,700 deaths yearly. Interestingly, if not antibiotic-treated, upon the resolution of acute disease, 1%-5% of patients become asymptomatic chronic carriers. Chronically infected hosts are not only critical reservoirs of infection that transmit the disease to naive individuals but are also predisposed to developing gallbladder carcinoma. Nevertheless, the molecular mechanisms involved in the early interactions between gallbladder epithelial cells and S. Typhi remain largely unknown. Based on our previous studies showing that closely related S. Typhi strains elicit distinct innate immune responses, we hypothesized that host molecular pathways activated by S. Typhi strains derived from acutely and chronically infected patients would differ. To test this hypothesis, we used a novel human organoid-derived polarized gallbladder monolayer model, and S. Typhi strains derived from acutely and chronically infected patients. We found that S. Typhi strains derived from acutely and chronically infected patients differentially regulate host mitogen-activated protein kinase (MAPK) and S6 transcription factors. These variations might be attributed to differential cytokine signaling, predominantly via TNF-α and IL-6 production and appear to be influenced by the duration the isolate was subjected to selective pressures in the gallbladder. These findings represent a significant leap in understanding the complexities behind chronic S. Typhi infections in the gallbladder and may uncover potential intervention targets.

Corrigendum: Evidence for the cytoplasmic localization of the L-α-Glycerophosphate oxidase in members of the "Mycoplasma mycoides cluster".

[This corrects the article DOI: 10.3389/fmicb.2019.01344.].

Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects.

Group B Streptococcus (GBS; S. agalactiae ) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δ cas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.

Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects.

Group B Streptococcus (GBS; S. agalactiae) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δcas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.

Influenza A-induced cystic fibrosis transmembrane conductance regulator dysfunction increases susceptibility to Streptococcus pneumoniae.

Influenza A virus (IAV) infection is commonly complicated by secondary bacterial infections that lead to increased morbidity and mortality. Our recent work demonstrates that IAV disrupts airway homeostasis, leading to airway pathophysiology resembling cystic fibrosis disease through diminished cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we use human airway organotypic cultures to investigate how IAV alters the airway microenvironment to increase susceptibility to secondary infection with Streptococcus pneumoniae (Spn). We observed that IAV-induced CFTR dysfunction and airway surface liquid acidification is central to increasing susceptibility to Spn. Additionally, we observed that IAV induced profound transcriptional changes in the airway epithelium and proteomic changes in the airway surface liquid in both CFTR-dependent and -independent manners. These changes correspond to multiple diminished host defense pathways and altered airway epithelial function. Collectively, these findings highlight both the importance of CFTR function during infectious challenge and demonstrate a central role for the lung epithelium in secondary bacterial infections following IAV.

Adaptation of Nontypeable Haemophilus influenzae in Human Airways in COPD: Genome Rearrangements and Modulation of Expression of HMW1 and HMW2.

Chronic obstructive pulmonary disease (COPD) is a common debilitating disorder that is the third most common cause of death globally. Chronic lower airway infection by nontypeable Haemophilus influenzae (NTHi) in adults with COPD increases airway inflammation, causes increased symptoms, and accelerates progressive loss of lung function. Little is known about the mechanisms by which NTHi survives in COPD airways. To explore this question, the present study analyzes, in detail, 14 prospectively collected, serial isolates of a strain that persisted for 543 days in a patient with COPD, including analysis of four gap-free complete genomes. The NTHi genome underwent inversion of a ~400-kb segment three times during persistence. This inversion event resulted in switching of expression of the HMW1A and HMW2A adhesins as the inversion sites are in the promoter regions of HMW1 and HMW2. Regulation of the level of expression of HMW 1 and HMW2 in the human airways was controlled by the ~400-kb inversion and by 7-bp repeats in the HMW promoters. Analysis of knockout mutants of the persistent strain demonstrated that HMW1 and HMW2 proteins both function in the adherence of NTHi to human respiratory epithelial cells during persistence and that HMW1 also facilitates invasion of epithelial cells. An inverse relationship between biofilm formation and HMW1 expression was observed during persistence. This work advances understanding of the mechanisms of persistence of NTHi in COPD airways, which can inform the development of novel interventions to treat and prevent chronic NTHi infection in COPD. IMPORTANCE Nontypeable Haemophilus influenzae (NTHi) persists in the lower airways of adults with chronic obstructive pulmonary disease (COPD) for months to years, increasing airway inflammation that accelerates the progressive loss of lung function. Understanding the mechanisms of persistence in human airways by NTHi is critical in developing novel interventions. Here, in detail, we studied longitudinally collected sequential isolates of a strain of NTHi that persisted in an adult with COPD, including analysis of four gap-free genomes and knockout mutants to elucidate how the genome adapts in human airways. The NTHi genome underwent a genome rearrangement during persistence and this inversion impacted regulation of expression of key virulence phenotypes, including adherence to respiratory epithelial cells, invasion of epithelial cells and biofilm formation. These novel observations advance our understanding of the mechanisms of persistence of NTHi in the airways of adults with COPD.

Influenza A virus modulation of Streptococcus pneumoniae infection using ex vivo transcriptomics in a human primary lung epithelial cell model reveals differential host glycoconjugate uptake and metabolism.

Background: Streptococcus pneumoniae (Spn) is typically an asymptomatic colonizer of the nasopharynx but it also causes pneumonia and disseminated disease affecting various host anatomical sites. Transition from colonization to invasive disease is not well understood. Studies have shown that such a transition can occur as result of influenza A virus coinfection. Methods: We investigated the pneumococcal (serotype 19F, strain EF3030) and host transcriptomes with and without influenza A virus (A/California/07 2009 pH1N1) infection at this transition. This was done using primary, differentiated Human Bronchial Epithelial Cells (nHBEC) in a transwell monolayer model at an Air-Liquid Interface (ALI), with multispecies deep RNA-seq. Results: Distinct pneumococcal gene expression profiles were observed in the presence and absence of influenza. Influenza coinfection allowed for significantly greater pneumococcal growth and triggered the differential expression of bacterial genes corresponding to multiple metabolic pathways; in totality suggesting a fundamentally altered bacterial metabolic state and greater nutrient availability when coinfecting with influenza. Surprisingly, nHBEC transcriptomes were only modestly perturbed by infection with EF3030 alone in comparison to that resulting from Influenza A infection or coinfection, which had drastic alterations in thousands of genes. Influenza infected host transcriptomes suggest significant loss of ciliary function in host nHBEC cells. Conclusions: Influenza A virus infection of nHBEC promotes pneumococcal infection. One reason for this is an altered metabolic state by the bacterium, presumably due to host components made available as result of viral infection. Influenza infection had a far greater impact on the host response than did bacterial infection alone, and this included down regulation of genes involved in expressing cilia. We conclude that influenza infection promotes a pneumococcal metabolic shift allowing for transition from colonization to disseminated disease.

COVID-19 vaccine design using reverse and structural vaccinology, ontology-based literature mining and machine learning.

Rational vaccine design, especially vaccine antigen identification and optimization, is critical to successful and efficient vaccine development against various infectious diseases including coronavirus disease 2019 (COVID-19). In general, computational vaccine design includes three major stages: (i) identification and annotation of experimentally verified gold standard protective antigens through literature mining, (ii) rational vaccine design using reverse vaccinology (RV) and structural vaccinology (SV) and (iii) post-licensure vaccine success and adverse event surveillance and its usage for vaccine design. Protegen is a database of experimentally verified protective antigens, which can be used as gold standard data for rational vaccine design. RV predicts protective antigen targets primarily from genome sequence analysis. SV refines antigens through structural engineering. Recently, RV and SV approaches, with the support of various machine learning methods, have been applied to COVID-19 vaccine design. The analysis of post-licensure vaccine adverse event report data also provides valuable results in terms of vaccine safety and how vaccines should be used or paused. Ontology standardizes and incorporates heterogeneous data and knowledge in a human- and computer-interpretable manner, further supporting machine learning and vaccine design. Future directions on rational vaccine design are discussed.

Anatomical Site-Specific Carbohydrate Availability Impacts Streptococcus pneumoniae Virulence and Fitness during Colonization and Disease.

Streptococcus pneumoniae colonizes the nasopharynx asymptomatically but can also cause severe life-threatening disease. Importantly, stark differences in carbohydrate availability exist between the nasopharynx and invasive disease sites, such as the bloodstream, which most likely impact S. pneumoniae's behavior. Herein, using chemically defined medium (CDM) supplemented with physiological levels of carbohydrates, we examined how anatomical site-specific carbohydrate availability impacted S. pneumoniae physiology and virulence. S. pneumoniae cells grown in CDM modeling the nasopharynx (CDM-N) had reduced metabolic activity and a lower growth rate, demonstrated mixed acid fermentation with marked H2O2 production, and were in a carbon-catabolite repression (CCR)-derepressed state versus S. pneumoniae cells grown in CDM modeling blood (CDM-B). Using transcriptome sequencing (RNA-seq), we determined the transcriptome for the S. pneumoniae wild-type (WT) strain and its isogenic CCR-deficient mutant in CDM-N and CDM-B. Genes with altered expression as a result of changes in carbohydrate availability or catabolite control protein deficiency, respectively, were primarily involved in carbohydrate metabolism, but also encoded established virulence determinants, such as polysaccharide capsule and surface adhesins. We confirmed that anatomical site-specific carbohydrate availability directly influenced established S. pneumoniae virulence traits. S. pneumoniae cells grown in CDM-B formed shorter chains, produced more capsule, were less adhesive, and were more resistant to macrophage killing in an opsonophagocytosis assay. Moreover, growth of S. pneumoniae in CDM-N or CDM-B prior to the challenge of mice impacted relative fitness in a colonization model and invasive disease model, respectively. Thus, anatomical site-specific carbohydrate availability alters S. pneumoniae physiology and virulence, in turn promoting anatomical site-specific fitness.

An in vivo atlas of host-pathogen transcriptomes during Streptococcus pneumoniae colonization and disease.

Streptococcus pneumoniae (Spn) colonizes the nasopharynx and can cause pneumonia. From the lungs it spreads to the bloodstream and causes organ damage. We characterized the in vivo Spn and mouse transcriptomes within the nasopharynx, lungs, blood, heart, and kidneys using three Spn strains. We identified Spn genes highly expressed at all anatomical sites and in an organ-specific manner; highly expressed genes were shown to have vital roles with knockout mutants. The in vivo bacterial transcriptome during colonization/disease was distinct from previously reported in vitro transcriptomes. Distinct Spn and host gene-expression profiles were observed during colonization and disease states, revealing specific genes/operons whereby Spn adapts to and influences host sites in vivo. We identified and experimentally verified host-defense pathways induced by Spn during invasive disease, including proinflammatory responses and the interferon response. These results shed light on the pathogenesis of Spn and identify therapeutic targets.

ReVac: a reverse vaccinology computational pipeline for prioritization of prokaryotic protein vaccine candidates.

BACKGROUND: Reverse vaccinology accelerates the discovery of potential vaccine candidates (PVCs) prior to experimental validation. Current programs typically use one bacterial proteome to identify PVCs through a filtering architecture using feature prediction programs or a machine learning approach. Filtering approaches may eliminate potential antigens based on limitations in the accuracy of prediction tools used. Machine learning approaches are heavily dependent on the selection of training datasets with experimentally validated antigens (positive control) and non-protective-antigens (negative control). The use of one or few bacterial proteomes does not assess PVC conservation among strains, an important feature of vaccine antigens. RESULTS: We present ReVac, which implements both a panoply of feature prediction programs without filtering out proteins, and scoring of candidates based on predictions made on curated positive and negative control PVCs datasets. ReVac surveys several genomes assessing protein conservation, as well as DNA and protein repeats, which may result in variable expression of PVCs. ReVac's orthologous clustering of conserved genes, identifies core and dispensable genome components. This is useful for determining the degree of conservation of PVCs among the population of isolates for a given pathogen. Potential vaccine candidates are then prioritized based on conservation and overall feature-based scoring. We present the application of ReVac, applied to 69 Moraxella catarrhalis and 270 non-typeable Haemophilus influenzae genomes, prioritizing 64 and 29 proteins as PVCs, respectively. CONCLUSION: ReVac's use of a scoring scheme ranks PVCs for subsequent experimental testing. It employs a redundancy-based approach in its predictions of features using several prediction tools. The protein's features are collated, and each protein is ranked based on the scoring scheme. Multi-genome analyses performed in ReVac allow for a comprehensive overview of PVCs from a pan-genome perspective, as an essential pre-requisite for any bacterial subunit vaccine design. ReVac prioritized PVCs of two human respiratory pathogens, identifying both novel and previously validated PVCs.

Evidence for the Cytoplasmic Localization of the L-α-Glycerophosphate Oxidase in Members of the "Mycoplasma mycoides Cluster".

Members of the "Mycoplasma mycoides cluster" are important animal pathogens causing diseases including contagious bovine pleuropneumonia and contagious caprine pleuropneumonia, which are of utmost importance in Africa or Asia. Even if all existing vaccines have shortcomings, vaccination of herds is still considered the best way to fight mycoplasma diseases, especially with the recent and dramatic increase of antimicrobial resistance observed in many mycoplasma species. A new generation of vaccines will benefit from a better understanding of the pathogenesis of mycoplasmas, which is very patchy up to now. In particular, surface-exposed virulence traits are likely to induce a protective immune response when formulated in a vaccine. The candidate virulence factor L-α-glycerophosphate oxidase (GlpO), shared by many mycoplasmas including Mycoplasma pneumoniae, was suggested to be a surface-exposed enzyme in Mycoplasma mycoides subsp. mycoides responsible for the production of hydrogen peroxide directly into the host cells. We produced a glpO isogenic mutant GM12::YCpMmyc1.1-ΔglpO using in-yeast synthetic genomics tools including the tandem-repeat endonuclease cleavage (TREC) technique followed by the back-transplantation of the engineered genome into a mycoplasma recipient cell. GlpO localization in the mutant and its parental strain was assessed using scanning electron microscopy (SEM). We obtained conflicting results and this led us to re-evaluate the localization of GlpO using a combination of in silico and in vitro techniques, such as Triton X-114 fractionation or tryptic shaving followed by immunoblotting. Our in vitro results unambiguously support the finding that GlpO is a cytoplasmic protein throughout the "Mycoplasma mycoides cluster." Thus, the use of GlpO as a candidate vaccine antigen is unlikely to induce a protective immune response.

Haemophilus influenzae genome evolution during persistence in the human airways in chronic obstructive pulmonary disease.

Nontypeable Haemophilus influenzae (NTHi) exclusively colonize and infect humans and are critical to the pathogenesis of chronic obstructive pulmonary disease (COPD). In vitro and animal models do not accurately capture the complex environments encountered by NTHi during human infection. We conducted whole-genome sequencing of 269 longitudinally collected cleared and persistent NTHi from a 15-y prospective study of adults with COPD. Genome sequences were used to elucidate the phylogeny of NTHi isolates, identify genomic changes that occur with persistence in the human airways, and evaluate the effect of selective pressure on 12 candidate vaccine antigens. Strains persisted in individuals with COPD for as long as 1,422 d. Slipped-strand mispairing, mediated by changes in simple sequence repeats in multiple genes during persistence, regulates expression of critical virulence functions, including adherence, nutrient uptake, and modification of surface molecules, and is a major mechanism for survival in the hostile environment of the human airways. A subset of strains underwent a large 400-kb inversion during persistence. NTHi does not undergo significant gene gain or loss during persistence, in contrast to other persistent respiratory tract pathogens. Amino acid sequence changes occurred in 8 of 12 candidate vaccine antigens during persistence, an observation with important implications for vaccine development. These results indicate that NTHi alters its genome during persistence by regulation of critical virulence functions primarily by slipped-strand mispairing, advancing our understanding of how a bacterial pathogen that plays a critical role in COPD adapts to survival in the human respiratory tract.

Transcriptional organization of pneumococcal psrP-secY2A2 and impact of GtfA and GtfB deletion on PsrP-associated virulence properties.

Pneumococcal serine-rich repeat protein (PsrP) is a glycoprotein that mediates Streptococcus pneumoniae attachment to lung cells and promotes biofilm formation. Herein, we investigated the transcriptional organization of psrP-secY2A2, the 37-kbp pathogenicity island encoding PsrP and its accessory genes. PCR amplification of cDNA and RNA-seq analysis found psrP-secY2A2 to be minimally composed of three operons: psrP-glyA, glyB, and glyC-asp5. Transcription of all three operons was greatest during biofilm growth and immunoblot analyses confirmed increased PsrP production by biofilm pneumococci. Using gas chromatography-mass spectrometry we identified monomeric N-acetylglucosamine as the primary glycoconjugate present on a recombinant intracellular version of PsrP, i.e. PsrP1-734. This finding was validated by immunoblot using lectins with known carbohydrate specificities. We subsequently deleted gtfA and gtfB, the GTFs thought to be responsible for addition of O-linked N-acetylglucosamine, and tested for PsrP and its associated virulence properties. These deletions negatively affected our ability to detect PsrP1-734 in bacterial whole cell lysates. Moreover, S. pneumoniae mutants lacking these genes pheno-copied the psrP mutant and were attenuated for: biofilm formation, adhesion to lung epithelial cells, and pneumonia in mice. Our studies identify the transcriptional organization of psrP-secY2A2 and show the indispensable role of GtfA and GtfB on PsrP-mediated pneumococcal virulence.