No abstract

The ability of the pathogenic Gram-positive bacterium Streptococcus pyogenes (group A streptococcus) to bind fibronectin and adhere to respiratory epithelial cells is dependent on a surface protein called protein F. In this study, we have examined the regulation of expression of protein F and have shown that it is environmentally regulated in response to alterations in atmosphere. In six recent clinical isolates expression of protein F was repressed during growth under reduced concentrations of O2. Expression in an anaerobic environment was induced by both superoxide-generating and redox-altering reagents. However, regulation did not involve mry, a gene that controls expression of several streptococcal surface proteins. Protein F was constitutively expressed in one of two laboratory-passaged strains analysed, and in a complementation analysis using an allele of the gene that encodes protein F (prtF) cloned from a regulated strain and expressed in a constitutive strain, the constitutive phenotype was shown to be dominant in trans. Regulation, as monitored by fusion of prtF to a promoterless chloramphenicol acetyltransferase gene, involved transcriptional control. Environmentally induced alterations in protein F expression affected the ability of the bacterium to adhere to epithelial cells, which suggests that the ability to regulate expression of protein F may be important during infection.

SU M M A RYThe success of mycobacteria as pathogens hinges on their ability to infect and persist within the macrophages of their host. However, activation of host macrophages by cytokines from a productive cellular immune response can stimulate the cells to kill their resident pathogens. This suggests that the interaction between host cell and microbe is in delicate balance, which can be tipped in favour of either organism. Biochemical analysis of mycobacterial vacuoles has shown them to be integral to the host cell's recycling endosomal system. As such they show limited acidi¢cation and hydrolytic activity despite possession of known lysosomal constituents such as cathepsins D, B and L, and LAMP 1. Even in established infections, they remain dynamic compartments accessible to several plasmalemma-derived constituents. Once the macrophage has been activated by IFN-and TNF-the vacuoles coalesce and acidify. This marks a distinct alteration in vacuole physiology and leads to stasis and death of the mycobacteria. Mycobacteria have developed several strategies to avoid this outcome. Most notably, live bacilli induce sustained release of IL-6 from infected macrophages. IL-6 blocks the ability of both polyclonal primary T cells and T-cell hybridomas to respond to appropriate stimuli. Such an activity could render the centres of infection foci, such as granulomas, anergic and thus avoid release of macrophage-activating cytokines. This paper discusses both the mechanisms by which mycobacteria try to ensure their success as intracellular pathogens and the relevance of these strategies to the overall understanding of mycobacterial diseases.

The virion host shutoff protein (Vhs) of herpes simplex virus type 1 induces destabilization of mRNA following infection. Our study of primary neurons from CD-1 mice demonstrates that vhs is functional in neurons but that more Vhs is required to mediate RNA degradation in neurons than in other susceptible cells.The virion host shutoff protein (Vhs) of herpes simplex virus type 1 (HSV-1) is a tegument phosphoprotein which upon infection induces the destabilization of mRNA (2-4, 6, 8). vhs-deficient viruses grow normally in cell culture but show significant attenuation in growth in the nervous system and other tissues (9,11,12). It was previously reported that Vhs cannot induce RNA degradation in neurons, a finding that is incompatible with the hypothesis that Vhs activity is critical for neuropathogenesis (7). Data reported in this study show that Vhs activity occurs in neurons but requires a higher viral inoculum than it does in other cell types.Vhs-mediated RNA degradation in neuron cultures was analyzed with the previously described RNA degradation assay (12). Primary superior cervical ganglion (SCG) cells were isolated from neonatal CD-1 mice according to procedures used previously for the isolation of neonatal rat SCG neurons (7). Briefly, 10 4 Vero cells or SCG neurons were cultured on collagen-coated 35-mm-diameter dishes, with one mouse pup yielding approximately 10 4 SCG neurons. Each sample in the Northern blot analysis required RNA extracted from eight plates of Vero cells or SCG neurons per virus type (7). Cells were mock infected or infected with wild-type virus (KOS) or a previously described vhs-null virus (UL41NHB) (12) at a multiplicity of infection (MOI) of 20 or 100. At 8 h postinfection, infected cells were treated with dissociation media (5), and total RNA was harvested with an RNeasy RNA preparation kit (QIAGEN, Valencia, Calif.) and analyzed by Northern blotting (12). Blots were probed for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA and for 28S rRNA as a loading control. Images were documented with a STORM phosphoimaging system (Amersham Biosciences, Piscataway, N.J.) and quantified with ImageQuant software. The value of each GAPDH measurement was normalized to the lowest 28S RNA value. The data were then expressed as the amount of GAPDH message remaining as a percentage of the amount remaining in mock-infected cells, which was set at a value of 100%. At MOIs of 20 and 100, infection with KOS resulted in the degradation of 25 to 35% of the GAPDH RNA in Vero cells (Fig. 1). As expected, the use of total RNA extracts, in contrast to the cytoplasmic extracts used previously (11), revealed more-modest levels of Vhs-mediated RNA degradation. This difference is found because Vhs mediates RNA degradation largely in the cytoplasm, and nuclear GAPDH RNA, while not immune to Vhs, is therefore protected from degradation due to its localization in the nucleus. The infection of SCG neurons with KOS at an MOI of 20 resulted in no RNA degradation, as found previously (9, 12). At an MOI of 100, ho...

The ability of Mycobacterium bovis Calmette-Guérin bacillus-infected bone marrow-derived macrophages to process and present exogenously added Ags to T cells and stimulate their growth and production of IL-2 was examined. The infected macrophages were inhibited in their ability to activate T cells, and this inhibition could be transferred to uninfected macrophages with filtered supernatants from mycobacteria-infected macrophages. The inhibition was not due to decreases in macrophage viability, Ag uptake, or cell surface expression of MHC class II or other accessory molecules necessary for Ag presentation. Other intracellular pathogens such as Listeria monocytogenes and Leishmania mexicana did not induce the soluble inhibitory factor, while Mycobacterium avium strain 101 did, suggesting the factor is specific to infection with mycobacteria. The inhibitory effect was reversed completely by preincubation with neutralizing Abs against IL-6, and rIL-6 partially restored the effect. Approximately 10,000-fold more IL-6 was produced by mycobacteria-infected macrophages compared with uninfected controls. Such sustained levels of IL-6 may account for the immune unresponsiveness apparent in both human and murine mycobacterial disease.