Characterization of a Unique ADP-Ribosyltransferase of Mycoplasma penetrans

Mycoplasma penetrans is a urogenital tract pathogen implicated in the deterioration of the immune system in human immunodeficiency virus-infected AIDS patients. Here, we describe a 78-kDa protein from M. penetrans, designated MYPE9110, that exhibits sequence similarity to known ADP-ribosyltransferases (ADPRTs) such as Bordetella pertussis pertussis toxin and Mycoplasma pneumoniae community-acquired respiratory distress syndrome toxin. MYPE9110 possesses key amino acid residues found in all ADPRTs that are essential for ADPRT activity. Several mammalian cell proteins are ADP-ribosylated by MYPE9110, and the full-length recombinant protein exhibits a strong auto-ADP-ribosylating activity. In the absence of target proteins, MYPE9110 demonstrates a NAD-glycohydrolase activity by hydrolyzing NAD. Furthermore, this toxin elicits cytopathology in HeLa cells by inducing cytoplasmic vacuolization in the presence of ammonium chloride. The deletion of the C-terminal region of MYPE9110 significantly diminishes its binding to host cells while still exhibiting an ADPRT activity, suggesting that MYPE9110 is a member of the family of A-B ADPRT toxins.The Mollicutes contain several human-pathogenic mycoplasmas including Mycoplasma pneumoniae, Mycoplasma genitalium, and Mycoplasma penetrans. M. penetrans GTU-54 was first isolated from the urine of a human immunodeficiency virus (HIV)-positive homosexual male (20). Subsequent studies reported a higher frequency of antibodies against M. penetrans (40%) in sera of HIV-infected AIDS patients than in sera of HIV-infected non-AIDS and HIV-negative control groups (20% and 0.3%, respectively) (36). It was previously hypothesized that M. penetrans could exist as either an opportunist or a cofactor in AIDS progression for several reasons, including the ability of M. penetrans to activate human T lymphocytes (31). Although considered predominately a urogenital tract pathogen, M. penetrans strains have also been isolated from blood (strain HF-1) and respiratory tract cultures (strain HF-2) of a non-HIV-infected patient with primary antiphospholipid syndrome and bacteremia (37).Among the genome-sequenced pathogenic mycoplasmas, M. penetrans strain HF-2 is the largest, at 1.4 Mbp, with a low G+C content of 25.7% and 1,038 predicted coding sequences (32). The genome consists of many paralogs, including the p35 gene family, which may account for its larger genome size than other pathogenic mycoplasmas. The p35 gene family encodes surface lipoproteins, including the immunodominant P35 protein, which is the basis of the serological diagnosis of M. penetrans infection (32). In silico analysis of the M. penetrans genome indicates the presence of a two-component response regulator (MYPE3960) and a putative sensory transduction histidine kinase (MYPE2360). No such regulatory elements have been found for other sequenced mollicute genomes, suggesting that M. penetrans is unique in that it may be able to sense and synthesize proteins in response to its survival niche (32).Following adherence to host cells, M. penetrans induces cytoskeleton rearrangement, as evidenced by the aggregation of tubulin and α-actinin (13). This event leads to the internalization of M. penetrans, where it resides in the host cell cytosol, membrane-bound vacuoles, or perinuclear region for extended periods of time (2, 10, 13). This intracellular environment offers many benefits to the bacterium, where it can avoid host immune responses and acquire essential nutrients (26). The mechanism by which M. penetrans triggers cytoskeletal rearrangements is still poorly understood. Manipulation of the cytoskeletal network by bacterial toxins, including ADP-ribosylating toxins, was previously reported for both gram-negative and gram-positive bacteria (26). Until recently, the only genome-encoded potential virulence factors of M. penetrans included endonucleases, hemolysins, and proteases (3, 18, 32), and their roles in pathogenesis remain unclear.Recently, an ADP-ribosylating and vacuolating toxin was described for M. pneumoniae (17). This toxin, designated community-acquired respiratory distress syndrome (CARDS) toxin, was first identified by its interaction with the human lung protein surfactant protein A and by its limited but critically relevant sequence similarity to the Bordetella pertussis pertussis toxin S1 subunit ADP-ribosyltransferase (ADPRT) domain (17, 19). ADPRTs are found in a wide range of bacterial pathogens and catalyze the transfer of a single ADP-ribosyl group from β-NAD onto specific amino acid residues of host cell proteins with the release of nicotinamide (24). Mono-ADP-ribosylation by these ADPRTs leads to the modification of various host cell target proteins and their activities, including the inhibition of host protein synthesis by diphtheria toxin (DT) (25), alterations in signal transduction pathways by pertussis toxin (21), and interference of actin polymerization by Clostridium iota and C2 toxins (1, 33). Many members of these ADPRTs, including pertussis toxin and DT, belong to the A-B family, which possesses an active ADPRT subunit, known as the A subunit, and a B subunit responsible for the binding and translocation of the active subunit across host cell membranes (11).Our discovery of CARDS toxin in M. pneumoniae prompted us to look at other mycoplasma genomes for the existence of a related protein. A hypothetical protein in the M. penetrans genome, annotated MYPE9110, shared sequence similarity with CARDS toxin and a conserved domain with the pertussis toxin S1 subunit. In this study we show that MYPE9110 is related to the family of classical A-B toxins, as it possesses an N-terminal ADPRT domain and a C-terminal cell-binding domain. We also show that MYPE9110 elicits cytopathology in host cells, indicating the virulence potential of this newly characterized M. penetrans protein.     

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.     

Mycoplasma penetrans and other mycoplasmas in urine of human immunodeficiency virus-positive children

Urine samples from children with human immunodeficiency virus (HIV) infection and healthy controls were examined for mycoplasmas by culture. Standard biochemical assays, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and PCR (16S and 16S-23S spacer rRNA region) were used for identification of isolates. Mycoplasmas were identified from 13 (87%) of 15 HIV-positive patients and 3 (20%) of 15 HIV-negative control patients. The frequency and type of mycoplasma varied with the severity of HIV infection. Mycoplasma penetrans, Mycoplasma pirum, Mycoplasma fermentans, and Mycoplasma genitalium were isolated from patients with severe immunodeficiency. Mycoplasma hominis and Ureaplasma urealyticum were isolated more frequently from children in the early stages of HIV infection and from HIV-negative patients. Mycoplasma penetrans was isolated from one (50%) of two patients in Centers for Disease Control and Prevention (CDC) group B and from five (55.5%) of nine pediatric patients with AIDS (CDC group C). This is the first report that indicates that "AIDS-associated" mycoplasmas are more common in HIV-infected children than in HIV-negative controls.     

Role of Mycoplasma penetrans Endonuclease P40 as a Potential Pathogenic Determinant

Recently, we reported the purification to homogeneity and characterization of Ca(2+)- and Mg(2+)-dependent endonuclease P40 produced by Mycoplasma penetrans (M. Bendjennat, A. Blanchard, M. Loutfi, L. Montagnier, and E. Bahraoui, J. Bacteriol. 179; 2210-2220, 1997), a mycoplasma which was isolated for the first time from the urine of human immunodeficiency virus-infected patients. To evaluate how this nuclease could interact with host cells, we tested its effect on CEM and Molt-4 lymphocytic cell lines and on peripheral blood mononuclear cells. We observed that 10(-7) to 10(-9) M P40 is able to mediate a cytotoxic effect. We found that 100% of cells were killed after 24 h of incubation with 10(-7) M P40 while only 40% cytotoxicity was obtained after 72 h of incubation with 10(-9) M P40. Phase-contrast microscopy observations of P40-treated cells revealed morphological changes, including pronounced blebbing of the plasma membrane and cytoplasmic shrinkage characteristic of programmed cell death, which is in agreement with the internucleosomal fragmentation of P40-treated cell DNA as shown by agarose gel electrophoresis. We showed that (125)I-radiolabeled or fluorescein isothiocyanate-labeled P40 was able to bind specifically in a dose-dependent manner to the cell membrane of CEM cells, which suggested that the cytotoxicity of P40 endonuclease was mediated by its interaction with the cell surface receptor(s). The concentration of unlabeled P40 required to inhibit by 50% the formation of (125)I-P40-CEM complexes was about 3 x 10(-9) M, indicating a high-affinity interaction. Both P40 interaction and cytotoxicity are Ca(2+) dependent. Our results suggest that the cytotoxicity of M. penetrans observed in vitro is mediated at least partially by secreted P40, which, after interaction with host cells, can induce an apoptosis-like death. These results strongly suggest a major role of mycoplasmal nucleases as potential pathogenic determinants.     

Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities.

A 46-kDa hemolytic protein, referred to as cystalysin, from Treponema denticola ATCC 35404 was overexpressed in Escherichia coli LC-67. Both the native and recombinant 46-kDa proteins were purified to homogeneity. Both proteins expressed identical biological and functional characteristics. In addition to its biological function of lysing erythrocytes and hemoxidizing the hemoglobin to methemoglobin, cystalysin was also capable of removing the sulfhydryl and amino groups from selected S-containing compounds (e.g., cysteine) producing H2S, NH3, and pyruvate. This cysteine desulfhydrase resulted in the following Michaelis-Menten kinetics: Km = 3.6 mM and k(cat) = 12 s(-1). Cystathionine and S-aminoethyl-L-cysteine were also substrates for the protein. Gas chromatography-mass spectrometry and high-performance liquid chromatography analysis of the end products revealed NH3, pyruvate, homocysteine (from cystathionine), and cysteamine (from S-aminoethyl-L-cysteine). The enzyme was active over a broad pH range, with highest activity at pH 7.8 to 8.0. The enzymatic activity was increased by beta-mercaptoethanol. It was not inhibited by the proteinase inhibitor TLCK (N alpha-p-tosyl-L-lysine chloromethyl ketone), pronase, or proteinase K, suggesting that the functional site was physically protected or located in a small fragment of the polypeptide. We hypothesize that cystalysin is a pyridoxal-5-phosphate-containing enzyme, with activity of an alphaC-N and betaC-S lyase (cystathionase) type. Since large amounts of H2S have been reported in deep periodontal pockets, cystalysin may also function in vivo as an important virulence molecule.     

Induced mouse spleen B-cell proliferation and secretion of immunoglobulin by lipid-associated membrane proteins of Mycoplasma fermentans incognitus and Mycoplasma penetrans.

Mycoplasmas have been implicated as a possible cofactor in AIDS pathogenesis. Mycoplasma fermentans and M. penetrans infect human immunodeficiency virus-positive patients at a significantly higher frequency than non-human immunodeficiency virus-infected control subjects. Various mycoplasmal membrane preparations are known to affect the functions of immune cells both in vitro and in vivo. A group of lipid-associated membrane proteins (LAMPs) extracted by Triton X-114 from mycoplasmas are major antigenic targets of human host antibody responses. In this study, LAMPs prepared from both M. fermentans and M. penetrans nonspecifically stimulated spleen cells of CBA/CaH mice to proliferate. LAMPs were also stimulatory to spleen cells from athymic mice. On the other hand, enriched splenic T cells from CBA/CaH mice with or without accessory cells responded poorly. Thus, the mitogenic effect of mycoplasmal LAMPs appeared mainly on B cells. High levels of immunoglobulin (Ig) M and low but detectable amounts of IgG were found in the supernatant of LAMP-treated splenic cell culture. M. penetrans LAMPs had a much more potent effect on murine spleen cells than did M. fermentans incognitus LAMPs in inducing both B-cell proliferation and Ig secretion. In conclusion, the mycoplasmal LAMPs contained an active component(s) with T-independent B-cell mitogenic effect.     

穿透支原体P35蛋白对细胞因子的诱生作用

目的　构建pQE31/p35原核细胞表达载体 ,研究穿透支原体 (Mpe)相对分子质量 (Mr)为 35× 10 3 蛋白 (P35 )诱生巨噬细胞 (MΦ)分泌TNF α、IL 1β、IL 6等细胞因子 (CKs)的作用。方法　PCR扩增p35全长 10 0 2bp基因片段 ,克隆至pQE31原核细胞表达载体 ,用PCR介导的突变将p35基因中两个“TGA”终止密码子定点突变为“TGG” ,使其在大肠杆菌 (E .coli)中编码表达色氨酸。IPTG诱导pQE31/p35在E .coli中表达目的蛋白rP35 ,用Ni NTASpin亲和纯化 ,Westernblot鉴定表达产物。用酶联免疫吸附试验 (ELISA)检测P35刺激小鼠MΦ分泌TNF α、IL 1β和IL 6的量。结果　PCR扩增出约 1kb的DNA片段 ,克隆至pQE31载体 ,筛选到阳性克隆pQE31/p35 ,p35基因中两个“TGA”终止密码子成功突变成“TGG”。IPTG诱导pQE31/p35在E .coli中表达出Mr 约 37× 10 3 的蛋白质 ,Westernblot鉴定其为rP35。ELISA结果表明 ,P35能刺激小鼠MΦ分泌大量的TNF α、IL 1β和IL 6。结论　P35能诱导MΦ分泌TNF α、IL 1β、IL 6等细胞因子     

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.     

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%).     

Lipid-associated membrane proteins of Mycoplasma fermentans and M. penetrans activate human immunodeficiency virus long-terminal repeats through Toll-like receptors

Mycoplasmas are known to enhance human immunodeficiency virus (HIV) replication, and mycoplasma-derived lipid extracts have been reported to activate nuclear factor-kappaB (NF-kappaB) through Toll-like receptors (TLRs). In this study, we examined the involvement of TLRs in the activation of HIV long-terminal repeats (LTR) by mycoplasma and their active components responsible for the TLR activation. Lipid-associated membrane proteins (LAMPs) from two species of mycoplasma (Mycoplasma fermentans and M. penetrans) that are associated with acquired immune-deficiency syndrome (AIDS), were found to activate HIV LTRs in a human monocytic cell line, THP-1. NF-kappaB deletion from the LTR resulted in inhibition of the activation. The LTR activation by M. fermentans LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and TLR6, whereas HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but not by DN TLR6. These results indicate that the activation of HIV LTRs by M. fermentans and M. penetrans LAMPs is dependent on NF-kappaB, and that the activation of HIV LTR by M. fermentans LAMPs is mediated through TLR1, TLR2 and TLR6. In contrast, the LTR activation by M. penetrans LAMPs is carried out through TLR1 and TLR2, but not TLR6. Subsequently, the active component of M. penetrans and M. fermentans LAMPs was purified by reverse-phase high-performance liquid chromatography (HPLC). Interestingly, the purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate NF-kappaB through TLR1 and TLR2. On the other hand, the activation of NF-kappaB by purified lipoprotein of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but not TLR1.     

Failure to detectMycoplasma fermentans,Mycoplasma penetrans,orMycoplasma pirum in the urethra of patients with acute nongonococcal urethritis

Urethral swab specimens collected from 108 male Japanese patients with acute nongonococcal urethritis (NGU) and from 50 Japanese men without NGU were examined for the presence ofMycoplasma fermentans, Mycoplasma penetrans, andMycoplasma pirum by means of polymerase chain reaction-based assays. These mycoplasmas were not detected in any of the specimens, which suggests that they are unlikely to have a pathogenic role in acute NGU.     

Identification and preliminary characterization of external membrane-bound nuclease activities in Mycoplasma pulmonis.

Mycoplasma pulmonis has substantial DNase activity exposed on the cell surface. At least part of this activity is attributable to an endonuclease. The activity is destroyed at 56 degrees C and inhibited by either 5 mM EDTA or 10 mM zinc chloride. It can also be eliminated by treatment of intact organisms with trypsin and is regenerated by incubation of the treated organisms in a medium that supports protein synthesis. DNase exposed at the cell surface constitutes 20% of the total DNase activity present in M. pulmonis extracts.     

自身免疫病患者血液中穿通支原体分离培养及其血清IL-6与TNF-α的检测

目的:探讨自身免疫病AID患者血液中穿通支原体(Mpe)的分离检出以及Mpe感染患者血清中IL-6与TNF-α的浓度水平.方法:采用分离培养共计从44例AID患者血液标本, 16例ASO或RF阳性体检人员及28例正常对照相应标本中进行支原体分离检测, 对培养阳性菌株采用穿通及发酵支原体套式PCR予以证实, 并用RIA对相应血清进行IL-6及TNF-α浓度检测.结果:于17例(38.6%)AID患者血液中分离到Mpe, 2例(12.5%)ASO或RF阳性体检人员中分离到Mpe, 而正常对照组中则无1例阳性, 组间支原体检出率有统计学意义(P<0.01).支原体阳性AID组血液IL-6浓度(3.30±1.49) μg/L与支原体阴性AID组(2.43±0.95) μg/L及正常对照组(1.14±0.32) μg/L之间差异有统计学意义(P<0.01);支原体阳性AID组血液TNF-α浓度(293.3±179.9) ng/L与支原体阴性AID组(173.9±73.9) ng/L及正常对照组(108.8±33.8) ng/L组之间也有统计学意义(P<0.01).结论:Mpe感染与AID的发生密切相关, Mpe感染患者血清中IL-6与TNF-α较非感染对照人群明显升高.     

Mycoplasma fermentans,but not M penetrans,detected by PCR assays in synovium from patients with rheumatoid arthritis and other rheumatic disorders.

AIM/BACKGROUND: Mycoplasmas, especially Mycoplasma fermentans, were suggested more than 20 years ago as a possible cause of rheumatoid arthritis but this hypothesis was never substantiated. In view of the superior sensitivity of the polymerase chain reaction (PCR) assay over culture, the aim was to use this method to seek M fermentans and M penetrans in synovial samples from patients with various arthritides. METHODS: Synovial fluid samples (n = 154) and synovial biopsy specimens (n = 20) from 133 patients with various rheumatic disorders were stored at -80 degrees C for between one and 40 months. Aliquots (500 microliters) of the synovial fluid samples were centrifuged and the deposit, and also the synovial biopsy specimens (approximately 1 g) were placed in lysis buffer with proteinase K for DNA extraction. The DNA was tested by using a semi-nested PCR assay for M fermentans and a single-round PCR for M penetrans. RESULTS: M fermentans was detected in the joints of eight (21%) of 38 patients with rheumatoid arthritis, two (20%) of 10 patients with spondyloarthropathy with peripheral arthritis, one (20%) of five patients with psoriatic arthritis, and four (13%) of 31 patients with unclassified arthritis. M fermentans was not found in the joints of the seven patients with reactive arthritis, the 29 with osteoarthritis or post-traumatic hydrarthrosis, the nine with gouty arthritis, nor the four with chronic juvenile arthritis. M penetrans was not detected in any sample. CONCLUSIONS: These findings show that the presence of M fermentans in the joint is associated with inflammatory rheumatic disorders of unknown cause, including rheumatoid arthritis. However, whether this organism triggers or perpetuates disease of behaves as a passenger remains conjectural.     

穿透支原体脂质相关膜蛋白激活核因子κB诱导小鼠巨噬细胞表达诱导性一氧化氮合酶

目的　了解穿透支原体潜在的致病性和穿透支原体脂质相关膜蛋白诱导小鼠巨噬细胞表达诱导性一氧化氮合酶 (iNOS)的分子机制。方法　用从穿透支原体提取的脂质相关膜蛋白刺激小鼠巨噬细胞 ,以RT PCR和Westernblot等方法分析诱导性一氧化氮合酶的表达和NO的产生。用间接免疫荧光和Westernblot检测经穿透支原体脂质相关膜蛋白处理的小鼠巨噬细胞中NF κB的激活和NF κB抑制剂PDTC(吡咯啉烷二甲基硫脲 )对iNOS的表达和NO产生的影响。结果　穿透支原体脂质相关膜蛋白以时间和剂量依赖方式刺激小鼠巨噬细胞产生NO ,且能激活NF κB诱导巨噬细胞表达i NOS的mRNA和蛋白 ,其mRNA和蛋白的表达水平能被PDTC所抑制。结论　由于穿透支原体脂质相关膜蛋白能通过激活NF κB诱导巨噬细胞表达诱导性一氧化氮合酶 ,因而可能是一个重要的致病因素。