Meningitis-Associated <i>Escherichia coli</i>
KWANG SIK KIM
[SECTION EDITORS: GORDON DOUGAN AND HARRY MOBLEY]
Posted October 12, 2006
Gram-negative bacillary meningitis continues to be an important cause of high mortality and morbidity throughout the world despite advances in antimicrobial chemotherapy and supportive care. Case fatality rates have ranged between 15 and 40%, and approximately 50% of the survivors sustain neurological sequelae (12, 17, 42, 68, 74). Both clinical and experimental data indicate limited efficacy with antimicrobial chemotherapy alone (33, 47). A major contributing factor to this high mortality and morbidity is our incomplete understanding of the pathogenesis of this disease.
E. coli is the most common gram-negative organism that causes meningitis, in particular during the neonatal period. Most cases of E. coli meningitis develop as a result of hematogenous spread, but it is incompletely understood how circulating E. coli cross the blood-brain barrier. Given the plethora of E. coli serotypes, it is striking that E. coli strains possessing the K1 capsular polysaccharide are predominant (approximately 80%) among isolates from neonatal E. coli meningitis (19, 45, 60), and most of these K1 isolates are associated with a limited number of O serotypes (e.g., O18, O7, O16, O1, O45) (5, 64). The basis of this association of K1 and certain O antigens with E. coli meningitis remains unclear.
The development of both in vitro and in vivo models of the blood-brain barrier has facilitated the understanding of the microbial translocation of the blood-brain barrier, a key step for the development of E. coli meningitis. Pathogens may cross the blood-brain barrier transcellularly, paracellularly, and/or by means of infected phagocytic cells ("Trojan horse" mechanism). Recent studies have demonstrated transcellular traversal of the blood-brain barrier by circulating E. coli K1 (34, 35, 36).
The blood-brain barrier is a structural and functional barrier that is formed by brain microvascular endothelial cells (BMECs), astrocytes and pericytes. It regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. BMECs possess distinct features such as tight junctions between them and low rates of pinocytosis (62). The blood-brain barrier usually protects the brain from microbes and toxins circulating in the blood, and astrocytes and pericytes help maintain the barrier property of BMEC. Recent studies, however, have shown that meningitis-causing pathogens such as E. coli K1 can cross the blood-brain barrier as live organisms and cause central nervous system (CNS) inflammation, resulting in meningitis (30, 34, 35, 36). The contributions of astrocytes and pericytes to E. coli translocation of the blood-brain barrier are shown to be minimal.
The in vitro blood-brain barrier model has been developed with human brain microvascular endothelial cells (HBMECs). Upon cultivation on collagen-coated Transwell inserts these HBMECs exhibit morphologic and functional properties of tight junction formation as well as polar monolayer. These are shown by the demonstrations of tight junction proteins (such as claudin 5 and ZO-1) and adherens junction proteins (such as VE-cadherin and β-catenin) and their spatial separation, limited transendothelial permeability to inulin (molecular weight, 4,000) and dextran (molecular weight, 70,000), and development of high transendothelial electrical resistance (41, 53, 63, 69, 70). Our previous studies with transmission electron microscopy revealed that E. coli K1 invades HBMECs and internalized bacteria are found within membrane-bound vacuoles of HBMECs (Fig. 1) and transmigrate HBMECs through an enclosed vacuole without intracellular multiplication and without any change in the integrity of HBMEC monolayers (50, 69). No free bacteria are found in the cytoplasm of HBMECs or between adjacent HBMECs.
Fig. 1Transmission electron micrographs of human brain microvascular endothelial cell monolayers infected with E. coli K1 strain RS218 (O18:K1). Scale bar = 1 μm. Modified with permission from the International Journal for Parasitology (36:607–614, 2006).
The in vivo blood-brain barrier model has been developed by inducing hematogenous meningitis in infant rats. In this animal model, E. coli is injected intracardially or subcutaneously, resulting in bacteremia and subsequent entry into the CNS (21, 22, 29, 37, 75, 76, 77). Studies in experimental hematogenous meningitis models indicate that the primary site of entry into the CNS for circulating E. coli is the cerebral vasculature, not the choroid plexus, and E. coli entry into the CNS was documented without any change in the blood-brain barrier permeability (40). Of note is that E. coli penetration of the blood-brain barrier occurred without the concomitant presence of host inflammatory cells (e.g., polymorphonuclear leukocyte, macrophage) (37), excluding the possibility that E. coli penetrates into the brain using the Trojan horse mechanism via transmigration of E. coli-infected phagocytic cells. Taken together, these findings indicate that E. coli K1 traverses the blood-brain barrier using a transcellular mechanism without altering the blood-brain barrier permeability.
Recent studies with the above-mentioned in vitro and in vivo models of the blood-brain barrier have revealed that successful crossing of the blood-brain barrier by circulating E. coli requires (i) a high degree of bacteremia, (ii) E. coli binding to and invasion of HBMEC, and (iii) traversal of the blood-brain barrier as live bacteria (30, 34, 35, 36) (Table 1). In addition, E. coli translocation of the blood-brain barrier involves host cell actin cytoskeletal rearrangements, most likely as the result of specific microbial-host interactions (34, 35, 36, 50).
Table 1Mechanisms involved in E. coli translocation of the blood-brain barrier and E. coli determinants contributing to translocation of the blood-brain barrier
Several studies in humans and experimental animals point to a relationship between the magnitude of bacteremia and the development of meningitis due to E. coli. For example, a significantly higher incidence of E. coli meningitis was noted in neonates who had bacterial counts in blood higher than 10³ CFU/ml (6 of 11, or 55%) compared with those with blood bacterial counts lower than 10³ CFU/ml (1 of 19, or 5%) (13). Similarly, a high degree of bacteremia was shown to be a primary determinant for meningeal invasion by circulating E. coli K1 (21, 22, 29, 37, 75, 76, 77). Thus, one of the reasons for the close association of E. coli K1 strains with meningitis is their ability to escape from host defenses and then to achieve a threshold level of bacteremia necessary for invasion of the meninges. These findings indicate that the prevention of bacterial multiplication in the blood that is required for entry into the CNS would be one potential approach to prevention of E. coli meningitis. Previous studies have indicated that the expression of K1 capsular polysaccharide and O-lipopolysaccharide (LPS) is critical for induction of a high degree of bacteremia (10, 38, 39), but the feasibility of using the K1 capsule and O-LPS for the prevention of E. coli bacteremia has been shown to be limited (9, 15, 67). Recent E. coli genomic studies have identified several E. coli structures that are shown to contribute to bacteremia (see "Functional Genomics," below), and studies are in progress to determine the utility of those E. coli structures for the prevention of E. coli bacteremia and meningitis.
Recent studies have shown that a high degree of bacteremia is necessary but not sufficient for the development of E. coli meningitis and that E. coli binding to and invasion of HBMECs is a prerequisite for penetration of the blood-brain barrier in vivo. This was shown by the demonstration in infant rats with experimental hematogenous meningitis that several isogenic mutants of E. coli K1 strain RS 218 that are deleted of structures contributing to HBMEC binding (e.g., OmpA) and invasion (e.g., Ibe, cytotoxic necrotizing factor 1 [CNF1]) were significantly less able to induce meningitis than the parent strain despite similar levels of bacteremia (Table 2). These findings indicate that those E. coli structures contributing to HBMEC binding and invasion are necessary for crossing the blood-brain barrier in vivo.
Table 2Development of bacteremia and meningitis (defined as positive CSF cultures) in newborn rats receiving the E. coli K1 strain RS 218 or its isogenic mutants
Infections caused by pathogenic E. coli are often initiated by the binding of the bacteria to the host cell surface, which may be important for circulating E. coli to withstand the blood flow in vivo and cross the blood-brain barrier. Several E. coli structures have been identified to be involved in HBMEC binding that subsequently affect invasion into HBMECs. These structures include type 1 fimbriae and outer membrane protein A (OmpA) (28, 59, 66, 72, 73). The roles of these E. coli structures in HBMEC binding have been verified by deletion and complementation experiments. For example, isogenic deletion mutants were significantly less able to bind HBMEC and their binding abilities were restored by complementation in trans with individual genes.
Pathogenic E. coli express many types of fimbrial adhesins, which can be divided into different groups by their affinity to specific receptor structures such as α-d-mannosides (type 1 fimbrial adhesins), α-d-Gal-(1-4)-β-d-Gal (P fimbrial adhesins), and NeuAc α2,3-galactose (S fimbrial adhesins) (27, 54). Our recent experiments to examine the gene expression patterns of HBMEC-associated E. coli K1 with E. coli DNA microarray revealed that type 1 fimbriae play an important role in E. coli K1 binding to HBMEC (72). For example, the HBMEC-associated E. coli K1 showed significantly higher expression levels of the fim cluster genes than the nonassociated bacteria. Expression of type 1 fimbriae in wild-type E. coli is regulated by phase variation in which each bacterium can alternate between fimbriated and nonfimbriated states, so-called phase-ON and phase-OFF, respectively. We have shown that E. coli K1 associated with HBMECs are predominantly type 1 fimbria phase-ON bacteria. We constructed the type 1 fimbria locked-ON and locked-OFF mutants of E. coli K1 strain RS218, whose fim promoters are fixed in the ON and OFF orientation, respectively, and showed that the binding to HBMECs is significantly greater with the locked-ON mutant than the wild-type strain, while it is significantly less with the locked-OFF mutant (72). Decreased binding as the result of the fimH deletion or the locked-OFF mutant resulted in decreased invasion into HBMECs. At present, it is unclear how type 1 fimbriae contribute to HBMEC binding and consequently affect HBMEC invasion. Note that locked-ON constructs of uropathogenic E. coli constitutively expressing type 1 fimbriae also display increased virulence in a murine model of urinary tract infection (20).
S fimbriae, which bind to terminal NeuAc α2,3-galactose sequences present on glycoproteins, have been implicated in E. coli binding to HBMECs. For example, purified S fimbriae or a recombinant E. coli strain HB101 expressing S fimbriae was shown to bind to the luminal surfaces of the brain vascular endothelium in neonatal rat brain tissues (55). We have previously shown, by using S fimbriated transformants of E. coli strain HB101, that S fimbriae allowed this laboratory strain of E. coli to bind to HBMECs (57, 71), suggesting that S fimbriae are the critical determinant contributing to E. coli binding to HBMECs. However, in-frame deletion of the S fimbria operon in E. coli K1 did not significantly affect E. coli binding to and invasion of HBMECs and also did not affect E. coli K1 penetration into the CNS in the experimental hematogenous meningitis animal model (76). These findings indicate that S fimbriae are not critical in E. coli K1 binding to HBMECs in vitro and traversal of the blood-brain barrier in vivo.
OmpA is one of the major outer membrane proteins in E. coli, and its N-terminal domain crosses the outer membrane eight times in antiparallel β-strands with four hydrophilic surface-exposed loops and short periplasmic turns. We have shown that the N-terminal portion of OmpA and its surface-exposed loops contribute to binding to HBMECs (66). We have also shown that OmpA interacts with HBMECs through N-acetylglucosamine (GlcNAc) residues of HBMEC glycoproteins, including gp 96 (28, 52, 56). The chitooligomers (GlcNAc β1,4-GlcNAc oligomers) block the E. coli K1 traversal of the blood-brain barrier in the newborn rat model of experimental hematogenous meningitis (51). Our recent study with E. coli DNA microarray comparing the ompA deletion mutant with its parent E. coli K1 strain RS218, however, revealed that the ompA deletion mutant exhibited significantly lower expression of the fim cluster genes and type 1 fimbriae on the bacterial surface (73), suggesting that decreased binding of the ompA deletion mutant may be related to its lower expression of type 1 fimbriae. The ompA deletion mutant was significantly less efficient in its penetration into the CNS in vivo than the parent E. coli K1 strain (76). Additional studies are needed to determine whether these in vitro and in vivo defects of the ompA deletion mutant are in part related to its decreased expression of type 1 fimbriae and also to understand how the deletion of ompA affects type 1 fimbria expression.
Previous studies using TnphoA mutagenesis, signature-tagged mutagenesis, and differential fluorescence induction with screening of a gfp fusion library identified several E. coli determinants contributing directly to invasion of HBMECs, which include Ibe proteins and CNF1 (1, 2, 21, 22, 23, 75). Isogenic deletion mutants were significantly less invasive in HBMECs and less able to cross the blood-brain barrier in vivo (Table 2), and their invasive abilities were restored by complementation with individual genes. Recombinant Ibe proteins inhibit E. coli invasion of HBMECs (23), suggesting that Ibe proteins contribute to HBMEC invasion by ligand-receptor interactions. This concept was supported by the demonstration of a 45-kDa HBMEC surface protein interactive with IbeA, and a polyclonal antibody raised against this receptor protein inhibited E. coli K1 invasion of HBMECs (49).
CNF1 is a bacterial virulence factor associated with pathogenic E. coli strains causing urinary tract infection and meningitis (6). CNF1 is an AB-type toxin, composed of the N-terminal cell binding domain and the C-terminal catalytic domain possessing a deaminase activity through the site-specific deamination of a Gln residue to Glu (16, 65). CNF1 has been shown to activate Rho GTPases such as Rho, Rac, and Cdc 42 and induce uptake of latex beads, bacteria, and apoptotic bodies into nonprofessional phagocytes such as epithelial and endothelial cells by macropinocytosis (14). CNF1 contributes to E. coli K1 invasion of HBMECs in vitro and traversal of the blood-brain barrier in vivo, and these in vitro and in vivo effects of CNF1 depend on RhoA activation (29). These were shown by (i) decreased invasion and RhoA activation with the cnf1 deletion mutant in HBMEC and (ii) restoration of the cnf1 mutant’s invasion rate to the level of the parent strain in HBMECs expressing constitutively active RhoA. CNF1 has been suggested to be internalized via receptor-mediated endocytosis upon binding to a cell surface receptor (6), but it is unclear how CNF1 enters the HBMEC and activates Rho GTPases. We have identified the HBMEC receptor for CNF1 by yeast two-hybrid screening of the HBMEC cDNA library using the N-terminal cell binding domain of CNF1 as bait (7). This receptor, 37-kDa laminin receptor precursor (LRP), interacted with the N-terminal CNF1 and full-length CNF1 but not with the C-terminal CNF1. CNF1-mediated RhoA activation and bacterial uptake were inhibited by exogenous LRP or LRP antisense oligodeoxynucleotides, whereas they were increased in LRP-overexpressing cells, demonstrating correlation between effects of CNF1 and levels of LRP expression in HBMEC (7). These findings indicate that CNF1 interaction with its receptor, 37-kDa LRP, is the initial step required for CNF1-mediated RhoA activation and bacterial uptake in eukaryotic cells. The 37-kDa LRP is a ribosome-associated cytoplasmic protein and shown to be a precursor of 67-kDa laminin receptor (LR). It is unclear how 67-kDa LR is matured and synthesized from the 37-kDa LRP, but mature 67-kDa LR is shown to be present on the cell surface and functions as a membrane receptor for the adhesive basement membrane protein laminin (46). Our recent studies have shown that incubation of HBMECs with CNF1-expressing E. coli K1 upregulates 67-kDa LR expression and recruits 67-kDa LR to the site of invading E. coli K1 in a CNF1-dependent manner (31). Increased expression of 67-kDa LR has been shown to be associated with invasive and metastatic properties of a variety of tumors (48), and it remains speculative whether CNF1-expressing E. coli has any role in malignant transformation of certain cancers. Although CNF1 is shown to interact with 37-kDa LRP/67-kDa LR on the cell surface of HBMEC, resulting in RhoA activation and increased internalization of CNF1-expressing E. coli K1, CNF1 is a bacterial cytoplasmic protein, and it remains unclear how it is secreted into the outer membrane and interacts with 37-kDa LRP/67-kDa LR. Preliminary experiments revealed that CNF1 can be translocated to the bacterial surface upon CNF1-expressing E. coli K1 interaction with HBMECs. Taken together, these findings indicate that E. coli K1 invades HBMECs through ligand-receptor interactions.
Pathogenic microbes have exploited various strategies to penetrate their host cells. Microbial internalization into nonprofessional phagocytes such as epithelial and endothelial cells is shown to occur mainly via two different mechanisms involving host cell actin cytoskeleton rearrangements, such as a zipper mechanism involving the formation of cell protrusions in contact with the pathogens and a trigger mechanism involving the formation of membrane ruffling around the pathogens (8, 43). Electron microscopy studies have shown that meningitis-causing E. coli K1 invasion of HBMECs is associated with microvillus-like protrusions at the entry site on the surface of HBMECs (36, 50) (Fig. 1), suggesting the involvement of host cell actin cytoskeleton rearrangement. This concept is supported by the demonstrations that the F-actin condensation occurs with invading bacteria and blockade of actin condensation with microfilament-disrupting agents such as cytochalasin D inhibits E. coli K1 invasion of HBMECs (52).
Several signal transduction pathways have been shown to be involved in bacterial invasion of HBMEC, most likely through their effects on actin cytoskeleton rearrangements. These include focal adhesion kinase (FAK); paxillin; phosphatidylinositol 3-kinase (PI3K); Src kinase; Rho GTPases; cytosolic phospholipase A2 (cPLA2); and ezrin, radixin, and moesin (ERM) (11, 28, 31, 34, 35, 36, 58, 59). Note that host cell actin cytoskeleton rearrangements are shown to be required for HBMEC invasion by meningitis-causing bacteria such as E. coli, group B Streptococcus, and Listeria monocytogenes, but the signaling mechanisms that are involved in actin cytoskeleton rearrangements and HBMEC invasion are shown to differ between meningitis-causing bacteria (Table 3). For example, host cell actin cytoskeleton rearrangements are required for HBMEC invasion by E. coli K1, group B Streptococcus, and L. monocytogenes, as shown by inhibition of their invasion into HBMEC by cytochalasin D (18, 50, 53). E. coli K1 invasion of HBMEC depends on activations of FAK, Src, PI3K, and cPLA2. In contrast, group B streptococcal invasion of HBMECs was independent of Src and cPLA2 activation and L. monocytogenes invasion of HBMECs was independent of FAK and cPLA2 activation. At present, the basis of microbial-host interactions contributing to E. coli K1 invasion of HBMECs and relevant signaling mechanisms has not been fully elucidated.
Table 3Comparison of host cell cytoskeleton and signaling mechanisms involved in bacterial invasion of human brain microvascular endothelial cells. Modified with permission from the International Journal for Parasitology (36:607–614, 2006)
Another crucial factor for the development of meningitis is the ability of E. coli to cross the blood-brain barrier as live bacteria. E. coli K1 has been shown to traverse the blood-brain barrier without altering the integrity of the HBMEC monolayer and without affecting the blood-brain barrier permeability (40, 69)
It has previously been shown that HBMECs have the complete trafficking machinery required to deliver the microbe-containing vacuoles to cathepsin D-containing components (i.e., lysosomes) (32). Vacuoles containing the E. coli K1 capsule deletion mutant interact sequentially with early endosomal marker proteins (e.g., early endosomal auto-antigen 1 and transferrin receptor) and late endosome and late endosome/lysosomal markers (e.g., Rab7 and lysosome-associated membrane proteins, respectively) and allow lysosomal fusion, with subsequent degradation inside vacuoles. In contrast, vacuoles containing E. coli K1+ (E. coli with the K1 capsule) obtained early and late endosomes without fusion with lysosomes, thereby allowing E. coli K1 to cross the blood-brain barrier as live bacteria (32), indicating that E. coli K1 modulates intracellular trafficking to avoid lysosomal fusion in HBMECs. E. coli K1 capsule is well recognized for its serum resistance and antiphagocytic properties (10, 38, 39), which are the essence of inducing a high degree of bacteremia. Another novel property of the K1 capsule is to modulate the maturation process of E. coli K1+-containing vacuoles and prevent their fusion with lysosomes, which is an event necessary for traversal of the blood-brain barrier as live bacteria. Additional studies are needed to understand how the K1 capsule is able to modulate intracellular trafficking of E. coli K1+-containing vacuoles to avoid fusion with lysosomes in HBMECs and whether similar events occur with other meningitis-causing microbes.
Genome sequencing of meningitis-causing microbes provides new tools for elucidating the pathogenesis of meningitis. For example, comparative genome analysis of the prototypic meningitis-causing E. coli K1 strain RS218 (O18:K1) versus laboratory K-12 strain MG1655 was carried out and identified 22 RS218-specific islands that are larger than 10 kb and are absent in strain MG1655 (78, 79). These RS218-specific islands are termed RSIs. The total length of these RSIs is approximately 793 kb, which replaced approximately 80 kb of MG1655-specific sequences. The actual chromosomal size difference between RS218 and MG1655 was approximately 450 kb, which is slightly smaller than the previously estimated genome size difference between RS218 and MG1655 (61). Previous studies using comparative macrorestriction mapping and subtractive hybridization of the chromosomes of meningitis-causing E. coli K1 (e.g., O18:K1 strains RS218 and C5) compared with nonpathogenic E. coli have identified 500 kb spread over at least 12 chromosome loci specific to E. coli K1 (4, 61). Mapping studies reveal that those E. coli loci are located at different regions of E. coli chromosome. Twenty-two RSIs have been shown to be located at different regions of E. coli K1 RS218 chromosome (78, 79).
By use of RSI deletion mutants, eight RSIs have been shown to be involved in the pathogenesis of meningitis (i.e., induction of a high degree of bacteremia and HBMEC binding/invasion). The size and characteristics of these eight RSIs are summarized in Table 4. Two RSIs include a P4-family integrase and are directly adjacent to tRNAs (RSI 4-serX and RSI 21-leuX), and four RSIs (RSI 7, RSI 16, RSI 21, and RSI 22) have markedly lower GC percentages compared with the whole RS218 genome, suggesting that these RSIs are acquired through horizontal gene transfer. Further identification and characterization of microbial determinants from these RSIs that are involved in the pathogenesis of E. coli meningitis should help in elucidating microbe-host interactions that are involved in meningitis.
Table 4Size and characteristics of eight RSIs relevant to the pathogenesis of E. coli meningitis
At present, relatively few virulence factors identified from prototypic meningitis-causing O18:K1 E. coli strains (e.g., strains RS218 and C5) have been used to understand the pathogenesis of meningitis (3, 25, 26, 44), but it is unclear whether the information derived from these E. coli K1 strains is relevant to other E. coli K1 strains isolated from cerebrospinal fluid (CSF). We have conducted a comparative genomic hybridization (CGH) with an E. coli DNA microarray to examine the basis of meningitis caused by representative E. coli K1 strains isolated from blood and CSF (80). These strains include RS218(O18:K1), C5(18:K1), IHE3034(O18:K1), EC10(O7:K1), A90(O1:K1), RS168(O1:K1), RS167(O16:K1), E253(O12:K1), E334(O12:K1), S88(O45:K1), and S95(O45:K1). Our hierarchical clustering revealed that these strains can be categorized into two groups. Group 1 includes strains RS218, C5, IHE3034, A90, RS167, E334, S88, and S95, while strains EC10, RS168, and E334 belong to group 2 (Fig. 2). All group 1 strains belong to the phylogenetic group B2, which is predominant in CSF isolates, and group 2 strains belong to less common phylogenetic groups A and D (3, 25, 26). We showed that all group 2 strains harbor some genes from E. coli type III secretion system 2 (ETT2), but none of group 1 strains harbor ETT2 (80). The existence of a degenerate ETT2 gene cluster has been shown in septicemic E. coli O78 strains (24). Sequence analysis of the ETT2 genes showed premature stop codons in eprI and eprJ encoding the needle structure and deletion of the invG gene, which encodes a conserved component of the outer membrane ring. This ETT2 lacks the gene (eivC) for the cytoplasmic ATPase that energizes secretion and some other conserved components of type III secretion system (e.g., epaS). However, a deletion mutant of genes coding for the putative inner membrane ring of the secretion complex showed significantly reduced virulence in a 1-day-old chick model, even though the mutation does not seem to affect the secreted proteome (24). E. coli K1 strain EC10 from group 2 that was isolated from the CSF of a neonate with meningitis was found to harbor all the genes needed to encode type III secretion apparatus proteins compared with the aforementioned septicemic E. coli O78 strain 789 (80). It remains speculative whether strain EC10 may utilize the type III secretion system to invade and subvert the signal transduction pathway in HBMECs to induce meningitis.
Fig. 2The clustering of 11 representative E. coli strains based on microarray data (80). 1 and 2 represent the phylogenetic groups and the O serotypes of 11 E. coli K1 strains. Modified with permission from Infection and Immunity (80).
We also examined with the CGH the distribution of the eight RSIs that are relevant to E. coli meningitis among representative E. coli K1 strains (79). RSI 16 harbors the K1 capsule biosynthesis gene cluster and, as expected, is present in all of these E. coli K1 strains. The other pathogenic RSIs are found to exist in strains belonging to our group1 and phylogenetic group B2. For example, RSI 1, 7, 13, 20, and 22 are widely distributed among this group of E. coli K1 strains. Previous studies using PCR, dot blot, and Southern blot suggest that PAI III536-like, PAI IIJ96-like, and GimA-like ectochromosomal DNA domains (ECDNAs) are prevalent among O18:K1 strains, the most common serogroup in meningitis-causing E. coli (5). Based on their virulence signatures, those ECDNAs correspond to RSI 4, 21, and 22, respectively. The distribution of these three RSIs among O18:K1 strains based on CGH is consistent with previous findings (5, 79).
In addition, microbial DNA microarrays offer new opportunities for exploring microbial gene expression profiles during microbe-host interactions. For example, using E. coli DNA microarray analysis with microarray-grade bacterial RNA isolated from E. coli K1 interacting with HBMECs, we showed that the expression of the type 1 fimbria genes are significantly higher for E. coli associated with HBMEC than for E. coli not associated with HBMECs (72). We subsequently showed that type 1 fimbriae play an important role in E. coli K1 binding to and invasion of HBMECs (72), indicating that microbial DNA microarray analysis has a potential for elucidating microbe-host interactions that are relevant to meningitis.
A major limitation to advances in prevention and therapy of E. coli meningitis is our incomplete understanding of the pathogenesis of this disease. For example, given the plethora of E. coli serotypes, it is unclear why K1 and limited numbers of O types account for most cases of meningitis. As indicated above, studies with the in vitro and in vivo blood-brain barrier models have shed light on the mechanisms of microbial translocation of the blood-brain barrier, a key step for the development of meningitis. We showed that E. coli K1 traverses the blood-brain barrier without altering the integrity of the HBMEC monolayer and without affecting blood-brain barrier permeability (40, 69). We subsequently showed that E. coli translocation of the blood-brain barrier is the result of specific bacterium-host interactions involving specific host cell signal transduction pathways. Complete understanding of microbe-host interactions that are involved in E. coli translocation of the blood-brain barrier should help in developing new strategies to prevent E. coli bacteremia and meningitis.
This work was supported by National Institutes of Health Grants.
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