Genomic analysis of cronobacter sakazakii and molecular study of hfq and CSK29544_02616 genes as virulence factors

Abstract

Cronobacter sakazakii is considered to be an opportunistic pathogen causing life-threatening diseases, including necrotizing enterocolitis, septicemia, and meningitis particularly to infants with high fatality. However, the mechanisms of its pathogenicity and virulence-associated factors remain largely unknown. To understand them, complete genome of C. sakazakii ATCC 29544, a type strain, was sequenced and analyzed using bioinformatics. The complete genome of C. sakazakii ATCC 29544 is composed of a circular chromosome (Genbank accession No. CP011047) and three plasmids, including pCSK29544_p1 (Genbank accession No. CP011048), pCSK29544_p2 (Genbank accession No. CP011049), and pCSK29544_p3 (Genbank accession No. CP011050). It has been known that C.sakazakii infection mainly occurs via contaminated reconstituted infant formula milk. In this regard, I tried to identify several gene clusters, predicted to be responsible for the survival of C.sakazakii in harsh environments, including dried infant formula. Three gene clusters were found on chromosome, which may confer advantages on C.sakazakii survival against extremely dried conditions. These gene clusters include biosynthesis of capsular proteins (CSK29544_00281-00284) as protectants from osmotic shock, cellulose (CSK29544_01124-01127) to form biofilms, and nanKTAR (CSK29544_00587-590) to utilize sialic acid for energy production. Moreover, the strain ATCC 29544 may have arsenic resistance activity for survival in formula milk powder (CSK29544_3p0051-0055), on the plasmid 3p. These gene clusters, in part, may contribute to C.sakazakii survival under dried infant formula. On human infection, C. sakazakii has invasins, OmpA (CSK29544_03699) for BMEC adhesion and the ibeB-homologous cusC (pCSK29544_3p0028) for the penetration of BMECs. For survival within the host, the strain ATCC 29544 has the privileged iron acquisition system, including siderophore biosynthesis system (iucABCD/iutA

CSK29544_1p0024- CSK29544_1p 0028) and an ABC-type iron transport system (eitCBAD

CSK29544_1p0056- CSK29544_1p 0059). The analysis of complete genome sequences would shed light on the understanding of virulence mechanisms by C. sakazakii. As hfq has been reported as a critical virulence factor in many pathogenic bacteria, it was chosen to see its effects on C. sakazakii virulence. Hfq is a global regulator conversed in many pathogens, but its contributions to virulence vary from one species to another. Therefore, the role of Hfq in C. sakazakii virulence was investigated for the first time. In the absence of hfq, C. sakazakii was highly attenuated in dissemination in vivo, showed defects in invasion into animal cells and survival within host cells, and exhibited low resistance to hydrogen peroxide. Remarkably, the loss of hfq led to hyper-motility on soft agar, which is contrary to what has been observed in other pathogenic bacteria. The hyper-flagellated bacteria were likely to be attributable to the increased transcription of genes associated with flagella biosynthesis in a strain lacking hfq. Together, these data strongly suggest that hfq plays important roles in the virulence of C. sakazakii by participating in the regulation of multiple genes. To identify new virulence factors required for invasiveness of C. sakazakii ATCC 29544, transposon-mediated random mutant library was constructed and screened for detection of mutants with reduced invasion ability into human intestine epithelial Caco-2 cells compared with wild type. One of the mutants showing the most reduced invasion ability but a comparable growth with WT was found to have transposon insertion in the CSK29544_02616, which is highly conserved in only Cronobacter species and encodes an unidentified protein. The deletion mutant of CSK29544_02616 was constructed and various virulence-associated traits, including invasion to intestinal Caco-2 cell, in vivo animal study, survival/phagocytosis within/into cultured-macrophages, motility, and biofilm formation, was characterized. The lack of CSK29544_02616 showed attenuations in invasiveness, in vivo colonization and dissemination, phagocytosis and survival into/within macrophage-like cells. Based on the observations, I questioned the function of CSK29544_02616 of C.sakazakii ATCC 29544 in terms of virulence. As it is an unidentified protein, I determined to perform ligand fishing assay to screen players which help the function of CSK29544_02616 to be unveiled. I found that LpxA, the first enzyme involved in lipid A biosynthesis, was shown to specifically interact with CSK29544_02616. The amount of lipid A-core was decreased in CSK29544_02616 mutation, whereas that of phospholipids was increased. This altered ratio between Lipid A and phospholipids in outer membrane influenced outer membrane assembly and changed cell surface to be more hydrophobic, resulting in increased cell autoaggregation and biofilm formation. Interestingly, the mutant was non-motile although it had flagella and torque generation, indicating loss of CSK295444_02616 caused paralyzed flagella. Taken together, CSK29544_02616 plays critical roles in the virulence of C. sakazakii by modulating LPS production. CSK29544_02616 would be a good target for biocontrol of C. sakazakii in the way that the deletion of CSK29544_02616 does not affect C. sakazakii growth, but influence its virulence as an antibacterial agent. In conclusion, understanding the pathogenesis of C. sakazakii ATCC 29544 by both genomic analysis and molecular studies of virulence factors would be the cornerstone for the development of a new strategy to control C. sakazakii pathogenesis.