A novel toxin-antitoxin system swpAB alters gene expression patterns and reduces virulence expression in enterohemorrhagic Escherichia coli.

Toxin-antitoxin (TA) systems are found widely among many bacteria, including enterohemorrhagic Escherichia coli (EHEC), but their functions are still poorly understood. In this study, we identified and characterized a novel TA system belonging to the relBE family, classified as a type II TA system, found in EHEC. The protein encoded by the toxin gene is homologous to RelE ribonuclease. Using various conditions for increasing the toxin activity, high-level induction of a toxin gene, and repression of an antitoxin gene in wild-type EHEC, we showed that the TA system, named swpAB (switching of gene expression profile), is involved in selective repression of a set of genes, including some virulence genes, and in the reduction of adherence capacity, rather than in suppression of bacterial growth. A detailed analysis of the profiles of RNA levels along sequences at 15 min after high expression of swpA revealed that two virulence genes, espA and tir, were direct targets of the SwpA toxin. These results suggested that the swpAB system can alter gene expression patterns and change bacterial physiological activity without affecting bacterial growth.

SlyA regulates virulence gene expressions through activation of pchA regulatory gene in enterohemorrhagic Escherichia coli.

SlyA is a DNA-binding protein that alters the nucleoid complex composed of histone-like nucleoid-structuring protein (H-NS) and activates gene expression. In enterohemorrhagic Escherichia coli (EHEC), the expression of virulence genes is repressed by H-NS but is up-regulated in response to environmental factors by releasing a nucleoid complex. This study examined the effect of slyA deletion mutation in EHEC and discovered that the production of the locus of enterocyte effacement (LEE)-encoded EspB and Tir, as well as the cell adherence ability, was reduced in the mutant compared with the wild type. The promoter activity of the LEE1 operon, including the regulatory gene, ler, was reduced by slyA mutation, but tac promoter-controlled expression of pchA, which is a regulatory gene of LEE1, abolished the effect. The promoter activity of pchA was down-regulated by the slyA mutation. Furthermore, the coding region was required for its regulation and was bound to SlyA, which indicates the direct regulation of pchA by SlyA. However, the slyA mutation did not affect the butyrate-induced increase in pchA promoter activity. Additionally, the pchA promoter activity was increased via induction of lrp, a regulatory gene for butyrate response, in the slyA mutant and, conversely, by introducing high copies of slyA into the lrp mutant. These results indicate that SlyA is a positive regulator of pchA and is independent of the Lrp regulatory system. SlyA may be involved in the virulence expression in EHEC, maintaining a certain level of expression in the absence of a butyrate response.

Multicenter prospective observational study of fungal keratitis in Japan: analyses of in vitro susceptibility tests for combinations of drugs.

PURPOSE: To determine the effects of a combination of two antifungal drugs against causative fungi of fungal keratitis in Japan. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eighteen isolates of yeast-like fungi and 22 isolates of filamentous fungi collected by the Multicenter Prospective Observational Study of Fungal Keratitis in Japan were studied. Specially manufactured minimum inhibitory concentration (MIC) measurement plates were used to test the effectiveness of 10 combinations of two antifungal drugs against the isolates. The combinations were pimaricin (PMR) + voriconazole (VRCZ), PMR + fluconazole (FLCZ), PMR + miconazole (MCZ), PMR + micafungin (MCFG), VRCZ + FLCZ, VRCZ + MCZ, VRCZ + MCFG, VRCZ + amphotericin-B (AMPH-B), MCZ + FLCZ, and MCZ + MCFG. The checkerboard microdilution method was used, and the fractional inhibitory concentration (FIC) index was calculated based on the guidelines of The Clinical & Laboratory Standards Institute (CLSI). RESULTS: In yeast-like fungi, additive effects were observed between PMR and MCFG in 77.8% of the isolates, and they were also observed between the azoles. Synergistic effects were observed on 11.1% of the isolates for MCZ and FLCZ. On the other hand, antagonistic effects were present between PMR and azoles with 88.9% between PMR and VRCZ, 72.2% between PMR and FLCZ, and 94.4% between PMR and MCZ. In filamentous fungi, additive effects were observed between PMR and MCFG in 40.9% of the isolates, and between VRCZ and MCZ in 40.9% of the isolates. Antagonistic effects were observed for PMR and the azoles. CONCLUSIONS: The combination of drugs prescribed for fungal keratitis incurs a possibility of synergistic, additive, indifferent, or antagonistic effects, depending on drug combinations and fungal strains.

Enterococcus spp. have higher fitness for survival, in a pH-dependent manner, in pancreatic juice among duodenal bacterial flora.

BACKGROUND AND AIM: Bacterial infection is involved in the progression of many gastrointestinal diseases, including those of pancreas; however, how and which bacteria colonize in pancreatic juice and tissue have yet to be elucidated. Recently, we reported that Enterococcus faecalis exists in the pancreatic juice and tissues of patients with chronic pancreatic disease. Here, we investigated the survival of E. faecalis in duodenal juice with different pH conditions. METHODS: Pancreatic juice samples from 62 patients with cancers of the duodeno-pancreato-biliary region were evaluated for the presence of E. faecalis. 16S ribosomal RNA polymerase chain reaction and 16S-based metagenome analyses were performed to determine the bacterial composition. The survival of E. faecalis in various pancreatic juice conditions was evaluated. RESULTS: Of 62 samples, 27% (17/62) were positive for Enterococcus spp., among which 71% (12/17) contained E. faecalis. Enterococcus spp. showed the highest fitness for survival in alkaline pancreatic juice among various bacterial species. The microbiome of pancreatic juice from patients with pancreatic and bile duct cancer showed diversity, but Enterococcus spp. were enriched among duodenal tumors and intraductal papillary mucinous neoplasms. CONCLUSIONS: Alkalinity is one of the important factors for the selective survival of E. faecalis among microbiota. E. faecalis can colonize the pancreatic duct when the pancreatic juice condition is altered.

Enterohaemorrhagic Escherichia coli activates nitrate respiration to benefit from the inflammatory response for initiation of microcolony-formation.

BACKGROUND: For successful colonization, enterohaemorrhagic Escherichia coli (EHEC) injects virulence factors, called effectors, into target cells through the type three secretion system (T3SS), which is composed of a needle and basal body. Under anaerobic conditions, the T3SS machinery remains immature and does not have a needle structure. However, activation of nitrate respiration enhances the completion of the T3SS machinery. Because nitric oxide released by the host inflammatory response increases nitrate concentration, we sought to determine the effect of the inflammatory response on initiation of EHEC microcolony-formation. RESULTS: The colony-forming capacity was increased in accordance with the increase of nitrate in the medium. The addition of the nitric oxide-producing agent NOR-4 also enhanced the adherence capacity, which was dependent on nitrate reductase encoded by the narGHJI genes. Culture supernatant of epithelial cells, which was stimulated by a cytokine mixture, enhanced the colony-forming capacity of wild-type EHEC but not of the narGHJI mutant. Finally, colony formation by wild-type EHEC on epithelial cells, which were preincubated with heat-killed bacteria, was higher than the narGHJI mutant, and this effect was abolished by aminoguanidine hydrochloride, which is an iNOS (inducible nitric oxide synthase) inhibitor. CONCLUSIONS: These results indicate that the inflammatory response enhances EHEC adherence by increasing nitrate concentration.

Risk for the occupational infection by cytomegalovirus among health-care workers.

BACKGROUND: Cytomegalovirus (CMV) are ubiquitously distributed worldwide, causing a wide range of clinical manifestations from congenital infection to a life-threatening disease in immunocompromised individuals. CMV can be transmitted via human-to-human contact through body fluids; however, the risk of CMV infection among healthcare workers (HCWs) has not been fully evaluated. AIM: This study aimed to assess the risk of CMV infection among HCWs through daily medical practices. METHODS: Serum samples from HCWs at Osaka University Hospital (Japan) were analysed. Initially, we compared CMV IgG seropositivity among HCWs (medical doctors, nurses, and others) in 2017, which was examined after 1 year to evaluate seroconversion rates among those with seronegative results. Then, we examined CMV seroconversion rates in HCWs who were exposed to blood and body fluids. FINDINGS: We analysed 1153 samples of HCWs (386 medical doctors, 468 nurses, and 299 others), of which CMV seropositivity rates were not significantly different (68.9%, 70.3%, and 70.9%, respectively). Of these, 63.9% (221/346) of CMV seronegative HCWs were followed after 1 year, with CMV seroconversion rates of 3.2% (7/221). Among 72 HCWs who tested negative for CMV IgG when exposed to blood and body fluids, the CMV seroconversion rate was 2.8% (2/72). The CMV seroconversion rates between the two situations were not significantly different. CONCLUSION: Our study indicated that CMV infection through daily patient care seems quite rare. Further well-designed studies with a large sample size are warranted to verify our finding.

Epidemiological and molecular characterization of invasive Streptococcus pneumoniae isolated following introduction of 7-valent conjugate vaccine in Kinki region, Japan, 2008-2013.

Streptococcus pneumoniae is one of the most common bacteria causing community-acquired pneumonia and meningitis. The use of 7-valent pneumococcal conjugate vaccine (PCV7) has reduced the incidence of pneumococcal disease while changing pneumococcal population through herd immunity and non-vaccine pneumococci replacement. This study investigated molecular epidemiologic characteristics of pneumococcal strains in the Kinki region of Japan from 2008 to 2013. A total of 159 invasive pneumococcal isolates were characterized by serotyping, antibiotic susceptibility testing, PCR analysis of penicillin-binding protein genes, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). In adult populations, pediatric PCV7 introduction decreased isolates expressing PCV7 serotypes via herd immunity and increased isolates expressing non-PCV7 serotypes. The rate of penicillin resistance and isolates with alterations in all three pbp genes was higher in PCV7 type isolates than in non-PCV7 type isolates. In MLST analysis, all of serotype 19F isolates were of the same sequence type, ST236, which is the antimicrobial-resistant clone Taiwan19F-14, and the majority of serotypes 23F and 19A isolates were of ST1437 and ST3111 respectively, which are the predominant clones of antimicrobial-resistant pneumococci in Japan. In PFGE profiles, serotype 6B-ST2224, serotype 19F-ST236, serotype 19A-ST3111, and serotype 23F-ST1437 formed six separate clusters composed of genetically identical strains, and genetically identical serotype 22F-ST433 formed two different clusters between the pre- and post-PCV7 period. The results of molecular analysis suggest the spread and persistence of these identical antimicrobial resistant clones in the Kinki region and genetic changes of epidemic clone serotype 22F-ST433 before and after pediatric PCV7 introduction.

Possible involvement of Enterococcus infection in the pathogenesis of chronic pancreatitis and cancer.

(Aim) Bacterial infection underlies the pathogenesis of many human diseases, including acute and chronic inflammation. Here, we investigated a possible role for bacterial infection in the progression of chronic pancreatitis. (Materials and Methods) Pancreatic juice was obtained from patients with pancreatic cancer (n = 20) or duodenal cancer/bile duct cancer (n = 16) and subjected to PCR using universal primers for the bacterial 16S ribosomal RNA gene. Bacterial species were identified by PCR using bile samples from four pancreatic cancer patients. PCR products were subcloned into T-vectors, and the sequences were then analyzed. Immunohistochemical and serologic analyses for Enterococcus faecalis infection were performed on a large cohort of healthy volunteers and patients with chronic pancreatitis or pancreatic cancer and on mice with caerulein-induced chronic pancreatitis. The effect of E. faecalis antigens on cytokine secretion by pancreatic cancer cells was also investigated. (Results) We found that 29 of 36 pancreatic juice samples were positive for bacterial DNA. Enterococcus and Enterobacter species were detected primarily in bile, which is thought to be a pathway for bacterial infection of the pancreas. Enterococcus faecalis was also detected in pancreatic tissue from chronic pancreatitis and pancreatic cancer patients; antibodies to E. faecalis capsular polysaccharide were elevated in serum from chronic pancreatitis patients. Enterococcus-specific antibodies and pancreatic tissue-associated E. faecalis were detected in mice with caerulein-induced chronic pancreatitis. Addition of Enterococcus lipoteichoic acid and heat-killed bacteria induced expression of pro-fibrotic cytokines by pancreatic cancer cells in vitro. (Conclusion) Infection with E. faecalis may be involved in chronic pancreatitis progression, ultimately leading to development of pancreatic cancer.

Laboratory surveillance of antimicrobial resistance and multidrug resistance among Streptococcus pneumoniae isolated in the Kinki region of Japan, 2001-2015.

The 7-valent pneumococcal conjugate vaccine (PCV7) was introduced among children in Japan in 2010. There are no long-term multicenter surveillance studies of antimicrobial resistance in S. pneumoniae before and after the introduction of PCV7. Therefore, we examined chronological trends in antimicrobial resistance among 4534 strains of S. pneumoniae isolated from both children and adults in the Kinki region of Japan during 2001-2015. High-level penicillin and third-generation cephalosporin resistance in S. pneumoniae increased among both children and adults during the period before the introduction of PCV7 (2001-2010). Besides penicillin and cephalosporin, pneumococcal carbapenem and macrolide resistance increased among children. The rate of resistance to these antibiotics was higher among children than among adults. The introduction of PCV7 decreased the rate of non-susceptibility to ß-lactam antibiotics and the rate of multidrug resistant S. pneumoniae among children, but not among adults.

Activation of lpxR gene through enterohaemorrhagic Escherichia coli virulence regulators mediates lipid A modification to attenuate innate immune response.

During the course of infection, pathogens must overcome a variety of host defence systems. Modulation of lipid A, which is a strong stimulant for host immune systems, is one of the strategies used by microorganisms to evade the host response. The lpxR gene, which encodes a lipid A 3'-O-deacylase, is commonly found in several pathogens and has been shown to reduce the inflammatory response. Here, we demonstrated that the lpxR gene of enterohaemorrhagic Escherichia coli (EHEC) was positively regulated by two virulence regulators, Pch and Ler, and that this regulation was coordinated with the locus of enterocyte effacement genes, which encode major virulence factors for colonisation. The lpxR promoter was repressed by the binding of H-NS, but the competitive binding of both regulators resulted in transcription activation. Next, we showed that lipid A from the lpxR mutant was more stimulatory of the inflammatory response in macrophage-like cells than lipid A from wild-type EHEC. Furthermore, phagocytic activity and phagosome maturation in host cells infected with the lpxR mutant were increased in a p38 mitogen-activated protein kinase-dependent manner in comparison with wild-type EHEC infection. Finally, we demonstrated that the pch mutant, which is deficient in activation of the locus of enterocyte effacement genes, was phagocytised more efficiently than the wild type. Thus, EHEC modulates lipid A to dampen the host immune response when activating virulence genes for colonisation.

Enterohaemorrhagic Escherichia coli produces outer membrane vesicles as an active defence system against antimicrobial peptide LL-37.

Antimicrobial peptides (AMPs) are important components of the innate immune system. Enterohaemorrhagic Escherichia coli (EHEC), a food-borne pathogen causing serious diarrheal diseases, must overcome attack by AMPs. Here, we show that resistance of EHEC against human cathelicidin LL-37, a primary AMP, was enhanced by butyrate, which has been shown to act as a stimulant for the expression of virulence genes. The increase of resistance depended on the activation of the ompT gene, which encodes the outer membrane protease OmpT for LL-37. The expression of the ompT gene was enhanced through the activation system for virulence genes. The increase in ompT expression did not result in an increase in OmpT protease in bacteria but in enhancement of the production of OmpT-loaded outer membrane vesicles (OMVs), which primarily contributed to the increase in LL-37-resistance. Furthermore, a sublethal dosage of LL-37 stimulated the production of OMVs. Finally, we showed that OMVs produced by OmpT-positive strains protect the OmpT-negative strain, which is susceptible to LL-37 by itself more efficiently than OMVs from the ompT mutant. These results indicate that EHEC enhances the secretion of OmpT-loaded OMVs in coordination with the activation of virulence genes during infection and blocks bacterial cell attack by LL-37.

Modulation of the Inflammasome Signaling Pathway by Enteropathogenic and Enterohemorrhagic Escherichia coli.

Innate immunity is an essential component in the protection of a host against pathogens. Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are known to modulate the innate immune responses of infected cells. The interference is dependent on their type III secretion system (T3SS) and T3SS-dependent effector proteins. Furthermore, these cytosolically injected effectors have been demonstrated to engage multiple immune signaling pathways, including the IFN/STAT, MAPK, NF-κB, and inflammasome pathways. In this review, recent work describing the interaction between EPEC/EHEC and the inflammasome pathway will be discussed.

Gene Activation through the Modulation of Nucleoid Structures by a Horizontally Transferred Regulator, Pch, in Enterohemorrhagic Escherichia coli.

The horizontally transferred chromosomal segments, which are the main source of genetic diversity among bacterial pathogens, are bound by the nucleoid protein H-NS, resulting in the formation of a nucleoprotein complex and the silencing of gene expression. The de-silencing or activation of virulence genes necessary for the colonization of enterohemorrhagic Escherichia coli is achieved mainly by the action of two regulators, Pch and Ler, which are encoded by horizontally transferred elements. Although Ler has been shown to activate transcription by counteracting H-NS silencing, the mechanism for Pch is poorly understood. We show here that Pch activates the LEE1 promoter and also enhances the Ler-mediated activation of other LEE promoters. Transcriptional activation was completely dependent on repression by the H-NS/StpA/Hha/YdgT complex, indicating that Pch-derived activation was achieved by alleviating H-NS-mediated silencing. Expression of pch reduced the binding of H-NS at LEE1 promoter and altered the nucleoprotein complex. Furthermore, in vitro reconstruction of the protein-DNA complex on LEE1 promoter DNA confirmed the exclusive effect of Pch on H-NS binding. These results demonstrated that Pch is another anti-silencing regulator and a modulator of H-NS-containing nucleoprotein complexes. Thus, the anti-silencing mechanism plays a key role in the coordinated regulation of virulence genes in EHEC.

H-NS Facilitates Sequence Diversification of Horizontally Transferred DNAs during Their Integration in Host Chromosomes.

Bacteria can acquire new traits through horizontal gene transfer. Inappropriate expression of transferred genes, however, can disrupt the physiology of the host bacteria. To reduce this risk, Escherichia coli expresses the nucleoid-associated protein, H-NS, which preferentially binds to horizontally transferred genes to control their expression. Once expression is optimized, the horizontally transferred genes may actually contribute to E. coli survival in new habitats. Therefore, we investigated whether and how H-NS contributes to this optimization process. A comparison of H-NS binding profiles on common chromosomal segments of three E. coli strains belonging to different phylogenetic groups indicated that the positions of H-NS-bound regions have been conserved in E. coli strains. The sequences of the H-NS-bound regions appear to have diverged more so than H-NS-unbound regions only when H-NS-bound regions are located upstream or in coding regions of genes. Because these regions generally contain regulatory elements for gene expression, sequence divergence in these regions may be associated with alteration of gene expression. Indeed, nucleotide substitutions in H-NS-bound regions of the ybdO promoter and coding regions have diversified the potential for H-NS-independent negative regulation among E. coli strains. The ybdO expression in these strains was still negatively regulated by H-NS, which reduced the effect of H-NS-independent regulation under normal growth conditions. Hence, we propose that, during E. coli evolution, the conservation of H-NS binding sites resulted in the diversification of the regulation of horizontally transferred genes, which may have facilitated E. coli adaptation to new ecological niches.

Enteropathogenic Escherichia coli Uses NleA to Inhibit NLRP3 Inflammasome Activation.

Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC) are related strains capable of inducing severe gastrointestinal disease. For optimal infection, these pathogens actively modulate cellular functions through the deployment of effector proteins in a type three secretion system (T3SS)-dependent manner. In response to enteric pathogen invasion, the Nod-like receptor pyrin domain containing (NLRP) inflammasome has been increasingly recognized as an important cytoplasmic sensor against microbial infection by activating caspase-1 and releasing IL-1ß. EPEC and EHEC are known to elicit inflammasome activation in macrophages and epithelial cells; however, whether the pathogens actively counteract such innate immune responses is unknown. Using a series of compound effector-gene deletion strains of EPEC, we screened and identified NleA, which could subdue host IL-1ß secretion. It was found that the reduction is not because of blocked NF-κB activity; instead, the reduction results from inhibited caspase-1 activation by NleA. Immunostaining of human macrophage-like cells following infection revealed limited formation of inflammasome foci with constituents of total caspase-1, ASC and NLRP3 in the presence of NleA. Pulldown of PMA-induced differentiated THP-1 lysate with purified MBP-NleA reveals that NLRP3 is a target of NleA. The interaction was verified by an immunoprecipitation assay and direct interaction assay in which purified MBP-NleA and GST-NLRP3 were used. We further showed that the effector interacts with regions of NLRP3 containing the PYD and LRR domains. Additionally, NleA was found to associate with non-ubiquitinated and ubiquitinated NLRP3 and to interrupt de-ubiquitination of NLRP3, which is a required process for inflammasome activation. Cumulatively, our findings provide the first example of EPEC-mediated suppression of inflammasome activity in which NieA plays a novel role in controlling the host immune response through targeting of NLRP3.

The dynamic balance of import and export of zinc in Escherichia coli suggests a heterogeneous population response to stress.

Zinc is essential for life, but toxic in excess. Thus all cells must control their internal zinc concentration. We used a systems approach, alternating rounds of experiments and models, to further elucidate the zinc control systems in Escherichia coli. We measured the response to zinc of the main specific zinc import and export systems in the wild-type, and a series of deletion mutant strains. We interpreted these data with a detailed mathematical model and Bayesian model fitting routines. There are three key findings: first, that alternate, non-inducible importers and exporters are important. Second, that an internal zinc reservoir is essential for maintaining the internal zinc concentration. Third, our data fitting led us to propose that the cells mount a heterogeneous response to zinc: some respond effectively, while others die or stop growing. In a further round of experiments, we demonstrated lower viable cell counts in the mutant strain tested exposed to excess zinc, consistent with this hypothesis. A stochastic model simulation demonstrated considerable fluctuations in the cellular levels of the ZntA exporter protein, reinforcing this proposal. We hypothesize that maintaining population heterogeneity could be a bet-hedging response allowing a population of cells to survive in varied and fluctuating environments.

LeuO enhances butyrate-induced virulence expression through a positive regulatory loop in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea and other severe symptoms such as haemorrhagic uraemic syndrome. The expression of virulence genes on the locus for enterocyte effacement (LEE) and associated genes is regulated by a variety of factors, including transcriptional regulators and environmental signals. Butyrate, one of the major short-chain fatty acids present in the intestine, enhances expression of LEE genes and flagella biosynthesis genes in EHEC O157:H7, resulting in increased bacterial adherence and motility. Here, we show that expression of the leuO gene, which encodes a LysR-type transcriptional regulator, is enhanced by butyrate via Lrp, which is also necessary for butyrate-induced responses of LEE genes. LeuO expression induces prolonged activation of the promoter of LEE1 operon, including the ler gene, as well as virulence mechanisms such as microcolony formation. Activation of the LEE1 promoter by LeuO depends on another regulator, called Pch. The response of the leuO promoter to butyrate requires two virulence regulators, Pch and Ler, in addition to Lrp. Pch, Ler and Lrp bind the upstream region of the leuO promoter. Thus, leuO is involved in butyrate-enhanced expression of LEE genes through a positive feedback mechanism, but its expression and action on the LEE1 promoter are dependent on the virulence regulators Pch and Ler.

Antisense transcription regulates the expression of the enterohemorrhagic Escherichia coli virulence regulatory gene ler in response to the intracellular iron concentration.

Enteric pathogens, such as enterohemorrhagic E. coli (EHEC) O157:H7, encounter varying concentrations of iron during their life cycle. In the gastrointestinal tract, the amount of available free iron is limited because of absorption by host factors. EHEC and other enteric pathogens have developed sophisticated iron-responsive systems to utilize limited iron resources, and these systems are primarily regulated by the Fur repressor protein. The iron concentration could be a signal that controls gene expression in the intestines. In this study, we explored the role of iron in LEE (locus for enterocyte effacement) virulence gene expression in EHEC. In contrast to the expression of Fur-regulated genes, the expression of LEE genes was greatly reduced in fur mutants irrespective of the iron concentration. The expression of the ler gene, the LEE-encoded master regulator, was affected at a post-transcription step by fur mutation. Further analysis showed that the loss of Fur affected the translation of the ler gene by increasing the intracellular concentration of free iron, and the transcription of the antisense strand was necessary for regulation. The results indicate that LEE gene expression is closely linked to the control of intracellular free iron homeostasis.

DNA looping-dependent autorepression of LEE1 P1 promoters by Ler in enteropathogenic Escherichia coli (EPEC).

Ler, a homolog of H-NS in enteropathogenic Escherichia coli (EPEC), plays a critical role in the expression of virulence genes encoded by the pathogenic island, locus of enterocyte effacement (LEE). Although Ler acts as an antisilencer of multiple LEE operons by alleviating H-NS-mediated silencing, it represses its own expression from two LEE1 P1 promoters, P1A and P1B, that are separated by 10 bp. Various in vitro biochemical methods were used in this study to elucidate the mechanism underlying transcription repression by Ler. Ler acts through two AATT motifs, centered at position -111.5 on the coding strand and at +65.5 on the noncoding strand, by simultaneously repressing P1A and P1B through DNA-looping. DNA-looping was visualized using atomic force microscopy. It is intriguing that an antisilencing protein represses transcription, not by steric exclusion of RNA polymerase, but by DNA-looping. We propose that the DNA-looping prevents further processing of open promoter complex (RPO) at these promoters during transcription initiation.