Combinatorial fluorescent labeling of live anaerobic bacteria via the incorporation of azide-modified sugars into newly synthesized macromolecules.

The human gut microbiome modulates physiological functions and pathologies; however, a mechanistic understanding of microbe-host and microbe-microbe interactions remains elusive owing to a lack of suitable approaches to monitor obligate anaerobic bacterial populations. Common genetically encoded fluorescent protein reporters, derived from the green fluorescent protein, require an oxidation step for fluorescent light emission and therefore are not suitable for use in anaerobic microbes residing in the intestine. Fluorescence in situ hybridization is a useful alternative to visualize bacterial communities in their natural niche; however, it requires tissue fixation. We therefore developed an approach for the real-time detection and monitoring of live communities of anaerobic gut commensals in their natural environment. We leverage the bacterial cells' reliance on sugars for macromolecule synthesis in combinatorial click chemistry labeling, where the addition of azide-modified sugars to the culturing media enables the fluorescence labeling of newly synthesized molecules via the addition of combinations of exogenous fluorophores conjugated to cyclooctynes. This process is suitable for labeling communities of live anaerobic gut bacteria with combinations of fluorophores that do not require oxygen to mature and fluoresce, and that can be detected over time in their natural environments. The labeling procedure requires 4-9 d, depending on the varying growth rates of different bacterial strains, and an additional 1-2 d for the detection and monitoring steps. The protocol can be completed by users with basic expertise in bacterial culturing.

Combinatorial Click Chemistry Labeling to Study Live Human Gut-Derived Microbiota Communities.

Gut bacteria were shown to exert pivotal effects on health and disease. However, mechanistic studies of gut bacterial communities are limited due to the lack of technologies for real-time studies on live bacteria. Here, we developed COMBInatorial cliCK-chemistry (COMBICK) labeling on human gut-derived bacteria, both aerobic and anaerobic strains, to enable dynamic tracing of live, heterogeneous bacterial communities on the strain level, including clinical isolates of the Enterobacteriaceae family. We further show that COMBICK labeling is applicable on anaerobic bacterial strains directly isolated from stool. In COMBICK, the number of labeled bacteria that can be simultaneously differentiated increases exponentially depending on the availability of fluorophores and machine capabilities. This method allows real-time studies of bacterial communities from a variety of ecosystems, and can significantly advance mechanistic research in the microbiome field.

Identification of bacteria-derived HLA-bound peptides in melanoma.

A variety of species of bacteria are known to colonize human tumours1-11, proliferate within them and modulate immune function, which ultimately affects the survival of patients with cancer and their responses to treatment12-14. However, it is not known whether antigens derived from intracellular bacteria are presented by the human leukocyte antigen class I and II (HLA-I and HLA-II, respectively) molecules of tumour cells, or whether such antigens elicit a tumour-infiltrating T cell immune response. Here we used 16S rRNA gene sequencing and HLA peptidomics to identify a peptide repertoire derived from intracellular bacteria that was presented on HLA-I and HLA-II molecules in melanoma tumours. Our analysis of 17 melanoma metastases (derived from 9 patients) revealed 248 and 35 unique HLA-I and HLA-II peptides, respectively, that were derived from 41 species of bacteria. We identified recurrent bacterial peptides in tumours from different patients, as well as in different tumours from the same patient. Our study reveals that peptides derived from intracellular bacteria can be presented by tumour cells and elicit immune reactivity, and thus provides insight into a mechanism by which bacteria influence activation of the immune system and responses to therapy.

Discovery of a highly potent novel rifampicin analog by preparing a hybrid of the precursors of the antibiotic drugs rifampicin and clofazimine.

Tuberculosis (TB) is an infectious disease caused by the bacillus Mycobacterium tuberculosis (Mtb). The present work reports the design and synthesis of a hybrid of the precursors of rifampicin and clofazimine, which led to the discovery of a novel Rifaphenazine (RPZ) molecule with potent anti-TB activity. In addition, the efficacy of RPZ was evaluated in-vitro using the reference strain Mtb H37Rv. Herein, 2,3 diamino phenazine, a precursor of an anti-TB drug clofazimine, was tethered to the rifampicin core. This 2,3 diamino phenazine did not have an inherent anti-TB activity even at a concentration of up to 2 µg/mL, while rifampicin did not exhibit any activity against Mtb at a concentration of 0.1 µg/mL. However, the synthesized novel Rifaphenzine (RPZ) inhibited 78% of the Mtb colonies at a drug concentration of 0.1 µg/mL, while 93% of the bacterial colonies were killed at 0.5 µg/mL of the drug. Furthermore, the Minimum Inhibitory Concentration (MIC) value for RPZ was 1 µg/mL. Time-kill studies revealed that all bacterial colonies were killed within a period of 24 h. The synthesized novel molecule was characterized using high-resolution mass spectroscopy and NMR spectroscopy. Cytotoxicity studies (IC50) were performed on human monocytic cell line THP-1, and the determined IC50 value was 96 µg/mL, which is non-cytotoxic.

Acetylation of Response Regulator Protein MtrA in M. tuberculosis Regulates Its Repressor Activity.

MtrA is an essential response regulator (RR) protein in M. tuberculosis, and its activity is modulated after phosphorylation from its sensor kinase MtrB. Interestingly, many regulatory effects of MtrA have been reported to be independent of its phosphorylation, thereby suggesting alternate mechanisms of regulation of the MtrAB two-component system in M. tuberculosis. Here, we show that RR MtrA undergoes non-enzymatic acetylation through acetyl phosphate, modulating its activities independent of its phosphorylation status. Acetylated MtrA shows increased phosphorylation and enhanced interaction with SK MtrB assessed by phosphotransfer assays and FRET analysis. We also observed that acetylated MtrA loses its DNA-binding ability on gene targets that are otherwise enhanced by phosphorylation. More interestingly, acetylation is the dominant post-translational modification, overriding the effect of phosphorylation. Evaluation of the impact of MtrA and its lysine mutant overexpression on the growth of H37Ra bacteria under different conditions along with the infection studies on alveolar epithelial cells further strengthens the importance of acetylated MtrA protein in regulating the growth of M. tuberculosis. Overall, we show that both acetylation and phosphorylation regulate the activities of RR MtrA on different target genomic regions. We propose here that, although phosphorylation-dependent binding of MtrA drives its repressor activity on oriC and rpf, acetylation of MtrA turns this off and facilitates division in mycobacteria. Our findings, thus, reveal a more complex regulatory role of RR proteins in which multiple post-translational modifications regulate the activities at the levels of interaction with SK and the target gene expression.

Acetylation of Response Regulator Proteins, TcrX and MtrA in M. tuberculosis Tunes their Phosphotransfer Ability and Modulates Two-Component Signaling Crosstalk.

Two-component signal transduction (TCS) cascades involve stimulus-dependent activation and phosphorylation of a sensor kinase (SK), which then transfers the phosphoryl moiety to the response regulator (RR) protein. The fidelity of this phosphotransfer reaction from the SK to the RR provides specificity to TCS signaling. In the present study, we show that for TcrX, a transcriptionally autoregulated RR of Mycobacterium tuberculosis, acetylation enhances its net phosphorylation from cognate SK TcrY and lowers it from a non-cognate SK MtrB. Similar acetylation mediated increase in phosphorylation was also observed for another RR MtrA from cognate SK MtrB. Thus, we establish a novel TCS signaling design wherein acetylation of RRs results in enhanced cognate phosphorylation and suppresses non-cognate phosphorylation. Using wild-type or acetylation-deficient TcrX proteins in M. tuberculosis H37Ra, we demonstrate that non-acetylated TcrX acts as a "phosphate sink" for MtrB and suppressing signal propagation from MtrB to MtrA in vivo, linking metabolism to TCS signaling. Overall, we report that acetylation of RRs shields TCSs from crosstalk, modulates the phosphatase activities and alters the DNA-binding activities of RRs, all of which are non-intuitive behavior of TCS systems.

The role of ω-subunit of Escherichia coli RNA polymerase in stress response.

ppGpp, an alarmone for stringent response, plays an important role in the reprogramming of the transcription complex at the time of stress. In Escherichia coli, ppGpp mediates its action by binding to at least two different sites on RNA polymerase (RNAP). One of the sites to which ppGpp binds to RNAP is at the ß'-ω interface; however, the underlying molecular mechanism and the physiological relevance of ppGpp binding to this site remain unclear. In this study, we have performed UV cross-linking experiments using 32 P azido-labeled ppGpp to probe its association with RNAP in the absence and presence of ω, and observed weaker binding of ppGpp to the RNAP without ω. Furthermore, we followed the binding kinetics of ppGpp to RNAP with and without ω by isothermal titration calorimetry and found it to be concurrent with the cross-linking results. Native ω is intrinsically disordered, and we have used a previously characterized structured mutant of ω, which affects the plasticity of the active site of RNAP. Results show that the flexibility conferred by the unstructured ω is a prerequisite for ppGpp binding to RNAP. We have analyzed the stress-associated phenotypes in an E. coli strain devoid of ω (∆rpoZ). ppGpp levels in ∆rpoZ strain were found to be similar to that of the wild-type strain. Interestingly, when the ∆rpoZ strain of E. coli was transferred after nutritional stress to an enriched media, the recovery of growth was compromised. We have identified a new phenotype of ∆rpoZ strain corresponding to defect in biofilm formation in minimal media.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome.

The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α2ßß'ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, ß', but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ-defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ-deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (ß', of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription was found to be altered as observed by qRT-PCR assay. All the observations here described indicate the involvement of RpoZ in recognition of some of the prophage genes. This study advances understanding of not only the regulatory role of omega subunit in the functions of RNAP but also the regulatory interplay between prophages and the host E. coli for adjustment of cellular physiology to a variety of environments in nature.

Synthetic (p)ppGpp Analogue Is an Inhibitor of Stringent Response in Mycobacteria.

Bacteria elicit an adaptive response against hostile conditions such as starvation and other kinds of stresses. Their ability to survive such conditions depends, in part, on stringent response pathways. (p)ppGpp, considered to be the master regulator of the stringent response, is a novel target for inhibiting the survival of bacteria. In mycobacteria, the (p)ppGpp synthetase activity of bifunctional Rel is critical for stress response and persistence inside a host. Our aim was to design an inhibitor of (p)ppGpp synthesis, monitor its efficiency using enzyme kinetics, and assess its phenotypic effects in mycobacteria. As such, new sets of inhibitors targeting (p)ppGpp synthesis were synthesized and characterized by mass spectrometry and nuclear magnetic resonance spectroscopy. We observed significant inhibition of (p)ppGpp synthesis by RelMsm in the presence of designed inhibitors in a dose-dependent manner, which we further confirmed by monitoring the enzyme kinetics. The Rel enzyme inhibitor binding kinetics were investigated by isothermal titration calorimetry. Subsequently, the effects of the compounds on long-term persistence, biofilm formation, and biofilm disruption were assayed in Mycobacterium smegmatis, where inhibition in each case was observed. In vivo, (p)ppGpp levels were found to be downregulated in M. smegmatis treated with the synthetic inhibitors. The compounds reported here also inhibited biofilm formation by the pathogen Mycobacterium tuberculosis The compounds were tested for toxicity by using an MTT assay with H460 cells and a hemolysis assay with human red blood cells, for which they were found to be nontoxic. The permeability of compounds across the cell membrane of human lung epithelial cells was also confirmed by mass spectrometry.

Influence of Flexible "ω" on the Activity of E. coli RNA Polymerase: A Thermodynamic Analysis.

The Escherichia coli RNA polymerase (RNAP) is a multisubunit protein complex containing the smallest subunit, ω. Despite the evolutionary conservation of ω and its role in assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of RNAP with the global chaperone protein GroEL. With an aim to get better insight into the structure and functional role of ω, we isolated a dominant negative mutant of ω (ω6), which is predominantly α-helical, in contrast to largely unstructured native ω, and then studied its assembly with reconstituted core1 (α2ßß') by a biophysical approach. The mutant showed higher binding affinity compared to native ω. We observed that the interaction between core1 and ω6 is driven by highly negative enthalpy and a small but unfavorable negative entropy term. Extensive structural alteration in ω6 makes it more rigid, the plasticity of the interacting domain formed by ω6 and core1 is compromised, which may be responsible for the entropic cost. Such tight binding of the structured mutant (ω6) affects initiation of transcription. However, once preinitiated, the complex elongates the RNA chain efficiently. The initiation of transcription requires recognition of appropriate σ-factors by the core enzyme (core2: α2ßß'ω). We found that the altered core enzyme (α2ßß'ω6) with mutant ω showed a decrease in binding affinity to the σ-factors (σ70, σ32 and σ38) compared to that of the core enzyme containing native ω. In the absence of unstructured ω, the association of σ-factors to the core is less efficient, suggesting that the flexible native ω plays a direct role in σ-factor recruitment.

Vitamin C targets (p)ppGpp synthesis leading to stalling of long-term survival and biofilm formation in Mycobacterium smegmatis.

Earlier, vitamin C was demonstrated to sterilize Mycobacterium tuberculosis culture via Fenton's reaction at high concentration. It alters the regulatory pathways associated with stress response and dormancy. Since (p)ppGpp is considered to be the master regulator of stress response and is responsible for bacterial survival under stress, we tested the effect of vitamin C on the formation of (p)ppGpp. In vivo estimation of (p)ppGpp showed a decrease in (p)ppGpp levels in vitamin C-treated M. smegmatis cells in comparison to the untreated cells. Furthermore, in vitro (p)ppGpp synthesis using RelMSM enzyme was conducted in order to confirm the specificity of the inhibition in the presence of variable concentrations of vitamin C. We observed that vitamin C at high concentration can inhibit the synthesis of (p)ppGpp. We illustrated binding of vitamin C to RelMSM by isothermal titration calorimetry. Enzyme kinetics was followed where K0.5 was found to be increased with the concomitant reduction of Vmax value suggesting mixed inhibition. Both long-term survival and biofilm formation were inhibited by vitamin C. The experiments suggest that vitamin C has the potential to be developed as the inhibitor of (p)ppGpp synthesis and stress response, at least in the concentration range used here.