RESUMO
Like for many bacteria, flagella are crucial for Campylobacter jejuni motility and virulence. Biogenesis of the flagellar machinery requires hierarchical transcription of early, middle (RpoN-dependent), and late (FliA-dependent) genes. However, little is known about post-transcriptional regulation of flagellar biogenesis by small RNAs (sRNAs). Here, we characterized two sRNAs with opposing effects on C. jejuni filament assembly and motility. We demonstrate that CJnc230 sRNA (FlmE), encoded downstream of the flagellar hook protein, is processed from the RpoN-dependent flgE mRNA by RNase III, RNase Y, and PNPase. We identify mRNAs encoding a flagella-interaction regulator and the anti-sigma factor FlgM as direct targets of CJnc230 repression. CJnc230 overexpression upregulates late genes, including the flagellin flaA, culminating in longer flagella and increased motility. In contrast, overexpression of the FliA-dependent sRNA CJnc170 (FlmR) reduces flagellar length and motility. Overall, our study demonstrates how the interplay of two sRNAs post-transcriptionally fine-tunes flagellar biogenesis through balancing of the hierarchically-expressed components.
Assuntos
Proteínas de Bactérias , Campylobacter jejuni , Flagelos , Regulação Bacteriana da Expressão Gênica , RNA Bacteriano , Pequeno RNA não Traduzido , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Flagelos/genética , Flagelos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Flagelina/metabolismo , Flagelina/genética , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Ribonuclease III/metabolismo , Ribonuclease III/genéticaRESUMO
The success of bacterial pathogens depends on the coordinated expression of virulence determinants. Regulatory circuits that drive pathogenesis are complex, multilayered, and incompletely understood. Here, we reveal that alterations in tRNA modifications define pathogenic phenotypes in the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that the enzymatic activity of GidA leads to the introduction of a carboxymethylaminomethyl modification in selected tRNAs. Modifications at the wobble uridine base (cmnm5U34) of the anticodon drives translation of transcripts containing rare codons. Specifically, in P. aeruginosa the presence of GidA-dependent tRNA modifications modulates expression of genes encoding virulence regulators, leading to a cellular proteomic shift toward pathogenic and well-adapted physiological states. Our approach of profiling the consequences of chemical tRNA modifications is general in concept. It provides a paradigm of how environmentally driven tRNA modifications govern gene expression programs and regulate phenotypic outcomes responsible for bacterial adaption to challenging habitats prevailing in the host niche.
Assuntos
Proteômica , Pseudomonas aeruginosa , Virulência/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Anticódon , Bactérias/metabolismoRESUMO
The soil-dwelling plant symbiont Sinorhizobium meliloti is a major model organism of Alphaproteobacteria. Despite numerous detailed OMICS studies, information about small open reading frame (sORF)-encoded proteins (SEPs) is largely missing, because sORFs are poorly annotated and SEPs are hard to detect experimentally. However, given that SEPs can fulfill important functions, identification of translated sORFs is critical for analyzing their roles in bacterial physiology. Ribosome profiling (Ribo-seq) can detect translated sORFs with high sensitivity, but is not yet routinely applied to bacteria because it must be adapted for each species. Here, we established a Ribo-seq procedure for S. meliloti 2011 based on RNase I digestion and detected translation for 60% of the annotated coding sequences during growth in minimal medium. Using ORF prediction tools based on Ribo-seq data, subsequent filtering, and manual curation, the translation of 37 non-annotated sORFs with ≤ 70 amino acids was predicted with confidence. The Ribo-seq data were supplemented by mass spectrometry (MS) analyses from three sample preparation approaches and two integrated proteogenomic search database (iPtgxDB) types. Searches against standard and 20-fold smaller Ribo-seq data-informed custom iPtgxDBs confirmed 47 annotated SEPs and identified 11 additional novel SEPs. Epitope tagging and Western blot analysis confirmed the translation of 15 out of 20 SEPs selected from the translatome map. Overall, by combining MS and Ribo-seq approaches, the small proteome of S. meliloti was substantially expanded by 48 novel SEPs. Several of them are part of predicted operons and/or are conserved from Rhizobiaceae to Bacteria, suggesting important physiological functions.
RESUMO
In contrast to extensively studied prokaryotic 'small' transcriptomes (encompassing all small noncoding RNAs), small proteomes (here defined as including proteins ≤70 aa) are only now entering the limelight. The absence of a complete small protein catalogue in most prokaryotes precludes our understanding of how these molecules affect physiology. So far, archaeal genomes have not yet been analyzed broadly with a dedicated focus on small proteins. Here, we present a combinatorial approach, integrating experimental data from small protein-optimized mass spectrometry (MS) and ribosome profiling (Ribo-seq), to generate a high confidence inventory of small proteins in the model archaeon Haloferax volcanii. We demonstrate by MS and Ribo-seq that 67% of the 317 annotated small open reading frames (sORFs) are translated under standard growth conditions. Furthermore, annotation-independent analysis of Ribo-seq data showed ribosomal engagement for 47 novel sORFs in intergenic regions. A total of seven of these were also detected by proteomics, in addition to an eighth novel small protein solely identified by MS. We also provide independent experimental evidence in vivo for the translation of 12 sORFs (annotated and novel) using epitope tagging and western blotting, underlining the validity of our identification scheme. Several novel sORFs are conserved in Haloferax species and might have important functions. Based on our findings, we conclude that the small proteome of H. volcanii is larger than previously appreciated, and that combining MS with Ribo-seq is a powerful approach for the discovery of novel small protein coding genes in archaea.
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Capturing an individual cell's transcriptional history is a challenge exacerbated by the functional heterogeneity of cellular communities. Here, we leverage reprogrammed tracrRNAs (Rptrs) to record selected cellular transcripts as stored DNA edits in single living bacterial cells. Rptrs are designed to base pair with sensed transcripts, converting them into guide RNAs. The guide RNAs then direct a Cas9 base editor to target an introduced DNA target. The extent of base editing can then be read in the future by sequencing. We use this approach, called TIGER (transcribed RNAs inferred by genetically encoded records), to record heterologous and endogenous transcripts in individual bacterial cells. TIGER can quantify relative expression, distinguish single-nucleotide differences, record multiple transcripts simultaneously and read out single-cell phenomena. We further apply TIGER to record metabolic bet hedging and antibiotic resistance mobilization in Escherichia coli as well as host cell invasion by Salmonella. Through RNA recording, TIGER connects current cellular states with past transcriptional states to decipher complex cellular responses in single cells.
Assuntos
Sistemas CRISPR-Cas , RNA , RNA/genética , RNA Guia de Sistemas CRISPR-Cas , Bactérias/genética , Bactérias/metabolismo , DNA/genética , Edição de Genes , RNA Bacteriano/genéticaRESUMO
CRISPR-Cas systems store fragments of foreign DNA, called spacers, as immunological recordings used to combat future infections. Of the many spacers stored in a CRISPR array, the most recent are known to be prioritized for immune defence. However, the underlying mechanism remains unclear. Here we show that the leader region upstream of CRISPR arrays in CRISPR-Cas9 systems enhances CRISPR RNA (crRNA) processing from the newest spacer, prioritizing defence against the matching invader. Using the CRISPR-Cas9 system from Streptococcus pyogenes as a model, we found that the transcribed leader interacts with the conserved repeats bordering the newest spacer. The resulting interaction promotes transactivating crRNA (tracrRNA) hybridization with the second of the two repeats, accelerating crRNA processing. Accordingly, disruption of this structure reduces the abundance of the associated crRNA and immune defence against targeted plasmids and bacteriophages. Beyond the S. pyogenes system, bioinformatics analyses revealed that leader-repeat structures appear across CRISPR-Cas9 systems. CRISPR-Cas systems thus possess an RNA-based mechanism to prioritize defence against the most recently encountered invaders.
Assuntos
Bacteriófagos , Proteínas Associadas a CRISPR , Bacteriófagos/genética , Bacteriófagos/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , RNA/genética , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismoRESUMO
Small proteins encoded by short open reading frames (ORFs) with 50 codons or fewer are emerging as an important class of cellular macromolecules in diverse organisms. However, they often evade detection by proteomics or in silico methods. Ribosome profiling (Ribo-seq) has revealed widespread translation in genomic regions previously thought to be non-coding, driving the development of ORF detection tools using Ribo-seq data. However, only a handful of tools have been designed for bacteria, and these have not yet been systematically compared. Here, we aimed to identify tools that use Ribo-seq data to correctly determine the translational status of annotated bacterial ORFs and also discover novel translated regions with high sensitivity. To this end, we generated a large set of annotated ORFs from four diverse bacterial organisms, manually labeled for their translation status based on Ribo-seq data, which are available for future benchmarking studies. This set was used to investigate the predictive performance of seven Ribo-seq-based ORF detection tools (REPARATION_blast, DeepRibo, Ribo-TISH, PRICE, smORFer, ribotricer and SPECtre), as well as IRSOM, which uses coding potential and RNA-seq coverage only. DeepRibo and REPARATION_blast robustly predicted translated ORFs, including sORFs, with no significant difference for ORFs in close proximity to other genes versus stand-alone genes. However, no tool predicted a set of novel, experimentally verified sORFs with high sensitivity. Start codon predictions with smORFer show the value of initiation site profiling data to further improve the sensitivity of ORF prediction tools in bacteria. Overall, we find that bacterial tools perform well for sORF detection, although there is potential for improving their performance, applicability, usability and reproducibility.
Assuntos
Benchmarking , Ribossomos , Bactérias/genética , Fases de Leitura Aberta , Reprodutibilidade dos Testes , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Bacterial small RNAs (sRNAs) are widespread post-transcriptional regulators that control bacterial stress responses and virulence. Nevertheless, little is known about how they arise and evolve. Homologs can be difficult to identify beyond the strain level using sequence-based approaches, and similar functionalities can arise by convergent evolution. Here, we found that the virulence-associated CJnc190 sRNA of the foodborne pathogen Campylobacter jejuni resembles the RepG sRNA from the gastric pathogen Helicobacter pylori. However, while both sRNAs bind G-rich sites in their target mRNAs using a C/U-rich loop, they largely differ in their biogenesis. RepG is transcribed from a stand-alone gene and does not require processing, whereas CJnc190 is transcribed from two promoters as precursors that are processed by RNase III and also has a cis-encoded antagonist, CJnc180. By comparing CJnc190 homologs in diverse Campylobacter species, we show that RNase III-dependent processing of CJnc190 appears to be a conserved feature even outside of C. jejuni. We also demonstrate the CJnc180 antisense partner is expressed in C. coli, yet here might be derived from the 3'UTR (untranslated region) of an upstream flagella-related gene. Our analysis of G-tract targeting sRNAs in Epsilonproteobacteria demonstrates that similar sRNAs can have markedly different biogenesis pathways.
Assuntos
Infecções por Campylobacter/microbiologia , Campylobacter jejuni/genética , Epsilonproteobacteria/genética , Infecções por Helicobacter/microbiologia , Helicobacter pylori/genética , Pequeno RNA não Traduzido/genética , Regiões 3' não Traduzidas/genética , Campylobacter jejuni/patogenicidade , Epsilonproteobacteria/patogenicidade , Flagelos/genética , Helicobacter pylori/patogenicidade , Regiões Promotoras Genéticas/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Ribonuclease III/genética , VirulênciaRESUMO
Small proteins encoded by open reading frames (ORFs) shorter than 50 codons (small ORFs [sORFs]) are often overlooked by annotation engines and are difficult to characterize using traditional biochemical techniques. Ribosome profiling has tremendous potential to empirically improve the annotations of prokaryotic genomes. Recent improvements in ribosome profiling methods for bacterial model organisms have revealed many new sORFs in well-characterized genomes. Antibiotics that trap ribosomes just after initiation have played a key role in these developments by allowing the unambiguous identification of the start codons (and, hence, the reading frame) for novel ORFs. Here, we describe these new methods and highlight critical controls and considerations for adapting ribosome profiling to different prokaryotic species.
Assuntos
Antibacterianos/farmacologia , Bactérias/metabolismo , Fases de Leitura Aberta , Ribossomos , Bactérias/genética , Códon , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Iniciação Traducional da Cadeia Peptídica , Terminação Traducional da Cadeia Peptídica , RNA Bacteriano , RNA RibossômicoRESUMO
CRISPR-Cas systems consist of a complex ribonucleoprotein (RNP) machinery encoded in prokaryotic genomes to confer adaptive immunity against foreign mobile genetic elements. Of these, especially the class 2, Type II CRISPR-Cas9 RNA-guided systems with single protein effector modules have recently received much attention for their application as programmable DNA scissors that can be used for genome editing in eukaryotes. While many studies have concentrated their efforts on improving RNA-mediated DNA targeting with these Type II systems, little is known about the factors that modulate processing or binding of the CRISPR RNA (crRNA) guides and the trans-activating tracrRNA to the nuclease protein Cas9, and whether Cas9 can also potentially interact with other endogenous RNAs encoded within the host genome. Here, we describe RIP-seq as a method to globally identify the direct RNA binding partners of CRISPR-Cas RNPs using the Cas9 nuclease as an example. RIP-seq combines co-immunoprecipitation (coIP) of an epitope-tagged Cas9 followed by isolation and deep sequencing analysis of its co-purified bound RNAs. This method can not only be used to study interactions of Cas9 with its known interaction partners, crRNAs and tracrRNA in native systems, but also to reveal potential additional RNA substrates of Cas9. For example, in RIP-seq analysis of Cas9 from the foodborne pathogen Campylobacter jejuni (CjeCas9), we recently identified several endogenous RNAs bound to CjeCas9 RNP in a crRNA-dependent manner, leading to the discovery of PAM-independent RNA cleavage activity of CjeCas9 as well as non-canonical crRNAs. RIP-seq can be easily adapted to any other effector RNP of choice from other CRISPR-Cas systems, allowing for the identification of target RNAs. Deciphering novel RNA-protein interactions for CRISPR-Cas proteins within host bacterial genomes will lead to a better understanding of the molecular mechanisms and functions of these systems and enable us to use the in vivo identified interaction rules as design principles for nucleic acid-targeting applications, fitted to each nuclease of interest.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sistemas CRISPR-Cas/genética , DNA , Endonucleases , RNA/genética , RNA Guia de Cinetoplastídeos/genéticaRESUMO
CRISPR-Cas systems provide bacteria with adaptive immunity against phages and plasmids; however, pathways regulating their activity are not well defined. We recently developed a high-throughput genome-wide method (SorTn-seq) and used this to uncover CRISPR-Cas regulators. Here, we demonstrate that the widespread Rsm/Csr pathway regulates the expression of multiple CRISPR-Cas systems in Serratia (type I-E, I-F and III-A). The main pathway component, RsmA (CsrA), is an RNA-binding post-transcriptional regulator of carbon utilisation, virulence and motility. RsmA binds cas mRNAs and suppresses type I and III CRISPR-Cas interference in addition to adaptation by type I systems. Coregulation of CRISPR-Cas and flagella by the Rsm pathway allows modulation of adaptive immunity when changes in receptor availability would alter susceptibility to flagella-tropic phages. Furthermore, we show that Rsm controls CRISPR-Cas in other genera, suggesting conservation of this regulatory strategy. Finally, we identify genes encoding RsmA homologues in phages, which have the potential to manipulate the physiology of host bacteria and might provide an anti-CRISPR activity.
Assuntos
Proteínas de Bactérias/genética , Sistemas CRISPR-Cas/genética , Serratia/genética , Transdução de Sinais/genética , Imunidade Adaptativa/genética , Bacteriófagos/genética , Bacteriófagos/patogenicidade , Flagelos/genética , Regulação Bacteriana da Expressão Gênica/genética , Plasmídeos/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA , Proteínas Repressoras , Virulência/genéticaRESUMO
The small, regulatory RNA RepG (Regulator of polymeric G-repeats) regulates the expression of the chemotaxis receptor TlpB in Helicobacter pylori by targeting a variable G-repeat in the tlpB mRNA leader. Here, we show that RepG additionally controls lipopolysaccharide (LPS) phase variation by also modulating the expression of a gene (hp0102) that is co-transcribed with tlpB. The hp0102 gene encodes a glycosyltransferase required for LPS O-chain biosynthesis and in vivo colonization of the mouse stomach. The G-repeat length defines a gradual (rather than ON/OFF) control of LPS biosynthesis by RepG, and leads to gradual resistance to a membrane-targeting antibiotic. Thus, RepG-mediated modulation of LPS structure might impact host immune recognition and antibiotic sensitivity, thereby helping H. pylori to adapt and persist in the host.
Assuntos
Farmacorresistência Bacteriana , Helicobacter pylori/fisiologia , Lipopolissacarídeos/biossíntese , RNA Bacteriano/metabolismo , Pequeno RNA não Traduzido/metabolismo , Regiões 5' não Traduzidas , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções por Helicobacter/microbiologia , Helicobacter pylori/efeitos dos fármacos , Lipopolissacarídeos/química , Camundongos , Antígenos O/biossíntese , Antígenos O/química , Polimixina B/farmacologia , RNA Bacteriano/genética , Pequeno RNA não Traduzido/genética , Sequências Repetitivas de Ácido Nucleico , Estresse Salino , Estômago/microbiologiaRESUMO
CRISPR-Cas systems recognize foreign genetic material using CRISPR RNAs (crRNAs). In type II systems, a trans-activating crRNA (tracrRNA) hybridizes to crRNAs to drive their processing and utilization by Cas9. While analyzing Cas9-RNA complexes from Campylobacter jejuni, we discovered tracrRNA hybridizing to cellular RNAs, leading to formation of "noncanonical" crRNAs capable of guiding DNA targeting by Cas9. Our discovery inspired the engineering of reprogrammed tracrRNAs that link the presence of any RNA of interest to DNA targeting with different Cas9 orthologs. This capability became the basis for a multiplexable diagnostic platform termed LEOPARD (leveraging engineered tracrRNAs and on-target DNAs for parallel RNA detection). LEOPARD allowed simultaneous detection of RNAs from different viruses in one test and distinguished severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its D614G (Asp614âGly) variant with single-base resolution in patient samples.
Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , RNA Guia de Cinetoplastídeos/genética , RNA Viral/análise , RNA/análise , RNA/genética , SARS-CoV-2/genética , Sequência de Bases , COVID-19/diagnóstico , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Sistemas CRISPR-Cas , Campylobacter jejuni , Humanos , Hibridização de Ácido Nucleico , RNA Bacteriano/genética , RNA Mensageiro/genética , RNA Viral/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
MOTIVATION: Ribosome profiling (Ribo-seq) is a powerful approach based on deep sequencing of cDNA libraries generated from ribosome-protected RNA fragments to explore the translatome of a cell, and is especially useful for the detection of small proteins (50-100 amino acids) that are recalcitrant to many standard biochemical and in silico approaches. While pipelines are available to analyze Ribo-seq data, none are designed explicitly for the automatic processing and analysis of data from bacteria, nor are they focused on the discovery of unannotated open reading frames (ORFs). RESULTS: We present HRIBO (High-throughput annotation by Ribo-seq), a workflow to enable reproducible and high-throughput analysis of bacterial Ribo-seq data. The workflow performs all required pre-processing and quality control steps. Importantly, HRIBO outputs annotation-independent ORF predictions based on two complementary bacteria-focused tools, and integrates them with additional feature information and expression values. This facilitates the rapid and high-confidence discovery of novel ORFs and their prioritization for functional characterization. AVAILABILITY AND IMPLEMENTATION: HRIBO is a free and open source project available under the GPL-3 license at: https://github.com/RickGelhausen/HRIBO.
Assuntos
Biossíntese de Proteínas , Ribossomos , Animais , Bactérias/genética , Sequenciamento de Nucleotídeos em Larga Escala , Cavalos , Fases de Leitura Aberta , RNA Ribossômico , Ribossomos/genética , Ribossomos/metabolismoRESUMO
Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE-respiring representatives of the genus, uncovered the genetic inactivation of a two-component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low-level expression of the TCS operon in fumarate-adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR-family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter-binding assays. The RR bound a cis-regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the -35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine-tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide-respiring bacteria.
Assuntos
Campylobacteraceae/genética , Campylobacteraceae/metabolismo , Oxirredutases/genética , Tetracloroetileno/metabolismo , Sequência de Bases , Biologia Computacional/métodos , Ensaio de Desvio de Mobilidade Eletroforética , Genoma Bacteriano/genética , Genômica/métodos , Regiões Promotoras Genéticas/genética , Alinhamento de Sequência , Transcriptoma/genéticaRESUMO
Many bacterial pathogens regulate their virulence genes via phase variation, whereby length-variable simple sequence repeats control the transcription or coding potential of those genes. Here, we have exploited this relationship between DNA structure and physiological function to discover a globally acting small RNA (sRNA) regulator of virulence in the gastric pathogen Helicobacter pylori. Our study reports the first sRNA whose expression is affected by a variable thymine (T) stretch in its promoter. We show the sRNA post-transcriptionally represses multiple major pathogenicity factors of H. pylori, including CagA and VacA, by base pairing to their mRNAs. We further demonstrate transcription of the sRNA is regulated by the nickel-responsive transcriptional regulator NikR (thus named NikS for nickel-regulated sRNA), thereby linking virulence factor regulation to nickel concentrations. Using in-vitro infection experiments, we demonstrate NikS affects host cell internalization and epithelial barrier disruption. Together, our results show NikS is a phase-variable, post-transcriptional global regulator of virulence properties in H. pylori.
Assuntos
Helicobacter pylori/genética , Helicobacter pylori/patogenicidade , RNA Bacteriano/genética , Sequências Repetitivas de Ácido Nucleico/genética , Fatores de Virulência/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Contagem de Colônia Microbiana , Endocitose/efeitos dos fármacos , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Helicobacter pylori/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Níquel/farmacologia , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Campylobacter spp. are one of the most important food-borne pathogens, which are quite susceptible to environmental or technological stressors compared to other zoonotic bacteria. This might be due to the lack of many stress response mechanisms described in other bacteria. Nevertheless, Campylobacter is able to survive in the environment and food products. Although some aspects of the heat stress response in Campylobacter jejuni are already known, information about the stress response in other Campylobacter species are still scarce. In this study, the stress response of Campylobacter coli and Campylobacter lari to elevated temperatures (46°C) was investigated by survival assays and whole transcriptome analysis. None of the strains survived at 46°C for more than 8 h and approximately 20% of the genes of C. coli RM2228 and C. lari RM2100 were differentially expressed. The transcriptomic profiles showed enhanced gene expression of several chaperones like dnaK, groES, groEL, and clpB in both strains, indicating a general involvement in the heat stress response within the Campylobacter species. However, the pronounced differences in the expression pattern between C. coli and C. lari suggest that stress response mechanisms described for one Campylobacter species might be not necessarily transferable to other Campylobacter species.
RESUMO
The Gram-negative Epsilonproteobacterium Campylobacter jejuni is currently the most prevalent bacterial foodborne pathogen. Like for many other human pathogens, infection studies with C. jejuni mainly employ artificial animal or cell culture models that can be limited in their ability to reflect the in-vivo environment within the human host. Here, we report the development and application of a human three-dimensional (3D) infection model based on tissue engineering to study host-pathogen interactions. Our intestinal 3D tissue model is built on a decellularized extracellular matrix scaffold, which is reseeded with human Caco-2 cells. Dynamic culture conditions enable the formation of a polarized mucosal epithelial barrier reminiscent of the 3D microarchitecture of the human small intestine. Infection with C. jejuni demonstrates that the 3D tissue model can reveal isolate-dependent colonization and barrier disruption phenotypes accompanied by perturbed localization of cell-cell junctions. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D model deviated from those obtained with 2D-monolayers, but recapitulated phenotypes previously observed in animal models. Moreover, we demonstrate the involvement of a small regulatory RNA pair, CJnc180/190, during infections and observe different phenotypes of CJnc180/190 mutant strains in 2D vs. 3D infection models. Hereby, the CJnc190 sRNA exerts its pathogenic influence, at least in part, via repression of PtmG, which is involved in flagellin modification. Our results suggest that the Caco-2 cell-based 3D tissue model is a valuable and biologically relevant tool between in-vitro and in-vivo infection models to study virulence of C. jejuni and other gastrointestinal pathogens.
Assuntos
Campylobacter jejuni/genética , Interações Hospedeiro-Patógeno/fisiologia , Modelos Biológicos , Células CACO-2 , Infecções por Campylobacter/microbiologia , Campylobacter jejuni/patogenicidade , Células Epiteliais/microbiologia , Matriz Extracelular/fisiologia , Humanos , Mucosa Intestinal/microbiologia , Intestino Delgado/patologia , Intestinos/microbiologia , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Alicerces Teciduais , VirulênciaRESUMO
Small proteins are an emerging class of gene products with diverse roles in bacterial physiology. However, a full understanding of their importance has been hampered by insufficient genome annotations and a lack of comprehensive characterization in microbes other than Escherichia coli. We have taken an integrative approach to accelerate the discovery of small proteins and their putative virulence-associated functions in Salmonella Typhimurium. We merged the annotated small proteome of Salmonella with new small proteins predicted with in silico and experimental approaches. We then exploited existing and newly generated global datasets that provide information on small open reading frame expression during infection of epithelial cells (dual RNA-seq), contribution to bacterial fitness inside macrophages (Transposon-directed insertion sequencing), and potential engagement in molecular interactions (Grad-seq). This integrative approach suggested a new role for the small protein MgrB beyond its known function in regulating PhoQ. We demonstrate a virulence and motility defect of a Salmonella ΔmgrB mutant and reveal an effect of MgrB in regulating the Salmonella transcriptome and proteome under infection-relevant conditions. Our study highlights the power of interpreting available 'omics' datasets with a focus on small proteins, and may serve as a blueprint for a data integration-based survey of small proteins in diverse bacteria.
RESUMO
BACKGROUND: We previously reported the identification of the aapA1/IsoA1 locus as part of a new family of toxin-antitoxin (TA) systems in the human pathogen Helicobacter pylori. AapA1 belongs to type I TA bacterial toxins, and both its mechanism of action towards the membrane and toxicity features are still unclear. METHODS: The biochemical characterization of the AapA1 toxic peptide was carried out using plasmid-borne expression and mutational approaches to follow its toxicity and localization. Biophysical properties of the AapA1 interaction with lipid membranes were studied by solution and solid-state NMR spectroscopy, plasmon waveguide resonance (PWR) and molecular modeling. RESULTS: We show that despite a low hydrophobic index, this toxin has a nanomolar affinity to the prokaryotic membrane. NMR spectroscopy reveals that the AapA1 toxin is structurally organized into three distinct domains: a positively charged disordered N-terminal domain (D), a single α-helix (H), and a basic C-terminal domain (R). The R domain interacts and destabilizes the membrane, while the H domain adopts a transmembrane conformation. These results were confirmed by alanine scanning of the minimal sequence required for toxicity. CONCLUSION: Our results have shown that specific amino acid residues along the H domain, as well as the R domain, are essential for the toxicity of the AapA1 toxin. GENERAL SIGNIFICANCE: Untangling and understanding the mechanism of action of small membrane-targeting toxins are difficult, but nevertheless contributes to a promising search and development of new antimicrobial drugs.