| Bacteria of Shigella spp are high contagious, severe hazardous and gram-negative facutative intracellular pathogens, responsible for Shigellosis which characterized by fever, abdominal cramping and scanty, loose stools containing blood and mucus in clinic. Shigellasis remain a leading cause of infant mortality in the world. Shigella belong to the family Enterobacteriaceae and the group Escherichiaeae. They are divided into four species and at least 47 serotypes: Shigella dysenteriae (13 serotypes), Shigella flexneri (15 serotypes), Shigella boydii (18 serotypes), and Shigella sonnei (1 serotype). Shigella flexneri, the prevalent species in developing countries, kill more than 25,000 lives annually. In China, about 2 to 3 million cases of shigellosis are identified every year, accounting for more than 80% of the reported bacillary dysentery. Shigella flexneri 2a has been found to be responsible for the major epidemics and pandemics of shigellosis in China, accounting for 50-70% of the isolated strains.The mechanism of pathogenicity of Shigella flexneri is based on the capacity of itself to reach the colonic mucusa and to invade colonic epithelial cells, leading to intracellula bacterial multiplication, spread to adjacent cells, cell death, and eventually inflammation and destruction of the colonic mucosa . Enteroinvasive strain Shigella flexneri contains a 220kb large virulence plasmid which encode a series of factors required for virulence. Expression of several plasmid-encoded proteins, such as IpaB, IpaC, IpaD, Spa/Mxi and VirG etc., are nessesary for the complete plasmid phenotype of Shigella flexneri. The Ipa proteins act as the invasine, while the Spa/Mxi proteins make up a type III secretion apparatus which are involved in the presentation and transport of the Ipa proteins to and beyond the bacterial surface. In the virulence plasmid, the genes for proteins Ipa, Spa, Mxi and VirG are tightly located in a 31kb area, which is named as "invasion region", including about 30 genes. The expression of the virulence of the plasmid is regulated by several genes located both on the virulence plasmid and on the chromosome, as well as environmental stimuli.Determination of the complete DNA sequence of the virulence plasmid and the complete genome sequence of S. flexneri 2a 301 not only offer the opportunity to understand themechanism of pathogenicity and drug resistance of Shigella flexneri, but also provide comparisons of the virulence to those of different Shigella serotypes and closely related species. It is important for the recognition of the relationship of pathogenicity and evolution of 5. flexneri, shigellosis control, new drug discovery and vaccine development. Based on the special mechanism of invading into the host cell, it is potential to apply this mechanism in intracellular target drug delivery systems and construction of supper pathogen in military.We determined the complete DNA sequence of the large virulence plasmid pCP301 and the complete genome of 5. flexneri 2a strain 301, as well as analysis of genes on the large virulence plasmid and the structural genomic properties associated with bacterial virulence. The mutations related to the quinolone resistance and genetics and evolution of the QRAR sequences of gyrA and parC genes were also carried out.It resulted that the entire DNA sequence of pCP301 compose of 221,618 bp. Sequence analysis identified 272 open reading frames (ORFs), which include 194 corresponding to proteins described previously, 61 have low identity (<60%) to known proteins and 17 have no regions of significant homology with proteins in database. The genes of pCP301 mainly include virulence associated genes, regulation associated genes and genes relating to plasmid maintenance, stability and DNA metabolism. Eighteen kind of insertion sequence (IS) elements, including 5 new IS elements were identified in pCP301, which account for 68 kb in length and 30 percent of the plasmid. It suggests that the multitime gene rearrangements have taken place in S. flexneri evolution hi...
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