Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools. In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M. pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 ؋ 10 ؊6 transformants/CFU/g of plasmid DNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region. Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.The mollicutes are the smallest free-living microorganisms capable of autoreplication (33). They are related to grampositive eubacteria from which they evolved by a drastic reduction of genome size, being thus considered the best representatives of the concept of a minimal cell. (964 kbp [5]). Postgenomic approaches aiming at deciphering the role of specific genes discovered by in silico analysis now require efficient strategies to genetically manipulate these organisms. The transposon Tn916 or Tn4001 can transpose in all the mollicute genomes, at least for the species amenable to transformation, such as M. genitalium, M. pneumoniae, and M. pulmonis. In M. genitalium and M. pneumoniae, a global approach of gene inactivation with the transposon Tn4001 was used to define the genes that are essential for the life of these bacteria (22). Derivatives of this transposon, obtained by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant, were also shown to transpose in M. pulmonis (12). The use of these genetic elements for gene inactivation, which relies on the random insertion of the transposon in the genome of the organism, does not allow specific targeting of a gene of interest. In addition, the complementation of mutants would require the cloning and expression of genes in...

Mycoplasma penetrans is a mycoplasma with unique morphology, recently identified in urine samples collected from HIV-infected patients. This mycoplasma has been found to be statistically associated with HIV infection, and to be cytopathic in vitro. The dominant antigen recognized during natural and experimental infections is an abundant lipoprotein, P35, which, upon extraction, segregates in the T r i t o n X-114 detergent phase. It is used as the basis of M. penetrans-specif ic serological assays. Although mycoplasma lipoproteins, including M. penetrans P35, are the main antigens recognized by the host humoral immune response, very little is known about the nature of the epitopes involved. lmmunoelectron microscopy revealed that all P35 is exposed a t the cell surface and is distributed all over the membrane. P35 linear B-epitopes were mapped by an ELISA approach based on a set of overlapping peptides covering the entire mature polypeptide. The immunoreactivity of the peptides was first tested with sera from immunized animals. The dominant Bepitopes were found a t the C-and N-terminal regions, in partial agreement with algorithmic predictions. Patient sera were evaluated with the same assay. Only some reacted with linear epitopes whereas others did not, indicating the importance of P35 nonsequential epitopes. Statistical analysis of the results allowed the definition of a set of peptides which were clearly immunodominant. Finally, the P35-encoding gene was modified by in vitro mutagenesis to allow the production and purification of a recombinant protein (rP35A0) in Escherichia coli. The antigenicity of rP35A0 was tested by Western blotting and compared to that of another recombinant product, rP35A3, a truncated P35 polypeptide. Although rP35A0 reacted with the M. penetransseropositive patient sera tested, rP35A3 was only immunoreactive with one of six sera. This result confirmed that P35-nonsequential epitopes dominate during M. penetrans infection. Our results have important implications for the understanding of lipoprotein antigenicity during mycoplasma infections. In addition, the P35-derived immunodominant synthetic peptides defined in this study, as well as the purified rP35A0, provide the antigenic material for the necessary improvement of M. penetrans serological assays.

We determined the prevalence of three emergent urogenital mycoplasma species (M. fermentans, M. penetrans, and M. genitalium) in comparison to the most common species (M. hominis and U. urealyticum). M. genitalium is probably the third most frequent agent of nongonococcal urethritis (NGU) in men. It has been suggested that M. fermentans and M. penetrans play a role in the development of AIDS. We analysed the urine and the urethral swab samples from 106 HIV‐1 infected individuals (HIV group) and 110 HIV‐negative patients with NGU (STD group) by using PCR and culture methods. M. genitalium was detected in 0.9% of the urine and in 1.9% of the urethral samples from the HIV group, compared to 9.1% found only in urethral swab samples from the STD group. M. fermentans was detected in 5.7% urethral swabs from the HIV group and in 0.9% from the STD group. M. penetrans was detected in 6.6% urine samples from the HIV group. M. hominis and U. urealyticum showed infection rates of 7.5 and 18.9% in the HIV group, and 0.9 and 13.6% in the STD group. Overall there was a higher prevalence of mycoplasmas in the HIV group than in the STD group, but the significance of these results remains unclear. J. Clin. Lab. Anal. 14:246–253, 2000. © 2000 Wiley‐Liss, Inc.

No abstract

No abstract

No abstract

We determined the prevalence of three emergent urogenital mycoplasma species (M. fermentans, M. penetrans, and M. genitalium) in comparison to the most common species (M. hominis and U. urealyticum). M. genitalium is probably the third most frequent agent of nongonococcal urethritis (NGU) in men. It has been suggested that M. fermentans and M. penetrans play a role in the development of AIDS. We analysed the urine and the urethral swab samples from 106 HIV-1 infected individuals (HIV group) and 110 HIV-negative patients with NGU (STD group) by using PCR and culture methods. M. genitalium was detected in 0.9% of the urine and in 1.9% of the urethral samples from the HIV group, compared to 9.1% found only in urethral swab samples from the STD group. M. fermentans was detected in 5.7% urethral swabs from the HIV group and in 0.9% from the STD group. M. penetrans was detected in 6.6% urine samples from the HIV group. M. hominis and U. urealyticum showed infection rates of 7.5 and 18.9% in the HIV group, and 0.9 and 13.6% in the STD group. Overall there was a higher prevalence of mycoplasmas in the HIV group than in the STD group, but the significance of these results remains unclear.

The ORF strain of Cysticercus longicollis represents an important model for the study of heterologous antigens in the immunodiagnosis of neurocysticcreosis (NC). The immunoperoxidase (IP) technique was standardized using a particulate antigen suspension of Cysticercus longicollis (Cl) and Cysitcercus cellulosae (Cc). Cerebrospinal fluid (CSF) samples were incubated on the antigen fixed to microscopy slides; the conjugate employed was anti-IgG-peroxidase and the enzymatic reaction was started by covering the slides with chromogen solution (diaminobenzidine/H2O2). After washing with distilled water, the slide was stained with 2% malachite green in water. Of the CSF samples from 21 patients with NC, 19 (90.5%) were positive, whereas the 8 CSF samples from the control group (100%) were negative. The results of the [P-C] test applied to 127 CSF samples from patients with suspected NC showed 28.3% reactivity as opposed to 29.1% for the IP-Cc test. The agreement index for the IP test (Cl x Cc) was 94.2%, with no significant difference between the two antigens.