Common and varied molecular responses of Escherichia coli to five different inhibitors of the lipopolysaccharide biosynthetic enzyme LpxC.

A promising yet clinically unexploited antibiotic target in difficult-to-treat Gram-negative bacteria is LpxC, the key enzyme in the biosynthesis of lipopolysaccharides, which are the major constituents of the outer membrane. Despite the development of dozens of chemically diverse LpxC inhibitor molecules, it is essentially unknown how bacteria counteract LpxC inhibition. Our study provides comprehensive insights into the response against five different LpxC inhibitors. All compounds bound to purified LpxC from Escherichia coli. Treatment of E. coli with these compounds changed the cell shape and stabilized LpxC suggesting that FtsH-mediated proteolysis of the inactivated enzyme is impaired. LpxC inhibition sensitized E. coli to vancomycin and rifampin, which poorly cross the outer membrane of intact cells. Four of the five compounds led to an accumulation of lyso-phosphatidylethanolamine, a cleavage product of phosphatidylethanolamine, generated by the phospholipase PldA. The combined results suggested an imbalance in lipopolysaccharides and phospholipid biosynthesis, which was corroborated by the global proteome response to treatment with the LpxC inhibitors. Apart from LpxC itself, FabA and FabB responsible for the biosynthesis of unsaturated fatty acids were consistently induced. Upregulated compound-specific proteins are involved in various functional categories, such as stress reactions, nucleotide, or amino acid metabolism and quorum sensing. Our work shows that antibiotics targeting the same enzyme do not necessarily elicit identical cellular responses. Moreover, we find that the response of E. coli to LpxC inhibition is distinct from the previously reported response in Pseudomonas aeruginosa.

Linalool controls the viability of Escherichia coli by regulating the synthesis and modification of lipopolysaccharide, the assembly of ribosome, and the expression of substrate transporting proteins.

Escherichia coli (E. coli) is a Gram-negative bacterium and some pathogenic types may cause serious diseases, foods or food environments were the primary routes for its infection. Citrus aurantium L. var. amara Engl., a variety of sour orange, were used as a kind of non-conventional edible plant in China, but its antimicrobial activity and mechanisms were not well studied. Thus, in this study, EO from the flower of Citrus aurantium L. var. amara Engl. (CAEO) were studied as a kind of natural antimicrobial agent to control E. coli, our results showed that both of CAEO and its main component (linalool) exhibited strong antibacterial efficacy. Further, transcriptomic and proteomic analysis were carried out to explore cell response under linalool treatment and the main results included: (1) The synthesis and modification of lipopolysaccharide (LPS) was significantly influenced. (2) Ribosomal assembly and protein synthesis were significantly inhibited. (3) The expression of proteins related to the uptake of several essential substances was significantly changed. In all, our results would supply a theoretical basis for the proper use of CAEO and linalool as a promising antimicrobial agent to prevent and control E. coli infection in the future.

Host- and Age-Dependent Transcriptional Changes in Mycobacterium tuberculosis Cell Envelope Biosynthesis Genes after Exposure to Human Alveolar Lining Fluid.

Tuberculosis (TB) infection, caused by the airborne pathogen Mycobacterium tuberculosis (M.tb), resulted in almost 1.4 million deaths in 2019, and the number of deaths is predicted to increase by 20% over the next 5 years due to the COVID-19 pandemic. Upon reaching the alveolar space, M.tb comes into close contact with the lung mucosa before and after its encounter with host alveolar compartment cells. Our previous studies show that homeostatic, innate soluble components of the alveolar lining fluid (ALF) can quickly alter the cell envelope surface of M.tb upon contact, defining subsequent M.tb-host cell interactions and infection outcomes in vitro and in vivo. We also demonstrated that ALF from 60+ year old elders (E-ALF) vs. healthy 18- to 45-year-old adults (A-ALF) is dysfunctional, with loss of homeostatic capacity and impaired innate soluble responses linked to high local oxidative stress. In this study, a targeted transcriptional assay shows that M.tb exposure to human ALF alters the expression of its cell envelope genes. Specifically, our results indicate that A-ALF-exposed M.tb upregulates cell envelope genes associated with lipid, carbohydrate, and amino acid metabolism, as well as genes associated with redox homeostasis and transcriptional regulators. Conversely, M.tb exposure to E-ALF shows a lesser transcriptional response, with most of the M.tb genes unchanged or downregulated. Overall, this study indicates that M.tb responds and adapts to the lung alveolar environment upon contact, and that the host ALF status, determined by factors such as age, might play an important role in determining infection outcome.

Characterization of a gene cluster containing four genes relevant to biosynthesis of inner core of lipopolysaccharide in Cronobacter sakazakii.

Many genes in the biosynthetic pathway of lipopolysaccharide in Cronobacter sakazakii have not been identified. In this study, we demonstrate that an operon containing four genes ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 is responsible for L-glycero-D-mannoheptose addition on the inner core of lipopolysaccharide in C. sakazakii. The proteins encoded by these four genes are homologous to E. coli WaaQ, WaaC, WaaF, and WaaD. Lipopolysaccharide from the deletion mutants of ESA_RS18945, ESA_RS18950, ESA_RS18955, and ESA_RS18960 (named as △RS18945, △RS18950, △RS18955 and △RS18960, respectively) were analyzed by SDS-PAGE. △RS18945 synthesized lipopolysaccharide with similar length to the wildtype BAA-894, whereas △RS18950, △RS18955, and △RS18960 synthesized much shorter lipopolysaccharide. This suggests that the enzyme encoded by ESA_RS18945 might function as E. coli WaaQ on the sidechain of lipopolysaccharide. When E. coli WaaC, WaaF, and WaaD were overexpressed in △RS18950, △RS18955, and △RS18960, respectively, the full length of lipopolysaccharide was recovered. Mass spectrometry analysis indicates that △RS18950 and △RS18960 only synthesized Kdo2 -lipid A, confirming that enzymes encoded by ESA_RS18950 and ESA_RS18960 have similar functions to E. coli WaaC and WaaD, respectively. Hep-Kdo2 -lipid A with a phosphoethanolamine was produced in △RS18955, suggesting that the enzyme encoded by ESA_RS18955 has similar function to E. coli WaaF.

Engineering for an HPV 9-valent vaccine candidate using genomic constitutive over-expression and low lipopolysaccharide levels in Escherichia coli cells.

BACKGROUND: The various advantages associated with the growth properties of Escherichia coli have justified their use in the production of genetically engineered vaccines. However, endotoxin contamination, plasmid vector instability, and the requirement for antibiotic supplementation are frequent bottlenecks in the successful production of recombinant proteins that are safe for industrial-scaled applications. To overcome these drawbacks, we focused on interrupting the expression of several key genes involved in the synthesis of lipopolysaccharide (LPS), an endotoxin frequently responsible for toxicity in recombinant proteins, to eliminate endotoxin contamination and produce better recombinant proteins with E. coli. RESULTS: Of 8 potential target genes associated with LPS synthesis, we successfully constructed 7 LPS biosynthesis-defective recombinant strains to reduce the production of LPS. The endotoxin residue in the protein products from these modified E. coli strains were about two orders of magnitude lower than that produced by the wild-type strain. Further, we found that 6 loci-lpxM, lpxP, lpxL, eptA, gutQ and kdsD-were suitable for chromosomal integrated expression of HPV L1 protein. We found that a single copy of the expression cassette conferred stable expression during long-term antibiotic-free cultivation as compared with the more variable protein production from plasmid-based expression. In large-scale fermentation, we found that recombinant strains bearing 3 to 5 copies of the expression cassette had 1.5- to 2-fold higher overall expression along with lower endotoxin levels as compared with the parental ER2566 strain. Finally, we engineered and constructed 9 recombinant E. coli strains for the later production of an HPV 9-valent capsid protein with desirable purity, VLP morphology, and antigenicity. CONCLUSIONS: Reengineering the LPS synthesis loci in the E. coli ER2566 strain through chromosomal integration of expression cassettes has potential uses for the production of a 9-valent HPV vaccine candidate, with markedly reduced residual endotoxin levels. Our results offer a new strategy for recombinant E. coli strain construction, engineering, and the development of suitable recombinant protein drugs.

Loss of YhcB results in dysregulation of coordinated peptidoglycan, LPS and phospholipid synthesis during Escherichia coli cell growth.

The cell envelope is essential for viability in all domains of life. It retains enzymes and substrates within a confined space while providing a protective barrier to the external environment. Destabilising the envelope of bacterial pathogens is a common strategy employed by antimicrobial treatment. However, even in one of the best studied organisms, Escherichia coli, there remain gaps in our understanding of how the synthesis of the successive layers of the cell envelope are coordinated during growth and cell division. Here, we used a whole-genome phenotypic screen to identify mutants with a defective cell envelope. We report that loss of yhcB, a conserved gene of unknown function, results in loss of envelope stability, increased cell permeability and dysregulated control of cell size. Using whole genome transposon mutagenesis strategies, we report the comprehensive genetic interaction network of yhcB, revealing all genes with a synthetic negative and a synthetic positive relationship. These genes include those previously reported to have a role in cell envelope biogenesis. Surprisingly, we identified genes previously annotated as essential that became non-essential in a ΔyhcB background. Subsequent analyses suggest that YhcB functions at the junction of several envelope biosynthetic pathways coordinating the spatiotemporal growth of the cell, highlighting YhcB as an as yet unexplored antimicrobial target.

Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention.

A study was conducted to determine the effects of a diet supplemented with fruits and vegetables (FV) on the host whole blood cell (WBC) transcriptome and the composition and function of the intestinal microbiome. Nine six-week-old pigs were fed a pig grower diet alone or supplemented with lyophilized FV equivalent to half the daily recommended amount prescribed for humans by the Dietary Guideline for Americans (DGA) for two weeks. Host transcriptome changes in the WBC were evaluated by RNA sequencing. Isolated DNA from the fecal microbiome was used for 16S rDNA taxonomic analysis and prediction of metabolomic function. Feeding an FV-supplemented diet to pigs induced differential expression of several genes associated with an increase in B-cell development and differentiation and the regulation of cellular movement, inflammatory response, and cell-to-cell signaling. Linear discriminant analysis effect size (LEfSe) in fecal microbiome samples showed differential increases in genera from Lachnospiraceae and Ruminococcaceae families within the order Clostridiales and Erysipelotrichaceae family with a predicted reduction in rgpE-glucosyltransferase protein associated with lipopolysaccharide biosynthesis in pigs fed the FV-supplemented diet. These results suggest that feeding an FV-supplemented diet for two weeks modulated markers of cellular inflammatory and immune function in the WBC transcriptome and the composition of the intestinal microbiome by increasing the abundance of bacterial taxa that have been associated with improved intestinal health.

Acinetobacter baumannii Can Survive with an Outer Membrane Lacking Lipooligosaccharide Due to Structural Support from Elongasome Peptidoglycan Synthesis.

Gram-negative bacteria resist external stresses due to cell envelope rigidity, which is provided by two membranes and a peptidoglycan layer. The outer membrane (OM) surface contains lipopolysaccharide (LPS; contains O-antigen) or lipooligosaccharide (LOS). LPS/LOS are essential in most Gram-negative bacteria and may contribute to cellular rigidity. Acinetobacter baumannii is a useful tool for testing these hypotheses as it can survive without LOS. Previously, our group found that strains with naturally high levels of penicillin binding protein 1A (PBP1A) could not become LOS deficient unless the gene encoding it was deleted, highlighting the relevance of peptidoglycan biosynthesis and suggesting that high PBP1A levels were toxic during LOS deficiency. Transposon sequencing and follow-up analysis found that axial peptidoglycan synthesis by the elongasome and a peptidoglycan recycling enzyme, ElsL, were vital in LOS-deficient cells. The toxicity of high PBP1A levels during LOS deficiency was clarified to be due to a negative impact on elongasome function. Our data suggest that during LOS deficiency, the strength of the peptidoglycan specifically imparted by elongasome synthesis becomes essential, supporting that the OM and peptidoglycan contribute to cell rigidity. IMPORTANCE Gram-negative bacteria have a multilayered cell envelope with a layer of cross-linked polymers (peptidoglycan) sandwiched between two membranes. Peptidoglycan was long thought to exclusively provide rigidity to the cell providing mechanical strength. Recently, the most outer membrane of the cell was also proposed to contribute to rigidity due to properties of a unique molecule called lipopolysaccharide (LPS). LPS is located on the cell surface in the outer membrane and is typically required for growth. By using Acinetobacter baumannii, a Gram-negative bacterium that can grow without LPS, we found that key features of the peptidoglycan structure also become essential. This finding supports that both the outer membrane and peptidoglycan contribute to cell rigidity.

Specific gut microbiome signatures and the associated pro-inflamatory functions are linked to pediatric allergy and acquisition of immune tolerance.

Understanding the functional potential of the gut microbiome is of primary importance for the design of innovative strategies for allergy treatment and prevention. Here we report the gut microbiome features of 90 children affected by food (FA) or respiratory (RA) allergies and 30 age-matched, healthy controls (CT). We identify specific microbial signatures in the gut microbiome of allergic children, such as higher abundance of Ruminococcus gnavus and Faecalibacterium prausnitzii, and a depletion of Bifidobacterium longum, Bacteroides dorei, B. vulgatus and fiber-degrading taxa. The metagenome of allergic children shows a pro-inflammatory potential, with an enrichment of genes involved in the production of bacterial lipo-polysaccharides and urease. We demonstrate that specific gut microbiome signatures at baseline can be predictable of immune tolerance acquisition. Finally, a strain-level selection occurring in the gut microbiome of allergic subjects is identified. R. gnavus strains enriched in FA and RA showed lower ability to degrade fiber, and genes involved in the production of a pro-inflammatory polysaccharide. We demonstrate that a gut microbiome dysbiosis occurs in allergic children, with R. gnavus emerging as a main player in pediatric allergy. These findings may open new strategies in the development of innovative preventive and therapeutic approaches. Trial: NCT04750980.

Enhanced Sensitivity of Salmonella to Antimicrobial Blue Light Caused by Inactivating rfaC Gene Involved in Lipopolysaccharide Biosynthesis.

Salmonella is a global foodborne pathogen that causes human diseases ranging from mild gastroenteritis to severe systemic infections. Recently, antimicrobial blue light (aBL) showed effective bactericidal activity against a variety of bacteria (e.g., Salmonella) with varying efficiency. However, the antimicrobial mechanism of aBL has not been fully elucidated. Our previous report showed that the outer membrane (OM) is a key target of aBL. The major component of the OM, lipopolysaccharide (LPS), may play a role in aBL bactericidal effect. Therefore, the influence of LPS truncation on the sensitivity of Salmonella Typhimurium SL1344 to aBL was investigated for the first time. First, the rfaC gene in the SL1344 strain likely involved in linking lipid A to the core region of LPS was inactivated and the influence on LPS structure was verified in the mutant strain SL1344ΔrfaC. SL1344ΔrfaC showed a significant increase in sensitivity to aBL, and the bactericidal efficiency exceeded 8 log CFU at an aBL dose of 383 J/cm2, while that of its parental SL1344 strain approached 4 log CFU. To discover the possible mechanism of higher sensitivity, the permeability of OM was determined. Compared to SL1344, SL1344ΔrfaC showed 2.7-fold higher permeability of the OM at 20 J/cm2, this may explain the higher vulnerability of the OM to aBL. Furthermore, the fatty acid profile was analyzed to reveal the detailed changes in the OM and inner membrane of the mutant. Results showed that the membrane lipids of SL1344ΔrfaC were markedly different to SL1344, indicating that change in fatty acid profile might mediate the enhancement of OM permeability and the increased sensitivity to aBL in SL1344ΔrfaC. Hence, we concluded that disruption of rfaC in Salmonella Typhimurium led to the formation of truncated LPS and thus enhanced the permeability of the OM, which contributed to the increased sensitivity to aBL.

Small RNA mediated gradual control of lipopolysaccharide biosynthesis affects antibiotic resistance in Helicobacter pylori.

The small, regulatory RNA RepG (Regulator of polymeric G-repeats) regulates the expression of the chemotaxis receptor TlpB in Helicobacter pylori by targeting a variable G-repeat in the tlpB mRNA leader. Here, we show that RepG additionally controls lipopolysaccharide (LPS) phase variation by also modulating the expression of a gene (hp0102) that is co-transcribed with tlpB. The hp0102 gene encodes a glycosyltransferase required for LPS O-chain biosynthesis and in vivo colonization of the mouse stomach. The G-repeat length defines a gradual (rather than ON/OFF) control of LPS biosynthesis by RepG, and leads to gradual resistance to a membrane-targeting antibiotic. Thus, RepG-mediated modulation of LPS structure might impact host immune recognition and antibiotic sensitivity, thereby helping H. pylori to adapt and persist in the host.

Molecular Basis of Essentiality of Early Critical Steps in the Lipopolysaccharide Biogenesis in Escherichia coli K-12: Requirement of MsbA, Cardiolipin, LpxL, LpxM and GcvB.

To identify the physiological factors that limit the growth of Escherichia coli K-12 strains synthesizing minimal lipopolysaccharide (LPS), we describe the first construction of strains devoid of the entire waa locus and concomitantly lacking all three acyltransferases (LpxL/LpxM/LpxP), synthesizing minimal lipid IVA derivatives with a restricted ability to grow at around 21 °C. Suppressors restoring growth up to 37 °C of Δ(gmhD-waaA) identified two independent single-amino-acid substitutions-P50S and R310S-in the LPS flippase MsbA. Interestingly, the cardiolipin synthase-encoding gene clsA was found to be essential for the growth of ΔlpxLMP, ΔlpxL, ΔwaaA, and Δ(gmhD-waaA) bacteria, with a conditional lethal phenotype of Δ(clsA lpxM), which could be overcome by suppressor mutations in MsbA. Suppressor mutations basS A20D or basR G53V, causing a constitutive incorporation of phosphoethanolamine (P-EtN) in the lipid A, could abolish the Ca++ sensitivity of Δ(waaC eptB), thereby compensating for P-EtN absence on the second Kdo. A single-amino-acid OppA S273G substitution is shown to overcome the synthetic lethality of Δ(waaC surA) bacteria, consistent with the chaperone-like function of the OppA oligopeptide-binding protein. Furthermore, overexpression of GcvB sRNA was found to repress the accumulation of LpxC and suppress the lethality of LapAB absence. Thus, this study identifies new and limiting factors in regulating LPS biosynthesis.

Helicobacter pylori GmhB enzyme involved in ADP-heptose biosynthesis pathway is essential for lipopolysaccharide biosynthesis and bacterial virulence.

Helicobacter pylori infection is linked to serious gastric-related diseases including gastric cancer. However, current therapies for treating H. pylori infection are challenged by the increased antibiotic resistance of H. pylori. Therefore, it is in an urgent need to identify novel targets for drug development against H. pylori infection. In this study, HP0860 gene from H. pylori predicted to encode a D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) involved in the synthesis of ADP-L-glycero-D-manno-heptose for the assembly of lipopolysaccharide (LPS) in the inner core region was cloned and characterized. We reported HP0860 protein is monomeric and functions as a phosphatase by converting D-glycero-D-manno-heptose-1,7-bisphosphate into D-glycero-D-manno-heptose-1-phosphate with a preference for the ß-anomer over the α-anomer of sugar phosphate substrates. Subsequently, a HP0860 knockout mutant and its complementary mutant were constructed and their phenotypic properties were examined. HP0860 knockout mutant contained both mature and immature forms of LPS and could still induce significant IL-8 secretion after gastric AGS cell infection, suggesting other enzymatic activities in HP0860 knockout mutant might be able to partially compensate for the loss of HP0860 activity. In addition, HP0860 knockout mutant was much more sensitive to antibiotic novobiocin, had decreased adherence abilities, and caused less classic hummingbird phenotype on the infected AGS cells, indicating H. pylori lacking HP0860 is less virulent. Furthermore, the disruption of HP0860 gene altered the sorting of cargo proteins into outer membrane vesicles (OMVs). The above findings confirm the importance of HP0860 in LPS core biosynthesis and shed light on therapeutic intervention against H. pylori infection.

The rfbN gene of Salmonella Typhimurium mediates phage adsorption by modulating biosynthesis of lipopolysaccharide.

The study of the interaction mechanism between bacteriophage and host is helpful in promoting development of bacteriophage applications. The mechanism of the interaction with the phage was studied by constructing the rfbN gene deletion and complemented with strains of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium, S. Typhimurium) D6. The rfbN gene deletion strain could not be lysed by phage S55 and led to a disorder of lipopolysaccharide (LPS) biosynthesis, which changed from the smooth type to rough type. Also, the RfbN protein lacking any of the three-segment amino acid (aa) sequences (90-120 aa, 121-158 aa, and 159-194 aa) produces the same result. Transmission electron microscopy and confocal microscopy assays demonstrated that phage S55 dramatically reduced adsorption to the rfbN deletion strain as compared to the wild strain D6. After co-incubation of the S55 with the purified smooth LPS, D6 could not be lysed, indicating that the smooth LPS binds to the S55 in vitro and then inhibits the cleavage activity of the S55. To sum up, the rfbN gene affects phage adsorption by regulating LPS synthesis. Furthermore, the functioning of the RfbN protein requires the involvement of multiple structures. To the best of our knowledge, this study is the first report of the involvement of the bacterial rfbN gene involved in the phage-adsorption process.

Spatial regulation of protein A in Staphylococcus aureus.

Surface proteins of Staphylococcus aureus play vital roles in bacterial physiology and pathogenesis. Recent work suggests that surface proteins are spatially regulated by a YSIRK/GXXS signal peptide that promotes cross-wall targeting at the mid-cell, though the mechanisms remain unclear. We previously showed that protein A (SpA), a YSIRK/GXXS protein and key staphylococcal virulence factor, mis-localizes in a ltaS mutant deficient in lipoteichoic acid (LTA) production. Here, we identified that SpA contains another cross-wall targeting signal, the LysM domain, which, in addition to the YSIRK/GXXS signal peptide, significantly enhances SpA cross-wall targeting. We show that LTA synthesis, but not LtaS, is required for SpA septal anchoring and cross-wall deposition. Interestingly, LTA is predominantly found at the peripheral cell membrane and is diminished at the septum of dividing staphylococcal cells, suggesting a restriction mechanism for SpA septal localization. Finally, we show that D-alanylation of LTA abolishes SpA cross-wall deposition by disrupting SpA distribution in the peptidoglycan layer without altering SpA septal anchoring. Our study reveals that multiple factors contribute to the spatial regulation and cross-wall targeting of SpA via different mechanisms, which coordinately ensures efficient incorporation of surface proteins into the growing peptidoglycan during the cell cycle.

Analysis of the Structure and Biosynthesis of the Lipopolysaccharide Core Oligosaccharide of Pseudomonas syringae pv. tomato DC3000.

Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, is important for bacterial viability in general and host-pathogen interactions in particular. Negative charges at its core oligosaccharide (core-OS) contribute to membrane integrity through bridging interactions with divalent cations. The molecular structure and synthesis of the core-OS have been resolved in various bacteria including the mammalian pathogen Pseudomonas aeruginosa. A few core-OS structures of plant-associated Pseudomonas strains have been solved to date, but the genetic components of the underlying biosynthesis remained unclear. We conducted a comparative genome analysis of the core-OS gene cluster in Pseudomonas syringae pv. tomato (Pst) DC3000, a widely used model pathogen in plant-microbe interactions, within the P. syringae species complex and to other plant-associated Pseudomonas strains. Our results suggest a genetic and structural conservation of the inner core-OS but variation in outer core-OS composition within the P. syringae species complex. Structural analysis of the core-OS of Pst DC3000 shows an uncommonly high phosphorylation and presence of an O-acetylated sugar. Finally, we combined the results of our genomic survey with available structure information to estimate the core-OS composition of other Pseudomonas species.

Multiple concurrent and convergent stages of genome reduction in bacterial symbionts across a stink bug family.

Nutritional symbioses between bacteria and insects are prevalent and diverse, allowing insects to expand their feeding strategies and niches. A common consequence of long-term associations is a considerable reduction in symbiont genome size likely influenced by the radical shift in selective pressures as a result of the less variable environment within the host. While several of these cases can be found across distinct insect species, most examples provide a limited view of a single or few stages of the process of genome reduction. Stink bugs (Pentatomidae) contain inherited gamma-proteobacterial symbionts in a modified organ in their midgut and are an example of a long-term nutritional symbiosis, but multiple cases of new symbiont acquisition throughout the history of the family have been described. We sequenced the genomes of 11 symbionts of stink bugs with sizes that ranged from equal to those of their free-living relatives to less than 20%. Comparative genomics of these and previously sequenced symbionts revealed initial stages of genome reduction including an initial pseudogenization before genome reduction, followed by multiple stages of progressive degeneration of existing metabolic pathways likely to impact host interactions such as cell wall component biosynthesis. Amino acid biosynthesis pathways were retained in a similar manner as in other nutritional symbionts. Stink bug symbionts display convergent genome reduction events showing progressive changes from a free-living bacterium to a host-dependent symbiont. This system can therefore be used to study convergent genome evolution of symbiosis at a scale not previously available.

Complementation in trans of Porphyromonas gingivalis Lipopolysaccharide Biosynthetic Mutants Demonstrates Lipopolysaccharide Exchange.

Porphyromonas gingivalis, a bacterial pathogen contributing to human periodontitis, exports and anchors cargo proteins to its surface, enabling the production of black pigmentation using a type IX secretion system (T9SS) and conjugation to anionic lipopolysaccharide (A-LPS). To determine whether T9SS components need to be assembled in situ for correct secretion and A-LPS modification of cargo proteins, combinations of nonpigmented mutants lacking A-LPS or a T9SS component were mixed to investigate in trans complementation. Reacquisition of pigmentation occurred only between an A-LPS mutant and a T9SS mutant, which coincided with A-LPS modification of cargo proteins detected by Western blotting and coimmunoprecipitation/quantitative mass spectrometry. Complementation also occurred using an A-LPS mutant mixed with outer membrane vesicles (OMVs) or purified A-LPS. Fluorescence experiments demonstrated that OMVs can fuse with and transfer lipid to P. gingivalis, leading to the conclusion that complementation of T9SS function occurred through A-LPS transfer between cells. None of the two-strain crosses involving only the five T9SS OM component mutants produced black pigmentation, implying that the OM proteins cannot be transferred in a manner that restores function and surface pigmentation, and hence, a more ordered temporal in situ assembly of T9SS components may be required. Our results show that LPS can be transferred between cells or between cells and OMVs to complement deficiencies in LPS biosynthesis and hemin-related pigmentation to reveal a potentially new mechanism by which the oral microbial community is modulated to produce clinical consequences in the human host.IMPORTANCEPorphyromonas gingivalis is a keystone pathogen contributing to periodontitis in humans, leading to tooth loss. The oral microbiota is essential in this pathogenic process and changes from predominantly Gram-positive (health) to predominantly Gram-negative (disease) species. P. gingivalis uses its type IX secretion system (T9SS) to secrete and conjugate virulence proteins to anionic lipopolysaccharide (A-LPS). This study investigated whether components of this secretion system could be complemented and found that it was possible for A-LPS biosynthetic mutants to be complemented in trans both by strains that had the A-LPS on the cell surface and by exogenous sources of A-LPS. This is the first known example of LPS exchange in a human bacterial pathogen which causes disease through complex microbiota-host interactions.

Checkpoints That Regulate Balanced Biosynthesis of Lipopolysaccharide and Its Essentiality in Escherichia coli.

The outer membrane (OM) of Gram-negative bacteria, such as Escherichia coli, is essential for their viability. Lipopolysaccharide (LPS) constitutes the major component of OM, providing the permeability barrier, and a tight balance exists between LPS and phospholipids amounts as both of these essential components use a common metabolic precursor. Hence, checkpoints are in place, right from the regulation of the first committed step in LPS biosynthesis mediated by LpxC through its turnover by FtsH and HslUV proteases in coordination with LPS assembly factors LapB and LapC. After the synthesis of LPS on the inner leaflet of the inner membrane (IM), LPS is flipped by the IM-located essential ATP-dependent transporter to the periplasmic face of IM, where it is picked up by the LPS transport complex spanning all three components of the cell envelope for its delivery to OM. MsbA exerts its intrinsic hydrocarbon ruler function as another checkpoint to transport hexa-acylated LPS as compared to underacylated LPS. Additional checkpoints in LPS assembly are: LapB-assisted coupling of LPS synthesis and translocation; cardiolipin presence when LPS is underacylated; the recruitment of RfaH transcriptional factor ensuring the transcription of LPS core biosynthetic genes; and the regulated incorporation of non-stoichiometric modifications, controlled by the stress-responsive RpoE sigma factor, small RNAs and two-component systems.

ADP-heptose: a bacterial PAMP detected by the host sensor ALPK1.

The innate immune response constitutes the first line of defense against pathogens. It involves the recognition of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors (PRRs), the production of inflammatory cytokines and the recruitment of immune cells to infection sites. Recently, ADP-heptose, a soluble intermediate of the lipopolysaccharide biosynthetic pathway in Gram-negative bacteria, has been identified by several research groups as a PAMP. Here, we recapitulate the evidence that led to this identification and discuss the controversy over the immunogenic properties of heptose 1,7-bisphosphate (HBP), another bacterial heptose previously defined as an activator of innate immunity. Then, we describe the mechanism of ADP-heptose sensing by alpha-protein kinase 1 (ALPK1) and its downstream signaling pathway that involves the proteins TIFA and TRAF6 and induces the activation of NF-κB and the secretion of inflammatory cytokines. Finally, we discuss possible delivery mechanisms of ADP-heptose in cells during infection, and propose new lines of thinking to further explore the roles of the ADP-heptose/ALPK1/TIFA axis in infections and its potential implication in the control of intestinal homeostasis.