A Novel Mechanism of Inactivating Antibacterial Nitro Compounds in the Human Pathogen Staphylococcus aureus by Overexpression of a NADH-Dependent Flavin Nitroreductase.

Recently, the nitro-substituted bisquaternary bisnaphthalimides were reported to have substantial anti-infective activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). Here, we selected resistant S. aureus clones by cultivation in increasing concentrations of the most active compound, MT02. Interestingly, MT02-resistant variants induced a diffusible red color of the broth. Chromatographic and spectroscopic investigations revealed a stepwise reduction of the bisquaternary bisnaphthalimides' nitro groups to amino groups. The corresponding derivatives were completely inactive against staphylococci. RNA sequencing experiments revealed a strong overexpression of a novel oxidoreductase in MT02-resistant strains. Deletion mutants of this enzyme did not produce the red color and were not able to develop resistance against bisquaternary bisnaphthalimides. Biochemical reactions confirmed an NADH-dependent deactivation of the nitro-substituted compounds. Thus, this is the first report of a nitroreductase-based antibiotic resistance mechanism in the human pathogen S. aureus.

Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy.

Staphylococcus aureus is the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase of S. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized all S. aureus strains tested, including hospital-acquired and community-acquired methicillin-resistant S. aureus strains. Therapeutic efficacy in vivo in mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection. Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases of S. aureus infections.

Transcriptome and functional analysis of the eukaryotic-type serine/threonine kinase PknB in Staphylococcus aureus.

The function of the Staphylococcus aureus eukaryotic-like serine/threonine protein kinase PknB was investigated by performing transcriptome analysis using DNA microarray technology and biochemical assays. The transcriptional profile revealed a strong regulatory impact of PknB on the expression of genes encoding proteins which are involved in purine and pyrimidine biosynthesis, cell wall metabolism, autolysis, and glutamine synthesis. Functional activity of overexpressed and purified PknB kinase was demonstrated using the myelin basic protein as a surrogate substrate. Phosphorylation occurred in a time-dependent manner with Mn(2+) as a preferred cofactor. Furthermore, biochemical characterization revealed regulation of adenylosuccinate synthase (PurA) activity by phosphorylation. Phosphorylated PurA showed a 1.8-fold decrease in enzymatic activity compared to unphosphorylated PurA. Loss of PknB led to formation of larger cell clusters, and a pknB deletion strain showed 32-fold-higher sensitivity to the cell wall-active antibiotic tunicamycin. The results of this study strongly indicate that PknB has a role in regulation of purine biosynthesis, autolysis, and central metabolic processes in S. aureus.

In vitro and in vivo validation of ligA and tarI as essential targets in Staphylococcus aureus.

A conditional expression system has been developed using the isopropyl-beta-d-thiogalactopyranoside (IPTG)-inducible Pspac promoter to validate essential genes of Staphylococcus aureus in vivo. The system has been applied to prove the essentiality of ligA and to evaluate the function of tarI, which was found to be essential in vitro but not in vivo.

In vivo and in vitro intragenomic rearrangement of TT viruses.

The in vitro replication of the Torque teno virus (TT virus) tth8 full-length genome and particle formation in a Hodgkin's lymphoma-derived cell line after transfection with cloned viral DNA were demonstrated. Analyses of the transcription patterns of tth8 and tth7 TT virus isolates in a number of lymphoma and T-cell leukemia cell lines indicated differential additional splicing events and intragenomic rearrangement generating open reading frames which could not be deducted from the genomic sequence. We also demonstrated the presence of rearranged TT virus genomes in vivo in sera taken from pregnant mothers whose children later developed childhood leukemia, as well as sera from control mothers. Control experiments using religated cloned genomic tth8 DNA mixed with cellular DNA did not result in such subviral molecules. These subviral isolates ranged from 172 bp to full-length TT virus genomes. Possible in vivo selection for specific rearranged molecules was indicated by the presence of one isolate (561 bp) in 11 serum samples. It remains to be clarified whether selected rearranged subviral components resulting from specific TT virus types may contribute to the initiation of disease. These data demonstrate new features of TT viruses suggesting possible similarities to plant viruses of the family Geminiviridae, as well as raise questions about the documented plurality and diversity of anelloviruses.

Staphylococcus aureus NfrA (SA0367) is a flavin mononucleotide-dependent NADPH oxidase involved in oxidative stress response.

The NfrA protein, a putative essential oxidoreductase in the soil bacterium Bacillus subtilis, is induced under heat shock and oxidative stress conditions. In order to characterize the function of an homologous NfrA protein in Staphylococcus aureus, an nfrA deletion strain was constructed, the protein was purified, the enzymatic activity was determined, and the transcriptional regulation was investigated. The experiments revealed that NfrA is not essential in S. aureus. The purified protein oxidized NADPH but not NADH, producing NADP in the presence of flavin mononucleotide, suggesting that NfrA is an NADPH oxidase in S. aureus. In addition, the NfrA enzyme showed nitroreductase activity and weak disulfide reductase activity. Transcription was strongly induced by ethanol, diamide, and nitrofurantoin. Hydrogen peroxide induced nfrA transcription only at high concentrations. The expression of nfrA was independent of the alternative sigma factor sigma(B). Furthermore, the transcriptional start site was determined, which allowed identification of a PerR box homologous sequence upstream of the nfrA promoter. The observations presented here suggest that NfrA is a nonessential NADPH oxidoreductase which may play a role in the oxidative stress response of S. aureus, especially in keeping thiol-disulfide stress in balance.