The Role of Coupled Positive Feedback in the Expression of the SPI1 Type Three Secretion System in Salmonella

Salmonella enterica serovar Typhimurium is a common food-borne pathogen that induces inflammatory diarrhea and invades intestinal epithelial cells using a type three secretion system (T3SS) encoded within Salmonella pathogenicity island 1 (SPI1). The genes encoding the SPI1 T3SS are tightly regulated by a network of interacting transcriptional regulators involving three coupled positive feedback loops. While the core architecture of the SPI1 gene circuit has been determined, the relative roles of these interacting regulators and associated feedback loops are still unknown. To determine the function of this circuit, we measured gene expression dynamics at both population and single-cell resolution in a number of SPI1 regulatory mutants. Using these data, we constructed a mathematical model of the SPI1 gene circuit. Analysis of the model predicted that the circuit serves two functions. The first is to place a threshold on SPI1 activation, ensuring that the genes encoding the T3SS are expressed only in response to the appropriate combination of environmental and cellular cues. The second is to amplify SPI1 gene expression. To experimentally test these predictions, we rewired the SPI1 genetic circuit by changing its regulatory architecture. This enabled us to directly test our predictions regarding the function of the circuit by varying the strength and dynamics of the activating signal. Collectively, our experimental and computational results enable us to deconstruct this complex circuit and determine the role of its individual components in regulating SPI1 gene expression dynamics

Peptidoglycan-Modifying Enzyme Pgp1 Is Required for Helical Cell Shape and Pathogenicity Traits in Campylobacter jejuni

The impact of bacterial morphology on virulence and transmission attributes of pathogens is poorly understood. The prevalent enteric pathogen Campylobacter jejuni displays a helical shape postulated as important for colonization and host interactions. However, this had not previously been demonstrated experimentally. C. jejuni is thus a good organism for exploring the role of factors modulating helical morphology on pathogenesis. We identified an uncharacterized gene, designated pgp1 (peptidoglycan peptidase 1), in a calcofluor white-based screen to explore cell envelope properties important for C. jejuni virulence and stress survival. Bioinformatics showed that Pgp1 is conserved primarily in curved and helical bacteria. Deletion of pgp1 resulted in a striking, rod-shaped morphology, making pgp1 the first C. jejuni gene shown to be involved in maintenance of C. jejuni cell shape. Pgp1 contributes to key pathogenic and cell envelope phenotypes. In comparison to wild type, the rod-shaped pgp1 mutant was deficient in chick colonization by over three orders of magnitude and elicited enhanced secretion of the chemokine IL-8 in epithelial cell infections. Both the pgp1 mutant and a pgp1 overexpressing strain – which similarly produced straight or kinked cells – exhibited biofilm and motility defects. Detailed peptidoglycan analyses via HPLC and mass spectrometry, as well as Pgp1 enzyme assays, confirmed Pgp1 as a novel peptidoglycan DL-carboxypeptidase cleaving monomeric tripeptides to dipeptides. Peptidoglycan from the pgp1 mutant activated the host cell receptor Nod1 to a greater extent than did that of wild type. This work provides the first link between a C. jejuni gene and morphology, peptidoglycan biosynthesis, and key host- and transmission-related characteristics

Regulation of the Salmonella Pathogenicity Island 1 Type Three Secretion System

175 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.The invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is mediated by a Type Three Secretion System (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). The SPI1 TTSS injects effector proteins into the cytosol of host cells where they promote actin rearrangement and engulfment of the bacteria. HilC, HilD, and RtsA activate expression of SPI1 genes by binding upstream of the master regulatory gene hilA to induce its expression. HilA activates the SPI1 T3SS structural genes. Here I present evidence that hilA expression, and hence the SPI1 T3SS, is controlled by a feedforward regulatory loop. I demonstrate that HilC, HilD, and RtsA are each capable of independently inducing expression of the hilC, hilD, and rtsA genes, and that each can independently activate hilA. Using competition assays in vivo, I show that each of the hilA regulators contribute to SPI1 induction in the intestine. Of the three, HilD has a predominant role, but apparently does not act alone either in vivo or in vitro to sufficiently activate SPI1. The two-component regulatory systems, SirA/BarA and OmpR/EnvZ, function through Hi1D, thus inducing hilC, rtsA, and hilA. However, the two-component systems are not responsible for environmental regulation of SPI1. Rather, "SPI1 inducing conditions" cause independent activation of the rtsA, hilC, and hilD genes in the absence of known regulators. One potential environmental signal is iron. Fur controls the iron response in the cell and activates hilA transcription in a HilD-dependent manner. Fur regulation of HilD does not appear to be simply at the transcriptional or translational level, but rather requires the presence of HilD protein. Fur activation of SPI1 is not mediated through the Fur regulated small RNAs, RfrA and RfrB, which are the Salmonella ortholog and paralog of RyhB (RfrA and RfrB, respectively) that control expression of sodB. The PhoP/PhoQ two-component regulatory system represses expression of SPI1 genes. This system works independently of HilD protein by negatively regulating hilA expression directly at the hilA promoter. Additionally, fatty acid degradation plays an undefined role in the regulation of SPI1. Although the exact mechanism of regulation for some of these factors is still unknown, and there is still much work to be done, this work provides a key step in understanding the SPI1 T3SS regulatory circuit.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD

Fur Regulates Expression of the Salmonella Pathogenicity Island 1 Type III Secretion System through HilD▿

The invasion of intestinal epithelial cells by Salmonella enterica serovar Typhimurium is mediated by a type III secretion system (T3SS) encoded on Salmonella pathogenicity island 1 (SPI1). Expression of the SPI1 T3SS is tightly regulated by the combined action of HilC, HilD, and RtsA, three AraC family members that can independently activate hilA, which encodes the direct regulator of the SPI1 structural genes. Expression of hilC, hilD, and rtsA is controlled by a number of regulators that respond to a variety of environmental signals. In this work, we show that one such signal is iron mediated by Fur (ferric uptake regulator). Fur activates hilA transcription in a HilD-dependent manner. Fur regulation of HilD does not appear to be simply at the transcriptional or translational level but rather requires the presence of the HilD protein. Fur activation of SPI1 is not mediated through the Fur-regulated small RNAs RfrA and RfrB, which are the Salmonella ortholog and paralog of RyhB that control expression of sodB. Fur regulation of HilD is also not mediated through the known SPI1 repressor HilE or the CsrABC system. Although understanding the direct mechanism of Fur action on HilD requires further analysis, this work is an important step toward elucidating how various global regulatory systems control SPI1

Intestinal Long-Chain Fatty Acids Act as a Direct Signal To Modulate Expression of the Salmonella Pathogenicity Island 1 Type III Secretion System

Salmonella enterica serovar Typhimurium uses the Salmonella pathogenicity island 1 (SPI1) type III secretion system (T3SS) to induce inflammatory diarrhea and bacterial uptake into intestinal epithelial cells. The expression of hilA, encoding the transcriptional activator of the T3SS structural genes, is directly controlled by three AraC-like regulators, HilD, HilC, and RtsA, each of which can activate hilD, hilC, rtsA, and hilA genes, forming a complex feed-forward regulatory loop. Expression of the SPI1 genes is tightly controlled by numerous regulatory inputs to ensure proper timing in production of the T3SS apparatus. Loss of FadD, an acyl coenzyme A (acyl-CoA) synthetase required for degradation of long-chain fatty acids (LCFAs), was known to decrease hilA expression. We show that free external LCFAs repress expression of hilA independently of FadD and the LCFA degradation pathway. Genetic and biochemical evidence suggests that LCFAs act directly to block primarily HilD activity. Further analyses show that in the absence of FadD, hilA expression is downregulated due to endogenous production of free LCFAs, which are excreted into the culture medium via TolC and then transported back into the bacterial cell via FadL. A fadL mutant is more virulent than the wild-type strain in mouse oral competition assays independently of LCFA degradation, showing that, in the host, dietary LCFAs serve as a signal for proper regulation of SPI1 expression, rather than an energy source

Towards a Computational Model of a Methane Producing Archaeum

Progress towards a complete model of the methanogenic archaeum Methanosarcina acetivorans is reported. We characterized size distribution of the cells using differential interference contrast microscopy, finding them to be ellipsoidal with mean length and width of 2.9 μm and 2.3 μm, respectively, when grown on methanol and 30% smaller when grown on acetate. We used the single molecule pull down (SiMPull) technique to measure average copy number of the Mcr complex and ribosomes. A kinetic model for the methanogenesis pathways based on biochemical studies and recent metabolic reconstructions for several related methanogens is presented. In this model, 26 reactions in the methanogenesis pathways are coupled to a cell mass production reaction that updates enzyme concentrations. RNA expression data (RNA-seq) measured for cell cultures grown on acetate and methanol is used to estimate relative protein production per mole of ATP consumed. The model captures the experimentally observed methane production rates for cells growing on methanol and is most sensitive to the number of methyl-coenzyme-M reductase (Mcr) and methyl-tetrahydromethanopterin:coenzyme-M methyltransferase (Mtr) proteins. A draft transcriptional regulation network based on known interactions is proposed which we intend to integrate with the kinetic model to allow dynamic regulation