Binding of IgA by Mycoplasma penetrans.

The current study shows that Mycoplasma penetrans strain GTU binds human serum immunoglobulin A (IgA) and secretory IgA but not IgG. Binding of IgA was associated almost exclusively with the lipoprotein fraction obtained by Triton X-114 fractionation of isolated M. penetrans membranes. Western immunoblot analysis of isolated membranes of M. penetrans strain GTU and of the Triton X-114 fraction showed that the major IgA-binding component was a lipoprotein with a molecular mass of 38 kDa, one of the major lipoproteins of this organism. The authors suggest that the high IgA-binding capacity of M. penetrans strain GTU may serve as a defense mechanism, conferring on this microorganism the ability to evade clearance mediated by specific IgA antibodies.

A mycoplasma peptide elicits heteroclitic CD4+ T cell responses against tumor antigen MAGE-A6.

PURPOSE: Although T-helper (Th) epitopes have been previously reported for many tumor antigens, including MAGE-A6, the relevant HLA-DR alleles that present these peptides are expressed by only a minority of patients. The identification of tumor antigenic epitopes presented promiscuously by many HLA-DR alleles would extend the clinical utility of these peptides in vaccines and for the immunomonitoring of cancer patients. EXPERIMENTAL DESIGN: A neural network algorithm and in vitro sensitization assays were employed to screen candidate peptides for their immunogenicity. RESULTS: The MAGE-A6(140-170), MAGE-A6(172-187), and MAGE-A6(280-302) epitopes were recognized by CD4+ T cells isolated from the majority of normal donors and melanoma patients evaluated. Peptide-specific CD4+ T cells also recognized autologous antigen-presenting cell pulsed with recombinant MAGE-A6 (rMAGE) protein, supporting the natural processing and MHC presentation of these epitopes. Given the strong primary in vitro sensitization of normal donor CD4+ T cells by the MAGEA6(172-187) epitope, suggestive of potential cross-reactivity against an environmental stimulus, we identified a highly homologous peptide within the Mycoplasma penetrans HF-2 permease (MPHF2) protein. MPHF2 peptide-primed CD4+ T cells cross-reacted against autologous APC pulsed with the MAGE-A6(172-187) peptide or rMAGE protein and recognized HLA-matched MAGE-A6+ melanoma cell lines. These responses seemed heteroclitic in nature because the functional avidity of MPHF2 peptide-primed CD4+ T cells for the MAGE-A6(172-187) peptide was approximately 1,000 times greater than that of CD4+ T cells primed with the corresponding MAGE-A6 peptide. CONCLUSIONS: We believe that these novel "promiscuous" MAGE-A6/MPHF2 Th epitopes may prove clinically useful in the treatment and/or monitoring of a high proportion of cancer patients.

Rapid and efficient preparation of monoclonal antibodies against 35 kDa lipoprotein of Mycoplasma penetrans.

To develop a rapid and efficient method for preparing monoclonal antibodies (MAb) against 35 kDa lipoprotein of Mycoplasma penetrans, BALB/c mice were injected into the footpads for immunization, and the popliteal lymph nodes were isolated 19 days later for MAb-producing hybridomas, from which the mAbs against the 35 kDa lipoprotein were screened. The identification of the mAb against the 35 kDa lipoprotein was performed using indirect enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Using popliteal lymph node procedures, we generated several positive clones, one of which we characterized by ELISA and immunoblot. The clone 1D41B8 was identified as the immunoglobulin G1 (IgG1) isotype, kappa chain with affinity constants (Ka) of 2.95 x 10(9) M(-1). The MAbs did not cross-react with a number of control bacteria, which included Mycoplasma fermentans, Mycoplasma hominis, and Mycoplasma genitalium. This is the first report on the preparation of mAbs against M. penetrans that is specific to 35 kDa lipoprotein using popliteal lymph nodes. The high-specificity and high-affinity MAbs produced by two methodologies used of hybridomas provide a basis for further research on the pathogenesis and early diagnosis of M. penetrans. This simple approach may become a method of choice for the generation and production of MAbs in a short period of time.

Mycoplasma penetrans infections and seroconversion in patients with AIDS: identification of major mycoplasmal antigens targeted by host antibody response.

We examined Mycoplasma penetrans-specific antibodies in sera of five male homosexual AIDS patients from whom M. penetrans was isolated during the disease process. No consistent immune reaction pattern could be recognized in Western blot using whole cell proteins. Serum samples obtained prior to M. penetrans isolation reacted with a number of M. penetrans proteins, most likely due to non-specific cross-reactions. Further analysis revealed that patients produced prominent antibody reaction to lipid-associated membrane proteins (LAMPs) of M. penetrans at the time of mycoplasma isolation, which could not be observed for serum samples obtained prior to M. penetrans isolation. The positive antibody reaction was mainly directed against two major LAMPs of M. penetrans with molecular mass of 35 and 38 kDa and produced a distinctive pattern of positive immunoreaction bands. Our observation suggested that, comparing with whole mycoplasmal proteins, LAMPs were more specific target antigens in serological assays for M. penetrans infection.

Intracellular location and survival of Mycoplasma penetrans within HeLa cells.

Mycoplasma penetrans invades HeLa cells and survives within them for prolonged periods of time. The intracellular distribution of M. penetrans within HeLa cells was studied utilizing the acidotropic dye LysoTracker (green), which permeates cell membranes and upon protonation remains trapped in acidic compartments. The excitation and emission spectra of the green LysoTracker are suitable for colocalization studies with rabbit anti- M. penetrans antibodies and red Cy5 goat anti-rabbit IgG. The images collected by confocal laser scanning microscopy revealed that in the infected HeLa cells almost all Cy5 fluorescent foci (red) were located within the LysoTrack-labelled intracellular compartments, apparently endosomes. Viable mycoplasmas were detected within endosomes for prolonged periods of time, apparently due to a potent antioxidant activity detected in M. penetrans.

Mycoplasma penetrans is capable of activating V gamma 9/V delta 2 T cells while other human pathogenic mycoplasmas fail to do so.

While most mycoplasma species appear to have evolutionarily lost the ability to synthesize isoprenoid precursors, Mycoplasma penetrans has retained the nonmevalonate pathway that proceeds via the immunogenic intermediate (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). Consequently, this pathogen is capable of stimulating human V gamma 9/V delta 2 T cells.

Multiple promoter inversions generate surface antigenic variation in Mycoplasma penetrans.

Mycoplasma penetrans is a newly identified species of the genus MYCOPLASMA: It was first isolated from a urine sample from a human immunodeficiency virus (HIV)-infected patient. M. penetrans changes its surface antigen profile with high frequency. The changes originate from ON<==>OFF phase variations of the P35 family of surface membrane lipoproteins. The P35 family lipoproteins are major antigens recognized by the human immune system during M. penetrans infection and are encoded by the mpl genes. Phase variations of P35 family lipoproteins occur at the transcriptional level of mpl genes; however, the precise genetic mechanisms are unknown. In this study, the molecular mechanisms of surface antigen profile change in M. penetrans were investigated. The focus was on the 46-kDa protein that is present in M. penetrans strain HF-2 but not in the type strain, GTU. The 46-kDa protein was the product of a previously reported mpl gene, pepIMP13, with an amino-terminal sequence identical to that of the P35 family lipoproteins. Nucleotide sequencing analysis of the pepIMP13 gene region revealed that the promoter-containing 135-bp DNA of this gene had the structure of an invertible element that functioned as a switch for gene expression. In addition, all of the mpl genes of M. penetrans HF-2 were identified using the whole-genome sequence data that has recently become available for this bacterium. There are at least 38 mpl genes in the M. penetrans HF-2 genome. Interestingly, most of these mpl genes possess invertible promoter-like sequences, similar to those of the pepIMP13 gene promoter. A model for the generation of surface antigenic variation by multiple promoter inversions is proposed.

The complete genomic sequence of Mycoplasma penetrans, an intracellular bacterial pathogen in humans.

The complete genomic sequence of an intracellular bacterial pathogen, Mycoplasma penetrans HF-2 strain, was determined. The HF-2 genome consists of a 1 358 633 bp single circular chromosome containing 1038 predicted coding sequences (CDSs), one set of rRNA genes and 30 tRNA genes. Among the 1038 CDSs, 264 predicted proteins are common to the Mycoplasmataceae sequenced thus far and 463 are M.penetrans specific. The genome contains the two-component system but lacks the essential cellular gene, uridine kinase. The relatively large genome of M.penetrans HF-2 among mycoplasma species may be accounted for by both its rich core proteome and the presence of a number of paralog families corresponding to 25.4% of all CDSs. The largest paralog family is the p35 family, which encodes surface lipoproteins including the major antigen, P35. A total of 44 genes for p35 and p35 homologs were identified and 30 of them form one large cluster in the chromosome. The genetic tree of p35 paralogs suggests the occurrence of dynamic chromosomal rearrangement in paralog formation during evolution. Thus, M.penetrans HF-2 may have acquired diverse repertoires of antigenic variation-related genes to allow its persistent infection in humans.

Phase variation among major surface antigens of Mycoplasma penetrans.

The pathogenicity and prevalence of Mycoplasma penetrans, a Mycoplasma species recently isolated from humans, are still debated. A major P35 antigen, which is used as target epitope in serological assays, was shown to be a phase-variable lipid-associated membrane protein (LAMP). In this study, we performed a comparative analysis of the LAMP patterns from five M. penetrans clinical isolates and from the type strain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles and immunoblots with sera serially collected from an M. penetrans-infected patient indicated that these strains expressed different LAMP repertoires. Furthermore, the intraclonal variation in the expression of LAMPs (P34A, P34B, P35, and P38) was monitored by immunoblot analysis with three specific monoclonal antibodies (MAbs) developed in this study and MAb 7 to P35. The phase variation of these LAMPs occurs in an independent manner, with frequencies of variation ranging from 10(-2) to 10(-4) per cell per generation. Consistent with their amphipathic nature, the P34B and P38 antigens were found exposed at the cell surface. The DNA sequence encoding the P38 antigen was defined and found to be related to those of the P35 gene and other putative LAMP-encoding genes, suggesting that these variable antigens are encoded by a family of related genes. Finally, the serum samples from an M. penetrans-infected patient contained antibodies that reacted with a P36 antigen expressed in different M. penetrans strains but not in the isolate recovered from this patient. This result suggested that in vivo phase variation of P36 occurred, which would support a role for these LAMP variations in avoiding the host's immune vigilance.

Evaluation of IgG, IgM, and IgA antibodies to mycoplasma penetrans detected by ELISA and immunoblot in HIV-1-infected and STD patients, in São Paulo, Prazil.

The aim of the present study was to determine the frequency of IgG, IgA, and IgM antibodies to Mycoplasma penetrans in HIV-1-infected patients and in patients with sexually transmitted diseases. We tested serum samples from 106 HIV-1-positive patients and 110 individuals with clinical symptoms of urethritis. ELISA and the immunoblot test were performed using M. penetrans lipid associated membrane proteins as antigen. By ELISA, we found a higher frequency (P < 0.05) of IgG against M. penetrans in HIV-1-infected and STD patients (25.5 and 17.3%) than in controls (1.2%), as well as a higher frequency of IgA (P < 0.05) (15.1 and 17.3% compared to 1.2%). For IgM, no differences were observed (P >/= 0.05) (3.8, 9.1, and 5. 8%, respectively). When the frequencies of IgG, IgM, and IgA antibodies of the HIV-1-infected patients were compared taking into account the CD4/CD8 cell ratios < 0.3 and >/= 0.3, no significant differences were observed between the two groups (13.3, 10, and 20%, compared to 20, 0, and 5%, respectively) (P > 0.05), possibly due to the low number of samples on which we could perform T-cell counts (53/106). The M. penetrans peptide of 38 kDa, considered immunodominant, was recognized in immunoblot by 51.8% of positive sera by ELISA for IgG, 50.0% for IgM, and 75% for IgA in the AIDS patients group, and by 47.4, 60.0, and 75.0%, respectively, in the sexually transmitted disease group. Cross-reactions in immunoblot for IgG were observed in sera from individuals infected with Mycoplasma pneumoniae and Mycoplasma hominis, and cross-reactions in immunoblot for IgA were observed in sera from individuals infected with M. hominis; all of them were ELISA negative to M. penetrans.

Phase variations of the Mycoplasma penetrans main surface lipoprotein increase antigenic diversity.

Mycoplasma penetrans is a recently identified mycoplasma, isolated from urine samples collected from human immunodeficiency virus (HIV)-infected patients. Its presence is significantly associated with HIV infection. The major antigen recognized during natural and experimental infections is an abundant P35 lipoprotein which, upon extraction, segregates in the Triton X-114 detergent phase and is the basis of M. penetrans-specific serological assays. We report here that the P35 antigen undergoes spontaneous and reversible phase variation at high frequency, leading to heterogeneous populations of mycoplasmas, even when derived from a clonal lineage. This variation was found to be determined at the transcription level, and although this property is not unique among the members of the class Mollicutes, the mechanism by which it occurs in M. penetrans differs from those previously described for other Mycoplasma species. Indeed, the P35 phase variation was due neither to a p35 gene rearrangement nor to point mutations within the gene itself or its promoter. The P35 phase variation in the different variants obtained was concomitant with modifications in the pattern of other expressed lipoproteins, probably due to regulated expression of selected members of a gene family which was found to potentially encode similar lipoproteins. M. penetrans variants could be selected on the basis of their lack of colony immunoreactivity with a polyclonal antiserum against a Triton X-114 extract, strongly suggesting that the mechanisms involved in altering surface antigen expression might allow evasion of the humoral immune response of the infected host.

Antigenic characterization and cytolocalization of P35, the major Mycoplasma penetrans antigen.

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 Triton X-114 detergent phase. It is used as the basis of M. penetrans-specific 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. Immunoelectron microscopy revealed that all P35 is exposed at 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 B-epitopes were found at 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 (rP35delta0) in Escherichia coil. The antigenicity of rP35delta0 was tested by Western blotting and compared to that of another recombinant product, rP35delta3, a truncated P35 polypeptide. Although rP35delta0 reacted with the M. penetrans-seropositive patient sera tested, rP35delta3 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 rP35delta0, provide the antigenic material for the necessary improvement of M. penetrans serological assays.

A longitudinal study of seroreactivity against Mycoplasma penetrans in HIV-infected homosexual men: association with disease progression.

We investigated the relationships between a putative cofactor of HIV infection, Mycoplasma penetrans, and the evolution of HIV disease. The evolution of titers of anti-M. penetrans antibodies in 58 randomly selected HIV-seropositive adult homosexual men was investigated. The median length of follow-up was 38 months. Thirty-six individuals was investigated. The median length of follow-up was 38 months. Thirty-six individuals (62.1%) remained M. penetrans seronegative (group 0). Fourteen patients (24.1%) had consistently low antibody titers or low antibody titer(s) in at least one sample and negative test(s) in the other(s). This pattern was possibly associated with latent or earlier infection (group 1). Eight patients (13.8%) had moderate to high antibody titers for long periods, indicating an active and persistent M. penetrans infection (group 2); four patients in this group presented a serological reactivation and thus probably developed an acute infection during the study; two had a stable and moderate level of antibody throughout the study; in two patients the antibody titers decreased substantially. Interestingly, CD4 cell counts declined more rapidly in group 2 than in group 0 (medians of -4.5 versus -2.1 cells/mm3/month, p < 0.05 and -0.16 versus 0 cell percentage/month, p < 0.05), whereas there was no significant difference between groups 1 and 0 (medians of -2.0 versus -2.1 cells/mm3/month and -0.15 versus 0 cell percentage/month). In patients with serological reactivation, the viral load was higher in sera with higher M. penetrans antibody titers. These findings suggest an association between active M. penetrans infection and progression of HIV disease.

Mycoplasmas and HIV infection, a possible interaction through immune activation.

The persistence of HIV in the host is associated with a state of chronic immune activation with deleterious effects. In addition to HIV, several infectious agents can contribute to this activation and, consequently, to an increased HIV load. We have postulated that mycoplasmas could act as cofactors of HIV, contributing to a faster progression of the disease through increased immune activation. Mycoplasma penetrans was shown to be associated with HIV infection and recent data have indicated the capacity of this mycoplasma to activate different functions of the immune system. However, its role as an HIV cofactor remains to be demonstrated.

Purification and characterization of Mycoplasma penetrans Ca2+/Mg2+-dependent endonuclease.

The major nuclease from Mycoplasma penetrans has been purified to homogeneity. The enzyme seems to be present as a membrane-associated precursor of 50 kDa and as a peripheral membrane monomeric polypeptide of 40 kDa that is easily removed by washing of cells with isotonic buffers and in the aqueous phase upon Triton partitioning of Triton X-114-solubilized protein. The 40-kDa nuclease was extracted from M. penetrans cells by Triton X-114 and phase fractionation and was further purified by chromatography on Superdex 75 and chelating Sepharose (Zn2+ form) columns. By gel filtration, the apparent molecular mass was 40 kDa. The purified enzyme exhibits both a nicking activity on superhelical and linear double-stranded DNA and a nuclease activity on RNA and single-stranded DNA. No exonuclease activity was found for this enzyme. This nuclease required both Mg2+ (optimum, 5 mM) and Ca2+ (optimum, 2 mM) for activity and exhibited a pH optimum between pH 7 and 8 for DNase activity. It was inhibited by Zn2+, Mn2+, heparin, sodium dodecyl sulfate, and chelator agents such EDTA and EGTA, but no effect was observed with ATP, 2-mercaptoethanol, N-ethylmaleimide, dithiothreitol, nonionic detergents, phenylmethylsulfonyl fluoride, and iodoacetamide. Nuclease activity was inhibited by diethylpyrocarbonate at both pH 6 and 8 and by pepstatin, suggesting the involvement of a histidine and an aspartate in the active site. When added to human lymphoblast nuclei, the purified M. penetrans endonuclease induced internucleosomal fragmentation of the chomatin into oligonucleosomal fragments. On the basis of this result, and taking into account the fact that M. penetrans has the capacity to invade eucaryotic cells, one can suggest, but not assert, that produced Ca2+/Mg2+-dependent endonuclease may alter the nucleic acid metabolism of host cells by DNA and/or RNA degradation and may act as a potential pathogenic determinant.

High prevalence of antibodies to Mycoplasma penetrans in human immunodeficiency virus-seronegative and -seropositive populations in Brazzaville, Congo.

In industrialized countries, the prevalence of antibodies to Mycoplasma penetrans is higher among human immunodeficiency virus (HIV)-seropositive homosexuals than other HIV-seropositive and HIV-seronegative groups. In an African heterosexual population, we found a higher prevalence of M. penetrans antibodies in HIV-seronegative blood donors (15.5%) than in France (0.9%) or the United States (0.3%) and a prevalence of 13.4% in HIV-seropositive individuals. HIV-seropositive individuals with less than 5% CD4 cells had a higher prevalence of M. penetrans antibodies than individuals with 5% or more CD4 cells (25.0 versus 8.5%).

PIP: Numerous studies in developed countries have revealed a higher prevalence of antibodies to Mycoplasma penetrans in homosexuals infected with HIV than in other HIV-positive and HIV-negative population groups. To confirm whether this association prevails in African countries, a cross-sectional analysis was conducted in Brazzaville, Congo, in 1993-94. Tested were 116 HIV-negative blood donors and 149 HIV-infected hospital patients. The prevalence of antibodies to M. penetrans was 13.4% in the HIV-positive and 15.5% in the HIV-negative group. Among HIV-infected patients, M. penetrans seropositivity was more frequent among patients under 30 years of age, those with CD4 lymphocyte counts below 200 cells/cu. mm, and those with CD4 lymphocyte percentages below 5%. This correlation between the prevalence of antibodies to M. penetrans and the severity of immunosuppression has been documented in studies from France and the US as well. The high prevalence of antibodies to M. penetrans in the late stages of HIV infection in Western homosexuals and Congolese heterosexuals may reflect a cohort effect in which the groups most exposed to HIV at the beginning of the epidemic were also those most exposed to M. penetrans infection. It is also possible that the development of M. penetrans is due to immunosuppression or, alternatively, infection influences HIV multiplication.

Adherence, fibronectin binding, and induction of cytoskeleton reorganization in cultured human cells by Mycoplasma penetrans.

Mycoplasma penetrans adhered to cultured human cells, forming clusters that localized to specific areas of the host cell surface. Adherence and cluster formation were inhibited by anti-M. penetrans antibodies, suggesting the involvement of specific adhesin-receptor interactions. Ultrastructural studies showed that after 2 h of infection, mycoplasmas attach to and penetrate the host cell surface. M. penetrans bound selectively to immobilized fibronectin, an interaction which was not inhibited by a 70-kDa fragment containing a heparin-gelatin-binding domain of fibronectin, other matrix glycoproteins, or an RGD tripeptide, suggesting the recognition of other specific binding sites on the fibronectin molecule. A ca. 65-kDa fibronectin-binding protein of M. penetrans was eluted following Sepharose-fibronectin affinity chromatography. Confocal, light, and immunofluorescence microscopy demonstrated that the interaction of M. penetrans with target cells triggers a signal that causes recruitment of several cytoskeletal components, including tubulin and alpha-actinin, and aggregation of phosphorylated proteins. Detergent-soluble mycoplasma proteins with apparent molecular masses of 18, 28, 32, 36, 39, and 41 kDa selectively bound to glutaraldehyde-fixed HEp-2 cells. Our findings offer new insights into understanding the interaction of this human mycoplasma with host target cells.

In vitro influence of Mycoplasma penetrans on activation of peripheral T lymphocytes from healthy donors or human immunodeficiency virus-infected individuals.

Mycoplasma penetrans is a mycoplasma species newly isolated from the urine of human immunodeficiency virus (HIV)-infected individuals and presents the only case in which an association has been found between antibodies against a mycoplasma and HIV infection. To further explore the effects of M. penetrans on the immune system, we studied the influence of this mycoplasma on peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-infected individuals. M. penetrans induced, in addition to blastogenesis of PBMCs, a significant proliferative response associated with the expression of some activation markers such as CD69, HLA-DR, and CD25. This M. penetrans-dependent lymphocyte activation was observed not only in healthy donors but also in HIV-infected persons at different stages of the disease. In addition, our study revealed that both CD4+ and CD8+ T lymphocytes were responsive to M. penetrans. Interestingly, the mitogenic activity of M. penetrans was associated with mycoplasma cells but not with the supernatants of mycoplasma culture. The potent stimulating activity of M. penetrans on T lymphocytes from HIV-infected individuals is of particular interest in view of the supposed contribution of immune activation to HIV replication and disease progression.