FtsN plays an essential role in promoting the inward synthesis of septal peptidoglycan (sPG) by the FtsWI complex during bacterial cell division. How it achieves this role is unclear. Here we use single-molecule tracking to investigate FtsN's dynamics during sPG synthesis in E. coli. We show that septal FtsN molecules move processively at ~9 nm s-1, the same as FtsWI molecules engaged in sPG synthesis (termed sPG-track), but much slower than the ~30 nm s-1 speed of inactive FtsWI molecules coupled to FtsZ's treadmilling dynamics (termed FtsZ-track). Importantly, processive movement of FtsN is exclusively coupled to sPG synthesis and is required to maintain active sPG synthesis by FtsWI. Our findings indicate that FtsN is part of the FtsWI sPG synthesis complex, and that while FtsN is often described as a "trigger" for the initiation for cell wall constriction, it must remain part of the processive FtsWI complex to maintain sPG synthesis activity.
by Tony Rady, Lorenzo Turelli, Marc Nothisen, Elisabetta Tobaldi, Stéphane Erb, Fabien Thoreau, Oscar Hernandez-Alba, Sarah Cianferani, François Daubeuf, Alain Wagner, and Guilhem Chaubet
Toxins (Basel). 2022 Aug 27;14(9):591. doi: 10.3390/toxins14090591.
ABSTRACT
Mycotoxin contaminations in the feed and food chain are common. Either directly or indirectly, mycotoxins enter the human body through the consumption of food of plant and animal origin. Bacteria with a high mycotoxin elimination capability can reduce mycotoxin contamination in feed and food. Four Gram-positive endospore-forming bacteria (Bacillus thuringiensis AMK10/1, Lysinibacillus boronitolerans AMK9/1, Lysinibacillus fusiformis AMK10/2, and Rummeliibacillus suwonensis AMK9/2) were isolated from fermented forages and tested for their deoxynivalenol (DON), aflatoxin B1 (AFB1), and zearalenone (ZEA) elimination potentials. Notably, the contribution of bacterial cell wall fractions to the observed outstanding ZEA elimination rates was demonstrated; however, the ZEA elimination differed considerably within the tested group of Gram-positive bacteria. It is worth noting that the purified cell wall of L. boronitolerans AMK9/1, L. fusiformis AMK10/2 and B. thuringiensis AMK10/1 were highly efficient in eliminating ZEA and the teichoic acid fractions of B. thuringiensis AMK10/1, and L. fusiformis AMK10/2 could also be successfully used in ZEA binding. The ZEA elimination capacity of viable R. suwonensis AMK9/2 cells was outstanding (40%). Meanwhile, R. suwonensis AMK9/2 and L. boronitolerans AMK9/1 cells produced significant esterase activities, and ZEA elimination of the cell wall fractions of that species did not correlate with esterase activity. DON and AFB1 binding capabilities of the tested bacterial cells and their cell wall fractions were low, except for B. thuringiensis AMK10/1, where the observed high 64% AFB1 elimination could be linked to the surface layer (S-layer) fraction of the cell wall.
Front Microbiol. 2022 Sep 6;13:985871. doi: 10.3389/fmicb.2022.985871. eCollection 2022.
ABSTRACT
Beta-lactams have been excluded from tuberculosis therapy due to the intrinsic resistance of Mycobacterium tuberculosis (Mtb) to this antibiotic class, usually attributed to a potent beta-lactamase, BlaC, and to an unusually complex cell wall. In this pathogen, the peptidoglycan is cross-linked by penicillin-binding proteins (PBPs) and L,D-transpeptidases, the latter resistant to inhibition by most beta-lactams. However, recent studies have shown encouraging results of beta-lactam/beta-lactamase inhibitor combinations in clinical strains. Additional research on the mechanisms of action and resistance to these antibiotics and other inhibitors of peptidoglycan synthesis, such as the glycopeptides, is crucial to ascertain their place in alternative regimens against drug-resistant strains. Within this scope, we applied selective pressure to generate mutants resistant to amoxicillin, meropenem or vancomycin in Mtb H37Rv or Mycolicibacterium smegmatis (Msm) mc2-155. These were phenotypically characterized, and whole-genome sequencing was performed. Mutations in promising targets or orthologue genes were inspected in Mtb clinical strains to establish potential associations between altered susceptibility to beta-lactams and the presence of key genomic signatures. The obtained isolates had substantial increases in the minimum inhibitory concentration of the selection antibiotic, and beta-lactam cross-resistance was detected in Mtb. Mutations in L,D-transpeptidases and major PBPs, canonical targets, or BlaC were not found. The transcriptional regulator PhoP (Rv0757) emerged as a common denominator for Mtb resistance to both amoxicillin and meropenem, while Rv2864c, a lipoprotein with PBP activity, appears to be specifically involved in decreased susceptibility to the carbapenem. Nonetheless, the mutational pattern detected in meropenem-resistant mutants was different from the yielded by amoxicillin-or vancomycin-selected isolates, suggesting that distinct pathways may participate in increased resistance to peptidoglycan inhibitors, including at the level of beta-lactam subclasses. Cross-resistance between beta-lactams and antimycobacterials was mostly unnoticed, and Msm meropenem-resistant mutants from parental strains with previous resistance to isoniazid or ethambutol were isolated at a lower frequency. Although cell-associated nitrocefin hydrolysis was increased in some of the isolates, our findings suggest that traditional assumptions of Mtb resistance relying largely in beta-lactamase activity and impaired access of hydrophilic molecules through lipid-rich outer layers should be challenged. Moreover, the therapeutical potential of the identified Mtb targets should be explored.
FEMS Microbiol Lett. 2022 Sep 7:fnac089. doi: 10.1093/femsle/fnac089. Online ahead of print.
ABSTRACT
Although probiotics have been isolated from different sources, few were isolated from traditional Chinese medicine. The current study firstly isolate Pulsatilla Radix-utilizing Pediococcus pentosaceus PR-1 from human faeces. Subsequently, the tolerance of PR-1 to low pH, bile salts, simulated gastric juice and succus entericus, antioxidant activity, antimicrobial activity, cholesterol-assimilation and antibiotics susceptibility were investigated. After 2 h incubation at pH 2.0, over 80% of PR-1 survived. The cell viability of PR-1 at 2 h under 0.1% bile salt condition was 99.2%. The survival rate of PR-1 in gastric juice and succus entericus were 64.48% and 81.86, respectively. Cell-free supernatant of PR-1 culture also showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium. Besides, antioxidant activity of PR-1 CFS was significantly greater than cell pellet. PR-1 was shown resistant to kanamycin, streptomycin, vancomycin, and norfloxacin and able to lower cholesterol level to 72.5 ± 1.5%. In addition, PR-1 displayed γ-hemolysis and was non-pathogenic.
Cell Host Microbe. 2022 Aug 25:S1931-3128(22)00395-X. doi: 10.1016/j.chom.2022.08.002. Online ahead of print.
ABSTRACT
The pattern-recognition receptor NOD2 senses bacterial muropeptides to regulate host immunity and maintain homeostasis. Loss-of-function mutations in NOD2 are associated with Crohn's disease (CD), but how the variations in microbial factors influence NOD2 signaling and host pathology is elusive. We demonstrate that the Firmicutes peptidoglycan remodeling enzyme, DL-endopeptidase, increased the NOD2 ligand level in the gut and impacted colitis outcomes. Metagenomic analyses of global cohorts (n = 857) revealed that DL-endopeptidase gene abundance decreased globally in CD patients and negatively correlated with colitis. Fecal microbiota from CD patients with low DL-endopeptidase activity predisposed mice to colitis. Administering DL-endopeptidase, but not an active site mutant, alleviated colitis via the NOD2 pathway. Therapeutically restoring NOD2 ligands with a DL-endopeptidase-producing Lactobacillus salivarius strain or mifamurtide, a clinical analog of muramyl dipeptide, exerted potent anti-colitis effects. Our study suggests that the depletion of DL-endopeptidase contributes to CD pathogenesis through NOD2 signaling, providing a therapeutically modifiable target.
Appl Environ Microbiol. 2022 Aug 30:e0084622. doi: 10.1128/aem.00846-22. Online ahead of print.
ABSTRACT
There is an urgent need to develop novel antibiotics since antibiotic resistance is an increasingly serious threat to global public health. Whole-cell biosensors are one of the promising strategies for new antibiotic discovery. The peptidoglycan (PG) of the bacterial cell wall is one of the most important targets for antibiotics. However, the biosensors for the detection of PG-targeting antibiotics in Gram-negative bacteria have not been developed, mainly because of the lack of the regulatory systems that sense and respond to PG stress. Recently, we identified a novel two-component signal transduction system (PghKR) that is responsible for sensing and responding to PG damage in the Gram-negative bacterium Shewanella oneidensis. Based on this system, we developed biosensors for the detection of PG-targeting antibiotics. Using ampicillin as an inducer for PG stress and the bacterial luciferase LuxCDABE as the reporter, we found that the PghKR biosensors are specific to antibiotics targeting PG synthesis, including β-lactams, vancomycin, and d-cycloserine. Deletion of genes encoding PG permease AmpG and β-lactamase BlaA improves the sensitivity of the biosensors substantially. The PghKR biosensor in the background of ΔblaA is also functional on agar plates, providing a simple method for screening bacteria that produce PG-targeting antibiotics. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative bacteria urgently needs new strategies so that researchers can develop novel antibiotics. Microbial whole-cell biosensors are capable of sensing various stimuli with a quantifiable output and show tremendous potential for the discovery of novel antibiotics. As the Achilles' heel of bacteria, the synthesis of the peptidoglycan (PG) is targeted by many antibiotics. However, the regulatory systems that sense and respond to PG-targeting stress in Gram-negative bacteria are reported rarely, restricting the development of biosensors for the detection of PG-targeting antibiotics. In this study, we developed a highly sensitive and specific biosensor based on a novel two-component system in the Gram-negative bacterium Shewanella oneidensis that is responsible for the sensing and responding to PG stress. Our biosensors have great potential for discovering novel antibiotics and determining the mode of action of antibiotics.
by Amber M. Frye,
Monir Ejemel,
Lisa Cavacini,
Yang Wang,
Michael J. Rudolph,
Renjie Song,
Nicholas J. Mantis
aDepartment of Biomedical Sciences, University at Albany, Albany, New York, USA
bMassBiologics, Boston, Massachusetts, USA
cNew York Structural Biology Centergrid.422632.3, New York, New York, USA
dDivision of Infectious Diseases, Wadsworth Center, New York State Department of Health, Albany, New York, USA
by Yujiao Gong,
Xianyong Liu,
Sixin Zhang,
Xinming Tang,
Jun Zou,
Xun Suo
aState Key Laboratory of Agrobiotechnology, China Agricultural Universitygrid.22935.3f, Beijing, China
bNational Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural Universitygrid.22935.3f, Beijing, China
cInstitute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing, China
dCenter for Inflammation, Immunity and Infection, Institute for Biomedical Sciences, Georgia State University, Atlanta, Georgia, USA
Vaccines (Basel). 2022 Aug 2;10(8):1239. doi: 10.3390/vaccines10081239.
ABSTRACT
The tick-borne bacterium, Borrelia burgdorferi has been implicated in Lyme disease-a deadly infection, formerly confined to North America, but currently widespread across Europe and Asia. Despite the severity of this disease, there is still no human Lyme disease vaccine available. A reliable immunoinformatic approach is urgently needed for designing a therapeutic vaccine against this Gram-negative pathogen. Through this research, we explored the immunodominant proteins of B. burgdorferi and developed a novel and reliable vaccine design with great immunological predictability as well as low contamination and autoimmunity risks. Our initial analysis involved proteome-wide analysis to filter out proteins on the basis of their redundancy, homology to humans, virulence, immunogenicity, and size. Following the selection of proteins, immunoinformatic tools were employed to identify MHC class I & II epitopes and B-cell epitopes, which were subsequently subjected to a rigorous screening procedure. In the final formulation, ten common MHC-I and II epitopes were used together with a suitable adjuvant. We predicted that the final chimeric multi-epitope vaccine could invoke B-cell responses and IFN-gamma-mediated immunity as well as being stable and non-allergenic. The dynamics simulations predicted the stable folding of the designed molecule, after which the molecular docking predicted the stability of the interaction between the potential antigenic epitopes and human immune receptors. Our studies have shown that the designed next-generation vaccine stimulates desirable immune responses, thus potentially providing a viable way to prevent Lyme disease. Nevertheless, further experimental studies in a wet lab are needed in order to validate the results.
by Li Xia, Tiffany R. Bellomo, Ruslan Gibadullin, Molly D. Congdon, Elijah F. Edmondson, Mi Li, Alexander Wlodawer, Crystal Li, J. Sebastian Temme, Pavan Patel, Donna Butcher, and Jeffrey C. Gildersleeve
by Xianfeng Zeng, Xi Xing, Meera Gupta, Felix C. Keber, Jaime G. Lopez, Ying-Chiang J. Lee, Asael Roichman, Lin Wang, Michael D. Neinast, Mohamed S. Donia, Martin Wühr, Cholsoon Jang, Joshua D. Rabinowitz
Isotope tracing into bacterial-specific protein sequences allows for a determination of nutrient preferences across gut microbes in vivo, and it reveals how diet alters microbiome composition.
Highlights
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Gut microbiome feedstocks mapped by isotope tracing into bacteria-specific peptides
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Major contributors are dietary fiber and protein and host lactate, urea, and mucins
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Microbiome composition shifts toward bacteria that are fed their preferred nutrients
Great progress has been made in understanding gut microbiomes’ products and their effects on health and disease. Less attention, however, has been given to the inputs that gut bacteria consume. Here, we quantitatively examine inputs and outputs of the mouse gut microbiome, using isotope tracing. The main input to microbial carbohydrate fermentation is dietary fiber and to branched-chain fatty acids and aromatic metabolites is dietary protein. In addition, circulating host lactate, 3-hydroxybutyrate, and urea (but not glucose or amino acids) feed the gut microbiome. To determine the nutrient preferences across bacteria, we traced into genus-specific bacterial protein sequences. We found systematic differences in nutrient use: most genera in the phylum Firmicutes prefer dietary protein, Bacteroides dietary fiber, and Akkermansia circulating host lactate. Such preferences correlate with microbiome composition changes in response to dietary modifications. Thus, diet shapes the microbiome by promoting the growth of bacteria that preferentially use the ingested nutrients.
Food Funct. 2022 Aug 30. doi: 10.1039/d2fo00001f. Online ahead of print.
ABSTRACT
L. plantarum 1.0386 repairs intestinal epithelial tight junction injury, and the present study was designed to further explore the role of its postbiotics, including the surface protein (1.0386-Slp), peptidoglycan (1.0386-PG) and exopolysaccharide (1.0386-EPS). The results showed that they all could improve the lipopolysaccharide (LPS)-induced decrease of transepithelial electrical resistance, increase of paracellular permeability, release of inflammatory factors, and disruption of tight junctions in Caco-2 cells, and the repairing effect of 1.0386-Slp was better than those of 1.0386-PG and 1.0386-EPS, and was similar to that of L. plantarum 1.0386. Moreover, either L. plantarum 1.0386 or 1.0386-Slp intervention significantly increased the expression of miR-200c inhibited by LPS, while the miR-200c inhibitor weakened the ability of 1.0386-Slp to promote the expression of tight junction proteins (ZO-1, occludin and claudin-1). Meanwhile, 1.0386-Slp restored the distribution of tight junction proteins and inhibited the increase of NF-κB p65, MLC and pMLC protein expression evoked by LPS. However, the addition of miR-200c inhibitors or mimics weakened or strengthened the down-regulation of MLCK-MLC pathway protein expression by 1.0386-Slp, respectively. In summary, 1.0386-Slp may be the main efficacy component of L. plantarum 1.0386, and miR-200c may be involved in the process of 1.0386-Slp inhibiting the MLCK pathway to repair intestinal epithelial tight junction injury.
Appl Environ Microbiol. 2022 Aug 30:e0084622. doi: 10.1128/aem.00846-22. Online ahead of print.
ABSTRACT
There is an urgent need to develop novel antibiotics since antibiotic resistance is an increasingly serious threat to global public health. Whole-cell biosensors are one of the promising strategies for new antibiotic discovery. The peptidoglycan (PG) of the bacterial cell wall is one of the most important targets for antibiotics. However, the biosensors for the detection of PG-targeting antibiotics in Gram-negative bacteria have not been developed, mainly because of the lack of the regulatory systems that sense and respond to PG stress. Recently, we identified a novel two-component signal transduction system (PghKR) that is responsible for sensing and responding to PG damage in the Gram-negative bacterium Shewanella oneidensis. Based on this system, we developed biosensors for the detection of PG-targeting antibiotics. Using ampicillin as an inducer for PG stress and the bacterial luciferase LuxCDABE as the reporter, we found that the PghKR biosensors are specific to antibiotics targeting PG synthesis, including β-lactams, vancomycin, and d-cycloserine. Deletion of genes encoding PG permease AmpG and β-lactamase BlaA improves the sensitivity of the biosensors substantially. The PghKR biosensor in the background of ΔblaA is also functional on agar plates, providing a simple method for screening bacteria that produce PG-targeting antibiotics. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative bacteria urgently needs new strategies so that researchers can develop novel antibiotics. Microbial whole-cell biosensors are capable of sensing various stimuli with a quantifiable output and show tremendous potential for the discovery of novel antibiotics. As the Achilles' heel of bacteria, the synthesis of the peptidoglycan (PG) is targeted by many antibiotics. However, the regulatory systems that sense and respond to PG-targeting stress in Gram-negative bacteria are reported rarely, restricting the development of biosensors for the detection of PG-targeting antibiotics. In this study, we developed a highly sensitive and specific biosensor based on a novel two-component system in the Gram-negative bacterium Shewanella oneidensis that is responsible for the sensing and responding to PG stress. Our biosensors have great potential for discovering novel antibiotics and determining the mode of action of antibiotics.
Appl Environ Microbiol. 2022 Aug 30:e0084622. doi: 10.1128/aem.00846-22. Online ahead of print.
ABSTRACT
There is an urgent need to develop novel antibiotics since antibiotic resistance is an increasingly serious threat to global public health. Whole-cell biosensors are one of the promising strategies for new antibiotic discovery. The peptidoglycan (PG) of the bacterial cell wall is one of the most important targets for antibiotics. However, the biosensors for the detection of PG-targeting antibiotics in Gram-negative bacteria have not been developed, mainly because of the lack of the regulatory systems that sense and respond to PG stress. Recently, we identified a novel two-component signal transduction system (PghKR) that is responsible for sensing and responding to PG damage in the Gram-negative bacterium Shewanella oneidensis. Based on this system, we developed biosensors for the detection of PG-targeting antibiotics. Using ampicillin as an inducer for PG stress and the bacterial luciferase LuxCDABE as the reporter, we found that the PghKR biosensors are specific to antibiotics targeting PG synthesis, including β-lactams, vancomycin, and d-cycloserine. Deletion of genes encoding PG permease AmpG and β-lactamase BlaA improves the sensitivity of the biosensors substantially. The PghKR biosensor in the background of ΔblaA is also functional on agar plates, providing a simple method for screening bacteria that produce PG-targeting antibiotics. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative bacteria urgently needs new strategies so that researchers can develop novel antibiotics. Microbial whole-cell biosensors are capable of sensing various stimuli with a quantifiable output and show tremendous potential for the discovery of novel antibiotics. As the Achilles' heel of bacteria, the synthesis of the peptidoglycan (PG) is targeted by many antibiotics. However, the regulatory systems that sense and respond to PG-targeting stress in Gram-negative bacteria are reported rarely, restricting the development of biosensors for the detection of PG-targeting antibiotics. In this study, we developed a highly sensitive and specific biosensor based on a novel two-component system in the Gram-negative bacterium Shewanella oneidensis that is responsible for the sensing and responding to PG stress. Our biosensors have great potential for discovering novel antibiotics and determining the mode of action of antibiotics.
Microbiol Spectr. 2022 Aug 30:e0069122. doi: 10.1128/spectrum.00691-22. Online ahead of print.
ABSTRACT
Diaminopimelic acid (DAP) is a unique component of the cell wall of Gram-negative bacteria. It is also an important component of organic matter and is widely utilized by microbes in the world's oceans. However, neither DAP concentrations nor marine DAP-utilizing microbes have been investigated. Here, DAP concentrations in seawater were measured and the diversity of marine DAP-utilizing bacteria and the mechanisms for their DAP metabolism were investigated. Free DAP concentrations in seawater, from surface to a 5,000 m depth, were found to be between 0.61 μM and 0.96 μM in the western Pacific Ocean. DAP-utilizing bacteria from 20 families in 4 phyla were recovered from the western Pacific seawater and 14 strains were further isolated, in which Pseudomonadota bacteria were dominant. Based on genomic and transcriptomic analyses combined with gene deletion and in vitro activity detection, DAP decarboxylase (LysA), which catalyzes the decarboxylation of DAP to form lysine, was found to be a key and specific enzyme involved in DAP metabolism in the isolated Pseudomonadota strains. Interrogation of the Tara Oceans database found that most LysA-like sequences (92%) are from Pseudomonadota, which are widely distributed in multiple habitats. This study provides an insight into DAP metabolism by marine bacteria in the ocean and contributes to our understanding of the mineralization and recycling of DAP by marine bacteria. IMPORTANCE DAP is a unique component of peptidoglycan in Gram-negative bacterial cell walls. Due to the large number of marine Gram-negative bacteria, DAP is an important component of marine organic matter. However, it remains unclear how DAP is metabolized by marine microbes. This study investigated marine DAP-utilizing bacteria by cultivation and bioinformational analysis and examined the mechanism of DAP metabolism used by marine bacteria. The results demonstrate that Pseudomonadota bacteria are likely to be an important DAP-utilizing group in the ocean and that DAP decarboxylase is a key enzyme involved in DAP metabolism. This study also sheds light on the mineralization and recycling of DAP driven by bacteria.
Insect Biochem Mol Biol. 2022 Aug 23;148:103827. doi: 10.1016/j.ibmb.2022.103827. Online ahead of print.
ABSTRACT
Peptidoglycan recognition proteins (PGRPs) detect invading bacteria to trigger or modulate immune responses in insects. While these roles are established in Drosophila, functional studies are not yet achieved at the PGRP family level in other insects. To attain this goal, we selected Manduca sexta PGRP12 and five of the nine secreted PGRPs for recombinant expression and biochemical characterization. We cloned PGRP2-5, 12 and 13 cDNAs, produced the proteins in full (PGRP2-5, 13) or in part (PGRP3s, 12e, 13N, 13C) in Sf9 cells, and tested their bindings of two muramyl pentapeptides by surface plasmon resonance, two soluble peptidoglycans by competitive ELISA, and four insoluble peptidoglycans and eight whole bacteria by a pull-down assay. Preferential binding of meso-diaminopimelic acid-peptidoglycans (DAP-PGs) was observed in all the proteins containing a peptidoglycan binding domain and, since PGRP6, 7 and 9 proteins were hardly detected in cell-free hemolymph, the reportoire of PGRPs (including PGRP1 published previously) in M. sexta hemolymph is likely adapted to mainly detect Gram-negative bacteria and certain Gram-positive bacteria with DAP-PGs located on their surface. After incubation with plasma from naïve larvae, PGRP2, 3f, 4, 5, 13f and 13N considerably stimulated prophenoloxidase activation in the absence of a bacterial elicitor. PGRP3s and 12e had much smaller effects. Inclusion of the full-length PGRPs and their regions in the plasma also led to proHP8 activation, supporting their connections to the Toll pathway, since HP8 is a Spӓtzle-1 processing enzyme in M. sexta. Together, these findings raised concerns on the common belief that the Toll-pathway is specific for Gram-positive bacteria in insects.
Int J Mol Sci. 2022 Aug 15;23(16):9141. doi: 10.3390/ijms23169141.
ABSTRACT
The gut is a well-established route of infection and target for viral damage by SARS-CoV-2. This is supported by the clinical observation that about half of COVID-19 patients exhibit gastrointestinal (GI) complications. We aimed to investigate whether the analysis of plasma could provide insight into gut barrier dysfunction in patients with COVID-19 infection. Plasma samples of COVID-19 patients (n = 146) and healthy individuals (n = 47) were collected during hospitalization and routine visits. Plasma microbiome was analyzed using 16S rRNA sequencing and gut permeability markers including fatty acid binding protein 2 (FABP2), peptidoglycan (PGN), and lipopolysaccharide (LPS) in both patient cohorts. Plasma samples of both cohorts contained predominately Proteobacteria, Firmicutes, Bacteroides, and Actinobacteria. COVID-19 subjects exhibit significant dysbiosis (p = 0.001) of the plasma microbiome with increased abundance of Actinobacteria spp. (p = 0.0332), decreased abundance of Bacteroides spp. (p = 0.0003), and an increased Firmicutes:Bacteroidetes ratio (p = 0.0003) compared to healthy subjects. The concentration of the plasma gut permeability marker FABP2 (p = 0.0013) and the gut microbial antigens PGN (p < 0.0001) and LPS (p = 0.0049) were significantly elevated in COVID-19 patients compared to healthy subjects. These findings support the notion that the intestine may represent a source for bacteremia and contribute to worsening COVID-19 outcomes. Therapies targeting the gut and prevention of gut barrier defects may represent a strategy to improve outcomes in COVID-19 patients.
Certain bacteria constitute a threat to humans due to their ability to escape host defenses as they easily develop drug resistance. Bacteria are classified into gram-positive and gram-negative according to the composition of the cell membrane structure. Gram-negative bacteria have an additional outer membrane (OM) that is not present in their gram-positive counterpart; the latter instead hold a thicker peptidoglycan (PG) layer. This review covers the main structural and functional properties of cell wall polysaccharides (CWPs) and PG. Drugs targeting CWPs are discussed, both noncarbohydrate-related (β-lactams, fosfomycin, and lipopeptides) and carbohydrate-related (glycopeptides and lipoglycopeptides). Bacterial resistance to these drugs continues to evolve, which calls for novel antibacterial approaches to be developed. The use of carbohydrate-based vaccines as a valid strategy to prevent bacterial infections is also addressed.
J Am Chem Soc. 2022 Aug 31;144(34):15885-15893. doi: 10.1021/jacs.2c07375. Epub 2022 Aug 17.
ABSTRACT
Binding via reversible covalent bond formation presents a novel and powerful mechanism to enhance the potency of synthetic inhibitors for therapeutically important proteins. Work on this front has yielded the anticancer drug bortezomib as well as the antisickling drug voxelotor. However, the rational design of reversible covalent inhibitors remains difficult even when noncovalent inhibitors are available as a scaffold. Herein, we report chemically modified phage libraries, both linear and cyclic, that incorporate 2-acetylphenylboronic acid (APBA) as a warhead to bind lysines via reversible iminoboronate formation. To demonstrate their utility, these APBA-presenting phage libraries were screened against sortase A of Staphylococcus aureus, as well as the spike protein of SARS-CoV-2. For both protein targets, peptide ligands were readily identified with single-digit micromolar potency and excellent specificity, enabling live-cell sortase inhibition and highly sensitive spike protein detection, respectively. Furthermore, our structure-activity studies unambiguously demonstrate the benefit of the APBA warhead for protein binding. Overall, this contribution shows for the first time that reversible covalent inhibitors can be developed via phage display for a protein of interest. The phage display platform should be widely applicable to proteins including those involved in protein-protein interactions.
Nature. 2022 Aug 24. doi: 10.1038/s41586-022-05125-x. Online ahead of print.
ABSTRACT
Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.
Front Immunol. 2022 Aug 5;13:949118. doi: 10.3389/fimmu.2022.949118. eCollection 2022.
ABSTRACT
Lyme disease (LD) infection is caused by Borrelia burgdorferi sensu lato (Bb). Due to the limited presence of this pathogen in the bloodstream in humans, diagnosis of LD relies on seroconversion. Immunoglobulins produced in response to infection are differentially glycosylated to promote or inhibit downstream inflammatory responses by the immune system. Immunoglobulin G (IgG) N-glycan responses to LD have not been characterized. In this study, we analyzed IgG N-glycans from cohorts of healthy controls, acute LD patient serum, and serum collected after acute LD patients completed a 2- to 3-week course of antibiotics and convalesced for 70-90 days. Results indicate that during the acute phase of Bb infection, IgG shifts its glycosylation profile to include structures that are not associated with the classic proinflammatory IgG N-glycan signature. This unexpected result is in direct contrast to what is reported for other inflammatory diseases. Furthermore, IgG N-glycans detected during acute LD infection discriminated between control, acute, and treated cohorts with a sensitivity of 75-100% and specificity of 94.7-100%.
FEMS Microbiol Lett. 2022 Aug 23:fnac080. doi: 10.1093/femsle/fnac080. Online ahead of print.
ABSTRACT
In this study, we analyzed the srtE gene from Corynebacterium glutamicum ATCC 13032, which codes for class E sortase, a transpeptidase involved in attaching surface proteins to the cell wall peptidoglycan. The surface proteins contain an N-terminal leader sequence and a C-terminal sorting signal which consist of a LAXTG motif, a transmembrane region, and a few positively charged amino acids. Sortase E deletion or its overexpression alters the attachment of the surface proteins to the cell wall peptidoglycan; however, the effects on morphology and bacterial physiology have not been studied. Thus, we constructed three C. glutamicum derivatives such as srtE deletion mutant, complemented and overexpressed strains to monitor the possible impact of the gene on cell growth, morphology, and physiological changes. Interestingly, deletion of the gene did not show any change in growth or morphology in C. glutamicum but showed a decrease in cell surface hydrophobicity and heat stress. However, the cells overexpressing the protein not only showed elongated cell morphology and a reduction in hydrophobicity when compared to wild-type and complemented strain, but also showed an increased sensitivity to heat. These results suggest that C. glutamicum sortase E deletion or overexpression causes sorting intermediates to accumulate, altering cellular morphology and physiology and adversely impacting the membrane integrity.
