Host-pathogen interaction profiling using self-assembling human protein arrays.

Host-pathogen protein interactions are fundamental to every microbial infection, yet their identification has remained challenging due to the lack of simple detection tools that avoid abundance biases while providing an open format for experimental modifications. Here, we applied the Nucleic Acid-Programmable Protein Array and a HaloTag-Halo ligand detection system to determine the interaction network of Legionella pneumophila effectors (SidM and LidA) with 10 000 unique human proteins. We identified known targets of these L. pneumophila proteins and potentially novel interaction candidates. In addition, we applied our Click chemistry-based NAPPA platform to identify the substrates for SidM, an effector with an adenylyl transferase domain that catalyzes AMPylation (adenylylation), the covalent addition of adenosine monophosphate (AMP). We confirmed a subset of the novel SidM and LidA targets in independent in vitro pull-down and in vivo cell-based assays, and provided further insight into how these effectors may discriminate between different host Rab GTPases. Our method circumvents the purification of thousands of human and pathogen proteins, and does not require antibodies against or prelabeling of query proteins. This system is amenable to high-throughput analysis of effectors from a wide variety of human pathogens that may bind to and/or post-translationally modify targets within the human proteome.

Bordetella pertussis fim3 gene regulation by BvgA: phosphorylation controls the formation of inactive vs. active transcription complexes.

Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and the Bordetella pertussis RR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at P(fim3), the promoter for the virulence gene fim3 (fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at P(fim3). However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence of fim3. We suggest that the presence of nonphosphorylated BvgA down-regulates P(fim3) activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating P(fim3) activation once the signal for BvgA phosphorylation is removed.

Transcription regulation at the core: similarities among bacterial, archaeal, and eukaryotic RNA polymerases.

Multisubunit RNA polymerases are complex protein machines that require a specificity factor for the recognition of a specific transcription start site. Although bacterial σ factors are thought to be quite different from the specificity factors employed in higher organisms, a comparison of the σ/RNA polymerase structures with recent structures of eukaryotic Pol II together with TFIIB highlights significant functional similarities. Other work reveals that both bacterial and eukaryotic promoters are composed of modular elements that are used in different combinations. Bacteria, archaea, and eukaryotes also utilize similar strategies to alter core promoter specificity, from specificity factor exchange to the employment of activators that bind close to or overlap core promoter sequences, directing the transcriptional machinery to a new start site. Here we examine the details of core promoter recognition in bacteria that reveal the transcriptional similarities throughout biology.

The Bordetella pertussis model of exquisite gene control by the global transcription factor BvgA.

Bordetella pertussis causes whooping cough, an infectious disease that is reemerging despite widespread vaccination. A more complete understanding of B. pertussis pathogenic mechanisms will involve unravelling the regulation of its impressive arsenal of virulence factors. Here we review the action of the B. pertussis response regulator BvgA in the context of what is known about bacterial RNA polymerase and various modes of transcription activation. At most virulence gene promoters, multiple dimers of phosphorylated BvgA (BvgA~P) bind upstream of the core promoter sequence, using a combination of high- and low-affinity sites that fill through cooperativity. Activation by BvgA~P is typically mediated by a novel form of class I/II mechanisms, but two virulence genes, fim2 and fim3, which encode serologically distinct fimbrial subunits, are regulated using a previously unrecognized RNA polymerase/activator architecture. In addition, the fim genes undergo phase variation because of an extended cytosine (C) tract within the promoter sequences that is subject to slipped-strand mispairing during replication. These sophisticated systems of regulation demonstrate one aspect whereby B. pertussis, which is highly clonal and lacks the extensive genetic diversity observed in many other bacterial pathogens, has been highly successful as an obligate human pathogen.

Different requirements for σ Region 4 in BvgA activation of the Bordetella pertussis promoters P(fim3) and P(fhaB).

Bordetella pertussis BvgA is a global response regulator that activates virulence genes, including adhesin-encoding fim3 and fhaB. At the fhaB promoter, P(fhaB), a BvgA binding site lies immediately upstream of the -35 promoter element recognized by Region 4 of the σ subunit of RNA polymerase (RNAP). We demonstrate that σ Region 4 is required for BvgA activation of P(fhaB), a hallmark of Class II activation. In contrast, the promoter-proximal BvgA binding site at P(fim3) includes the -35 region, which is composed of a tract of cytosines that lacks specific sequence information. We demonstrate that σ Region 4 is not required for BvgA activation at P(fim3). Nonetheless, Region 4 mutations that impair its typical interactions with core and with the -35 DNA affect P(fim3) transcription. Hydroxyl radical cleavage using RNAP with σD581C-FeBABE positions Region 4 near the -35 region of P(fim3); cleavage using RNAP with α276C-FeBABE or α302C-FeBABE also positions an α subunit C-terminal domain within the -35 region, on a different helical face from the promoter-proximal BvgA~P dimer. Our results suggest that the -35 region of P(fim3) accommodates a BvgA~P dimer, an α subunit C-terminal domain, and σ Region 4. Molecular modeling suggests how BvgA, σ Region 4, and α might coexist within this DNA in a conformation that suggests a novel mechanism of activation.

The secret to 6S: regulating RNA polymerase by ribo-sequestration.

Regulating transcription under different conditions is vital to all organisms. As Escherichia coli shifts from exponential to stationary growth, regulation of transcription is achieved in large part by the tight binding of 6S RNA to Esigma(70), RNA polymerase with the sigma(70) specificity subunit. Ribo-sequestration of Esigma(70) by 6S RNA serves to downregulate sigma(70)-dependent transcription, which is needed for exponential growth. This facilitates transcription from promoters dependent on stationary phase sigma, sigma(s). Previous work has suggested that the 6S RNA binding to Esigma(70) simply mimics the Esigma(70)/promoter interaction. In this issue of Molecular Microbiology, Klocko and Wassarman demonstrate that many of the contacts between residues within sigma(70) region 4 and 6S RNA are distinct from those between region 4 and promoter DNA. Several residues that interact with 6S RNA are ones previously known to interact with protein activators of Esigma(70). Their work adds 6S RNA to the growing list of factors that can regulate Esigma(70) by interacting with region 4.

Seasonal phenology and natural enemies of the squash bug (Hemiptera: Coreidae) in Kentucky.

The squash bug, Anasa tristis (De Geer), is a major indigenous pest of Cucurbita species across the United States and a vector of cucurbit yellow vine disease. The seasonal phenology of the squash bug in central Kentucky and its natural enemies were studied using summer squash planted sequentially throughout the 2005 and 2006 growing seasons. The squash bug was first detected on 5 June 2005 and 3 June 2006. In both years, peak numbers of all squash bug stages occurred in July and August. Our field data, substantiated by published degree-day models for squash bug development, suggest one complete and a partial second generation of squash bugs in 2005 and one complete generation of squash bugs in 2006. The most abundant ground-active predators in squash fields included Araneae, Carabidae, Staphylinidae, and Geocoridae. Coleomegilla maculata (De Geer) and Geocoris punctipes (Say) were the most abundant foliage-inhabiting predators. Direct field observations of predators feeding on squash bugs or their eggs included G. punctipes, Pagasa fusca (Stein), and Nabis sp. The parasitoids Trichopoda pennipes (Fabricius) and Gyron pennsylvanicum (Ashmead) were found also. Squash bug egg masses were monitored to determine predation and parasitism rates in the field. In four studies during 2005 and 2006, predation rates were low (7% or less), and parasitism ranged from 0 to 31%. Overall, squash bug egg mortality increased as the season progressed.