An Innovative Protocol for Metaproteomic Analyses of Microbial Pathogens in Cystic Fibrosis Sputum

Hallmarks of cystic fibrosis (CF) are increased viscosity of mucus and impaired mucociliary clearance within the airways due to mutations of the cystic fibrosis conductance regulator gene. This facilitates the colonization of the lung by microbial pathogens and the concomitant establishment of chronic infections leading to tissue damage, reduced lung function, and decreased life expectancy. Although the interplay between key CF pathogens plays a major role during disease progression, the pathophysiology of the microbial community in CF lungs remains poorly understood. Particular challenges in the analysis of the microbial population present in CF sputum is (I) the inhomogeneous, viscous, and slimy consistence of CF sputum, and (II) the high number of human proteins masking comparably low abundant microbial proteins. To address these challenges, we used 21 CF sputum samples to develop a reliable, reproducible and widely applicable protocol for sputum processing, microbial enrichment, cell disruption, protein extraction and subsequent metaproteomic analyses. As a proof of concept, we selected three sputum samples for detailed metaproteome analyses and complemented and validated metaproteome data by 16S sequencing, metabolomic as well as microscopic analyses. Applying our protocol, the number of bacterial proteins/protein groups increased from 199-425 to 392-868 in enriched samples compared to nonenriched controls. These early microbial metaproteome data suggest that the arginine deiminase pathway and multiple proteases and peptidases identified from various bacterial genera could so far be underappreciated in their contribution to the CF pathophysiology. By providing a standardized and effective protocol for sputum processing and microbial enrichment, our study represents an important basis for future studies investigating the physiology of microbial pathogens in CF in vivo – an important prerequisite for the development of novel antimicrobial therapies to combat chronic recurrent airway infection in CF

Real-time imaging of the bacillithiol redox potential in the human pathogen Staphylococcus aureus using a genetically encoded bacilliredoxin-fused redox biosensor

Aims: Bacillithiol (BSH) is utilized as major thiol-redox buffer in the human pathogen Staphylococcus aureus. Under oxidative stress, BSH forms mixed disulfides with proteins, termed as S-bacillithiolation which can be reversed by bacilliredoxins (Brx). In eukaryotes, glutaredoxin-fused roGFP2 biosensors have been applied for dynamic live-imaging of the glutathione redox potential. Here, we have constructed a genetically encoded bacilliredoxin-fused redox biosensor (Brx-roGFP2) to monitor dynamic changes in the BSH redox potential in S. aureus. Results: The Brx-roGFP2 biosensor showed a specific and rapid response to low levels bacillithiol disulphide (BSSB) in vitro which required the active-site Cys of Brx. Dynamic live-imaging in two methicillin-resistant S. aureus (MRSA) USA300 and COL strains revealed fast and dynamic responses of the Brx-roGFP2 biosensor under hypochlorite and H2O2 stress and constitutive oxidation of the probe in different BSH-deficient mutants. Furthermore, we found that the Brx-roGFP2 expression level and the dynamic range is higher in S. aureus COL compared to the USA300 strain. In phagocytosis assays with THP-1 macrophages, the biosensor was 87 % oxidized in S. aureus COL. However, no changes in the BSH redox potential were measured after treatment with different antibiotics classes indicating that antibiotics do not cause oxidative stress in S. aureus. Conclusion and Innovation: This Brx-roGFP2 biosensor catalyzes specific equilibration between the BSH and roGFP2 redox couples and can be applied for dynamic live imaging of redox changes in S. aureus and other BSH-producing Firmicutes

Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hinzke, T., Kleiner, M., Meister, M., Schlueter, R., Hentschker, C., Pane-Farre, J., Hildebrandt, P., Felbeck, H., Sievert, S. M., Bonn, F., Voelker, U., Becher, D., Schweder, T., & Markert, S. Bacterial symbiont subpopulations have different roles in a deep-sea symbiosis. Elife, 10, (2021): e58371, https://doi.org/10.7554/eLife.58371.The hydrothermal vent tubeworm Riftia pachyptila hosts a single 16S rRNA phylotype of intracellular sulfur-oxidizing symbionts, which vary considerably in cell morphology and exhibit a remarkable degree of physiological diversity and redundancy, even in the same host. To elucidate whether multiple metabolic routes are employed in the same cells or rather in distinct symbiont subpopulations, we enriched symbionts according to cell size by density gradient centrifugation. Metaproteomic analysis, microscopy, and flow cytometry strongly suggest that Riftia symbiont cells of different sizes represent metabolically dissimilar stages of a physiological differentiation process: While small symbionts actively divide and may establish cellular symbiont-host interaction, large symbionts apparently do not divide, but still replicate DNA, leading to DNA endoreduplication. Moreover, in large symbionts, carbon fixation and biomass production seem to be metabolic priorities. We propose that this division of labor between smaller and larger symbionts benefits the productivity of the symbiosis as a whole.This work was supported by the German Research Foundation DFG (grant MA 6346/2–1 to SM), fellowships of the Institute of Marine Biotechnology Greifswald (TH, MM), a German Academic Exchange Service (DAAD) grant (TH), the NC State Chancellor’s Faculty Excellence Program Cluster on Microbiomes and Complex Microbial Communities (MK), the USDA National Institute of Food and Agriculture, Hatch project 1014212 (MK), the U.S. National Science Foundation (grants OCE-1131095 and OCE-1559198 to SMS), and The WHOI Investment in Science Fund (to SMS). We furthermore acknowledge support for article processing charges from the DFG (Grant 393148499) and the Open Access Publication Fund of the University of Greifswald

A Proteomic View of an Important Human Pathogen – Towards the Quantification of the Entire Staphylococcus aureus Proteome

The genome sequence is the “blue-print of life,” but proteomics provides the link to the actual physiology of living cells. Because of their low complexity bacteria are excellent model systems to identify the entire protein assembly of a living organism. Here we show that the majority of proteins expressed in growing and non-growing cells of the human pathogen Staphylococcus aureus can be identified and even quantified by a metabolic labeling proteomic approach. S. aureus has been selected as model for this proteomic study, because it poses a major risk to our health care system by combining high pathogenicity with an increasing frequency of multiple antibiotic resistance, thus requiring the development of new anti-staphylococcal therapy strategies. Since such strategies will likely have to target extracellular and surface-exposed virulence factors as well as staphylococcal survival and adaptation capabilities, we decided to combine four subproteomic fractions: cytosolic proteins, membrane-bound proteins, cell surface-associated and extracellular proteins, to comprehensively cover the entire proteome of S. aureus. This quantitative proteomics approach integrating data ranging from gene expression to subcellular localization in growing and non-growing cells is a proof of principle for whole-cell physiological proteomics that can now be extended to address physiological questions in infection-relevant settings. Importantly, with more than 1700 identified proteins (and 1450 quantified proteins) corresponding to a coverage of about three-quarters of the expressed proteins, our model study represents the most comprehensive quantification of a bacterial proteome reported to date. It thus paves the way towards a new level in understanding of cell physiology and pathophysiology of S. aureus and related pathogenic bacteria, opening new avenues for infection-related research on this crucial pathogen

<i>Staphylococcus aureus </i>Transcriptome Architecture:From Laboratory to Infection-Mimicking Conditions

Staphylococcus aureus is a major pathogen that colonizes about 20% of the human population. Intriguingly, this Gram-positive bacterium can survive and thrive under a wide range of different conditions, both inside and outside the human body. Here, we investigated the transcriptional adaptation of S. aureus HG001, a derivative of strain NCTC 8325, across experimental conditions ranging from optimal growth in vitro to intracellular growth in host cells. These data establish an extensive repertoire of transcription units and non-coding RNAs, a classification of 1412 promoters according to their dependence on the RNA polymerase sigma factors SigA or SigB, and allow identification of new potential targets for several known transcription factors. In particular, this study revealed a relatively low abundance of antisense RNAs in S. aureus, where they overlap only 6% of the coding genes, and only 19 antisense RNAs not co-transcribed with other genes were found. Promoter analysis and comparison with Bacillus subtilis links the small number of antisense RNAs to a less profound impact of alternative sigma factors in S. aureus. Furthermore, we revealed that Rho-dependent transcription termination suppresses pervasive antisense transcription, presumably originating from abundant spurious transcription initiation in this A+T-rich genome, which would otherwise affect expression of the overlapped genes. In summary, our study provides genome-wide information on transcriptional regulation and non-coding RNAs in S. aureus as well as new insights into the biological function of Rho and the implications of spurious transcription in bacteria

Physical and antibiotic stresses require activation of the RsbU phosphatase to induce the general stress response in Listeria monocytogenes

Among pathogenic strains of Listeria monocytogenes, the σB transcription factor has a pivotal role in the outcome of food-borne infections. This factor is activated by diverse stresses to provide general protection against multiple challenges, including those encountered during gastrointestinal passage. It also acts with the PrfA regulator to control virulence genes needed for entry into intestinal lumen cells. Environmental and nutritional signals modulate σB activity via a network that operates by the partner switching mechanism, in which protein interactions are controlled by serine phosphorylation. This network is well characterized in the related bacterium Bacillus subtilis. A key difference in Listeria is the presence of only one input phosphatase, RsbU, instead of the two found in B. subtilis. Here, we aim to determine whether this sole phosphatase is required to convey physical, antibiotic and nutritional stress signals, or if additional pathways might exist. To that end, we constructed L. monocytogenes 10403S strains bearing single-copy, σB-dependent opuCA–lacZ reporter fusions to determine the effects of an rsbU deletion under physiological conditions. All stresses tested, including acid, antibiotic, cold, ethanol, heat, osmotic and nutritional challenge, required RsbU to activate σB. This was of particular significance for cold stress activation, which occurs via a phosphatase-independent mechanism in B. subtilis. We also assayed the effects of the D80N substitution in the upstream RsbT regulator that activates RsbU. The mutant had a phenotype consistent with low and uninducible phosphatase activity, but nonetheless responded to nutritional stress. We infer that RsbU activity but not its induction is required for nutritional signalling, which would enter the network downstream from RsbU

A search for small noncoding RNAs in Staphylococcus aureus reveals a conserved sequence motif for regulation

Bioinformatic analysis of the intergenic regions of Staphylococcus aureus predicted multiple regulatory regions. From this analysis, we characterized 11 novel noncoding RNAs (RsaA‐K) that are expressed in several S. aureus strains under different experimental conditions. Many of them accumulate in the late-exponential phase of growth. All ncRNAs are stable and their expression is Hfq-independent. The transcription of several of them is regulated by the alternative sigma B factor (RsaA, D and F) while the expression of RsaE is agrA-dependent. Six of these ncRNAs are specific to S. aureus, four are conserved in other Staphylococci, and RsaE is also present in Bacillaceae. Transcriptomic and proteomic analysis indicated that RsaE regulates the synthesis of proteins involved in various metabolic pathways. Phylogenetic analysis combined with RNA structure probing, searches for RsaE‐mRNA base pairing, and toeprinting assays indicate that a conserved and unpaired UCCC sequence motif of RsaE binds to target mRNAs and prevents the formation of the ribosomal initiation complex. This study unexpectedly shows that most of the novel ncRNAs carry the conserved C−rich motif, suggesting that they are members of a class of ncRNAs that target mRNAs by a shared mechanism

The bacterial stressosome:a modular system that has been adapted to control secondary messenger signaling

SummaryThe stressosome complex regulates downstream effectors in response to environmental signals. In Bacillus subtilis, it activates the alternative sigma factor σB, leading to the upregulation of the general stress regulon. Herein, we characterize a stressosome-regulated biochemical pathway in Moorella thermoacetica. We show that the presumed sensor, MtR, and the scaffold, MtS, form a pseudo-icosahedral structure like that observed in B. subtilis. The N-terminal domain of MtR is structurally homologous to B. subtilis RsbR, despite low sequence identity. The affinity of the switch kinase, MtT, for MtS decreases following MtS phosphorylation and not because of structural reorganization. Dephosphorylation of MtS by the PP2C type phosphatase MtX permits the switch kinase to rebind the stressosome to reset the response. We also show that MtT regulates cyclic di-GMP biosynthesis through inhibition of a GG(D/E)EF-type diguanylate cyclase, demonstrating that secondary messenger levels are regulated by the stressosome

Vagal control of the heart decreases during increasing imminence of interoceptive threat in patients with panic disorder and agoraphobia

Theoretically, panic disorder and agoraphobia pathology can be conceptualized as a cascade of dynamically changing defensive responses to threat cues from inside the body. Guided by this trans‑diagnostic model we tested the interaction between defensive activation and vagal control as a marker of prefrontal inhibition of subcortical defensive activation. We investigated ultra‑short‑term changes of vagally controlled high frequency heart rate variability (HRV) during a standardized threat challenge (entrapment) in n = 232 patients with panic disorder and agoraphobia, and its interaction with various indices of defensive activation. We found a strong inverse relationship between HRV and heart rate during threat, which was stronger at the beginning of exposure. Patients with a strong increase in heart rate showed a deactivation of prefrontal vagal control while patients showing less heart rate acceleration showed an increase in vagal control. Moreover, vagal control collapsed in case of imminent threat, i.e., when body symptoms increase and seem to get out of control. In these cases of defensive action patients either fled from the situation or experienced a panic attack. Active avoidance, panic attacks, and increased sympathetic arousal are associated with an inability to maintain vagal control over the heart suggesting that teaching such regulation strategies during exposure treatment might be helpful to keep prefrontal control, particularly during the transition zone from post‑encounter to circa strike defense

Short- and Long-Term Biomarkers for Bacterial Robustness: A Framework for Quantifying Correlations between Cellular Indicators and Adaptive Behavior

The ability of microorganisms to adapt to changing environments challenges the prediction of their history-dependent behavior. Cellular biomarkers that are quantitatively correlated to stress adaptive behavior will facilitate our ability to predict the impact of these adaptive traits. Here, we present a framework for identifying cellular biomarkers for mild stress induced enhanced microbial robustness towards lethal stresses. Several candidate-biomarkers were selected by comparing the genome-wide transcriptome profiles of our model-organism Bacillus cereus upon exposure to four mild stress conditions (mild heat, acid, salt and oxidative stress). These candidate-biomarkers—a transcriptional regulator (activating general stress responses), enzymes (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)—were quantitatively determined at transcript, protein and/or activity level upon exposure to mild heat, acid, salt and oxidative stress for various time intervals. Both unstressed and mild stress treated cells were also exposed to lethal stress conditions (severe heat, acid and oxidative stress) to quantify the robustness advantage provided by mild stress pretreatment. To evaluate whether the candidate-biomarkers could predict the robustness enhancement towards lethal stress elicited by mild stress pretreatment, the biomarker responses upon mild stress treatment were correlated to mild stress induced robustness towards lethal stress. Both short- and long-term biomarkers could be identified of which their induction levels were correlated to mild stress induced enhanced robustness towards lethal heat, acid and/or oxidative stress, respectively, and are therefore predictive cellular indicators for mild stress induced enhanced robustness. The identified biomarkers are among the most consistently induced cellular components in stress responses and ubiquitous in biology, supporting extrapolation to other microorganisms than B. cereus. Our quantitative, systematic approach provides a framework to search for these biomarkers and to evaluate their predictive quality in order to select promising biomarkers that can serve to early detect and predict adaptive traits