Acid adaptation in Streptococcus mutans UA159 alleviates sensitization to environmental stress due to RecA deficiency.

A RecA-deficient stain of Streptococcus mutans, isolated previously, was found to be more susceptible than the prototroph organism to acid killing and also showed reduced colony-forming ability on sucrose-containing medium. The deficient strain was able to grow in chemostat culture at a low pH value of 5 and did not show reduced capacity to produce acid in standard pH-drop experiments with excess glucose. Moreover, it was able to undergo an adaptive response when grown at a low pH to become more resistant to acid killing and also to killing by ultraviolet radiation or hydrogen peroxide. In fact, after adaptation, it was nearly as resistant as the prototroph strain. These findings were interpreted, in part, in terms of an acid-inducible DNA repair system which functions independently of RecA.

Human papillomavirus type in anal epithelial lesions is influenced by human immunodeficiency virus.

BACKGROUND AND OBJECTIVE: Infection with human immunodeficiency virus (HIV) increases the risk for human papillomavirus (HPV)-associated genital neoplasia. Human immunodeficiency virus-infected patients also have higher rates of treatment failure and more rapid neoplastic progression. Impaired immune function does not entirely explain these clinical observations. This pilot project was designed to investigate the hypothesis that HIV infection is associated with changes in HPV type and integration within anogenital lesions that could explain the increased risk of neoplastic progression. METHODS: Anal neoplastic lesions from patients with and without HIV infection were analyzed for the presence, type, and integration status of HPV by colorimetric in situ hybridization. Tissue localization of HIV was evaluated by p24 immunohistochemistry and HIV-1 DNA polymerase chain reaction. Results for matched histology were compared for the two patient groups. RESULTS: For all lesions, the presence of high-risk HPV types and multiple HPV types was strongly associated with HIV infection (P = .003 and .0003, respectively). For lesions with matched histology there was no association of HPV integration with HIV status. Tissue localization of HIV did not significantly influence HPV type or integration. CONCLUSIONS: The presence of high-risk HPV types and multiple types within low-grade lesions may explain the increased risk of neoplastic progression in HIV patients. Colocalization of HIV and HPV does not appear to be required for this effect. There is no evidence that HPV integration is influenced by HIV infection.

Sequence variation in the capsid protein genes of human papillomavirus type 16 and type 31.

The sequences of the capsid genes of a human papillomavirus type 16 (HPV 16) DNA and an HPV 31 DNA were determined. The HPV 16 DNA contained genes coding for the most variable HPV 16 capsid proteins yet identified (17 variable amino acids). Three of six coding changes in the HPV 31 DNA occurred at positions equivalent to ones where variable amino acids in HPV 16 have been observed. Variable amino acids in both viruses occurred predominantly in regions which showed amino acid variation when closely related types of HPV were compared; thus, most of the factors which determined the intratypic variation in the capsid proteins of the viruses described here were likely the same as those which determined differences between the capsid proteins of different HPV types.

Streptococcus salivarius urease: genetic and biochemical characterization and expression in a dental plaque streptococcus.

The hydrolysis of urea by urease enzyme of oral bacteria is believed to have a major impact on oral microbial ecology and to be intimately involved in oral health and diseases. To begin to understand the biochemistry and genetics of oral ureolysis, a study of the urease of Streptococcus salivarius, a highly ureolytic organism which is present in large numbers on the soft tissues of the oral cavity, has been initiated. By using as a probe a 0.6-kpb internal fragment of the S. salivarius 57.I ureC gene, two clones from subgenomic libraries of S. salivarius 57.I in an Escherichia coli plasmid vector were identified. Nucleotide sequence analysis revealed the presence of one partial and six complete open reading frames which were most homologous to ureIAB-CEFGD of other ureolytic bacteria. Plasmid clones were generated to construct a complete gene cluster and used to transform E. coli and Streptococcus gordonii DL1, a nonureolytic, dental plaque microorganism. The recombinant organisms expressed high levels of urease activity when the growth medium was supplemented with NiCl2. The urease enzyme was purified from E. coli, and its biochemical properties were compared with those of the urease produced by S. salivarius and those of the urease produced by S. gordonii carrying the plasmid-borne ure genes. In all cases, the enzyme had a Km of 3.5 to 4.1 mM, a pH optimum near 7.0, and a temperature optimum near 60 degrees C. S. gordonii carrying the urease genes was then demonstrated to have a significant capacity to temper glycolytic acidification in vitro in the presence of concentrations of urea commonly found in the oral cavity. The ability to genetically engineer plaque bacteria that can modulate environmental pH through ureolysis will open the way to using recombinant ureolytic organisms to test hypotheses regarding the role of oral ureolysis in dental caries, calculus formation, and periodontal diseases. Such recombinant organisms may eventually prove useful for controlling dental caries by replacement therapy.

Characterization of recombinant, ureolytic Streptococcus mutans demonstrates an inverse relationship between dental plaque ureolytic capacity and cariogenicity.

Dental caries results from prolonged plaque acidification that leads to the establishment of a cariogenic microflora and demineralization of the tooth. Urease enzymes of oral bacteria hydrolyze urea to ammonia, which can neutralize plaque acids. To begin to examine the relationship between plaque ureolytic activity and the incidence of dental caries, recombinant, ureolytic strains of Streptococcus mutans were constructed. Specifically, the ureABCEFGD operon from Streptococcus salivarius 57.I was integrated into the S. mutans chromosome in such a way that the operon was transcribed from a weak, cognate promoter in S. mutans ACUS4 or a stronger promoter in S. mutans ACUS6. Both strains expressed NiCl(2)-dependent urease activity, but the maximal urease levels in ACUS6 were threefold higher than those in ACUS4. In vitro pH drop experiments demonstrated that the ability of the recombinant S. mutans strains to moderate a decrease in pH during the simultaneous metabolism of glucose and urea increased proportionately with the level of urease activity expressed. Specific-pathogen-free rats that were infected with ACUS6 and fed a cariogenic diet with drinking water containing 25 mM urea and 50 microM NiCl(2) had relatively high levels of oral urease activity, as well as dramatic decreases in the prevalence of smooth-surface caries and the severity of sulcal caries, relative to controls. Urease activity appears to influence plaque biochemistry and metabolism in a manner that reduces cariogenicity, suggesting that recombinant, ureolytic bacteria may be useful to promote dental health.

A longitudinal study of human papillomavirus DNA detection in human immunodeficiency virus type 1-seropositive and -seronegative women.

Cericovaginal lavage samples from 124 human immunodeficiency virus type 1 (HIV-1)-seropositive and 126 HIV-1-seronegative women were collected monthly for 8 months and tested for human papillomavirus (HPV) DNA. The estimated prevalence of HPV was 42.8% in HIV-1-seropositive and 13.4% in -seronegative women (P < .001). There was no significant difference in HPV DNA detection in HIV-1-seropositive women with CD4 cell counts of < 300/mm3 (50% HPV-positive), 300-499/mm3 (36.4% HPV-positive), or > or = to 500/mm3 (40.5% HIV-positive). However, HIV-1-seropositive women who were more immunocompromised, as indicated by lower CD4 cell counts, were more likely to shed HPV persistently. The quantity of HPV DNA detected in cervicovaginal lavage samples was similar in HIV-1-seropositive and -seronegative women. This study further defined the characteristics of HPV infections in HIV-1-infected women.

Correlation of human immunodeficiency virus type 1 RNA levels in blood and the female genital tract.

In this study, the correlations of human immunodeficiency virus type 1 (HIV-1) RNA levels in blood plasma, vaginal secretions, and cervical mucus of 52 HIV-1-infected women were determined. The amount of cell-free HIV-1 RNA in blood plasma was correlated with that in vaginal secretions (Spearman's rank correlation coefficient (r) = 0.64, P<.001). In both blood plasma and vaginal secretions, the amounts of cell-free and cell-associated HIV-1 RNA were highly correlated (r=0.76, P<.01 and r=0.85, P<.01, respectively). Cell-free HIV-1 RNA levels in blood plasma and vaginal secretions were negatively correlated with CD4+ T lymphocyte count (r=-0.44, P<.01 and r=-0.40, P<.01, respectively). Similar to the effect observed in blood plasma, initiation of antiretroviral therapy significantly reduced the amount of HIV-1 RNA in vaginal secretions. These findings suggest that factors that lower blood plasma virus load may also reduce the risk of perinatal and female-to-male heterosexual transmission by lowering vaginal virus load.

Cellular replication of human immunodeficiency virus type 1 occurs in vaginal secretions.

Most human immunodeficiency virus type 1 (HIV-1) transmission worldwide is the result of exposure to infectious virus in genital secretions. However, current vaccine candidates are based on virus isolates from blood. In this study, vaginal secretions from HIV-1-infected women were examined for evidence of cellular viral replication that produced virus with properties different from that in blood. Multiply spliced HIV-1 messenger RNA, which is found only in cells replicating virus, was detected in all vaginal lavage samples tested. There was a strong correlation between the amounts of multiply spliced HIV-1 messenger RNA and of cell-free HIV-1 RNA in the lavage samples. In addition, significant genotypic differences were found in cell-free virus from matched blood plasma and vaginal secretions. Moreover, drug resistance-associated mutations appeared in plasma virus several months before appearing in vaginal virus. These findings indicate that cellular replication of HIV-1 occurs in vaginal secretions and can result in a virus population with important differences from that in blood.