Study On The Role Of Polyphosphate Kinase 1 In The Pathopoiesis Of Meningitis Escherichia Coli K1

Escherichia coli (E. coli) Kl is the most common gram-negative organism causing neonatal bacterial meningitis. Due to our incomplete knowledge about the pathogenesis of E. coli meningitis, this disease has remained high mortality and morbidity despite the advances in antimicrobial chemotherapy and supportive care. Most cases of neonatal E. coli meningitis develop as a result of hematogenous spread, and a critical step for the occurrence of E. coli meningitis is the circulating bacteria crossing blood-brain barrier (BBB), which is mainly composed of brain microvascular endothelial cells (BMECs). To across the BBB successfully for E. coli, it needs to meet some certain prerequisites including a high degree of bacteremia, binding to and invasion of HBMEC, host cell cytoskeleton rearrangement and related signal pathway activation.E. coli K1 invasion of human BMEC (HBMEC) has been shown to be significantly higher in the stationary-phase (SP) than in the exponential-phase, suggesting that the SP is important for expression of E. coli Kl invasion-associated virulence genes. Polyphosphate (Poly P) kinasel (PPK1), which is the major enzyme for the synthesis of inorganic polyphosphate (poly P) from ATP and encoded by the ppkl gene, has been identified to be associated with stationary-phase survival in many bacteria species. A ppkl mutant of E. coli K-12 was deficient in responses to stresses and failed to survive in the stationary phase. The Vibrio cholerae ppkl mutants were defective in growth, motility, and surface attachment. Pseudomonas aeruginosa with ppkl knockout was aberrant in quorum sensing and biofilm formation. The ppkl mutants of Shigella and Salmonella spp. were insufficient in long-term survival and virulence. It has been shown that Poly P is associated with the expression of RpoS, the sigma factor responsible for regulating the expression of survival and many virulence genes during the stationary-phase (SP) in various bacteria, such as E. coli O157:H7, Salmonella typhimurium, Shigella flexneri, Yersinia enterocolitica, Vibrio cholera, and Borrelia burgdorfer. It is worth noting, however, that a loss-of-function mutation was found in the SP regulatory gene rpoS of E. coli K1 strain E44, and IbeR has been identified by us as an RpoS-like regulator contributing to the expression of SP genes (including many virulence and stress adaption related genes) in E44.Methods and results:Because the role of ppkl/poly P in the pathogenesis of meningitic Escherichia coli K1, and the connection between the ppk1/poly P and SP regulation gene ibeR are both unkwon, an isogenic in-frame ppkl deletion mutant (PD44) of E. coli K1 strain has been made and characterized. The expression level of the important invasion gene ibeA, which locates in the same operon of ibeRAT with ibeR was also tested in this study.1. Isogenic deletion of ppkl gene in E. coli K1 strain E44In order to determine the role of the ppkl gene in the pathogenesis of E. coli meningitis, a ppkl isogenic in-frame deletion mutant was constructed. As shown in Fig 1A, two PCR DNA fragments (1.14 kb and 1.28 kb) flanking the ppkl coding region (2.06 kb) were obtained and ligated together into the suicide plasmid pCVD442 to produce the recombinant plasmid pCVDPKl. Then the plasmid pCVDPK1 was transformed into SM10γpir, and the ppkl deletion mutant was obtained by mating E44 with SM10γpir carrying pCVDPKl as described in Materials and Methods. The deletion of the ppkl gene was confirmed by colony PCR and DNA sequencing.2. Examination of poly P levels The growth profiles of E44, PD44 and the ppkl complemented strain in rich medium were identical (data not shown).To examine the poly P levels in the cells, poly P was extrcated with glassmilk and quantitated by using the Toluidine Blue O dye as described in Materials and Methods.The poly P levels in PD44 were singnifcantly lower than those of the E44 and the ppkl complemented strain at both time point of 3h and 18h. Especially in stationary phase, the poly P level in the E44 and the ppkl complemented strain amounted to 13.3 and 12.6 nmol poly P/mg of total celluar protein respectively. However, the PD44 exhibited barely detectable level of poly P (<1.5 nmol poly P/ mg of total celluar protein). These results show that the ppkl gene plays an important role in the poly P production of E. coli E44.3. Survival abilities of E44 and PD44 under hypertonic or acid stress conditionTo test survival under osmotic stress, bacteria were exposed to 2.5 M NaCl for 2 hours and plated on LB agar plates to determine the survival rates. After 2 hours, the survival rates of E44, PD44 and PD44 carrying ppk1 was 39.62%,9.84% and 38.37% respectively. For acid stress assay, overnight cultures were collected and suspended in acidic LB (pH 2.8) for 60 minutes. Similar with the results of hypertonic stress assay, the ppkl mutant strain PD44 was significantly defective in survival in the acidic LB when compared to E44 and PD44 carrying ppkl. After 1 hour under this condition, the survival rate of PD44 was only about 1/6 of E44 and the ppkl complemented strain. These results suggest that poly P is required for stress tolerance of E44 in stationary phase.4. Bacteria adhesion and invasion of HBMECThe in vitro adhesion and invasion assay were performed as described in Materials and Methods. Because the adhesion of E. coli to the HBMEC is the initial step of bacterial crossing of the BBB during the development of meningitis, the effect of ppkl deletion in E. coli E44 adhesion of HBMEC was examined. The HBMEC were incubated with E44 and PD44 for 1,2,3 hours. After three times of washing, the bacteria adhering to HBMEC were enumerated and the adhesion rates were calculated. The adhesion rates of E44 at 1,2, and 3h were 3.97%,9.80% and 26.80% respectively. However, the adhesion rates of PD44 reduced to 1.73%,3.87% and 9.23%. In order to test whether the complementation of ppkl can restore the adhesion phenotype, E44 carrying the plasmid pGEM-T, PD44 carrying either pGEM-T or pGEMPKl that contains the complete coding region of ppkl were incubated with the HBMEC monolayers for 2 h, and the adhesion rates were analyzed. The adhesion rates of PD44 carrying pGEM-T and E44 carrying pGEM-T were 3.20% and 8.43% respectively. The adhesion rate of PD44 complemented wit ppk1was 7.30% which raised to about 90% when compared to that of the parent strain E44. Similarly, the ppkl deletion led to a significant reduction of invasion ability for E44. Following the initial adhesion to HBMEC, part of E. coli will be internalized druing the E. coli accrosing the BBB. And this can be analyzed with the invasion assay as described previously. The in vitro invasion results showed that the invasion rates of PD44 (0.041%) and ZD1 (0.029%) were significantly lower than that of their parent strain E44 (0.137%), and the plasmid pGEMPK1 containing the ppkl gene was able to complement the less invasive phenotype of PD44.5. Cytoskeleton changes in HBMEC induced by the bacteriaAfter treatment with bacteria, the HBMEC were stained with Rhodamine-labelled phalloidin, which makes the the organization of actin be easily and clearly observed under a confocal fluorescence microscope. In the untreated group, the actin filament evenly distributed throughout the internal compartment of the cytoplasm. In the groups incubated with E44 and PD44 (pGEMPK1), the actin filament in the cytoplasm reduced obviously, and it could be observed a denser actin aggregates in the margin of the cells. A long protrusion of actin filament staining in HBMEC also can often be observed in these two groups. In the cells treated with PD44, a less internal reduction and marginal aggregates of actin filament than that of the other two groups treated with E. coli was observed.6. Neonatal rat model of hematogenous E. coli K1 meningitisThe in vitro experiments demonstrated that the ppkl deletion mutant PD44 was less adherent and invasive than its parent strain E44 in the HBMEC cell culture model. To further investigate the role of ppkl on the development of neonatal meningitis, the infant rat model of experimental hematogenesis meningitis was used to determine the in vivo virulence phenotype. The blood and CSF specimen were cultured for indication of bacteremia and meningitis, respectively. The magnitude of bacteremia induced by the mutant strain was similar to that of the wild-type strain. However, the occurrence of meningitis (defined as positive CSF cultures) in animals receiving PD44 (9 of 23, or 39%), was significantly lower (P< 0.05) than in those receiving the parent strain E44 (17 of 23, or 74%). Our in vitro and in vivo studies demonstrated that the ppkl gene was required for E. coli E44 penetration across the BBB in vitro and in vivo.7. The mRNA expression levels of IbeR and IbeA in E. coli E44 and PD44In order to determine whether loss of ppkl modulated the stationary phase regulator IbeR and the major virulence factor IbeA in E. coli E44, real time RT-PCR was used to detect the mRNA expression levels of IbeR and IbeA as described above. After normalization with the copy number of the GAPDH gene, the relative copy number of ibeR in PD44 (13.35±2.11) was significantly less than that in E44 (31.10±7.90), and the relative copy number of ibeR in the ppkl complemented strain raised to 23.76±3.71. The relative copy number of ibeA in PD44 (32.12±7.33) was also less than that in E44(57.60±10.31) and the ppkl complemented strain(49.71±8.41). The result suggested that the ppkl deletion led to a decline expression of ibeR and ibeA in E. coli K1 E44.Statistical analysisAll values are expressed as mean±standard deviation. Statistical analysis was performed with Student's t-test for comparison of two groups, and with ANOVA for multiple comparisons. In vivo experiments data were analyzed by Pearson Chi-Square test. Differences with P<0.05 were considered to be statistically significant.ConclusionThe ppkl gene is a highly conserved gene among many bacterial species, including bacterial pathogens, Mycobacterium tuberculosis, Neisseria meningitides, Helicobacter pylori, Vibrio cholerae, Salmonella typhimurium, etc.. Due to the absence of the PPK1 homologue in mammalian cells, PPK1 exhibits potential to be an attractive target for chemotherapy. In order to determine the role of poly P in the pathogenesis of E. coli Kl meningitis, we constructed the ppkl deletion mutants to test its biological functions using the in vitro and in vivo models of the BBB and genetic complementation. We showed that the mutant PD44 was considerably less adherent and invasive for HBMEC, and significantly less virulent in the entry into the CNS in the newborn rat model of experimental hematogenous meningitis. The less adherent and invasive phenotype of the PD44 mutant were complemented by the ppkl gene. Meanwhile, the poly P level of the PD44 in stationary phase was also found to be obviously lower than that of the parent strain E44 and the ppkl complemented strain by using the nonradioactive method. It should also be noticed that, the phenomenon of poly P accumulation still exists in ppkl mutant of P. aeruginosa and Campylobacter jejuni, which may be due to the harbouring of not only ppkl but also ppk2 mediating poly P-driven generation of GTP in these orgnisms.Remodelling of the host-cell actin cytoskeleton has been observed during pathogenic invasion. The E. coli adhesion and invasion of the HBMEC leading to cytoskeletal reorganization and downstrem signal activiation is important for the bacteria to accross the HBMEC. To test the cytoskeletal reorganization in the HBMEC induced by ppkl deletion mutant and the wild-type strain, the rhodamine-labelled phalloidin was used to make the rearrangement of actin filaments induced by E. coli visible under the confocal fluorescence microscope. Our findings showed that the actin filaments rearrangement in the HBMEC induced by PD44 was significantly weaker than that induced by the parent strain E44 and the ppkl complemented strain.During the development process of neonatal E. coli meningitis, it's an important pathogenesis for the pathogen to endure the hyperosmotic of fecal in intestine and acid stress in the host's stomach or vagina of puerpera. As a product of the reaction catalyzed by the PPK1, poly P has been reported to be associated with the expression of the SP regulator RpoS, which governs expression of over 50 genes and plays a central role on stationary-phase adaptations in most E. coli strains. The adaptation processes modulated by RpoS is important for the bacteria to survival in kinds of environments. However, the SP regulation in meningitic E. coli K1 carrying the ibeR gene was drastically different from that in many other E. coli strains. There is a nonsense mutation in the rpoS gene in E. coli K1 strain E44, which can keep its stress resistance independent of RpoS. The phenomenon of rpoS mutation is not unique to E. coli E44, but also exists in some other enteric pathogens. We have demonstrated that IbeR is an RpoS-like regulator contributing to the E. coli E44 entry into HBMEC and stress tolerance in the stationary-phase. Some proteins which are related to environmental modifications or central metabolism can be regulated by IbeR in E44, such as TnaA, LpdA, OmpC, TufB, GapA, OmpA, AhpC.As the product of PPK1, poly P, plays an important role in the RpoS induction in other E. coli strains, we further examined whether PPK1 contributed to regulation of IbeR expression and stress tolerance in E44. The ppkl deletion mutant PD44 was tested for its survival rates under stress conditions in comparison with the parent strain E44. The survival rates were significantly reduced after the ppk1 mutant receiving stress treatments compared to that of E44. These results suggest that ppk1/poly P is important for the E. coli E44 to survival in stress environments. Real time RT-PCR was used to test the mRNA expression of IbeR. The expression levels of the major virulence gene ibeA in PD44 and E44 were also compared. The results showed that the IbeR and IbeA expression levels of ppk1 mutant were both lower than those of the parent strain E44, and the complementation of ppk1 gene could significantly improve the mRNA levels of IbeR and IbeA in PD44.Taken together, our findings suggest that the ppk1/poly P plays an important role in stress resistance and virulence in E. coli K1 E44. And the ppk1 deletion will result in a significant decline expression of the SP-regulator IbeR and important virulence factor IbeA, which is crucial for the E44 to survival in stress environment and cross the blood brain barrier.