scholarly journals In Vivo Dual RNA-Seq Analysis Reveals the Basis for Differential Tissue Tropism of Clinical Isolates of Streptococcus pneumoniae

2019 ◽  
Author(s):  
Vikrant Minhas ◽  
Rieza Aprianto ◽  
Lauren J. McAllister ◽  
Hui Wang ◽  
Shannon C. David ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Single Cell Analysis ◽  
Regulatory Gene ◽  
Systemic Infection ◽  
Rna Seq ◽  
Transcriptional Responses ◽  
Disease Outcomes ◽  
Significant Difference ◽  
Genes Encoding

ABSTRACTStreptococcus pneumoniae is a genetically diverse human-adapted pathogen commonly carried asymptomatically in the nasopharynx. We have recently shown that a single nucleotide polymorphism (SNP) in the raffinose pathway regulatory gene rafR accounts for a significant difference in the capacity of clonally-related strains to cause localised versus systemic infection. Here we have used dual RNA-seq to show that this SNP extensively impacts both bacterial and host transcriptomes in infected lungs. It affects expression of bacterial genes encoding multiple sugar transporters, and fine-tunes carbohydrate metabolism, along with extensive rewiring of host transcriptional responses to infection, particularly expression of genes encoding cytokine and chemokine ligands and receptors. The dual RNA-seq data predicted a crucial role for differential neutrophil recruitment in the distinct virulence profiles of the infecting strains and single cell analysis revealed that while reduced expression of the RafR regulon driven by a single rafR SNP provides a clear advantage for pneumococci to colonize the ear, in the lung it leads to massive recruitment of neutrophils and bacterial clearance. Importantly, the observed disease outcomes were confirmed by in vivo neutrophil depletion showing that early detection of bacteria by the host in the lung environment is crucial for effective clearance. Thus, dual RNA-seq provides a powerful tool for understanding complex host-pathogen interactions and revealed how a single bacterial SNP can drive differential disease outcomes.

scholarly journals Exploration of bacterial bottlenecks and Streptococcus pneumoniae pathogenesis by CRISPRi-seq

2020 ◽  
Author(s):  
Xue Liu ◽  
Jacqueline M. Kimmey ◽  
Vincent de Bakker ◽  
Victor Nizet ◽  
Jan-Willem Veening
Keyword(s):  
Streptococcus Pneumoniae ◽  
Post Infection ◽  
Systemic Infection ◽  
The Body ◽  
Genetic Screens ◽  
Animal Pathogens ◽  
Genes Encoding ◽  
One Step

AbstractStreptococcus pneumoniae is a commensal bacterium of the human nasopharynx, but can cause harmful infections if it spreads to other parts of the body, such as pneumonia, sepsis or meningitis. To facilitate pathogenesis studies, we constructed a doxycycline-inducible pooled CRISPR interference (CRISPRi) library targeting all operons in protypical S. pneumoniae strain D39V. Our library design allows fitness within the pool to be assessed by a one-step PCR reaction directly followed by Illumina sequencing (CRISPRi-seq). The doxycycline-inducible CRISPRi system is tightly controllable and suitable for both bottleneck exploration and evaluation of gene fitness in vitro and in vivo. Here, we applied CRISPRi-seq to identify genetic factors important for causing pneumococcal pneumonia. Mice were infected intratracheally with our CRISPRi library and bacteria collected at 24 h (from lung) and 48 h (from both lung and blood) post-infection. CRISPRi-seq showed a critical bottleneck at 48 h after intratracheal infection, with only a few bacteria surviving the brunt of the innate immune response to cause systemic infection. However, earlier at 24 h post-infection, many significant differences in gene fitness cost between in vitro and in vivo conditions were identified, including genes encoding known and putative novel virulence factors, genes essential only in vivo, and genes essential only in vitro. A key advantage of CRISPRi-seq over traditional transposon-based genetic screens is that all genes, including essential genes, can be tested for their role in virulence and pathogenicity. The approaches developed here should be generally applicable to study infection bottlenecks and in vivo fitness for other important human and animal pathogens.

scholarly journals In vivo dual RNA-seq reveals that neutrophil recruitment underlies differential tissue tropism of Streptococcus pneumoniae

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Vikrant Minhas ◽  
Rieza Aprianto ◽  
Lauren J. McAllister ◽  
Hui Wang ◽  
Shannon C. David ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Neutrophil Recruitment ◽  
Tissue Tropism ◽  
Rna Seq ◽  
Differential Tissue

scholarly journals The Tat Substrate SufI Is Critical for the Ability of Yersinia pseudotuberculosis To Cause Systemic Infection

2017 ◽  
Vol 85 (4) ◽  
Author(s):  
Ummehan Avican ◽  
Tugrul Doruk ◽  
Yngve Östberg ◽  
Anna Fahlgren ◽  
Åke Forsberg
Keyword(s):  
Stress Responses ◽  
Pathogenic Bacteria ◽  
Yersinia Pseudotuberculosis ◽  
Systemic Infection ◽  
Mesenteric Lymph Nodes ◽  
Content Type ◽  
Twin Arginine Translocation ◽  
Genes Encoding ◽  
Folded Proteins

ABSTRACT The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important human-pathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a ΔsufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosis. In vivo bioluminescent imaging of orally infected mice revealed that both the ΔsufI and ΔtatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the ΔtatC mutant nor the ΔsufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the ΔtatC and ΔsufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the ΔtatC mutant.

scholarly journals Characterization of Pit, a Streptococcus pneumoniae Iron Uptake ABC Transporter

2002 ◽  
Vol 70 (8) ◽  
pp. 4389-4398 ◽  
Author(s):  
Jeremy S. Brown ◽  
Sarah M. Gilliland ◽  
Javier Ruiz-Albert ◽  
David W. Holden
Keyword(s):  
Streptococcus Pneumoniae ◽  
Abc Transporter ◽  
Iron Uptake ◽  
Systemic Infection ◽  
Iron Chelator ◽  
Iron Deficient ◽  
Deficient Medium ◽  
Mutant Strains

ABSTRACT Bacteria frequently have multiple mechanisms for acquiring iron, an essential micronutrient, from the environment. We have identified a four-gene Streptococcus pneumoniae operon, named pit, encoding proteins with similarity to components of a putative Brachyspira hyodysenteriae iron uptake ABC transporter, Bit. An S. pneumoniae strain containing a defined mutation in pit has impaired growth in medium containing the iron chelator ethylenediamine di-o-hydroxyphenylacetic acid, reduced sensitivity to the iron-dependent antibiotic streptonigrin, and impaired virulence in a mouse model of S. pneumoniae systemic infection. Furthermore, addition of a mutation in pit to a strain containing mutations in the two previously described S. pneumoniae iron uptake ABC transporters, piu and pia, resulted in a strain with impaired growth in two types of iron-deficient medium, a high degree of resistance to streptonigrin, and a reduced rate of iron uptake. Comparison of the susceptibilities to streptonigrin of the individual pit, piu, and pia mutant strains and comparison of the growth in iron-deficient medium and virulence of single and double mutant strains suggest that pia is the dominant iron transporter during in vitro and in vivo growth.

scholarly journals Single Cell Sequencing Reveals Early PGC-like Intermediates During Mouse Primed to Naïve Transition

2021 ◽  
Author(s):  
Jing Liu ◽  
Shengyong Yu ◽  
Chunhua Zhou ◽  
Jiangping He ◽  
Xingnan Huang ◽  
...  
Keyword(s):  
Germ Cell ◽  
Single Cell ◽  
Cell Fate ◽  
Single Cell Analysis ◽  
Primordial Germ Cell ◽  
Model System ◽  
Cell Analysis ◽  
Rna Seq ◽  
Single Cell Sequencing

Abstract Single cell analysis provides clarity unattainable with bulk approaches. Here we apply single cell RNA-seq to a newly established BMP4 induced mouse primed to naive transition (Bi-PNT) system and show that the reset is not a direct reversal of cell fate but through developmental intermediates. We first show that mEpiSCs bifurcate into c-Kit+ naïve and c-Kit- placenta-like cells, among which, the naive branch undergoes further transition through a primordial germ cell-like cells (PGCLCs) intermediate capable of spermatogenesis in vivo. Indeed, deficiency of Prdm1/Blimp1, the key regulator for PGC specification, blocks the induction of PGCLCs and naïve cells. Instead, Gata2 knockout arrests placenta-like fate, but facilitates the generation of PGCLCs. Our results not only reveal a newly cell fate dynamics between primed and naive states at single-cell resolution, but also provide a model system to explore mechanisms involved in regaining germline competence from primed pluripotency.

scholarly journals Isolation of Streptococcus pneumoniae Biofilm Mutants and Their Characterization during Nasopharyngeal Colonization

2008 ◽  
Vol 76 (11) ◽  
pp. 5049-5061 ◽  
Author(s):  
Ernesto J. Muñoz-Elías ◽  
Joan Marcano ◽  
Andrew Camilli
Keyword(s):  
Cell Wall ◽  
Streptococcus Pneumoniae ◽  
Biofilm Formation ◽  
Bacterial Infections ◽  
Nasopharyngeal Colonization ◽  
Genes Encoding ◽  
Transposon Mutants ◽  
Bona Fide

ABSTRACT Asymptomatic colonization of the nasopharynx by Streptococcus pneumoniae precedes pneumococcal disease, yet pneumococcal colonization factors remain poorly understood. Many bacterial infections involve biofilms which protect bacteria from host defenses and antibiotics. To gain insight into the genetics of biofilm formation by S. pneumoniae, we conducted an in vitro screen for biofilm-altered mutants with the serotype 4 clinical isolate TIGR4. In a first screen of 6,000 mariner transposon mutants, we repeatedly isolated biofilm-overproducing acapsular mutants, suggesting that the capsule was antagonistic to biofilm formation. Therefore, we screened 6,500 additional transposon mutants in an S. pneumoniae acapsular background. Following this approach, we isolated 69 insertions in 49 different genes. The collection of mutants includes genes encoding bona fide and putative choline binding proteins, adhesins, synthases of membrane and cell wall components, extracellular and cell wall proteases, efflux pumps, ABC and PTS transporters, and transcriptional regulators, as well as several conserved and novel hypothetical proteins. Interestingly, while four insertions mapped to rrgA, encoding a subunit of a recently described surface pilus, rrgB and rrgC (encoding the other two pilus subunits) mutants had no biofilm defects, implicating the RrgA adhesin but not the pilus structure per se in biofilm formation. To correlate our findings to the process of colonization, we transferred a set of 29 mutations into the wild-type encapsulated strain and then tested the fitness of the mutants in vivo. Strikingly, we found that 23 of these mutants were impaired for nasopharyngeal colonization, thus establishing a link between biofilm formation and colonization.

scholarly journals TheIn VitroRedundant Enzymes PurN and PurT Are Both Essential for Systemic Infection of Mice in Salmonella enterica Serovar Typhimurium

2016 ◽  
Vol 84 (7) ◽  
pp. 2076-2085 ◽  
Author(s):  
Lotte Jelsbak ◽  
Mie I. B. Mortensen ◽  
Mogens Kilstrup ◽  
John E. Olsen
Keyword(s):  
Salmonella Enterica ◽  
Single Gene ◽  
Systemic Infection ◽  
Gene Deletions ◽  
Content Type ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Glycine Cleavage

Metabolic enzymes show a high degree of redundancy, and for that reason they are generally ignored in searches for novel targets for anti-infective substances. The enzymes PurN and PurT are redundantin vitroinSalmonella entericaserovar Typhimurium, in which they perform the third step of purine synthesis. Surprisingly, the results of the current study demonstrated that single-gene deletions of each of the genes encoding these enzymes caused attenuation (competitive infection indexes [CI] of <0.03) in mouse infections. While the ΔpurTmutant multiplied as fast as the wild-type strain in cultured J774A.1 macrophages, net multiplication of the ΔpurNmutant was reduced approximately 50% in 20 h. The attenuation of the ΔpurTmutant was abolished by simultaneous removal of the enzyme PurU, responsible for the formation of formate, indicating that the attenuation was related to formate accumulation or wasteful consumption of formyl tetrahydrofolate by PurU. In the process of further characterization, we disclosed that the glycine cleavage system (GCV) was the most important for formation of C1unitsin vivo(CI = 0.03 ± 0.03). In contrast, GlyA was the only important enzyme for the formation of C1unitsin vitro. The results with the ΔgcvTmutant further revealed that formation of serine by SerA and further conversion of serine into C1units and glycine by GlyA were not sufficient to ensure C1formation inS. Typhimuriumin vivo. The results of the present study call for reinvestigations of the concept of metabolic redundancy inS. Typhimuriumin vivo.

scholarly journals Activity of Tedizolid Phosphate (TR-701) in Murine Models of Infection with Penicillin-Resistant and Penicillin-Sensitive Streptococcus pneumoniae

2012 ◽  
Vol 56 (9) ◽  
pp. 4713-4717 ◽  
Author(s):  
Sunghak Choi ◽  
Weonbin Im ◽  
Ken Bartizal
Keyword(s):  
Streptococcus Pneumoniae ◽  
Inflammatory Cell ◽  
Systemic Infection ◽  
Murine Models ◽  
Once Daily ◽  
Content Type ◽  
Lung Histopathology ◽  
Phosphate Treatment

ABSTRACTThein vitroactivity of tedizolid (previously known as torezolid, TR-700) against penicillin-resistantStreptococcus pneumoniae(PRSP) clinical isolates and thein vivoefficacy of tedizolid phosphate (torezolid phosphate, TR-701) in murine models of PRSP systemic infection and penicillin-susceptibleS. pneumoniae(PSSP) pneumonia were examined using linezolid as a comparator. The MIC90against 28 PRSP isolates was 0.25 μg/ml for tedizolid, whereas it was 1 μg/ml for linezolid. In mice infected systemically with a lethal inoculum of PRSP 1 h prior to a single administration of either antimicrobial, oral tedizolid phosphate was equipotent to linezolid (1 isolate) to 2-fold more potent than linezolid (3 isolates) for survival at day 7, with tedizolid phosphate 50% effective dose (ED50) values ranging from 3.19 to 11.53 mg/kg of body weight/day. In the PSSP pneumonia model, the ED50for survival at day 15 was 2.80 mg/kg/day for oral tedizolid phosphate, whereas it was 8.09 mg/kg/day for oral linezolid following 48 h of treatment with either agent. At equivalent doses (10 mg/kg once daily tedizolid phosphate or 5 mg/kg twice daily linezolid), pneumococcal titers in the lungs at 52 h postinfection were approximately 3 orders of magnitude lower with tedizolid phosphate treatment than with linezolid treatment or no treatment. Lung histopathology showed less inflammatory cell invasion into alveolar spaces in mice treated with tedizolid phosphate than in untreated or linezolid-treated mice. These results demonstrate that tedizolid phosphate is effective in murine models of PRSP systemic infection and PSSP pneumonia.

scholarly journals Arsenite Oxidase Also Functions as an Antimonite Oxidase

2015 ◽  
Vol 81 (6) ◽  
pp. 1959-1965 ◽  
Author(s):  
Qian Wang ◽  
Thomas P. Warelow ◽  
Yoon-Suk Kang ◽  
Christine Romano ◽  
Thomas H. Osborne ◽  
...  
Keyword(s):  
Environmental Protection Agency ◽  
Large Subunit ◽  
Environmental Pollutants ◽  
Transcriptional Responses ◽  
Oxidation Activity ◽  
Content Type ◽  
Genes Encoding ◽  
Basic Features

ABSTRACTArsenic and antimony are toxic metalloids and are considered priority environmental pollutants by the U.S. Environmental Protection Agency. Significant advances have been made in understanding microbe-arsenic interactions and how they influence arsenic redox speciation in the environment. However, even the most basic features of how and why a microorganism detects and reacts to antimony remain poorly understood. Previous work withAgrobacterium tumefaciensstrain 5A concluded that oxidation of antimonite [Sb(III)] and arsenite [As(III)] required different biochemical pathways. Here, we show within vivoexperiments that a mutation inaioA[encoding the large subunit of As(III) oxidase] reduces the ability to oxidize Sb(III) by approximately one-third relative to the ability of the wild type. Further,in vitrostudies with the purified As(III) oxidase fromRhizobiumsp. strain NT-26 (AioA shares 94% amino acid sequence identity with AioA ofA. tumefaciens) provide direct evidence of Sb(III) oxidation but also show a significantly decreasedVmaxcompared to that of As(III) oxidation. TheaioBAgenes encoding As(III) oxidase are induced by As(III) but not by Sb(III), whereasarsRgene expression is induced by both As(III) and Sb(III), suggesting that detection and transcriptional responses for As(III) and Sb(III) differ. While Sb(III) and As(III) are similar with respect to cellular extrusion (ArsB or Acr3) and interaction with ArsR, they differ in the regulatory mechanisms that control the expression of genes encoding the different Ars or Aio activities. In summary, this study documents an enzymatic basis for microbial Sb(III) oxidation, although additional Sb(III) oxidation activity also is apparent in this bacterium.

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