A Novel Facultative Intracellular Bacterium-HLK1～T: The Ability And Mechanism Of Invasion And Persistent Infection In Human Cells

Bacterial pathogens are the leading cause of death worldwide. Not only new Bacterial pathogens emerging, but also the re-emergence of deadly infectious diseases, and the increasing prevalence of antimicrobial resistant strains, present a formidable threat to public health and welfare. So, studies on the detection of the potential bacterial pathogens and the mechanisms of pathogenetic are of significant imporntace.K562 is a human leukemia cell line and is used in the laboratories all over the world. During karyotyping K562 using Giemsa staining, to our surprise, we repeatedly observed rod-bacteria like structures within some of the cells. We then checked if the culture was contaminated by the environmenatal bacteria, as this could occur in cell culture. We collected the medium and cells separately from the same culture and plated them separately on the blood agar. Bacterial colonies were observed on the blood agar plated with the cells but not the medium. It is interesting to note that the K562 cells in the culture grow healthily with perfect morphology, and the culture looks clean without any signs of bacterial contamination. It would be quite unusual if the cells indeed carry intracellular bacteria, because most of the known intracellular microbes in human cells eventually damage and kill host cells after invasion. On the basis of these observations, we hypothesized that the K562 cells may carry an unidentified intracellular bacterial species, named as HLK1~T. Since HLK1~T is a novel species, it is essential that its biological characteristics be studied.Part 1: Isolation and characterization of the HLK1TObjective: To isolate and characterize the HLK1~T from the human erythro leukemia cell line K562.Methods: HLK1~T was isolated from K562 cells by plated on the blood agar. The morphology of HLKl was examined under scanning and transmission electron microscope. The bacterium was studied by standard conventional biochemical methods and the API 20NE test kit. Genomic DNA of HLK1~T was prepared using a bacterial genomic DNA purification kit and the G + C content of the DNA was determined by HPLC. The 16S RNA and gyrB genes of this microbe were sequenced. The phylogenetic relationships between the isolated strain and closely related bacteria were determined using MEGA2 software. The bootstrap values were obtained with the MEGA2 software package.Results: HLK1~T is a gram-negative rod (0.3 to 0.5 by 0.5 to 2 um), and motile with a polar flagellum. It is nonfermentative, aerobic, and oxidase and catalase positive. Phylogenetically, although it shares 98% homology of the 16S rRNA with Phenylobacterium lituiforme, the 2 species only have 89% homology of gyrB. It has the DNA G + C content of 71.2 ± 0.2 mol%, slightly higher than that (66.5 ± 0.5 mol%) of P. lituiforme. HLK1T also differs from the P. lituiforme on some aspects of growth and biochemical characteristics. On the basis of the phylogenetic and phenotypic characterisation, HLK1T is proposed to be classified in the genus Phenylobacterium, as zucineum sp. nov.Conclusion: HLK1T from K562 cells was successfully isolated from K562 cells. HLK1T is a novel species, and named as Phenylobacterium zucineum sp. nov..Part 2: Characterization of the invasion mechanisms and life-styles of theintracellular HLK1TObjiective: To characterize the ability and mechanism of HLK1T invasion into human host cells, and to identify the life-styles of HLK1T in host cells-Methods: Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were applied to examine the HLK1T invasion and life-styles in SW480, L02 and Jurkat cells. Confocal laser scanning microscopy (CLSM) was used to identify the function of actin and tubulin in HLK1T invasion and motility. Immunochemistry and CFU counting were used to indentify the enteroinvasive ability into SW480 and Jurkat cells. HLK1T genome was sequenced. The whole genome sequence was analyzed by bioinformatics to identify the virulence factors in bacteria invasion and intracellular infection.Results: HLK1T can tightly adhere to the surface of SW480, L02 and Jurkat cell, and induce phagocytic cup formation and invave into cells. The process of HLK1T invasion into host cells depends on the polymerization of F-actin and microtube. The TEM results indicate that HLK1T can escape from the phagocitic vacuole, replicate in the cytosol, and move to the region where there is abundance of reticulum around the nucleus. HLK1 can form protrusions that endocytosed by neighboring cells. The ability of enteroinvasive into SW480 and Jurkat cells is time and concentration dependent. The results of bioinformatics analysis imply that, there are many genes in HLK1T which encode the adheren and invasion proteins suchaspilus assembly protein, fimbriae, components of ype IVpilus pilin subunit, P60 family protein, invasion protein A, invasin protein et al., and the lipase or esterase protein that involve in the phagocitic vacuole lysis. In HLK1T, there are also have the type IV secretion systems - VirB/VirD4, which areessential for some intracellular bacteria surviving in host cells. The genes encode the VirB/VirD4 systems are in the same operon. And the VirB/VirD4 systems are highly homologue with the systems in the facultative intracellular bacteria - Brucella and Bartonella.Conclusions: The facultative intracellular bacterium - HLK1T can tightly adhere to the host cell surface and invade human cells via a "Zipper" entry mechanism. The process of invasion is dependent on the HLK1 viability and host cell cytoskeleton. HLK1T disseminated through the cytosol can spread from cell to cell. The invasion of HLK1T into SW480 and Jurkat cells were time and density dependent. HLK1T have the virulence factors that essential for invasion into host cells and surviving in intracellular.Part 3: The toxicity and persistent infection of HLK1 to human host cells Objective: To study the toxicity of HLKlTto human host cells, and the ability and types of persistent infection in human colon cancer cell line SW480 and human lymphoma cell line Jurkat.Methods: Flow cytometry was used to identify the cell growth rate and cell cycle distribution. DNA electrophoresis was used to detect DNA-ladder. The host cell death and intracellular bacterium cell were identified by TEM. CFU assay was used to detect the intracellular HLK1T' and persistent infection.Results: At invasion stage, the growth rates of the host cells were impaired in a time and MOI dependent manner. In the presence of HLK1T at MOI of 500 for 72 hours, although greatly reduced in comparison with cells not treated with the microbe, the tatal cell numbers of SW480 and Jurkat cells were increased modestly (63.75 +16.25)% and (93.75 + 11.84)%, respectivley. HLK1T can simultaneously induce apoptosis and necrosis of SW480, characterized by blockade at S-phase. However, in the stage of chonic infection, i.e., cells recovered from acute infection after removing extracelular bacteria, neither S W480 nor Jurkat were affected by the presence of the intracellular HLK1 on the aspect of cell cycle distribution and cell growth rates. Under electron microscope, intracellular HLK1T were present in cytoplasm, and most of them appeared to be featured with an L-phase-like structure, without typical 3-layer outer structure of the Gram-negative bacterium. The L-phase-like bodies constituted for (91.8 ± 5.1)% and (63.75 + 16.25)% of the total numbers of the intracellular HLK1 in Jurkat and SW480, respectively.Conclusion: At invasion stage, HLK1T has modest toxicity to SW480 and Jurkat cells, through inhibiting cell growth, blocking cell cycle at S-phase, inducing cell apoptosis and necrosis simultaneously. Persistent HLK1T are transited to L-phase spheroplast in host cells, and keeps a pace with the growth rate of the host cells. Persistent presence of intracellular HLK1T doesn't affect the growth of the host cells.Part 4: The mechanisms of HLK1T persistent infection in human host cells Objective: CtrA is a master response regulator found in many alpha-proteobacteria. In C. crescentus, CtrA is the most important master regulator controlling multiple cell cycle events, such as DNA rnethylation, DNA replication, flagella and pili biogenesis and septation. CtrA itself is also regulated in cell cycles at the levels of transcription, phosphorylation, and proteolysis. If CtrA remains constitutive expressed, C. cresenctus could not complete the cell cycle and would remain at predivisional status. In the genome of HLK1T, we found a gene (ctrZ), which was highly similar to ctrA. Since CtrA is a master regulator which is of critical importance in controlling cell cycle, we hyposized that the gene (ctrZ) is probablythe master regulator contolling the cell cycle of HLK1T , and hence may contribute to the persistent infection of HLK 1 in the host cells.Methods: CLUSTALW was used to compare the homologous of CtrZ with CtrA. SWISS-MODEL was used to predict the tertiary structure of CtrA and CtrZ. The gene of ctrZ was cloned by PCR. The vectors of pSAL-ctrZ and pSAL-pro-ctrZ were constructed. The vectors of pSALl.4, pSAL-ctrZ and pSAL-pro-ctrZ were introduced into LS2195 from E. coli SI7-1 by conjugation separetely. TEM and Gimsa staining were used to identify the shape of LS2195 transfected with the indicated plasmids containing ctrA or ctrZ insert. LS2195 is a mutant of C. crescentus with a point mutation of ctrA, which lead the the strain non-divivional at 37 °C.Results: CtrZ belongs to a unique subfamily of the OmpR family of helix-turn-helix transcriptional regulators, have 94 percent of homologue with CtrA. The primary sequence and tertiary structure of CtrZ were highly similar to CtrA. The ctrZ and ctrA promoters are strikingly similar. After successfully introduce the plasmid [LS2195(pSAL114), LS2195(pSAL-ctrZ) and LS2195(pSAL-pro-ctrZ)] into LS2195, the temperature sensitive LS2195 with CtrA or CtrZ could grow at 37°C the same as the wild type C. crescentus, indicating that CtrZ could complement the function of CtrA. The results suggested that CtrZ is a master regulator of HLK1T.Conclusions: CtrZ has 94 percent of homologuous with CtrA. CtrZ can complement the C. crescentus ctrA mutant at the restrictive temperature. The promoters of ctrZ and ctrA confer the same mechanism in regulation of gene transcription.