Hfq-licensed RNA-RNA interactome in Pseudomonas aeruginosa reveals a keystone sRNA.

The RNA chaperone Hfq plays important regulatory roles in many bacteria by facilitating the base pairing between small RNAs (sRNAs) and their cognate mRNA targets. In the gram-negative opportunistic pathogen Pseudomonas aeruginosa, over a hundred putative sRNAs have been identified but for most, their regulatory targets remained unknown. Using RIL-seq with Hfq in P. aeruginosa, we identified the mRNA targets for dozens of previously known and unknown sRNAs. Strikingly, hundreds of the RNA-RNA interactions we discovered involved PhrS. This sRNA was thought to mediate its effects by pairing with a single target mRNA and regulating the abundance of the transcription regulator MvfR required for the synthesis of the quorum sensing signal PQS. We present evidence that PhrS controls many transcripts by pairing with them directly and employs a two-tiered mechanism for governing PQS synthesis that involves control of an additional transcription regulator called AntR. Our findings in P. aeruginosa expand the repertoire of targets for previously known sRNAs, reveal potential regulatory targets for previously unknown sRNAs, and suggest that PhrS may be a keystone sRNA with the ability to pair with an unusually large number of transcripts in this organism.

Sequestration of a dual function DNA-binding protein by Vibrio cholerae CRP.

Although the mechanism by which the cyclic AMP receptor protein (CRP) regulates global gene transcription has been intensively studied for decades, new discoveries remain to be made. Here, we report that, during rapid growth, CRP associates with both the well-conserved, dual-function DNA-binding protein peptidase A (PepA) and the cell membrane. These interactions are not present under nutrient-limited growth conditions, due to post-translational modification of three lysines on a single face of CRP. Although coincident DNA binding is rare, dissociation from CRP results in increased PepA occupancy at many chromosomal binding sites and differential regulation of hundreds of genes, including several encoding cyclic dinucleotide phosphodiesterases. We show that PepA represses biofilm formation and activates motility/chemotaxis. We propose a model in which membrane-bound CRP interferes with PepA DNA binding. Under nutrient limitation, PepA is released. Together, CRP and free PepA activate a transcriptional response that impels the bacterium to seek a more hospitable environment. This work uncovers a function for CRP in the sequestration of a regulatory protein. More broadly, it describes a paradigm of bacterial transcriptome modulation through metabolically regulated association of transcription factors with the cell membrane.

Acinetobacter baumannii ATCC 17978 encodes a microcin system with antimicrobial properties for contact-independent competition.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen that persists in the hospital environment and causes various clinical infections, primarily affecting immunocompromised patients. A. baumannii has evolved a wide range of mechanisms to compete with neighbouring bacteria. One such competition strategy depends on small secreted peptides called microcins, which exert antimicrobial effects in a contact-independent manner. Here, we report that A. baumannii ATCC 17978 (AB17978) encodes the class II microcin 17 978 (Mcc17978) with antimicrobial activity against closely related Acinetobacter, and surprisingly, also Escherichia coli strains. We identified the genetic locus encoding the Mcc17978 system in AB17978. Using classical bacterial genetic approaches, we determined that the molecular receptor of Mcc17978 in E. coli is the iron-catecholate transporter Fiu, and in Acinetobacter is Fiu's homolog, PiuA. In bacteria, the Ferric uptake regulator (Fur) positively regulates siderophore systems and microcin systems under iron-deprived environments. We found that the Mcc17978 system is upregulated under low-iron conditions commonly found in the host environment and identified a putative Fur binding site upstream of the mcc17978 gene. When we tested the antimicrobial activity of Mcc17978 under different levels of iron availability, we observed that low iron levels not only triggered transcriptional induction of the microcin, but also led to enhanced microcin activity. Taken together, our findings suggest that A. baumannii may utilize microcins to compete with other microbes for resources during infection.

Colonization of the Caenorhabditis elegans gut with human enteric bacterial pathogens leads to proteostasis disruption that is rescued by butyrate.

Protein conformational diseases are characterized by misfolding and toxic aggregation of metastable proteins, often culminating in neurodegeneration. Enteric bacteria influence the pathogenesis of neurodegenerative diseases; however, the complexity of the human microbiome hinders our understanding of how individual microbes influence these diseases. Disruption of host protein homeostasis, or proteostasis, affects the onset and progression of these diseases. To investigate the effect of bacteria on host proteostasis, we used Caenorhabditis elegans expressing tissue-specific polyglutamine reporters that detect changes in the protein folding environment. We found that colonization of the C. elegans gut with enteric bacterial pathogens disrupted proteostasis in the intestine, muscle, neurons, and the gonad, while the presence of bacteria that conditionally synthesize butyrate, a molecule previously shown to be beneficial in neurodegenerative disease models, suppressed aggregation and the associated proteotoxicity. Co-colonization with this butyrogenic strain suppressed bacteria-induced protein aggregation, emphasizing the importance of microbial interaction and its impact on host proteostasis. Further experiments demonstrated that the beneficial effect of butyrate depended on the bacteria that colonized the gut and that this protective effect required SKN-1/Nrf2 and DAF-16/FOXO transcription factors. We also found that bacteria-derived protein aggregates contribute to the observed disruption of host proteostasis. Together, these results reveal the significance of enteric infection and gut dysbiosis on the pathogenesis of protein conformational diseases and demonstrate the potential of using butyrate-producing microbes as a preventative and treatment strategy for neurodegenerative disease.

Widespread targeting of nascent transcripts by RsmA in Pseudomonas aeruginosa.

In the opportunistic pathogen Pseudomonas aeruginosa, RsmA is an RNA-binding protein that plays critical roles in the control of virulence, interbacterial interactions, and biofilm formation. Although RsmA is thought to exert its regulatory effects by binding full-length transcripts, the extent to which RsmA binds nascent transcripts has not been addressed. Moreover, which transcripts are direct targets of this key posttranscriptional regulator is largely unknown. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing, with cells grown in the presence and absence of the RNA polymerase inhibitor rifampicin, we identify hundreds of nascent transcripts that RsmA associates with in P. aeruginosa We also find that the RNA chaperone Hfq targets a subset of those nascent transcripts that RsmA associates with and that the two RNA-binding proteins can exert regulatory effects on common targets. Our findings establish that RsmA associates with many transcripts as they are being synthesized in P. aeruginosa, identify the transcripts targeted by RsmA, and suggest that RsmA and Hfq may act in a combinatorial fashion on certain transcripts. The binding of posttranscriptional regulators to nascent transcripts may be commonplace in bacteria where distinct regulators can function alone or in concert to achieve control over the translation of transcripts as soon as they emerge from RNA polymerase.

H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription.

The H-NS-like proteins MvaT and MvaU act coordinately as global repressors in Pseudomonas aeruginosa by binding to AT-rich regions of the chromosome. Although cells can tolerate loss of either protein, identifying their combined regulatory effects has been challenging because the loss of both proteins is lethal due to induction of prophage Pf4 and subsequent superinfection of the cell. In other bacteria, H-NS promotes the cellular fitness by inhibiting intragenic transcription from AT-rich target regions, preventing them from sequestering RNA polymerase; however, it is not known whether MvaT and MvaU function similarly. Here, we utilize a parental strain that cannot be infected by Pf4 phage to define the collective MvaT and MvaU regulon and demonstrate that the combined loss of both MvaT and MvaU leads to increased intragenic transcription from loci directly controlled by these proteins. We further show that the loss of MvaT and MvaU leads to a striking redistribution of RNA polymerase containing σ70 to genomic regions vacated by these proteins. Our findings suggest that the ability of H-NS-like proteins to repress intragenic transcription is a universal function of these proteins and point to a second mechanism by which MvaT and MvaU may contribute to the growth of P. aeruginosa.

GigC, a LysR Family Transcription Regulator, Is Required for Cysteine Metabolism and Virulence in Acinetobacter baumannii.

A critical facet of mammalian innate immunity involves the hosts' attempts to sequester and/or limit the availability of key metabolic products from pathogens. For example, nutritional immunity encompasses host approaches to limit the availability of key heavy metal ions such as zinc and iron. Previously, we identified several hundred genes in a multidrug-resistant isolate of Acinetobacter baumannii that are required for growth and/or survival in the Galleria mellonella infection model. In the present study, we further characterize one of these genes, a LysR family transcription regulator that we previously named GigC. We show that mutant strains lacking gigC have impaired growth in the absence of the amino acid cysteine and that gigC regulates the expression of several genes involved in the sulfur assimilation and cysteine biosynthetic pathways. We further show that cells harboring a deletion of the gigC gene are attenuated in two murine infection models, suggesting that the GigC protein, likely through its regulation of the cysteine biosynthetic pathway, plays a key role in the virulence of A. baumannii.

GigA and GigB are Master Regulators of Antibiotic Resistance, Stress Responses, and Virulence in Acinetobacter baumannii.

A critical component of bacterial pathogenesis is the ability of an invading organism to sense and adapt to the harsh environment imposed by the host's immune system. This is especially important for opportunistic pathogens, such as Acinetobacter baumannii, a nutritionally versatile environmental organism that has recently gained attention as a life-threatening human pathogen. The emergence of A. baumannii is closely linked to antibiotic resistance, and many contemporary isolates are multidrug resistant (MDR). Unlike many other MDR pathogens, the molecular mechanisms underlying A. baumannii pathogenesis remain largely unknown. We report here the characterization of two recently identified virulence determinants, GigA and GigB, which comprise a signal transduction pathway required for surviving environmental stresses, causing infection and antibiotic resistance. Through transcriptome analysis, we show that GigA and GigB coordinately regulate the expression of many genes and are required for generating an appropriate transcriptional response during antibiotic exposure. Genetic and biochemical data demonstrate a direct link between GigA and GigB and the nitrogen phosphotransferase system (PTSNtr), establishing a novel connection between a novel stress response module and a well-conserved metabolic-sensing pathway. Based on the results presented here, we propose that GigA and GigB are master regulators of a global stress response in A. baumannii, and coupling this pathway with the PTSNtr allows A. baumannii to integrate cellular metabolic status with external environmental cues.IMPORTANCE Opportunistic pathogens, including Acinetobacter baumannii, encounter many harsh environments during the infection cycle, including antibiotic exposure and the hostile environment within a host. While the development of antibiotic resistance in A. baumannii has been well studied, how this organism senses and responds to environmental cues remain largely unknown. Herein, we investigate two previously identified virulence determinants, GigA and GigB, and report that they are required for in vitro stress resistance, likely comprising upstream elements of a global stress response pathway. Additional experiments identify a connection between GigA/GigB and a widely conserved metabolic-sensing pathway, the nitrogen phosphotransferase system. We propose that coordination of these two pathways allows A. baumannii to respond appropriately to changing environmental conditions, including those encountered during infection.

Targeting Staphylococcus aureus α-toxin as a novel approach to reduce severity of recurrent skin and soft-tissue infections.

Staphyococcus aureus frequently causes recurrent skin and soft-tissue infection (SSTI). In the pediatric population, elevated serum antibody targeting S. aureus α-toxin is correlated with a reduced incidence of recurrent SSTI. Using a novel model of recurrent SSTI, we demonstrated that expression of α-toxin during primary infection increases the severity of recurrent disease. Antagonism of α-toxin by either a dominant-negative toxin mutant or a small molecule inhibitor of the toxin receptor ADAM10 during primary infection reduces reinfection abscess severity. Early neutralization of α-toxin activity during S. aureus SSTI therefore offers a new therapeutic strategy to mitigate primary and recurrent disease.

Genome-wide protein-DNA interaction site mapping in bacteria using a double-stranded DNA-specific cytosine deaminase.

DNA-protein interactions are central to fundamental cellular processes, yet widely implemented technologies for measuring these interactions on a genome scale in bacteria are laborious and capture only a snapshot of binding events. We devised a facile method for mapping DNA-protein interaction sites in vivo using the double-stranded DNA-specific cytosine deaminase toxin DddA. In 3D-seq (DddA-sequencing), strains containing DddA fused to a DNA-binding protein of interest accumulate characteristic mutations in DNA sequence adjacent to sites occupied by the DNA-bound fusion protein. High-depth sequencing enables detection of sites of increased mutation frequency in these strains, yielding genome-wide maps of DNA-protein interaction sites. We validated 3D-seq for four transcription regulators in two bacterial species, Pseudomonas aeruginosa and Escherichia coli. We show that 3D-seq offers ease of implementation, the ability to record binding event signatures over time and the capacity for single-cell resolution.

The gigA/gigB Genes Regulate the Growth, Stress Response, and Virulence of Acinetobacter baumannii ATCC 17978 Strain.

Acinetobacter baumannii is an important pathogen of nosocomial infection. Recently, a group of genes, named "gig" (for Growth in Galleria), have been identified in a contemporary multi-drug resistant clinical isolate of A. baumannii-strain AB5075. Among these so-called gig genes, gigA and gigB were found to promote antibiotic resistance, stress survival, and virulence of AB5075 by interacting with the nitrogen phosphotransferase system (PTSNtr). This study aimed to investigate the roles of gigA/gigB, which appear to comprise a stress-signaling pathway (encoding for an atypical two-component system response regulator and a predicted anti-anti-sigma factor, respectively), and the involvement of ptsP (encoding the Enzyme I component of the PTSNtr) in the growth, stress resistance, and virulence of the widely studied A. baumannii strain ATCC 17978. Genetic analyses of strains harboring mutations of gigA and gigB were performed to investigate the roles of these genes in bacterial growth, stress resistance, evading macrophage defense, and killing of Galleria mellonella larva. In contrast with findings from strain AB5075 where gigA and gigB contribute to aminoglycoside resistance, the data presented herein indicate that the loss of gigA/gigB does not impact antibiotic resistance of strain ATCC 17978. Interestingly, however, we found that deletion of gigA/gigB in the ATCC 17978 background imparts a general growth in laboratory medium and also conferred growth and replication defects within murine macrophages and an inability to kill G. mellonella larvae. Importantly, studies as well as the loss of ptsP restored the phenotypes of the gigA/gigB mutant to that of the wild-type. The data presented herein indicate that in A. baumannii ATCC 17978, the gigA/gigB genes play a key role in both growth and virulence traits, but are dispensable for other stress-resistance survival phenotypes, including aminoglycoside resistance. Our findings thus highlight several similarities and also important differences between the gigA/gigB stress-signaling pathway in two commonly studied isolates of this troublesome pathogen.

Seeing red; the development of pON.mCherry, a broad-host range constitutive expression plasmid for Gram-negative bacteria.

The development of plasmid-mediated gene expression control in bacteria revolutionized the field of bacteriology. Many of these expression control systems rely on the addition of small molecules, generally metabolites or non-metabolized analogs thereof, to the growth medium to induce expression of the genes of interest. The paradigmatic example of an expression control system is the lac system from Escherichia coli, which typically relies on the Ptac promoter and the Lac repressor, LacI. In many cases, however, constitutive gene expression is desired, and other experimental approaches require the coordinated control of multiple genes. While multiple systems have been developed for use in E. coli and its close relatives, the utility and/or functionality of these tools does not always translate to other species. For example, for the Gram-negative pathogen, Legionella pneumophila, a causative agent of Legionnaires' Disease, the aforementioned Ptac system represents the only well-established expression control system. In order to enhance the tools available to study bacterial gene expression in L. pneumophila, we developed a plasmid, pON.mCherry, which confers constitutive gene expression from a mutagenized LacI binding site. We demonstrate that pON.mCherry neither interferes with other plasmids harboring an intact LacI-Ptac expression system nor alters the growth of Legionella species during intracellular growth. Furthermore, the broad-host range plasmid backbone of pON.mCherry allows constitutive gene expression in a wide variety of Gram-negative bacterial species, making pON.mCherry a useful tool for the greater research community.

Cuticular microstructures turn specular black into matt black in a stick insect.

The stick insect Peruphasma schultei stands out from other insects by its deep matt black cuticle. We tested whether the appearance of P. schultei is due to microstructures of the cuticle, a phenomenon that has recently been described for the velvet black scales of the Gaboon viper. The shiny black stick insect Anisomorpha paromalus served as a control. We found that the P. schultei cuticle is characterised by two different types of microstructures, tall elevations with a maximum size of 18 µm and small structures with a height of 4 µm. Other than in the snake, P. schultei microstructures do not bear nanostructures. The microstructures scatter light independently of the viewing angle. This causes the matt appearance of the cuticle, whereas pigments are responsible for the black colouration, resulting in a maximum reflectance of 2.8% percent. The microstructures also cause the hydrophobic properties of the cuticle with contact angles near 130°. Resin replicas and bleaching of the cuticle strongly support these results. Moreover, the matt black cuticle has a higher heat absorption compared to the control. We discuss the selective benefit of the matt black appearance of P. schultei in the context of behaviour, ecology and phylogeny.

Joint Transcriptional Control of Virulence and Resistance to Antibiotic and Environmental Stress in Acinetobacter baumannii.

UNLABELLED: The increasing emergence of antibiotic-resistant bacterial pathogens represents a serious risk to human health and the entire health care system. Many currently circulating strains of Acinetobacter baumannii exhibit resistance to multiple antibiotics. A key limitation in combating A. baumannii is that our understanding of the molecular mechanisms underlying the pathogenesis of A. baumannii is lacking. To identify potential virulence determinants of a contemporary multidrug-resistant isolate of A. baumannii, we used transposon insertion sequencing (TnSeq) of strain AB5075. A collection of 250,000 A. baumannii transposon mutants was analyzed for growth within Galleria mellonella larvae, an insect-based infection model. The screen identified 300 genes that were specifically required for survival and/or growth of A. baumannii inside G. mellonella larvae. These genes encompass both known, established virulence factors and several novel genes. Among these were more than 30 transcription factors required for growth in G. mellonella. A subset of the transcription factors was also found to be required for resistance to antibiotics and environmental stress. This work thus establishes a novel connection between virulence and resistance to both antibiotics and environmental stress in A. baumannii. IMPORTANCE: Acinetobacter baumannii is rapidly emerging as a significant human pathogen, largely because of disinfectant and antibiotic resistance, causing lethal infection in fragile hosts. Despite the increasing prevalence of infections with multidrug-resistant A. baumannii strains, little is known regarding not only the molecular mechanisms that allow A. baumannii to resist environmental stresses (i.e., antibiotics and disinfectants) but also how these pathogens survive within an infected host to cause disease. We employed a large-scale genetic screen to identify genes required for A. baumannii to survive and grow in an insect disease model. While we identified many known virulence factors harbored by A. baumannii, we also discovered many novel genes that likely play key roles in A. baumannii survival of exposure to antibiotics and other stress-inducing chemicals. These results suggest that selection for increased resistance to antibiotics and environmental stress may inadvertently select for increased virulence in A. baumannii.