Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk

Baddal, Buket; Muzzi, Alessandro; Censini, Stefano; Calogero, Raffaele; Torricelli, Giulia; Guidotti, Silvia; Taddei, Anna Rita; Covacci, Antonello; Pizza, Mariagrazia; Rappuoli, Rino; Soriani, Marco; Pezzicoli, Alfredo

doi:10.1128/mbio.01765-15

Cited by 104 publications

(118 citation statements)

References 58 publications

(56 reference statements)

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“…Thus, conventional RNA-seq methods, such as Tru-seq, are readily applicable to host-fungal pathogen analysis. Other current available method that allows simultaneous analysis of host and bacterial pathogen by RNA-seq is the differential RNA-Seq (dRNA-Seq) 16,17 as well as [6][7][8] . Compared to our protocol, these methods require a high amount of input RNA (e.g.,at least 15 µg total RNA of high quality is required for dRNA-seq 16 ), and thus cannot be used for analyzing samples with limited RNA quantity (e.g., FACS sorted populations or limiting clinical samples).…”

Section: Comparison To Other Currently Available Methodsmentioning

confidence: 99%

“…The original RNAtag-seq protocol 10 was optimized for bacterial transcript analysis, and lysis methods involved enzymatic pretreatment and mechanical disruption, both of which can be deleterious to host RNA and inadequate for low yield samples. In this protocol, we have optimized the lysis of both host and bacterial cells either by using gentle bead disruption (for higher cell number; >10 6 ) or by enzymatic treatment (for lower cell numbers; <10 5 ). It is important to follow the lysis instructions detailed here as other bead beating methods or other cell wall lytic enzymes may degrade the host RNA, will not provide efficient bacterial lysis, or are not suitable for downstream RNA processing.…”

Section: Comparison To Other Currently Available Methodsmentioning

confidence: 99%

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A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

et al. 2016

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EDITORIAL SUMMARY:This protocol enables simultaneous analysis of host and bacterial transcripts by RNA-Seq. Including procedures for efficient host and bacterial cell lysis, barcoding of samples, and analysis of both mammalian and microbial reads from mixed host-pathogen RNA-Seq data.TWEET: Simultaneous analysis of host and pathogen transcriptomes by RNA-Seq. Abstract The ability to simultaneously characterize the bacterial and host expression programs during infection would facilitate a comprehensive understanding of pathogen-host interactions. While RNA-Seq has greatly advanced our ability to study the transcriptomes of prokaryote and eukaryotes separately, limitations in existing protocols for generating and analyzing RNA-Seq data have hindered simultaneous profiling of host and bacterial pathogen transcripts from the same sample. Here we provide a detailed protocol for simultaneous analysis of host and bacterial transcripts by RNA-Seq. Importantly, this protocol details the steps required for efficient host and bacteria lysis, barcoding of samples, technical advances in sample preparation for low yield sample inputs and a computational pipeline to analyze both mammalian and microbial reads from mixed hostpathogen RNA-Seq data. Sample preparation takes 3 d from cultured cells to pooled libraries. Data analysis takes an additional day. Compared with previous methods, the protocol detailed here provides a sensitive, facile and generalizable approach, suitable for large-scale studies, which will enable the field to obtain in-depth analysis of hostpathogen interactions in infection models. Introduction Intracellular bacterial pathogens, such as Mycobacterium tuberculosis, Salmonella enterica, Legionella pneumophilia, and Neisseria gonorrhea, spend a significant portion of their life-cycle surviving and replicating within host cells. The cellular interaction entails both a complex virulence program executed by the bacterial pathogen during infection 1 and activation of an orchestrated defense response by the host to counter the pathogen 2 . Genomic approaches have been employed in recent years to uncover substantial molecular details of this rich host-pathogen biology 3, 4 . However, technical constraints limit these studies to profiling either the host or the pathogen -while a comprehensive understanding of host-pathogen interactions requires simultaneous analysis of the associated gene expression changes in both the pathogen and the host 5 . The limitations of conventional protocols for simultaneous analysis of host and pathogen transcripts include 1) the inability to obtain high quality RNA with efficient lysis of both bacterial and mammalian host cells; 2) inefficient depletion of both microbial and mammalian ribosomal RNA species; 3) inability to simultaneously process polyadenylated and non-polyadenylated transcripts from low yield samples (>10ng of RNA); and 4) a lack of robust computational approaches for analyzing the often small subset of bacterial transcripts in infected cells or tissue. Recently, sever...

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Section: Comparison To Other Currently Available Methodsmentioning

confidence: 99%

Section: Comparison To Other Currently Available Methodsmentioning

confidence: 99%

A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

et al. 2016

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“…Typhimurium [30], Staphylococcus aureus [46], Uropathogenic Escherichia coli (UPEC) [47], Chlamydia trachomatis [48], Mycobacterium bovis [49] and Haemophilus influenzae [50]. More recently, dual RNAseq was also used to simultaneously study host and pathogen gene expression profiles in in vivo infection models (reviewed in [19]).…”

Section: Figure 1| Macromolecular Differences Between Cells Of Eukarymentioning

confidence: 99%

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

Fels¹,

Gevaert²,

Damme³

2017

Preprint

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By providing useful tools to study host-pathogen interactions, next-generation omics has recently enabled the study of gene expression changes in both pathogen and infected host simultaneously. However, since great discriminative power is required to study pathogen and host simultaneously throughout the infection process, the depth of quantitative gene expression profiling has proven to be unsatisfactory when focusing on bacterial pathogens, thus preferentially requiring specific strategies or the development of novel methodologies based on complementary omics approaches. In this review, we focus on the difficulties encountered when making use of omics approaches to study bacterial pathogenesis. Besides, we review different omics strategies (i.e. transcriptomics, proteomics and secretomics) and their applications for studying interactions of pathogens with their host.

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“…Studies have demonstrated that VapBC1 and VapBC2 are significantly upregulated in NTHi during infection, where they function to regulate growth and enhance survival (34,35). The VapC toxin contains a PIN domain, a roughly 100-amino-acid domain that has an active site containing four conserved acidic amino acids (36).…”

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confidence: 99%

Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

Walling

Butler

2016

J Bacteriol

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Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Under normal growth conditions, the antitoxin neutralizes the toxin. However, under conditions of stress, such as nutrient starvation or antibiotic treatment, cellular proteases degrade the antitoxin, and the toxin functions to arrest bacterial growth. We characterized the specificity determinants of the interactions between VapB antitoxins and VapC toxins from nontypeable Haemophilus influenzae (NTHi) in an effort to gain a better understanding of how antitoxins control toxin activity and bacterial persistence. We studied truncated and full-length antitoxins with single amino acid mutations in the toxin-binding domain. Coexpressing the toxin and antitoxin in Escherichia coli and measuring bacterial growth by dilution plating assayed the ability of the mutant antitoxins to neutralize the toxin. Our results identified two single amino acid residues (W48 and F52) in the C-terminal region of the VapB2 antitoxin necessary for its ability to neutralize its cognate VapC2 toxin. Additionally, we attempted to alter the specificity of VapB1 by making a mutation that would allow it to neutralize its noncognate toxin. A mutation in VapB1 to contain the tryptophan residue identified herein as important in the VapB2-VapC2 interaction resulted in a VapB1 mutant (the T47W mutant) that binds to and neutralizes both its cognate VapC1 and noncognate VapC2 toxins. This represents the first example of a single mutation causing relaxed specificity in a type II antitoxin. IMPORTANCEToxin-antitoxin systems are of particular concern in pathogenic organisms, such as nontypeable Haemophilus influenzae, as they can elicit dormancy and persistence, leading to chronic infections and failure of antibiotic treatment. Despite the importance of the TA interaction, the specificity determinants for VapB-VapC complex formation remain uncharacterized. Thus, our understanding of how antitoxins control toxin-induced dormancy and bacterial persistence requires thorough investigation of antitoxin specificity for its cognate toxin. This study characterizes the crucial residues of the VapB2 antitoxin from NTHi necessary for its interaction with VapC2 and provides the first example of a single amino acid change altering the toxin specificity of an antitoxin.T oxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Originally discovered as addictive elements involved in postsegregational killing during cell division, they have since been identified as playing a critical role in responding to stress (1-3). TA systems are typically encoded by a single operon that produces a stable protein toxin and its cognate labile antitoxin, with the gene encoding the antitoxin located upstream of the toxin in most cases (4). Under normal growth conditions, the antitoxin, which may be a protein (types II, IV, and V) or an RNA...

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Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk

Cited by 104 publications

References 58 publications

A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

A highly multiplexed and sensitive RNA-seq protocol for simultaneous analysis of host and pathogen transcriptomes

Omics in Aid of Host-pathogen Interaction Studies: A Bacterial Perspective

Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

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