Major outer membrane protein Omp25 of Brucella suis is involved in inhibition of tumor necrosis factor alpha production during infection of human macrophages.

Brucella spp. can establish themselves and cause disease in humans and animals. The mechanisms by which Brucella spp. evade the antibacterial defenses of their host, however, remain largely unknown. We have previously reported that live brucellae failed to induce tumor necrosis factor alpha (TNF-alpha) production upon human macrophage infection. This inhibition is associated with a nonidentified protein that is released into culture medium. Outer membrane proteins (OMPs) of gram-negative bacteria have been shown to modulate macrophage functions, including cytokine production. Thus, we have analyzed the effects of two major OMPs (Omp25 and Omp31) of Brucella suis 1330 (wild-type [WT] B. suis) on TNF-alpha production. For this purpose, omp25 and omp31 null mutants of B. suis (Deltaomp25 B. suis and Deltaomp31 B. suis, respectively) were constructed and analyzed for the ability to activate human macrophages to secrete TNF-alpha. We showed that, in contrast to WT B. suis or Deltaomp31 B. suis, Deltaomp25 B. suis induced TNF-alpha production when phagocytosed by human macrophages. The complementation of Deltaomp25 B. suis with WT omp25 (Deltaomp25-omp25 B. suis mutant) significantly reversed this effect: Deltaomp25-omp25 B. suis-infected macrophages secreted significantly less TNF-alpha than did macrophages infected with the Deltaomp25 B. suis mutant. Furthermore, pretreatment of WT B. suis with an anti-Omp25 monoclonal antibody directed against an epitope exposed at the surface of the bacteria resulted in substancial TNF-alpha production during macrophage infection. These observations demonstrated that Omp25 of B. suis is involved in the negative regulation of TNF-alpha production upon infection of human macrophages.

Identification of the nik gene cluster of Brucella suis: regulation and contribution to urease activity.

Analysis of a Brucella suis 1330 gene fused to a gfp reporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous to nikA, the first gene of the Escherichia coli operon encoding the specific transport system for nickel. DNA sequence analysis of the corresponding B. suis nik locus showed that it was highly similar to that of E. coli except for localization of the nikR regulatory gene, which lies upstream from the structural nikABCDE genes and in the opposite orientation. Protein sequence comparisons suggested that the deduced nikABCDE gene products belong to a periplasmic binding protein-dependent transport system. The nikA promoter-gfp fusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl(2) excess. Insertional inactivation of nikA strongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, the nikA mutant of B. suis was functionally complemented with the E. coli nik gene cluster, leading to the recovery of urease activity. Reciprocally, an E. coli strain harboring a deleted nik operon recovered hydrogenase activity by heterologous complementation with the B. suis nik locus. Taking into account these results, we propose that the nik locus of B. suis encodes a nickel transport system. The results further suggest that nickel could enter B. suis via other transport systems. Intracellular growth rates of the B. suis wild-type and nikA mutant strains in human monocytes were similar, indicating that nikA was not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.

Genetic characterization of the nef gene from human immunodeficiency virus type 1 group M strains representing genetic subtypes A, B, C, E, F, G, and H.

Most efforts to characterize sequence variation of HIV isolates has been directed toward the structural envelope gene. Few studies have evaluated the sequence variability of auxiliary genes such as nef. In this study 41 new HIV-1 strains, representing the majority of the described envelope subtypes of HIV-1 (A to H), were genetically characterized in the nef region. Phylogenetic analysis showed that 34 strains could be classified in the same subtype in nef and env, and 7 (19%) of the 41 new viruses were recombinants. For two of the seven strains, recombination occurred upstream of the nef gene, whereas for five of the seven strains recombination occurred within the nef gene with a crossover close to the 5' end of the LTR (long terminal repeat). The low intersubtype distance between subtype B and D in the nef gene confirms previous observations in the pol, env, and gag genes, which suggest a common ancestor for these subtypes. The majority of all the previously described functional domains in the nef gene were relatively conserved among the different subtypes, with only minor differences being observed. The myristoylation signal among the different subtypes, with only minor differences being observed. The myristoylation signal was less conserved for subtype C, with one or more amino acid changes being observed at positions 3, 4, and 5. The highly conserved acidic region (positions 62 to 65), critical for the enhancement of viral synthesis with an increased virus growth rate, was less conserved among the subtype G strains from our study. At least three epitopic regions of the nef gene have been defined and each can be recognized by CTLs under a variety of HLA restrictions; all were also relatively well conserved between the different genetic subtypes. Despite the relatively important genetic variation in nef sequences obtained among the different genetic subtypes, functional domains and CTL epitopes were relatively well conserved. In vitro and/or in vivo studies are necessary to study the relevance of the observed differences.

Regulatory genes of simian immunodeficiency viruses from west African green monkeys (Cercopithecus aethiops sabaeus).

The high seroprevalence of simian immunodeficiency viruses (SIVs) in African green monkeys (AGMs) without immunological defects in their natural hosts has prompted consideration of SIV-infected AGMs as a model of apathogenic SIV infection. Study of the molecular mechanisms of SIVagm asymptomatic infection could thus provide clues for understanding the pathogenesis of human immunodeficiency viruses. Regulatory genes could be candidates for genetic control of SIVagm apathogenicity. We have characterized Vpr, Tat, Rev, and Nef genes of two SIVagm strains isolated from naturally infected sabaeus monkeys captured in Senegal. The results provide further evidence that SIVagm from West African green monkeys is the most divergent class of AGM viruses, with structural features in long terminal repeat sequences and Vpr and Tat genes that distinguish them from viruses isolated from other AGM species (vervet, grivet, and tantalus monkeys).

A new 5' sequence associated with mouse L1 elements is representative of a major class of L1 termini.

Full-length L1 elements have been shown to possess, at their 5' end, tandem repeats called "A" or "F" types. By sequencing the 5' region of two large L1 copies that did not hybridize to A or F probes, we have identified a new sequence that is found at the 5' end of many L1 elements and that we call "V." The element characterized has no 200-bp tandem repetitive structure, and the new 5' sequence is not similar to the A or F sequences. The study of the relationships between the V and L1 sequences has shown that only half of the V (i.e., V-specific 5') sequences in the genome are linked to the 5' end of L1 copies. In related rodent species, a comparative study by Southern blot and PCR analysis of the V sequence suggests that this L1 subfamily has an ancient origin and that V sequence isolated from the remainder of the L1 element has been amplified during the evolution of the mouse genome.

Unrelated sequences at the 5' end of mouse LINE-1 repeated elements define two distinct subfamilies.

Some full length members of the mouse long interspersed repeated DNA family L1Md have been shown to be associated at their 5' end with a variable number of tandem repetitions, the A repeats, that have been suggested to be transcription controlling elements. We report that the other type of repeat, named F, found at the 5' end of a few L1 elements is also an integral part of full length L1 copies. Sequencing shows that the F repeats are GC rich, and organized in tandem. The L1 copies associated with either A or F repeats can be correlated with two different subsets of L1 sequences distinguished by a series of variant nucleotides specific to each and by unassociated but frequent restriction sites. These findings suggest that sequence replacement has occurred at least once in 5' of L1Md, and is related to the generation of specific subfamilies.

The relationships between the 5' end repeats and the largest members of the L1 interspersed repeated family in the mouse genome.

Analysis of a few large L1 elements has revealed two types of tandem repeats at the 5' end: A and F. In this study, the relationships between these repeats and a series of large L1 elements has been analysed. Most of cloned L1 repeats were shown to lack either A or F sequences at their 5' ends. F sequences are found less frequently associated than A sequences to the 5' ends of L1 and an evolutionary comparison shows that the A type was introduced more recently during the evolution of the mouse genome than the F type.

Distributions of two recently inserted long interspersed elements of the L1 repetitive family at the Alb and beta h3 loci in wild mice populations.

The presence of the L1 sequences, L1Md4 next to the pseudogene beta h3 and I12 found in the twelfth intron of the albumin gene, in certain strains of laboratory mice but not of others has led to the suggestion that these sequences were recent insertions into the Mus mus domesticus genome. To be sure that they are really recent insertions and not relics of an ancestral chromosome, we investigated the presence or absence of these sequences in populations of wild mice belonging to the semispecies M. m. domesticus and M. m. musculus as well as in other species of the genus Mus and in related murids. The sequence I12 in the albumin gene was found in 34% of the chromosomes of the wild mice belonging to M. m. domesticus and to a lesser extent (6%) in M. m. musculus. Of 114 M. m. domesticus chromosomes, L1Md4 was found in only nine, seven of which came from the same locality. Its presence was associated with the haplotype Hbbp, which is relatively rare in European populations of M. musculus. Since there was no evidence for the presence of these two L1 sequences in more distantly related species, we conclude that they are recent insertions in the M. musculus genome.

Comparative study of the L1 family in the genus Mus. Possible role of retroposition and conversion events in its concerted evolution.

The long interspersed repetitive family L1 was analysed in different species belonging to the genus Mus. It is shown to be highly conserved even in M.n. setulosus, which diverged from the other species around ten million years ago. The study of the linkage between diagnostic restriction sites in the various species and the sequence variations of different regions of the L1Md repeat shows that the L1 family undergoes concerted changes involving subsets of repeats. The rate at which this homogenization process occurs does not appear to be the same for all the subfamilies detected. The L1Md repeat in the twelfth intron of the serum albumin gene of Balb/c mice is shown to be a recent insertion. The role retroposon- and gene conversion-like events may play in the concerted evolution of the L1 family is discussed.