Mechanisms of Mycoplasma-induced inflammation and cellular transformation

There is a growing interest in “medical gasses” for their antibacterial and anti-inflammatory properties. Hydrogen sulfide (H2S), a member of the family of gasotransmitters, is in fact increasingly being recognized as an important signaling molecule, but its precise role in the regulation of the inflammatory response is still not clear. For this reason, the aim of the first part of this thesis was to investigate the effects of H2S on the expression of pro-inflammatory cytokines, such as MCP-1, by using an in vitro model composed by both primary monocytes-derived macrophages cultures and the human monocytic cell line U937 infected with Mycoplasma fermentans, a well-known pro-inflammatory agent. In our experiments, we observed a marked increase in the production of pro-inflammatory cytokines in infected cells. In particular, MCP-1 was induced both at the RNA and at the protein level. To test the effects of H2S on infected cells, we treated the cells with two different H2S donors (NaHS and GYY4137), showing that both H2S treatments had anti-inflammatory effects in Mycoplasma-infected cells: the levels of MCP-1, both mRNA expression and protein production, were reduced. Our subsequent studies aimed at understanding the molecular mechanisms responsible for these effects, focused on two specific molecular pathways, both involved in inflammation: the NF-κB and the Nrf2 pathway. After treatment with pharmacological inhibitors, we demonstrated that Mycoplasma fermentans induces MCP-1 expression through the TLR-NF-κB pathway with the nuclear translocation of its subunits, while treatment with H2S completely blocked the nuclear translocation of NF-κB heterodimer p65/p50. Then, once infected cells were treated with H2S donors, we observed an increased protective effect of Nrf2 and also a decrease in ROS production. These results highlight the importance of H2S in reducing the inflammatory process caused by Mycoplasma fermentans. To this regard, it should be noted that several projects are currently ongoing to develop H2S-releasing compounds as candidate drugs capable of alleviating cell deterioration and to reduce the rate of decline in organ function. In the second part of this study, we investigated the role of Mycoplasma infection in cellular transformation. Infectious agents are involved in the etiology of many different cancers and a number of studies are still investigating the role of microbiota in tumor development. Mycoplasma has been associated with some human cancers, such as prostate cancer and non-Hodgkin’s lymphoma in HIV-seropositive people, and its potential causative role and molecular mechanisms involved are being actively investigated. To this regard, in vitro studies demonstrated that, upon infection, Mycoplasma suppresses the transcriptional activity of p53, key protein in the cancer suppression. As a consequence, infected cells were less susceptible to apoptosis and proliferated more than the uninfected cells. The mechanism(s) responsible for the Mycoplasma-induced inhibitory effect on p53 were not determined. Aim of the second part of this thesis was to better understand the tumorigenic role of the microorganism, by investigating more in details the effect(s) of Mycoplasma on p53 activity in an adenocarcinoma HCT116 cell line. Treatment of Mycoplasma-infected cells with 5FU or with Nutlin, two molecules that induce p53 activity, resulted in cellular proliferation comparable to untreated controls. These results suggested that Mycoplasma infection inhibited p53 activity. Immunoprecipitation of p53 with specific antibodies, and subsequent Gas Chromatography and Mass Spectroscopy (GC-MS) assays, allowed us to identify several Mycoplasma-specific proteins interacting with p53, such as DnaK, a prokaryotic heat shock protein and stress inducible chaperones. In cells transfected with DnaK we observed i) reduced p53 protein levels; ii) reduced activity and expression of p21, Bax and PUMA, iii) a marked increase in cells leaving G1 phase. Taken together, these data show an interaction between the human p53 and the Mycoplasma protein DnaK, with the consequent decreased p53 activity and decreased capability to respond to DNA damage and prevent cell proliferation. Our data indicate that Mycoplasma could be involved in cancer formation and the mechanism(s) has the potential to be a target for cancer diagnosis and treatment(s).

Examination of the role of Mycoplasma bovis in bovine pneumonia and a mathematical model for its evaluation

The authors screened 34 large cattle herds for the presence of Mycoplasma bovis infection by examining slaughtered cattle for macroscopic lung lesions, by culturing M. bovis from lung lesions and at the same time by testing sera for the presence of antibodies against M. bovis. Among the 595 cattle examined, 33.9% had pneumonic lesions, mycoplasmas were isolated from 59.9% of pneumonic lung samples, and 10.9% of sera from those animals contained antibodies to M.bovis. In 25.2% of the cases M. bovis was isolated from lungs with no macroscopic lesions. The proportion of seropositive herds was 64.7%. The average seropositivity rate of individuals was 11.3% but in certain herds it exceeded 50%. A probability model was developed for examining the relationship among the occurrence of pneumonia, the isolation of M. bovis from the lungs and the presence of M. bovis specific antibodies in sera.

Anti-N-methyl-D-Aspartate Receptor Encephalitis with Positive Serum Antithyroid Antibodies, IgM Antibodies Against Mycoplasma Pneumoniae and Human Herpesvirus 7 PCR in the CSF

We report the case of a boy with an encephalopathy associated with extrapyramidal and psychiatric symptoms and anti-N-methyl-D-aspartate receptor antibodies. He had positive serum antithyroid antibodies, IgM antibodies against Mycoplasma pneumoniae and human herpesvirus 7 polymerase chain reaction in the cerebrospinal fluid. He was successfully treated with rituximab, after steroids, intravenous immunoglobulin and plasma exchange. The pathophysiology of this disorder may be post-infectious and autoimmune.

Th1/Th2 Cytokine Profile and Its Diagnostic Value in Mycoplasma pneumoniae Pneumonia

Background: The levels of Th1/Th2 cytokine can alter in pathogenic infection in children with pneumonia. Objectives: To evaluate Th1/Th2 cytokine profile and its diagnostic value in M. pneumoniae pneumonia in children. Patients and Methods: Children with M. pneumoniae mono-infection and 30 healthy children were tested with cytokines assay. We used real time PCR to detect M. pneumoniae in children with pneumonia. Results: M. pneumoniae test was positive in 2188 (16.62%) out of 13161 pneumonia children. Children aged 5 - 9 years had the highest rate and summer was a season with high rate of M. pneumoniae incidence in Zhejiang province. During the course of study, in 526 pneumonia children with M. pneumoniae mono-infection and 30 healthy children cytokines assay was performed. IL-2 level of M. pneumoniae pneumonia children was lower than that of healthy children (median levels, pg/mL: IL-2: 3.2 vs. 5.7, P = 0.00), while IL-4, IL-10 and IFN-γ were higher than in healthy children (median levels, pg/mL: IL-4: 3.2 vs. 1.5, P = 0.00; IL-10: 5.6 vs. 2.5, P = 0.001; IFN-γ: 20.4 vs. 4.8, P = 0.001). Conclusions: IL-2 decreases and IL-4, IL-10 and IFN-γ increase in children with M. pneumoniae pneumonia, which has a promising prospect in diagnosis of this disease in clinical practice.

Etiologic diagnosis of bovine infectious abortion by PCR

Infectious abortion is a significant cause of reproductive failure and economic losses in cattle. The goal of this study was to detect nucleic acids of several infectious agents known to cause abortion including Arcanobacterium pyogenes, Bovine Herpesvirus 1, Brucella abortus, Campylobacter fetus subsp. venerealis, Chlamydophila abortus, Leptospira sp., Listeria monocytogenes, Salmonella sp., Mycoplasma bovis, Mycoplasma bovigenitalium, Neospora caninum, and Tritrichomonas foetus. Tissue homogenates from 42 fetuses and paraffin-embedded tissues from 28 fetuses and 14 placentas/endometrium were included in this study. Brucella abortus was detected in 14.2% (12/84) of the samples. Salmonella sp. DNA was amplified from 2 fetuses, and there was one positive for Neospora caninum, and another for Listeria monocytogenes. This PCR-based approach resulted in identification of the etiology in 19% of samples, or 20% if considered fetal tissues only.

Serosurvey of selected avian pathogens in brazilian commercial Rheas (Rhea americana) and Ostriches (Struthio camelus)

Ratite farming of has expanded worldwide. Due to the intensive farming methods used by ratite producers, preventive medicine practices should be established. In this context, the surveillance and control of some avian pathogens are essential for the success of the ratite industry; however, little is known on the health status of ratites in Brazil. Therefore, the prevalence of antibodies against Newcastle Disease virus, Chlamydophila psittaci, Mycoplasma gallisepticum, Mycoplasma synoviae, and Salmonella Pullorum were evaluated in 100 serum samples collected from commercial ostriches and in 80 serum samples from commercial rheas reared in Brazil. All sampled animals were clinically healthy. The results showed that all ostriches and rheas were serologically negative to Newcastle disease virus, Chlamydophila psittaci, Mycoplasma gallisepticum, and Mycoplasma synoviae. Positive antibody responses against Salmonella Pullorum antigen were not detected in ostrich sera, but were detected in two rhea serum samples. These results can be considered as a warning as to the presence of Salmonella spp. in ratite farms. Therefore, the implementation of good health management and surveillance programs in ratite farms may contribute to improve not only animal production, but also public health conditions.

Invasion of Ureaplasma diversum in Hep-2 cells

Background: Understanding mollicutes is challenging due to their variety and relationship with host cells. Invasion has explained issues related to their opportunistic role. Few studies have been done on the Ureaplasma diversum mollicute, which is detected in healthy or diseased bovine. The invasion in Hep-2 cells of four clinical isolates and two reference strains of their ureaplasma was studied by Confocal Laser Scanning Microscopy and gentamicin invasion assay. Results: The isolates and strains used were detected inside the cells after infection of one minute without difference in the arrangement for adhesion and invasion. The adhesion was scattered throughout the cells, and after three hours, the invasion of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% of the ATCC strains were inside the infected Hep-2 cells in contrast to 10% to the clinical isolates. A high level of phospholipase C activity was also detected in all studied ureaplasma. Conclusions: The results presented herein will help better understand U. diversum infections, aswell as cellular attachment and virulence.

Hemoplasma Infection in HIV-positive Patient, Brazil

Hemotrophic mycoplasmas infect a variety of mammals. Although infection in humans is rarely reported, an association with an immunocompromised state has been suggested. We report a case of a Mycoplasma haemofelis-like infection in an HIV-positive patient co-infected with Bartonella henselae.

HEMOPLASMAS IN WILD CANIDS AND FEUDS IN BRAZIL

Hemotropic mycoplasmas, epicellular erythrocytic bacterial parasites lacking a cell wall, are the causative agents of infectious anemia in numerous mammalian species. The presence of hemotropic mycoplasmas in blood samples of neotropical and exotic wild canids and felids from Brazilian zoos were recorded using molecular techniques. Blood samples were collected from 146 Brazilian wild felids, 19 exotic felids, 3 European wolves (Canis lupus), and from 97 Brazilian wild canids from zoos in the Brazilian states of Sao Paulo and Mato Grosso and the Federal District. Using conventional polymerase chain reaction (PCR), this work found 22 (13%) wild felids positive to Candidatus Mycoplasma haemominutum [4 jaguars (Panthera onca); 3 pumas (Puma concolor); 10 ocelots (Leopardus pardalis); 2 jaguarondis (Puma yagouaroundi); and 3 little spotted cats (Leopardus tigrinus)]. Only one little spotted cat (Leopardus tigrinus) was positive to Mycoplasma haemofelis, and none was positive to Candidatus Mycoplasma turicensis. Two bush dogs (Speothos venaticus) were positive for a Mycoplasma sp. closely related to Candidatus Mycoplasma haematoparvum, and two European wolves were positive for a Mycoplasma sp. closely related to candidatus Mycoplasma haemominutum. This is the first study regarding the molecular detection of hemotropic mycoplasmas in wild canids.

Serosurvey Of Selected Avian Pathogens In Brazilian Commercial Rheas (Rhea americana) And Ostriches (Struthio camelus)

Ratite farming of has expanded worldwide. Due to the intensive farming methods used by ratite producers, preventive medicine practices should be established. In this context, the surveillance and control of some avian pathogens are essential for the success of the ratite industry; however, little is known on the health status of ratites in Brazil. Therefore, the prevalence of antibodies against Newcastle Disease virus, Chlamydophila psittaci, Mycoplasma gallisepticum, Mycoplasma synoviae, and Salmonella Pullorum were evaluated in 100 serum samples collected from commercial ostriches and in 80 serum samples from commercial rheas reared in Brazil. All sampled animals were clinically healthy. The results showed that all ostriches and rheas were serologically negative to Newcastle disease virus, Chlamydophila psittaci, Myco plasma gallisepticum, and Myco plasma synoviae. Positive antibody responses against Salmonella Pullorum antigen were not detected in ostrich sera, but were detected in two rhea serum samples. These results can be considered as a warning as to the presence of Salmonella spp. in ratite farms. Therefore, the implementation of good health management and surveillance programs in ratite farms may contribute to improve not only animal production, but also public health conditions.

Drug targets and mechanisms of resistance in the anaerobic protozoa

The anaerobic protozoa Giardia duodenalis, Trichomonas vaginalis, and Entamoeba histolytica infect up to a billion people each year. G. duodenalis and E. histolytica are primarily pathogens of the intestinal tract, although E. histolytica can form abscesses and invade other organs, where it can be fatal if left untreated. T. vaginalis infection is a sexually transmitted infection causing vaginitis and acute inflammatory disease of the genital mucosa. T. vaginalis has also been reported in the urinary tract fallopian tubes, and pelvis and can cause pneumonia, bronchitis, and oral lesions. Respiratory infections can be acquired perinatally. T. vaginalis infections have been associated with preterm delivery, low birth weight, and increased mortality as well as predisposing to human immunodeficiency virus infection, AIDS, and cervical cancer. All three organisms lack mitochondria and are susceptible to the nitroimidazole metronidazole because of similar low-redox-potential anaerobic metabolic pathways. Resistance to metronidazole and other drugs has been observed clinically and in the laboratory. Laboratory studies have identified the enzyme that activates metronidazole, pyruvate:ferredoxin oxidoreductase, to its nitroso form and distinct mechanisms of decreasing drug susceptibility that are induced in each organism. Although the nitroimidazoles have been the drug family of choice for treating the anaerobic protozoa, G. duodenalis is less susceptible to other antiparasitic drugs, such as furazolidone, albendazole, and quinacrine. Resistance has been demonstrated for each agent and the mechanism of resistance has been investigated. Metronidazole resistance in T. vaginalis is well documented, and the principal mechanisms have been defined Bypass metabolism, such as alternative oxidoreductases, have been discovered in both organisms. Aerobic versus anaerobic resistance in T. vaginalis is discussed. Mechanisms of metronidazole resistance in E. histolytica have recently been investigated ruing laboratory-induced resistant isolates. Instead of downregulation of the pyruvate:ferredoxin oxidoreductase and ferredoxin pathway as seen in G. duodenalis and T. vaginalis, E. histolytica induces oxidative stress mechanisms, including superoxide dismutase and peroxiredoxin. The review examines the value of investigating both clinical and laboratory-induced syngeneic drug-resistant isolates and dissection of the complementary data obtained. Comparison of resistance mechanisms in anaerobic bacteria and the parasitic protozoa is discussed as well as the value of studies of the epidemiology of resistance.

Phase variable restriction-modification systems in Moraxella catarrhalis

A repetitive DNA motif was used as a marker to identify novel genes in the mucosal pathogen Moraxella catarrhalis. There is a high prevalence of such repetitive motifs in virulence genes that display phase variable expression. Two repeat containing loci were identified using a digoxigenin-labelled 5'-(CAAC)(6)-3' oligonucleotide probe. The repeats are located in the methylase components of two distinct type III restriction-modification (R-M) systems. We suggest that the phase variable nature of these R-M systems indicates that they have an important role in the biology of M. catarrhalis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.