In silico MS/MS prediction for peptidoglycan profiling uncovers novel anti-inflammatory peptidoglycan fragments of the gut microbiota.

Peptidoglycan is an essential exoskeletal polymer across all bacteria. Gut microbiota-derived peptidoglycan fragments (PGNs) are increasingly recognized as key effector molecules that impact host biology. However, the current peptidoglycan analysis workflow relies on laborious manual identification from tandem mass spectrometry (MS/MS) data, impeding the discovery of novel bioactive PGNs in the gut microbiota. In this work, we built a computational tool PGN_MS2 that reliably simulates MS/MS spectra of PGNs and integrated it into the user-defined MS library of in silico PGN search space, facilitating automated PGN identification. Empowered by PGN_MS2, we comprehensively profiled gut bacterial peptidoglycan composition. Strikingly, the probiotic Bifidobacterium spp. manifests an abundant amount of the 1,6-anhydro-MurNAc moiety that is distinct from Gram-positive bacteria. In addition to biochemical characterization of three putative lytic transglycosylases (LTs) that are responsible for anhydro-PGN production in Bifidobacterium, we established that these 1,6-anhydro-PGNs exhibit potent anti-inflammatory activity in vitro, offering novel insights into Bifidobacterium-derived PGNs as molecular signals in gut microbiota-host crosstalk.

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments.

The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas.

Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis.

The approval of bedaquiline has placed energy metabolism in the limelight as an attractive target space for tuberculosis antibiotic development. While bedaquiline inhibits the mycobacterial F1 F0 ATP synthase, small molecules targeting other components of the oxidative phosphorylation pathway have been identified. Of particular interest is Telacebec (Q203), a phase 2 drug candidate inhibitor of the cytochrome bcc:aa3 terminal oxidase. A functional redundancy between the cytochrome bcc:aa3 and the cytochrome bd oxidase protects M. tuberculosis from Q203-induced death, highlighting the attractiveness of the bd-type terminal oxidase for drug development. Here, we employed a facile whole-cell screen approach to identify the cytochrome bd inhibitor ND-011992. Although ND-011992 is ineffective on its own, it inhibits respiration and ATP homeostasis in combination with Q203. The drug combination was bactericidal against replicating and antibiotic-tolerant, non-replicating mycobacteria, and increased efficacy relative to that of a single drug in a mouse model. These findings suggest that a cytochrome bd oxidase inhibitor will add value to a drug combination targeting oxidative phosphorylation for tuberculosis treatment.

Features and Functional Importance of Key Residues of the Mycobacterium tuberculosis Cytochrome bd Oxidase.

Cytochrome bd (cyt-bd) oxygen reductases have a high affinity to oxygen and use the two electrons provided by ubiquinol or menaquinol, like in mycobacteria, to reduce oxygen to water. Although they do not pump protons from the cytoplasmic to the periplasmic side, they generate a proton motive force due to the release of protons after quinol oxidation. Here, we show that the mycobacterial cyt-bd has a number of specific features, including a 17-residue stretch (307SGVTLQGIRDLQQEYQQ323) near the Q-loop of the Mycobacterium tuberculosis subunit CydA and a 412QLVRLTVKA420 region on the periplasmic side. Site directed mutagenesis and whole-bacteria assays demonstrated that these mycobacteria-specific stretches are essential for the oxidase's function. Single amino acid substitutions around the 307SGVTLQGIRDLQQEYQQ323 stretch revealed the importance of the aromatic residue Y330 in oxygen consumption and consequently in ATP synthesis. A moderate reduction and no effect was observed for mutants F325 and Y321, respectively, while the double mutant CydAY321/F325 drastically reduced enzyme activity. In addition, single mutants of the mycobacterial cyt-bd were generated to probe the role of proposed critical residues for proton shuffling. Further data demonstrate that amino acids W64 and F18 in the CydB subunit might be important as any slight destabilization of the hydrophobic environment near them makes the enzyme inactive. Finally, the potential of the mycobacterial cyt-bd as a drug target is discussed.

Targeting the cytochrome oxidases for drug development in mycobacteria.

Mycobacterium tuberculosis strictly depends on oxygen to multiply, and the terminal oxidases are a vital part of the oxidative phosphorylation pathway. The bacterium possesses two aerobic respiratory branches: a cytochrome bcc-aa3 and a bacteria-specific cytochrome bd oxidase. The identification of small-molecule inhibitors of the cytochrome bcc-aa3 under numerous experimental conditions reflects the essentiality of the pathway for the optimum growth of M. tuberculosis. Recent findings on the biology of the cytochrome bcc-aa3 as well as the report of the first high-resolution structure of a mycobacterial cytochrome bcc-aa3 complex will help in the characterization and further development of potent inhibitors. Although the aerobic cytochrome bd respiratory branch is not strictly essential for growth, the discovery of a strong synthetic lethal interaction with the cytochrome bcc-aa3 placed the cytochrome bd oxidase under the spotlight as an attractive drug target for its synergistic role in potentiating the efficacy of cytochrome bcc-aa3 inhibitors and other drugs targeting oxidative phosphorylation. In this review, we are discussing current knowledge about the two mycobacterial aerobic respiratory branches, their potential as drug targets, as well as potential drawbacks.

Minor C allele of the SNP rs7873784 associated with rheumatoid arthritis and type-2 diabetes mellitus binds PU.1 and enhances TLR4 expression.

Toll-like receptor 4 (TLR4) is an innate immunity receptor predominantly expressed on myeloid cells and involved in the development of various diseases, many of them with complex genetics. Here we present data on functionality of single nucleotide polymorphism rs7873784 located in the 3'-untranslated region (3'-UTR) of TLR4 gene and associated with various pathologies involving chronic inflammation. We demonstrate that TLR4 3'-UTR strongly enhanced the activity of TLR4 promoter in U937 human monocytic cell line while minor rs7873784(C) allele created a binding site for transcription factor PU.1 (encoded by SPI1 gene), a known regulator of TLR4 expression. Increased binding of PU.1 further augmented the TLR4 transcription while PU.1 knockdown or complete disruption of the PU.1 binding site abrogated the effect. We hypothesize that additional functional PU.1 site may increase TLR4 expression in individuals carrying minor C variant of rs7873784 and modulate the development of certain pathologies, such as rheumatoid arthritis and type-2 diabetes mellitus.

Hypoacylated LPS from Foodborne Pathogen Campylobacter jejuni Induces Moderate TLR4-Mediated Inflammatory Response in Murine Macrophages.

Toll-like receptor 4 (TLR4) initiates immune response against Gram-negative bacteria upon specific recognition of lipid A moiety of lipopolysaccharide (LPS), the major component of their cell wall. Some natural differences between LPS variants in their ability to interact with TLR4 may lead to either insufficient activation that may not prevent bacterial growth, or excessive activation which may lead to septic shock. In this study we evaluated the biological activity of LPS isolated from pathogenic strain of Campylobacter jejuni, the most widespread bacterial cause of foodborne diarrhea in humans. With the help of hydrophobic chromatography and MALDI-TOF mass spectrometry we showed that LPS from a C. jejuni strain O2A consists of both hexaacyl and tetraacyl forms. Since such hypoacylation can result in a reduced immune response in humans, we assessed the activity of LPS from C. jejuni in mouse macrophages by measuring its capacity to activate TLR4-mediated proinflammatory cytokine and chemokine production, as well as NFκB-dependent reporter gene transcription. Our data support the hypothesis that LPS acylation correlates with its bioactivity.

p63 and p73 repress CXCR5 chemokine receptor gene expression in p53-deficient MCF-7 breast cancer cells during genotoxic stress.

Many types of chemotherapeutic agents induce of DNA-damage that is accompanied by activation of p53 tumor suppressor, a key regulator of tumor development and progression. In our previous study we demonstrated that p53 could repress CXCR5 chemokine receptor gene in MCF-7 breast cancer cells via attenuation of NFkB activity. In this work we aimed to determine individual roles of p53 family members in the regulation of CXCR5 gene expression under genotoxic stress. DNA-alkylating agent methyl methanesulfonate caused a reduction in CXCR5 expression not only in parental MCF-7 cells but also in MCF-7-p53off cells with CRISPR/Cas9-mediated inactivation of the p53 gene. Since p53 knockout was associated with elevated expression of its p63 and p73 homologues, we knocked out p63 using CRISPR/Cas9 system and knocked down p73 using specific siRNA. The CXCR5 promoter activity, CXCR5 expression and CXCL13-directed migration in MCF-7 cells with inactivation of all three p53 family genes were completely insensitive to genotoxic stress, while pairwise p53+p63 or p53+p73 inactivation resulted in partial effects. Using deletion analysis and site-directed mutagenesis, we demonstrated that effects of NFkB on the CXCR5 promoter inversely correlated with p63 and p73 levels. Thus, all three p53 family members mediate the effects of genotoxic stress on the CXCR5 promoter using the same mechanism associated with attenuation of NFkB activity. Understanding of this mechanism could facilitate prognosis of tumor responses to chemotherapy.

Structural Relationship of the Lipid A Acyl Groups to Activation of Murine Toll-Like Receptor 4 by Lipopolysaccharides from Pathogenic Strains of Burkholderia mallei, Acinetobacter baumannii, and Pseudomonas aeruginosa.

Toll-like receptor 4 (TLR4) is required for activation of innate immunity upon recognition of lipopolysaccharide (LPS) of Gram-negative bacteria. The ability of TLR4 to respond to a particular LPS species is important since insufficient activation may not prevent bacterial growth while excessive immune reaction may lead to immunopathology associated with sepsis. Here, we investigated the biological activity of LPS from Burkholderia mallei that causes glanders, and from the two well-known opportunistic pathogens Acinetobacter baumannii and Pseudomonas aeruginosa (causative agents of nosocomial infections). For each bacterial strain, R-form LPS preparations were purified by hydrophobic chromatography and the chemical structure of lipid A, an LPS structural component, was elucidated by HR-MALDI-TOF mass spectrometry. The biological activity of LPS samples was evaluated by their ability to induce production of proinflammatory cytokines, such as IL-6 and TNF, by bone marrow-derived macrophages. Our results demonstrate direct correlation between the biological activity of LPS from these pathogenic bacteria and the extent of their lipid A acylation.