Julie L. Badger

Motility is required to initiate host cell invasion by Yersinia enterocolitica.

ABSTRACT Invasin-mediated invasion of host cells by the pathogenYersinia enterocolitica was shown to be affected by flagellar-dependent motility. Motility appears to be required to ensure the bacterium migrates to and contacts the host cell. Nonmotile strains of Y. enterocolitica were less invasive than motile strains, but the reduction in invasion could be overcome by artificially bringing the bacteria into host cell contact by centrifugation. Mutations in known regulatory genes of the flagellar regulon, flhDC and fliA, resulted in lessinv expression but did not have a significant effect on invasin levels. However, invasin levels were reduced for strains that harbored flhDC on a multicopy plasmid, apparently as a result of increased proteolysis of invasin.

Application of signature-tagged mutagenesis for identification of escherichia coli K1 genes that contribute to invasion of human brain microvascular endothelial cells

ABSTRACT Escherichia coli K1 is the leading cause of gram-negative bacterial meningitis in neonates. It is principally due to our limited understanding of the pathogenesis of this disease that the morbidity and mortality rates remain unacceptably high. To identify genes required for E. coli K1 penetration of the blood-brain barrier (BBB), we used the negative selection strategy of signature-tagged transposon mutagenesis (STM) to screen mutants for loss or decreased invasion of human brain microvascular endothelial cells (HBMEC) which comprise the BBB. A total of 3,360 insertion mutants of E. coli K1 were screened, and potential HBMEC invasion mutants were subjected to a secondary invasion screen. Those mutants that failed to pass the serial invasion screens were then tested individually. Seven prototrophic mutants were found to exhibit significantly decreased invasive ability in HBMEC. We identifiedtraJ and five previously uncharacterized loci whose gene products are necessary for HBMEC invasion by E. coli K1. In addition, cnf1, a gene previously shown to play a role in bacterial invasion, was identified. More importantly, atraJ mutant was attenuated in penetration of the BBB in the neonatal rat model of experimental hematogenous meningitis. This is the first in vivo demonstration that traJ is involved in the pathogenesis of E. coli K1 meningitis.

Escherichia coli K1 aslA contributes to invasion of brain microvascular endothelial cells in vitro and in vivo.

ABSTRACT Neonatal Escherichia coli meningitis remains a devastating disease, with unacceptably high morbidity and mortality despite advances in supportive care measures and bactericidal antibiotics. To further our ability to improve the outcome of affected neonates, a better understanding of the pathogenesis of the disease is necessary. To identify potential bacterial genes which contribute toE. coli invasion of the blood-brain barrier, a cerebrospinal fluid isolate of E. coli K1 was mutagenized with TnphoA. TnphoA mutant 27A-6 was found to have a significantly decreased ability to invade brain microvascular endothelial cells compared to the wild type. In vivo, 32% of the animals infected with mutant 27A-6 developed meningitis, compared to 82% of those infected with the parent strain, despite similar levels of bacteremia. The DNA flanking the TnphoA insertion in 27A-6 was cloned and sequenced and determined to be homologous toE. coli K-12 aslA (arylsulfatase-like gene). The deduced amino acid sequence of the E. coli K1aslA gene product shows homology to a well-characterized arylsulfatase family of enzymes found in eukaryotes, as well as prokaryotes. Two additional aslA mutants were constructed by targeted gene disruption and internal gene deletion. Both of these mutants demonstrated decreased invasion phenotypes, similar to that of TnphoA mutant 27A-6. Complementation of the decreased-invasion phenotypes of these mutants was achieved whenaslA was supplied in trans. This is the first demonstration that this locus contributes to invasion of the blood-brain barrier by E. coli K1.

Citrobacter koseri Brain Abscess in the Neonatal Rat: Survival and Replication within Human and Rat Macrophages

ABSTRACT A unique feature of Citrobacter koseri is the extremely high propensity to initiate brain abscesses during neonatal meningitis. Previous clinical reports and studies on infant rats have documented many Citrobacter-filled macrophages within the ventricles and brain abscesses. It has been hypothesized that intracellular survival and replication within macrophages may be a mechanism by which C. koseri subverts the host response and elicits chronic infection, resulting in brain abscess formation. In this study, we showed that C. koseri causes meningitis and brain abscesses in the neonatal rat model, and we utilized histology and magnetic resonance imaging technology to visualize brain abscess formation. Histology and electron microscopy (EM) revealed that macrophages (and not fibroblasts, astrocytes, oligodendrocytes, or neurons) were the primary target for long-term C. koseri infection. To better understand C. koseri pathogenesis, we have characterized the interactions of C. koseri with human macrophages. We found that C. koseri survives and replicates within macrophages in vitro and that uptake of C. koseri increases in the presence of human pooled serum in a dose-dependent manner. EM studies lend support to the hypothesis that C. koseri uses morphologically different methods of uptake to enter macrophages. FcγRI blocking experiments show that this receptor primarily facilitates the entry of opsonized C. koseri into macrophages. Further, confocal fluorescence microscopy demonstrates that C. koseri survives phagolysosomal fusion and that more than 90% of intracellular C. koseri organisms are colocalized within phagolysosomes. The ability of C. koseri to survive phagolysosome fusion and replicate within macrophages may contribute to the establishment of chronic central nervous system infection including brain abscesses.

TraJ-Dependent Escherichia coli K1 Interactions with Professional Phagocytes Are Important for Early Systemic Dissemination of Infection in the Neonatal Rat

ABSTRACT Escherichia coli is a major cause of neonatal bacterial sepsis and meningitis. We recently identified a gene, traJ, which contributes to the ability of E. coli K1 to penetrate the blood-brain barrier in the neonatal rat. Because very little is known regarding the most critical step in disease progression, translocation to the gut and dissemination to the lymphoid tissues after a natural route of infection, we assessed the ability of a traJ mutant to cause systemic disease in the neonatal rat. Our studies determined that the traJ mutant is significantly less virulent than the wild type in the neonatal rat due to a decreased ability to disseminate from the mesenteric lymph nodes to the deeper tissues of the liver and spleen and to the blood during the early stages of systemic disease. Histopathologic studies determined that although significantly less or no mutant bacteria were recovered from the spleen and livers of infected neonatal rats, the inflammatory response was considerably greater than that in wild-type-colonized tissues. In vitro studies revealed that macrophages internalize the traJ mutant less frequently than they do the wild type and by a morphologically distinct process. Furthermore, we determined that tissue macrophages and dendritic cells within the liver and spleen are the major cellular targets of E. coli K1 and that TraJ significantly contributes to the predominantly intracellular nature of E. coli K1 within these professional phagocytes exclusively during the early stages of systemic disease. These data indicate that, contrary to earlier indications, E. coli K1 resides within professional phagocytes, and this is essential for the efficient progression of systemic disease.

Invasion of Intestinal Epithelial Cells In Vitro by Escherichia coli Is Influenced by Microaerophilic Conditions and the Presence of ibe Genes † 890

Enteric bacteria are an important cause of sepsis in neonates, children with chronic gastrointestinal diseases (such as short bowel syndrome), immunocompromised hosts, and children with central venous catheters. It is unclear what microbial factors may mediate the translocation of enteric bacteria across the intestinal epithelial barrier. E. coli RS218 is a K1 encapsulated clinical isolate from the CSF of a newborn infant with meningitis, which has been used to elucidate mechanisms of invasion of brain microvascular endothelial cells. The aims of our study were to evaluate (1) the ability of RS218 and Tn phoA transposon mutants of RS218 to invade two intestinal epithelial cell lines, and (2) the influence of growth conditions on RS218 invasion. Materials and Methods: Monolayers of Caco-2 and C2BBe1 cells were allowed to fully differentiate, expressing dome formation characteristic for transporting epithelia, and were infected at a minimum of 15 days post-confluency. Bacterial strains were grown overnight under either aerobic or microaerophilic conditions, and approximately 107 bacteria were added to each monolayer. Cells were incubated at 37°C for 1.5 hours, washed, and then incubated with a gentamicin containing medium for one hour to eliminate extracellular bacteria. Cells were lysed and viable bacteria were enumerated by plating onto sheep blood agar plates. Results: RS218 was invasive in both Caco-2 and C2BBe1 cell lines. RS218 was 3 to 6.5-fold more invasive than HB101, a known noninvasive E. coli, in both cell lines. RS218 grown under microaerophilic conditions showed a 10-fold increase in invasion in both cell lines. Tn phoA mutants, which have been shown to be noninvasive in brain microvascular endothelial cells, were at least 16 to 300-fold less invasive than RS218 in both cell lines.Conclusions: (1) The E. coli genes involved in the invasion of brain microvascular endothelial cells (such as ibe10) also play a role in the in vitro invasion of intestinal epithelial cells. (2) The microaerophilic environment enhances E. coli invasion of gut epithelial cells in vitro.