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ABSTRACTLipid A structure is a critical determinant of the interaction between pathogens and the innate immune system. Previously, we demonstrated the presence of non- and monophosphorylated tetra-acylated lipid A structures in the outer membrane of Porphyromonas gingivalis, an agent of human periodontal disease. These modifications to lipid A structure lead to evasion and suppression of innate defenses mediated by Toll-like receptor 4 (TLR4) and cationic antimicrobial peptides. In this investigation, we examined the influence of growth temperature on P. gingivalislipid A structure and recognition by TLR4 as an example of an environmental influence which is known to vary between healthy and diseased sites in the periodontium. We demonstrate that P. gingivalisgrown at a normal body temperature produces mainly nonphosphorylated and monophosphorylated tetra-acylated lipid A structures, whereas bacteria grown at 39°C and 41°C intended to mimic increasing levels of inflammation, producing increasing proportions of monophosphorylated, penta-acylated lipid A. The temperature-dependent alteration in lipid A renders the bacterium significantly more potent for activating TLR4 and more susceptible to killing by β-defensins 2 and 3. This is the first report of a lipid A remodeling system linked to temperature shifts associated with a deregulated inflammatory response. Temperature elevation at sites of inflammation in the periodontium may be a significant environmental regulator of the lipid A modification systems of P. gingivalis, which will influence the interaction of this organism with the innate host defense.

Lipid A structure is a critical determinant of the interaction between pathogens and the innate immune system. Previously, we demonstrated the presence of non- and monophosphorylated tetra-acylated lipid A structures in the outer membrane of Porphyromonas gingivalis, an agent of human periodontal disease. These modifications to lipid A structure lead to evasion and suppression of innate defenses mediated by Toll-like receptor 4 (TLR4) and cationic antimicrobial peptides. In this investigation, we examined the influence of growth temperature on P. gingivalis lipid A structure and recognition by TLR4 as an example of an environmental influence which is known to vary between healthy and diseased sites in the periodontium. We demonstrate that P. gingivalis grown at a normal body temperature produces mainly nonphosphorylated and monophosphorylated tetra-acylated lipid A structures, whereas bacteria grown at 39°C and 41°C intended to mimic increasing levels of inflammation, producing increasing proportions of monophosphorylated, penta-acylated lipid A. The temperature-dependent alteration in lipid A renders the bacterium significantly more potent for activating TLR4 and more susceptible to killing by β-defensins 2 and 3. This is the first report of a lipid A remodeling system linked to temperature shifts associated with a deregulated inflammatory response. Temperature elevation at sites of inflammation in the periodontium may be a significant environmental regulator of the lipid A modification systems of P. gingivalis, which will influence the interaction of this organism with the innate host defense.

ABSTRACTImmune responses in the guinea pig model are understudied because of a lack of commercial reagents. We have developed a custom-made guinea pig oligonucleotide microarray (81 spots) and have examined the gene expression profile of splenocytes restimulated in vitro from Mycobacterium bovisBCG-vaccinated and naive animals. Eleven genes were significantly (P< 0.05) up-regulated following vaccination, indicating a Th1-type response. These results show that microarrays can be used to more fully define immune profiles of guinea pigs.

ABSTRACTProtection of cattle against bovine tuberculosis by vaccination could be an important control strategy in countries where there is persistent Mycobacterium bovisinfection in wildlife and in developing countries where it is not economical to implement a tuberculin test and slaughter control program. The main aim of such a vaccination strategy would be to reduce transmission of infection by reducing the lung pathology caused by infection and preventing seeding of the organism to organs from which M. boviscould be excreted. Recent reports of successful DNA vaccination against Mycobacterium tuberculosisin small-animal models have suggested that DNA vaccines act by reducing lung pathology without sensitizing animals to tuberculin testing. We therefore evaluated the ability of vaccines consisting of DNA encoding the mycobacterial antigens MPB83 and 85A to reduce lung pathology and prevent hematogenous spread in guinea pigs challenged with a low dose of aerosolized M. bovis. Vaccination with MPB83 DNA reduced the severity of pulmonary lesions, as assessed by histopathology, and resembled M. bovisBCG vaccination in this respect. However, unlike BCG vaccination, MPB83 DNA vaccination did not protect challenged guinea pigs from hematogenous spread of organisms to the spleen. In contrast, vaccination with antigen 85A DNA, a promising DNA vaccine for human tuberculosis, had no measurable protective effect against infection with M. bovis.

ABSTRACTTuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovisand despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosisrelative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovisand protection of the spleens of guinea pigs infected with M. tuberculosisin the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.

ABSTRACTPorphyromonas gingivalisis a gram-negative, black-pigmented anaerobe that has been associated with advanced periodontal disease. The genome of P. gingivalishas the potential to produce a number of virulence determinants including proteases, hemagglutinins, hemolysin, invasion-associated proteins, and products of the pathogenicity island ragAB; however, little is known about how their expression is controlled. Periodontal pockets experience a higher temperature during inflammation, and this elevated temperature may influence the pathogenicity of P. gingivalisby changing its patterns of gene expression. In this study, RNA has been isolated from cells of P. gingivalisgrown to steady state at temperatures of 37, 39, and 41°C under hemin excess conditions (pH 7.0) in a chemostat. The RNA was subjected to PCR amplification following reverse transcription, using various combinations of randomly selected oligonucleotide primers. Reproducible RNA fingerprints have been obtained; however, differences were demonstrated in the RNA profiles of cells grown at the three temperatures, indicating differences in gene expression. Several PCR fragments were isolated that appeared to represent temperature-regulated genes. The nucleotide sequence of one of these has been identified as part of the ragABlocus, which codes for both a 55-kDa immunodominant antigen (RagB) and a homologue of the family of TonB-linked outer membrane receptors (RagA). These data indicate that expression of ragABmay be modulated in response to changes in temperature and that this may suggest a mechanism of evading the host response in the inflamed periodontal pocket.

A comparison was made of acid production by cells of Streptococcus mutansIngbritt and S. sanguisNCTC 7865 that had been washed twice and incubated in different concentrations of sodium and potassium ions. Organisms were grown under defined conditions in a chemostat under both glucose limitation and glucose excess conditions at a dilution rate of 0.1 h−1(mean generation time, 6.9 h). Acid production after a pulse of glucose, sucrose, and fructose was measured by pH fall experiments and as a rate at pH 7.0. S. mutansproduced more acid than S. sanguisas measured by either criterion, although statistically faster rates of acid production and lower terminal pH values were obtained when cells of both species were suspended in KCl rather than in NaCl, with 200 mM KCl resulting in the lowest terminal pH in pH fall experiments. Sodium ions inhibited acid production: 183 mM NaCl reduced the glycolytic rates of S. mutansand S. sanguismetabolizing glucose at pH 7.0 in 135 mM KCl by 39 and 33%, respectively. The most pronounced stimulatory effect of potassium on acid production was by washed cells of S. sanguisthat had been grown under arginine and under phosphate limitation. The pH fell by a further 0.86 and 1.21 pH units, respectively, and to below the critical pH for enamel demineralization when these cells were metabolizing glucose in 135 mM KCl compared with the same concentration of NaCl. This enhancement of acid production was not due to potassium translocation, as had been suggested previously, because no movement of potassium ions across the cell membrane could be detected. An alternative explanation is proposed in which sodium ions are excluded from the cell at the expense of membrane energy, i.e., the proton motive force, which could otherwise be used for the transport of sugars.

ABSTRACTCoaggregation is a well-characterized phenomenon by which specific pairs of oral bacteria interact physically. The aim of this study was to examine the patterns of coaggregation between obligately anaerobic and oxygen-tolerant species that coexist in a model oral microbial community. Obligate anaerobes other than Fusobacterium nucleatumcoaggregated only poorly with oxygen-tolerant species. In contrast, F. nucleatumwas able to coaggregate not only with both oxygen-tolerant and other obligately anaerobic species but also with otherwise-noncoaggregating obligate anaerobe–oxygen-tolerant species pairs. The effects of the presence or absence of F. nucleatumon anaerobe survival in both the biofilm and planktonic phases of a complex community of oral bacteria grown in an aerated (gas phase, 200 ml of 5% CO2in air · min−1) chemostat system were then investigated. In the presence of F. nucleatum, anaerobes persisted in high numbers (>107· ml−1in the planktonic phase and >107· cm−2in 4-day biofilms). In an equivalent culture in the absence of F. nucleatum, the numbers of black-pigmented anaerobes (Porphyromonas gingivalisand Prevotella nigrescens) were significantly reduced (P≤ 0.001) in both the planktonic phase and in 4-day biofilms, while the numbers of facultatively anaerobic bacteria increased in these communities. Coaggregation-mediated interactions between F. nucleatumand other species facilitated the survival of obligate anaerobes in aerated environments.