Interaction of Mycoplasma hominis PG21 with Human Dendritic Cells: Interleukin-23-Inducing Mycoplasmal Lipoproteins and Inflammasome Activation of the Cell.

Mycoplasma hominis lacks a cell wall, and lipoproteins anchored to the extracellular side of the plasma membrane are in direct contact with the host components. A Triton X-114 extract of M. hominis enriched with lipoproteins was shown to stimulate the production of interleukin-23 (IL-23) by human dendritic cells (hDCs). The inflammasome activation of the host cell has never been reported upon M. hominis infection. We studied here the interaction between M. hominis PG21 and hDCs by analyzing both the inflammation-inducing mycoplasmal lipoproteins and the inflammasome activation of the host cell. IL-23-inducing lipoproteins were determined using a sequential extraction strategy with two nondenaturing detergents, Sarkosyl and Triton X-114, followed by SDS-PAGE separation and mass spectrometry identification. The activation of the hDC inflammasome was assessed using PCR array and enzyme-linked immunosorbent assay (ELISA). We defined a list of 24 lipoproteins that could induce the secretion of IL-23 by hDCs, 5 with a molecular mass between 20 and 35 kDa and 19 with a molecular mass between 40 and 100 kDa. Among them, lipoprotein MHO_4720 was identified as potentially bioactive, and a synthetic lipopeptide corresponding to the N-terminal part of the lipoprotein was subsequently shown to induce IL-23 release by hDCs. Regarding the hDC innate immune response, inflammasome activation with caspase-dependent production of IL-1ß was observed. After 24 h of coincubation of hDCs with M. hominis, downregulation of the NLRP3-encoding gene and of the adaptor PYCARD-encoding gene was noticed. Overall, this study provides insight into both protagonists of the interaction of M. hominis and hDCs.IMPORTANCEMycoplasma hominis is a human urogenital pathogen involved in gynecologic and opportunistic infections. M. hominis lacks a cell wall, and its membrane contains many lipoproteins that are anchored to the extracellular side of the plasma membrane. In the present study, we focused on the interaction between M. hominis and human dendritic cells and examined both sides of the interaction, the mycoplasmal lipoproteins involved in the activation of the host cell and the immune response of the cell. On the mycoplasmal side, we showed for the first time that M. hominis lipoproteins with high molecular mass were potentially bioactive. On the cell side, we reported an activation of the inflammasome, which is involved in the innate immune response.

The MAP3K ZAK, a novel modulator of ERK-dependent migration, is upregulated in colorectal cancer.

Often described as a mediator of cell cycle arrest or as a pro-apoptotic factor in stressful conditions, the MAP3K ZAK (Sterile alpha motif and leucine zipper-containing kinase) has also been proven to positively regulate epidermal growth factor receptor (EGFR) and WNT signaling pathways, cancer cell proliferation and cellular neoplastic transformation. Here, we show that both isoforms of ZAK, ZAK-α and ZAK-ß are key factors in cancer cell migration. While ZAK depletion reduced cell motility of HeLa and HCT116 cells, its overexpression triggered the activation of all three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-JUN N-terminal kinase (JNK) and p38, as well as an increase in cell motion. On the contrary, the kinase-dead mutants, ZAK-α K45M and ZAK-ß K45M, were not able to provoke such events, and instead exerted a dominant-negative effect on MAPK activation and cell migration. Pharmacological inhibition of ZAK by nilotinib, preventing ZAK-autophosphorylation and thereby auto-activation, led to the same results. Activated by epidermal growth factor (EGF), we further showed that ZAK constitutes an essential element of the EGF/ERK-dependent cell migration pathway. Using public transcriptomic databases and tissue microarrays, we finally established that, as strong factors of the EGFR signaling pathway, ZAK-α and/or ZAK-ß transcripts and protein(s) are frequently upregulated in colorectal adenoma and carcinoma patients. Notably, gene set enrichment analysis disclosed a significant correlation between ZAK+ colorectal premalignant lesions and gene sets belonging to the MAPK/ERK and motility-related signaling pathways of the reactome database, strongly suggesting that ZAK induces such pro-tumoral reaction cascades in human cancers.

Antimicrobial resistance of bacteria isolated from patients with bloodstream infections at a tertiary care hospital in the Democratic Republic of the Congo.

BACKGROUND: Bloodstream infection (BSI) is a life-threatening condition that requires rapid antimicrobial treatment. METHODS: We determined the prevalence of bacterial isolates associated with BSI at Bukavu General Hospital (BGH), South Kivu Province, Democratic Republic of the Congo, and their patterns of susceptibility to antimicrobial drugs, from February 2013 to January 2014. RESULTS: We cultured 112 clinically relevant isolates from 320 blood cultures. Of these isolates, 104 (92.9%) were Gram-negative bacteria (GNB), with 103 bacilli (92.0%) and one coccus (0.9%). Among GNB, Escherichia coli (51.9%), Klebsiella spp. (20.2%), Enterobacter spp. (6.7%), Shigella spp. (5.8%) and Salmonella spp. (4.8%) were the most frequent agents causing BSIs. Other GNB isolates included Proteus spp., Citrobacter spp. and Pseudomonas aeruginosa (both 2.9%), and Acinetobacter spp. and Neisseria spp. (both 0.9%). High rates of resistance to co-trimoxazole (100%), erythromycin (100%) and ampicillin (66.7-100%) and moderate to high resistance to ciprofloxacin, ceftazidime, ceftriaxone, cefuroxime and cefepime were observed among GNB. Furthermore, there were high rates of multidrug resistance and of extended-spectrum ß-lactamase (ESBL) production phenotype among Enterobacteriaceae. Gram-positive bacteria included three Staphylococcus aureus isolates (2.7%), four oxacillin-resistant coagulase-negative staphylococci (CoNS) isolates (3.6%) and one Streptococcus pneumoniae (0.9%). No oxacillin-resistant S. aureus was isolated. Among clinically relevant staphylococci, susceptibility to co-trimoxazole and ampicillin was low (0-25%). In addition, 58 contaminant CoNS were isolated from blood cultures, and the calculated ratio of contaminants to pathogens in blood cultures was 1:2. CONCLUSIONS: Multidrug-resistant and ESBL-producing GNB are the leading cause of BSI at BGH.

Functional dynamic compartmentalization of respiratory chain intermediate substrates: implications for the control of energy production and mitochondrial diseases.

Activity defects in respiratory chain complexes are responsible for a large variety of pathological situations, including neuromuscular diseases and multisystemic disorders. Their impact on energy production is highly variable and disproportional. The same biochemical or genetic defect can lead to large differences in clinical symptoms and severity between tissues and patients, making the pathophysiological analysis of mitochondrial diseases difficult. The existence of compensatory mechanisms operating at the level of the respiratory chain might be an explanation for the biochemical complexity observed for respiratory defects. Here, we analyzed the role of cytochrome c and coenzyme Q in the attenuation of complex III and complex IV pharmacological inhibition on the respiratory flux. Spectrophotometry, HPLC-EC, polarography and enzymology permitted the calculation of molar ratios between respiratory chain components, giving values of 0.8:61:3:12:6.8 in muscle and 1:131:3:9:6.5 in liver, for CII:CoQ:CIII:Cyt c:CIV. The results demonstrate the dynamic functional compartmentalization of respiratory chain substrates, with the existence of a substrate pool that can be recruited to maintain energy production at normal levels when respiratory chain complexes are inhibited. The size of this reserve was different between muscle and liver, and in proportion to the magnitude of attenuation of each respiratory defect. Such functional compartmentalization could result from the recently observed physical compartmentalization of respiratory chain substrates. The dynamic nature of the mitochondrial network may modulate this compartmentalization and could play a new role in the control of mitochondrial respiration as well as apoptosis.

Physiological diversity of mitochondrial oxidative phosphorylation.

To investigate the physiological diversity in the regulation and control of mitochondrial oxidative phosphorylation, we determined the composition and functional features of the respiratory chain in muscle, heart, liver, kidney, and brain. First, we observed important variations in mitochondrial content and infrastructure via electron micrographs of the different tissue sections. Analyses of respiratory chain enzyme content by Western blot also showed large differences between tissues, in good correlation with the expression level of mitochondrial transcription factor A and the activity of citrate synthase. On the isolated mitochondria, we observed a conserved molar ratio between the respiratory chain complexes and a variable stoichiometry for coenzyme Q and cytochrome c, with typical values of [1-1.5]:[30-135]:[3]:[9-35]:[6.5-7.5] for complex II:coenzyme Q:complex III:cytochrome c:complex IV in the different tissues. The functional analysis revealed important differences in maximal velocities of respiratory chain complexes, with higher values in heart. However, calculation of the catalytic constants showed that brain contained the more active enzyme complexes. Hence, our study demonstrates that, in tissues, oxidative phosphorylation capacity is highly variable and diverse, as determined by different combinations of 1) the mitochondrial content, 2) the amount of respiratory chain complexes, and 3) their intrinsic activity. In all tissues, there was a large excess of enzyme capacity and intermediate substrate concentration, compared with what is required for state 3 respiration. To conclude, we submitted our data to a principal component analysis that revealed three groups of tissues: muscle and heart, brain, and liver and kidney.

What do mitochondrial diseases teach us about normal mitochondrial functions...that we already knew: threshold expression of mitochondrial defects.

This paper shows how metabolic control analysis (MCA) can help to explain two important features of mitochondrial diseases: (i) the existence of a threshold in the expression of the complex deficiencies on the respiratory flux or on ATP synthesis, i.e. the fact that it is necessary to have a large complex deficiency in order to observe a substantial decrease in these fluxes; (ii) the tissue specificity, i.e. the fact that all tissues are not affected, even if the complex deficiency is present in all of them. We also show the limits of MCA, particularly when considering the in vivo situation. However, MCA offers a new way to consider mitochondrial diseases. The fact that fluxes only slightly change, when a complex is affected, is done at the expense of great changes in intermediate metabolite concentrations; intermediate metabolites situated upstream from the deficient complex are more reduced, leading to a greater generation of free radicals. This could bring an explanation for the diseases observed in conditions where the mitochondrial rate of ATP synthesis is only slightly affected.