Murein lipoprotein is a critical outer membrane component involved in Salmonella enterica serovar typhimurium systemic infection.

Lipopolysaccharide (LPS) and Braun (murein) lipoprotein (Lpp) are major components of the outer membrane of gram-negative enteric bacteria that function as potent stimulators of inflammatory and immune responses. In a previous paper, we provided evidence that two functional copies of the lipoprotein gene (lppA and lppB) located on the chromosome of Salmonella enterica serovar Typhimurium contributed to bacterial virulence. In this study, we characterized lppA and lppB single-knockout (SKO) mutants and compared them with an lpp double-knockout (DKO) mutant using in vitro and in vivo models. Compared to the lpp DKO mutant, which was nonmotile, the motility of the lpp SKO mutants was significantly increased (73 to 77%), although the level of motility did not reach the level of wild-type (WT) S. enterica serovar Typhimurium. Likewise, the cytotoxicity was also significantly increased when T84 human intestinal epithelial cells and RAW264.7 murine macrophages were infected with the lpp SKO mutants compared to the cytotoxicity when cells were infected with the lpp DKO mutant. The level of interleukin-8 (IL-8) in polarized T84 cells infected with the lppB SKO mutant was significantly higher (two- to threefold higher), reaching the level in cells infected with WT S. enterica serovar Typhimurium, than the level in host cells infected with the lppA SKO mutant. The lpp DKO mutant induced minimal levels of IL-8. Similarly, sera from mice infected with the lppB SKO mutant contained 4.5- to 10-fold-higher levels of tumor necrosis factor-alpha and IL-6; the levels of these cytokines were 1.7- to 3.0-fold greater in the lppA SKO mutant-infected mice than in animals challenged with the lpp DKO mutant. The increased cytokine levels observed with the lppB SKO mutant in mice correlated with greater tissue damage in the livers and spleens of these mice than in the organs of animals infected with the lppA SKO and lpp DKO mutants. Moreover, the lppB SKO mutant-infected mice had increased susceptibility to death. Since the lpp DKO mutant retained intact LPS, we constructed an S. enterica serovar Typhimurium triple-knockout (TKO) mutant in which the lppA and lppB genes were deleted from an existing msbB mutant (msbB encodes an enzyme required for the acylation of lipid A). Compared to the lpp DKO and msbB SKO mutants, the lpp-msbB TKO mutant was unable to induce cytotoxicity and to produce cytokines and chemokines in vitro and in vivo. These studies provided the first evidence of the relative contributions of Lpp and lipid A acylation to Salmonella pathogenesis.

The two murein lipoproteins of Salmonella enterica serovar Typhimurium contribute to the virulence of the organism.

Septic shock due to Salmonella and other gram-negative enteric pathogens is a leading cause of death worldwide. The role of lipopolysaccharide in sepsis is well studied; however, the contribution of other bacterial outer membrane components, such as Braun (murein) lipoprotein (Lpp), is not well defined. The genome of Salmonella enterica serovar Typhimurium harbors two copies of the lipoprotein (lpp) gene. We constructed a serovar Typhimurium strain with deletions in both copies of the lpp gene (lpp1 and lpp2) by marker exchange mutagenesis. The integrity of the cell membrane and the secretion of the effector proteins through the type III secretion system were not affected in the lpp double-knockout mutant. Subsequently, the virulence potential of this mutant was examined in a cell culture system using T84 intestinal epithelial and RAW264.7 macrophage cell lines and a mouse model of salmonellosis. The lpp double-knockout mutant was defective in invading and inducing cytotoxic effects in T84 and RAW264.7 cells, although binding of the mutant to the host cell was not affected when compared to the wild-type (WT) serovar Typhimurium. The motility of the mutant was impaired, despite the finding that the number of flagella was similar in the lpp double knockout mutant and the WT serovar Typhimurium. Deletion in the lpp genes did not affect the intracellular survival and replication of Salmonella in macrophages and T84 cells. Induction of the proinflammatory cytokines tumor necrosis factor alpha and interleukin-8 (IL-8) was significantly reduced in macrophages and T84 cells infected with the lpp double-knockout mutant. The levels of IL-8 remained unaffected in T84 cells when infected with either live or heat-killed WT and lpp mutant, indicating that invasion was not required for IL-8 production and that Toll-like receptor 2 signaling might be affected in the Lpp double-knockout mutant. These effects of the Lpp protein could be restored by complementation of the isogenic mutant. The lpp double-knockout mutant was avirulent in mice, and animals infected with this mutant were protected from a lethal challenge dose of WT serovar Typhimurium. The severe combined immunodeficient mice, on the other hand, were susceptible to infection by the lpp double-knockout mutant. The serovar Typhimurium mutants from which only one of the lpp (lpp1 or lpp2) genes was deleted were also avirulent in mice. Taken together, our data indicated that Lpp specifically contributed to the virulence of the organism.

Streptococcus pneumoniae PstS production is phosphate responsive and enhanced during growth in the murine peritoneal cavity.

Differential display-PCR (DDPCR) was used to identify a Streptococcus pneumoniae gene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment as pstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the complete pstS gene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identify pstS expression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation of pstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.

Identification and characterization of an in vivo regulated D15/Oma87 homologue in Shigella flexneri using differential display polymerase chain reaction.

Shigella genes expressed during infection likely contribute to adaptation and virulence in the host. Using differential display PCR (DDPCR), a cDNA fragment from Shigella flexneri serotype 5 that showed enhanced expression in a murine model was identified, cloned and sequenced. Enhanced expression was verified by RNA dot blot. The full-length gene was cloned using PCR and sequenced. The complete gene sequence was BLAST searched against GenBank, and exhibited strong homology to genes encoding Haemophilus influenzae D15 and Pasteurella multocida Oma87 protective outer membrane antigens. The S. flexneri gene putatively encodes a approximately 90-kDa protein and was termed oma90. The deduced amino acid sequence from oma90 was analyzed and compared to the D15/Oma87 antigens. Additionally, oma90 mapped to a cluster of orthologous groups, and probably contains an ancient conserved domain. The chromosomal organization of oma90 was similar to that for H. influenzae and P. multocida as well as for other known homologues. Northern blot revealed that the oma90 transcript encoded only oma90. This report represents the first description of a S. flexneri gene identified based on enhanced expression in the host. Furthermore, we report the first evidence demonstrating in vivo regulation of a member of the d15/oma87 gene family.

Peritoneal culture alters Streptococcus pneumoniae protein profiles and virulence properties.

We have examined the properties of Streptococcus pneumoniae cultured in the murine peritoneal cavity and compared its virulence-associated characteristics to those of cultures grown in vitro. Analysis of mRNA levels for specific virulence factors demonstrated a 2.8-fold increase in ply expression and a 2.2-fold increase in capA3 expression during murine peritoneal culture (MPC). Two-dimensional gels and immunoblots using convalescent-phase patient sera and murine sera revealed distinct differences in protein production in vivo (MPC). MPC-grown pneumococci adhered to A549 epithelial cell lines at levels 10-fold greater than those cultured in vitro.

Neutrophil activation by bacterial lipoprotein versus lipopolysaccharide: differential requirements for serum and CD14.

Neutrophil activation plays an important role in the inflammatory response to Gram-negative bacterial infections. LPS has been shown to be a major mediator of neutrophil activation which is accompanied by an early down-regulation of L-selectin and up-regulation of CD1lb/CD18. In this study, we investigated whether lipoprotein (LP), the most abundant protein in the outer membrane of bacteria from the family Enterobacteriaceae, can activate neutrophils and whether this activation is mediated by mechanisms that differ from those used by LPS or Escherichia coli diphosphoryl lipid A (EcDPLA). Neutrophil activation was assessed by measuring down-regulation of L-selectin and up-regulation of CD11b/CD18. When comparing molar concentrations of LP vs EcDPLA, LP was more potent (four times) at activating neutrophils. In contrast to LPS/EcDPLA, LP activation of neutrophils was serum independent. However, LP activation of neutrophils was enhanced by the addition of soluble CD14 and/or LPS-binding protein. In the presence of serum, LP activation of neutrophils was inhibited by different mAbs to CD14. This inhibition was significantly reduced or absent when performed in the absence of serum. Diphosphoryl lipid A from Rhodobacter spheroides (RaDPLA) completely inhibited LPS/EcDPLA activation of neutrophils but only slightly inhibited LP activation of neutrophils. These results suggest that LP activation of human neutrophils can be mediated by a mechanism that is different from LPS activation and that LP is a potentially important component in the development of diseases caused by Gram-negative bacteria of the family Enterobacteriaceae.

Role of Salmonella enterotoxin in overall virulence of the organism.

In this study, the Salmonella enterotoxin gene (stn) was mutated by marker exchange mutagenesis, and the overall virulence of the organism was evaluated. Salmonella marker exchange mutants evoked significantly less fluid secretion in mouse intestinal loops compared to that seen with wild-type S. typhimurium. Salmonella mutants were as invasive as wild-type bacteria for HeLa cells; however, their capacity to cause destruction of the intestinal mucosa was impaired, when compared with wild-type bacteria by electron microscopy. Upon oral challenge of mice, the LD(50)of the Salmonella mutants was greater than that for the wild-type bacteria. The fluid secretory potential, as well as a reduction in the LD(50)of these mutants was restored when the mutated stn gene was replaced by the native stn gene sequence. These mutations had no effect on the aerobic growth of these bacteria in minimal or complete medium; anaerobic growth was also not affected. With these studies, we demonstrated that the presence of an intact stn gene contributed significantly to the overall virulence of S. typhimurium in a murine model.

Lipoprotein release by bacteria: potential factor in bacterial pathogenesis.

Lipoprotein (LP) is a major component of the outer membrane of bacteria in the family Enterobacteriaceae. LP induces proinflammatory cytokine production in macrophages and lethal shock in LPS-responsive and -nonresponsive mice. In this study, the release of LP from growing bacteria was investigated by immuno-dot blot analysis. An immuno-dot blot assay that could detect LP at levels as low as 100 ng/ml was developed. By using this assay, significant levels of LP were detected in culture supernatants of growing Escherichia coli cells. During mid-logarithmic growth, approximately 1 to 1.5 microgram of LP per ml was detected in culture supernatants from E. coli. In contrast, these culture supernatants contained 5 to 6 microgram/ml of lipopolysaccharide (LPS). LP release was not unique to E. coli. Salmonella typhimurium, Yersinia enterocolitica, and two pathogenic E. coli strains also released LP during in vitro growth. Treatment of bacteria with the antibiotic ceftazidime significantly enhanced LP release. Culture supernatants from 5-h cultures of E. coli were shown to induce in vitro production of interleukin-6 (IL-6) by macrophages obtained from LPS-nonresponsive C3H/HeJ mice. In contrast, culture supernatants from an E. coli LP-deletion mutant were significantly less efficient at inducing IL-6 production in C3H/HeJ macrophages. These results suggest, for the first time, that LP is released from growing bacteria and that this released LP may play an important role in the induction of cytokine production and pathologic changes associated with gram-negative bacterial infections.

Bacterial lipoprotein and lipopolysaccharide act synergistically to induce lethal shock and proinflammatory cytokine production.

Septic shock is a major cause of death in the world. Although much is known about the role of LPS in septic shock, little is known about the role of other bacterial components. Lipoprotein (LP) is a major component of bacteria in the family Enterobacteriaceae. LP purified from Escherichia coli was shown to induce TNF-alpha and IL-6 production in peritoneal exudate macrophages obtained from LPS-responsive (C3H/HeOuJ) and LPS-nonresponsive (C3H/HeJ) mice. LP and LPS acted synergistically to induce cytokine production not only in C3H/HeOuJ macrophages but also in C3H/HeJ macrophages. These results suggest that LPS can induce cellular signaling in C3H/HeJ macrophages, and that LPS and LP activate macrophages via different receptors and/or signaling pathways. The role LP plays in septic shock was investigated using the mouse D-galactosamine model. LP induced lethal shock and in vivo production of TNF-alpha and IL-6 in both LPS-responsive and LPS-nonresponsive mice. LPS failed to induce lethal shock or in vivo cytokine production in C3H/HeJ mice. However, LP and LPS acted synergistically in inducing lethal shock and in vivo cytokine production in both LPS-responsive and LPS-nonresponsive mice. Finally, a heat-killed preparation of an E. coli mutant strain that lacked LP was shown to be less efficient than heat-killed wild-type E. coli at inducing lethal shock in C3H/HeJ mice. Collectively, these results suggest that LP and LPS induce cytokine production via different mechanisms and that LP plays an important role in septic shock induced by bacteria in the family Enterobacteriaceae.

Lipoprotein from Yersinia enterocolitica contains epitopes that cross-react with the human thyrotropin receptor.

Yersinia enterocolitica has recently been shown to produce a low molecular mass envelope protein that contains an epitope(s) that is cross-reactive with the extracellular domain of the human thyrotropin receptor (ETSHR). In this study, we have generated mAb to this cross-reactive protein and have obtained amino acid sequences for peptide fragments obtained from Lys-c digestion of the protein. The amino acid sequences of these peptides were identical to sequences present in bacterial lipoprotein (LP). All bacteria of the Enterobacteriaceae family produce LP as a major outer membrane protein. However, the ETSHR cross-reactive epitope(s) was shown to be unique to LP produced by Yersinia species. This was shown by Western blot analysis using a mAb specific for LP and with affinity-purified Ab specific for either LP or ETSHR and obtained from mouse antiserum generated to Y. enterocolitica. LPs from different Gram-negative bacteria were shown to be mitogenic for C3H/HeJ spleen cells and induced production and secretion of significant levels of Ig. Production of Ab that recognized the ETSHR was only induced in spleen cells stimulated with the LP obtained from Yersinia. In contrast, LP was not mitogenic for either human PBMC or human B cells. However, LP did induce IL6 and IL8 production in human monocytes at levels equivalent to that seen after LPS activation. These results identify, for the first time, the Yersinia envelope protein that is cross-reactive with the ETSHR and show that it can activate human monocytes. These findings are potentially important for advancing our understanding of the role molecular mimicry plays in the induction of autoimmunity to the thyrotropin receptor.

Treatment of HeLa cells with bacterial water extracts inhibits Shigella flexneri invasion.

Pathogenesis mediated by Shigella flexneri requires invasion of the gastrointestinal epithelium. It has been previously shown that HeLa cells challenged with S. flexneri show alterations in their phosphotyrosine-containing protein profile. In this report, we demonstrated that bacterial water extracts (WE) abrogated the invasion of HeLa cells by S. flexneri in a dose-dependent manner. A proteinaceous component of S. flexneri was shown to be responsible for this inhibitory activity. Proteins encoded on the 140-MDa plasmid were not responsible for the observed inhibition. WE from other Gram-negative bacteria also inhibited Shigella invasion of HeLa cells pretreated with WE showed changes in the profile and the intensity of phosphotyrosine-containing protein bands. These data were consistent with a surface protein component in WE which initiated aberrant host cell signaling at the membrane which may account for the inhibition of bacterial entry.

Yersinia enterocolitica envelope proteins that are crossreactive with the thyrotropin receptor (TSHR) also have B-cell mitogenic activity.

Autoantibodies to the thyrotropin receptor (TSHR) have been shown to mediate the hyperthyroidism associated with Graves' disease (GD). A number of hypotheses have been proposed which link an infectious agent to the mechanism(s) involved in the induction of GD. Several studies have suggested that the development of GD may be linked to infection with the enteric pathogen Yersinia enterocolitica. We have recently identified two low molecular weight (5.5 and 8 kDa) envelope proteins of Y. enterocolitica that are cross-reactive with the extracellular domain of human TSHR (ETSHR). In this study, we have purified these ETSHR-crossreactive Yersinia proteins (TSHR-CRP) and have further characterized their immunoreactivity. Both the 5.5 and 8 kDa TSHR-CRPs were shown to be mitogenic for mouse spleen cells. This mitogenic activity was specific for B cells and was not due to lipopolysaccharide (LPS) contamination. TSHR-CRPs were mitogenic for LPS-non-responsive spleen cells obtained from C3H/Hej mice, and polymyxin B did not inhibit the mitogenic activity of the TSHR-CRPs. TSHR-CRPs also induced high levels of IL-6 production in B cells and induced production and secretion of significant levels of IgG and IgM. Finally, culture supernatants from TSHR-CRP-stimulated spleen cells were shown by Western blot analysis to contain antibodies that recognized the ETSHR These results identify for the first time two envelope proteins of Yersinia that have mitogenic activity and therefore could represent important proteins involved in the pathogenesis of Yersinia infections. Because these mitogenic proteins also contain epitopes crossreactive with the TSHR, they are potentially important for advancing our understanding of the role molecular mimicry plays in the induction of autoimmunity to the TSHR.

A role for stem cell factor (SCF): c-kit interaction(s) in the intestinal tract response to Salmonella typhimurium infection.

Cholera toxin (CT) has been shown to induce stem cell factor (SCF) production in mouse ligated intestinal loops. Further, SCF interaction(s) with its receptor (c-kit) was shown to be important for the intestinal tract secretory response after CT exposure. In this study, we have investigated whether SCF production is induced in the intestinal tract after exposure to Salmonella typhimurium and whether this production could be an important intestinal tract response to Salmonella infection. Using a mouse ligated intestinal loop model, increased levels of SCF mRNA were detected at 2-4 h post-Salmonella challenge. Intestinal fluid obtained from Salmonella-challenged loops contained high levels of SCF by ELISA. Human and murine intestinal epithelial cell lines were also shown to have increased levels of SCF mRNA after exposure to Salmonella. Inhibition of Salmonella invasion of epithelial cells was shown to be one potentially important role for SCF:c-kit interactions in host defense to Salmonella infection. Pretreatment of human or murine intestinal cell lines with SCF resulted in a cellular state that was resistant to Salmonella invasion. Finally, mice having mutations in the white spotting (W) locus, which encodes the SCF-receptor (c-kit), were significantly more susceptible to oral Salmonella challenge than their control littermates. Taken together, the above results suggest that an important intestinal tract response to Salmonella infection is an enhanced production of SCF and its subsequent interactions with c-kit.

Human T-cell recognition of Listeria monocytogenes: recognition of listeriolysin O by TcR alpha beta + and TcR gamma delta + T cells.

The cell-mediated immune response to Listeria monocytogenes has been well characterized in the mouse. Listeriolysin O (LLO) is a major antigen in murine T-cell recognition of L. monocytogenes. In this study, we show that LLO is also recognized by human TcR alpha beta T cells and TcR gamma delta T cells. Human peripheral blood mononuclear cells (PBMC) cultured in vitro with live listeriae and then expanded with interleukin 2 were shown to respond to purified LLO. The generation of LLO-responsive T cells was dependent on the use of live bacteria during the initial in vitro challenge. LLO-induced proliferation of T cells expanded by exposure of PBMC to live listeriae was major histocompatibility complex restricted. PBMC cultured with formalin-fixed listeriae and subsequently expanded by interleukin 2 gave high proliferative responses to fixed bacteria but failed to respond to LLO. PBMC stimulated in vitro with fixed listeriae contained predominantly TcR alpha beta + T cells. In contrast, PBMC obtained from 85% of the donors studied generated high numbers of TcR gamma delta + T cells following in vitro culture with live listeriae. Using a panel of synthetic amphipathic LLO peptides, we found that LLO-specific T cells from different individuals recognized both common and unique peptides. LLO 470-508 was recognized by three of five individuals, while LLO 203-226 and LLO 107-126 were recognized by two of six individuals. A TcR gamma delta + T-cell line was established from PBMC stimulated with live listeriae and was shown to recognize LLO 470-508. Proliferative responses could be induced in this cell line by peptide-pulsed autologous PBMC but not by peptide-pulsed allogeneic PBMC. Our results establish the importance of LLO in human T-cell recognition of listeriae and show that both TcR alpha beta + T cells and TcR gamma delta + T cells recognize this antigen. Finally, since LLO 470-508 has a high degree of homology with other gram-positive bacterial toxins, the recognition of this peptide by TcR gamma delta + T cells suggests that an important role of these T cells in host defense is the recognition of bacterium-derived toxins.

Cholera toxin and Salmonella typhimurium induce different cytokine profiles in the gastrointestinal tract.

Salmonella infection of the gastrointestinal tract (GT) results in fluid secretion and inflammation. In contrast, cholera toxin (CT) induces fluid secretion but no inflammation. Using a murine ligated intestinal loop model, we investigated cytokine production (interleukin-1 [IL-1], IL-2, IL-4, IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha) in the GT following exposure to these agents. Salmonella typhimurium induced a Th1-like cytokine profile in loops obtained from either nonimmune mice or Salmonella-immunized mice. CT induced only IL-6 and IL-10 production in ligated loops from nonimmune mice but induced a Th2-like cytokine profile in ligated loops obtained from CT-immunized mice. These results show that CT and S. typhimurium induce very different cytokine profiles in the GT.

Shigella flexneri-HeLa cell interactions: a putative role for host cell protein kinases.

Epithelial cell invasion has been shown to be a prerequisite for Shigella flexneri virulence. Recently, we have documented the induction of transcription factor DNA binding activities as a result of S. flexneri challenge of HeLa cells. In this report, we show that HeLa cells challenged with S. flexneri display differences in phosphotyrosine-containing proteins. These changes are detected as early as 5 min post-challenge. Challenge with a noninvasive ipaB mutant strain resulted in the induction of a similar, but less intense, profile of phosphotyrosine-containing host cell proteins. Phosphotyrosine-containing proteins could be detected in S. flexneri, but were unique from those detected following HeLa cell challenge. S. flexneri invasion of HeLa cell monolayers was reduced by treatment with protein kinase inhibitors. These data suggest a role for protein kinases in the initial response of host cells to S. flexneri.

Purification and characterization of Yersinia enterocolitica envelope proteins which induce antibodies that react with human thyrotropin receptor.

Graves' disease is an autoimmune disease mediated by autoantibodies to the thyrotropin receptor (TSHR). Several studies have suggested that the development of Graves' disease may be linked to infection with the enteric pathogen Yersinia enterocolitica. Using the purified recombinant extracellular domain of human TSHR (ETSHR), we have recently shown that immunization of mice with Y. enterocolitica results in the production of antibodies capable of reacting with the ETSHR. In this study, we identify two low molecular weight (5.5 kDa and 8 kDa) envelope proteins of Yersinia containing epitopes that are crossreactive with the TSHR. Identification of these crossreactive envelope proteins was achieved by Western blotting using affinity-purified anti-Y. enterocolitica antibodies that specifically react with the TSHR and, conversely, for envelope proteins of Yersinia. Confirmation that these Yersinia proteins contained crossreactive epitopes with the ETSHR was obtained by immunizing mice with partially purified envelope proteins, which resulted in the production of Abs that recognized the ETSHR. Further, some of the cross-reactive envelope proteins were purified with SDS-PAGE and HPLC. The crossreactive envelope proteins were shown to be chromosomally encoded, exposed on the surface of bacteria, and produced by virulent as well as avirulent strains of Yersinia (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica VW+, and Y. enterocolitica VW-). These results identify for the first time the Yersinia envelope proteins that are crossreactive with the ETSHR. Availability of these proteins will allow future studies to determine whether patients with Graves' disease have a unique immune response against these proteins when compared with healthy individuals.

Shigella flexneri invasion of HeLa cells induces NF-kappa B DNA-binding activity.

Although information about the genetic basis and mechanisms of Shigella flexneri cellular invasion is accumulating, little is known about changes in cell signaling and their consequences following bacterium-host cell interactions. A general result of signal transduction is alterations in the levels and/or activities of transcription factors. Alterations in transcription factor binding activities were observed following challenge with S. flexneri. Changes in the DNA-binding activities of cellular transcription factors to AP1, AP2, cyclic AMP response element, CTF1/NF1, NF-kappa B/Rel, OCT1, and SP1 DNA-binding sites were investigated by electrophoretic mobility shift assays. NF-kappa B/Rel DNA-binding activity was enhanced more than 11-fold by cellular invasion; noninvasive S. flexneri strains induced low levels of kappa B DNA binding. Both subunits of the NF-kappa B transcription factor, p50 and p65, but not c-Rel (p85), are components of the kappa B DNA-binding activity. These data suggest that changes in cellular transcription factor binding activity are a consequence of S. flexneri invasion, and these changes could play a role in the initial host response or in the pathogenesis of the disease.

Tumor necrosis factor (TNF alpha) regulates intestinal mucus production during salmonellosis.

Mucus production by goblet cells in the gastrointestinal tract following Salmonella typhimurium infection using a ligated ileal loop model in mice was investigated. Assessment of the morphology of the loop tissue after Salmonella challenge revealed generalized tissue inflammation, characterized by edema and an inflammatory cell infiltrate. Villi were shortened and blunted, and crypts contained an increased number of cells with mitotic figures. Production of TNF alpha in the loops followed Salmonella challenge and occurred at the same time as the pathological sequelae. A nearly 50% decrease in the number of goblet cells in infected tissue compared to tissue from noninfected controls was observed at these same times. The sulfation of mucins produced by the goblet cells in infected tissues was increased in the villi but was unchanged in the crypts compared to uninfected tissues. Treatment of mice with antibody to TNF alpha before Salmonella challenge abrogated tissue pathology and returned goblet cell numbers and mucin profiles to those observed in noninfected controls. Our results indicate that TNF alpha may mediate changes in goblet cell expression and mucin sulfation in response to Salmonella challenge.