Mycoplasma genitalium Lipoproteins Induce Human Monocytic Cell Expression of Proinflammatory Cytokines and Apoptosis by Activating Nuclear Factor κB

This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins (LPs) prepared from Mycoplasma genitalium. Cultured cells were stimulated with M. genitalium LP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide (PI) staining and acridine orange (AO)-ethidium bromide (EB) staining. The DNA-binding activity of nuclear factor-κB (NF-κB) was assessed by electrophoretic mobility shift assay (EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-κB, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-κB. Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-κB

Targeting RNA Polymerase Primary σ70 as a Therapeutic Strategy against Methicillin-Resistant Staphylococcus aureus by Antisense Peptide Nucleic Acid

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) causes threatening infection-related mortality worldwide. Currently, spread of multi-drug resistance (MDR) MRSA limits therapeutic options and requires new approaches to "druggable" target discovery, as well as development of novel MRSA-active antibiotics. RNA polymerase primary σ⁷⁰ (encoded by gene rpoD) is a highly conserved prokaryotic factor essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying potential for antisense inhibition. METHODOLOGY/PRINCIPAL FINDINGS: By synthesizing a serial of cell penetrating peptide conjugated peptide nucleic acids (PPNAs) based on software predicted parameters and further design optimization, we identified a target sequence (234 to 243 nt) within rpoD mRNA conserved region 3.0 being more sensitive to antisense inhibition. A (KFF)₃K peptide conjugated 10-mer complementary PNA (PPNA2332) was developed for potent micromolar-range growth inhibitory effects against four pathogenic S. aureus strains with different resistance phenotypes, including clinical vancomycin-intermediate resistance S. aureus and MDR-MRSA isolates. PPNA2332 showed bacteriocidal antisense effect at 3.2 fold of MIC value against MRSA/VISA Mu50, and its sequence specificity was demonstrated in that PPNA with scrambled PNA sequence (Scr PPNA2332) exhibited no growth inhibitory effect at higher concentrations. Also, PPNA2332 specifically interferes with rpoD mRNA, inhibiting translation of its protein product σ⁷⁰ in a concentration-dependent manner. Full decay of mRNA and suppressed expression of σ⁷⁰ were observed for 40 µM or 12.5 µM PPNA2332 treatment, respectively, but not for 40 µM Scr PPNA2332 treatment in pure culture of MRSA/VISA Mu50 strain. PPNA2332 (≥1 µM) essentially cleared lethal MRSA/VISA Mu50 infection in epithelial cell cultures, and eliminated viable bacterial cells in a time- and concentration- dependent manner, without showing any apparent toxicity at 10 µM. CONCLUSIONS: The present result suggested that RNAP primary σ⁷⁰ is a very promising candidate target for developing novel antisense antibiotic to treat severe MRSA infections

Mycoplasma genitalium lipoproteins inhibit tumour necrosis factor α-induced apoptosis in HeLa cells

This aim of this study was to investigate the effects of Mycoplasma genitalium lipoproteins on the tumour necrosis factor α (TNF-α)-induced apoptosis in HeLa cells. We separated the M. genitalium lipid-associated membrane proteins (LAMPs) from M. genitalium and used them to treat HeLa cells. Apoptosis was induced by TNF-α treatment. Cell cycle and apoptosis were detected by flow cytometry. Pro-inflammatory cytokine contents were determined by enzyme-linked immunosorbent assay (ELISA). Mitochondrial membrane potential and caspase-3 protease activity were also measured. There was no significant difference in the viable cell percentage in HeLa cells treated with M. genitalium LAMPs at 0–20 μg/mL. However, when treated with 40 μg/mL LAMPs, cytocidal activity was observed during the first 24 h. M. genitalium LAMPs induced cell cycle arrest at the G1 phase in HeLa cells. When apoptosis was induced by TNF-α in HeLa cells, M. genitalium LAMPs significantly reduced the percentage of apoptotic cells. In addition, M. genitalium LAMPs significantly reduced the loss of mitochondrial membrane potential and caspase-3 protease activity in TNF-α-treated HeLa cells. However, the blocking of NF-κB significantly increased the mitochondrial membrane potential and caspase-3 protease activity in TNF-α-induced HeLa cells treated with M. genitalium LAMPs. The obtained results suggest that M. genitalium LAMPs could inhibit TNF-α-induced apoptosis in HeLa cells, which would contribute to the understanding of the pathogenesis of M. genitalium

Sneaky tactics: Ingenious immune evasion mechanisms of Bartonella

ABSTRACTGram-negative Bartonella species are facultative intracellular bacteria that can survive in the harsh intracellular milieu of host cells. They have evolved strategies to evade detection and degradation by the host immune system, which ensures their proliferation in the host. Following infection, Bartonella alters the initial immunogenic surface-exposed proteins to evade immune recognition via antigen or phase variation. The diverse lipopolysaccharide structures of certain Bartonella species allow them to escape recognition by the host pattern recognition receptors. Additionally, the survival of mature erythrocytes and their resistance to lysosomal fusion further complicate the immune clearance of this species. Certain Bartonella species also evade immune attacks by producing biofilms and anti-inflammatory cytokines and decreasing endothelial cell apoptosis. Overall, these factors create a challenging landscape for the host immune system to rapidly and effectively eradicate the Bartonella species, thereby facilitating the persistence of Bartonella infections and creating a substantial obstacle for therapeutic interventions. This review focuses on the effects of three human-specific Bartonella species, particularly their mechanisms of host invasion and immune escape, to gain new perspectives in the development of effective diagnostic tools, prophylactic measures, and treatment options for Bartonella infections

Binding parameters predicted by software Oligo walk 5.0 and PNALIGHT.

a<p>Numbering from the first base of the gene <i>rpoD</i>;</p>b<p>ΔG means free energy; index for each parameter: GC%≤60%, overall ΔG<−10 kcal/mol, Duplex ΔG<−25 kcal/mol, oligo-self ΔG≥−1.1 kcal/mol, oligo-oligo ΔG≥−8 kcal/mol, Tm>50°C;</p>c<p>“—” means not determined;</p>d<p>Calculated melting temperature in [°C] of matching PNA/DNA hybrids with no dangling ends.</p

MIC of anti-<i>rpoD</i> peptide-PNAs for quality control and clinical strains of <i>Staphyloccus aureus</i> in M-H broth culture.

a<p><i>rpoD</i>, RNA polymerase sigma 70; PNA, peptide nucleic acid; PPNA means peptide conjugated PNA; The PNAs are written from their N to their C terminus, and the N terminus corresponds to the 5′ end of a conventional oligonucleotide; “K” indicates lysine, F indicates phenylalanine, “X” indicates 6-aminohexanoic acid, “B” indicates β-alanine, and eg¹ indicates glycine; Scr means PNA with a scrambled base sequence (as control);</p>b<p>Minimal inhibitory concentrations (MIC) were the lowest PNA concentrations that prevented bacterial growth by visual inspection after overnight (24 h) growth from an inoculum of 10⁵ CFU/mL. “VISA” is abbreviation for vancomycin-intermediate resistance <i>Staphyloccus aureus</i>; “MRSA” is abbreviation for methicillin-resistant <i>Staphyloccus aureus</i>; XIJING means clinical MRSA isolate from patients in Fourth Military Medical University affiliated XIJING hospital.</p

Light micrographs of epithelial cell cultures.

<p>The images show epithelial cell cultures grown in DMEM/HIGH Glucose, 10% FCS. The column at left shows epithelial cell cultures without added MRSA/VISA Mu50; the right column shows epithelial cell cultures that were inoculated with MRSA/VISA Mu50. The top panels of each column show cultures not treated with PPNA, and the rows below show cultures treated with increasing amounts of anti-<i>rpoD</i> PPNA2332 (1, 5, and 10 µM). Magnification, ×100.</p

Effects of anti-<i>rpoD</i> PPNA2332 on the growth of MRSA/VISA Mu50 in pure culture.

<p>Anti-<i>rpoD</i> PPNA2332 was added to cell cultures containing 1.0×10⁵ CFU/mL MRSA/VISA Mu50 to a final concentration of 6.25, 12.5, or 25 M. Additional cell cultures were treated with free MH broth, scrambled PPNA2332 (final concentration of 40 µM), and peptide (KFF)₃K (final concentration of 40 µM) in a volume equal to that of the PPNA2332 preparation as controls. The growth of different groups of MRSA/VISA Mu50 cells was monitored by using OD measurements. The data are shown as means for 2 samples from 2 independent tests.</p

Homologies of gene <i>rpoD</i> among <i>Staphylococcus</i> species.

<p>Homologies of gene <i>rpoD</i> among <i>Staphylococcus</i> species.</p