The peptidoglycan (PG), as the exoskeleton of most prokaryotes, maintains a defined shape and ensures cell integrity against the high internal turgor pressure. These important roles have attracted ...researchers to target PG metabolism in order to control bacterial infections. Most studies, however, have been performed in bacteria grown under laboratory conditions, leading to only a partial view on how the PG is synthetized in natural environments. As a case in point, PG metabolism and its regulation remain poorly understood in symbiotic and pathogenic bacteria living inside eukaryotic cells. This review focuses on the PG metabolism of intracellular bacteria, emphasizing the necessity of more in vivo studies involving the analysis of enzymes produced in the intracellular niche and the isolation of PG from bacteria residing within eukaryotic cells. The review also points to persistent infections caused by some intracellular bacterial pathogens and the extent at which the PG could contribute to establish such physiological state. Based on recent evidences, I speculate on the idea that certain structural features of the PG may facilitate attenuation of intracellular growth. Lastly, I discuss recent findings in endosymbionts supporting a cooperation between host and bacterial enzymes to assemble a mature PG.
The peptidoglycan (PG), as main component of the bacterial cell wall, has attracted for decades researchers interested in basic aspects of microbial physiology and the control of microbial infections. Despite the knowledge of PG metabolism gained from bacteria grown under laboratory conditions, the structure and enzymology of the PG in bacteria residing within eukaryotic cells remain poorly characterized. This review compiles the available information about PG metabolism in intracellular bacteria.
Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria ...face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan purified from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropeptides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)-meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This peptidoglycan has a reduced glycan chain average length and ~30% increase in the L,D-crosslink, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D-transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L,D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhimurium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remarkably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-containing muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D-alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.
The Candidate Phyla Radiation (CPR) encompasses widespread uncultivated bacteria with reduced genomes and limited metabolic capacities. Most CPR bacteria lack the minimal set of enzymes required for ...peptidoglycan (PG) synthesis, leaving it unclear how these bacteria produce this essential envelope component. In this study, we analysed the distribution of d‐amino acid racemases that produce the universal PG components d‐glutamate (d‐Glu) or d‐alanine (d‐Ala). We also examined moonlighting enzymes that synthesize d‐Glu or d‐Ala. Unlike other phyla in the domain Bacteria, CPR bacteria do not exhibit these moonlighting activities and have, at most, one gene encoding either a Glu or Ala racemase. One of these ‘orphan’ racemases is a predicted Glu racemase (MurICPR) from the CPR bacterium Candidatus Saccharimonas aalborgenesis. The expression of MurICPR restores the growth of a Salmonella d‐Glu auxotroph lacking its endogenous racemase and results in the substitution of l‐Ala by serine as the first residue in a fraction of the PG stem peptides. In vitro, MurICPR exclusively racemizes Glu as a substrate. Therefore, Ca. Saccharimonas aalborgensis may couple Glu racemization to serine and d‐Glu incorporation into the stem peptide. Our findings provide the first insights into the synthesis of PG by an uncultivated environmental bacterium and illustrate how to experimentally test enzymatic activities from CPR bacteria related to PG metabolism.
Bacteria from the uncultivated Candidate Phyla Radiation (CPR) dominate various habitats, despite possessing limited metabolic capacities. These bacteria have incomplete sets of enzymes, which include pathways for producing structures that are essential for life. This supports the hypothesis of moonlighting activities in the remaining enzymes. In this study, we characterized a CPR enzyme in vivo using a Salmonella strain that is auxotrophic for d‐glutamate (d‐Glu), and a predicted racemase from Candidatus Saccharimonas aalborgensis. Our findings suggest that metabolic complementation assays could be extended to other enzymes from uncultivated bacteria that are suspected to catalyse specific reactions.
Iron is an essential oligoelement that incorporates into proteins as a biocatalyst or electron carrier. The intracellular pathogen Salmonella enterica serovar Typhimurium (
Typhimurium) takes iron as ...free reduced ferrous cation or as oxidized ferric cation complexed to siderophores or ferrichromes. Deficiencies in ferrous or ferric iron uptake attenuate
Typhimurium virulence, but how the uptake systems are used in the intracellular environment remains poorly understood. Here, using
Typhimurium mutants deficient in multiple iron uptake systems, we show that SitABCD and FeoABC, involved in ferrous iron uptake, are central for this pathogen to persist within vacuoles of fibroblasts. Assays at the protein level showed that components of these two uptake systems, SitD and FeoB, are produced at high levels by intravacuolar bacteria. Despite not being essential for viability inside the vacuole, intracellular bacteria also upregulate transporters involved in ferric iron uptake such as IroN, FepA, and CirA. In addition, an unprecedented cleavage at the N-terminal region of FepA was observed as a distinctive feature of nonproliferating intravacuolar bacteria. Collectively, our findings indicate that SitABCD and FeoABC contribute to
Typhimurium virulence by promoting iron acquisition within the vacuolar compartment.
Nucleotide-binding oligomerization domain 1 (NOD1), a pattern recognition receptor (PRR) that detects bacterial peptidoglycan fragments and other danger signals, has been linked to inflammatory ...pathologies. NOD1, which is expressed by immune and non-immune cells, is activated after recognizing microbe-associated molecular patterns (MAMPs). This recognition triggers host defense responses and both immune memory and tolerance can also be achieved during these processes. Since the gut microbiota is currently considered a master regulator of human physiology central in health and disease and the intestine metabolizes a wide range of nutrients, drugs and hormones, it is a fact that dysbiosis can alter tissues and organs homeostasis. These systemic alterations occur in response to gastrointestinal immune adaptations that are not yet fully understood. Even if previous evidence confirms the connection between the microbiota, the immune system and metabolic disorders, much remains to be discovered about the contribution of NOD1 to low-grade inflammatory pathologies such as obesity, diabetes and cardiovascular diseases. This review compiles the most recent findings in this area, while providing a dynamic and practical framework with future approaches for research and clinical applications on targeting NOD1. This knowledge can help to rate the consequences of the disease and to stratify the patients for therapeutic interventions.
Mechanisms of activation of NOD1 by bacterial products. NOD1 is expressed in specific cells, including immune cells, and is located in the cytoplasm. NOD1 recognizes molecular patterns from the microbiota (MAMPs), pathogenic (PAMPs) or beneficial, and danger associated molecular patterns (DAMPs) that reach the circulation and activates the innate and adaptive immune responses. Display omitted
•NOD1 mediates immunometabolic responses to the gastrointestinal microbiota.•NOD1 activation depends on the release of bacterial peptidoglycans.•NOD1 expression exhibits a cell-specific profile.•Pharmacological inhibition of NOD1 can prevent adverse effects of the microbiota.
Salmonella enterica serovar Typhimurium infects eukaryotic cells residing within membrane‐bound phagosomes. In this compartment, the pathogen replaces the morphogenetic penicillin‐binding proteins 2 ...and 3 (PBP2/PBP3) with PBP2SAL/PBP3SAL, two proteins absent in Escherichia coli. The basis for this switch is unknown. Here, we show that PBP3 protein levels drop drastically when S. Typhimurium senses acidity, high osmolarity and nutrient scarcity, cues that activate virulence functions required for intra‐phagosomal survival and proliferation. The protease Prc and the transcriptional regulator OmpR contribute to lower PBP3 levels whereas OmpR stimulates PBP2SAL/PBP3SAL production. Surprisingly, despite being essential for division in E. coli, PBP3 levels also drop in non‐pathogenic and pathogenic E. coli exposed to phagosome cues. Such exposure alters E. coli morphology resulting in very long bent and twisted filaments indicative of failure in the cell division and elongation machineries. None of these aberrant shapes are detected in S. Typhimurium. Expression of PBP3SAL restores cell division in E. coli exposed to phagosome cues although the cells retain elongation defects in the longitudinal axis. By switching the morphogenetic program, OmpR and Prc allow S. Typhimurium to properly divide and elongate inside acidic phagosomes maintaining its cellular dimensions and the rod shape.
Salmonella switches morphogenetic peptidoglycan synthases inside eukaryotic cells, a phenomenon triggered by three cues: acid pH, high osmolarity and nutrient scarcity. OmpR responds to acid pH upregulating alternative synthases PBP2SAL and PBP3SAL. Prc fine‐tunes PBP3 levels and prevents escape of PBP2SAL/PBP3SAL production in extracellular bacteria, in which PBP2/PBP3 direct rod shape. Escherichia coli, which lacks PBP2SAL/PBP3SAL, displays gross morphological alterations if exposed to phagosome cues due to decrease in levels of its morphogenetic peptidoglycan synthases.
Bacterial virulence relies on a delicate balance of signals interchanged between the invading microbe and the host. This communication has been extensively perceived as a battle involving harmful ...molecules produced by the pathogen and host defenses. In this review, we focus on a largely unexplored element of this dialogue, as are toxin–antitoxin (TA) systems of the pathogen. TA systems are reported to respond to stresses that are also found in the host and, as a consequence, could modulate the physiology of the intruder microbe. This view is consistent with recent studies that demonstrate a contribution of distinct TA systems to virulence since their absence alters the course of the infection. TA loci are stress response modules that, therefore, could readjust pathogen metabolism to favor the generation of slow-growing or quiescent cells ‘before’ host defenses irreversibly block essential pathogen activities. Some toxins of these TA modules have been proposed as potential weapons used by the pathogen to act on host targets. We discuss all these aspects based on studies that support some TA modules as important regulators in the pathogen–host interface.
In this review, the authors discuss toxin–antitoxin systems due to their capacity to modulate bacterial physiology in response to stress, which, as a consequence, can influence host–pathogen interactions.
Graphical Abstract Figure.
In this review, the authors discuss toxin–antitoxin systems due to their capacity to modulate bacterial physiology in response to stress, which, as a consequence, can influence host–pathogen interactions.
Abstract
RcsB is a transcriptional regulator that controls expression of numerous genes in enteric bacteria. RcsB accomplishes this role alone or in combination with auxiliary transcriptional factors ...independently or dependently of phosphorylation. To understand the mechanisms by which RcsB regulates such large number of genes, we performed structural studies as well as in vitro and in vivo functional studies with different RcsB variants. Our structural data reveal that RcsB binds promoters of target genes such as rprA and flhDC in a dimeric active conformation. In this state, the RcsB homodimer docks the DNA-binding domains into the major groove of the DNA, facilitating an initial weak read-out of the target sequence. Interestingly, comparative structural analyses also show that DNA binding may stabilize an active conformation in unphosphorylated RcsB. Furthermore, RNAseq performed in strains expressing wild-type or several RcsB variants provided new insights into the contribution of phosphorylation to gene regulation and assign a potential role of RcsB in controlling iron metabolism. Finally, we delimited the RcsB box for homodimeric active binding to DNA as the sequence TN(G/A)GAN4TC(T/C)NA. This RcsB box was found in promoter, intergenic and intragenic regions, facilitating both increased or decreased gene transcription.
is a ubiquitous environmental bacterium and intracellular pathogen that responds to stress using predominantly the alternative sigma factor SigB. Stress is sensed by a multiprotein complex, the ...stressosome, extensively studied in bacteria grown in nutrient media. Following signal perception, the stressosome triggers a phosphorylation cascade that releases SigB from its anti-sigma factor. Whether the stressosome is activated during the intracellular infection, is unknown. Here, we analysed the subcellular distribution of stressosome proteins in
located inside epithelial cells following their immunodetection in membrane and cytosolic fractions prepared from intracellular bacteria. Unlike bacteria in laboratory media, intracellular bacteria have a large proportion of the core stressosome protein RsbR1 associated with the membrane. Another core protein, RsbS, is however undetectable. Despite the absence of RsbS, a SigB-dependent reporter revealed that SigB activity increases gradually from early (1 h) to late (6 h) post-infection times. We also found that RsbR1 paralogues attenuate the intensity of the SigB response and that the miniprotein Prli42, reported to tether the stressosome to the membrane in response to oxidative stress, plays no role in associating RsbR1 to the membrane of intracellular bacteria. Altogether, these data indicate that, once inside host cells, the
stressosome may adopt a unique configuration to sense stress and to activate SigB in the intracellular eukaryotic niche.
The response to stress mediated by the alternative sigma factor SigB has been extensively characterized in
and
These bacteria sense stress using a supra-macromolecular complex, the stressosome, which triggers a cascade that releases SigB from its anti-sigma factor. Despite much structural data of the complex available and analyses performed in mutants lacking components of the stressosome or the signalling cascade, the integration of the stress signal and the dynamics of stressosome proteins following environmental changes remain poorly understood. Our study provides data at the protein level on essential stressosome components and SigB activity when
, normally a saprophytic bacterium, adapts to an intracellular lifestyle. Our results support activation of the stressosome complex in intracellular bacteria. The apparent loss of the stressosome core protein RsbS in intracellular
also challenges current models, favouring the idea of a unique stressosome architecture responding to intracellular host cues.
Listeria monocytogenes is a saprophytic gram-positive bacterium, and an opportunistic foodborne pathogen that can produce listeriosis in humans and animals. It has evolved an exceptional ability to ...adapt to stress conditions encountered in different environments, resulting in a ubiquitous distribution. Because some food preservation methods and disinfection protocols in food-processing environments cannot efficiently prevent contaminations, L. monocytogenes constitutes a threat to human health and a challenge to food safety. In the host, Listeria colonizes the gastrointestinal tract, crosses the intestinal barrier, and disseminates through the blood to target organs. In immunocompromised individuals, the elderly, and pregnant women, the pathogen can cross the blood-brain and placental barriers, leading to neurolisteriosis and materno-fetal listeriosis. Molecular and cell biology studies of infection have proven L. monocytogenes to be a versatile pathogen that deploys unique strategies to invade different cell types, survive and move inside the eukaryotic host cell, and spread from cell to cell. Here, we present the multifaceted Listeria life cycle from a comprehensive perspective. We discuss genetic features of pathogenic Listeria species, analyze factors involved in food contamination, and review bacterial strategies to tolerate stresses encountered both during food processing and along the host's gastrointestinal tract. Then we dissect host-pathogen interactions underlying listerial pathogenesis in mammals from a cell biology and systemic point of view. Finally, we summarize the epidemiology, pathophysiology, and clinical features of listeriosis in humans and animals. This work aims to gather information from different fields crucial for a comprehensive understanding of the pathogenesis of L. monocytogenes.
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