Mycobacterium avium-intracellulare induces interleukin-6 from human monocytes and large granular lymphocytes.

Mycobacterium avium-intracellulare (MAI) is an opportunistic pathogen commonly found in acquired immunodeficiency syndrome patients, whose immune systems are severely compromised. However, normal responses to this bacterium are apparently sufficient to prevent disseminated infection because disease is rarely found unless an immunocompromised state is present. Because interleukin-6 (IL-6) is an inflammatory cytokine with a multitude of activities, we investigated the potential of MAI to induce IL-6 from normal human leukocytes. Peripheral blood mononuclear cells were fractionated into monocytes (Mo), large granular lymphocytes (LGL), and T cells and stimulated with bacteria. Culture supernatants were collected and assayed for IL-6 activity by bioassay. Mo and LGL, but not T cells, were found to release IL-6 within 12 hours of stimulation, with optimal production occurring by 2 days of culture. Production of IL-6 from human leukocyte subsets was confirmed by Northern blot analysis and by neutralization of biologic function of the culture supernatants with specific antisera. Taken together, these results indicate that production of IL-6 is a key response of Mo and LGL to MAI. The role of IL-6 in MAI infection, therefore, needs to be further investigated.

Tandem insertion sequence-like elements define the expression site for variable antigen genes of Borrelia hermsii.

The spirochete Borrelia hermsii avoids the immune response of its mammalian host through multiphasic antigenic variation. Serotype specificity is determined by variable antigens, Vmp proteins, in the outer membrane. Through nonreciprocal recombination between linear plasmids, a formerly silent vmp gene replaces another vmp gene downstream from a common expression site. To further characterize this activating site, we determined the nucleotide sequence of 6.9 kb of the common upstream expression region of strain HS1 of B. hermsii. Preceding the vmp gene promoter and a poly(dT.dA) run were three imperfectly repeated segments of 2 kb. Each of the 2-kb segments contained 1-kb elements with inverted repeats of approximately 0.2 kb each at their termini. The potential of the 1-kb elements to form stem-and-loop structures was demonstrated by heteroduplex analysis. There was no evidence of the presence of the elements elsewhere in the genome of B. hermsii. One or more of these elements may confer the unidirectionality that characterizes vmp gene switches.

Fimbrial phase variation and DNA rearrangements in uropathogenic isolates of Escherichia coli.

Having previously shown that the oscillating on-off expression (phase variation) of type 1 fimbriae in Escherichia coli is regulated genetically by an invertible element of DNA, we wished to determine whether E. coli isolates recovered from infected humans behaved in similar fashion. We examined four different clinical isolates that expressed type 1 fimbriae, P fimbriae, or both. Using, in Southern blot analysis, a DNA probe from the type 1 fimbrial switch, that hybridized to one DNA band from phase-on bacteria and to two DNA bands from phase-off bacteria, we found that the three clinical isolates expressing type 1 fimbriae contained the same invertible switch previously seen in the K-12 isolate. Employing a similar approach to characterize the on-off expression of P fimbriae, we used a DNA probe containing the known transcriptional signals for P fimbriae. Although we detected DNA rearrangement in the two strains expressing P fimbriae, unlike the case for type 1 fimbriae, the rearrangement did not correlate with the on-off state of the P fimbriae. Rather, the DNA rearrangement correlated with the environmental conditions of growth of the bacteria from which the DNA was isolated. These results confirm the notion that P fimbriae expression and type 1 fimbriae expression are controlled differently.

An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli.

The expression of type 1 fimbriae (pili) of Escherichia coli is turned on and off at the transcriptional level at a high frequency (10(-3) per cell per generation) in a process termed phase variation. Using Southern blot and DNA sequence analysis, we have detected a genomic rearrangement in the switch region immediately upstream of the fimbrial structural gene. This rearrangement involves an invertible 314-base-pair segment of DNA whose alternating orientation apparently results in the on-and-off activation of a promoter that determines the state of fimbrial expression.

Genetic analysis of the phase variation control of expression of type 1 fimbriae in Escherichia coli.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation, whereby individual cells can alternate between states of organelle expression (Fim+) and nonexpression (Fim-). Strains with a fimD-lac operon fusion, in which lac, rather than fimD, expression is under the control of the fimD promoter, undergo Lac+ in equilibrium Lac- phase variation, instead. After positioning a lambda prophage adjacent to the operon fusion, we were able to isolate specialized lambda phage carrying both the fimD-lac fusion and the phase variation control region. Introduction of such phage into an Fim+ strain resulted in construction of a strain with a double, independently switching phenotype (Fim+ in equilibrium Fim- and Lac+ in equilibrium Lac-), demonstrating that the region controlling phase variation is contiguous with the fimD-lac operon fusion and is cis acting. When the specialized lambda phage was propagated on a delta lac delta fim strain, phase variation occurred within the plaques, confirming that the phase variation control region is carried on the specialized transducing phage. All lysogens acquired the Lac+ in equilibrium Lac- phenotype, except for two nonswitching Lac+ recombinants, which acquired Lac+ in equilibrium Lac- phase variation only by trans complementation with fim. Phase variation of type 1 fimbriae, therefore, appears to involve both a cis-active element, which is cloned on a specialized lambda phage, and a trans-active permissive factor, which is not present on the phage, but rather must be supplied by the recipient strain in the transduction.

Genetic mapping and transcriptional orientation of the fimD gene.

Expression of type 1 fimbriae in Escherichia coli exhibits phase variation whereby individual cells can alternate between states of organelle expression and nonexpression. Strains carrying fim-lac operon fusions in which lac operon expression is under the control of a fim promoter undergo Lac+ in equilibrium Lac- phase variation. We have determined the genetic map location and direction of transcription of a fim-lac operon fusion which was obtained by insertion of lac into a locus we have named fimD. We found the gene order to be as follows: valS fimD uxuA serB. The direction of transcription of fimD was found to be clockwise on the E. coli chromosome.

Isolation and characterization of cAMP suppressor mutants of Escherichia coli K12.

We have isolated spontaneous and chemically induced revertants of cya mutant strains of Escherichia coli. Three different classes of revertants were obtained. One class consisted of primary site revertants; a second class was pseudorevertants that had phenotypically reverted to wild type but retaining the original cya mutant and the third class of revertants, designated csm, were pseudorevertants hypersensitive to exogenous cAMP. Transductional analysis of the csm mutation indicated the mechanism of suppression in these strains was intergenic. The csm mutation and hypersensitivity to cAMP map in or near the crp gene. Growth of the csm strains on PTS (phosphoenolpyruvate phosphotransferase system) and non-PTS substrates was inhibited by 5 mM cAMP. The csm strains were found to accumulate toxic levels of methylglyoxal when grown on non-PTS substrates in the presence of exogenous cAMP. All csm strains were sensitive to catabolite repression mediated by alpha-methylglucoside. Revertants selected as resistant to cAMP fell into four major classes that could be distinguished by their fermentation patterns in the presence and absence of cAMP as well as by their growth response to streptomycin in the presence of cAMP.