Christina Morris

Salmonella enterica Serovar Typhi Uses Type IVB Pili To Enter Human Intestinal Epithelial Cells

ABSTRACT DNA sequencing upstream of the Salmonella entericaserovar Typhi pilV and rci genes previously identified in the ca. 118-kb major pathogenicity island (X.-L. Zhang, C. Morris, and J. Hackett, Gene 202:139–146, 1997) identified a further 10 pil genes apparently forming a piloperon. The product of the pilS gene, prePilS protein (a putative type IVB structural prepilin) was purified, and an anti-prePilS antiserum was raised in mice. Mutants of serovar Typhi either lacking the whole pil operon or with an insertion mutation in the pilS gene were constructed, as was a strain in which the pilN to pilV genes were driven by the tac promoter. The pil+ strains synthesized type IVB pili, as judged by (i) visualization in the electron microscope of thin pili in culture supernatants of one such strain and (ii) the presence of PilS protein (smaller than the prePilS protein by removal of the leader peptide) on immunoblotting of material pelleted by high-speed centrifugation of either the culture supernatant or sonicates of pil+ strains. Controlpil mutants did not express the PilS protein. ApilS mutant of serovar Typhi entered human intestinal INT407 cells in culture to levels only 5 to 25% of those of the wild-type strain, and serovar Typhi entry was strongly inhibited by soluble prePilS protein (50% inhibition of entry at 1.4 μM prePilS).

The Type IVB Pili of Salmonella enterica Serovar Typhi Bind to the Cystic Fibrosis Transmembrane Conductance Regulator

ABSTRACT Salmonella enterica serovar Typhi expresses type IVB pili. We show that the prePilS protein (the soluble precursor form of the structural pilin) interacts with a 15-mer peptide representing the first extracellular domain of the cystic fibrosis transmembrane conductance regulator (CFTR), a recognized human epithelial cell receptor for serovar Typhi (G. B. Pier et al., Nature 393:79-82, 1998). This indicates that after mediating bacterial self-association (C. Morris et al., Infect. Immun. 71:1141-1146, 2003), the pili then act to attach the bacterial clumps to CFTR in the membrane of gut epithelial cells. These sequential type IVB pilus-mediated events cannot be performed by (for example) S. enterica serovar Typhimurium, which may explain why only serovar Typhi causes epidemics of enteric fever in humans.

Molecular analysis of mxbD and mxbM, a putative sensor-regulator pair required for oxidation of methanol in Methylobacterium extorquens AM1

Five genes are thought to be required for transcription of methanol oxidation genes in Methylobacterium strains. These putative regulatory genes include mxcQE, which encode a putative sensor-regulator pair, and mxbDM and mxaB, whose functions are less well-understood. In this study, mxbDM in Methylobacterium extorquens AM1 were shown to be required for expression of a xylE transcriptional fusion to the structural gene for the large subunit of methanol dehydrogenase (mxaF), confirming the role of these genes in transcriptional regulation of mxaF. The nucleotide sequence suggests that mxbD encodes a histidine protein kinase with two transmembrane domains and that mxbM encodes a DNA-binding response regulator. A xylE transcriptional fusion to the putative mxbD promoter showed low-level expression in wild-type cells grown on one-carbon (C1) compounds and no detectable expression in cells grown on succinate. Deletion analysis of this promoter construct showed that the region 229-129 bp upstream of the start of mxbD is required for expression. The expression of the mxbD-xylE fusion was examined in each of the five known regulatory mutant classes. xylE expression was reduced to non-detectable levels in MxcQ and MxcE mutants, but was not affected in the other regulatory mutants or in non-regulatory mutants defective in methanol oxidation. These results suggest a regulatory hierarchy in which the sensor-regulator pair MxcQE control expression of the sensor-regulator pair MxbDM, and MxbDM in turn control expression of a number of genes involved in methanol oxidation.

The shufflon of Salmonella enterica serovar Typhi regulates type IVB pilus-mediated bacterial self-association

ABSTRACT Previously, it was shown that type IVB pili encoded by the Salmonella enterica serovar Typhi pil operon are used to facilitate bacterial entry into human intestinal epithelial cells in vitro and that such entry is inhibited by purified prepilin (pre-PilS) protein (X.-L. Zhang, I. S. M. Tsui, C. M. C. Yip, A. W. Y. Fung, D. K.-H. Wong, X. Dai, Y. Yang, J. Hackett, and C. Morris, Infect. Immun. 68:3067-3073, 2000). The pil operon concludes with a simple shufflon, and a recombinase gene product (Rci) inverts DNA in the C-terminal region of the pilV gene to allow synthesis of two distinct PilV proteins, PilV1 and PilV2, which are presumptive minor pilus proteins. We show here that the type IVB pili mediate bacterial self-association, but only when the PilV1 and PilV2 proteins are not expressed. This may be achieved in wild-type serovar Typhi by rapid DNA inversion activity of the shufflon. We show that the inversion activity inhibits the expression of genes inserted between the 19-bp inverted repeats used for Rci-mediated recombination and that the activity of Rci increases when DNA is supercoiled. The data suggest that serovar Typhi self-associates under conditions (such as low oxygen tension in the gut) that favor DNA supercoiling. These results explain (i) the function of the serovar Typhi shufflon and (ii) why there are only two possible shufflon states, in contrast to the many possible states of other shufflon systems. The data further indicate that a very early step in serovar Typhi pathogenesis may be type IVB pilus-mediated self-association of bacteria in the anaerobic human small intestine prior to invasion of the human gut epithelium. The suggested type IVB pilus-dependent step in typhoid fever pathogenesis may partially explain the enhanced invasiveness of serovar Typhi for humans.

Multiple Gi Proteins Participate in Nerve Growth Factor-Induced Activation of c-Jun N-terminal Kinases in PC12 Cells

Nerve growth factor (NGF)-mediated activation of mitogen-activated protein kinases (MAPK) is critical for differentiation and apoptosis of PC12 cells. Since NGF employs stress-activated c-Jun N-terminal kinase (JNK) to regulate both programmed cell death and neurite outgrowth of PC12 cells, we examined NGF-regulated JNK activity and the role of Gi/o proteins. Induction of JNK phosphorylation by NGF occurred in a time- and dose-dependent manner and was partially inhibited by pertussis toxin (PTX). To discern the participation of various signaling intermediates, PC12 cells were treated with specific inhibitors prior to NGF challenge. NGF-elevated JNK activity was abolished by inhibitors of JNK, p38 MAPK, Src, JAK3 and MEK1/2. NGF-dependent JNK phosphorylation became insensitive to PTX treatment upon transient expressions of Gαz or the PTX-resistant mutants of Gαi1–3 and GαoA. Collectively, these studies indicate that NGF-dependent JNK activity may be mediated via Gi1–3 proteins, JAK3, Src, p38 MAPK and the MEK/ERK cascade.

Salmonella enterica serovar Paratyphi C carries an inactive shufflon

ABSTRACT Salmonella enterica serovar Typhi uses type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. This pilus-mediated event may be important in typhoid fever pathogenesis. We initially show that S. enterica serovar Paratyphi C strains harbor a pil operon very similar to that of serovar Typhi. An important difference, however, is located in the shufflon which concludes the pil operon. In serovar Typhi, the Rci recombinase acts upon two 19-bp inverted repeats to invert the terminal region of the pilV gene, thereby disrupting PilV synthesis and permitting bacterial self-association. In serovar Paratyphi C, however, the shufflon is essentially inactive because each of the Rci 19-bp substrates has acquired a single base pair insertion. A PilV protein is thus synthesized whenever the pil operon is active, and bacterial self-association therefore does not occur in serovar Paratyphi C. The data thus suggest that serovar Typhi bacterial self-association using type IVB pili may be important in the pathogenesis of epidemic enteric fever.

The Salmonella enterica serovar Typhi type IVB self-association pili are detached from the bacterial cell by the PilV minor pilus proteins.

ABSTRACT Salmonella enterica serovar Typhi and some strains (Vi+) of serovar Dublin use type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. Pilus-mediated self-association may be important in the pathogenesis of enteric fever. We have shown previously that the extent of DNA supercoiling controls the rate of Rci-catalyzed inversion of a DNA fragment which includes the C-terminal portions of the PilV proteins. This inversion therefore controls PilV synthesis as a high inversion rate prohibits transcription of pilV-encoding DNA. Here, we describe the manner in which PilV protein expression inhibits bacterial self-association and present data which suggest that incorporation of one or a few PilV protein molecules into a growing pilus, comprised of PilS subunits, causes the pilus to detach at the bacterial membrane. The bacteria are then unable to self-associate. We suggest that this phenomenon may be relevant to the pathogenesis of typhoid fever.

Identification of O-antigen polymerase transcription and translation start signals and visualization of the protein in Salmonella enterica serovar Typhimurium.

The wzy/rfc gene, encoding the O-antigen polymerase, of Salmonella enterica serovar Typhimurium has been previously cloned and sequenced. In the present work, the wzy transcriptional startpoint was initially identified by primer extension. Next, wzy promoter strength in Escherichia coli K-12 was measured, and was found to be greater than that of the induced lac promoter. To define the Wzy translational startpoint, DNA including the wzy promoter and the putative first five residues of the Wzy protein was fused to the N-terminus of glutathione-S-transferase, and the fusion protein purified by affinity chromatography. N-terminal amino acid sequencing yielded the Wzy translational startpoint. Next, the Wzy protein was C-terminally tagged with the FLAG peptide, and immunoblotting of an S. typhimurium strain expressing a low-copy wzy-FLAG gene (five copies per cell) localized the intact Wzy protein in the cytoplasmic membrane of S. typhimurium cells. The Wzy protein was not well-expressed from a multi-copy wzy-FLAG+ plasmid in S. typhimurium, or in E. coli K-12.

Rate of Inversion of the Salmonella enterica Shufflon Regulates Expression of Invertible DNA

ABSTRACT Salmonella enterica serovar Typhi and some strains (Vi+) of serovar Dublin use type IVB pili to facilitate bacterial self-association, but only when the PilV proteins (potential minor pilus proteins) are not synthesized. Pilus-mediated self-association may be important in the pathogenesis of enteric fever. We have suggested that the rate of Rci-catalyzed inversion of DNA encoding the C-terminal portions of the PilV proteins controls PilV protein synthesis. This potentially represents a novel means of transcriptional control. Here, it is initially shown that DNA inversion per se is required for inhibition of gene expression from invertible DNA. Binding, without DNA scission, of Rci to its substrate sequences on DNA cannot explain the data obtained. Next, it is shown that inversion frequencies of xylE-encoding DNA, bracketed by Rci substrate sequences, may be modulated by changes in the 19-bp consensus sequences which are essential components of Rci substrate DNA. The affinity of Rci for these sequences affects inversion frequencies, so that a greater affinity is predictive of faster inversion, and therefore less synthesis of product encoded by invertible DNA. Inversion events may inhibit transcription of DNA from external promoters. In vivo, the frequency of Rci-mediated inversion is influenced by the extent of DNA supercoiling, with increasing levels of expression of invertible genes as novobiocin inhibits DNA supercoiling and thus Rci action. This inhibition of DNA supercoiling results in increased synthesis of PilV proteins as Rci activity decreases, and, in turn, bacterial self-association (particularly in serovar Dublin) decreases.

Activator of G protein signaling 3 forms a complex with resistance to inhibitors of cholinesterase-8A without promoting nucleotide exchange on Gα(i3).

Activator of G protein signaling 3 (AGS3) is a guanine nucleotide dissociation inhibitor (GDI) which stabilizes the Gαi/o subunits as an AGS3/Gαi/o-GDP complex. It has recently been demonstrated in reconstitution experiments that the AGS3/Gαi/o-GDP complex may act as a substrate of resistance to inhibitors of cholinesterase 8A (Ric-8A), a guanine exchange factor (GEF) for heterotrimeric Gα proteins. Since the ability of Ric-8A to activate Gαi/o subunits that are bound to AGS3 in a cellular environment has not been confirmed, we thus examined the effect of Ric-8A on cAMP accumulation in HEK293 cells expressing different forms of AGS3 and Gαi3. Co-immunoprecipitation assays indicate that full-length AGS3 and its N- and C-terminal truncated mutants can interact with Ric-8A in HEK293 cells. Yeast two-hybrid assay further confirmed that Ric-8A can directly bind to AGS3S, a short form of AGS3 which is endogenously expressed in heart. However, Ric-8A failed to facilitate Gαi-induced suppression of adenylyl cyclase, suggesting that it may not serve as a GEF for AGS3/Gαi/o-GDP complex in a cellular environment.