Ching Hao Teng

Escherichia coli K1 RS218 Interacts with Human Brain Microvascular Endothelial Cells via Type 1 Fimbria Bacteria in the Fimbriated State

ABSTRACT Escherichia coli K1 is a major gram-negative organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMEC) are a prerequisite for E. coli penetration into the central nervous system in vivo. In the present study, we showed using DNA microarray analysis that E. coli K1 associated with HBMEC expressed significantly higher levels of the fim genes compared to nonassociated bacteria. We also showed that E. coli K1 binding to and invasion of HBMEC were significantly decreased with its fimH deletion mutant and type 1 fimbria locked-off mutant, while they were significantly increased with its type 1 fimbria locked-on mutant. E. coli K1 strains associated with HBMEC were predominantly type 1 fimbria phase-on (i.e., fimbriated) bacteria. Taken together, we showed for the first time that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMEC and that type 1 fimbria phase-on E. coli is the major population interacting with HBMEC.

Effects of ompA Deletion on Expression of Type 1 Fimbriae in Escherichia coli K1 Strain RS218 and on the Association of E. coli with Human Brain Microvascular Endothelial Cells

ABSTRACT We have previously shown that outer membrane protein A (OmpA) and type 1 fimbriae are the bacterial determinants involved in Escherichia coli K1 binding to human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. In investigating the role of OmpA in E. coli K1 binding to HBMEC, we showed for the first time that ompA deletion decreased the expression of type 1 fimbriae in E. coli K1. Decreased expression of type 1 fimbriae in the ompA deletion mutant was largely the result of driving the fim promoter toward the type 1 fimbrial phase-OFF orientation. mRNA levels of fimB and fimE were found to be decreased with the OmpA mutant compared to the parent strain. Of interest, the ompA deletion further decreased the abilities of E. coli K1 to bind to and invade HBMEC under the conditions of fixing type 1 fimbria expression in the phase-ON or phase-OFF status. These findings suggest that the decreased ability of the OmpA mutant to interact with HBMEC is not entirely due to its decreased type 1 fimbrial expression and that OmpA and type 1 fimbriae facilitate the interaction of E. coli K1 with HBMEC at least in an additive manner.

HbiF regulates type 1 fimbriation independently of FimB and FimE

ABSTRACT Type 1 fimbriae have been suggested to play a role in the pathogenesis of extraintestinal Escherichia coli infection. Type 1 fimbriation in E. coli is phase variable and known to be dependent upon FimB and FimE, the two recombinases that invert the molecular switch fimS and control the expression of the downstream fim operon. Here we showed that HbiF, a novel site-specific recombinase, inverted fimS independently of FimB and FimE. HbiF-mediated fimS inversion appeared to be predominantly switching from “off” (termed OFF) to “on” (termed ON) orientation. This is different from the fimS inversion mediated by either FimB (bidirectional ON to OFF and OFF to ON) or FimE (unidirectional ON to OFF). Constitutive expression of the hbiF gene in E. coli resulted in a fimS-locked-ON phenotype, which resulted in the pathogenic E. coli K1 strain being incapable of inducing a high degree of bacteremia in neonatal rats. Discovery of HbiF-mediated OFF-to-ON fimS switching provides a new opportunity to develop a strategy for the prevention and therapy of extraintestinal E. coli infection including bacteremia and meningitis.

Loss of Outer Membrane Protein C in Escherichia coli Contributes to Both Antibiotic Resistance and Escaping Antibody-Dependent Bactericidal Activity

ABSTRACT Outer membrane proteins (OMPs) serve as the permeability channels for nutrients, toxins, and antibiotics. In Escherichia coli, OmpA has been shown to be involved in bacterial virulence, and OmpC is related to multidrug resistance. However, it is unclear whether OmpC also has a role in the virulence of E. coli. The aims of this study were to characterize the role of OmpC in antimicrobial resistance and bacterial virulence in E. coli. The ompC deletion mutant showed significantly decreased susceptibility to carbapenems and cefepime. To investigate the survival of E. coli exposed to the innate immune system, a human blood bactericidal assay showed that the ompC mutant increased survival in blood and serum but not in complement-inactivated serum. These effects were also demonstrated in the natural selection of OmpC mutants. Also, C1q interacted with E. coli through a complex of antibodies bound to OmpC as a major target. Bacterial survival was increased in the wild-type strain in a dose-dependent manner by adding free recombinant OmpC protein or anti-C1q antibody to human serum. These results demonstrated that the interaction of OmpC-specific antibody and C1q was the key step in initiating the antibody-dependent classical pathway for the clearance of OmpC-expressing E. coli. Anti-OmpC antibody was detected in human sera, indicating that OmpC is an immunogen. These data indicate that the loss of OmpC in E. coli is resistant to not only antibiotics, but also the serum bactericidal effect, which is mediated from the C1q and anti-OmpC antibody-dependent classical pathway.

NlpI contributes to Escherichia coli K1 strain RS218 interaction with human brain microvascular endothelial cells

ABSTRACT Escherichia coli K1 is the most common Gram-negative bacillary organism causing neonatal meningitis. E. coli K1 binding to and invasion of human brain microvascular endothelial cells (HBMECs) is a prerequisite for its traversal of the blood-brain barrier (BBB) and penetration into the brain. In the present study, we identified NlpI as a novel bacterial determinant contributing to E. coli K1 interaction with HBMECs. The deletion of nlpI did not affect the expression of the known bacterial determinants involved in E. coli K1-HBMEC interaction, such as type 1 fimbriae, flagella, and OmpA, and the contribution of NlpI to HBMECs binding and invasion was independent of those bacterial determinants. Previous reports have shown that the nlpI mutant of E. coli K-12 exhibits growth defect at 42°C at low osmolarity, and its thermosensitive phenotype can be suppressed by a mutation on the spr gene. The nlpI mutant of strain RS218 exhibited similar thermosensitive phenotype, but additional spr mutation did not restore the ability of the nlpI mutant to interact with HBMECs. These findings suggest the decreased ability of the nlpI mutant to interact with HBMECs is not associated with the thermosensitive phenotype. NlpI was determined as an outer membrane-anchored protein in E. coli, and the nlpI mutant was defective in cytosolic phospholipase A2α (cPLA2α) phosphorylation compared to the parent strain. These findings illustrate the first demonstration of NlpIs contribution to E. coli K1 binding to and invasion of HBMECs, and its contribution is likely to involve cPLA2α.

Bacterial characteristics and glycemic control in diabetic patients with Escherichia coli urinary tract infection

BACKGROUND Patients with diabetes mellitus have an increased risk of infection. The roles of bacterial characteristics and glycemic control in diabetic patients with Escherichia coli infection have not been well investigated. The aims of this study were to examine the bacterial characteristics and glycemic control in diabetic patients with E. coli infections arising in the urinary tract. METHODS A total of 271 E. coli isolates were collected from urine and bloodstream. Phylogenetic groups, the presence of virulence genes, and antimicrobial susceptibility of E. coli isolates were determined. RESULTS There were few differences in E. coli bacterial characteristics between 190 diabetic and 81 nondiabetic patients. In diabetic patients with urosepsis, there was a higher hemoglobin A(1C) level, and the related E. coli strains had more neuA, papG II, afa and hlyA genes, and a lower prevalence of antimicrobial resistance to cephalosporins and fluoroquinolones than those with asymptomatic bacteriuria and urinary tract infection. Multivariate logistic regression analysis revealed that increased hemoglobin A(1C) and presence of papG II and afa genes were independent factors associated with development of urosepsis in diabetic patients. CONCLUSION This study demonstrated that more virulent E. coli isolates, especially with papG II and afa genes, and poorer glycemic control were important determinants for development of urosepsis in diabetic patients.

Clinical and Microbiological Characteristics of Community-Acquired Staphylococcus lugdunensis Infections in Southern Taiwan

ABSTRACT Most Staphylococcus lugdunensis strains (49/59, 83%) were related to clinical infections, were susceptible to most antimicrobial agents with an overall oxacillin-resistant rate of 5% (3/58), and carried relatively great genetic diversity. Community-acquired infections (41/49, 84%) were dominant, often developed in patients with comorbidity, and had rather benign clinical courses without mortality.

Identification of Escherichia coli Genes Associated with Urinary Tract Infections

ABSTRACT Escherichia coli is the most common cause of urinary tract infections (UTIs). E. coli genes epidemiologically associated with UTIs are potentially valuable in developing strategies for treating and/or preventing such infections as well as differentiating uropathogenic E. coli from nonuropathogenic E. coli. To identify E. coli genes associated with UTIs in humans, we combined microarray-based and PCR-based analyses to investigate different E. coli source groups derived from feces of healthy humans and from patients with cystitis, pyelonephritis, or urosepsis. The cjrABC-senB gene cluster, sivH, sisA, sisB, eco274, and fbpB, were identified to be associated with UTIs. Of these, cjrABC-senB, sisA, sisB, and fbpB are known to be involved in urovirulence in the mouse model of ascending UTI. Our results provide evidence to support their roles as urovirulence factors in human UTIs. In addition, the newly identified UTI-associated genes were mainly found in members of phylogenetic groups B2 and/or D.

NlpI facilitates deposition of C4bp on Escherichia coli by blocking classical complement-mediated killing, which results in high-level bacteremia.

ABSTRACT Neonatal meningitis Escherichia coli (NMEC) is the most common Gram-negative organism that is associated with neonatal meningitis, which usually develops as a result of hematogenous spread of the bacteria. There are two key pathogenesis processes for NMEC to penetrate into the brain, the essential step for the development of E. coli meningitis: a high-level bacteremia and traversal of the blood-brain barrier (BBB). Our previous study has shown that the bacterial outer membrane protein NlpI contributes to NMEC binding to and invasion of brain microvascular endothelial cells, the major component cells of the BBB, suggesting a role for NlpI in NMEC crossing of the BBB. In this study, we showed that NlpI is involved in inducing a high level of bacteremia. In addition, NlpI contributed to the recruitment of the complement regulator C4bp to the surface of NMEC to evade serum killing, which is mediated by the classical complement pathway. NlpI may be involved in the interaction between C4bp and OmpA, which is an outer membrane protein that directly interacts with C4bp on the bacterial surface. The involvement of NlpI in two key pathogenesis processes of NMEC meningitis may make this bacterial factor a potential target for prevention and therapy of E. coli meningitis.

Immunostimulatory and antigen delivery properties of liposomes made up of total polar lipids from non-pathogenic bacteria leads to efficient induction of both innate and adaptive immune responses

Novel liposomes prepared from total polar lipids of non-pathogenic bacteria, viz. Leptospira biflexa serovar Potac (designated leptosomes) and Mycobacterium smegmatis (designated smegmosomes) were evaluated for their adjuvant effects with various antigen presenting cells (APCs), viz. murine macrophage cell line, J774A.1 and bone marrow derived dendritic cells (BMDCs). These liposomes induced strong membrane fusion as evident from resonance energy transfer (RET) assays and effectively transferred the fluorescent probe to the membrane of these APCs. Moreover, both vesicles caused significant activation of APCs as revealed by release of proinflammatory cytokines (IL-6, IL-12, TNF-α) and enhanced expression of co-stimulatory signals and maturation markers (CD80, CD86, MHCII), which was significantly higher for smegmosomes as compared to leptosomes. Additionally, activation of APCs by liposomes correlated with their ability to stimulate allospecific T cell proliferation and IFN-γ release. In contrast, conventional PC/chol liposomes failed to fuse and induced only a very low level of APC activation. Interestingly, the stimulatory activity of these lipid vesicles was restricted to APCs as they did not cause any significant activation or mitogenic effect on lymphocytes (B and T cells) in vitro. Overall, the activation of APCs by both leptosomes and smegmosomes correlated with activation of strong humoral and cell mediated immune responses in C57/BL6 mice to entrapped ovalbumin (OVA) and was significantly higher than those induced by conventional liposomes and alum, which failed to activate cytotoxic T lymphocytes (CTLs). Taken together these results demonstrate the adjuvant potential of these novel lipid vesicles that may simultaneously induce both innate and adaptive immune responses due to their immune stimulatory and antigen delivery properties.