Pathogenic potential of environmental Vibrio cholerae strains carrying genetic variants of the toxin-coregulated pilus pathogenicity island

The major virulence factors of toxigenic Vibrio cholerae are cholera toxin (CT), which is encoded by a lysogenic bacteriophage (CTXPhi), and toxin-coregulated pilus (TCP), an essential colonization factor which is also the receptor for CTXPhi. The genes for the biosynthesis of TCP are part of a larger genetic element known as the TCP pathogenicity island. To assess their pathogenic potential, we analyzed environmental strains of V. cholerae carrying genetic variants of the TCP pathogenicity island for colonization of infant mice, susceptibility to CTXPhi, and diarrheagenicity in adult rabbits. Analysis of 14 environmental strains, including 3 strains carrying a new allele of the tcpA gene, 9 strains carrying a new allele of the toxT gene, and 2 strains carrying conventional tcpA and toxT genes, showed that all strains colonized infant mice with various efficiencies in competition with a control El Tor biotype strain of V. cholerae O1. Five of the 14 strains were susceptible to CTXPhi, and these transductants produced CT and caused diarrhea in adult rabbits. These results suggested that the new alleles of the tcpA and toxT genes found in environmental strains of V. cholerae encode biologically active gene products. Detection of functional homologs of the TCP island genes in environmental strains may have implications for understanding the origin and evolution of virulence genes of V. cholerae.     

Inhibition of Prostate Cancer Cell Colony Formation by the Flavonoid Quercetin Correlates with Modulation of Specific Regulatory Genes

The natural product quercetin is a flavonoid found in many fruits and vegetables. Previous research has shown that quercetin has antitumor, anti-inflammatory, antiallergic, and antiviral activities. In the present investigation we studied the effect of quercetin on the ability of prostate cancer cell lines with various degrees of aggressive potential to form colonies in vitro. Specifically, we examined the molecular mechanisms underlying this effect, including the expression of cell cycle and tumor suppressor genes as well as oncogenes. We observed that quercetin at concentrations of 25 and 50 μM significantly inhibited the growth of the highly aggressive PC-3 prostate cancer cell line and the moderately aggressive DU-145 prostate cancer cell line, whereas it did not affect colony formation by the poorly aggressive LNCaP prostate cancer cell line or the normal fibroblast cell line BG-9. Using the gene array methodology, we found that quercetin significantly inhibited the expression of specific oncogenes and genes controlling G₁, S, G₂, and M phases of the cell cycle. Moreover, quercetin reciprocally up-regulated the expression of several tumor suppressor genes. In conclusion, our results demonstrate that the antitumor effects of quercetin directly correlate with the aggressive potential of prostate cancer cells and that the mechanism(s) of quercetin-mediated antitumor effects may involve up-regulation of tumor suppressor genes and reciprocal down-regulation of oncogenes and cell cycle genes. The results of these studies provide a scientific basis for the potential use of flavonoids as nutraceuticals in the chemoprevention of cancer.     

Optimization of plasmid maintenance in the attenuated live vector vaccine strain Salmonella typhi CVD 908-htrA

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.     

New variants of Vibrio cholerae O1 biotype El Tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in Bangladesh

The sixth pandemic of cholera and, presumably, the earlier pandemics were caused by the classical biotype of Vibrio cholerae O1, which was progressively replaced by the El Tor biotype representing the seventh cholera pandemic. Although the classical biotype of V. cholerae O1 is extinct, even in southern Bangladesh, the last of the niches where this biotype prevailed, we have identified new varieties of V. cholerae O1, of the El Tor biotype with attributes of the classical biotype, from hospitalized patients with acute diarrhea in Bangladesh. Twenty-four strains of V. cholerae O1 isolated between 1991 and 1994 from hospitalized patients with acute diarrhea in Matlab, a rural area of Bangladesh, were examined for the phenotypic and genotypic traits that distinguish the two biotypes of V. cholerae O1. Standard reference strains of V. cholerae O1 belonging to the classical and El Tor biotypes were used as controls in all of the tests. The phenotypic traits commonly used to distinguish between the El Tor and classical biotypes, including polymyxin B sensitivity, chicken cell agglutination, type of tcpA and rstR genes, and restriction patterns of conserved rRNA genes (ribotypes), differentiated the 24 strains of toxigenic V. cholerae O1 into three types designated the Matlab types. Although all of the strains belonged to ribotypes that have been previously found among El Tor vibrios, type I strains had more traits of the classical biotype while type II and III strains appeared to be more like the El Tor biotype but had some classical biotype properties. These results suggest that, although the classical and El Tor biotypes have different lineages, there are possible naturally occurring genetic hybrids between the classical and El Tor biotypes that can cause cholera and thus provide new insight into the epidemiology of cholera in Bangladesh. Furthermore, the existence of such novel strains may have implications for the development of a cholera vaccine.     

Identification of a fibronectin-binding protein from Staphylococcus epidermidis

Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research S. epidermidis genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain embp. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of S. epidermidis, but not the binding of Staphylococcus aureus, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the S. aureus fibronectin-binding protein FnBPB, blocked binding of S. aureus to fibronectin but had a negligible effect on the binding of S. epidermidis.     

Inhibition of prostate cancer cell colony formation by the flavonoid quercetin correlates with modulation of specific regulatory genes

The natural product quercetin is a flavonoid found in many fruits and vegetables. Previous research has shown that quercetin has antitumor, anti-inflammatory, antiallergic, and antiviral activities. In the present investigation we studied the effect of quercetin on the ability of prostate cancer cell lines with various degrees of aggressive potential to form colonies in vitro. Specifically, we examined the molecular mechanisms underlying this effect, including the expression of cell cycle and tumor suppressor genes as well as oncogenes. We observed that quercetin at concentrations of 25 and 50 micro M significantly inhibited the growth of the highly aggressive PC-3 prostate cancer cell line and the moderately aggressive DU-145 prostate cancer cell line, whereas it did not affect colony formation by the poorly aggressive LNCaP prostate cancer cell line or the normal fibroblast cell line BG-9. Using the gene array methodology, we found that quercetin significantly inhibited the expression of specific oncogenes and genes controlling G(1), S, G(2), and M phases of the cell cycle. Moreover, quercetin reciprocally up-regulated the expression of several tumor suppressor genes. In conclusion, our results demonstrate that the antitumor effects of quercetin directly correlate with the aggressive potential of prostate cancer cells and that the mechanism(s) of quercetin-mediated antitumor effects may involve up-regulation of tumor suppressor genes and reciprocal down-regulation of oncogenes and cell cycle genes. The results of these studies provide a scientific basis for the potential use of flavonoids as nutraceuticals in the chemoprevention of cancer.     

Immunoregulatory effects of morphine on human lymphocytes.

It is now well established that parenteral drug abuse is a significant risk factor for contracting human immunodeficiency virus type 1 (HIV-1) infection and subsequently developing AIDS. Earlier studies have shown that morphine can modulate various immune responses and therefore support the premise that morphine is a cofactor in susceptibility to and progression of HIV infection. Dysregulation of interferon (IFN) production, nonspecific apoptosis of T cells, and the immune response to soluble HIV gene products have been associated with potential mechanisms of pathogenesis in HIV disease. The present study was undertaken to examine the immunomodulatory role of morphine on HIV protein-induced lymphocyte proliferative responses, Sendai and Newcastle disease virus-induced alpha IFN (IFN-alpha) and IFN-beta production by lymphocytes and fibroblast cells, respectively, and induction of apoptosis of normal lymphocytes in vitro. Our results demonstrate that HIV protein-induced human lymphocyte proliferative responses were significantly inhibited by morphine in a dose-dependent manner. Furthermore, morphine significantly inhibited both IFN-alpha and IFN-beta production by normal lymphocytes and fibroblasts but induced apoptosis of normal lymphocytes. Inhibition of IFN-alpha production by morphine could be reversed by the opiate receptor antagonist naloxone. This suggests that the immunomodulatory effects of morphine are mediated through the opioid receptor. These studies support a role of morphine as a cofactor in the pathogenesis of HIV infection and describe some of the possible pathologic mechanisms which underlie the immunoregulatory effects of morphine.     

RS1 element of Vibrio cholerae can propagate horizontally as a filamentous phage exploiting the morphogenesis genes of CTXphi.

In toxigenic Vibrio cholerae, cholera toxin is encoded by the CTX prophage, which consists of a core region carrying ctxAB genes and genes required for CTXPhi morphogenesis, and an RS2 region encoding regulation, replication, and integration functions. Integrated CTXPhi is often flanked by another genetic element known as RS1 which carries all open reading frames (ORFs) found in RS2 and an additional ORF designated rstC. We identified a single-stranded circularized form of the RS1 element, in addition to the CTXPhi genome, in nucleic acids extracted from phage preparations of 32 out of 83 (38.5%) RS1-positive toxigenic V. cholerae strains analyzed. Subsequently, the corresponding double-stranded replicative form (RF) of the RS1 element was isolated from a representative strain and marked with a kanamycin resistance (Km(r)) marker in an intergenic site to construct pRS1-Km. Restriction and PCR analysis of pRS1-Km and sequencing of a 300-bp region confirmed that this RF DNA was the excised RS1 element which formed a novel junction between ig1 and rstC. Introduction of pRS1-Km into a V. cholerae O1 classical biotype strain, O395, led to the production of extracellular Km(r) transducing particles, which carried a single-stranded form of pRS1-Km, thus resembling the genome of a filamentous phage (RS1-KmPhi). Analysis of V. cholerae strains for susceptibility to RS1-KmPhi showed that classical biotype strains were more susceptible to the phage compared to El Tor and O139 strains. Nontoxigenic (CTX(-)) O1 and O139 strains which carried genes encoding the CTXPhi receptor toxin-coregulated pilus (TCP) were also more susceptible (>1,000-fold) to the phage compared to toxigenic El Tor or O139 strains. Like CTXPhi, the RS1Phi genome also integrated into the host chromosomes by using the attRS sequence. However, only transductants of RS1-KmPhi which also harbored the CTXPhi genome produced a detectable level of extracellular RS1-KmPhi. This suggested that the core genes of CTXPhi are also required for the morphogenesis of RS1Phi. The results of this study showed for the first time that RS1 element, which encodes a site-specific recombination system in V. cholerae, can propagate horizontally as a filamentous phage, exploiting the morphogenesis genes of CTXPhi.