Desialylation of peripheral blood mononuclear cells promotes growth of HIV-1.

Activation of peripheral blood CD4+ helper T lymphocytes establishes a permissive state for growth of HIV-1. Activated T lymphocytes expressed increased sialidase (neuraminidase) activity and were hyposialylated. Treatment of freshly isolated peripheral blood mononuclear cells (PBMCs) with microbial neuraminidase (NANase) or phytohemagglutinin (PHA) prior to infection at low multiplicity with T cell line-adapted HIV-1IIIB resulted in production of large amounts of p24 antigen and reverse transcriptase. In contrast, neither viral component was detected in the medium of mock-treated cells infected at a similar multiplicity through 21 days in culture. The titer of a stock solution of HIV-1IIIB was 1.4 +/- 0.18 log10 greater in NANase-treated PBMCs than in mock-treated cells; the titer was similarly raised 1.5 to 1.76 +/- 0.18 log10 in PHA-treated cells. Growth of the primary isolate HIV-1(91/US/056) was also enhanced in NANase-treated PBMCs; the titer of a stock solution of HIV-1(91/US/056 was 1.0 +/- 0.16 log10 greater in NANase-treated PBMCs than in mock-treated cells 7 days after infection. No enhancement of viral growth in PBMCs was detected when NANase was heat-inactivated or specifically inhibited with 2,3-dehydro-2-desoxy-N-acetyl-neuraminic acid prior to use. Treatment of PBMCs with NANase did not alter the distribution of lymphocyte subsets nor change the density of CD4 antigen per cell after 7 days in culture. Whereas PHA treatment of PBMCs was mitogenic, pretreatment with NANase was not; the amount of [3H]thymidine incorporated into DNA and culture growth characteristics were similar for NANase- and mock-treated cells. Thus, desialylation of PBMCs promoted a permissive state for growth of HIV-1 without affecting the rate of DNA synthesis or relative number of target CD4+ cells.

Detection and characterization of Leishmania species and strains from mammals and vectors by hybridization and restriction endonuclease digestion of kinetoplast DNA.

Leishmania parasites from animals, man or insect vectors were characterized by the gel electrophoresis of restriction endonuclease enzyme-produced mitochondrial (kinetoplast) DNA (kDNA) fragments and/or by DNA-DNA hybridization with 32P-labelled cloned, or uncloned, kDNA fragment probes from type isolates. The electrophoretic separation of kDNA fragments is a sensitive method for detecting genetic similarities and differences among Leishmania. Parasites with similar kDNA restriction fragment patterns belong to the same schizodeme and schizodeme analysis is useful for studying Leishmania populations. Cloned, species-specific kDNA probes detected Leishmania in sandflies and in liver, spleen or blood preparations from infected animals. Cloned DNA probes also hybridized to immobilized kDNA from in vitro cultivated promastigotes and detected as few as 100 parasites in a species-specific manner. Sensitive DNA hybridization probes should be useful in research on the immunology, chemotherapy or epidemiology of animal and human leishmaniasis.

Restriction endonuclease analysis of Leishmania kinetoplast DNA characterizes parasites responsible for visceral and cutaneous disease.

The kinetoplast DNA (kDNA) from promastigotes of Leishmania responsible for Old and New World cutaneous and visceral disease was characterized to determine if species and strains causing similar or different diseases could be identified. Restriction enzymes were used to digest kDNA into fragments that were separated into characteristic banding patterns after electrophoresis in agarose or linear gradient polyacrylamide gels. Hybridization was conducted with a 32P-kDNA probe and kDNA fragments transferred from agarose gels to nitrocellulose paper. Leishmania species causing cutaneous diseases in the New and Old Worlds all had different kDNA digest patterns. Visceralizing Leishmania from the New and Old Worlds also had different kDNA restriction fragment patterns although Leishmania donovani parasites with similar fragment patterns were isolated from several humans from central Kenya. Nucleotide sequences were shared among kDNA networks from L. donovani, Leishmania d. chagasi, Leishmania d. infantum, Leishmania tropica, and Leishmania major as determined by hybridization with a 32P-kDNA probe from L. donovani. However, no hybridization was detected between the L. donovani 32P-kDNA probe and kDNA from Leishmania aethiopica or Leishmania braziliensis panamensis. Leishmania characterization results for the same isolates from the published literature were compared and kinetoplast DNA analysis was found to be one of the most sensitive procedures for species and strain identification.

Presence of a virulence-associated plasmid in Yersinia pseudotuberculosis.

We have shown that Yersinia pseudotuberculosis can possess plasmids which are similar in size and function to the previously described Vwa plasmids of Y. enterocolitica. These plasmids are associated with the production of V and W antigens (calcium dependency) and pathogenicity of the organism. Further investigation of these plasmids from Y. pseudotuberculosis and Y. enterocolitica with restriction endonucleases revealed significant differences in their fragmentation pattern.

lambdaimm P22dis: a hybrid of coliphage lambda with both immunity regions of Salmonella phage P22.

Genetically marked lambda and P22 phages were recombined in Escherichia coli-Salmonella typhimurium hybrid WR4028, a host sensitive to infection by both of these phages. Hybrid phages that acquired the immC region of P22, but retained the genes for the lambda protein coat were selected on WR4027 (lambda), a lambda-immune, P22-resistant derivative of WR4028. In these lambdaimmP22 hybrids, at least the c through P genes of lambda were replaced with functionally related P22 genes. Phage recombinants with more extensive regions of the P22 genome were selected on the double lysogen WR4027 (lambda, lambdaimmP22). One such hybrid, lambdaimmP22dis, was determined by heteroduplex analysis to contain approximately 40% of the P22 genome. Genetic studies established that lambdaimmP22dis possesses the two widely separated immunity control regions of P22 (immC and immI) and that these loci are expressed in E. coli K-12 lysogenic for lambdaimmP22dis. In addition, lambdaimmP22dis contains the P22 a 1 locus responsible for somatic 0--1 antigen conversion in Salmonella. Although the lambdaimmP22dis phage particle has the lambda head and tail, the phage genome also carried P22 tail gene 9 as evidenced by the production of free P22 tails. It also has the P22 att site as indicated by the integration of the lambdaimmP22dis prophage near the proA locus on the bacterial chromosome.

Interaction between bacteriophage Sf6 and Shigella flexner.

The Shigella flexneri phage Sf6 has an isometric head with hexagonal symmetry 53nm in diameter. The noncontractile tails in 16 nm long and terminates with a base plate containing six spikes. Sf6 is typical of the C phages in the morphological classification of Bradley. Phage Sf6 processes alpha-1,3-endorhamnosidase activity as demonstrated by methylation and reducing end group sugar analyses of the products obtained on interaction with the O-polysaccharide chain of S.flexneri strains which have the O-group 3,4 antigen. The major end product was an octasaccharide with the following structure: Rha III-GlcNAc-Rha I-Rha II-Rha III-GlcNAc-Rha I-Rha II. Acetylation of 0-2 of rhamnose III of the O-polysaccharide chain, either brought about by Sf6 lysogenization or found in wild-type S. flexneri (3b) strains, prevented enzymatic hydrolysis. O-deacetylation of the polysaccharide chain again made it susceptible to the S6f endorhamnosidase.

Cellular release of heat-labile enterotoxin of Escherichia coli by bacteriophage induction.

Treatment of some enterotoxigenic Escherichia coli strains with the antibiotic mitomycin C resulted in lysis of the bacteria. Heat-labile enterotoxin (LT) activity of culture filtrates, determined by means of the Y-1 adrenal cell assay, increased dramatically as lysis of the culture proceeded. Further studies with E. coli strains 263 and B21-4 revealed that lysis is due to mitomycin C induction of vetetative development of a temperature bacteriophage. These findings suggest that the elevated levels of LT detected after mitomycin C treatment reflect the lytic release of cell-bound LT rather than the induction by mitomycin C of de novo toxin biosynthesis. Comparable increases in LT activity also resulted from thermal induction of a phage P1Cm lysogen of strain 263 or from sonic disruption of enterotoxigenic strains.

Transfer of plasmid-borne beta-lactamase in Neisseria gonorrhoeae.

Neisseria gonorrhoeae strain GC82 contains a plasmid specifying a beta-lactamase (beta-Lam(+)). Mixed incubation of strain GC82 with a penicillin-susceptible (beta-Lam(-)), streptomycin-resistant mutant of strain GC9 results in the expression of beta-lactamase activity and streptomycin resistance in the transcipients. The frequency of transfer of the plasmid-specified resistance to penicillin seems to be proportional to the initial input ratio of the mating mixture of donor to recipient and to correlate positively with bacterial density. Cell-to-cell transmission of the deoxyribonucleic acid (DNA) appears to be by a conjugal mechanism or, alternatively, by an as yet undescribed transducing phage. Additionally, whole-cell DNA from a beta-lactamase-producing strain could be used to transform streptomycin-resistant recipients, resulting in the expression of both beta-lactamase activity and streptomycin resistance in the transformants, and purified gonococcal plasmid DNA transformed Escherichia coli but not the gonococcus. Circular DNA extracted from donor GC82 comprised three molecular species (approximately 2.7, 4.8, and 25 megadaltons [Mdal]), whereas the recipients GC9-S (Str(r)) contained only the 2.7-Mdal cryptic DNA species. DNA from the GC9-S82 (Str(r), beta-Lam(+)) transcipient contained a 4.8-Mdal species in addition to the cryptic molecular species (2.7 Mdal). The finding that the transcipient will not retransfer beta-lactamase is consistent with the hypothesis that the 25-Mdal plasmid promotes mobilization of the smaller 4.8-Mdal R plasmid.

Plasmid-determined ability of a Salmonella tennessee strain to ferment lactose and sucrose.

The ability of a Salmonella tennessee strain to ferment both lactose and sucrose was attributed to a conjugally transmissible plasmid, deoxyribonucleic acid molecular weight 164 x 10(6), bearing the genetic determinants of both fermentation characters.

Extensive segments of the Escherichia coli K12 chromosome in Proteus mirabilis diploids.

Various Escherichia coli K12 Hfr donors transfer at low frequency portions of the E. coli genome to Proteus mirabilis. By remating such Proteus hybrids with the same or a different E. coli Hfr strain, other genetic characters could be added to yield diploid Proteus hybrids which contained more than 30 percent of the E. coli genome. The extent of the E. coli genetic material in these unstable Proteus diploid hybrids included segments with the following selected markers: gal, lac, ara, mel, mtl, and malA. Unselected markers known to map throughout this region of the chromosome were also detected in these hybrids. Among the markers expressed in Proteus hybrids with the E. coli malA region was the receptor site for coliphage lambda. Although plaques were not seen, lambda was adsorbed by the Proteus hybrids. Examination of DNA from the various Proteus hybrids by CsCl density gradient centrifugation showed a satellite component of E. coli DNA with a size that corresponded to the extent of the E. coli genome present as determined by genetic analysis.

Characterization of transmissible genetic elements from sucrose-fermenting Salmonella strains.

Two of seven sucrose-fermenting Salmonella strains obtained from clinical sources were found capable of conjugal transfer of the sucrose fermentation (Scr+) property to the Escherichia coli K-12 strain WR3026. The genetic elements conferring this Scr+ property, designated scr-53 and scr-94, were then conjugally transmissible from Escherichia coli WR3026 Scr+ exconjugants to other strains of Escherichia coli at frequences of 5 times 10- minus 6 to 5 times 10- minus 3 for the scr-53 element and 10- minus 6 to 10- minus 5 for the scr-94 element. In Escherichia coli hosts, both of these elements were compatible with F-lac and with each of six previously characterized transmissible lac elements. No antibiotic resistance characteristics or colicin production were discovered to be associated with either scr-53 or scr-94. Neither scr element generated a male host response to the female-specific phage phiII, but the scr-53 element rendered its Escherichia coli host sensitive to the male-specific phage R-17. Escherichia coli hosts containing scr-53 were susceptible to lysis by P1vir, and transduction of the scr-53 element was accomplished with this phage. The scr-53 element was isolated from Escherichia coli WR3026, Scr+ transductants, and Escherichia coli WR2036 Scr+ exconjugants as a covalently closed circular deoxyribonucleic acid molecule with a molecular weight (determined by electron microscopy) of approximately 52 times 10-6. Receipt of the scr-94 element rendered Escherichia coli hosts of this element unsusceptible to lysis by P1vir, although adsorption of the phage by an Escherichia coli WR3026 exconjugant containing scr-94 occurred as efficiently as it did on WR3026 itself. Repeated examination of Escherichia coli strains harboring scr-94, as well as of the Salmonella strain which initially contained it, did not reveal the presence of circular deoxyribonucleic acid. The synthesis of the sucrose cleaving enzyme was inducible in Escherichia coli exconjugants containing either scr-53 or scr-94.

Isolation and characterization of circular deoxyribonucleic acid obtained from lactose-fermenting Salmonella strains.

Six lac elements originally contained in Salmonella strains were transferred to Escherichia coli WR3026. All of the six E. coli strains that received one of the lac elements were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Segregants of each of these E. coli WR3026 strains that had lost the ability to utilize lactose, when examined in the same manner as the lactose-fermenting strains, were not observed to contain these supercoiled, circular DNA molecules. Thus the DNA of the lac elements is maintained in E. coli WR3026 in the supercoiled, circular form. Molecular weights of the supercoiled, circular molecules isolated from strains carrying the lac elements were determined by sucrose density gradient centrifugation to be 30 million to 56 million. The calculated number of copies per chromosome of the lac elements varied from 1.4 to 3.7, depending upon the particular lac element examined. Each of the elements was determined to have a guanine plus cytosine composition of 50%. All six of the E. coli WR3026 strains containing a transmissible lac element were tested with the E. coli male-specific phage, R-17, and the E. coli female-specific phage, phiII, and did not respond to either of these phages as do F-containing derivatives of E. coli K-12.

Conservation of Salmonella typhimurium deoxyribonucleic acid in partially diploid hybrids of Escherichia coli.

Partially diploid Escherichia coli K-12 hybrids recovered from mating with a Salmonella typhimurium Hfr strain were found to differ with respect to the manner in which they conserved the added Salmonella deoxyribonucleic acid (DNA). Five of the diploid hybrids examined appeared to maintain the Salmonella DNA as part of a functional F-merogenote; these hybrids were sensitive to the male-specific phage, R-17, responded as males to the female-specific phage, phiII, and transferred their inherited Salmonella genetic markers at high frequency in conjugation experiments. Six diploid hybrids were observed which were not sensitive to R-17, and from which the added Salmonella DNA was not transmissible in conjugation tests; nevertheless, these hybrids responded as males to phiII, and the Salmonella chromosomal fragments were conserved in them as parts of supercoiled, circular DNA elements. It was concluded that these circular DNA elements were defective F-merogenotes, unable to direct the synthesis of F-pili. Three diploid hybrids were found which were not sensitive to R-17, and which responded as females to phiII; no circular DNA was found in them, and it was concluded that their conservation of the Salmonella genetic fragments was accomplished in some manner which did not involve association with F or assumption of the supercoiled circular configuration. Other partially diploid hybrids were observed which appeared similar to these latter three hybrids with regard to their conservation of the Salmonella DNA, but which also contained an infecting F-factor; in these hybrids, both genetic and molecular experiments indicated that the unstably conserved Salmonella DNA was not associated physically with the F-factor.

Isolation and characterization of the fertility factor P of Vibrio cholerae.

Vibrio cholerae strains with the transmissible fertility factor P contained a supercoiled circular deoxyribonucleic acid (DNA) component amounting to between 2 and 6% of the total DNA obtained from the cells. Such a component was not observed in V. cholerae strains lacking the fertility factor. This supercoiled circular DNA was isolated from P(+) cells, and the molecular weight was determined by sedimentation velocity experiments and electron microscopy to be approximately 80 million daltons. These supercoiled circular DNA molecules, which have a guanine plus cytosine (G + C) composition of 42%, were concluded to be the extrachromosomal P factor. It was calculated that there is approximately one copy of the P factor per chromosome. A small amount of supercoiled circular DNA was occasionally isolated from the P(-) strains of V. cholerae. The function of this component, which has a molecular weight of 40 million daltons, is not known. The molecules found in the P(-) strains were readily distinguished from the P(+) circular molecules by their smaller molecular weight and different G + C composition.

Isolation of circular deoxyribonucleic acid from Salmonella typhosa hybrids obtained from matings with Escherichia coli Hfr donors.

Heterozygous, partial diploid Salmonella typhosa hybrids obtained from matings with Escherichia coli K-12 Hfr strains were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Examination of one such S. typhosa hybrid after its loss, by segregation, of the inherited E. coli genetic markers revealed a concurrent loss of its supercoiled circular DNA. Subsequent remating of this segregant with various E. coli Hfr strains resulted in the reappearance of the circular DNA. Molecular weight determinations of circular DNA molecules isolated from a number of S. typhosa partial diploid hybrids were made by sucrose density gradient ultracentrifugation and electron microscopy. These studies revealed a range of molecular sizes among the various hybrids examined, but each hybrid exhibited only a single characteristic size for its contained circular DNA. The range of size is consistent with the presence in each hybrid of a different length of E. coli chromosome. It was concluded that the E. coli Hfr genetic segments transferred to these S. typhosa hybrids were conserved, in the diploid state, in the form of supercoiled, circular DNA molecules.

Nature of lactose-fermenting Salmonella strains obtained from clinical sources.

Six of seven lactose-fermenting (lac(+)) Salmonella strains obtained from clinical sources were found to be capable of transferring the lac(+) property by conjugation to Salmonella typhosa WR4204. All of the six S. typhosa strains which received the lac(+) property transferred it in turn to S. typhimurium WR5000 at the high frequencies typical of extrachromosomal F-merogenotes. These six lac elements were also transmissible from S. typhosa WR4204 to Proteus mirabilis and to some strains of Escherichia coli K-12; moreover, they were capable of promoting low frequency transfer of chromosomal genes from S. typhimurium WR5000 to S. typhosa WR4204. One of these lac elements was shown also to be capable of promoting low frequency chromosome transfer in E. coli K-12. E. coli K-12 strains harboring these lac elements exhibited sensitivity to the male specific phage R-17. Sensitivity to R-17 was not detected in Salmonella strains containing the elements. Examination of the lac elements in P. mirabilis by cesium chloride density gradient centrifugation showed that each element had a guanine plus cytosine content of 50%. The sizes of the elements varied from 0.8 to 3% of the total Proteus deoxyribonucleic acid. The amount of beta-galactosidase produced by induced and uninduced cultures of S. typhimurium WR5000 and S. typhosa WR4204 containing the lac elements was lower than that produced by these strains with the F-lac episome. The heat sensitivity of beta-galactosidase produced by the lac elements in their original Salmonella hosts indicated that the enzyme made by these strains differs from E. coli beta-galactosidase.