Global analysis of the RNA-RNA interactome in Acinetobacter baumannii AB5075 uncovers a small regulatory RNA repressing the virulence-related outer membrane protein CarO.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to characterize environmental adaptation, antibiotic resistance and pathogenicity and their genetic regulators to inform intervention strategies. Critical to adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. Forty sRNAs were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii. In-depth characterization uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including the transcript encoding outer membrane protein CarO. Aar initiates base-pairing with these mRNAs using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in multiple stress conditions suggesting a role in fine-tuning translation of the Aar-target molecules. Our study provides mechanistic insights into sRNA-mediated gene regulation in A. baumannii and represents a valuable resource for future RNA-centric research endeavours.

Salmonella enterica serovar Typhimurium ST313 sublineage 2.2 has emerged in Malawi with a characteristic gene expression signature and a fitness advantage.

Invasive non-typhoidal Salmonella (iNTS) disease is a serious bloodstream infection that targets immune-compromised individuals, and causes significant mortality in sub-Saharan Africa. Salmonella enterica serovar Typhimurium ST313 causes the majority of iNTS in Malawi. We performed an intensive comparative genomic analysis of 608 S. Typhimurium ST313 isolates dating between 1996 and 2018 from Blantyre, Malawi. We discovered that following the arrival of the well-characterized S. Typhimurium ST313 lineage 2 in 1999, two multidrug-resistant variants emerged in Malawi in 2006 and 2008, designated sublineages 2.2 and 2.3, respectively. The majority of S. Typhimurium isolates from human bloodstream infections in Malawi now belong to sublineages 2.2 or 2.3. To understand the emergence of the prevalent ST313 sublineage 2.2, we studied two representative strains, D23580 (lineage 2) and D37712 (sublineage 2.2). The chromosome of ST313 lineage 2 and sublineage 2.2 only differed by 29 SNPs/small indels and a 3 kb deletion of a Gifsy-2 prophage region including the sseI pseudogene. Lineage 2 and sublineage 2.2 had distinctive plasmid profiles. The transcriptome was investigated in 15 infection-relevant in vitro conditions and within macrophages. During growth in physiological conditions that do not usually trigger S. Typhimurium SPI2 gene expression, the SPI2 genes of D37712 were transcriptionally active. We identified down-regulation of flagellar genes in D37712 compared with D23580. Following phenotypic confirmation of transcriptomic differences, we discovered that sublineage 2.2 had increased fitness compared with lineage 2 during mixed growth in minimal media. We speculate that this competitive advantage is contributing to the emergence of sublineage 2.2 in Malawi.

Hierarchic regulation of a metabolic pathway: H-NS, CRP, and SsrB control myo-inositol utilization by Salmonella enterica.

IMPORTANCE: The capacity to utilize myo-inositol (MI) as sole carbon and energy source is widespread among bacteria, among them the intestinal pathogen S. Typhimurium. This study elucidates the complex and hierarchical regulation that underlies the utilization of MI by S. Typhimurium under substrate limitation. A total of seven regulatory factors have been identified so far, allowing the pathogen an environment-dependent, efficient, and fine-tuned regulation of a metabolic property that provides growth advantages in different environments.

Plasmid-encoded lactose metabolism and mobilized colistin resistance (mcr-9) genes in Salmonella enterica serovars isolated from dairy facilities in the 1980s.

Horizontal gene transfer by plasmids can confer metabolic capabilities that expand a host cell's niche. Yet, it is less understood whether the coalescence of specialized catabolic functions, antibiotic resistances and metal resistances on plasmids provides synergistic benefits. In this study, we report whole-genome assembly and phenotypic analysis of five Salmonella enterica strains isolated in the 1980s from milk powder in Munich, Germany. All strains exhibited the unusual phenotype of lactose-fermentation and encoded either of two variants of the lac operon. Surprisingly, all strains encoded the mobilized colistin resistance gene 9 (mcr-9), long before the first report of this gene in the literature. In two cases, the mcr-9 gene and the lac locus were linked within a large gene island that formed an IncHI2A-type plasmid in one strain but was chromosomally integrated in the other strain. In two other strains, the mcr-9 gene was found on a large IncHI1B/IncP-type plasmid, whereas the lac locus was encoded on a separate chromosomally integrated plasmidic island. The mcr-9 sequences were identical and genomic contexts could not explain the wide range of colistin resistances exhibited by the Salmonella strains. Nucleotide variants did explain phenotypic differences in motility and exopolysaccharide production. The observed linkage of mcr-9 to lactose metabolism, an array of heavy-metal detoxification systems, and other antibiotic resistance genes may reflect a coalescence of specialized phenotypes that improve the spread of colistin resistance in dairy facilities, much earlier than previously suspected.

IKK2/NF-κB Activation in Astrocytes Reduces amyloid ß Deposition: A Process Associated with Specific Microglia Polarization.

Alzheimer's disease (AD) is a common neurodegenerative disease that is accompanied by pronounced neuroinflammatory responses mainly characterized by marked microgliosis and astrogliosis. However, it remains open as to how different aspects of astrocytic and microglial activation affect disease progression. Previously, we found that microglia expansion in the spinal cord, initiated by IKK2/NF-κB activation in astrocytes, exhibits stage-dependent beneficial effects on the progression of amyotrophic lateral sclerosis. Here, we investigated the impact of NF-κB-initiated neuroinflammation on AD pathogenesis using the APP23 mouse model of AD in combination with conditional activation of IKK2/NF-κB signaling in astrocytes. We show that NF-κB activation in astrocytes triggers a distinct neuroinflammatory response characterized by striking astrogliosis as well as prominent microglial reactivity. Immunohistochemistry and Congo red staining revealed an overall reduction in the size and number of amyloid plaques in the cerebral cortex and hippocampus. Interestingly, isolated primary astrocytes and microglia cells exhibit specific marker gene profiles which, in the case of microglia, point to an enhanced plaque clearance capacity. In contrast, direct IKK2/NF-κB activation in microglia results in a pro-inflammatory polarization program. Our findings suggest that IKK2/NF-κB signaling in astrocytes may activate paracrine mechanisms acting on microglia function but also on APP processing in neurons.

Draft Genome Sequence and Annotation of Acinetobacter soli AS15, Isolated from an Irish Hospital.

We report the draft genome sequence of Acinetobacter soli AS15, which was isolated in 2018 from a rectal screen of a patient at St. Vincent's University Hospital (Dublin, Ireland). The draft genome sequence is 3,589,002 bp and was assembled into 82 contigs.

A window into lysogeny: revealing temperate phage biology with transcriptomics.

Prophages are integrated phage elements that are a pervasive feature of bacterial genomes. The fitness of bacteria is enhanced by prophages that confer beneficial functions such as virulence, stress tolerance or phage resistance, and these functions are encoded by 'accessory' or 'moron' loci. Whilst the majority of phage-encoded genes are repressed during lysogeny, accessory loci are often highly expressed. However, it is challenging to identify novel prophage accessory loci from DNA sequence data alone. Here, we use bacterial RNA-seq data to examine the transcriptional landscapes of five Salmonella prophages. We show that transcriptomic data can be used to heuristically enrich for prophage features that are highly expressed within bacterial cells and represent functionally important accessory loci. Using this approach, we identify a novel antisense RNA species in prophage BTP1, STnc6030, which mediates superinfection exclusion of phage BTP1. Bacterial transcriptomic datasets are a powerful tool to explore the molecular biology of temperate phages.

The diversity, evolution and ecology of Salmonella in venomous snakes.

BACKGROUND: Reptile-associated Salmonella bacteria are a major, but often neglected cause of both gastrointestinal and bloodstream infection in humans globally. The diversity of Salmonella enterica has not yet been determined in venomous snakes, however other ectothermic animals have been reported to carry a broad range of Salmonella bacteria. We investigated the prevalence and diversity of Salmonella in a collection of venomous snakes and non-venomous reptiles. METHODOLOGY/PRINCIPLE FINDINGS: We used a combination of selective enrichment techniques to establish a unique dataset of reptilian isolates to study Salmonella enterica species-level evolution and ecology and used whole-genome sequencing to investigate the relatedness of phylogenetic groups. We observed that 91% of venomous snakes carried Salmonella, and found that a diverse range of serovars (n = 58) were carried by reptiles. The Salmonella serovars belonged to four of the six Salmonella enterica subspecies: diarizonae, enterica, houtanae and salamae. Subspecies enterica isolates were distributed among two distinct phylogenetic clusters, previously described as clade A (52%) and clade B (48%). We identified metabolic differences between S. diarizonae, S. enterica clade A and clade B involving growth on lactose, tartaric acid, dulcitol, myo-inositol and allantoin. SIGNIFICANCE: We present the first whole genome-based comparative study of the Salmonella bacteria that colonise venomous and non-venomous reptiles and shed new light on Salmonella evolution. Venomous snakes examined in this study carried a broad range of Salmonella, including serovars which have been associated with disease in humans such as S. Enteritidis. The findings raise the possibility that venomous snakes could be a reservoir for Salmonella serovars associated with human salmonellosis.

Draft Genome Sequence and Annotation of Acinetobacter junii MHI21018, Isolated from Bovine Colostrum.

We report here the draft genome sequence of Acinetobacter junii MHI21018, isolated in 2009 from bovine colostrum. The draft genome sequence is composed of 3,267,995 bp, has a GC content of 38.54%, and was assembled into 114 contigs (contig size, >500 bp) with an N 50 value of 72,566 bp.

Adding function to the genome of African Salmonella Typhimurium ST313 strain D23580.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

The small RNA RssR regulates myo-inositol degradation by Salmonella enterica.

Small noncoding RNAs (sRNAs) with putative regulatory functions in gene expression have been identified in the enteropathogen Salmonella enterica serovar Typhimurium (S. Typhimurium). Two sRNAs are encoded by the genomic island GEI4417/4436 responsible for myo-inositol (MI) degradation, suggesting a role in the regulation of this metabolic pathway. We show that a lack of the sRNA STnc2160, termed RssR, results in a severe growth defect in minimal medium (MM) with MI. In contrast, the second sRNA STnc1740 was induced in the presence of glucose, and its overexpression slightly attenuated growth in the presence of MI. Constitutive expression of RssR led to an increased stability of the reiD mRNA, which encodes an activator of iol genes involved in MI utilization, via interaction with its 5'-UTR. SsrB, a response regulator contributing to the virulence properties of salmonellae, activated rssR transcription by binding the sRNA promoter. In addition, the absence of the RNA chaperone Hfq resulted in strongly decreased levels of RssR, attenuated S. Typhimurium growth with MI, and reduced expression of several iol genes required for MI degradation. Considered together, the extrinsic RssR allows fine regulation of cellular ReiD levels and thus of MI degradation by acting on the reiD mRNA stability.

The primary transcriptome, small RNAs and regulation of antimicrobial resistance in Acinetobacter baumannii ATCC 17978.

We present the first high-resolution determination of transcriptome architecture in the priority pathogen Acinetobacter baumannii. Pooled RNA from 16 laboratory conditions was used for differential RNA-seq (dRNA-seq) to identify 3731 transcriptional start sites (TSS) and 110 small RNAs, including the first identification in A. baumannii of sRNAs encoded at the 3' end of coding genes. Most sRNAs were conserved among sequenced A. baumannii genomes, but were only weakly conserved or absent in other Acinetobacter species. Single nucleotide mapping of TSS enabled prediction of -10 and -35 RNA polymerase binding sites and revealed an unprecedented base preference at position +2 that hints at an unrecognized transcriptional regulatory mechanism. To apply functional genomics to the problem of antimicrobial resistance, we dissected the transcriptional regulation of the drug efflux pump responsible for chloramphenicol resistance, craA. The two craA promoters were both down-regulated >1000-fold when cells were shifted to nutrient limited medium. This conditional down-regulation of craA expression renders cells sensitive to chloramphenicol, a highly effective antibiotic for the treatment of multidrug resistant infections. An online interface that facilitates open data access and visualization is provided as 'AcinetoCom' (http://bioinf.gen.tcd.ie/acinetocom/).

Role of a single noncoding nucleotide in the evolution of an epidemic African clade of Salmonella.

Salmonella enterica serovar Typhimurium ST313 is a relatively newly emerged sequence type that is causing a devastating epidemic of bloodstream infections across sub-Saharan Africa. Analysis of hundreds of Salmonella genomes has revealed that ST313 is closely related to the ST19 group of S Typhimurium that cause gastroenteritis across the world. The core genomes of ST313 and ST19 vary by only ∼1,000 SNPs. We hypothesized that the phenotypic differences that distinguish African Salmonella from ST19 are caused by certain SNPs that directly modulate the transcription of virulence genes. Here we identified 3,597 transcriptional start sites of the ST313 strain D23580, and searched for a gene-expression signature linked to pathogenesis of Salmonella We identified a SNP in the promoter of the pgtE gene that caused high expression of the PgtE virulence factor in African S. Typhimurium, increased the degradation of the factor B component of human complement, contributed to serum resistance, and modulated virulence in the chicken infection model. We propose that high levels of PgtE expression by African S Typhimurium ST313 promote bacterial survival and dissemination during human infection. Our finding of a functional role for an extragenic SNP shows that approaches used to deduce the evolution of virulence in bacterial pathogens should include a focus on noncoding regions of the genome.

Broad-scale redistribution of mRNA abundance and transcriptional machinery in response to growth rate in Salmonella enterica serovar Typhimurium.

We have investigated the connection between the four-dimensional architecture of the bacterial nucleoid and the organism's global gene expression programme. By localizing the transcription machinery and the transcriptional outputs across the genome of the model bacterium Salmonella enterica serovar Typhimurium at different stages of the growth cycle, a surprising disconnection between gene dosage and transcriptional output was revealed. During exponential growth, gene output occurred chiefly in the Ori (origin), Ter (terminus) and NSL (non-structured left) domains, whereas the Left macrodomain remained transcriptionally quiescent at all stages of growth. The apparently high transcriptional output in Ter was correlated with an enhanced stability of the RNA expressed there during exponential growth, suggesting that longer mRNA half-lives compensate for low gene dosage. During exponential growth, RNA polymerase (RNAP) was detected everywhere, whereas in stationary phase cells, RNAP was concentrated in the Ter macrodomain. The alternative sigma factors RpoE, RpoH and RpoN were not required to drive transcription in these growth conditions, consistent with their observed binding to regions away from RNAP and regions of active transcription. Specifically, these alternative sigma factors were found in the Ter macrodomain during exponential growth, whereas they were localized at the Ori macrodomain in stationary phase.

Small RNA, Big Effect: Control of Flagellin Production.

Many bacteria move in their environment using a remarkable, rotating nanomachine - the flagellum. In a recent publication, Choi et al. report a new addition to the group of flagellar regulators, a trans-acting small RNA (sRNA).

If it transcribes, we can sequence it: mining the complexities of host-pathogen-environment interactions using RNA-seq.

Host-pathogen interactions are exceedingly complex because they involve multiple host tissues, often occur in the context of normal microflora, and can span diverse microenvironments. Although decades of gene expression studies have provided detailed insights into infection processes, technical challenges have restricted experiments to single pathogenic species or host tissues. RNA-sequencing (RNA-seq) has revolutionized the study of gene expression because in addition to quantifying transcriptional output, it allows detection and characterization of all transcripts in a genome. Here, we review how refined approaches to RNA-seq are used to map the transcriptional networks that control host-pathogen interactions. These enhanced techniques include dRNA-seq and term-seq for the fine-scale mapping of transcriptional start and termination sites, and dual RNA-seq for simultaneous sequencing of host and bacterial pathogen transcriptomes. Dual RNA-seq experiments are currently limited to in vitro infection systems that do not fully reflect the complexities of the in vivo environment, thus a challenge is to develop in vivo model systems and experimental approaches that address the biological heterogeneity of host environments, followed by the integration of RNA-seq with other genome-scale datasets to identify the transcriptional networks that mediate host-pathogen interactions.

Whole-Genome Sequence and Annotation of Salmonella enterica subsp. enterica Serovar Enteritidis Phage Type 8 Strain EN1660.

The genome of Salmonella enterica subspecies enterica serovar Enteritidis phage type 8 strain EN1660, isolated from an outbreak in Thunder Bay, Canada, was sequenced to 46-fold coverage using an Illumina MiSeq with 300-bp paired-end sequencing chemistry to produce 28 contigs with an N50 value of 490,721 bp.

Genetic Regulation of Virulence and Antibiotic Resistance in Acinetobacter baumannii.

Multidrug resistant microorganisms are forecast to become the single biggest challenge to medical care in the 21st century. Over the last decades, members of the genus Acinetobacter have emerged as bacterial opportunistic pathogens, in particular as challenging nosocomial pathogens because of the rapid evolution of antimicrobial resistances. Although we lack fundamental biological insight into virulence mechanisms, an increasing number of researchers are working to identify virulence factors and to study antibiotic resistance. Here, we review current knowledge regarding the regulation of virulence genes and antibiotic resistance in Acinetobacter baumannii. A survey of the two-component systems AdeRS, BaeSR, GacSA and PmrAB explains how each contributes to antibiotic resistance and virulence gene expression, while BfmRS regulates cell envelope structures important for pathogen persistence. A. baumannii uses the transcription factors Fur and Zur to sense iron or zinc depletion and upregulate genes for metal scavenging as a critical survival tool in an animal host. Quorum sensing, nucleoid-associated proteins, and non-classical transcription factors such as AtfA and small regulatory RNAs are discussed in the context of virulence and antibiotic resistance.

The Impact of 18 Ancestral and Horizontally-Acquired Regulatory Proteins upon the Transcriptome and sRNA Landscape of Salmonella enterica serovar Typhimurium.

We know a great deal about the genes used by the model pathogen Salmonella enterica serovar Typhimurium to cause disease, but less about global gene regulation. New tools for studying transcripts at the single nucleotide level now offer an unparalleled opportunity to understand the bacterial transcriptome, and expression of the small RNAs (sRNA) and coding genes responsible for the establishment of infection. Here, we define the transcriptomes of 18 mutants lacking virulence-related global regulatory systems that modulate the expression of the SPI1 and SPI2 Type 3 secretion systems of S. Typhimurium strain 4/74. Using infection-relevant growth conditions, we identified a total of 1257 coding genes that are controlled by one or more regulatory system, including a sub-class of genes that reflect a new level of cross-talk between SPI1 and SPI2. We directly compared the roles played by the major transcriptional regulators in the expression of sRNAs, and discovered that the RpoS (σ38) sigma factor modulates the expression of 23% of sRNAs, many more than other regulatory systems. The impact of the RNA chaperone Hfq upon the steady state levels of 280 sRNA transcripts is described, and we found 13 sRNAs that are co-regulated with SPI1 and SPI2 virulence genes. We report the first example of an sRNA, STnc1480, that is subject to silencing by H-NS and subsequent counter-silencing by PhoP and SlyA. The data for these 18 regulatory systems is now available to the bacterial research community in a user-friendly online resource, SalComRegulon.

RNA-seq Brings New Insights to the Intra-Macrophage Transcriptome of Salmonella Typhimurium.

Salmonella enterica serovar Typhimurium is arguably the world's best-understood bacterial pathogen. However, crucial details about the genetic programs used by the bacterium to survive and replicate in macrophages have remained obscure because of the challenge of studying gene expression of intracellular pathogens during infection. Here, we report the use of deep sequencing (RNA-seq) to reveal the transcriptional architecture and gene activity of Salmonella during infection of murine macrophages, providing new insights into the strategies used by the pathogen to survive in a bactericidal immune cell. We characterized 3583 transcriptional start sites that are active within macrophages, and highlight 11 of these as candidates for the delivery of heterologous antigens from Salmonella vaccine strains. A majority (88%) of the 280 S. Typhimurium sRNAs were expressed inside macrophages, and SPI13 and SPI2 were the most highly expressed pathogenicity islands. We identified 31 S. Typhimurium genes that were strongly up-regulated inside macrophages but expressed at very low levels during in vitro growth. The SalComMac online resource allows the visualisation of every transcript expressed during bacterial replication within mammalian cells. This primary transcriptome of intra-macrophage S.-Typhimurium describes the transcriptional start sites and the transcripts responsible for virulence traits, and catalogues the sRNAs that may play a role in the regulation of gene expression during infection.