Molecular Pathogenesis And Molecular Diagnosis Study Of Human Mucosal Pathogens

Human microbial pathogens including some commensals (normal microflora, indigenous microbiota) consist of those microorganisms both are present on body surfaces covered by epithelial cells and are exposed to the external environment (gastrointestinal and respiratory tract, vagina, skin, etc.). The number of bacteria colonizing mucosal and skin surfaces exceeds the number of cells forming human body. These microbial pathogens co-evolved with their hosts. It is therefore not surprising that they have developed a large array of virulence factors well suited to interfere with or stimulate a variety of host-cell responses in order to invade, survive, replicate and some of them even cause cancer within their hosts. So the identification and characterization of the virulence factors involved in their pathogenesis and early diagnosis of their existence in human body are crucial in providing the basis for development of novel preventive and therapeutic approaches against the infection cause by them. Taking a multidisciplinary approach involving bacterial genetics, cell biology, biochemistry, molecular biology, and immunology, we sought to study the molecular pathogenesis of two important human mucosal pathogens, group Bstreptococcus (GBS) and Bacteroides fragilis (B. fragilis) as well as the early diagnosis of human papillomavirus (HPV) infection combing restriction display (RD) technique and DNA chip technology.GBS is a gram-positive commensal bacterium colonizing the intestinal tract of a significant proportion of the human population and it is also a major cause of life-threatening infection in newborn infants, causing sepsis, meningitis and pneumonia. The capsular polysaccharide (CPS) of GBS is both a major virulence determinant and an important vaccine antigen. To start with, we focused on the molecular genetics and biochemistry of capsular polysaccharide production in GBS. Previous work had identified CpsL as a protein required for capsular polysaccharide synthesis, although its precise function was undefined. We used a novel system of genetic complementation to demonstrate the function of a pupative polysaccharide repeating unit transporter CpsL in GBS. The results show that:1. A non-polar deletion mutation was introduced into cpsIaL, a conserved gene in the capsule gene cluster of type la GBS.2. Inactivation of cpsIaL completely abrogated production of capsular polysaccharide without disrupting transcription of downstream genes in the capsule synthesis operon.3. Expression of cps3L, the corresponding gene from type III GBS, from the chromosomal tetM locus in the AlaL mutant restored type la capsular polysaccharide production.In conclusion: This work established that the function of CpsL was independent of the repeating unit structure of the polysaccharide and supported its role as part of the polysaccharide export machinery.In further work on CpsL, we made use of alkaline phosphatase translational fusions to map the topology of CpsL in type la GBS strain 515. Based on the hypothesized computer model, we constructed and expressed a series of more than 19 recombinant fusion proteins and measured their enzymatic activity as an index of exposure to the interior or exterior of the bacterial cell.In order to further investigate the molecular pathogenesis of GBS, we expanded our study to global regulation of virulence gene expression and quorum sensing in GBS. Microorganisms live in environments subject to rapid changes in the availability of the carbon and nitrogen compounds necessary to provide energy and building blocks for the synthesis of cell material. Their survival depends on their ability to regulate the expression of genes coding for the enzymes and transport proteins required for growth in the altered environment. PhoB, a member of the two-component signal transduction regulatory system in some microorganisms, is known to be a transcriptional activator of the Pho regulon, expression of which is activated during phosphate starvation. We found that GBS also possess a phoB homologous gene, in order to investigate the mechanism of global virulence gene expression regulation inGBS, we created phoB deletion mutants in GBS la 515 and V 2603 backgounds, and performed preliminary study for their genotypic and phenotypic characteristics. These mutants provide powerful tools for further investigation of Pho regulator target genes, molecular interaction of Pho regulator with other regulators, as well as the role of Pho regulator in GBS virulence global regulation.Certain GBS products have been implicated in its virulence, but very little is known about the factors that mediate the transition from asymptomatic colonization to invasive infection. Bacteria often respond to specific environmental signals that trigger the expression of factors required for infection. One system many pathogenic organisms use is cell density -dependent response, or quorum sensing. In gram-negative organisms, accumulation of a specific molecule, Autoinducer-1 (AI-1), activates expression of a variety of virulence factors. A second system, Autoinducer-2 (AI-2), appears to be used by both gram positive and negative bacteria for cell-cell communication. An essential component of the AI-2 pathway is LuxS. So, to start with, we tested the ability of GBS to induce bioluminescence in V. harveyi reporter strain BB170 and BB960. Then, we constructed and characterized luxS deletion mutants in the GBS la 515 and V 2603 background. After that, we assayed the effect of the luxS deletion on bioluminescence. We detected an approximately two-fold decrease of bioluminescence inducing activity in the luxS deltion mutants comparedto the wild-parent strains. These results suggest the decreased bioluminescence in strain BB170 is due to the luxS deletion. However, similar to findings in S. pyogenes, it appears that some other molecules in GBS may also be able to induce bioluminescence in V. harvyie strain BB170.B. fragilis is an gram-negative obligate anaerobic bacterium found in the mammalian gastrointestinal tract and it palys a crucial role both in severe intraabdominal infections and in mucosal immunity. As with GBS, polysaccharides also are the major virulence factor in B. fragilis. In order to investigate the role of bacterial polysaccharides in eliciting immune responses during host-bacterial interactions, we generated and confirmed a series of bacterial deletion mutants deleted in the production of known immunomodulatory polysaccharides in the organism B. fargilis. Then we studied the effect of the deletion of one or more specific polysaccharides on the other polysacchande expression in different mutants by Immunoblot analysis. The results show that:1. Normally, PSA and PSB are the dominant polysaccharides in B. fragilis.2. The expression of PSA is decreased in the absence of PSB.3. The expression of PSG needs the presence of either PSA or PSB.4. PSG expression is increased in the absence PSD.5. In animals colonized with APSA mutants, overtime, we see increased expression of PSB and PSG.As zwitterionic polysaccharides (ZPSs) with both positively and negatively charged motifs produced by B. fragilis are essential for abscess formation, we analyzed zwitterionic characteristics of the mutants by IEP-Immunoblot technique based on their migration direction and distance. The results show that the APSA/APSB/APSG triple mutant migrates more slowly than WT strain and other mutants. Combining previous data, we speculate that excep the known ZPSs PSA and PSB, there are other ZPSs out of the 8 capsular polysaccharides synthesized by B. fragilis.With a view to determine the function of the polysaccharides in B. fragilis, wedeveloped a co-culture model whereby bacterial strains are used to infect dendritic cells (DCs) in vitro. Combining the confocol Microscopy technology, the B. fragilis-DC in vitro co-culture experiments provided the data showing that the bacterial strains are internalized differently into DCs based on the presence of certain polysaccharides and the DCs can support the B. fragilis growth in the co-culture system.HPV infection has been shown to cause virtually all cases of cervical cancer and its preinvasive cytopathoglogic precursors, and that virtually all cases of invasive cervical cancer worldwide contain DNA of the oncogenic HPV types. So the early detection of the high-risk oncogenic HPV molecules has important clinical value. A relatively newly developed technology for gene detection and analysis, DNA chip technology, provides a very powerful tool which may probably be of great value for the clinical diagnosis of infectious diseases and the monitoring of the therapeutics. To explore the application of DNA chip technology for detection and typing of HPV, we prepared HPV DNA chip by isolating gene fragments from HPV6, 11, 16, and 18 genome and then printing them onto aminosilane-coated glass slides as probes. Then, labeled the HPV samples with fluorescence dye by restriction display (RD) technique and hybridized them with the HPV DNA chip. The results of the study demonstrated the high sensibility and reproducibility of using restriction display and DNA chip technology to detect and genotype HPV, suggesting that this method may be expanded to early detection of other pathogens as well.To conclude, we have shown that CpsL is important in the biosynthesis of capsular polysaccharides in GBS and we also mapped the topology of CpsL in GBS. These studies provided some of the most definitive data available on the function of the polysaccharide synthesis and export machinery in this important bacterial pathogen. We created phoB deletion mutants in GBS, providing powerful tools for further virulence gene global regulation in GBS. We identified the quorum sensing activity in GBS and found the deletion of luxS causes a decrease of this activity. These results shed new light on regulatory pathways that control virulence in GBS. We created and characterized a series of polysaccharide deletion mutants of B.fragilis and developed an in vitro co-culture model for their functional study, the work done so far is very important in understanding how B. fragilis interacts with immune cells. Using HPV as a test pathogen, we demonstrated the feasibility and optimistic prospect of combining DNA chip technology and restriction display technique in pathogen early detection and genotyping.